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Shikonin inhibits cancer cell cycling by targeting Cdc25s

Shikonin inhibits cancer cell cycling by targeting Cdc25s Background: Shikonin, a natural naphthoquinone, is abundant in Chinese herb medicine Zicao (purple gromwell) and has a wide range of biological activities, especially for cancer. Shikonin and its analogues have been reported to induce cell-cycle arrest, but target information is still unclear. We hypothesized that shikonin, with a structure similar to that of quinone-type compounds, which are inhibitors of cell division cycle 25 (Cdc25) phosphatases, will have similar effects on Cdc25s. To test this hypothesis, the effects of shikonin on Cdc25s and cell-cycle progression were determined in this paper. Methods: The in vitro effects of shikonin and its analogues on Cdc25s were detected by fluorometric assay kit. The binding mode between shikonin and Cdc25B was modelled by molecular docking. The dephosphorylating level of cyclin-dependent kinase 1 (CDK1), a natural substrate of Cdc25B, was tested by Western blotting. The effect of shikonin on cell cycle progression was investigated by flow cytometry analysis. We also tested the anti-proliferation activity of shikonin on cancer cell lines by MTT assay. Moreover, in vivo anti-proliferation activity was tested in a mouse xenograft tumour model. Results: Shikonin and its analogues inhibited recombinant human Cdc25 A, B, and C phosphatase with IC values ranging from 2.14 ± 0.21 to 13.45 ± 1.45 μM irreversibly. The molecular modelling results showed that shikonin bound to the inhibitor binding pocket of Cdc25B with a favourable binding mode through hydrophobic interactions and hydrogen bonds. In addition, an accumulation of the tyrosine 15-phosphorylated form of CDK1 was induced by shikonin in a concentration-dependent manner in vitro and in vivo. We also confirmed that shikonin showed an anti-proliferation effect on three cancer cell lines with IC values ranging from 6.15 ± 0.46 to 9.56 ± 1.03 μM. Furthermore, shikonin showed a promising anti-proliferation effect on a K562 mouse xenograph tumour model. Conclusion: In this study, we provide evidence for how shikonin induces cell cycle arrest and functions as a Cdc25s inhibitor. It shows an anti-proliferation effect both in vitro and in vivo by mediating Cdc25s. Keywords: Shikonin, Cdc25s, Anticancer, Cell cycle progression Background promising anticancer target [2–4]. Several inhibitors of Dual-specificity protein phosphatases (DSP) Cdc25s Cdc25s with antitumour activity have been reported [5, 6]. (Cdc25A, Cdc25B, and Cdc25C) have an essential role in Most potent small molecule inhibitors of the Cdc25 phos- cell cycle progression via controlling the phosphorylation phatases are quinone-derived compounds, such as mena- state of their natural substrates cyclin-dependent kinases dione (vitamin K )[7], compound 2 [8], NSC95397 [9], (CDKs). Overexpression of Cdc25s and over-activation of compound 5 [10], and NSC668394 [11](Fig. 1). This type CDKs are involved in tumour-associated cell-cycle aberra- of compound was hypothesized to inhibit the activity of tion [1]. Therefore, Cdc25s is currently considered to be a Cdc25s by oxidation of the catalytically essential cysteine residue in the enzyme’s active site through production of reactive oxygen species [12]. * Correspondence: Shoude.zhang@qhu.edu.cn; suzhanhai@foxmail.com The naphthoquinone-type natural product shikonin was State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, derived from the root of Lithospermum erythrorhizon, 251# Ningda Road, Xining 810016, Qinghai, China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. BMC Cancer (2019) 19:20 Page 2 of 9 Fig. 1 Structures of quinone-type inhibitors of Cdc25s and shikonin which has broad applications in Traditional Chinese quinone-type inhibitor of Cdc25s [7] that was used as a Medicine [13, 14]. Over the past few decades, a number of positive control here. studies have demonstrated multiple biological effects of For the dialysis assay, the enzyme-inhibitor complex shikonin. It has been reported to have anti-HIV [14], including 0.2 μM Cdc25B and 50 μM shikonin was dia- anti-inflammatory [15, 16], antibacterial, and anticancer lyzed against 5000-fold of the assay buffer for the indi- [17–20] activities. Among these activities, the anticancer cated period of time. At the end of each dialysis, Cdc25B activity, especially the induction of apoptosis and necrop- activity was determined as described above. tosis, is well reported [13, 19–23]. However, the key target is still unclear. Shikonin has a similar chemical skeleton to Molecular modelling that of the quinone-type inhibitors of Cdc25s. There- The docking method used is described in a previous fore, we hypothesized that shikonin will have similar work [24]. In summary, molecular modelling was per- effects on Cdc25s. To test this hypothesis, the effects formed using Maestro 9.0. The X-ray structure of of shikonin on Cdc25s and related biofunction were Cdc25B (PDB ID: 1QB0) was downloaded from the Pro- confirmed in this paper. tein Data Bank (PDB, https://www.rcsb.org) and pre- pared with the “Protein Preparation Wizard” workflow Methods with default settings. The grid-enclosing box was gener- Chemicals ated within 10 Å of Cys473 in the refined crystal struc- Shikonin and its analogues are natural products that ture. The ligand structure was prepared with the Ligprep were purchased from Herbest, Inc. (Baoji, Shanxi, China). module. Finally, shikonin was docked into Cdc25B using All other chemicals were purchased from Sigma-Aldrich Glide (version 5.5) in extra precision (XP) mode with de- (Shanghai, China) unless otherwise noted. fault settings [25]. Favourable binding poses were se- lected according to the docking score and view check. Measurement of phosphatase inhibitory activity of shikonin and its analogues Cell lines and culture conditions CycLex® protein phosphatase Cdc25A, -B and -C fluoro- K562 cells (myelogenous leukaemia cell line), MCF-7 metric assay Kit (CycLex, Cat. No. CY-1352, CY-1353, cells (breast cancer cell line) and HeLa cells (cervical CY-1354) was used to test the enzyme inhibition rate of cancer cells) were obtained from the Chinese Academy shikonin and its analogues for Cdc25A, -B and -C. In of Sciences Cell Bank (Shanghai, China). The catalogue summary, dual-specificity phosphatase activity was numbers of these cell lines are TCHu191, TCHu74 and measured in a 96-well microtiter plate using O-methyl- TCHu187, respectively. The temperature-sensitive FT210 fluorescein phosphate (OMFP) as a substrate. 