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. 2020 Jan;53(1):e12706.
doi: 10.1111/cpr.12706. Epub 2019 Oct 23.

Withaferin A triggers G2/M arrest and intrinsic apoptosis in glioblastoma cells via ATF4-ATF3-CHOP axis

Qin Tang  1   2 , Liwen Ren  1   2 , Jinyi Liu  1   2 , Wan Li  1   2 , Xiangjin Zheng  1   2 , Jinhua Wang  1   2 , Guanhua Du  1   2
Affiliations

Withaferin A triggers G2/M arrest and intrinsic apoptosis in glioblastoma cells via ATF4-ATF3-CHOP axis

Qin Tang et al. Cell Prolif. 2020 Jan.

Erratum in

Abstract

Objective: Withaferin A (WA) is a bioactive compound with a remarkable anti-cancer effect derived from Withania somnifera, commonly known as ashwagandha. However, the anti-cancer mechanisms of WA in glioblastoma multiforme (GBM) are still unclear.

Materials and methods: Cell viability assays and xenografted nude mice were used to evaluate the effects of WA, along with flow cytometry to detect apoptosis and cell cycle of GBM. RNA-seq analysis, Western blotting, immunofluorescence staining, qRT-PCR and siRNA gene silencing were carried out to determine the signalling pathways affected by WA.

Results: Withaferin A significantly inhibited the growth of GBM in vitro and in vivo and triggered the intrinsic apoptosis of GBM cells by up-regulating expression of Bim and Bad. WA arrested GBM cells at the G2/M phase of the cell cycle through dephosphorylating Thr161 of CDK1 by activating p53-independent p21 up-regulation. Knockdown of p21 restored cell cycle progression and cell viability by down-regulating the expression of Bad rather than Bim. We demonstrated that endoplasmic reticulum (ER) stress induced by WA through the ATF4-ATF3-CHOP axis, initiated apoptosis and G2/M arrest in GBM cells.

Conclusion: We revealed a novel pathway that elucidated WA activation of apoptosis and G2/M arrest in GBM cells through the ATF4-ATF3-CHOP axis. This discovery is important for optimization of WA-based regimens for prevention and/or treatment of GBM.

Keywords: apoptosis; cell cycle; endoplasmic reticulum; glioblastoma; unfolded protein response.

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Conflict of interest statement

The authors declare no conflicts of interest that pertain to this work.

