Autophagy modulates temozolomide-induced cell death in alveolar Rhabdomyosarcoma cells

Adel Moghadam; Simone da Silva Rosa; Ehsan Samiei; Javad Alizadeh; Jared Field; Philip Kawalec; James Thliveris; Mohsen Akbari; Saeid Ghavami; Joseph Gordon

doi:10.1038/s41420-018-0115-9

Autophagy modulates temozolomide-induced cell death in alveolar Rhabdomyosarcoma cells

Moghadam, Adel; da Silva Rosa, Simone; Samiei, Ehsan; Alizadeh, Javad; Field, Jared; Kawalec, Philip; Thliveris, James; Akbari, Mohsen; Ghavami, Saeid; Gordon, Joseph 2018-10-25 00:00:00 Rhabdomyosarcoma (RMS) is a muscle-derived tumor. In both pre-clinical and clinical studies Temozolomide (TMZ) has been recently tested against RMS; however, the precise mechanism of action of TMZ in RMS remains unclear. Here we demonstrate that TMZ decreases the cell viability of the RH30 RMS and C2C12 cell line, where cells display evidence of mitochondrial outer membrane permeability. Interestingly, the C2C12 mouse myoblast line was relatively more resistant to TMZ-induced apoptosis. Moreover, we observed that TMZ activated biochemical and morphological markers of autophagy in both cell lines. Autophagy inhibition in both RH30 and C2C12 cells signiﬁcantly increased TMZ-induced cell death. In RH30 cells, TMZ increased Mcl-1 and Bax protein expression compared to corresponding time match controls while in C2C12 Mcl-1, Bcl-2, Bcl-XL, and Bax protein expression were not changed. Baf-A1 co- treatment with TMZ signiﬁcantly decrease Mcl-1 expression compared to TMZ while increase Bax expression in C2C12 cells (Bcl2 and Bcl-XL do not signiﬁcantly change in Baf-A1/TMZ co-treatment). Using a three-dimensional (3D) C2C12 and RH30 culture model we demonstrated that TMZ is signiﬁcantly more toxic in RH30 cells (live/dead assay). Additionally, we have observed in our 3D culture model that TMZ induced both apoptosis (cleavage of PARP) and autophagy (LC3-puncta and localization of LC3/p62). Therefore, our data demonstrate that TMZ induces simultaneous autophagy and apoptosis in both RH30 and C2C12 cells in 2D and 3D culture model, where RH30 cells are more sensitive to TMZ-induced death. Furthermore, autophagy serves to protect RH30 cells from TMZ-induced death. Introduction years, emphasizing an urgent need to develop new stra- Rhabdomyosarcoma (RMS) is an aggressive soft-tissue tegies to treat and prevent this disease . Four subgroups of malignant tumor that occurs in both children and adults , RMS have been described based on histological, genetic, but comprises up to 50% of all childhood soft tissue and clinical criteria : embryonal RMS, pleomorphic RMS, 2,3 sarcomas . Clinically, survival among patients with spindle cell/sclerosing RMS, and alveolar RMS (ARMS). metastatic RMS has not improved appreciably in the past ARMS is an aggressive subtype of RMS suffered by ado- lescents and young adults . In addition, the high-mortality rate in ARMS has been attributed to the presence of Correspondence: Saeid Ghavami (saeid.ghavami@umanitoba.ca)or oncogenic fusion proteins (i.e., PAX3-FOXO1 and PAX7- Joseph W. Gordon (joseph.gordon@umanitoba.ca) 6 FOXO1) generated by chromosomal translocations . Department of Human Anatomy and Cell Science, Max Rady College of Recently, oral alkylating agents such as TMZ have Medicine, Rady Faculty of Health Science, University of Manitoba, Winnipeg, Canada received considerable attention in RMS pre-clinical and Laboratory for Innovations in Microengineering (LiME), Department of clinical studies . TMZ has a broad spectrum of antitumor Mechanical Engineering, University of Victoria, Victoria, BC, Canada activity while being well-tolerated by the patient due to its Full list of author information is available at the end of the article Edited by: I. Amelio © 2018 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. 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The mechanism of action of family of proteins plays a crucial role in the regulation of TMZ results in the production of a highly reactive the cross-talk between apoptosis and autophagy, consist- methyldiazonium cation that transfers its methyl group ing of both pro-survival and pro-death family mem- 36,37 to purine bases of DNA resulting in double-stranded bers . However, the role of the BCL-2 family in 12–15 breaks during repair . This process leads to G2/M cell regulating apoptosis and autophagy in ARMS is not well 16–18 cycle arrest and activation of apoptosis . However, the dissected. cellular response to TMZ also involves alterations in gene In this report, we demonstrate that TMZ decreased cell expression that have been shown to be cancer-cell spe- viability of the RH30 RMS cell line and C2C12 cell line in ciﬁc. Thus, the pathways involved in apoptosis induction presence of autophagy activation. TMZ induces outer may be different for each type of sarcoma, and there is mitochondrial membrane permeabilization without little information regarding how TMZ affects ARMS at changes in mitochondrial membrane potential. We also the cellular level. In our studies, we explored the role of show that treatment of RH30 and C2C12 cells with low autophagy in RH30 cells to further elucidate the concentrations of the autophagy ﬂux inhibitor Baf-A1, mechanism of action of TMZ. signiﬁcantly increased TMZ-induced cell death. Collec- Autophagy is a conserved physiological process of cel- tively, these observations help deﬁne the role of autophagy lular self-eating, which plays an essential role in cellular in RH30 cells and contribute to our knowledge regarding housekeeping activity by degrading protein aggregates, the mechanism of action of TMZ in PAX3-FOXO1 cytoplasmic components, and damaged or dysfunctional positive sarcomas. organelles. At least three distinct forms of autophagy can be activated depending on the route that cytoplasmic Results material is delivered to lysosomes, such as chaperone- TMZ induces apoptotic cell death in C2C12 and RH30 cell mediated autophagy, microautophagy, and macro- lines 19–22 autophagy (from here on referred to as autophagy) . We treated both the RH30 RMS cell line and C2C12 The role of autophagy in cancer cell biology is compli- myoblasts with TMZ (0, 50, 100, 250, 500, and 1000 μMin cated and evolves throughout tumorigenesis. For instance, 48, 72, and 96 h) and cell viability was examined by MTT autophagy has been shown to promote cancer cell survival assay. TMZ-induced cell death in a dose- and time- during conditions of a nutrient or hypoxic stress and dependent manner in C2C12 cells (Fig. 1a–c) and RH30 contribute to cell demise through autophagic cell death cells (Fig. 1d–f), where RH30 cells appeared more sensi- (i.e., type II programmed cell death) tive to the effects of TMZ. We conﬁrmed that TMZ (100 . More recently, autophagy has also been shown to contribute to epithe- μM, 72 h) increases apoptotic cell death in both C2C12 lium to mesenchymal transition (EMT) and promote and RH30 cell lines using a propidium iodide (PI)- 21,24 cancer metastasis in different cancer models . In RH30 dependent method (i.e. Nicoletti assay). Following TMZ cells, autophagy is known to be a crucial process in the treatment, the percentage of apoptotic cells was maintenance of cellular viability and proliferation . Fur- approximately three times more than control C2C12 cells thermore, inhibition of autophagy by the Atg7 knock- (Fig. 1g). However, in RH30 cells, TMZ-induced apoptosis down, or pharmacological inhibition with chloroquine or by 17.5 times compared to the control cells (Fig. 1g). Baf-A1 treatment, has been demonstrated to decrease cell Therefore, TMZ signiﬁcantly activated higher levels of 26,27 growth and reduced viability in RMS cell lines . apoptosis in RH30 cells than in C2C12 cells (Fig. 1h). We Autophagy and apoptosis are two independent pro- also performed ﬂow cytometry analysis where cells were cesses, but under certain conditions, they cooperate in a stained with Annexin-V and PI (Fig. 1i, j). In C2C12 cells hierarchical relationship to regulate the turnover of treated with TMZ, we observed an increase in Annexin-V organelles and proteins within cells, and of cells within positive (A+/P−) cells, but no change in double positive 28,29 organisms . However, within a given cell, considerable cells (A+/P+). Interestingly, we also observed a decrease cross-talk exists between apoptosis and autophagy, and in PI positive cells (A−/P+) (Fig. 1i). Conversely, in RH30 nature of this cross-talk can change in a cell context- cells treated with TMZ we observed signiﬁcant increases 30–32 dependent manner . Generally, autophagy is a rapidly in Annexin-V positive and double positive cells, with a induced survival pathway activated by sublethal stress, corresponding decreased in double negative cells (A−/P whereas apoptosis is initiated at lethal doses of stress . −) (Fig. 1j). These observations are consistent with our However, in certain conditions, autophagy may also MMT and Nicoletti ﬁndings, and suggest that RH30 cells contribute to the induction of cell death by either the are more sensitive to the apoptosis inducing effects of activation of programmed cell death type II, activation of TMZ compared to C2C12 cells. mitochondrial-dependent cell death pathways (i.e. apop- To investigate the cellular mechanisms behind the dif- tosis or necrosis), or by providing substrates to promote ferential response to TMZ treatment, we performed a 33–35 ATP-dependent apoptotic mechanisms . The BCL-2 series of experiments to evaluate intrinsic cell death Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 3 of 16 Fig. 1 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 4 of 16 (see ﬁgure on previous page) Fig. 1 TMZ induces apoptotic cell death in C2C12 and RH30 cell lines. a–c C2C12 cells were treated with TMZ (50, 100, 250, and 1000 μM) and cell viability was assessed 48, 72, and 96 h after that by MTT assay. Control cells for each time point were treated with the solvent control (DMSO). Results are expressed as a percentage of corresponding time point control and represent the means ± SD of 15 replicates in three independent experiments (**P < 0.01; ***P < 0.0001). d–f C2C12 cells were treated TMZ (50, 100, 250, and 1000 μM) and cell viability was assessed 48, 72, and 96 h after that by MTT assay. Control cells for each time point were treated with the solvent control (DMSO). Results are expressed as a percentage of corresponding time point control and represent the means ± SD of 15 replicates in three independent experiments (**P < 0.01; ****P < 0.0001). g Representative ﬁgures of the ﬂow cytometry histogram for C2C12 and RH30 are shown. Cells were treated with TMZ (100 μM, 72 h) and Percent sub- G1 abundance induced by TMZ (100 μM) or DMSO solvent control after 72 h. The Sub-G1 population showed an abundance of apoptotic cell death in each treatment. h The average of the sub-G1 population of C2C12 and RH30 cells which were treated with TMZ (100 μM, 72 h) and DMSO solvent control has been measured. Results represent the means ± SD of six replicates in three independent experiments (****P < 0.0001). i, j C2C12 and RH30 cells were treated with TMZ and stained with Annexin-V-FITC (A) and PI (P) and analyzed by ﬂow cytometry (n = 3)(*P < 0.05). k TMRM staining of C2C12 and RH30 cells treated with TMZ (100 μM, 48 h). l MitoTracker staining of C2C12 and RH30 cells treated with TMZ (100 μM, 48 h). m C2C12 and RH30 cells were transfected with cytochrome C-GFP (CytoC-GFP) and treated with TMZ (100 μM, 48 h)(*P < 0.05). n, o C2C12 and RH30 cells were treated with 100 μM TMZ for 48 h and then Caspase-9 activity was measured using Caspase-Glo luminescence assay. (**P < 0.01) pathways initiated by mitochondria. We stained cells with 9 activity was unchanged in C2C12s at this dose and time the mitochondrial membrane potential-sensitive dye (P > 0.05). 38,39 TMRM , as rapid dissipation of mitochondrial mem- brane potential has been recently associated with mito- TMZ induces autophagy activation in C2C12 and RH30 chondrial permeability transition. Surprisingly, we cells observed that TMZ treatment did not lead to mitochon- Previous reports have shown that TMZ induces simul- drial depolarization at this dose and time in either cell line taneous apoptosis and autophagy in glioblastoma 22,47 (Fig. 1k). No change in MitoTracker staining was cells . Thus, we evaluated if differential activation of observed in RH30 cells, indicating that mitochondrial autophagy could explain the increased toxicity to TMZ in content was not altered by TMZ treatment, however we RH30 cells compared to C2C12 cells. Our results did observe a modest, but signiﬁcant reduction in Mito- demonstrate that TMZ induces autophagy in both C2C12 Tracker staining in C2C12 cells (Fig. 1l). Next, we trans- and RH30 cell lines. Immunoblotting results showed fected cells with a cytochrome C-GFP fusion construct lipidation of LC3B, Atg5-12 conjugation, and increase in that has been previously shown to accumulate in the Beclin-1 expression in both cell lines, indicating autop- mitochondrial intermembrane space and be released into hagy activation by TMZ (100 µM) (Fig. 2a). In RH30 and the cytosol during mitochondrial outer membrane per- C2C12 cells, ﬂuorescent microscopy conﬁrmed that TMZ meabilization (MOMP) . Following transfection, we increased the number of LC3-GFP puncta and lysosomal observed ﬂuorescent mitochondrial puncta in both activation (LysoTracker red ﬂuorescence intensity), which untreated cell lines (Fig. 1m). When RH30 and C2C12 co-localized in TMZ treated cells (Fig. 2b, c). Also, cells were treated with TMZ, we observed release of transmission electron microscopy (TEM) further con- cytochrome C-GFP into the cytosol. In RH30 cells, no ﬁrmed autophagy activation in RH30 and C2C12 cells ﬂuorescent puncta remained following TMZ treatment; (double membrane autophagosome formation), after 72 h however, in C2C12 cells residual puncta remained in up treatment with TMZ (100 µM) (Fig. 2d). to 20% of cells (Fig. 1m). Collectively, these ﬁndings To investigate whether the accumulation of LC3B-II is indicate that TMZ induces MOMP without mitochon- due to an increase in autophagic