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Objective Gastric cancer (GC) remains a prevailing threat to life. Baicalein exhibits anti-cancer properties. This study estimated the mechanism of baicalein in GC cell apoptosis by mediating endoplasmic reticulum stress (ERS) through the PI3K/AKT pathway. Methods After treatment with different concentrations of baicalein, GC cell (HGC-27 and AGS) viability was detected by MTT assay. AGS cells more sensitive to baicalein treatment were selected as study subjects. The IC50 of baicalein on AGS cells was determined. Colony formation, cell cycle, and apoptosis were detected using crystal violet staining and flow cytometry. Levels of ERS-related and BTG3/PI3K/AKT pathway-related proteins were determined by Western blot. 2+ Intracellular Ca level was measured using Fluo-3 AM fluorescence working solution. GC mouse models were estab - lished by subcutaneously injecting AGS cells into the right rib and were intragastrically administrated with baicalein. Tumor volume and weight were recorded. Expression of Ki67 in tumor tissues and positive expression of apoptotic cells were detected by immunohistochemistry and TUNEL staining. Results Baicalein inhibited cell proliferation and induced G0/G1 arrest and apoptosis by regulating the cell cycle, and triggered ERS in GC cells. Baicalein impeded the PI3K/AKT pathway by activating BTG3, thereby triggering ERS and inducing apoptosis. BTG3 inhibition reversed baicalein-induced apoptosis and ERS. Baicalein regulated GC cells in a concentration-dependent manner. Moreover, in xenograft mice, baicalein prevented tumor growth, decreased Ki67- positive cells, activated BTG3, and inhibited the PI3K/AKT pathway, thus activating ERS and increasing apoptotic cells. Conclusion Baicalein facilitates GC cell apoptosis by triggering ERS via repression of the PI3K/AKT pathway. 2+ Keywords Baicalein, Gastric cancer, PI3K/AKT, Endoplasmic reticulum stress, BTG3, Cell apoptosis, Ca , Ki67 Introduction Gastric cancer (GC) ranks the 5th most prevailing can- cer globally, which accounts for approximately 1 million new cases and above 720 000 deaths annually . Most *Correspondence: human GC occurs following long-term Helicobacter Mingxing Hou pylori infection through the Correa pathway, involv- MingxingHou1108@163.com 1 ing the following processes: gastritis, atrophy, intestinal Nanjing University of Chinese Medicine, Nanjing 210029, Jiangsu province, China metaplasia, dysplasia, and finally cancer . Individuals Gastrointestinal Surgery, Affiliated Hospital of Inner Mongolia Medical with newly diagnosed GC frequently present with dys- University, Hohhot 010050, Inner Mongolia, China 3 pepsia and reflux, and the patients at the advanced stage The Affiliated Hospital of Inner Mongolia Medical University, No. 1, Datong North Street, Huimin District, 010050 Hohhot, Inner Mongolia, may exhibit symptoms including weight loss, dysphagia, China emesis, anemia, and gastrointestinal bleeding . The © The Author(s) 2023. 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 the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Shen et al. Applied Biological Chemistry (2023) 66:10 Page 2 of 14 common malnutrition occurring at advanced or meta- the underlying mechanism remains elusive. The PI3K/ static GC can impact the life quality, enhance the toxicity AKT pathway represents one of the most vital pathways of chemotherapy, and decrease overall survival . Con- that regulate cell proliferation, growth, and apoptosis sequently, the 5-year survival for advanced GC individu- in assorted cancers . PI3K/AKT pathway also trig- als is only 5-20% . Herein, it is extremely paramount to gers stem cell-like properties in GC cells . Besides, explore new valid treatments and medicines for GC. B-cell translocation gene 3 (BTG3) overexpression can Baicalein, a bioactive constituent found in O. indicum block the PI3K/AKT/mTOR pathway activation , and medicinal plant, exhibits numerous biological activities, BTG3 can regulate GC cell proliferation, migration, and including anti-cancer, anti-inflammatory, antibacterial, apoptosis . Recently, PI3K/AKT pathway is consid- anti-adipogenesis, cardioprotective, neurogenesis, anti- ered paramount in controlling cell survival by repress- hyperglycemia, and wound healing effects . Baicalein ing ERS-induced cell apoptosis , so we speculated is a representative flavonoid in Scutellaria baicalensis, that PI3K/AKT pathway may also be implicated with and in modern clinical studies, Scutellaria baicalensis the occurrence of ERS in GC cells. Therefore, this study is widely adopted to treat an array of diseases, includ- probed