Figures

Figure 1
Figure 1
WA‐induced apoptosis of U87 and U251 cells via an intrinsic pathway. Cells were treated with WA at various concentrations for the indicated time, and harvested for MTT, Flow cytometry and Western blotting. Cells were pre‐treated with Emricasan (50 μmol/L) for 24 h and then treated with WA (1 or 3 μmol/L) for 48 h. A, The structure of Withaferin A (WA). B, The effects of WA on U87, U251 and HA1800 cells. *P < .05, **P < .01 and ***P < .01 represented significant differences between U87 and U251 cells vs HA1800 cells. C. Cell viability was measured by MTT assay. *P < .05, **P < .01 and ***P < .001 represented significant differences between the WA‐treated group and control group. D, The apoptotic rates were determined by Flow cytometry after staining for Annexin V conjugated with FITC and PI dyes. The Q1, Q2, Q3 and Q4 quadrants represented dead, late‐apoptotic, early‐apoptotic and normal cells, respectively. *P < .05 and **P < .01 indicated significant differences between the WA‐treated group and control group. E, Apoptotic proteins were identified by Western blotting. F, Cell viability treated with 3 μmol/L WA was measured by MTT assay after pre‐treated with Emiricasan (50 μmol/L) for 24 h. *P < .05 and **P < .01 represented the significant differences between the Emiricasan‐treated group and corresponding non‐Emiricasan‐treated group. G. Western blot was used to check the alteration of apoptotic proteins after pre‐treatment with Emricasan. WA, Withaferin A
Figure 2
Figure 2
WA‐induced apoptosis of U87 and U251 cells partly by up‐regulating expression of Bim and Bad. After treatment with WA at the indicated concentrations for the indicated times, cells were separately harvested for Flow cytometry, Western blotting and MTT assay. For siRNA silencing, U251 cells were transfected with siRNA of Bim or Bad for 48 h and treated with 3 μmol/L WA for an additional 24 h. A, Mitochondrial membrane potential was determined by Flow cytometry after staining with JC‐1 dyes. The Q2 and Q3 quadrants represented normal cells and cells of decreased mitochondrial membrane potential, respectively. *P < .05, **P < .01 and ***P < .001 represented significant differences between the WA‐treated group and control group. B, Proteins regulating the intrinsic apoptotic pathway were detected by Western blotting. C, Cell viability of U251 was determined by MTT assay after transfection with siRNA of Bim or Bad. *P < .05 and **P < .01 represented significant differences between the siRNA‐treated group and corresponding non‐siRNA‐treated group. D, The changes of regulated proteins in intrinsic apoptotic pathway in U251 cells were determined by Western blotting after transfection with siRNA of Bim or Bad. WA, Withaferin A
Figure 3
Figure 3
The cell cycle was arrested at the G2/M phase by WA through p53‐independent p21 up‐regulation. Cells were treated with WA at the indicated concentration for the indicated time and then assayed by flow cytometry and Western immunoassay. For siRNA silencing, U251 cells were transfected with siRNA of p21 for 48 h then treated with 3 μmol/L WA for 24 h longer, and finally analysed by MTT, Flow cytometry and Western blotting. A, Cell cycle was determined by Flow cytometry after staining with PI. *P < .05, and **P < .01 represented significant differences of cells at G2/M phase vs control group. B, Proteins regulating the G2/M phase were detected by Western blotting. C, Cell cycle analysis after p21 knockout was performed by Flow cytometry. **P < .01 represented significant differences between the sip21‐treated group and NC‐treated group. D, Cell viability of U251 was measured by MTT assay after transfection with siRNA of p21. *P < .05 represented significant differences between the siRNA‐treated group and the corresponding non‐siRNA‐treated group. E, The changes of Bim, Bad and cleaved PARP1 proteins in U251 cells were checked by Western blotting after transfection with siRNA of p21. WA, Withaferin A
Figure 4
Figure 4
Expression of HMOX1, DNJB1 and ATF3 was induced by WA at an early stage of treatment. After treatment with 3 μmol/L WA for 12 h, cells were harvested and analysed by RNA‐seq. A, The differential gene expression caused by WA in U251 and U87 cells was determined by RNA‐seq. B, The cellular signalling pathways triggered by WA in U251 and U87 cells were obtained by GO enrichment at DAVID website. C, The top ten differentially expressed genes in both U251 and U87 cells are listed. *P < .05 and **P < .01 represented significant differences between U251 cells and U87 cells at the same gene. D, The changes of HMOX1, PPP1R15A, DNJB1, ATF3 and SH3BGR in U251 and U87 cells were detected at the mRNA level after WA treatment for 0.45, 1.5, 3, 6, 12 and 24 h. * P < .05, **P < .01 and ***P < .001 represented significant differences between U251 cells and U87 cells. E, The changes in HMOX1, PPP1R15A, DNJB1 and ATF3 in U251 and U87 cells were detected from expression levels after WA treatment for 3, 6, 12, 24 and 48 h. F, Immunofluorescence was used to observe the nuclear translocation of HMOX1, PPP1R15A, DNJB1 and ATF3 in U251 and U87 cells caused by WA. WA, Withaferin A
Figure 5
Figure 5
WA‐induced apoptosis and G2/M arrest of GBM cells by ATF4‐ATF3‐CHOP axis. Cells were treated with 3 μmol/L WA for the indicated time and harvested for qRT‐PCR and Western blotting. For siRNA silencing, U251 cells were separately transfected with siRNAs of HMOX1, DNAJB1, ATF3, ATF4, XBP1 and CHOP for 48 h, then treated with 3 μmol/L WA for an additional 24 h, and finally analysed by MTT, Western blotting and Flow cytometry. A, Genes associated with ER stress were determined by qRT‐PCR. *P < .05, **P < .01 and ***P < .001 represented significant differences between U251 cells and U87 cells. B, Proteins associated with ER stress were identified by Western blotting. C, Cell viability of U251 cells treated by 3 μmol/L WA was measured by MTT assay after transfection with siRNAs of HMOX1, DNAJB1, ATF3, ATF4, XBP1 and CHOP, respectively. **P < .01 represented significant differences between the siRNA‐treated group and the corresponding negative control group. D, Apoptosis of U251 treated with 3 μmol/L WA was measured by Fow cytometry after transfection with siRNAs of ATF3, ATF4 and CHOP, respectively. *P < .05 and **P < .01 represented significant differences between the siRNA‐treated group and the corresponding negative control group. E, Mitochondrial membrane potentials of U251 treated with 3 μmol/L WA were measured by Flow cytometry after transfection with siRNAs of ATF3, ATF4 and CHOP, respectively. *P < .05 and **P < .01 represented significant differences between the siRNA‐treated group and the corresponding negative control group. F, The cell cycle stage of U251 treated with 3 μmol/L WA was determined by Flow cytometry after transfection with siRNAs of ATF3, ATF4 and CHOP, respectively. **P < .01 and ***P < .01 represented significant differences between the siRNA‐treated group and the corresponding negative control group. G, The changes of p21, Bim, Bad and cleaved‐PARP1, caspase 3/7/9 and cleaved caspase 3/7/9 proteins in U251 cells treated with 3 μmol/L WA were determined by Western blotting after transfection with siRNAs of ATF3, ATF4 and CHOP, respectively. H, The overall mechanism of WA activity in GBM cells. WA, Withaferin A
Figure 6
Figure 6
WA inhibited the growth of subcutaneous U87 xenografts. U87 cells (5 × 106) were subcutaneously injected into the right flank of nude mice. After the tumour reached a volume of 40‐50 mm3, the mice were injected with vehicle or 5 mg/kg WA in the tail vein every day for 1 month. A, WA had no effect on the weight of the mice. B, WA reduced the tumour volume. C, WA decreased the tumour weight. **P < .01 represented significant difference between the WA group and the vehicle group. D, The photographs of tumours were collected from different groups of mice at the end of treatment (day 27). WA, Withaferin A
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