ﬂux or prevention of drial permeability transition. autophagic proteolysis, we used Baf-A1 (100 nM) for Caspase-9 is an important effector of intrinsic or different time points (2, 3, 4 h) to study autophagy ﬂux. It 41–44 mitochondrial-regulated apoptosis . It has been has been previously shown that a high concentration of recently reported that TMZ induces caspase-dependent Baf-A1 with short time point exposures decreases lyso- 45,46 apoptosis in several cancer cells . Thus, we determined some numbers, causes fusion block, and suppresses the effects of TMZ-induced MOMP on caspase-9 acti- autolysosome destruction by interfering with late-stage 19,48 vation. Following treatment of TMZ 100 μM for 48 h autophagosome-lysosome fusion . As shown by wes- (Fig. 1n, o), we observed a signiﬁcant activation of tern blot, treatment with Baf-A1 (100 nM) 2, and 4 h caspase-9 in RH30 cells (**P < 0.01); however, the caspase- markedly increased the accumulation levels of LC3-II and Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 5 of 16 Fig. 2 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 6 of 16 (see ﬁgure on previous page) Fig. 2 TMZ induces autophagy in C2C12 and RH30 cell lines. a C2C12 and RH30 cells were treated with TMZ (100 μM, 36, 72, 96 h) and autophagy hallmark was detected in both C2C12 and RH30 cell lines using immunoblotting. TMZ-induced LC3β lipidation, Atg5-12 conjugation, and Beclin-1 expression in both cell lines. Beta-actin was used as loading control. Data are representative of three independent experiments using different cultures. b, c RH30 and C2C12 cells were treated with TMZ (100 μM, 72 h) and using immunocytochemistry LC3 puncta and changes in lysosomal activity (LysoTracker red staining) has been investigated. The results showed that TMZ increased LC3 puncta and LysoTracker red ﬂuorescence intensity and co-localization of LC3 puncta and LysoTracker in both RH30 and C2C12 cells. d Transmission electron microscopy showed that in treated RH30 and C2C12 cells there are accumulated autophagosome-like structures compared to control and normal cells after 72 h treatment. Arrows show the autophagolysosomes containing the cargo (magniﬁcation ×11,600). e C2C12 cells were treated with TMZ (100 µM, 72 h) and then treated with Baf-A1 (100 nM, 1, 2, 3, 4 h) to evaluate for autophagy ﬂux. TMZ+ Baf-A1 treatment induces more lipidated LC3β and reduces the degradation of p62 in RH30 cells. Beta-actin was used as the loading control. Data are representative of three independent experiments. f C2C12 cells were treated with TMZ (100 μM, 72 h) and Baf-A1 (100 nM, +4 h) followed by immunocytochemistry to evaluate LC3 puncta and changes in lysosomal activity (LysoTracker red staining). The results showed that TMZ increased LC3 puncta and LysoTracker red ﬂuorescence intensity in C2C12 cells. On the other hand, Baf-A1 and TMZ + Baf-A1 signiﬁcantly decreased the LysoTracker red ﬂuorescence in the presence of an accumulation of LC3 puncta showing the inhibition of autophagy ﬂux by Baf-A1. g, h Representative ﬁgures of LC3 puncta and ﬂuorescence intensity for LysoTracker in C2C12 cells in the presence of TMZ, Baf-A1, and TMZ/Baf-A1 treatment. These results showed that the number of LC3 puncta is signiﬁcantly higher in cells which are treated with Baf-A1 and TMZ + Baf-A1. However, the ﬂuorescence intensity of LysoTracker was lower in Baf-A1, and TMZ + Baf-A1 treated cells. i RH30 cells were treated with TMZ (100 µM, 72 h) and then treated with Baf-A1 (100 nM, 1, 2, 3, 4 h) to evaluate for autophagy ﬂux. TMZ/ Baf-A1 treatment induces more LC3β lipidation and reduces the degradation of p62 in RH30 cells compared to TMZ and Baf-A1 single treatment. Beta-actin was used as the loading control. Data are representative of three independent experiments. j RH30 cells were treated with TMZ (100 μM, 72 h) and Baf-A1 (100 nM, +4 h) followed by immunocytochemistry to evaluate LC3 puncta and changes in lysosomal activity (LysoTracker red staining). The results showed that TMZ increased LC3 puncta and LysoTracker ﬂuorescence intensity in RH30 cells. On the other hand, Baf-A1 and TMZ+ Baf-A1 signiﬁcantly decreased the LysoTracker red ﬂuorescence showing the inhibition of autophagy by Baf-A1. k, l Representative ﬁgures of LC3 puncta and ﬂuorescence intensity for LysoTracker in RH30 cells in the presence of TMZ, Baf-A1, and TMZ/Baf-A1 treatment. These results showed that many LC3 puncta are signiﬁcantly higher in cells which are treated with Baf-A1 and TMZ+ Baf-A1. However, the ﬂuorescence intensity of LysoTracker was lower in Baf-A1, and TMZ+ Baf-A1 treated cells P62 in both C2C12 and RH30 cells. Combination treat- inhibits autophagy, but cell toxicity can occur at higher 49,50 ment of cells with TMZ (100 µM, 72 h) and Baf-A1 (100 doses . Therefore, we performed a cell viability assay nM) at 1, 2, 3, and 4 h showed a time-dependent accu- (MTT) to establish the appropriate concentration of mulation of LC3B-II compared to cells treated with Baf- Baf-A1 to inhibit autophagy with the least cytotoxic A1 (100 nM) alone (C2C12 cells Fig. 2e; RH30 cellsFig. 2i). effect on C2C12 and RH30 cells. C2C12 and RH30 cells These results indicate that TMZ induces autophagy ﬂux were treated with different concentration of Baf-A1 in both cell lines. Consistent results were obtained using (0.1–10 nM) for 48 and 72 h. Our results revealed that GFP-LC3, where in cells treated with a combination of Baf-A1 (4 and 6 nM) had low cytotoxicity at 72 h in both TMZ and Baf-A1 (3 h, 100 nM) we observed increased cell lines (Fig. 3a, b), thus we evaluated the effectiveness GFP-LC3 puncta, compared to cells treated with Baf-A1 of these two Baf-A1 concentrations to inhibit autop- (100 nM) alone (C2C12 cells Fig. 2e; RH30 cells Fig. 2i). hagy. Cells were treated with Baf-A1 (4 and 6 nM) for Lysosomal activity was found to be lower in cells after 48, and 72 h and autophagy markers (LC3 lipidation and treatment with Baf-A1 (100 nM) alone, as well in com- P62 degradation/accumulation) were investigated in bination with TMZ (100 µM, 60 h) compared to the both cell lines using western blotting. We observed that control and TMZ alone group, conﬁrming lysosomal Baf-A1 (4 and6nM)inhibitsautophagy,determinedby acidiﬁcation inhibition and blockage of the autophagy ﬂux the accumulation of lipidated LC3B-II and P62 (Fig. 3c) (C2C12 cells Fig. 2f–h; RH30 cells Fig. 2i–l). Moreover, in both C2C12 and RH30 cells. Next, we conﬁrmed the we demonstrate that the number of autophagic C2C12 effect of Baf-A1 (4 and 6 nM) on autophagy markers in cells is higher in cells treated with Baf-A1 with and C2C12 and RH30 cells treated with TMZ (100 µM/ml; without TMZ. However, the ﬂuorescence intensity of Fig. 3d, e). Finally, we evaluated apoptosis by ﬂow LysoTracker was lower in Baf-A1 and in TMZ + Baf-A1 cytometry analysis with PI staining in cells treated with treated cells (C2C12 cells Fig. 2f–h; RH30 cells Fig. 2i–l). TMZ (100 μM) and Baf-A1 (4 nM) for 72 h. Both TMZ Overall, these ﬁndings suggest that TMZ induces autop- and hagy ﬂux in C2C12 and RH30 cells. Baf-A1 increased the apoptotic cell population com- pared to control; however, the percentage of apoptosis Autophagy inhibition increases TMZ-induced apoptosis in in cells treated with the combination of TMZ and Baf- C2C12 and RH30 A1 was greater than either TMZ or Baf-A1 alone Next, we examined the effect of chemical inhibition of (Fig. 3f–h). Collectively, these ﬁndings demonstrate that autophagy on TMZ-induced apoptosis. Previously, we autophagy inhibition increases TMZ-induced apoptosis demonstrated that low dose concentrations of Baf-A1 in both C2C12 and RH30 cells. Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 7 of 16 Fig. 3 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 8 of 16 (see ﬁgure on previous page) Fig. 3 Autophagy inhibition increases TMZ-induced apoptosis in C2C12 and RH30 cells. a, b C2C12 and RH30 cells were treated with Baf-A1 (2, 4, 6, 8, and 10 nM) and cell viability was assessed after 72 h using MTT assay. Control cells for each time point were treated with the solvent control (DMSO). Results are expressed as a percentage of corresponding time point control and represent the means ± SD of 15 replicates in three independent experiments (**P < 0.01; ***P < 0.0001). c C2C12 and RH30 cells were treated with Baf-A1 (4 and 6 nM, 48, 72 h) and autophagy hallmark proteins were detected in both C2C12 and RH30 cell lines using immunoblotting. Baf-A1 induces accumulation of lipidated LC3β and decreases the degradation of p62 in both cell lines. Beta-actin was used as loading control. Data are representative of three independent experiments using different cultures. d, e C2C12 and RH30 cells were treated with TMZ (100 µM, 72 h) and then treated with Baf-A1 (4, 6 nM, 72 h) to evaluate autophagy inhibition in the presence of Baf-A1 treatment. TMZ+ Baf-A1 treatment induces LC3β lipidation and reduces the degradation of p62 in C2C12 and RH30 cells. Beta-actin was used as the loading control. Data are representative of three independent experiments. f–h Representative ﬁgures of the ﬂow cytometry histogram for RH30 are shown. Cells were treated with TMZ (100 μM, 72 h), Baf-A1 (4 nM, 72 h) and Baf-A1/TMZ. Percentage of sub-G1 abundance induced by TMZ (100 μM, 72 h), Baf-A1 (4 nM, 72 h) and Baf-A1/TMZ after 72 h has been showed. The Sub-G1 population showed an abundance of apoptotic cell death in each treatment. g, h C2C12 and RH30 cells were treated with TMZ (100 µM, 72 h) and Baf-A1 (4 nM, 72 h) and then cell lysates were collected to examine the effect of TMZ, Baf-A1, and TMZ/Baf-A treatment on expression of Bcl2 family proteins (Bcl-2, Bcl-XL, Mcl-1, and Bax). k–r, k, l TMZ does not signiﬁcantly change Mcl-1 expression in both C2C12 and RH30 cells. It is notable that Baf-A1 (6 nM)/TMZ co- treatment signiﬁcantly (P < 0.01) decrease Mcl-1 expression in C2C12 cells while does not have any effect in RH30 cells. m, n TMZ does not signiﬁcantly change Bcl-2 expression in both C2C12 and RH30 cells. TMZ/Baf-A combination treatment also does not signiﬁcantly change Bcl-XL expression in both C2C12 and RH30 cells. o, p TMZ does not signiﬁcantly change Bcl-XL expression in both C2C12 and RH30 cells. TMZ/Baf-A combination treatment also does not signiﬁcantly change Bcl-2 expression in both C2C12 and RH30 cells. q, r TMZ does not signiﬁcantly change Bax expression in both C2C12 and RH30 cells. TMZ/Baf-A combination treatment signiﬁcantly increase Bax-expression in C2C12 cells (P < 0.01) while does not have a signiﬁcant effect on its expression in RH30 cells. s–u We have evaluated changes in mitochondrial membrane potential in the presence of TMZ (100 µM, 60 h), Baf-A1 (4 nM, 60 h), and TMZ/Baf-A1 combination in C2C12 and RH30 cells. s Representative images show the mitochondrial membrane potential measured by TMRM. Red color denotes TMRM staining. t, u Measurement of the mean of TMRM ﬂuorescence intensity shows that TMZ (100 µM, 60 h), Baf-A1 (4 nM, 60 h), and TMZ/Baf-A1 combination does not change mitochondrial membrane potential in C2C12 t and RH30 u cells. The data are representative of the mean ﬂuorescence in at least 100 cells in each cell type. The data were analyzed by Student’s t-test or ANOVA, followed by post hoc analysis. If p < 0.05, results were considered statistically signiﬁcant To further dissect the mechanisms by which 5 mm and thickness = 1 mm µm) to evaluate the effect of TMZ-induces apoptosis in presence of autophagy inhi- TMZ. These disks were fabricated by mixing C2C12 and bition, we evaluated the expression of Bcl2 family proteins RH30 cells with collagen type I (3 mg/mL) at a ﬁnal by western blot in RH30 and C2C12 cells treated with density of 2 × 10 cells/mL and casting the disks in poly- 31,35–37 TMZ and/or Baf-A1, as previously described .We dimethylsiloxane (PDMS) molds. After 24 h, cells were evaluated Bcl-2, Bcl-xl, Mcl-1 (anti-apoptotic), and Bax treated with TMZ (0–500 µM) for 48 and 96 h. Cell via- (pro-apoptotic). As shown in Fig. 3i, j, Baf-A1 (6 nM) co- bility was evaluated by staining cells with treatment with TMZ (100 µM) signiﬁcantly decreased green-ﬂuorescent calcein-AM (live) and red-ﬂuorescent Mcl-1 expression (P < 0.01) compared to TMZ (100 µM) ethidium homodimer-1 (dead) dyes (Fig. 4c, d). Our with increased Bax expression (P < 0.01) in C2C12 cells. results showed a signiﬁcant decrease in the number of live Bcl-2 and Bcl-XL did not signiﬁcantly change in Baf-A1/ cells in TMZ-treated groups in both C2C12 and RH30 TMZ co-treatment (Figure K, M, N, and Q). Interestingly, cells (P < 0.001), while TMZ induces more signiﬁcant cell we did not see any signiﬁcant changes in RH30 cells with death in RH30 cells (Fig. 4e, f). Bright ﬁeld images regards to levels of Mcl-1, Bcl-2, Bcl-XL, or Bax expres- revealed signiﬁcant changes in cell morphology of TMZ sion after co-treatment of Baf-A1/TMZ (Fig. 3l, n, p, r). treated cells compared to control group in both C2C12 We also evaluated the effect of TMZ (100 µM, 60 h) and (Fig. 4a) and RH30 cells (Fig. 4b). Additionally, TMZ autophagy inhibition (Baf-A1, 4 nM, 60 h) on mitochon- treatment also induced higher PARP cleavage compared drial membrane potential in C2C12 and RH30 cells. to control in both C2C12 (Fig. 4g) and RH30 cells Interestingly, the results showed that TMZ, Baf-A1, and (Fig. 4h). Labeling of C2C12 and RH30 cells by using TMZ/Baf-A co-treatment do not affect mitochondrial autophagy markers demonstrated an increase in the membrane potential in either C2C12 and RH30 cells number of autophagosomes in TMZ-treated groups as (Fig. 3s–u). compared to corresponding time match controls (Fig. 4i, j). TMZ induces apoptosis and autophagy in C2C12 and RH30 3D culture Discussion Three-dimensional (3D) cellular models have been In the present study, we demonstrate that TMZ differ- extensively used to mimic the 3D microenvironment of entially induces apoptotic cell death in C2C12 and RH30 cells and to study the effects of extracellular matrix as a cells in both 2D and 3D culture models, and that the cell 51,52 barrier against drug diffusion . We used 3D models of death is negatively regulated by