5 μL cell line (tsFT210) is a mouse breast cancer cell line that is (0.1 μg/μL) of purified recombinant Cdc25s (Cdc25A, -B widely used for studying cell cycle progression. Intracellu- and -C) was mixed with 40 μL of assay mixture and in- lar CDK1 protein of tsFT210 is inactive at 39 °C because cubated with 5 μL of the test compound at various con- of two point mutations on the cdc2 gene, which leads to it centrations in a well. Then, 25 μL of stop solution was being easily controlled at different cell cycle phases added. Fluorescence was measured at an excitation through changes in temperature [26]. This cell line was wavelength of 485 nm and an emission wavelength of kindly provided by Dr. Rongcai Yue from his laboratory 530 nm using a fluorescence microplate reader (BioTek (School of Pharmacy, Second Military Medical University). Instruments, Inc., Winooski, VT, USA). Menadione is a All cell lines were kept in the logarithmic growth phase in Zhang et al. BMC Cancer (2019) 19:20 Page 3 of 9 5% CO at 37 °C with RPMI-1640 medium supplemented negative control. Nocodazole, a potent mitotic blocker with 10% FBS and 1% penicillin G-streptomycin in a hu- that arrests cells at the G /M phase, was used as a posi- midified chamber at 5% CO . tive control at a concentration of 100 nM. Western blotting Anti-proliferation assay The phosphorylation status of CDK1 was analysed by The anti-proliferation activity of shikonin against cancer Western blotting as described in our previous work [24]. cells was tested by the MTT method in 96-well plates. 6 3 In summary, tsFT210 cells (1 × 10 ) were treated with First, 4 × 10 cells were seeded per well and treated with shikonin (0, 1, 5 and 25 μM) for 4 h followed by collec- the shikonin with serial concentrations for 48 h. Then, tion and suspension in lysis buffer (150 mM NaCl, 50 the cells were incubated with 10 μL of MTT solution (5 mM Tris, 0.02% NaN , 0.01% phenylmethylsulfonyl mg/mL in PBS) for an additional 2–4 h at 37 °C. The for- fluoride, 0.2% aprotinin, and 1% TritonX-100, pH 8.0) mazan crystals were dissolved with 100 μLofDMSO after containing a protease inhibitor cocktail (Thermo Scien- removing the supernatant. The absorbance at 570 nm was tific) for 30 min at 4 °C. The protein concentration in measured by a BioTek Synergy 2 plate reader (BioTek cell lysates was confirmed by the Bradford method. Fifty Instruments, Inc., Winooski, VT, USA). Doxorubicin, a micrograms of protein per lane was resolved on 10% chemotherapy medication used to treat cancer and having SDS polyacrylamide gels, and the bands were transferred a broad spectrum of anticancer activities [29], was used as to PVDF membranes (Millipore). Nonspecific reactivity a positive control here. was blocked by 5% non-fat milk prepared in TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) at Xenograph tumour model in mice room temperature for 1 h. The protein signals were cap- The mouse model was constructed according to our pre- tured with primary antibodies and the secondary anti- vious work [24]. Balb/c mice (female, 5 weeks old), pur- bodies according to the manufacturers’ instructions. In chased from Shanghai SLAC Laboratory Animal Co., this process, the protein β-actin was used to normalize LTD (Shanghai, China), were subcutaneously injected target protein. All antibodies used in this paper were into a 100 μL K562 cell suspension (5 × 10 /mL, PBS) in purchased from Cell Signaling Technology (Inc, China). the right flank of mice. We started treating the mice with shikonin at 1, 5, or 10 mg/kg or vehicle containing Intracellular reactive oxygen species assay 5% DMSO (control group) by intraperitoneal injection Reactive oxygen species (ROS) were detected with the every other day when the tumour’s volume reached ap- fluorescent probe 2′,7′-dichlorofluorescin diacetate proximately 200 mm . The long and short dimensions of (DCFH-DA) as described by others [18, 27]. Briefly, tumour size were measured by a digital calliper at days MCF-7 cells or tsFT210 (synchronized at G2 phase) cells 1, 4, 8, 11, 16, 21, and 24 after treatment. The volume were plated in 96-well plates and loaded with 20 μM was calculated with the formula (long dimension) × (short DCFH-DA for 30 min at 37 °C in culture medium. Cells dimension) /2. The mice were euthanized 2 days after the were washed with PBS three times after incubation and last treatment by the method of cervical dislocation. then treated with shikonin at concentrations of 5 μM and 25 μM for 4 h. Untreated cells were used as control. Statistical analysis Fluorescence was measured using a fluorescence micro- Statistical analyses were performed using PRISM soft- plate reader (BioTek Instruments, excitation wavelength: ware. All quantitative values are given as the means ± 485 nm, emission wavelength: 528 nm). The ROS levels SEM. An unpaired Student’s t test was used to evaluate were expressed as RFU (relative fluorescence unit). the difference between two different treatments, and p < 0.05 was considered statistically significant. The statis- Cell cycle analysis tical significance of differences in the survival of mice Cell cycle analysis was performed as described previ- from the different groups was determined by the log-rank ously [28]. Briefly, tsFT210 cells were plated at 1 × 10 test using the same program. cells/well (24-well plate) and maintained at 32 °C. Cell proliferation was blocked at the G2 phase by increasing Results the temperature from 32 to 39 °C and treating for 17 h. Shikonin induce irreversible inhibition of human recombinant Then, the cells were released at 32 °C and immediately CDC25 phosphatases treated with shikonin. The harvested cells were stained Using the protein phosphatase Cdc25 combo fluoromet- with staining buffer (50 μg/ml propidium iodide, 0.1% ric assay kit, shikonin and its analogues in vitro inhibited sodium citrate and 0.2% NP-40) and analysed by Flow recombinant human Cdc25A, -B, and -C phosphatases Cytometry (BD Biosciences). A final concentration of in a concentration-dependent manner with IC values 0.5% DMSO was used for all compounds and as a ranging from 2.14 to 14.32 μM (Table 1). The positive Zhang et al. BMC Cancer (2019) 19:20 Page 4 of 9 Table 1 IC values of shikonin and analogues for inhibition of recombinant human protein phosphatases Structure R Name Cdc25A (μM) Cdc25B (μM) Cdc25C (μM) -OH Shikonin 2.14 ± 0.21 5.82 ± 0.37 4.78 ± 0.18 -H Deoxyshikonin 3.22 ± 0.76 7.32 ± 0.45 6.33 ± 0.65 -OCOCH Acetylshikonin 3.92 ± 0.66 3.87 ± 0.68 4.67 ± 0.34 -OCOCH(CH ) Isobutylshikonin 6.79 ± 1.02 7. 86 ± 0.23 5.89 ± 0.43 3 2 -OCOCH CH(CH ) Isovalerylshikonin 9.53 ± 0.78 11. 23 ± 1.22 10.98 ± 0.97 2 3 2 -OCOCH=CH(CH ) β,β-Dimethylacrylshikonin 4.67 ± 0.85 8.56 ± 0.67 10. 