into the regulation of baicalein in GC cell apopto- ing acute respiratory infection, hypertension, acute gas- sis by mediating ERS via the PI3K/AKT pathway. troenteritis, trachoma hepatitis, infantile diarrhoea, and vomiting during pregnancy . Shuang-Huang-Lian oral Materials and methods liquid, with Scutellaria baicalensis as one of the main Ethics statement ingredients, is considered a symptomatic treatment for All animal experiments were approved by the ethics com- coronavirus disease 2019 in China . PHY906 contain- mittee of Affiliated Hospital of Inner Mongolia Medical ing Scutellaria baicalensis can enhance the therapeutic University. Considerable efforts were conducted to mini - effect of anticancer drugs as the adjuvant to chemother - mize the animal number and their pain. apy, with promising results examined in clinical studies for pancreatic, colorectal, and liver cancers . Compel- ling evidence has vindicated the anti-cancer properties of Cell lines and culture baicalein by inducing cancer cell apoptosis through inhi- GC cell lines HGC-27 and AGS provided by Cell Bank of bition of phosphatidylinositol-3 kinase/protein kinase B Type Culture Collection of Chinese Academy of Sciences (PI3K/AKT), such as breast cancer  and liver cancer (Shanghai, China) were cultured in RPMI-1640 medium . Moreover, baicalein can enhance cisplatin sensitivity containing 10% fetal bovine serum, 100 U/mL penicillin, of GC cells by stimulating cell apoptosis and autophagy and 100 µg/mL streptomycin at 37 °C. by affecting AKT/mTOR and Nrf2/Keap 1 pathways . Although preceding reports have revealed the potential Drug of baicalein in GC therapy, there still lacks systematic Baicalein provided by Sigma-Aldrich (Merck KGaA, studies on the mechanism of baicalein in regulating GC. Darmstadt, Germany) was dissolved in dimethyl sul- The stimulation of internal pressure (including onco - phoxide (DMSO, Sigma-Aldrich) at 100 mmol/L as the genic activation) and external adverse environmental stock solution and kept at − 20 °C in the dark. Baicalein factors (such as hypoxia and nutritional deficiencies) was diluted in high-glucose Dulbecco’s modified Eagle that tumor cells are constantly subjected to may pose a medium to treat GC cells at indicated dosages. serious threat to proteostasis . Endoplasmic reticu- lum stress (ERS), a fundamental cellular stress response, sustains cellular protein homeostasis under exogenous Cell viability assay or endogenous stimuli, which is also involved in the Drug sensitivity was estimated using the 3-(4,5-dimeth- onset, progression, and drug resistance of tumors . ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) ERS response and the unfolded protein response (UPR) assay. Briefly, HGC-27 or AGS cells were trypsinized and keep protein homeostasis in cells by increasing protein 3 plated into 96-well plates (Corning, NY, USA) at 5 × 10 folding capacity and decreasing the intracellular load of cells per well. The cells were cultured overnight and next secretory proteins or triggering cell apoptosis that cannot cultured with a fresh medium containing baicalein at dif- recover . Furthermore, ERS determines the fate of ferent concentrations (0, 15, 30, 60, and 120 µM). After cancer cells by modulating cell signaling networks during 48 h of incubation, 20 µL MTT (Sigma-Aldrich) was sup- GC progression , identifying the substantial involve- plemented directly at the indicated times and dissolved in ment of ERS in GC. phosphate-buffered saline (PBS) at 5 mg/mL. Afterwards, Although the function of ERS to induce apoptosis the dishes were incubated for 4 h at 37 °C to conduct the has been demonstrated by many researchers [17, 18], MTT reaction. The supernatant was removed after 4 h. Shen et al. Applied Biological Chemistry (2023) 66:10 Page 3 of 14 Subsequently, the formed formazan crystals were dis- with 10% formalin and stained for 1 h with 0.1% crys- solved via addition of 100 µL DMSO, and the optical den- tal violet (Sigma-Aldrich). Thereafter, cells were rinsed sity (OD) at 490 nm was determined using a microplate and dried. Digital images were obtained using a micro- reader (Bio-Tek, Norcross, GA, USA). Cell survival rate scope (Leica, Solms, Germany). (%) = (OD of treatment group/OD of control group)* 100%. Cell cycle analysis The collected AGS cells were fixed overnight with 70% Cell transfection and grouping cold ethanol at − 20 °C. Next, after washing and resus- AGS cells were selected to carry out in vitro stud- pension in cold PBS, cells were incubated with 10 mg/ ies since their cell viability was greatly affected by mL RNase and 1 mg/mL propidium iodide (PI, Sigma- baicalein. Next, siRNA-negative