autophagy. Interestingly, C2C12 and RH30 made of cell collagen disks (diameter = TMZ treatment does not change mitochondrial Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 9 of 16 Fig. 4 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 10 of 16 (see ﬁgure on previous page) Fig. 4 TMZ induces apoptosis and autophagy in C2C12 and RH30 3D culture. a, b Bright ﬁeld image of C2C12 a and RH30 b 3D culture which shows the morphology of untreated and TMZ treated cells (100, 250 µM, 4 h) in 3D culture. c–f Viability assay was done by adding the live/dead solution to cells 48 and 96 h after treatment with TMZ (0–500 µM). Cells were incubated for 2 h in the dark at room temperature, rinsed twice with DPBS, and confocal microscopy was used to capture live/dead cell images in C2C12 c and RH30 d cells. e, f Quantiﬁcation of live/dead assay was measured by calculating the ratio of live: total cells which showed a signiﬁcant decrease in viability of C2C12 e and RH30 f cells treated with different concentrations of TMZ. The data showed TMZ signiﬁcantly induces cell death in both C2C12 and RH30 cells (P < 0.001) while TMZ induces more cell death in RH30 compared to C2C12 cells. g, h IF labeling of C2C12 cells g and RH30 cells h by cleaved PARP following treatment with TMZ (100 µM, 72 h) increased number of cells with cleaved PARP in TMZ treated cells in comparison to control cells which is the hallmark of increase of apoptosis in these cells. i, j After treatment of C2C12 i and RH30 j cells with TMZ (100 µM, 72 h), cells were IF labeled with autophagosome markers, LC3 and P62. Data showed that TMZ increases LC3 puncta (green) which is localized with p62 compared to corresponding time match control, a hallmark of autophagy induction in C2C12 and RH30 3D culture membrane potential in either RH30 or C2C12 cells, and increase in caspase-9 activation) while TMZ-induced the apoptosis is not caspase-9 dependent in C2C12 cells. It differential TMZ-induced apoptosis in C2C12 and RH30 has been reported that TMZ-induced apoptosis is caspase 68– cells cannot be entirely explained by changes in Bcl-2 dependent in different models, including glioma cells 70 71 anti-apoptotic (Bcl-2, Bcl-XL, and Mcl-1) and/or , and human glioma stem cells . It has been previously pro-apoptotic (Bax) protein expression. reported that TMZ induces endoplasmic reticulum (ER)- There are currently a few therapeutic options available stress with subsequent unfolded protein response in many for patients with metastatic RMS . To improve the cell models. We are currently investigating whether dif- therapeutic effect of chemotherapy in RMS, TMZ ferences in the ER-stress response or the extrinsic apop- has recently received considerable interest as a factor tosis pathway underlies the differential responsiveness of 22,72 for combination therapy because it is a well-tolerated C2C12 and RH30 cells to TMZ treatment . oral alkylating agent with a broad spectrum of antitumor Mitochondrial Bcl-2 family proteins are considered to 12,13 activity and relatively low toxicity . Several Phase I be one of the critical components in the regulation of 73–75 trials suggest that using TMZ alone or in combination apoptotic cell death and known to be essential for the with other drugs is well tolerated in RMS when used in apoptotic response in chemotherapy, emphasizing that 54 76,77 relapse settings these proteins are potential therapeutic targets . The combination of irinotecan and . TMZ, for instance, has shown synergistic antitumor Interestingly our results showed that TMZ-induced 54–57 activity against RMS . Several studies have pointed apoptosis is not associated with changes in Bcl-2, out that autophagy acts as a pro-survival pathway which Bcl-XL, Mcl-1, and Bax expression in RH30 and C2C12 allows cancer cells to survive the existing stressed present cell lines. It has been previously reported that 23,58,59 in the tumor microenvironment , such as the TMZ-induced apoptosis decreases mitochondrial mem- 60 78,79 stresses caused by anticancer agents . It has been found brane potential in glioma cell line while our results that autophagy and apoptosis have multiple direct and showed that TMZ-induced apoptosis had not affected the 31,61,62 indirect interactions with each other . In the present mitochondrial membrane potential in both RH30 and study, we observed that inhibition of autophagy could C2C12 cells. This observation can be explained by our enhance the apoptotic effect of TMZ in C2C12 and RH30 ﬁnding that TMZ has not affected the expression of anti- cells; therefore, autophagy is a negative regulator of TMZ- apoptotic (Bcl-2, Bcl-XL, and Mcl-1), and pro-apoptotic induced apoptosis in both C2C12 and RH30 cells. (Bax) Bcl-2 family proteins. In addition, our data show We have also determined that TMZ can activate that inhibition of autophagy by using Baﬁlomycin-A1 apoptosis in a time and dose-dependent manner in RH30 increased TMZ-induced apoptosis in RH30 and C2C12 cells. The enhancement of apoptosis was found to be cell lines without affecting mitochondrial membrane associated with higher expression of histone H2A variant potential in these cells H2AX (data not shown). The phosphorylation of histone This pilot study has expanded our understanding of the H2A variant H2AX at Ser139 is a marker of DNA double- cross-talk between apoptosis and autophagy controlling strand breaks, a type of DNA damage that can lead to cell TMZ-induced apoptosis. It has been shown that the 63,64 death, if unrepaired . This mechanism of TMZ- maintenance of cellular viability in RH30 cells is depen- induced apoptosis has been validated in previous studies dent on baseline autophagy , while loss of ATG7 or Baf- in other cancer cell lines which is consistent with our A1 treatment can negatively regulate proliferation of 11,65–67 26,27 results . Our results demonstrated that intrinsic or RH30 cells . We found that Baf-A1 inhibited baseline mitochondrial pathway is involved in the induction of autophagy in RH30 and C2C12 cells and decreased their apoptosis after TMZ treatment in RH30 cells (signiﬁcant viability. More importantly, our study showed that the Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 11 of 16 combination treatment of RH30 cells with Baf-A1 and Sigma-Aldrich (St. Louis, MO, USA). Autophagy inhibitor TMZ has a synergic effect on Baﬁlomycin-A1 (Baf-A1), rabbit anti-human/mouse/rat TMZ-induced apoptosis compared to treatment with LC3 (L8918, 1:3,000), anti-mouse IgG (A8924, 1:3000), either TMZ or Baf-A1 alone. This suggests that autophagy Temozolomide, anti-rabbit IgG (A6154, 1:5000), propi- has a survival role in RH30 and C2C12 cells treated with dium iodide (PI), and 3-(4,5-dimethyl-2-thiazolyl)-2,5- TMZ and the autophagy inhibitor enhances the anti- diphenyl-2H-tetrazolium bromide) (MTT) were pur- chased from Sigma-Aldrich Canada Co, Oakville, CA. tumor effect of TMZ against RH30 cells by activation of apoptosis. Caspase-Glo®-9 assay was purchased from Promega We have reproduced two-dimensional (2D) RH30 and (Toronto, ON, Canada). Enhanced chemiluminescence C2C12 culture results in a 3D culture models (ECL) prime regents (western blotting detection reagent) (Fig. 4) which showed that TMZ induces higher apoptotic were purchased from Amersham-Pharmacia Biotech. cell death in RH30 cells in the presence of autophagy Hyperﬁlm™ ECL was purchased from Fisher Scientiﬁc. activation in both cell models. These discoveries are an Polydimethylsiloxane (PDMS) was purchased from Dow essential step for translational application towards devel- Corning®™, 5 mm diameter sterile biopsy punches were oping new therapies for RMS. The ﬁndings in most cell- purchased from Stevens Company, 5 mg/mL bovine col- based assays are demonstrated in monolayer or suspen- lagen type 1 with neutralized pH was purchased from sion culture models, which may give false negative or Advanced BioMatrix. Penicillin-streptomycin (Pen-Strep, positive results due to an artiﬁcial cellular environment. It 10,000 units/mL penicillin and 10,000 µg/mL streptomy- also represents restricted signiﬁcance for in vivo studies cin) was purchased from Gibco®, fetal bovine serum (FBS) and often little value in foreseeing clinical effectiveness of was purchased from Millipore Sigma. Secondary anti- both generally cytotoxic and molecularly targeted drugs . bodies with conjugated ﬂuorescence (Alexa Fluor® 488 3D cultures have received more attention and become AfﬁniPure Donkey Anti-Rabbit IgG, and Alexa Fluor® 647 more accepted in the last few years as an essential stra- AfﬁniPure Donkey Anti-Mouse IgG) were purchased the 80,81 tegic toolkit for developing new cancer therapy . Fur- from Jackson ImmunoResearch Inc. DAPI (4’,6-Diami- thermore, 3D in vitro culture systems mimic different dino-2-Phenylindole, Dihydrochloride) was purchased aspects of human tumor tissue environment, therefore from Thermo Fisher Scientiﬁc. IgG-free bovine serum should be considered as an advanced model for routine albumin (BSA) was purchased from Jackson ImmunoR- anti-tumor drug testing . In our current ﬁndings, we esearch Inc. Live/dead viability kit was purchased from Millipore Sigma; formaldehyde 37% solution was pur- conﬁrmed that 3D RMS culture can be used to dissect the role of TMZ in RMS therapy. chased from VWR, Triton X-100 was purchased from Bio Our ﬁndings revealed that autophagy is a negative Basic Canada Inc. pCytochrome C-GFP was a gift from regulator of TMZ-induced apoptosis in both RH30 and Douglas Green (Addgene plasmid # 41182) . C2C12 cells. In addition, we have shown that TMZ- induces MOMP and apoptosis that is not readily Cell lines and cell culture explained by changes in Bcl-2, Bcl-XL, Mcl-1, or Bax Cell culture plastic ware, penicillin, and streptomycin expression, and is not accompanied by changes in mito- were purchased from VWR (Toronto, ON, Canada). Cells chondrial membrane potential. were cultured in Roswell Park Memorial Institute (RPMI- 1640) with L-glutamine and 25 mM HEPES (BioWhit- Materials and methods taker; Cat #: 12-115Q) and Dulbecco’s Modiﬁed Eagle’s Drugs and reagents Medium (DMEM) (CORNING; Cat #: 50-003-PB) with Antibodies against human p62 (5114, 1:1000), Bax 10% fetal bovine serum (FBS) (Gibco™; Cat #: 16000044). (5023, 1:1000), Bcl-2 (3498, 1:1000), Bcl-xl (2762,1:1000) The human rhabdomyosarcoma cell line (RH30) [RC13, Mcl-1 (94296, 1:1000) Bak (12105, 1:1000) Nix (12396, RMS 13, SJRH30] (ATCC® CRL¬ 2061™) (Human muscle 1:1000) Beclin-1(3495, 1:1000), SQSTM1/p62 (D5L7G) cancer cells) and mouse muscle cell line (C2C12) (ATCC® (88588, 1:100 for ICC), LC3B (D11) XP® (3868, 1:100 for CRL¬1772™) were used in this project. RH30 cell lines ICC), Cleaved PARP (Asp214) (D64E10) XP® (5625, 1:100 were cultured in (RPMI-1640) with L-glutamine and 25 for ICC), and Bid (2002, 1:1000) were purchased from Cell mM HEPES media, and C2C12 cells were cultured in Signaling Technology (Beverly, MA, USA); LC3 was (DMEM) with high glucose media. Both media were purchased from Sigma-Aldrich (St. Louis, MO, USA); supplemented with FBS (10%), penicillin (1%), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) streptomycin (1%). Cells were grown to 35–40% con- and Actin were purchased from Santa Cruz Biotechnol- ﬂuency on a 100 mm cell culture plate, 6-well plates, and ogy, Inc. (Dallas, TX, USA). Anti-rabbit IgG (whole 96-well plates. Cells were maintained in a humidiﬁed molecule) and anti-mouse IgG (Fab speciﬁc) peroxidase- incubator with 95% air and 5% CO at 37 °C and were conjugated secondary antibodies were purchased from passaged once every 2–3 days. Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 12 of 16 Cell viability assay three times with 1X TBST (0.025% Tween) for 20 min, The RH30 cells (30,000 cells/ml) and C2C12 (20,000 and membranes were incubated with secondary anti- cells/ml) were seeded in 96-well plates and treated with bodies (HRP) for 2 h on the shaker at RT. Membranes different concentrations of TMZ in different time points were rewashed three times for 20 min and incubated with (0–1000 μM, 0–96 h). Cells were also treated with various enhanced chemiluminescence (ECL) reagents (Amer- concentrations (0.1, 1, 2.5, 5, or 10 nM) of Baf-A1, an sham-Pharmacia Biotech) for 2–3 min. Autoradiography visualized the signals. Obtained protein bands were eval- autophagy inhibitor, and cell viability was assessed after 48 and 72 h. In each time point, 20 µl of MTT uated for changes in the autophagy and apoptosis sig- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- naling pathways. To assess even protein loading, mide (5 mg/ml), is the aqueous solubility of the reduced membranes were incubated in milk 1% with primary formazan product, was added into each well and incu- antibodies against GAPDH or Actin overnight, washed bated for 3 h. Then, media gently were removed from three times and probed with a secondary antibody to each well using pipette and 200 µl of solvent control visualize the signals. In the instances of re-probing of (DMSO) was added to each well and mixed very well by other proteins on the same membrane, blots were incu- pipetting to solubilize the MTT formazan. The plates bated with stripping solution containing 200 nM glycine, were analyzed at a test wavelength of 570 nm on a plate pH 2.5, 0.005 Tween 20 for 15 min at RT and followed the 83,84 reader, following a 20 min incubation at room tempera- same instruction as after blocking for these blots . ture (RT) . Measurement of apoptosis by ﬂow cytometry Immunoblotting Apoptotic cells were assessed by ﬂow cytometry with 85,86 Western blot analysis was used to assess markers of propidium iodide (PI), using the Nicoletti method . apoptosis and autophagy in RH30 and C2C12 cells. We RH30 and C2C12 cells were treated with TMZ (100 μM, examined hallmarks of intrinsic or extrinsic apoptotic 72 h) in cells cultured in 12-well plates. In each time point pathway and autophagy signaling pathways, while cells were detached by EDTA buffer and centrifuged at GAPDH or actin was used to normalize the results. After 1500×g for 5 min at 4 °C. Then, cells were washed by PBS treatment, cells were collected, and protein