58 ± 1.13 3 2 -OCOCH(CH )CH CH α-Methyl-η-butylshikonin 11.24 ± 1.45 14.32 ± 1.27 13.45 ± 1.45 3 2 3 All values are micromolar concentrations and are the mean ± S.E.M. of three or more independent determinations control menadione led to an inhibition of 4.12 ± 0.87, motif [32]. The phosphorylated amino acids of the sub- 5.37 ± 0.45, and 5.13 ± 0.24 μM, respectively. strate usually bind to this loop through forming hydro- Previous research revealed that the quinone-type in- gen bonds to arginine R. The quinone-type inhibitor was hibitors of Cdc25 phosphatases play a role through irre- supposed to bind the inhibitor binding pocket, which versible oxidation of the catalytic cysteine of Cdc25B was beside the active site (Fig. 3), and induce oxidation [30, 31]. To identify the inhibition pattern of shikonin, of the catalytic cysteine through redox cycling reaction dialysis was used to study the reversibility of shikonin [1]. After docking the shikonin into the above binding action. As shown in Fig. 2a, after dialysis for 2, 4, 6, and loop around cys473, we found that the shikonin bound even 24 h, the activity of Cdc25B was not rescued from to the inhibitor binding pocket similar to other quinone- inhibition by shikonin, indicating that the Cdc25B was type inhibitors. Figure 2 shows the most energetically inhibited irreversibly. In addition, to confirm whether favourable binding pose from docking results. In such a the shikonin has similar mechanism with other quinone- binding mode, the benzene ring of shikonin is involved in type inhibitors, the ROS content was measured in cells hydrophobic interactions with the pocket residues. In treated with or without shikonin. After treating with shi- addition, a hydrogen bond between one oxygen of the konin, intracellular ROS in MCF-7 and tsFT210 cells in- naphthoquinone core and Tyr428 was predicted. creased significantly compared with those of control cells (Fig. 2b), suggesting that Cdc25s inhibition by shi- Shikonin inhibits cell cycle progression konin could be caused by redox cycling of the shikonin Inhibition of Cdc25s results in hyper-phosphorylation of similar to that of other quinones. CDKs and cell cycle arrest. Therefore, the impact of shi- konin on cell cycle progress was investigated. The Molecular model of shikonin interactions with the Cdc25B tsFT210 cell line has been widely used for studying cell catalytic domain cycle progression because it can be easily controlled at In order to evaluate the binding mode and affinity of different cell cycle phases through changing temperature shikonin with Cdc25B, molecular modelling was per- [26]. The synchronized tsFT210 cells were then treated formed using the docking program Glide. X-ray crystal- with the indicated concentration of shikonin and noco- lography showed that the catalytic domain of Cdc25B dazole (a potent mitotic blocker) or 1% DMSO for 6 h. contains the canonical HCX R PTPase catalytic-site The positive control nocodazole arrested the cells at the Fig. 2 Shikonin inhibits Cdc25B irreversibly in vitro and induces ROS production in MCF-7 cells. a Activity of shikonin-treated Cdc25B after dialysis. b ROS production in MCF-7 and tsFT210 cells after treatment with shikonin. Data points represent fold change of three independent experiments. *P < 0.05; **P < 0.01 compared with the control group Zhang et al. BMC Cancer (2019) 19:20 Page 5 of 9 cells (8.97 ± 0.87 μM) compared with the negative con- trol. Doxorubicin was used as a positive control and showed normal anticancer activity according to a previ- ous report. Shikonin has anti-tumour effects in vivo by inhibiting CDK1 dephosphorylation Xenograph tumour model mice were used to evaluate if shikonin can inhibit tumour growth in vivo. K562-bear- ing mice were treated with shikonin at different doses and vehicle for 21 days. The tumour sizes were recorded in this process. As shown in Fig. 5a, shikonin concentration-dependently inhibited tumour growth. Higher doses of shikonin had a better inhibitory effect and longer observed survival time (Fig. 6a, 6b). In the process of this experiment, the tumour growth of mice from higher dose groups was significantly inhibited com- pared with those of the control and low-dose groups. Fig. 3 Binding mode of shikonin in the Cdc25B binding cavity. Shikonin These results suggest that shikonin has profound anti- (yellow) and key interacting residues (green) are represented as stick cancer activity in vivo. Moreover, the protein levels of models with the protein as a surface. H-bonds are shown as dashed phosphorylated CDK1 in primary tumour tissues ob- black lines. The figure was generated using Pymol from Protein Data tained from the mice were significantly increased after Bank ID 1QB0 treatment by shikonin compared with that of the control group, especially for the 5 and 10 mg/kg groups (Fig. 6c). G /M phase. Comparatively, the cells treated with shikonin This means that the shikonin still participates in the cell were blocked at the G /M phase in a concentration- cycle process in vivo. dependent manner, whereas the DMSO-treated control cells had completed mitosis and had entered the following Discussion cell cycle (Fig. 4). Such results indicate that shikonin can Overexpression of CDC25s, which leads to an irregular target and delay cell cycle progression at the G2/M phase. cell cycle, is often present in cancer cells. They control the cell cycle by activating CDKs. Therefore, CDC25s Shikonin inhibits CDK1 dephosphorylation are considered attractive targets for anticancer drug dis- Endogenous Cdc25s controls the cell cycle through de- covery. Several potent CDC25s inhibitors have been phosphorylation of their natural substrate CDKs [2]. identified; however, few of them have entered clinical tri- Therefore, CDK1 protein will be hyper-phosphorylated if als. Shikonin, a natural naphthoquinone, has a similar the CDC25s are inhibited. In order to confirm whether structure to that of the well-reported quinone-type in- shikonin inhibits the activity of intracellular Cdc25 phos- hibitors of CDC25s. Hence, we hypothesized that shiko- phatases, the phosphorylation status of CDK1 was ana- nin will have similar effects on Cdc25s. To test this lysed by Western blotting. As shown in Fig. 5, shikonin hypothesis, the effects of shikonin on Cdc25s and related at 1 μM, 5 μΜ, and 25 μM induced an accumulation of biofunction were proved in this work. the tyrosine 15-phosphorylated form of CDK1. These re- Shikonin and six analogues showed considerable in- sults suggested that shikonin downregulated the activity hibition for CDC25s using OMFP as substrate. The main of Cdc25s, leading to hyper-phosphorylation of CDK1 in difference between these analogues and shikonin is the cultured cells. R groups (Table 1), but they have similar inhibition for CDC25s. Therefore, we suggest that the R groups have Shikonin inhibits in vitro tumour cell proliferation little effect on the activity and that the pharmacological To evaluate the anti-proliferation activity of shikonin for group is the skeleton of naphthoquinone. Finally, shiko- different cancer cells, four cancer cell lines (MCF-7, nin was selected for further research because it showed HeLa, K562, and tsFT210) were selected to investigate the best inhibition for CDC25s compared with other an- the anti-proliferation activity. The viability of cells was alogues and can be generated from other analogues by evaluated by the MTT method. As shown in Table 2, hydrolysing the R group. Shikonin binds