control (NC) and Aldrich) for 30 min at 37 °C. DNA content was meas- siRNA-BTG3 (GenePharma, Shanghai, China) were ured utilizing the flow cytometer (BD Biosciences, San respectively transfected into AGS cells (above 80% Diego, CA, USA). Cell Quest acquisition software (BD confluence) using the Lipofectamine 2000 (11668-019, Biosciences) was employed to analyze the percentage of Invitrogen, Carlsbad, CA, USA), and subsequent exper- cells in different cell cycle phases. iments were performed after 48 h of transfection. Experiments were performed using different con - Flow cytometric analysis of cell apoptosis centrations of baicalein or 4-PBA, LY294002 (all from The Annexin V/PI assay was performed based on the Sigma-Aldrich), or DMSO (baicalein, 4-PBA, and manufacturer’s instructions (Invitrogen). AGS cells Ly294002 were all dissolved in DMSO). AGS cells were were collected and next rinsed with cold PBS, cen- treated and grouped as follows: blank group (with- trifuged, and resuspended in 100 µL binding buffer out any treatment), DMSO group (treated with 0.1% containing 2.5 µL fluorescein isothiocyanate-labeled DMSO, served as a solvent control group of baicalein Annexin V and 1 µL 100 µg/mL PI, followed by incu- group), Bai (15 µM) group (treated with 15 µM baica- bation for 15 min in the dark. Afterwards, cells were lein), Bai (30 µM) group (treated with 30 µM baica- detected utilizing the flow cytometer. lein), Bai (60 µM) group (treated with 60 µM baicalein), Bai (30 µM) + DMSO group (treated with 30 µM bai- Reverse transcription quantitative polymerase chain calein and supplemented with solvent of 4-PBA, 0.1% reaction (RT‑qPCR) DMSO), Bai (30 µM) + 4-PBA group (after treatment Total RNA was separated from cultured cells using of 30 µM baicalein, added with 10 µM ERS inhibitor Rneasy Mini kits (Qiagen, Valencia, CA, USA). 4-PBA), Bai (30 µM) + siRNA-NC group (transfected qPCR analyses were processed on a 96-well plate ABI with si-NC and then supplemented with 30 µM baica- Prism7500 System (ABI, Foster City, CA, USA) uti- lein), Bai (30 µM) + siRNA-BTG3 group (delivered with lizing SYBR Green PCR Master Mix (Takara, Otsu, si-BTG3 and later added with 30 µM baicalein), Bai (30 Japan). The primers used were as follows: the forward µM) + siRNA-BTG3 + DMSO group (after transfec- of BTG3 was 5′-CTC CTC CTG TTC CAT TTG GT-3’ and tion with si-BTG3, supplemented with 30 µM baicalein the reverse was 5′-TAA TCC AGT GAT TCC GGT CA-3′. and solvent of LY294002, 0.1% DMSO), and Bai (30 Cycling conditions were as follows: 5 s at 94°C, 34 s at µM) + siRNA-BTG3 + LY294002 group (after transfec- 60°C, and 72°C for 40 cycles. Glyceraldehyde-3-phos- tion with si-BTG3, added with 30 µM baicalein and 10 phate dehydrogenase (GAPDH) acted as internal con- µM PI3K/AKT pathway inhibitor LY294002). The doses trol. The forward of GAPDH was 5′-GTC TTA CTC CTT of 4-PBA and LY294002 were determined according to GGA GGC C-3′ and the reverse was 5′-TCA TTT CCT the product instructions. The subsequent indicators GGT ATG ACA ACGA-3′. Quantitative expression was were detected after 48 h of treatment. −ΔΔCt computed utilizing the 2 method . Colony formation assay 2+ Ca detection AGS cells were detached into single-cell suspension 2+ Intracellular Ca level was measured using the Fluo-3 using trypsin-ethylene diamine tetraacetic acid (Gibco, AM fluorescence working solution (Beyotime, Shang - Grand Island, NY, USA) solution. Subsequently, 2 hai, China). Briefly, AGS cells treated differently were mL cells were seeded onto 6-well plates (Corning) at collected and subsequently added with Fluo-3 AM fluo - 2 × 10 cells/mL. After attachment, cells were treated rescence working solution at 37 °C for another 30 min. with baicalein (0 and 30 µmol/L) for 48 h. The superna - Cells were rinsed thrice with PBS buffer and photo - tant was substituted by fresh medium and subsequently graphs were obtained using a fluorescence microscope cells were cultured for 15 days. Next, cells were fixed Shen et al. Applied Biological Chemistry (2023) 66:10 Page 4 of 14 and rehydrated in graded alcohols and distilled water. (magnification: 200× ; Nikon, Tokyo, Japan). Intracel- 2+ Endogenous peroxidase was blocked for 5 min with 3% lular Ca concentration was monitored through flow hydrogen peroxide in distilled water. Nonspecific bind - cytometry. ing was blocked for 30 min with normal horse serum at 37 °C. Sections were subsequently incubated with Ki67 Western blot (1:500, ab16667, Abcam). Ki67 levels were detected Cells were treated with radio-immunoprecipita- using VETASTAIN ABC kits (Vector Laboratories, Burl- tion assay buffer (89,901; Thermo Fisher Scientific, ingame, CA, USA). Waltham, MA, USA) at 4 °C to extract proteins from cells. Protein concentration was determined by bicin- TUNEL assay choninic acid kits (Thermo Fisher Scientific). Proteins TUNEL assay was conducted in line with the manufac- (30 µg/lane) were isolated by 10% sodium dodecyl sul- turer’s protocol (In Situ Cell Death Detection kit, POD, fate-polyacrylamide gel electrophoresis and transferred Roche, Basel, Switzerland). In brief, after fixing with 4% to polyvinylidene fluoride membranes. Subsequently, paraformaldehyde in PBS and washing thrice with PBS, membranes were incubated at 4 °C with 5% nonfat dry cells were rinsed with 3% H O in methanol for 10 min. milk overnight and with primary antibodies against 2 2 Following incubation for 2 min with 0.1% Triton X-100 glucose-regulated protein 78 (Grp78, 1:1000, ab21685, in 0.1% sodium citrate on ice, cells were then incubated Abcam, Cambridge, UK), C/EBP homologous pro- for 1 h with the TUNEL reaction mixture and later with tein (CHOP, 1:1000, 5554, Cell signaling technology, 4’,6-diamidino-2-phenylindole for 5 min in the dark. A USA), BTG3 (1:1000, ab92309, Abcam), PI3K (1:2000; fluorescence microscope (Eclipse 80I, Nikon) was applied ab140307, Abcam), p-PI3K (1:1000; ab182651, Abcam), for data analyses. AKT (1:1000, ab8805, Abcam), p-AKT (1:1000, ab38449, Abcam), and GAPDH (1:1000, ab9485, Statistical analysis Abcam) for 24 h. After washing with PBS-containing All cell experiments were independently conducted in Tween-20, membranes were incubated for 2 h with goat triplicate. Data were exhibited as mean ± standard devia- anti-rabbit immunoglobulin G (1:2000, ab6721, Abcam) tion (SD). SPSS 21.0 software (IBM Corp. Armonk, NY, horseradish peroxidase-labeled secondary antibody at USA) was applied for data analyses. Firstly, the normal- 4 °C. Next, enhanced chemiluminescence substrates ity and homogeneity of variance tests were performed (Generay Biotech, Shanghai, China) were used to detect and the tests conformed to the normal distribution and the membranes. the variances were homogeneous. The measurement data were displayed as mean ± SD. The comparisons Animal experiments among multiple groups were analyzed by the one-way Total 24 five-week-old male BALB/c nude mice pro - ANOVA, followed by Tukey’s test. GraphPad Prism 8 was vided by Shanghai Laboratory Animal Center (Shang- employed for data plotting. The p < 0.05 indicated statisti - hai, China) were reared in the specific-pathogen-free cally significant. laboratory animal room and provided with a sterile diet under a controlled temperature of 26 ± 1 °C, humidity of Results 40–60%, and light exposure for 10 h/24 h. After one week Baicalein inhibited GC cell proliferation and induced G0/G1 of adaptive feeding, the xenograft model was established arrest and apoptosis by regulating cell cycle by subcutaneously injecting 100 µL AGS cells (contain- Firstly, the action of baicalein on GC cell growth was ing approximately 1 × 10 cells) into the right rib of each detected by the MTT assay and GC cell lines HGC-27 mouse. Three weeks later, the nude mice were intragas - and AGS were cultured using different concentrations of trically administered with 0.2 mL normal saline (control baicalein. We noted that baicalein effectively repressed group), or baicalein at 15 mg/kg/day or 50 mg/kg/day for GC cell proliferation in a concentration-dependent man- 4 weeks, with 8 nude mice per group. Tumor size was ner, while there was no evident difference in the sup - measured weekly after treatment and calculated as length pression of 120 µM baicalein and 60 µM baicalein on × width × width/2. Later, 2 h after the last treatment, GC cell viability (Fig. 1A). Moreover, the repression of mice were euthanized and the tumors were removed and baicalein on AGS cell viability was better than that of weighed for subsequent experiments. HGC-27 cells, and therefore AGS cells were used as the study subjects. The IC50 (50% inhibitory concentration) Immunohistochemical study of baicalein against AGS cells was approximately 30 µM The formalin-fixed paraffin-embedded (FFPE) sections via the cell viability curve (Fig. 1A). The AGS cells were were subjected to immunohistochemistry. FFPE GC assigned into the blank group, DMSO group, and Bai (30 tumor sections were cut (3 μm), deparaffinized in xylene, Shen et al. Applied Biological Chemistry (2023) 66:10 Page 5 of 14 Fig. 1 Baicalein inhibited GC cell proliferation and induced G0/G1 arrest and apoptosis by regulating cell cycle. A Cell viability detected by the MTT assay; B Colony number, colony diameter, and colony area were detected by the colony formation assay; C Cell cycle detected using flow cytometry; D Apoptosis detected using flow cytometry. Cell experiment was repeated thrice, and data were expressed as mean ± SD. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001 µM) groups. The results evinced that baicalein markedly GC cell apoptosis (Fig. 1D, p < 0.001). Additionally, com- reduced colony number, colony diameter, and colony area pared with the blank group, DMSO showed no obvious (Fig. 1B, all p < 0.05). To gain insight into the mechanism effects on AGS cells (Fig. 1B−D, all p > 0.05). Overall, bai- by which baicalein prevented GC cell growth, we adopted calein prevented GC cell proliferation and induced G0/ flow cytometry to detect the cell cycle of baicalein- G1 arrest and GC cell apoptosis by mediating the cell treated AGS cells. Baicalein treatment noticeably caused cycle. more cells arrested in G0/G1 phase and fewer cells in the S phase (Fig. 1C, p < 0.05). To test whether baicalein could induce GC cell death, we detected apoptosis using flow cytometry, which indicated the promotion of baicalein in Shen et al. Applied Biological Chemistry (2023) 66:10 Page 6 of 14 Fig. 2 Baicalein triggered ERS in GC cells. A Cell morphology observed under a microscope; B Expression levels of ERS markers Grp78 and CHOP determined using Western blot; C Intracellular Ca2 + levels measured by Fluo-3 AM fluorescent probes; D Intercellular Ca2 + levels measured by flow cytometry. Cell experiment was duplicated thrice, and data were exhibited as mean ± SD. One-way ANOVA was adopted for comparisons among multiple groups, followed by Tukey’s test. *p < 0.05, **p < 0.01, ***p < 0.001 Baicalein triggered ERS in GC cells that, compared with GC cells without baicalein treat- During baicalein-induced apoptosis, we observed the ment, the expression levels of Grp78 and CHOP were presence of cellular vacuolization in 30 µM baicalein- increased after baicalein treatment (Fig. 2B, p < 0.05), treated AGS cells using microscopy, but not in normal indicating the triggering of ERS in GC cells. Cellular cells (Fig. 2A). Hence we speculated that these cyto- calcium homeostasis remains one of the functions of 2+ plasmic vacuoles might be the dilated ER lumen under ER and elevated intracellular Ca level is considered a stress . Therefore, we used Western blot to detect vital indicator of ERS . The results unveiled that the 2+ the ERS markers Grp78 and CHOP , with tunica-intracellular Ca levels were prominently raised in bai- mycin (TM, 5 µg/mL)-treated cells as positive controls calein-induced cells, reaching more than 10-fold higher for ERS-induced GC cells. The results demonstrated than that of control cells (Fig. 2C, D, all p < 0.05). Rela- tive to the blank group, DMSO showed no significant Shen et al. Applied Biological Chemistry (2023) 66:10 Page 7 of 14 effects on ERS in AGS cells (Fig. 2A−D, all p > 0.05). 30 µM baicalein for 48 h. Firstly, Western blot revealed Altogether, baicalein could trigger ERS in GC cells. that ERS inhibitor 4-PBA partly annulled the effects of baicalein on upregulating Grp78 and CHOP levels 2+ Baicalein induced apoptosis by triggering ERS in GC cells (Fig. 3A, all p < 0.01). Additionally, intracellular C a con- To assess whether baicalein-induced GC cell apoptosis centration was measured by Fluo-3 AM calcium-sensitive was affected by ERS, we used the ERS inhibitor 4-PBA to fluorescent probes and flow cytometry, which revealed pre-treat GC cells and subsequently treated the cells with that 4-PBA partially counteracted the promotion role of Fig. 3 Baicalein induced apoptosis by triggering ERS in GC cells. A Expression levels of ERS markers Grp78 and CHOP measured using Western blot; B Intracellular Ca2 + levels determined by Fluo-3 AM fluorescent probes; C Intercellular Ca2 + levels measured by flow cytometry; D Cell apoptosis detected by flow cytometry. Cell experiment was replicated thrice, and the data were presented as mean ± SD. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey’s test. **p < 0.01, ***p < 0.001 Shen et al. Applied Biological Chemistry (2023) 66:10 Page 8 of 14 2+ Furthermore, flow cytometry revealed that LY294002 baicalein in Ca concentration (Fig. 3B, C, all p < 0.01). significantly increased the proportion of apoptotic cells The induced ERS can trigger the UPR to respond to (Fig. 5E, all p < 0.01). The above results demonstrated environmental factors. Afterwards, flow cytometry was that LY294002 partly reversed the antagonistic effects employed to verify GC cell apoptosis, which unraveled of BTG3 downregulation on baicalein. In brief, baicalein that 4-PBA could partially reverse the promotion of bai- inhibited the PI3K/AKT pathway by activating BTG3, calein on apoptosis (Fig. 3D, all p < 0.01). Briefly, baicalein thereby triggering ERS and inducing apoptosis. induced apoptosis by triggering ERS in GC cells. Baicalein effectively prevented GC progression in vivo Baicalein triggered ERS in GC cells to induce apoptosis To validate whether baicalein could inhibit GC in vivo, we by activating BTG3 established subcutaneous xenograft models. The tumor BTG3, a candidate tumor suppressor, prevents cell pro- growth curve revealed the repression of baicalein at 15 liferation, induces apoptosis, and mediates cell cycle and 50 mg/kg/d on xenograft tumor growth (Fig. 6A, in various tumors [22, 28]. Firstly, we found that BTG3 p < 0.01), and smaller tumors were observed after 50 mg/ expression presented a concentration-dependent kg/d baicalein treatment. Additionally, mice in the treat- increase in GC cells under 15, 30, and 60 µM baicalein ment groups showed markedly lower tumor weight than treatment (Fig. 4A, B). To investigate the role of BTG3 in the control mice (Fig. 6B, p < 0.001). The expression of baicalein-induced apoptosis, we inhibited BTG3 expres- Ki67 in tumors was detected by immunohistochemistry sion in GC cells. The expression levels of BTG3 in GC to reveal the cell proliferation in tumors, and the staining cells of each treatment group were determined by RT- results showed that the number and staining degree of qPCR and Western blot, which unveiled the successful Ki67-positive cells were notably decreased upon 50 mg/ transfection of BTG3-siRNA into GC cells (Fig. 4A, B, all kg/d baicalein treatment (Fig. 6C), indicating that baica- p < 0.01). BTG3 silencing partially invalidated the facili- lein treatment could relieve cell proliferation in the trans- tation effects of baicalein on Grp78, CHOP (Fig. 4C, all 2+ planted tumors to some extent. Moreover, TUNEL assay p < 0.01), and Ca levels (Fig. 4D, E, all p < 0.01). Moreo- unveiled that the number of tumor apoptosis-positive ver, BTG3 silencing lowered the proportion of apoptotic cells was prominently higher in the 50 mg/kg/d baica- cells and counteracted the baicalein-induced apoptosis lein group than in the 15 mg/kg/d baicalein and control (Fig. 4F, all p < 0.01). Taken together, baicalein triggered groups (Fig. 6D). These aforesaid results indicated that ERS in GC cells via the activation of BTG3, thus inducing baicalein effectively inhibited GC progression in vivo. apoptosis. To further validate the findings in vitro, after tumor homogenization, we determined the ERS-related pro- Baicalein triggered ERS to induce apoptosis by impeding teins Grp78, CHOP, and the BTG3/PI3K/AKT pathway- the PI3K/AKT pathway via the activation of BTG3 related proteins BTG3, PI3K, AKT, p-PI3K, and p-AKT PI3K/AKT is an imperative intracellular pathway; when using Western blot, which revealed that BTG3 was aberrantly activated, it can activate downstream mol- remarkably elevated and the levels of p-PI3K and p-AKT ecules, thus affecting GC development [29, 30]. Herein, were diminished in nude mice treated with 50 mg/kg/d we guessed that BTG3 could participate in ERS-initiated baicalein compared with the 15 mg/kg/d baicalein group apoptosis via the PI3K/AKT pathway. Firstly, we demon- (Fig. 6E, F, all p < 0.001). Altogether, baicalein treatment strated that after adding baicalein (15, 30, and 60 µM) to triggered ERS by impeding the PI3K/AKT pathway and GC cells, PI3K/AKT pathway activity was inhibited and activating BTG3, thus inducing cell apoptosis. PI3K and AKT phosphorylation levels were prominently reduced in a concentration-dependent manner (Fig. 5A, Discussion all p < 0.05). In addition, we silenced BTG3 by cell trans- GC represents a lethal disease with poor overall sur- fection and then added 30 µM baicalein. BTG3 knock- vival and most cases are attributed to diverse pathogenic down partially annulled the suppression of baicalein on infections . Baicalein possesses efficient anti-tumor the PI3K/AKT pathway. Subsequently, we supplemented properties and can promote the apoptosis of different the PI3K inhibitor (10 µM LY294002) to baicalein-treated human cancer cell lines . Surprisingly, tumor micro- siRNA-BTG3-transfected GC cells. Western blot results environment stresses that break proteostasis can arise illustrated that LY294002 diminished p-PI3K and p-AKT ERS, which is perceived as a trigger for apoptosis [33, 34]. levels in GC cells (Fig. 5A, all p < 0.01), indicating the Moreover, preceding evidence unravels the participation blocking of the PI3K/AKT pathway. However, Grp78 of the PI3K/AKT pathway in human GC cell autophagy and