extracts were once. The cells were permeabilized and treated with a made using NP-40 lysis buffer (20 mM Tris-HCl (pH 7.5), ﬂuorescent dye that stains DNA quantitatively, using 0.5% Nonidet P-40, 0.5 mM PMSF, 100 µM β-glycerol 3- hypotonic PI lysis buffer (0.1% Triton X-100, 1% sodium phosphate and 0.5% protease inhibitor cocktail). The citrate, 0.5 mg/ml RNase A, 40 μg/ml propidium iodide). extracts were kept in −20 °C until all extracts from dif- Before ﬂow cytometry analysis, cells were incubated for at ferent time points were collected. Samples were then least 1 h, at 4 °C, and in the dark to prevent photo- sonicated in ﬁve times/ﬁve cycles using ultrasound soni- bleaching. The measurement was in red ﬂuorescence cator, followed by centrifugation at 13,000×g for 10 min to (460 nm) for 10,000 cells. Flow cytometer was adequately collect the supernatant protein. Protein content was then calibrated to gate out debris accurately. Finally, after determined via a Lowry protein assay, and protein sam- elimination of residual debris, the percentage of normal ples were made. Prepared samples, of a volume between and apoptotic nuclei were estimated by analysis of the 86,87 15 and 20 µl, were heated at 90 °C for 5 min before loading DNA histogram . The nuclei of apoptotic cells were into 10–15% polyacrylamide gels (depending on the located on the left side of the G1 peak. Apoptotic nuclei molecular weight of the proteins). Additionally, 10 µl of a have less DNA compared to nuclei of healthy G0/G1cells, standard molecular weight marker (Thermo Fischer Sci- causing an increase in sub-G1 section in the ﬂuorescence entiﬁc, ON, Canada) was loaded on each gel, as an histogram which can be applied to distinguish apoptotic approximate indicator of molecular protein weights. cells in samples. In each sample, the sub-G1 peak was Proteins were immediately transferred under reducing measured and statistically compared with other samples . conditions in transfer buffer (500 nM glycine, 50 mM Annexin-V FITC and PI staining was performed accord- Tris-HCl, and 20% methanol) to Immuno-Blot PVDF ing to manufacturer’s instructions (BD Biosciences Membranes (Bio-Rad; #1620177), at RT and 100 volts for 556547). Stained cells were analyzed on a Thermo Sci- 2–2.5 h. Upon transferring completion, membranes were entiﬁc Attune NxT ﬂow cytometer with a 488 nm laser. carefully transferred into 5% non-fat dried milk in 1X Tris-buffered saline containing Tween (TBS/0.025% Live cell imaging: LC3-GFP tween 20; TBST) and placed on the shaker in the cold GFP-LC3 is a speciﬁc marker for the occurrence of 88,89 room overnight or RT for 2 h. Following blocking, autophagosomes formation . GFP-LC3 is the fusion of membranes were incubated with the proper dilution of the green ﬂuorescent protein (GFP) and LC3 and can 90,91 primary antibodies in 1% milk made in 1X TBST and kept behave similarly as endogenous LC3 . The GFP-LC3 is in cold room (4 °C) overnight. Membranes were washed localized on the autophagosome membrane, and green Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 13 of 16 punctate signals are observed . To conﬁrm By removing cellular growth media before staining, as well TMZ-induced autophagy and autophagy ﬂux inhibition as well by washing C2C12s with phosphate buffered saline through Baf-A1 (100 nM), cells were transfected with a (1×, Hyclone), but not RH30, as RH30 cells can be easily green ﬂuorescent protein plasmid called LC3-GFP detached, staining sensitivity was increased. (Addgene, #24920), a vector to visualize autophagosome TMRM (Tetramethylrhodamine Methyl Ester Per- formation in real time. C2C12s were transfected using chlorate), a mitochondrial membrane dye, and Hoechst, a nuclear dye, at concentrations of 100 nM and 10 μM, JetPrime Polyplus reagent, while RH30 cell line was transfected using Qiagen’s Effectene reagent, as per respectively, were diluted in media and added to cells for manufacturer’s instructions. After 48 h of transfection, 30 min at 37 °C. Cells were imaged using an Olympus cells were treated with TMZ (60 h) and Baf-A1 (3 h before epiﬂuorescence microscope. Fluorescence intensity was imaging). LysoTracker red staining (Molecular Probes™; measured with ImageJ (NIH, Bethesda, MD, USA), LysoTracker® Red DND-99; L7528) was used to detect minimum of 20 cells per condition. In each condition, lysosomal activity and MitoTracker Red CMXRos at a ﬂuorescence intensity for 20 cells (one by one) randomly concentration of 50 nM to detect active mitochondrial was measured, and then intensity was averaged out for 20 membrane potential. Cells were stained for 30 min in 37 ° cells and quantiﬁed . C incubator. Using this approach, instances, where LC3- GFP puncta co-localized with LysotTracker, were con- Transmission electron microscopy (TEM) sidered to be autophagic, while LC3-GFP co-localization TEM was used to evaluate autophagy activation in both with MitoTracker was interpreted as mitophagy . cell lines following treatment with TMZ or Baf-A1 or combination. TEM imaging was performed according to a Immunocytochemistry protocol described previously . Brieﬂy, RH30 and C2C12 For detection of autophagy ﬂux, we used immunocy- cells were seeded in 100 mm plates (300,000 cells/dish) in tochemistry (ICC) in C2C12 and RH30 cells . Brieﬂy, RPMI and DMEM (high glucose) media, respectively, RH30 cells were cultured on coverslips in 6-well plates in supplemented with 10% FBS. Cells were treated with RPMI media with 10% FBS. The cells were then treated TMZ and Baf-A1 and then collected using EDTA for cell with TMZ (100 µM) or vehicle control and for 72 h. Four detachment. Cells were centrifuged three times (1500×g) hours before time point Baf-A1 (100 nM) was added and then ﬁxed (3% glutaraldehyde in PBS, pH 7.4) for 3 h which already has been treated with TMZ. At the indi- at room temperature. Additionally, cells were treated with cated time point, ICC was performed using the protocol a post-ﬁxation step using 1% osmium tetroxide in phos- described previously (Lysosomes were stained with phate buffer for 2 h at room temperature, followed by an LysoTracker red (Molecular Probes; 100 nM, 10 min) alcohol dehydration series, before embedding in Epon. 31,92 before ﬁxation and permeabilization) . GFP-LC3 TEM was performed with a Philips CM10, at 80 kV, on punctuate co-localized with activated LysoTracker redd ultra-thin sections (100 nm on 200 mesh grids) 72 h after were identiﬁed as autophagic cells . treatment. Cells were stained with uranyl acetate and counterstained with lead citrate for 3 min sequentially. TMRM staining for mitochondrial membrane potential Finally, grids were washed with water for 1 min and dried measurement utterly to be ready for imaging. TEM was done to conﬁrm Healthy mitochondrial membranes hold the electrical the autophagy induction by TMZ and autophagy sup- potential difference between the exterior and interior of pression by Baf-A1 in the cells. the cell, well known as membrane potential. This is an important process, which is linked to a multitude of Three-dimensional (3D) culture mitochondrial function. Tetramethylrhodamine methyl Fabrication of cell-loaded collagen disks ester (TMRM), a cell-permeant dye, can accumulate Disks of collagen with 5 mm diameter and 1 mm inside the healthy and active mitochondria with intact thickness loaded with 2 million cells/mL were used to 93,94 membrane potential which then becomes ﬂuorescent . perform 3D culture. The disks were made by curing the TMRM ﬂuorescent signal disappears when there is a loss suspension of cells in collagen in PDMS holders placed in of mitochondrial membrane potential. TMRM ﬂuores- 12-well plates. To fabricate the PDMS holders, the base cence can be detected with ﬂuorescence microscopy PDMS elastomer and the curing agent were mixed with a which allows quantiﬁcation of mitochondrial membrane ratio of 10/1 and degassed using a vacuum chamber. The potential. RH30 and C2C12 cells were cultured in 6-well solution was then poured on a microscope slide and cured plates (30,000 cells/ml) and treated with TMZ (100 μM, on a hot plate at 70 °C for 2 h to form a uniform 1 mm 60 h), Baﬁlomycin-A1 (Baf-A1, 4 nM) and TMZ/Baf-A1. layer. The PDMS ﬁlm was then cut to 15 mm square Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 14 of 16 pieces, and a 5 mm hole was punched through them using Primary antibodies were diluted in DPBS with 1% BSA a biopsy punch. To sterilize the PDMS holders, they were and 0.3% Triton, in a dilution factor of 1/300. The incubated in pure ethanol for 1 h and baked at 80 °C for 4 blocking solution was removed, and the primary antibody h to remove the ethanol. The PDMS holders were then solutions were added to the samples and incubated placed in 12-well plates. overnight at 4 °C. LC3 and p62 were incubated simulta- neously for co-staining and PARP were incubated sepa- rately. Next day, primary antibody solutions were RH30 and C2C12 culturing in 3D RH30 and C2C12 cells were cultured in a T75 culture removed, and samples were washed three times using ﬂask in a 5% CO incubator at 37 °C, supplied with culture DPBS for 5 min each. The secondary antibody solutions media (DMEM with 10% FBS and 0.5% Pen-Strep). The were made by diluting the secondary antibody in DPBS media was replaced every 24 h and the cells were collected with 1% BSA and 0.3% Triton with a ratio of 1/300. at a conﬂuency of 80%. To harvest cells, media were Secondary antibodies were added to the samples and removed and cells were rinsed once with 4 mL of trypsin- incubated for 2 h at room temperature in the dark. Sec- EDTA, followed by 5 mL of trypsin-EDTA incubation for ondary antibodies were later removed, and the solution of 5 min. Ten mililiters of media were added to the cell DAPI was added to the samples and incubated for 1 h at suspension and centrifuged at 200×g for 5 min at 4 °C. room temperature in the dark. Finally, DAPI solution was The supernatant was removed, and the cells were re- removed, and the samples were washed three times with suspended in fresh media and gently mixed with collagen DPBS for 5 min each. Confocal microscopy was per- at 4 °C to reach a ﬁnal collagen concentration of 3 mg/mL formed right after the samples were stained. and the cell density of 2 million cells/mL. Twenty microliters of the solution was added to each well in the Statistical analysis PDMS holder and placed in the incubator for 45 min to All results were presented as mean ± SD, and the dif- cure the collagen. Then 2 mL of media was added to each ferences between the groups were tested by one-way well, and the cells were cultured overnight. Treatments ANOVA or two-way ANOVA analysis (non-parametric, were started after the incubation overnight. Two condi- Brown–Forsythe test), using GraphPad Prism 7.0. The tions were considered for the study which includes con- conﬁdence interval in each analysis was 95%, and P < 0.05 trol (media) and TMZ (0, 100, 250, and 500 µM) was considered statistically signiﬁcant. treatment. The cells were treated for a total of 48, 72, and 96 h and then the viability and immunocytochemistry Acknowledgements were performed. All experiments were performed in This work was supported by a Children’s Hospital Research Institute of triplicates. Manitoba (CHRIM) operating grant and the University of Manitoba Collaborative Research Program to S.G. and J.W.G. Research Manitoba (RM) New Investigator operating grant also supported S.G. J.W.G. is supported by an Live dead assay in 3D culture NSERC Discovery grant. J.A. was supported by a RM studentship grant and Live/dead solution was prepared as per supplier’s pro- NIMAD operating grant. J.F. is supported by studentships from NSERC and RM/ tocol, where 5 µL of calcein AM and 20 µL of ethidium CHRIM, and S.C.d.S.R. is supported by studentships from the University of Manitoba and RM/CHRIM. P.K. is supported by an NSERC summer studentship. homodimer-1 were added to 10 mL of DPBS. After M.A., and E.S. thank NSERC (Discovery program) and BC Cancer Foundation for treatment, media were removed from the wells and live/ their ﬁnancial support. M.A. also thanks Canadian Foundation for Innovations dead solution was added followed by 2 h incubation at and B.C. Knowledge Development Fund for supporting this project. room temperature in the dark. Next, the solution was removed, and the wells were gently rinsed with DPBS Author details twice. Confocal microscopy was performed right after the Department of Human Anatomy and Cell Science, Max Rady College of samples were stained. For quantifying viability, an image Medicine, Rady Faculty of Health Science, University of Manitoba, Winnipeg, of each test case was considered to count the number of Canada. Laboratory for Innovations in Microengineering (LiME), Department of Mechanical Engineering, University of Victoria, Victoria, BC, Canada. Health live and dead cells. The viability was quantiﬁed based on Policy Research Centre, Shiraz University of Medical Sciences, Shiraz, Iran. the ratio of the number of live cells to the total number of Colleges of Medicine and Nursing, Rady Faculty of Health Science, University cells in each image. of Manitoba, Winnipeg, Canada. Center for Biomedical Research, University of Victoria, Victoria, Canada; Center for Advanced Materials and Related Technology (CAMTEC), University of Victoria, Victoria, Canada. The Biology of 3D immunocytochemistry Breathing Theme, Children’s Hospital Research Institute of Manitoba, University Cells were ﬁxed using 3.7% formaldehyde in DPBS, after of Manitoba, Winnipeg, Canada. The Diabetes Research Envisioned and Accomplished in Manitoba (DREAM) Theme, Children’s Hospital Research removing media, for 40 min at room temperature. 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Publisher: Springer Journals
Copyright: Copyright © 2018 by The Author(s)
Subject: Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
eISSN: 2058-7716
DOI: 10.1038/s41420-018-0115-9
Publisher site: See Article on Publisher Site

Abstract