to the same shikonin markedly inhibits the proliferation of MCF-7 pocket of Cdc25B as other quinone-type inhibitors ac- cells (IC : 6.82 ± 0.76 μM), HeLa cells (IC : 9.56 ± cording to the molecular modelling results. Together 50 50 1.03 μM), K562 cells (IC : 6.15 ± 0.46 μM) and tsFT210 with the unaltered activity of Cdc25B after dialysis and 50 Zhang et al. BMC Cancer (2019) 19:20 Page 6 of 9 Fig. 4 Cell cycle analysis of tsFT210 cells in the absence or presence of shikonin. a G2/M-arrested cells after a temperature shift for 17 h at 39 °C. b DMSO-treated cells after a temperature shift for 4 h at 32 °C. c Cells treated with 100 nM nocodazole. d-f Cells treated with 1–25 μM shikonin. Data are representative of two independent experiments increasing ROS content after treatment with shikonin, phosphorylation state of CDK1 were investigated. We we suggested that shikonin inhibited Cdc25B similar to proved that shikonin could block the cell cycle of other quinone-type inhibitors, which were hypothe- tsFT210 at the G2/M phase and effectively inhibit de- sized to disrupt Cdc25s phosphatase activity by oxida- phosphorylation of CDK1 in vitro. tion of the catalytically essential cysteine residue in We also evaluated the anti-proliferation activity of shi- the enzyme’s active site through production of react- konin both in vitro and in vivo. Shikonin showed broad ive oxygen species [23]. spectrum anticancer activity after evaluating the anti- Endogenous Cdc25s control the cell cycle through de- proliferation activity for four cancer cell lines. Moreover, phosphorylating their natural substrate CDKs. Therefore, tumour growth of xenograph tumour model mice was the impacts of shikonin on cell cycle progress and significantly inhibited by shikonin. To confirm the in vivo activity of shikonin was through inhibition of the Cdc25s, we also checked the phosphorylation state of CDK1 in tumour tissue from xenograph tumour model mice, and the results showed that shikonin could inhibit dephosphorylation of CDK1 in tumour tissue also, which suggested that shikonin could also participate in the Cdc25s-related cell cycle pathway in vivo. Table 2 Inhibitory activity of shikonin on tumour cells via MTT assay (IC , μM, n = 3, mean ± SD) Fig. 5 Inhibition of CDK1 dephosphorylation caused by shikonin. Cells Compound Cell Line in G2/M phase were treated with the indicated concentrations of MCF-7 HeLa K562 tsFT210 shikonin and DMSO for 4 h and then harvested. Samples were Shikonin 6.82 ± 0.76 9.56 ± 1.03 6.15 ± 0.46 8.97 ± 0.87 processed for Western blot analysis. Data are representative of two independent experiments Doxorubicin 0.28 ± 0.03 0.43 ± 0.06 0.51 ± 0.12 0.56 ± 0.14 Zhang et al. BMC Cancer (2019) 19:20 Page 7 of 9 Fig. 6 Shikonin inhibits tumour growth in vivo in the K562-bearing mice by affecting the phosphorylation of CDK1 (n = 10/group). a Tumour volume plot of K562-bearing mice treated with vehicle or shikonin at 1, 5, or 10 mg/kg by oral gavage for 21 days. The tumours were measured twice per week. The data are represented as the mean ± SEM. Tumour growth was inhibited significantly after treatment with shikonin compared with the control group. *P < 0.05; †P < 0.01; ‡P < 0.001 compared with the control group. b Kaplan-Meier survival plot of the K562-bearing nude mice. Survival of the K562-bearing nude mice was prolonged in the shikonin-treated groups compared with control group. c The phosphorylation level of CDK1 is affected by shikonin. Shikonin reportedly exerts anticancer effects with less death when the cancer cells were co-treated with shi- drug resistance [33], which can be attribute to its mul- konin and necroptotic inhibitor Nec-1 [39]. Moreover, tiple targets. ROS generation, inactivation of NF-κB, up- shikonin and its analogues have been reported can in- regulation of p53 and p21, and activation of caspases duce cell cycle arrest, but the mechanism is still un- may be involved in the anticancer mechanisms of shiko- clear [27]. Here, we identified shikonin as an inhibitor nin [34–38]. In brief, shikonin disrupts the cancer cell of Cdc25s that plays an essential role in cell cycle lines by inhibiting either the necroptosis or apoptosis progression. pathway. Cancer cells died predominantly through the The normal development of tissue depends on the necroptotic pathway when they were co-treated with homeostasis and balance between cell proliferation and shikonin and the apoptosis inhibitor Z-VAD-FMK, death (Fig. 7). When out of balance, as generated by in- while apoptosis became a selective route for cell appropriate proliferation or inhibition of apoptosis and Fig. 7 Schematic of the coordination between cell proliferation and death. ↓: inhibited by shikonin, ↑: promoted by shikonin Zhang et al. BMC Cancer (2019) 19:20 Page 8 of 9 necroptosis pathways, cancer usually results [40]. If one Received: 12 November 2017 Accepted: 13 December 2018 pathway was inhibited or key proteins of it mutated, shi- konin can also play an anticancer role through two other References pathways, which could explain the lower drug resistance 1. Kristjansdottir K, Rudolph J. Cdc25 phosphatases and cancer. Chemistry & of shikonin. biology. 2004;11(8):1043–51. 2. 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Identification of a potent and selective Funding pharmacophore for Cdc25 dual specificity phosphatase inhibitors. Molecular This work was supported by the Project of Qinghai Science & Technology pharmacology. 2002;61(4):720–8. Department (2016-ZJ-Y01, 2018-ZJ-948Q) and the Open Project of State Key 10. Tamura K, Southwick EC, Kerns J, Rosi K, Carr BI, Wilcox C, Lazo JS. Cdc25 Laboratory of Plateau Ecology and Agriculture, Qinghai University (2017-ZZ-02). inhibition and cell cycle arrest by a synthetic thioalkyl vitamin K analogue. The first funding source was mainly used in the collection, analysis, and inter- Cancer research. 2000;60(5):1317–25. pretation of experimental data. The second funding source mainly supported 11. Lazo JS, Aslan DC, Southwick EC, Cooley KA, Ducruet AP, Joo B, Vogt A, the drafting and revisions of the manuscript. Wipf P. Discovery and biological evaluation of a new family of potent inhibitors of the dual specificity protein phosphatase Cdc25. J Med Chem. 2001;44(24):4042–9. Availability of data and materials 12. Brisson M, Foster C, Wipf P, Joo B, Tomko RJ Jr, Nguyen T, Lazo JS. Independent All data generated or analysed during this study are included in this mechanistic inhibition of cdc25 phosphatases by a natural product published article. caulibugulone. Mol Pharmacol. 2007;71(1):184–92. 13. Gong K, Li W. Shikonin, a Chinese plant-derived naphthoquinone, induces apoptosis in hepatocellular carcinoma cells through reactive oxygen Authors’ contributions species: A potential new treatment for hepatocellular carcinoma. Free Designed the experiments: SDZ, QG, SS and ZHS; Performed the experiments radical biology & medicine. 