CHOP levels were noticeably elevated (Fig. 5B, all 2+ and apoptosis . This study demonstrated the p < 0.01) and Ca levels were also raised (Fig. 5C, D, all p < 0.05), suggesting the initiation of ERS in GC cells. Shen et al. Applied Biological Chemistry (2023) 66:10 Page 9 of 14 Fig. 4 Baicalein triggered ERS in GC cells to induce apoptosis by activating BTG3. A, B BTG3 mRNA and protein expression determined by RT-qPCR and Western blot; C Expression levels of Grp78 and CHOP determined by Western blot; D Intracellular Ca2 + levels measured by Fluo-3 AM fluorescent probes; E: Intercellular Ca2 + levels measured by flow cytometry; F: Apoptosis assessed by flow cytometry. Cell experiment was repeated thrice, and the data were showed as mean ± SD. One-way ANOVA was employed for comparisons among multiple groups, followed by Tukey ’s test. *p < 0.05, **p < 0.01, ***p < 0.001 Shen et al. Applied Biological Chemistry (2023) 66:10 Page 10 of 14 Fig. 5 Baicalein triggered ERS to induce apoptosis by inhibiting the PI3K/AKT pathway via the activation of BTG3. A PI3K, p-PI3K, AKT, and p-AKT proteins determined by Western blot; B Expressions levels of Grp78, and CHOP determined by Western blot; C Intracellular Ca2 + levels measured using Fluo-3 AM fluorescent probes; D Intercellular Ca2 + levels measured using flow cytometry; E Apoptosis detected by flow cytometry. Cell experiment was duplicated thrice, and the data were expressed as mean ± SD. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey’s test. *p < 0.05, **p<0.01, ***p < 0.001 Shen et al. Applied Biological Chemistry (2023) 66:10 Page 11 of 14 Fig. 6 Baicalein effectively prevented GC progression in vivo. A Tumor volume during baicalein treatment; B Tumor weight after baicalein treatment; C Immunohistochemical staining of Ki67; D Apoptotic cells detected using TUNEL staining; E, F ERS-related and BTG3/PI3K/AKT pathway-related proteins detected by Western blot. The data were exhibited as mean ± SD. One-way ANOVA was adopted for comparisons among multiple groups, followed by Tukey’s test. *p<0.05, **p<0.01, ***p < 0.001 regulation of baicalein in GC cell apoptosis by triggering p38 pathway . Baicalein potently reduces Bcl-2 and ERS through suppression of the PI3K/AKT pathway. raises Bax, which impedes colony formation and growth Firstly, we treated GC cells with baicalein and found of GC cells and might elicit apoptosis via the mito- the inhibition of baicalein on cell growth. More specifi - chondrial pathway . Moreover, ERS is implicated in cally, baicalein prevented GC cell proliferation, resulted inhibiting GC tumorigenesis via activation of Grp78 and in more cells arrested in G0/G1 phases, and induced CHOP that facilitate tumor cell growth and cell cycle 2+ apoptosis by regulating cell cycle. Consistently, baica- arrest . Likewise, Ca is an imperative cytokine in lein can inhibit proliferation and invasive capability, the CaMK II pathway and can be construed as a media- and stimulate autophagy and apoptosis in GC cells . tor of ERS apoptosis pathway . Hence, we subse- Interestingly, baicalein suppresses GC cell invasion by quently estimated the ERS in GC cells. Our results noted 2+ lowering cell migration and motility via repression of the that levels of Grp78 and CHOP and Ca concentration Shen et al. Applied Biological Chemistry (2023) 66:10 Page 12 of 14 were elevated in baicalein-treated GC cells, and the adenocarcinoma . Next, we added baicalein into trend was partially abrogated by 4-PBA. Much in line BTG3-downregulated GC cells and observed that BTG3 with our finding, prior studies have noted that baicalein silencing abrogated the suppression of baicalein on the 2+ elicits apoptosis via Ca generation, which also induces PI3K/AKT pathway. BTG3 overexpression suppresses ERS through the Grp78 in breast cancer cells . Bai- the activation of the PI3K/AKT/mTOR pathway; in 2+ calein potentiates cytosolic Ca activity, presumably due contrast, BTG3 silencing promotes it . Afterwards, to stimuli of cation channels in the cell membrane . the PI3K inhibitor (LY294002) was further supple- Additionally, baicalein induces the ERS response and mented to these GC cells and the detection results illus- up-regulation of the CHOP protein in C2C12 myotubes trated the initiation of ERS and enhanced cell apoptosis . The above information highlighted that baicalein after inhibition of the PI3K/AKT pathway. Intriguingly, can trigger apoptosis and ERS in GC cells. Furthermore, p-AKT level is elevated in SGC7901 and AGS cells and to estimate whether baicalein induced GC cell apopto- GC tissues, and blockade of PI3K/AKT pathway inhib- sis through ERS, we pre-treated