Rhabdomyosarcoma (RMS) is a muscle-derived tumor. In both pre-clinical and clinical studies Temozolomide (TMZ) has been recently tested against RMS; however, the precise mechanism of action of TMZ in RMS remains unclear. Here we demonstrate that TMZ decreases the cell viability of the RH30 RMS and C2C12 cell line, where cells display evidence of mitochondrial outer membrane permeability. Interestingly, the C2C12 mouse myoblast line was relatively more resistant to TMZ-induced apoptosis. Moreover, we observed that TMZ activated biochemical and morphological markers of autophagy in both cell lines. Autophagy inhibition in both RH30 and C2C12 cells signiﬁcantly increased TMZ-induced cell death. In RH30 cells, TMZ increased Mcl-1 and Bax protein expression compared to corresponding time match controls while in C2C12 Mcl-1, Bcl-2, Bcl-XL, and Bax protein expression were not changed. Baf-A1 co- treatment with TMZ signiﬁcantly decrease Mcl-1 expression compared to TMZ while increase Bax expression in C2C12 cells (Bcl2 and Bcl-XL do not signiﬁcantly change in Baf-A1/TMZ co-treatment). Using a three-dimensional (3D) C2C12 and RH30 culture model we demonstrated that TMZ is signiﬁcantly more toxic in RH30 cells (live/dead assay). Additionally, we have observed in our 3D culture model that TMZ induced both apoptosis (cleavage of PARP) and autophagy (LC3-puncta and localization of LC3/p62). Therefore, our data demonstrate that TMZ induces simultaneous autophagy and apoptosis in both RH30 and C2C12 cells in 2D and 3D culture model, where RH30 cells are more sensitive to TMZ-induced death. Furthermore, autophagy serves to protect RH30 cells from TMZ-induced death. Introduction years, emphasizing an urgent need to develop new stra- Rhabdomyosarcoma (RMS) is an aggressive soft-tissue tegies to treat and prevent this disease . Four subgroups of malignant tumor that occurs in both children and adults , RMS have been described based on histological, genetic, but comprises up to 50% of all childhood soft tissue and clinical criteria : embryonal RMS, pleomorphic RMS, 2,3 sarcomas . Clinically, survival among patients with spindle cell/sclerosing RMS, and alveolar RMS (ARMS). metastatic RMS has not improved appreciably in the past ARMS is an aggressive subtype of RMS suffered by ado- lescents and young adults . In addition, the high-mortality rate in ARMS has been attributed to the presence of Correspondence: Saeid Ghavami (saeid.ghavami@umanitoba.ca)or oncogenic fusion proteins (i.e., PAX3-FOXO1 and PAX7- Joseph W. Gordon (joseph.gordon@umanitoba.ca) 6 FOXO1) generated by chromosomal translocations . Department of Human Anatomy and Cell Science, Max Rady College of Recently, oral alkylating agents such as TMZ have Medicine, Rady Faculty of Health Science, University of Manitoba, Winnipeg, Canada received considerable attention in RMS pre-clinical and Laboratory for Innovations in Microengineering (LiME), Department of clinical studies . TMZ has a broad spectrum of antitumor Mechanical Engineering, University of Victoria, Victoria, BC, Canada activity while being well-tolerated by the patient due to its Full list of author information is available at the end of the article Edited by: I. Amelio © 2018 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Ofﬁcial journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Moghadam et al. Cell Death Discovery (2019) 5:52 Page 2 of 16 8–10 relatively low toxicity . The mechanism of action of family of proteins plays a crucial role in the regulation of TMZ results in the production of a highly reactive the cross-talk between apoptosis and autophagy, consist- methyldiazonium cation that transfers its methyl group ing of both pro-survival and pro-death family mem- 36,37 to purine bases of DNA resulting in double-stranded bers . However, the role of the BCL-2 family in 12–15 breaks during repair . This process leads to G2/M cell regulating apoptosis and autophagy in ARMS is not well 16–18 cycle arrest and activation of apoptosis . However, the dissected. cellular response to TMZ also involves alterations in gene In this report, we demonstrate that TMZ decreased cell expression that have been shown to be cancer-cell spe- viability of the RH30 RMS cell line and C2C12 cell line in ciﬁc. Thus, the pathways involved in apoptosis induction presence of autophagy activation. TMZ induces outer may be different for each type of sarcoma, and there is mitochondrial membrane permeabilization without little information regarding how TMZ affects ARMS at changes in mitochondrial membrane potential. We also the cellular level. In our studies, we explored the role of show that treatment of RH30 and C2C12 cells with low autophagy in RH30 cells to further elucidate the concentrations of the autophagy ﬂux inhibitor Baf-A1, mechanism of action of TMZ. signiﬁcantly increased TMZ-induced cell death. Collec- Autophagy is a conserved physiological process of cel- tively, these observations help deﬁne the role of autophagy lular self-eating, which plays an essential role in cellular in RH30 cells and contribute to our knowledge regarding housekeeping activity by degrading protein aggregates, the mechanism of action of TMZ in PAX3-FOXO1 cytoplasmic components, and damaged or dysfunctional positive sarcomas. organelles. At least three distinct forms of autophagy can be activated depending on the route that cytoplasmic Results material is delivered to lysosomes, such as chaperone- TMZ induces apoptotic cell death in C2C12 and RH30 cell mediated autophagy, microautophagy, and macro- lines 19–22 autophagy (from here on referred to as autophagy) . We treated both the RH30 RMS cell line and C2C12 The role of autophagy in cancer cell biology is compli- myoblasts with TMZ (0, 50, 100, 250, 500, and 1000 μMin cated and evolves throughout tumorigenesis. For instance, 48, 72, and 96 h) and cell viability was examined by MTT autophagy has been shown to promote cancer cell survival assay. TMZ-induced cell death in a dose- and time- during conditions of a nutrient or hypoxic stress and dependent manner in C2C12 cells (Fig. 1a–c) and RH30 contribute to cell demise through autophagic cell death cells (Fig. 1d–f), where RH30 cells appeared more sensi- (i.e., type II programmed cell death) tive to the effects of TMZ. We conﬁrmed that TMZ (100 . More recently, autophagy has also been shown to contribute to epithe- μM, 72 h) increases apoptotic cell death in both C2C12 lium to mesenchymal transition (EMT) and promote and RH30 cell lines using a propidium iodide (PI)- 21,24 cancer metastasis in different cancer models . In RH30 dependent method (i.e. Nicoletti assay). Following TMZ cells, autophagy is known to be a crucial process in the treatment, the percentage of apoptotic cells was maintenance of cellular viability and proliferation . Fur- approximately three times more than control C2C12 cells thermore, inhibition of autophagy by the Atg7 knock- (Fig. 1g). However, in RH30 cells, TMZ-induced apoptosis down, or pharmacological inhibition with chloroquine or by 17.5 times compared to the control cells (Fig. 1g). Baf-A1 treatment, has been demonstrated to decrease cell Therefore, TMZ signiﬁcantly activated higher levels of 26,27 growth and reduced viability in RMS cell lines . apoptosis in RH30 cells than in C2C12 cells (Fig. 1h). We Autophagy and apoptosis are two independent pro- also performed ﬂow cytometry analysis where cells were cesses, but under certain conditions, they cooperate in a stained with Annexin-V and PI (Fig. 1i, j). In C2C12 cells hierarchical relationship to regulate the turnover of treated with TMZ, we observed an increase in Annexin-V organelles and proteins within cells, and of cells within positive (A+/P−) cells, but no change in double positive 28,29 organisms . However, within a given cell, considerable cells (A+/P+). Interestingly, we also observed a decrease cross-talk exists between apoptosis and autophagy, and in PI positive cells (A−/P+) (Fig. 1i). Conversely, in RH30 nature of this cross-talk can change in a cell context- cells treated with TMZ we observed signiﬁcant increases 30–32 dependent manner . Generally, autophagy is a rapidly in Annexin-V positive and double positive cells, with a induced survival pathway activated by sublethal stress, corresponding decreased in double negative cells (A−/P whereas apoptosis is initiated at lethal doses of stress . −) (Fig. 1j). These observations are consistent with our However, in certain conditions, autophagy may also MMT and Nicoletti ﬁndings, and suggest that RH30 cells contribute to the induction of cell death by either the are more sensitive to the apoptosis inducing effects of activation of programmed cell death type II, activation of TMZ compared to C2C12 cells. mitochondrial-dependent cell death pathways (i.e. apop- To investigate the cellular mechanisms behind the dif- tosis or necrosis), or by providing substrates to promote ferential response to TMZ treatment, we performed a 33–35 ATP-dependent apoptotic mechanisms . The BCL-2 series of experiments to evaluate intrinsic cell death Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 3 of 16 Fig. 1 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 4 of 16 (see ﬁgure on previous page) Fig. 1 TMZ induces apoptotic cell death in C2C12 and RH30 cell lines. a–c C2C12 cells were treated with TMZ (50, 100, 250, and 1000 μM) and cell viability was assessed 48, 72, and 96 h after that by MTT assay. Control cells for each time point were treated with the solvent control (DMSO). Results are expressed as a percentage of corresponding time point control and represent the means ± SD of 15 replicates in three independent experiments (**P < 0.01; ***P < 0.0001). d–f C2C12 cells were treated TMZ (50, 100, 250, and 1000 μM) and cell viability was assessed 48, 72, and 96 h after that by MTT assay. Control cells for each time point were treated with the solvent control (DMSO). Results are expressed as a percentage of corresponding time point control and represent the means ± SD of 15 replicates in three independent experiments (**P < 0.01; ****P < 0.0001). g Representative ﬁgures of the ﬂow cytometry histogram for C2C12 and RH30 are shown. Cells were treated with TMZ (100 μM, 72 h) and Percent sub- G1 abundance induced by TMZ (100 μM) or DMSO solvent control after 72 h. The Sub-G1 population showed an abundance of apoptotic cell death in each treatment. h The average of the sub-G1 population of C2C12 and RH30 cells which were treated with TMZ (100 μM, 72 h) and DMSO solvent control has been measured. Results represent the means ± SD of six replicates in three independent experiments (****P < 0.0001). i, j C2C12 and RH30 cells were treated with TMZ and stained with Annexin-V-FITC (A) and PI (P) and analyzed by ﬂow cytometry (n = 3)(*P < 0.05). k TMRM staining of C2C12 and RH30 cells treated with TMZ (100 μM, 48 h). l MitoTracker staining of C2C12 and RH30 cells treated with TMZ (100 μM, 48 h). m C2C12 and RH30 cells were transfected with cytochrome C-GFP (CytoC-GFP) and treated with TMZ (100 μM, 48 h)(*P < 0.05). n, o C2C12 and RH30 cells were treated with 100 μM TMZ for 48 h and then Caspase-9 activity was measured using Caspase-Glo luminescence assay. (**P < 0.01) pathways initiated by mitochondria. We stained cells with 9 activity was unchanged in C2C12s at this dose and time the mitochondrial membrane potential-sensitive dye (P > 0.05). 38,39 TMRM , as rapid dissipation of mitochondrial mem- brane potential has been recently associated with mito- TMZ induces autophagy activation in C2C12 and RH30 chondrial permeability transition. Surprisingly, we cells observed that TMZ treatment did not lead to mitochon- Previous reports have shown that TMZ induces simul- drial depolarization at this dose and time in either cell line taneous apoptosis and autophagy in glioblastoma 22,47 (Fig. 1k). No change in MitoTracker staining was cells . Thus, we evaluated if differential activation of observed in RH30 cells, indicating that mitochondrial autophagy could explain the increased toxicity to TMZ in content was not altered by TMZ treatment, however we RH30 cells compared to C2C12 cells. Our results did observe a modest, but signiﬁcant reduction in Mito- demonstrate that TMZ induces autophagy in both C2C12 Tracker staining in C2C12 cells (Fig. 1l). Next, we trans- and RH30 cell lines. Immunoblotting results showed fected cells with a cytochrome C-GFP fusion construct lipidation of LC3B, Atg5-12 conjugation, and increase in that has been previously shown to accumulate in the Beclin-1 expression in both cell lines, indicating autop- mitochondrial intermembrane space and be released into hagy activation by TMZ (100 µM) (Fig. 2a). In RH30 and the cytosol during mitochondrial outer membrane per- C2C12 cells, ﬂuorescent microscopy conﬁrmed that TMZ meabilization (MOMP) . Following transfection, we increased the number of LC3-GFP puncta and lysosomal observed ﬂuorescent mitochondrial puncta in both activation (LysoTracker red ﬂuorescence intensity), which untreated cell lines (Fig. 1m). When RH30 and C2C12 co-localized in TMZ treated cells (Fig. 2b, c). Also, cells were treated with TMZ, we observed release of transmission electron microscopy (TEM) further con- cytochrome C-GFP into the cytosol. In RH30 cells, no ﬁrmed autophagy activation in RH30 and C2C12 cells ﬂuorescent puncta remained following TMZ treatment; (double membrane autophagosome formation), after 72 h however, in C2C12 cells residual puncta remained in up treatment with TMZ (100 µM) (Fig. 2d). to 20% of cells (Fig. 1m). Collectively, these ﬁndings To investigate whether the accumulation of LC3B-II is indicate that TMZ induces MOMP without mitochon- due to an increase in autophagic ﬂux or prevention of drial permeability transition. autophagic proteolysis, we used Baf-A1 (100 nM) for Caspase-9 is an important effector of intrinsic or different time points (2, 3, 4 h) to study autophagy ﬂux. It 41–44 mitochondrial-regulated apoptosis . It has been has been previously shown that a high concentration of recently reported that TMZ induces caspase-dependent Baf-A1 with short time point exposures decreases lyso- 45,46 apoptosis in several cancer cells . Thus, we determined some numbers, causes fusion block, and suppresses the effects of TMZ-induced MOMP on caspase-9 acti- autolysosome