2011;51(12):2259–71. and analysed data: WL, QHS, SF, JMJ, LWZ and QQJ; Wrote the manuscript: 14. Chen X, Yang L, Zhang N, Turpin JA, Buckheit RW, Osterling C, Oppenheim SDZ and QG; Edited the manuscript: ZHS and SS; SDZ, QG, WL, QHS, SF, JMJ, JJ, Howard OM. Shikonin, a component of chinese herbal medicine, inhibits LWZ, QQJ, SS, ZHS have given final approval of this version to be published. chemokine receptor function and suppresses human immunodeficiency virus type 1. Antimicrob Agents Chemother. 2003;47(9):2810–6. Ethics approval 15. Sun WX, Liu Y, Zhou W, Li HW, Yang J, Chen ZB. Shikonin inhibits TNF-alpha All cell lines used in this work are non-primary cells and do not required eth- production through suppressing PKC-NF-kappaB-dependent decrease of ics approval for their use. All animal experiments were approved by the animal IL-10 in rheumatoid arthritis-like cell model. J Nat Med. 2017;71(2):349–56. care committee of the Second Military Medical University in accordance with 16. Liao PL, Lin CH, Li CH, Tsai CH, Ho JD, Chiou GC, Kang JJ, Cheng YW. Anti- institutional and Chinese government guidelines for animal care and use. inflammatory properties of shikonin contribute to improved early-stage diabetic retinopathy. 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Shikonin inhibits cancer cell cycling by targeting Cdc25s

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Springer Journals
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Copyright © The Author(s). 2019
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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine, general; Medicine/Public Health, general
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Abstract

Background: Shikonin, a natural naphthoquinone, is abundant in Chinese herb medicine Zicao (purple gromwell) and has a wide range of biological activities, especially for cancer. Shikonin and its analogues have been reported to induce cell-cycle arrest, but target information is still unclear. We hypothesized that shikonin, with a structure similar to that of quinone-type compounds, which are inhibitors of cell division cycle 25 (Cdc25) phosphatases, will have similar effects on Cdc25s. To test this hypothesis, the effects of shikonin on Cdc25s and cell-cycle progression were determined in this paper. Methods: The in vitro effects of shikonin and its analogues on Cdc25s were detected by fluorometric assay kit. The binding mode between shikonin and Cdc25B was modelled by molecular docking. The dephosphorylating level of cyclin-dependent kinase 1 (CDK1), a natural substrate of Cdc25B, was tested by Western blotting. The effect of shikonin on cell cycle progression was investigated by flow cytometry analysis. We also tested the anti-proliferation activity of shikonin on cancer cell lines by MTT assay. Moreover, in vivo anti-proliferation activity was tested in a mouse xenograft tumour model. Results: Shikonin and its analogues inhibited recombinant human Cdc25 A, B, and C phosphatase with IC values ranging from 2.14 ± 0.21 to 13.45 ± 1.45 μM irreversibly. The molecular modelling results showed that shikonin bound to the inhibitor binding pocket of Cdc25B with a favourable binding mode through hydrophobic interactions and hydrogen bonds. In addition, an accumulation of the tyrosine 15-phosphorylated form of CDK1 was induced by shikonin in a concentration-dependent manner in vitro and in vivo. We also confirmed that shikonin showed an anti-proliferation effect on three cancer cell lines with IC values ranging from 6.15 ± 0.46 to 9.56 ± 1.03 μM. Furthermore, shikonin showed a promising anti-proliferation effect on a K562 mouse xenograph tumour model. Conclusion: In this study, we provide evidence for how shikonin induces cell cycle arrest and functions as a Cdc25s inhibitor. It shows an anti-proliferation effect both in vitro and in vivo by mediating Cdc25s. Keywords: Shikonin, Cdc25s, Anticancer, Cell cycle progression Background promising anticancer target [2–4]. Several inhibitors of Dual-specificity protein phosphatases (DSP) Cdc25s Cdc25s with antitumour activity have been reported [5, 6]. (Cdc25A, Cdc25B, and Cdc25C) have an essential role in Most potent small molecule inhibitors of the Cdc25 phos- cell cycle progression via controlling the phosphorylation phatases are quinone-derived compounds, such as mena- state of their natural substrates cyclin-dependent kinases dione (vitamin K )[7], compound 2 [8], NSC95397 [9], (CDKs). Overexpression of Cdc25s and over-activation of compound 5 [10], and NSC668394 [11](Fig. 1). This type CDKs are involved in tumour-associated cell-cycle aberra- of compound was hypothesized to inhibit the activity of tion [1]. Therefore, Cdc25s is currently considered to be a Cdc25s by oxidation of the catalytically essential cysteine residue in the enzyme’s active site through production of reactive oxygen species [12]. * Correspondence: Shoude.zhang@qhu.edu.cn; suzhanhai@foxmail.com The naphthoquinone-type natural product shikonin was State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, derived from the root of Lithospermum erythrorhizon, 251# Ningda Road, Xining 810016, Qinghai, China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. BMC Cancer (2019) 19:20 Page 2 of 9 Fig. 1 Structures of quinone-type inhibitors of Cdc25s and shikonin which has broad applications in Traditional Chinese quinone-type inhibitor of Cdc25s [7] that was used as a Medicine [13, 14]. Over the past few decades, a number of positive control here. studies have demonstrated multiple biological effects of For the dialysis assay, the enzyme-inhibitor complex shikonin. It has been reported to have anti-HIV [14], including 0.2 μM Cdc25B and 50 μM shikonin was dia- anti-inflammatory [15, 16], antibacterial, and anticancer lyzed against 5000-fold of the assay buffer for the indi- [17–20] activities. Among these activities, the anticancer cated period of time. At the end of each dialysis, Cdc25B activity, especially the induction of apoptosis and necrop- activity was determined as described above. tosis, is well reported [13, 19–23]. However, the key target is still unclear. Shikonin has a similar chemical skeleton to Molecular modelling that of the quinone-type inhibitors of Cdc25s. There- The docking method used is described in a previous fore, we hypothesized that shikonin will have similar work [24]. In summary, molecular modelling was per- effects on Cdc25s. To test this hypothesis, the effects formed using Maestro 9.0. The X-ray structure of of shikonin on Cdc25s and related biofunction were Cdc25B (PDB ID: 1QB0) was downloaded from the Pro- confirmed in this paper. tein Data Bank (PDB, https://www.rcsb.org) and pre- pared with the “Protein Preparation Wizard” workflow Methods with default settings. The grid-enclosing box was gener- Chemicals ated within 10 Å of Cys473 in the refined crystal struc- Shikonin and its analogues are natural products that ture. The ligand structure was prepared