GC cells using an ERS its GC metastasis and induces apoptosis [51, 52]. ERS inhibitor 4-PBA, followed by baicalein treatment. Later, triggers cell apoptosis in bladder cancer, ovarian cancer, we observed that the ERS inhibitor nullified the promo - and lung cancer by repressing the PI3K/AKT/mTOR 2+ tion of baicalein on Grp78 and CHOP levels, C a con- pathway [23, 53, 54]. Moreover, we performed in vivo centration, and apoptosis. Excessive ERS contributes to experiments, which revealed that baicalein effectively apoptosis and blockage of ERS attenuated scoulerine- inhibited GC growth by elevating BTG3 and decreas- induced apoptosis in colorectal cancer cells . Consist- ing levels of p-PI3K and p-AKT. Collectively, baicalein ently, schizandrin A stimulates apoptosis and impedes blocked the PI3K/AKT pathway by activating BTG3, GC cell growth by activating ERS . Baicalein induces thus triggering ERS to induce apoptosis. hepatocellular carcinoma apoptosis by triggering ERS, ERS can be induced by various pathological stimuli, presumably by lowering the anti-apoptotic regulators and including glucose starvation, hypoxia, and oxidative 2+ elevating intracellular Ca . In consistency with the stress . The ERS response has been identified to aforementioned findings, our results ascertained that bai - have adaptive and apoptotic pathways, and the per- calein could induce GC cell apoptosis by initiating ERS. sistent ERS could initiate cancer cell apoptosis . Compelling evidence has unraveled the tumor-sup- Therein regulating ERS presumably be an anti-cancer pressive properties of BTG3 in numerous tumors: for strategy. Since it is temporarily unknown by which instance, overexpression of BTG3 prevents the GC cell molecular mechanism baicalein regulates cancer cells, invasion . Therefore, we determined BTG3 expres - this study determined GC cell viability following treat- sion and found up-regulated BTG3 levels in baicalein- ment with various concentrations of baicalein, and the treated GC cells in a baicalein concentration-dependent results unraveled that baicalein impeded the PI3K/AKT manner. Afterwards, GC cells were transfected with pathway, activated ERS, reduced cell viability, and facil- BTG3-siRNA to explore the function of BTG3 in baica- itated apoptosis in a dose-dependent pattern. However, lein-elicited apoptosis. The results revealed that BTG3 baicalein may cause other changes such as autophagy knockdown noticeably diminished Grp78, CHOP, and in GC cells by activating ERS, and these changes also 2+ Ca levels, and counteracted the inducement of baica- exhibit some therapeutic effects on cancer cells. Future lein on apoptosis. BTG3 is weakly expressed in GC tis- studies should examine the morphology and activity of sues and prevents GC cell growth by lowering cell-cycle cancer cells affected by baicalein and explore other pos - progression and augmenting apoptosis . Overall, bai- sible alterations in cancer cells resulted from baicalein calein triggered ERS in GC cells to induce apoptosis by by activating ERS. activating BTG3. Acknowledgements GC represents one of the most influenced cancers Not applicable. through the PI3K/AKT pathway , thus arising an Author contributions assumption that BTG3 is involved in the ERS-trig- JS contributed to the study concepts and study design, ZY contributed to gered apoptosis via the PI3K/AKT pathway. In our the guarantor of integrity of the entire study; XW contributed to the literature study, the phosphorylation of PI3K and AKT in GC research; JS, GY contributed to the experimental studies and data acquisition; JS, ZY, XW contributed to the manuscript preparation and MH contributed to cells was lowered after baicalein treatment in a baica- the manuscript editing and review. All authors read and approved the final lein concentration-dependent manner. Growing stud- manuscript. ies have elucidated the anti-cancer roles of baicalein Funding by impeding the PI3K/AKT pathway in cervical can- Not applicable. cer , undifferentiated thyroid cancer , and lung Shen et al. Applied Biological Chemistry (2023) 66:10 Page 13 of 14 Availability of data and materials 16. Wang Y, Wang K, Jin Y, Sheng X (2019) Endoplasmic reticulum proteo- All the data generated or analyzed during thisstudy are included in this stasis control and gastric cancer. Cancer Lett 449:263–271 published article. 17. 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Applied Biological Chemistry – Springer Journals
Published: Feb 13, 2023
Keywords: Baicalein; Gastric cancer; PI3K/AKT; Endoplasmic reticulum stress; BTG3; Cell apoptosis; Ca2+; Ki67
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