destruction by interfering with late-stage 19,48 vation. Following treatment of TMZ 100 μM for 48 h autophagosome-lysosome fusion . As shown by wes- (Fig. 1n, o), we observed a signiﬁcant activation of tern blot, treatment with Baf-A1 (100 nM) 2, and 4 h caspase-9 in RH30 cells (**P < 0.01); however, the caspase- markedly increased the accumulation levels of LC3-II and Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 5 of 16 Fig. 2 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 6 of 16 (see ﬁgure on previous page) Fig. 2 TMZ induces autophagy in C2C12 and RH30 cell lines. a C2C12 and RH30 cells were treated with TMZ (100 μM, 36, 72, 96 h) and autophagy hallmark was detected in both C2C12 and RH30 cell lines using immunoblotting. TMZ-induced LC3β lipidation, Atg5-12 conjugation, and Beclin-1 expression in both cell lines. Beta-actin was used as loading control. Data are representative of three independent experiments using different cultures. b, c RH30 and C2C12 cells were treated with TMZ (100 μM, 72 h) and using immunocytochemistry LC3 puncta and changes in lysosomal activity (LysoTracker red staining) has been investigated. The results showed that TMZ increased LC3 puncta and LysoTracker red ﬂuorescence intensity and co-localization of LC3 puncta and LysoTracker in both RH30 and C2C12 cells. d Transmission electron microscopy showed that in treated RH30 and C2C12 cells there are accumulated autophagosome-like structures compared to control and normal cells after 72 h treatment. Arrows show the autophagolysosomes containing the cargo (magniﬁcation ×11,600). e C2C12 cells were treated with TMZ (100 µM, 72 h) and then treated with Baf-A1 (100 nM, 1, 2, 3, 4 h) to evaluate for autophagy ﬂux. TMZ+ Baf-A1 treatment induces more lipidated LC3β and reduces the degradation of p62 in RH30 cells. Beta-actin was used as the loading control. Data are representative of three independent experiments. f C2C12 cells were treated with TMZ (100 μM, 72 h) and Baf-A1 (100 nM, +4 h) followed by immunocytochemistry to evaluate LC3 puncta and changes in lysosomal activity (LysoTracker red staining). The results showed that TMZ increased LC3 puncta and LysoTracker red ﬂuorescence intensity in C2C12 cells. On the other hand, Baf-A1 and TMZ + Baf-A1 signiﬁcantly decreased the LysoTracker red ﬂuorescence in the presence of an accumulation of LC3 puncta showing the inhibition of autophagy ﬂux by Baf-A1. g, h Representative ﬁgures of LC3 puncta and ﬂuorescence intensity for LysoTracker in C2C12 cells in the presence of TMZ, Baf-A1, and TMZ/Baf-A1 treatment. These results showed that the number of LC3 puncta is signiﬁcantly higher in cells which are treated with Baf-A1 and TMZ + Baf-A1. However, the ﬂuorescence intensity of LysoTracker was lower in Baf-A1, and TMZ + Baf-A1 treated cells. i RH30 cells were treated with TMZ (100 µM, 72 h) and then treated with Baf-A1 (100 nM, 1, 2, 3, 4 h) to evaluate for autophagy ﬂux. TMZ/ Baf-A1 treatment induces more LC3β lipidation and reduces the degradation of p62 in RH30 cells compared to TMZ and Baf-A1 single treatment. Beta-actin was used as the loading control. Data are representative of three independent experiments. j RH30 cells were treated with TMZ (100 μM, 72 h) and Baf-A1 (100 nM, +4 h) followed by immunocytochemistry to evaluate LC3 puncta and changes in lysosomal activity (LysoTracker red staining). The results showed that TMZ increased LC3 puncta and LysoTracker ﬂuorescence intensity in RH30 cells. On the other hand, Baf-A1 and TMZ+ Baf-A1 signiﬁcantly decreased the LysoTracker red ﬂuorescence showing the inhibition of autophagy by Baf-A1. k, l Representative ﬁgures of LC3 puncta and ﬂuorescence intensity for LysoTracker in RH30 cells in the presence of TMZ, Baf-A1, and TMZ/Baf-A1 treatment. These results showed that many LC3 puncta are signiﬁcantly higher in cells which are treated with Baf-A1 and TMZ+ Baf-A1. However, the ﬂuorescence intensity of LysoTracker was lower in Baf-A1, and TMZ+ Baf-A1 treated cells P62 in both C2C12 and RH30 cells. Combination treat- inhibits autophagy, but cell toxicity can occur at higher 49,50 ment of cells with TMZ (100 µM, 72 h) and Baf-A1 (100 doses . Therefore, we performed a cell viability assay nM) at 1, 2, 3, and 4 h showed a time-dependent accu- (MTT) to establish the appropriate concentration of mulation of LC3B-II compared to cells treated with Baf- Baf-A1 to inhibit autophagy with the least cytotoxic A1 (100 nM) alone (C2C12 cells Fig. 2e; RH30 cellsFig. 2i). effect on C2C12 and RH30 cells. C2C12 and RH30 cells These results indicate that TMZ induces autophagy ﬂux were treated with different concentration of Baf-A1 in both cell lines. Consistent results were obtained using (0.1–10 nM) for 48 and 72 h. Our results revealed that GFP-LC3, where in cells treated with a combination of Baf-A1 (4 and 6 nM) had low cytotoxicity at 72 h in both TMZ and Baf-A1 (3 h, 100 nM) we observed increased cell lines (Fig. 3a, b), thus we evaluated the effectiveness GFP-LC3 puncta, compared to cells treated with Baf-A1 of these two Baf-A1 concentrations to inhibit autop- (100 nM) alone (C2C12 cells Fig. 2e; RH30 cells Fig. 2i). hagy. Cells were treated with Baf-A1 (4 and 6 nM) for Lysosomal activity was found to be lower in cells after 48, and 72 h and autophagy markers (LC3 lipidation and treatment with Baf-A1 (100 nM) alone, as well in com- P62 degradation/accumulation) were investigated in bination with TMZ (100 µM, 60 h) compared to the both cell lines using western blotting. We observed that control and TMZ alone group, conﬁrming lysosomal Baf-A1 (4 and6nM)inhibitsautophagy,determinedby acidiﬁcation inhibition and blockage of the autophagy ﬂux the accumulation of lipidated LC3B-II and P62 (Fig. 3c) (C2C12 cells Fig. 2f–h; RH30 cells Fig. 2i–l). Moreover, in both C2C12 and RH30 cells. Next, we conﬁrmed the we demonstrate that the number of autophagic C2C12 effect of Baf-A1 (4 and 6 nM) on autophagy markers in cells is higher in cells treated with Baf-A1 with and C2C12 and RH30 cells treated with TMZ (100 µM/ml; without TMZ. However, the ﬂuorescence intensity of Fig. 3d, e). Finally, we evaluated apoptosis by ﬂow LysoTracker was lower in Baf-A1 and in TMZ + Baf-A1 cytometry analysis with PI staining in cells treated with treated cells (C2C12 cells Fig. 2f–h; RH30 cells Fig. 2i–l). TMZ (100 μM) and Baf-A1 (4 nM) for 72 h. Both TMZ Overall, these ﬁndings suggest that TMZ induces autop- and hagy ﬂux in C2C12 and RH30 cells. Baf-A1 increased the apoptotic cell population com- pared to control; however, the percentage of apoptosis Autophagy inhibition increases TMZ-induced apoptosis in in cells treated with the combination of TMZ and Baf- C2C12 and RH30 A1 was greater than either TMZ or Baf-A1 alone Next, we examined the effect of chemical inhibition of (Fig. 3f–h). Collectively, these ﬁndings demonstrate that autophagy on TMZ-induced apoptosis. Previously, we autophagy inhibition increases TMZ-induced apoptosis demonstrated that low dose concentrations of Baf-A1 in both C2C12 and RH30 cells. Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 7 of 16 Fig. 3 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 8 of 16 (see ﬁgure on previous page) Fig. 3 Autophagy inhibition increases TMZ-induced apoptosis in C2C12 and RH30 cells. a, b C2C12 and RH30 cells were treated with Baf-A1 (2, 4, 6, 8, and 10 nM) and cell viability was assessed after 72 h using MTT assay. Control cells for each time point were treated with the solvent control (DMSO). Results are expressed as a percentage of corresponding time point control and represent the means ± SD of 15 replicates in three independent experiments (**P < 0.01; ***P < 0.0001). c C2C12 and RH30 cells were treated with Baf-A1 (4 and 6 nM, 48, 72 h) and autophagy hallmark proteins were detected in both C2C12 and RH30 cell lines using immunoblotting. Baf-A1 induces accumulation of lipidated LC3β and decreases the degradation of p62 in both cell lines. Beta-actin was used as loading control. Data are representative of three independent experiments using different cultures. d, e C2C12 and RH30 cells were treated with TMZ (100 µM, 72 h) and then treated with Baf-A1 (4, 6 nM, 72 h) to evaluate autophagy inhibition in the presence of Baf-A1 treatment. TMZ+ Baf-A1 treatment induces LC3β lipidation and reduces the degradation of p62 in C2C12 and RH30 cells. Beta-actin was used as the loading control. Data are representative of three independent experiments. f–h Representative ﬁgures of the ﬂow cytometry histogram for RH30 are shown. Cells were treated with TMZ (100 μM, 72 h), Baf-A1 (4 nM, 72 h) and Baf-A1/TMZ. Percentage of sub-G1 abundance induced by TMZ (100 μM, 72 h), Baf-A1 (4 nM, 72 h) and Baf-A1/TMZ after 72 h has been showed. The Sub-G1 population showed an abundance of apoptotic cell death in each treatment. g, h C2C12 and RH30 cells were treated with TMZ (100 µM, 72 h) and Baf-A1 (4 nM, 72 h) and then cell lysates were collected to examine the effect of TMZ, Baf-A1, and TMZ/Baf-A treatment on expression of Bcl2 family proteins (Bcl-2, Bcl-XL, Mcl-1, and Bax). k–r, k, l TMZ does not signiﬁcantly change Mcl-1 expression in both C2C12 and RH30 cells. It is notable that Baf-A1 (6 nM)/TMZ co- treatment signiﬁcantly (P < 0.01) decrease Mcl-1 expression in C2C12 cells while does not have any effect in RH30 cells. m, n TMZ does not signiﬁcantly change Bcl-2 expression in both C2C12 and RH30 cells. TMZ/Baf-A combination treatment also does not signiﬁcantly change Bcl-XL expression in both C2C12 and RH30 cells. o, p TMZ does not signiﬁcantly change Bcl-XL expression in both C2C12 and RH30 cells. TMZ/Baf-A combination treatment also does not signiﬁcantly change Bcl-2 expression in both C2C12 and RH30 cells. q, r TMZ does not signiﬁcantly change Bax expression in both C2C12 and RH30 cells. TMZ/Baf-A combination treatment signiﬁcantly increase Bax-expression in C2C12 cells (P < 0.01) while does not have a signiﬁcant effect on its expression in RH30 cells. s–u We have evaluated changes in mitochondrial membrane potential in the presence of TMZ (100 µM, 60 h), Baf-A1 (4 nM, 60 h), and TMZ/Baf-A1 combination in C2C12 and RH30 cells. s Representative images show the mitochondrial membrane potential measured by TMRM. Red color denotes TMRM staining. t, u Measurement of the mean of TMRM ﬂuorescence intensity shows that TMZ (100 µM, 60 h), Baf-A1 (4 nM, 60 h), and TMZ/Baf-A1 combination does not change mitochondrial membrane potential in C2C12 t and RH30 u cells. The data are representative of the mean ﬂuorescence in at least 100 cells in each cell type. The data were analyzed by Student’s t-test or ANOVA, followed by post hoc analysis. If p < 0.05, results were considered statistically signiﬁcant To further dissect the mechanisms by which 5 mm and thickness = 1 mm µm) to evaluate the effect of TMZ-induces apoptosis in presence of autophagy inhi- TMZ. These disks were fabricated by mixing C2C12 and bition, we evaluated the expression of Bcl2 family proteins RH30 cells with collagen type I (3 mg/mL) at a ﬁnal by western blot in RH30 and C2C12 cells treated with density of 2 × 10 cells/mL and casting the disks in poly- 31,35–37 TMZ and/or Baf-A1, as previously described .We dimethylsiloxane (PDMS) molds. After 24 h, cells were evaluated Bcl-2, Bcl-xl, Mcl-1 (anti-apoptotic), and Bax treated with TMZ (0–500 µM) for 48 and 96 h. Cell via- (pro-apoptotic). As shown in Fig. 3i, j, Baf-A1 (6 nM) co- bility was evaluated by staining cells with treatment with TMZ (100 µM) signiﬁcantly decreased green-ﬂuorescent calcein-AM (live) and red-ﬂuorescent Mcl-1 expression (P < 0.01) compared to TMZ (100 µM) ethidium homodimer-1 (dead) dyes (Fig. 4c, d). Our with increased Bax expression (P < 0.01) in C2C12 cells. results showed a signiﬁcant decrease in the number of live Bcl-2 and Bcl-XL did not signiﬁcantly change in Baf-A1/ cells in TMZ-treated groups in both C2C12 and RH30 TMZ co-treatment (Figure K, M, N, and Q). Interestingly, cells (P < 0.001), while TMZ induces more signiﬁcant cell we did not see any signiﬁcant changes in RH30 cells with death in RH30 cells (Fig. 4e, f). Bright ﬁeld images regards to levels of Mcl-1, Bcl-2, Bcl-XL, or Bax expres- revealed signiﬁcant changes in cell morphology of TMZ sion after co-treatment of Baf-A1/TMZ (Fig. 3l, n, p, r). treated cells compared to control group in both C2C12 We also evaluated the effect of TMZ (100 µM, 60 h) and (Fig. 4a) and RH30 cells (Fig. 4b). Additionally, TMZ autophagy inhibition (Baf-A1, 4 nM, 60 h) on mitochon- treatment also induced higher PARP cleavage compared drial membrane potential in C2C12 and RH30 cells. to control in both C2C12 (Fig. 4g) and RH30 cells Interestingly, the results showed that TMZ, Baf-A1, and (Fig. 4h). Labeling of C2C12 and RH30 cells by using TMZ/Baf-A co-treatment do not affect mitochondrial autophagy markers demonstrated an increase in the membrane potential in either C2C12 and RH30 cells number of autophagosomes in TMZ-treated groups as (Fig. 3s–u). compared to corresponding time match controls (Fig. 4i, j). TMZ induces apoptosis and autophagy in C2C12 and RH30 3D culture Discussion Three-dimensional (3D) cellular models have been In the present study, we demonstrate that TMZ differ- extensively used to mimic the 3D microenvironment of entially induces apoptotic cell death in C2C12 and RH30 cells and to study the effects of extracellular matrix as a cells in both 2D and 3D culture models, and that the cell 51,52 barrier against drug diffusion . We used 3D models of death is negatively regulated by autophagy. Interestingly, C2C12 and RH30 made of cell collagen disks (diameter = TMZ treatment does not change mitochondrial Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 9 of 16 Fig. 4 (See legend on next page.) Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 10 of 16 (see ﬁgure on previous page) Fig. 4 TMZ induces apoptosis and autophagy in C2C12 and RH30 3D culture. a, b Bright ﬁeld image of C2C12 a and RH30 b 3D culture which shows the morphology of untreated and TMZ treated cells (100, 250 µM, 4 h) in 3D culture. c–f Viability assay was done by adding the live/dead solution to cells 48 and 96 h after treatment with TMZ (0–500 µM). Cells were incubated for 2 h in the dark at room temperature, rinsed twice with DPBS, and confocal microscopy was used to capture live/dead cell images in C2C12 c and RH30 d cells. e, f Quantiﬁcation of live/dead assay was measured by calculating the ratio of live: total cells which showed a signiﬁcant decrease in viability of C2C12 e and RH30 f cells treated with different concentrations of TMZ. The data showed TMZ signiﬁcantly induces cell death in both C2C12 and RH30 cells (P < 0.001) while TMZ induces more cell death in RH30 compared to C2C12 cells. g, h IF labeling of C2C12 cells g and RH30 cells h by cleaved PARP following treatment with TMZ (100 µM, 72 h) increased number of cells with cleaved PARP in TMZ treated cells in comparison to control cells which is the hallmark of increase of apoptosis in these cells. i, j After treatment of C2C12 i and RH30 j cells with TMZ (100 µM, 72 h), cells were IF labeled with autophagosome markers, LC3 and P62. Data showed that TMZ increases LC3 puncta (green) which is localized with p62 compared to corresponding time match control, a hallmark of autophagy induction in C2C12 and RH30 3D culture membrane potential in either RH30 or C2C12 cells, and increase in caspase-9 activation) while TMZ-induced the apoptosis is not caspase-9 dependent in C2C12 cells. It differential TMZ-induced apoptosis in C2C12 and RH30 has been reported that TMZ-induced apoptosis is caspase 68– cells cannot be entirely explained by changes in Bcl-2 dependent in different models, including glioma cells 70 71 anti-apoptotic (Bcl-2, Bcl-XL, and Mcl-1) and/or , and human glioma stem cells . It has been previously pro-apoptotic (Bax) protein expression. reported that TMZ induces endoplasmic reticulum (ER)- There are currently a few therapeutic options available stress with subsequent unfolded protein response in many for patients with metastatic RMS . To improve the cell models. We are currently investigating whether dif- therapeutic effect of chemotherapy in RMS, TMZ ferences in the ER-stress response or the extrinsic apop- has recently received considerable interest as a factor tosis pathway underlies the differential responsiveness of 22,72 for combination therapy because it is a well-tolerated C2C12 and RH30 cells to TMZ treatment . oral alkylating agent with a broad spectrum of antitumor Mitochondrial Bcl-2 family proteins are considered to 12,13 activity and relatively low toxicity . Several Phase I be one of the critical components in the regulation of 73–75 trials suggest that using TMZ alone or in combination apoptotic cell death and known to be essential for the with other drugs is well tolerated in RMS when used in apoptotic response in chemotherapy, emphasizing that 54 76,77 relapse settings these proteins are potential therapeutic targets . The combination of irinotecan and . TMZ, for instance, has shown synergistic antitumor Interestingly our results showed that TMZ-induced 54–57 activity against RMS . Several studies have pointed apoptosis is not associated with changes in Bcl-2, out that autophagy acts as a pro-survival pathway which Bcl-XL, Mcl-1, and Bax expression in RH30 and C2C12 allows cancer cells to survive the existing stressed present cell lines. It has been previously reported that 23,58,59 in the tumor microenvironment , such as the TMZ-induced apoptosis decreases mitochondrial mem- 60 78,79 stresses caused by anticancer agents . It has been found brane potential in glioma cell line while our results that autophagy and apoptosis have multiple direct and showed that TMZ-induced apoptosis had not affected the 31,61,62 indirect interactions with each other . In the present mitochondrial membrane potential in both RH30 and study, we observed that inhibition of autophagy could C2C12 cells. This observation can be explained by our enhance the apoptotic effect of TMZ in C2C12 and RH30 ﬁnding that TMZ has not affected the expression of anti- cells; therefore, autophagy is a negative regulator of TMZ- apoptotic (Bcl-2, Bcl-XL, and Mcl-1), and pro-apoptotic induced apoptosis in both C2C12 and RH30 cells. (Bax) Bcl-2 family proteins. In addition, our data show We have also determined that TMZ can activate that inhibition of autophagy by using Baﬁlomycin-A1 apoptosis in a time and dose-dependent manner in RH30 increased TMZ-induced apoptosis in RH30 and C2C12 cells. The enhancement of apoptosis was found to be cell lines without affecting mitochondrial membrane associated with higher expression of histone H2A variant potential in these cells H2AX (data not shown). The phosphorylation of histone This pilot study has expanded our understanding of the H2A variant H2AX at Ser139 is a marker of DNA double- cross-talk between apoptosis and autophagy controlling strand breaks, a type of DNA damage that can lead to cell TMZ-induced apoptosis. It has been shown that the 63,64 death, if unrepaired . This mechanism of TMZ- maintenance of cellular viability in RH30 cells is depen- induced apoptosis has been validated in previous studies dent on baseline autophagy , while loss of ATG7 or Baf- in other cancer cell lines which is consistent with our A1 treatment can negatively regulate proliferation of 11,65–67 26,27 results . Our results demonstrated that intrinsic or RH30 cells . We found that Baf-A1 inhibited baseline mitochondrial pathway is involved in the induction of autophagy in RH30 and C2C12 cells and decreased their apoptosis after TMZ treatment in RH30 cells (signiﬁcant viability. More importantly, our study showed that the Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 11 of 16 combination treatment of RH30 cells with Baf-A1 and Sigma-Aldrich (St. Louis, MO, USA). Autophagy inhibitor TMZ has a synergic effect on Baﬁlomycin-A1 (Baf-A1), rabbit anti-human/mouse/rat TMZ-induced apoptosis compared to treatment with LC3 (L8918, 1:3,000), anti-mouse IgG (A8924, 1:3000), either TMZ or Baf-A1 alone. This suggests that autophagy Temozolomide, anti-rabbit IgG (A6154, 1:5000), propi- has a survival role in RH30 and C2C12 cells treated with dium iodide (PI), and 3-(4,5-dimethyl-2-thiazolyl)-2,5- TMZ and the autophagy inhibitor enhances the anti- diphenyl-2H-tetrazolium bromide) (MTT) were pur- chased from Sigma-Aldrich Canada Co, Oakville, CA. tumor effect of TMZ against RH30 cells by activation of apoptosis. Caspase-Glo®-9 assay was purchased from Promega We have reproduced two-dimensional (2D) RH30 and (Toronto, ON, Canada). Enhanced chemiluminescence C2C12 culture results in a 3D culture models (ECL) prime regents (western blotting detection reagent) (Fig. 4) which showed that TMZ induces higher apoptotic were purchased from Amersham-Pharmacia Biotech. cell death in RH30 cells in the presence of autophagy Hyperﬁlm™ ECL was purchased from Fisher Scientiﬁc. activation in both cell models. These discoveries are an Polydimethylsiloxane (PDMS) was purchased from Dow essential step for translational application towards devel- Corning®™, 5 mm diameter sterile biopsy punches were oping new therapies for RMS. The ﬁndings in most cell- purchased from Stevens Company, 5 mg/mL bovine col- based assays are demonstrated in monolayer or suspen- lagen type 1 with neutralized pH was purchased from sion culture models, which may give false negative or Advanced BioMatrix. Penicillin-streptomycin (Pen-Strep, positive results due to an artiﬁcial cellular environment. It 10,000 units/mL penicillin and 10,000 µg/mL streptomy- also represents restricted signiﬁcance for in vivo studies cin) was purchased from Gibco®, fetal bovine serum (FBS) and often little value in foreseeing clinical effectiveness of was purchased from Millipore Sigma. Secondary anti- both generally cytotoxic and molecularly targeted drugs . bodies with conjugated ﬂuorescence (Alexa Fluor® 488 3D cultures have received more attention and become AfﬁniPure Donkey Anti-Rabbit IgG, and Alexa Fluor® 647 more accepted in the last few years as an essential stra- AfﬁniPure Donkey Anti-Mouse IgG) were purchased the 80,81 tegic toolkit for developing new cancer therapy . Fur- from Jackson ImmunoResearch Inc. DAPI (4’,6-Diami- thermore, 3D in vitro culture systems mimic different dino-2-Phenylindole, Dihydrochloride) was purchased aspects of human tumor tissue environment, therefore from Thermo Fisher Scientiﬁc. IgG-free bovine serum should be considered as an advanced model for routine albumin (BSA) was purchased from Jackson ImmunoR- anti-tumor drug testing . In our current ﬁndings, we esearch Inc. Live/dead viability kit was purchased from Millipore Sigma; formaldehyde 37% solution was pur- conﬁrmed that 3D RMS culture can be used to dissect the role of TMZ in RMS therapy. chased from VWR, Triton X-100 was purchased from Bio Our ﬁndings revealed that autophagy is a negative Basic Canada Inc. pCytochrome C-GFP was a gift from regulator of TMZ-induced apoptosis in both RH30 and Douglas Green (Addgene plasmid # 41182) . C2C12 cells. In addition, we have shown that TMZ- induces MOMP and apoptosis that is not readily Cell lines and cell culture explained by changes in Bcl-2, Bcl-XL, Mcl-1, or Bax Cell culture plastic ware, penicillin, and streptomycin expression, and is not accompanied by changes in mito- were purchased from VWR (Toronto, ON, Canada). Cells chondrial membrane potential. were cultured in Roswell Park Memorial Institute (RPMI- 1640) with L-glutamine and 25 mM HEPES (BioWhit- Materials and methods taker; Cat #: 12-115Q) and Dulbecco’s Modiﬁed Eagle’s Drugs and reagents Medium (DMEM) (CORNING; Cat #: 50-003-PB) with Antibodies against human p62 (5114, 1:1000), Bax 10% fetal bovine serum (FBS) (Gibco™; Cat #: 16000044). (5023, 1:1000), Bcl-2 (3498, 1:1000), Bcl-xl (2762,1:1000) The human rhabdomyosarcoma cell line (RH30) [RC13, Mcl-1 (94296, 1:1000) Bak (12105, 1:1000) Nix (12396, RMS 13, SJRH30] (ATCC® CRL¬ 2061™) (Human muscle 1:1000) Beclin-1(3495, 1:1000), SQSTM1/p62 (D5L7G) cancer cells) and mouse muscle cell line (C2C12) (ATCC® (88588, 1:100 for ICC), LC3B (D11) XP® (3868, 1:100 for CRL¬1772™) were used in this project. RH30 cell lines ICC), Cleaved PARP (Asp214) (D64E10) XP® (5625, 1:100 were cultured in (RPMI-1640) with L-glutamine and 25 for ICC), and Bid (2002, 1:1000) were purchased from Cell mM HEPES media, and C2C12 cells were cultured in Signaling Technology (Beverly, MA, USA); LC3 was (DMEM) with high glucose media. Both media were purchased from Sigma-Aldrich (St. Louis, MO, USA); supplemented with FBS (10%), penicillin (1%), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) streptomycin (1%). Cells were grown to 35–40% con- and Actin were purchased from Santa Cruz Biotechnol- ﬂuency on a 100 mm cell culture plate, 6-well plates, and ogy, Inc. (Dallas, TX, USA). Anti-rabbit IgG (whole 96-well plates. Cells were maintained in a humidiﬁed molecule) and anti-mouse IgG (Fab speciﬁc) peroxidase- incubator with 95% air and 5% CO at 37 °C and were conjugated secondary antibodies were purchased from passaged once every 2–3 days. Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 12 of 16 Cell viability assay three times with 1X TBST (0.025% Tween) for 20 min, The RH30 cells (30,000 cells/ml) and C2C12 (20,000 and membranes were incubated with secondary anti- cells/ml) were seeded in 96-well plates and treated with bodies (HRP) for 2 h on the shaker at RT. Membranes different concentrations of TMZ in different time points were rewashed three times for 20 min and incubated with (0–1000 μM, 0–96 h). Cells were also treated with various enhanced chemiluminescence (ECL) reagents (Amer- concentrations (0.1, 1, 2.5, 5, or 10 nM) of Baf-A1, an sham-Pharmacia Biotech) for 2–3 min. Autoradiography visualized the signals. Obtained protein bands were eval- autophagy inhibitor, and cell viability was assessed after 48 and 72 h. In each time point, 20 µl of MTT uated for changes in the autophagy and apoptosis sig- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- naling pathways. To assess even protein loading, mide (5 mg/ml), is the aqueous solubility of the reduced membranes were incubated in milk 1% with primary formazan product, was added into each well and incu- antibodies against GAPDH or Actin overnight, washed bated for 3 h. Then, media gently were removed from three times and probed with a secondary antibody to each well using pipette and 200 µl of solvent control visualize the signals. In the instances of re-probing of (DMSO) was added to each well and mixed very well by other proteins on the same membrane, blots were incu- pipetting to solubilize the MTT formazan. The plates bated with stripping solution containing 200 nM glycine, were analyzed at a test wavelength of 570 nm on a plate pH 2.5, 0.005 Tween 20 for 15 min at RT and followed the 83,84 reader, following a 20 min incubation at room tempera- same instruction as after blocking for these blots . ture (RT) . Measurement of apoptosis by ﬂow cytometry Immunoblotting Apoptotic cells were assessed by ﬂow cytometry with 85,86 Western blot analysis was used to assess markers of propidium iodide (PI), using the Nicoletti method . apoptosis and autophagy in RH30 and C2C12 cells. We RH30 and C2C12 cells were treated with TMZ (100 μM, examined hallmarks of intrinsic or extrinsic apoptotic 72 h) in cells cultured in 12-well plates. In each time point pathway and autophagy signaling pathways, while cells were detached by EDTA buffer and centrifuged at GAPDH or actin was used to normalize the results. After 1500×g for 5 min at 4 °C. Then, cells were washed by PBS treatment, cells were collected, and protein extracts were once. The cells were permeabilized and treated with a made using NP-40 lysis buffer (20 mM Tris-HCl (pH 7.5), ﬂuorescent dye that stains DNA quantitatively, using 0.5% Nonidet P-40, 0.5 mM PMSF, 100 µM β-glycerol 3- hypotonic PI lysis buffer (0.1% Triton X-100, 1% sodium phosphate and 0.5% protease inhibitor cocktail). The citrate, 0.5 mg/ml RNase A, 40 μg/ml propidium iodide). extracts were kept in −20 °C until all extracts from dif- Before ﬂow cytometry analysis, cells were incubated for at ferent time points were collected. Samples were then least 1 h, at 4 °C, and in the dark to prevent photo- sonicated in ﬁve times/ﬁve cycles using ultrasound soni- bleaching. The measurement was in red ﬂuorescence cator, followed by centrifugation at 13,000×g for 10 min to (460 nm) for 10,000 cells. Flow cytometer was adequately collect the supernatant protein. Protein content was then calibrated to gate out debris accurately. Finally, after determined via a Lowry protein assay, and protein sam- elimination of residual debris, the percentage of normal ples were made. Prepared samples, of a volume between and apoptotic nuclei were estimated by analysis of the 86,87 15 and 20 µl, were heated at 90 °C for 5 min before loading DNA histogram . The nuclei of apoptotic cells were into 10–15% polyacrylamide gels (depending on the located on the left side of the G1 peak. Apoptotic nuclei molecular weight of the proteins). Additionally, 10 µl of a have less DNA compared to nuclei of healthy G0/G1cells, standard molecular weight marker (Thermo Fischer Sci- causing an increase in sub-G1 section in the ﬂuorescence entiﬁc, ON, Canada) was loaded on each gel, as an histogram which can be applied to distinguish apoptotic approximate indicator of molecular protein weights. cells in samples. In each sample, the sub-G1 peak was Proteins were immediately transferred under reducing measured and statistically compared with other samples . conditions in transfer buffer (500 nM glycine, 50 mM Annexin-V FITC and PI staining was performed accord- Tris-HCl, and 20% methanol) to Immuno-Blot PVDF ing to manufacturer’s instructions (BD Biosciences Membranes (Bio-Rad; #1620177), at RT and 100 volts for 556547). Stained cells were analyzed on a Thermo Sci- 2–2.5 h. Upon transferring completion, membranes were entiﬁc Attune NxT ﬂow cytometer with a 488 nm laser. carefully transferred into 5% non-fat dried milk in 1X Tris-buffered saline containing Tween (TBS/0.025% Live cell imaging: LC3-GFP tween 20; TBST) and placed on the shaker in the cold GFP-LC3 is a speciﬁc marker for the occurrence of 88,89 room overnight or RT for 2 h. Following blocking, autophagosomes formation . GFP-LC3 is the fusion of membranes were incubated with the proper dilution of the green ﬂuorescent protein (GFP) and LC3 and can 90,91 primary antibodies in 1% milk made in 1X TBST and kept behave similarly as endogenous LC3 . The GFP-LC3 is in cold room (4 °C) overnight. Membranes were washed localized on the autophagosome membrane, and green Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 13 of 16 punctate signals are observed . To conﬁrm By removing cellular growth media before staining, as well TMZ-induced autophagy and autophagy ﬂux inhibition as well by washing C2C12s with phosphate buffered saline through Baf-A1 (100 nM), cells were transfected with a (1×, Hyclone), but not RH30, as RH30 cells can be easily green ﬂuorescent protein plasmid called LC3-GFP detached, staining sensitivity was increased. (Addgene, #24920), a vector to visualize autophagosome TMRM (Tetramethylrhodamine Methyl Ester Per- formation in real time. C2C12s were transfected using chlorate), a mitochondrial membrane dye, and Hoechst, a nuclear dye, at concentrations of 100 nM and 10 μM, JetPrime Polyplus reagent, while RH30 cell line was transfected using Qiagen’s Effectene reagent, as per respectively, were diluted in media and added to cells for manufacturer’s instructions. After 48 h of transfection, 30 min at 37 °C. Cells were imaged using an Olympus cells were treated with TMZ (60 h) and Baf-A1 (3 h before epiﬂuorescence microscope. Fluorescence intensity was imaging). LysoTracker red staining (Molecular Probes™; measured with ImageJ (NIH, Bethesda, MD, USA), LysoTracker® Red DND-99; L7528) was used to detect minimum of 20 cells per condition. In each condition, lysosomal activity and MitoTracker Red CMXRos at a ﬂuorescence intensity for 20 cells (one by one) randomly concentration of 50 nM to detect active mitochondrial was measured, and then intensity was averaged out for 20 membrane potential. Cells were stained for 30 min in 37 ° cells and quantiﬁed . C incubator. Using this approach, instances, where LC3- GFP puncta co-localized with LysotTracker, were con- Transmission electron microscopy (TEM) sidered to be autophagic, while LC3-GFP co-localization TEM was used to evaluate autophagy activation in both with MitoTracker was interpreted as mitophagy . cell lines following treatment with TMZ or Baf-A1 or combination. TEM imaging was performed according to a Immunocytochemistry protocol described previously . Brieﬂy, RH30 and C2C12 For detection of autophagy ﬂux, we used immunocy- cells were seeded in 100 mm plates (300,000 cells/dish) in tochemistry (ICC) in C2C12 and RH30 cells . Brieﬂy, RPMI and DMEM (high glucose) media, respectively, RH30 cells were cultured on coverslips in 6-well plates in supplemented with 10% FBS. Cells were treated with RPMI media with 10% FBS. The cells were then treated TMZ and Baf-A1 and then collected using EDTA for cell with TMZ (100 µM) or vehicle control and for 72 h. Four detachment. Cells were centrifuged three times (1500×g) hours before time point Baf-A1 (100 nM) was added and then ﬁxed (3% glutaraldehyde in PBS, pH 7.4) for 3 h which already has been treated with TMZ. At the indi- at room temperature. Additionally, cells were treated with cated time point, ICC was performed using the protocol a post-ﬁxation step using 1% osmium tetroxide in phos- described previously (Lysosomes were stained with phate buffer for 2 h at room temperature, followed by an LysoTracker red (Molecular Probes; 100 nM, 10 min) alcohol dehydration series, before embedding in Epon. 31,92 before ﬁxation and permeabilization) . GFP-LC3 TEM was performed with a Philips CM10, at 80 kV, on punctuate co-localized with activated LysoTracker redd ultra-thin sections (100 nm on 200 mesh grids) 72 h after were identiﬁed as autophagic cells . treatment. Cells were stained with uranyl acetate and counterstained with lead citrate for 3 min sequentially. TMRM staining for mitochondrial membrane potential Finally, grids were washed with water for 1 min and dried measurement utterly to be ready for imaging. TEM was done to conﬁrm Healthy mitochondrial membranes hold the electrical the autophagy induction by TMZ and autophagy sup- potential difference between the exterior and interior of pression by Baf-A1 in the cells. the cell, well known as membrane potential. This is an important process, which is linked to a multitude of Three-dimensional (3D) culture mitochondrial function. Tetramethylrhodamine methyl Fabrication of cell-loaded collagen disks ester (TMRM), a cell-permeant dye, can accumulate Disks of collagen with 5 mm diameter and 1 mm inside the healthy and active mitochondria with intact thickness loaded with 2 million cells/mL were used to 93,94 membrane potential which then becomes ﬂuorescent . perform 3D culture. The disks were made by curing the TMRM ﬂuorescent signal disappears when there is a loss suspension of cells in collagen in PDMS holders placed in of mitochondrial membrane potential. TMRM ﬂuores- 12-well plates. To fabricate the PDMS holders, the base cence can be detected with ﬂuorescence microscopy PDMS elastomer and the curing agent were mixed with a which allows quantiﬁcation of mitochondrial membrane ratio of 10/1 and degassed using a vacuum chamber. The potential. RH30 and C2C12 cells were cultured in 6-well solution was then poured on a microscope slide and cured plates (30,000 cells/ml) and treated with TMZ (100 μM, on a hot plate at 70 °C for 2 h to form a uniform 1 mm 60 h), Baﬁlomycin-A1 (Baf-A1, 4 nM) and TMZ/Baf-A1. layer. The PDMS ﬁlm was then cut to 15 mm square Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 14 of 16 pieces, and a 5 mm hole was punched through them using Primary antibodies were diluted in DPBS with 1% BSA a biopsy punch. To sterilize the PDMS holders, they were and 0.3% Triton, in a dilution factor of 1/300. The incubated in pure ethanol for 1 h and baked at 80 °C for 4 blocking solution was removed, and the primary antibody h to remove the ethanol. The PDMS holders were then solutions were added to the samples and incubated placed in 12-well plates. overnight at 4 °C. LC3 and p62 were incubated simulta- neously for co-staining and PARP were incubated sepa- rately. Next day, primary antibody solutions were RH30 and C2C12 culturing in 3D RH30 and C2C12 cells were cultured in a T75 culture removed, and samples were washed three times using ﬂask in a 5% CO incubator at 37 °C, supplied with culture DPBS for 5 min each. The secondary antibody solutions media (DMEM with 10% FBS and 0.5% Pen-Strep). The were made by diluting the secondary antibody in DPBS media was replaced every 24 h and the cells were collected with 1% BSA and 0.3% Triton with a ratio of 1/300. at a conﬂuency of 80%. To harvest cells, media were Secondary antibodies were added to the samples and removed and cells were rinsed once with 4 mL of trypsin- incubated for 2 h at room temperature in the dark. Sec- EDTA, followed by 5 mL of trypsin-EDTA incubation for ondary antibodies were later removed, and the solution of 5 min. Ten mililiters of media were added to the cell DAPI was added to the samples and incubated for 1 h at suspension and centrifuged at 200×g for 5 min at 4 °C. room temperature in the dark. Finally, DAPI solution was The supernatant was removed, and the cells were re- removed, and the samples were washed three times with suspended in fresh media and gently mixed with collagen DPBS for 5 min each. Confocal microscopy was per- at 4 °C to reach a ﬁnal collagen concentration of 3 mg/mL formed right after the samples were stained. and the cell density of 2 million cells/mL. Twenty microliters of the solution was added to each well in the Statistical analysis PDMS holder and placed in the incubator for 45 min to All results were presented as mean ± SD, and the dif- cure the collagen. Then 2 mL of media was added to each ferences between the groups were tested by one-way well, and the cells were cultured overnight. Treatments ANOVA or two-way ANOVA analysis (non-parametric, were started after the incubation overnight. Two condi- Brown–Forsythe test), using GraphPad Prism 7.0. The tions were considered for the study which includes con- conﬁdence interval in each analysis was 95%, and P < 0.05 trol (media) and TMZ (0, 100, 250, and 500 µM) was considered statistically signiﬁcant. treatment. The cells were treated for a total of 48, 72, and 96 h and then the viability and immunocytochemistry Acknowledgements were performed. All experiments were performed in This work was supported by a Children’s Hospital Research Institute of triplicates. Manitoba (CHRIM) operating grant and the University of Manitoba Collaborative Research Program to S.G. and J.W.G. Research Manitoba (RM) New Investigator operating grant also supported S.G. J.W.G. is supported by an Live dead assay in 3D culture NSERC Discovery grant. J.A. was supported by a RM studentship grant and Live/dead solution was prepared as per supplier’s pro- NIMAD operating grant. J.F. is supported by studentships from NSERC and RM/ tocol, where 5 µL of calcein AM and 20 µL of ethidium CHRIM, and S.C.d.S.R. is supported by studentships from the University of Manitoba and RM/CHRIM. P.K. is supported by an NSERC summer studentship. homodimer-1 were added to 10 mL of DPBS. After M.A., and E.S. thank NSERC (Discovery program) and BC Cancer Foundation for treatment, media were removed from the wells and live/ their ﬁnancial support. M.A. also thanks Canadian Foundation for Innovations dead solution was added followed by 2 h incubation at and B.C. Knowledge Development Fund for supporting this project. room temperature in the dark. Next, the solution was removed, and the wells were gently rinsed with DPBS Author details twice. Confocal microscopy was performed right after the Department of Human Anatomy and Cell Science, Max Rady College of samples were stained. For quantifying viability, an image Medicine, Rady Faculty of Health Science, University of Manitoba, Winnipeg, of each test case was considered to count the number of Canada. Laboratory for Innovations in Microengineering (LiME), Department of Mechanical Engineering, University of Victoria, Victoria, BC, Canada. Health live and dead cells. The viability was quantiﬁed based on Policy Research Centre, Shiraz University of Medical Sciences, Shiraz, Iran. the ratio of the number of live cells to the total number of Colleges of Medicine and Nursing, Rady Faculty of Health Science, University cells in each image. of Manitoba, Winnipeg, Canada. Center for Biomedical Research, University of Victoria, Victoria, Canada; Center for Advanced Materials and Related Technology (CAMTEC), University of Victoria, Victoria, Canada. The Biology of 3D immunocytochemistry Breathing Theme, Children’s Hospital Research Institute of Manitoba, University Cells were ﬁxed using 3.7% formaldehyde in DPBS, after of Manitoba, Winnipeg, Canada. The Diabetes Research Envisioned and Accomplished in Manitoba (DREAM) Theme, Children’s Hospital Research removing media, for 40 min at room temperature. Next, Institute of Manitoba, University of Manitoba, Winnipeg, Canada the formaldehyde solution was removed, and the samples were washed with DPBS three times for 5 min each. Samples were then blocked using a solution of 5% BSA in Conﬂict of interest DPBS with 0.3% Triton for 2 h at room temperature. The authors declare that they have no conﬂict of interest. Ofﬁcial journal of the Cell Death Differentiation Association Moghadam et al. Cell Death Discovery (2019) 5:52 Page 15 of 16 Publisher’s note 22. Hombach-Klonisch, S. et al. Glioblastoma and chemoresistance to alkylating Springer Nature remains neutral with regard to jurisdictional claims in agents: involvement of apoptosis, autophagy, and unfolded protein response. published maps and institutional afﬁliations. Pharmacol. Ther. 184,13–41 (2018). 23. Mokarram, P. et al. 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Autophagy modulates temozolomide-induced cell death in alveolar Rhabdomyosarcoma cells

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