with the Ligprep were purchased from Herbest, Inc. (Baoji, Shanxi, China). module. Finally, shikonin was docked into Cdc25B using All other chemicals were purchased from Sigma-Aldrich Glide (version 5.5) in extra precision (XP) mode with de- (Shanghai, China) unless otherwise noted. fault settings [25]. Favourable binding poses were se- lected according to the docking score and view check. Measurement of phosphatase inhibitory activity of shikonin and its analogues Cell lines and culture conditions CycLex® protein phosphatase Cdc25A, -B and -C fluoro- K562 cells (myelogenous leukaemia cell line), MCF-7 metric assay Kit (CycLex, Cat. No. CY-1352, CY-1353, cells (breast cancer cell line) and HeLa cells (cervical CY-1354) was used to test the enzyme inhibition rate of cancer cells) were obtained from the Chinese Academy shikonin and its analogues for Cdc25A, -B and -C. In of Sciences Cell Bank (Shanghai, China). The catalogue summary, dual-specificity phosphatase activity was numbers of these cell lines are TCHu191, TCHu74 and measured in a 96-well microtiter plate using O-methyl- TCHu187, respectively. The temperature-sensitive FT210 fluorescein phosphate (OMFP) as a substrate. 5 μL cell line (tsFT210) is a mouse breast cancer cell line that is (0.1 μg/μL) of purified recombinant Cdc25s (Cdc25A, -B widely used for studying cell cycle progression. Intracellu- and -C) was mixed with 40 μL of assay mixture and in- lar CDK1 protein of tsFT210 is inactive at 39 °C because cubated with 5 μL of the test compound at various con- of two point mutations on the cdc2 gene, which leads to it centrations in a well. Then, 25 μL of stop solution was being easily controlled at different cell cycle phases added. Fluorescence was measured at an excitation through changes in temperature [26]. This cell line was wavelength of 485 nm and an emission wavelength of kindly provided by Dr. Rongcai Yue from his laboratory 530 nm using a fluorescence microplate reader (BioTek (School of Pharmacy, Second Military Medical University). Instruments, Inc., Winooski, VT, USA). Menadione is a All cell lines were kept in the logarithmic growth phase in Zhang et al. BMC Cancer (2019) 19:20 Page 3 of 9 5% CO at 37 °C with RPMI-1640 medium supplemented negative control. Nocodazole, a potent mitotic blocker with 10% FBS and 1% penicillin G-streptomycin in a hu- that arrests cells at the G /M phase, was used as a posi- midified chamber at 5% CO . tive control at a concentration of 100 nM. Western blotting Anti-proliferation assay The phosphorylation status of CDK1 was analysed by The anti-proliferation activity of shikonin against cancer Western blotting as described in our previous work [24]. cells was tested by the MTT method in 96-well plates. 6 3 In summary, tsFT210 cells (1 × 10 ) were treated with First, 4 × 10 cells were seeded per well and treated with shikonin (0, 1, 5 and 25 μM) for 4 h followed by collec- the shikonin with serial concentrations for 48 h. Then, tion and suspension in lysis buffer (150 mM NaCl, 50 the cells were incubated with 10 μL of MTT solution (5 mM Tris, 0.02% NaN , 0.01% phenylmethylsulfonyl mg/mL in PBS) for an additional 2–4 h at 37 °C. The for- fluoride, 0.2% aprotinin, and 1% TritonX-100, pH 8.0) mazan crystals were dissolved with 100 μLofDMSO after containing a protease inhibitor cocktail (Thermo Scien- removing the supernatant. The absorbance at 570 nm was tific) for 30 min at 4 °C. The protein concentration in measured by a BioTek Synergy 2 plate reader (BioTek cell lysates was confirmed by the Bradford method. Fifty Instruments, Inc., Winooski, VT, USA). Doxorubicin, a micrograms of protein per lane was resolved on 10% chemotherapy medication used to treat cancer and having SDS polyacrylamide gels, and the bands were transferred a broad spectrum of anticancer activities [29], was used as to PVDF membranes (Millipore). Nonspecific reactivity a positive control here. was blocked by 5% non-fat milk prepared in TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) at Xenograph tumour model in mice room temperature for 1 h. The protein signals were cap- The mouse model was constructed according to our pre- tured with primary antibodies and the secondary anti- vious work [24]. Balb/c mice (female, 5 weeks old), pur- bodies according to the manufacturers’ instructions. In chased from Shanghai SLAC Laboratory Animal Co., this process, the protein β-actin was used to normalize LTD (Shanghai, China), were subcutaneously injected target protein. All antibodies used in this paper were into a 100 μL K562 cell suspension (5 × 10 /mL, PBS) in purchased from Cell Signaling Technology (Inc, China). the right flank of mice. We started treating the mice with shikonin at 1, 5, or 10 mg/kg or vehicle containing Intracellular reactive oxygen species assay 5% DMSO (control group) by intraperitoneal injection Reactive oxygen species (ROS) were detected with the every other day when the tumour’s volume reached ap- fluorescent probe 2′,7′-dichlorofluorescin diacetate proximately 200 mm . The long and short dimensions of (DCFH-DA) as described by others [18, 27]. Briefly, tumour size were measured by a digital calliper at days MCF-7 cells or tsFT210 (synchronized at G2 phase) cells 1, 4, 8, 11, 16, 21, and 24 after treatment. The volume were plated in 96-well plates and loaded with 20 μM was calculated with the formula (long dimension) × (short DCFH-DA for 30 min at 37 °C in culture medium. Cells dimension) /2. The mice were euthanized 2 days after the were washed with PBS three times after incubation and last treatment by the method of cervical dislocation. then treated with shikonin at concentrations of 5 μM and 25 μM for 4 h. Untreated cells were used as control. Statistical analysis Fluorescence was measured using a fluorescence micro- Statistical analyses were performed using PRISM soft- plate reader (BioTek Instruments, excitation wavelength: ware. All quantitative values are given as the means ± 485 nm, emission wavelength: 528 nm). The ROS levels SEM. An unpaired Student’s t test was used to evaluate were expressed as RFU (relative fluorescence unit). the difference between two different treatments, and p < 0.05 was considered statistically significant. The statis- Cell cycle analysis tical significance of differences in the survival of mice Cell cycle analysis was performed as described previ- from the different groups was determined by the log-rank ously [28]. Briefly, tsFT210 cells were plated at 1 × 10 test using the same program. cells/well (24-well plate) and maintained at 32 °C. Cell proliferation was blocked at the G2 phase by increasing Results the temperature from 32 to 39 °C and treating for 17 h. Shikonin induce irreversible inhibition of human recombinant Then, the cells were released at 32 °C and immediately CDC25 phosphatases treated with shikonin. The harvested cells were stained Using the protein phosphatase Cdc25 combo fluoromet- with staining buffer (50 μg/ml propidium iodide, 0.1% ric assay kit, shikonin and its analogues in vitro inhibited sodium citrate and 0.2% NP-40) and analysed by Flow recombinant human Cdc25A, -B, and -C phosphatases Cytometry (BD Biosciences). A final concentration of in a concentration-dependent manner with IC values 0.5% DMSO was used for all compounds and as a ranging from 2.14 to 14.32 μM (Table 1). The positive Zhang et al. BMC Cancer (2019) 19:20 Page 4 of 9 Table 1 IC values of shikonin and analogues for inhibition of recombinant human protein phosphatases Structure R Name Cdc25A (μM) Cdc25B (μM) Cdc25C (μM) -OH Shikonin 2.14 ± 0.21 5.82 ± 0.37 4.78 ± 0.18 -H Deoxyshikonin 3.22 ± 0.76 7.32 ± 0.45 6.33 ± 0.65 -OCOCH Acetylshikonin 3.92 ± 0.66 3.87 ± 0.68 4.67 ± 0.34 -OCOCH(CH ) Isobutylshikonin 6.79 ± 1.02 7. 86 ± 0.23 5.89 ± 0.43 3 2 -OCOCH CH(CH ) Isovalerylshikonin 9.53 ± 0.78 11. 23 ± 1.22 10.98 ± 0.97 2 3 2 -OCOCH=CH(CH ) β,β-Dimethylacrylshikonin 4.67 ± 0.85 8.56 ± 0.67 10. 58 ± 1.13 3 2 -OCOCH(CH )CH CH α-Methyl-η-butylshikonin 11.24 ± 1.45 14.32 ± 1.27 13.45 ± 1.45 3 2 3 All values are micromolar concentrations and are the mean ± S.E.M. of three or more independent determinations control menadione led to an inhibition of 4.12 ± 0.87, motif [32]. The phosphorylated amino acids of the sub- 5.37 ± 0.45, and 5.13 ± 0.24 μM, respectively. strate usually bind to this loop through forming hydro- Previous research revealed that the quinone-type in- gen bonds to arginine R. The quinone-type inhibitor was hibitors of Cdc25 phosphatases play a role through irre- supposed to bind the inhibitor binding pocket, which versible oxidation of the catalytic cysteine of Cdc25B was beside the active site (Fig. 3), and induce oxidation [30, 31]. To identify the inhibition pattern of shikonin, of the catalytic cysteine through redox cycling reaction dialysis was used to study the reversibility of shikonin [1]. After docking the shikonin into the above binding action. As shown in Fig. 2a, after dialysis for 2, 4, 6, and loop around cys473, we found that the shikonin bound even 24 h, the activity of Cdc25B was not rescued from to the inhibitor binding pocket similar to other quinone- inhibition by shikonin, indicating that the Cdc25B was type inhibitors. Figure 2 shows the most energetically inhibited irreversibly. In addition, to confirm whether favourable binding pose from docking results. In such a the shikonin has similar mechanism with other quinone- binding mode, the benzene ring of shikonin is involved in type inhibitors, the ROS content was measured in cells hydrophobic interactions with the pocket residues. In treated with or without shikonin. After treating with shi- addition, a hydrogen bond between one oxygen of the konin, intracellular ROS in MCF-7 and tsFT210 cells in- naphthoquinone core and Tyr428 was predicted. creased significantly compared with those of control cells (Fig. 2b), suggesting that Cdc25s inhibition by shi- Shikonin inhibits cell cycle progression konin could be caused by redox cycling of the shikonin Inhibition of Cdc25s results in hyper-phosphorylation of similar to that of other quinones. CDKs and cell cycle arrest. Therefore, the impact of shi- konin on cell cycle progress was investigated. The Molecular model of shikonin interactions with the Cdc25B tsFT210 cell line has been widely used for studying cell catalytic domain cycle progression because it can be easily controlled at In order to evaluate the binding mode and affinity of different cell cycle phases through changing temperature shikonin with Cdc25B, molecular modelling was per- [26]. The synchronized tsFT210 cells were then treated formed using the docking program Glide. X-ray crystal- with the indicated concentration of shikonin and noco- lography showed that the catalytic domain of Cdc25B dazole (a potent mitotic blocker) or 1% DMSO for 6 h. contains the canonical HCX R PTPase catalytic-site The positive control nocodazole arrested the cells at the Fig. 2 Shikonin inhibits Cdc25B irreversibly in vitro and induces ROS production in MCF-7 cells. a Activity of shikonin-treated Cdc25B after dialysis. b ROS production in MCF-7 and tsFT210 cells after treatment with shikonin. Data points represent fold change of three independent experiments. *P < 0.05; **P < 0.01 compared with the control group Zhang et al. BMC Cancer (2019) 19:20 Page 5 of 9 cells (8.97 ± 0.87 μM) compared with the negative con- trol. Doxorubicin was used as a positive control and showed normal anticancer activity according to a previ- ous report. Shikonin has anti-tumour effects in vivo by inhibiting CDK1 dephosphorylation Xenograph tumour model mice were used to evaluate if shikonin can inhibit tumour growth in vivo. K562-bear- ing mice were treated with shikonin at different doses and vehicle for 21 days. The tumour sizes were recorded in this process. As shown in Fig. 5a, shikonin concentration-dependently inhibited tumour growth. Higher doses of shikonin had a better inhibitory effect and longer observed survival time (Fig. 6a, 6b). In the process of this experiment, the tumour growth of mice from higher dose groups was significantly inhibited com- pared with those of the control and low-dose groups. Fig. 3 Binding mode of shikonin in the Cdc25B binding cavity. Shikonin These results suggest that shikonin has profound anti- (yellow) and key interacting residues (green) are represented as stick cancer activity in vivo. Moreover, the protein levels of models with the protein as a surface. H-bonds are shown as dashed phosphorylated CDK1 in primary tumour tissues ob- black lines. The figure was generated using Pymol from Protein Data tained from the mice were significantly increased after Bank ID 1QB0 treatment by shikonin compared with that of the control group, especially for the 5 and 10 mg/kg groups (Fig. 6c). G /M phase. Comparatively, the cells treated with shikonin This means that the shikonin still participates in the cell were blocked at the G /M phase in a concentration- cycle process in vivo. dependent manner, whereas the DMSO-treated control cells had completed mitosis and had entered the following Discussion cell cycle (Fig. 4). Such results indicate that shikonin can Overexpression of CDC25s, which leads to an irregular target and delay cell cycle progression at the G2/M phase. cell cycle, is often present in cancer cells. They control the cell cycle by activating CDKs. Therefore, CDC25s Shikonin inhibits CDK1 dephosphorylation are considered attractive targets for anticancer drug dis- Endogenous Cdc25s controls the cell cycle through de- covery. Several potent CDC25s inhibitors have been phosphorylation of their natural substrate CDKs [2]. identified; however, few of them have entered clinical tri- Therefore, CDK1 protein will be hyper-phosphorylated if als. Shikonin, a natural naphthoquinone, has a similar the CDC25s are inhibited. In order to confirm whether structure to that of the well-reported quinone-type in- shikonin inhibits the activity of intracellular Cdc25 phos- hibitors of CDC25s. Hence, we hypothesized that shiko- phatases, the phosphorylation status of CDK1 was ana- nin will have similar effects on Cdc25s. To test this lysed by Western blotting. As shown in Fig. 5, shikonin hypothesis, the effects of shikonin on Cdc25s and related at 1 μM, 5 μΜ, and 25 μM induced an accumulation of biofunction were proved in this work. the tyrosine 15-phosphorylated form of CDK1. These re- Shikonin and six analogues showed considerable in- sults suggested that shikonin downregulated the activity hibition for CDC25s using OMFP as substrate. The main of Cdc25s, leading to hyper-phosphorylation of CDK1 in difference between these analogues and shikonin is the cultured cells. R groups (Table 1), but they have similar inhibition for CDC25s. Therefore, we suggest that the R groups have Shikonin inhibits in vitro tumour cell proliferation little effect on the activity and that the pharmacological To evaluate the anti-proliferation activity of shikonin for group is the skeleton of naphthoquinone. Finally, shiko- different cancer cells, four cancer cell lines (MCF-7, nin was selected for further research because it showed HeLa, K562, and tsFT210) were selected to investigate the best inhibition for CDC25s compared with other an- the anti-proliferation activity. The viability of cells was alogues and can be generated from other analogues by evaluated by the MTT method. As shown in Table 2, hydrolysing the R group. Shikonin binds to the same shikonin markedly inhibits the proliferation of MCF-7 pocket of Cdc25B as other quinone-type inhibitors ac- cells (IC : 6.82 ± 0.76 μM), HeLa cells (IC : 9.56 ± cording to the molecular modelling results. Together 50 50 1.03 μM), K562 cells (IC : 6.15 ± 0.46 μM) and tsFT210 with the unaltered activity of Cdc25B after dialysis and 50 Zhang et al. BMC Cancer (2019) 19:20 Page 6 of 9 Fig. 4 Cell cycle analysis of tsFT210 cells in the absence or presence of shikonin. a G2/M-arrested cells after a temperature shift for 17 h at 39 °C. b DMSO-treated cells after a temperature shift for 4 h at 32 °C. c Cells treated with 100 nM nocodazole. d-f Cells treated with 1–25 μM shikonin. Data are representative of two independent experiments increasing ROS content after treatment with shikonin, phosphorylation state of CDK1 were investigated. We we suggested that shikonin inhibited Cdc25B similar to proved that shikonin could block the cell cycle of other quinone-type inhibitors, which were hypothe- tsFT210 at the G2/M phase and effectively inhibit de- sized to disrupt Cdc25s phosphatase activity by oxida- phosphorylation of CDK1 in vitro. tion of the catalytically essential cysteine residue in We also evaluated the anti-proliferation activity of shi- the enzyme’s active site through production of react- konin both in vitro and in vivo. Shikonin showed broad ive oxygen species [23]. spectrum anticancer activity after evaluating the anti- Endogenous Cdc25s control the cell cycle through de- proliferation activity for four cancer cell lines. Moreover, phosphorylating their natural substrate CDKs. Therefore, tumour growth of xenograph tumour model mice was the impacts of shikonin on cell cycle progress and significantly inhibited by shikonin. To confirm the in vivo activity of shikonin was through inhibition of the Cdc25s, we also checked the phosphorylation state of CDK1 in tumour tissue from xenograph tumour model mice, and the results showed that shikonin could inhibit dephosphorylation of CDK1 in tumour tissue also, which suggested that shikonin could also participate in the Cdc25s-related cell cycle pathway in vivo. Table 2 Inhibitory activity of shikonin on tumour cells via MTT assay (IC , μM, n = 3, mean ± SD) Fig. 5 Inhibition of CDK1 dephosphorylation caused by shikonin. Cells Compound Cell Line in G2/M phase were treated with the indicated concentrations of MCF-7 HeLa K562 tsFT210 shikonin and DMSO for 4 h and then harvested. Samples were Shikonin 6.82 ± 0.76 9.56 ± 1.03 6.15 ± 0.46 8.97 ± 0.87 processed for Western blot analysis. Data are representative of two independent experiments Doxorubicin 0.28 ± 0.03 0.43 ± 0.06 0.51 ± 0.12 0.56 ± 0.14 Zhang et al. BMC Cancer (2019) 19:20 Page 7 of 9 Fig. 6 Shikonin inhibits tumour growth in vivo in the K562-bearing mice by affecting the phosphorylation of CDK1 (n = 10/group). a Tumour volume plot of K562-bearing mice treated with vehicle or shikonin at 1, 5, or 10 mg/kg by oral gavage for 21 days. The tumours were measured twice per week. The data are represented as the mean ± SEM. Tumour growth was inhibited significantly after treatment with shikonin compared with the control group. *P < 0.05; †P < 0.01; ‡P < 0.001 compared with the control group. b Kaplan-Meier survival plot of the K562-bearing nude mice. Survival of the K562-bearing nude mice was prolonged in the shikonin-treated groups compared with control group. c The phosphorylation level of CDK1 is affected by shikonin. Shikonin reportedly exerts anticancer effects with less death when the cancer cells were co-treated with shi- drug resistance [33], which can be attribute to its mul- konin and necroptotic inhibitor Nec-1 [39]. Moreover, tiple targets. ROS generation, inactivation of NF-κB, up- shikonin and its analogues have been reported can in- regulation of p53 and p21, and activation of caspases duce cell cycle arrest, but the mechanism is still un- may be involved in the anticancer mechanisms of shiko- clear [27]. Here, we identified shikonin as an inhibitor nin [34–38]. In brief, shikonin disrupts the cancer cell of Cdc25s that plays an essential role in cell cycle lines by inhibiting either the necroptosis or apoptosis progression. pathway. Cancer cells died predominantly through the The normal development of tissue depends on the necroptotic pathway when they were co-treated with homeostasis and balance between cell proliferation and shikonin and the apoptosis inhibitor Z-VAD-FMK, death (Fig. 7). When out of balance, as generated by in- while apoptosis became a selective route for cell appropriate proliferation or inhibition of apoptosis and Fig. 7 Schematic of the coordination between cell proliferation and death. ↓: inhibited by shikonin, ↑: promoted by shikonin Zhang et al. BMC Cancer (2019) 19:20 Page 8 of 9 necroptosis pathways, cancer usually results [40]. If one Received: 12 November 2017 Accepted: 13 December 2018 pathway was inhibited or key proteins of it mutated, shi- konin can also play an anticancer role through two other References pathways, which could explain the lower drug resistance 1. Kristjansdottir K, Rudolph J. Cdc25 phosphatases and cancer. Chemistry & of shikonin. biology. 2004;11(8):1043–51. 2. 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Journal

BMC Cancer – Springer Journals

Published: Jan 7, 2019

Keywords: Shikonin; Cdc25s; Anticancer; Cell cycle progression

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