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. 2015 Jan 30;6(3):1640-51.
doi: 10.18632/oncotarget.2746.

Synergistic anticancer effect of cisplatin and Chal-24 combination through IAP and c-FLIPL degradation, Ripoptosome formation and autophagy-mediated apoptosis

Shaoqing Shi  1   2 , Qiong Wang  2   3 , Jennings Xu  2 , Jun-Ho Jang  2   4 , Mabel T Padilla  2 , Toru Nyunoya  2   4 , Chengguo Xing  5 , Lin Zhang  1   3 , Yong Lin  2
Affiliations

Synergistic anticancer effect of cisplatin and Chal-24 combination through IAP and c-FLIPL degradation, Ripoptosome formation and autophagy-mediated apoptosis

Shaoqing Shi et al. Oncotarget. .

Abstract

Drug resistance is a major hurdle in anticancer chemotherapy. Combined therapy using drugs with distinct mechanisms of function may increase anticancer efficacy. We have recently identified the novel chalcone derivative, chalcone-24 (Chal-24), as a potential therapeutic that kills cancer cells through activation of an autophagy-mediated necroptosis pathway. In this report, we investigated if Chal-24 can be combined with the frontline genotoxic anticancer drug, cisplatin for cancer therapy. The combination of Chal-24 and cisplatin synergistically induced apoptotic cytotoxicity in lung cancer cell lines, which was dependent on Chal-24-induced autophagy. While cisplatin slightly potentiated the JNK/Bcl2/Beclin1 pathway for autophagy activation, its combination with Chal-24 strongly triggered proteasomal degradation of the cellular inhibitor of apoptosis proteins (c-IAPs) and formation of the Ripoptosome complex that contains RIP1, FADD and caspase 8. Furthermore, the cisplatin and Chal-24 combination induced dramatic degradation of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (cFLIPL) which suppresses Ripoptosome-mediated apoptosis activation. These results establish a novel mechanism for potentiation of anticancer activity with the combination of Chal-24 and cisplatin: to enhance apoptosis signaling through Ripoptosome formation and to release the apoptosis brake through c-FLIPL degradation. Altogether, our work suggests that the combination of Chal-24 and cisplatin could be employed to improve chemotherapy efficacy.

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Figures

Figure 1
Figure 1. Combination of Chal-24 and cisplatin results in synergistic apoptotic cell death in lung cancer cells
(A) A549 cells were treated with cisplatin (10–30 μM) and Chal-24 (0.5 μM) for 48 h. Cell death were measured by LDH release assay. Data shown are mean ± SD, representative of three independent experiments. (B) A549 cells were treated with cisplatin (10 μM) and Chal-24 (0.125–1 μM) for 48 h. Cell death were measured as described in A. (C) A549 cells were treated with cisplatin (10 μM) and Chal-24 (1 μM) for indicated time periods. Cell extracts were resolved in 12% SDS-PAGE gels. PARP and active caspase-3 were detected by Western blot. GAPDH was detected as an input control. (D) A549 cells were pretreated with z-VAD (10 μM) for 30 min, followed by 48 h treatment with cisplatin (10 μM) and Chal-24 (1 μM), cell death were detected as described in A. (E) the cells were transfected with indicated siRNA for 24 h, and treated with (10 μM) and Chal-24 (1 μM) for additional 48 h. Cell death was measured as described in A. **p < 0.01. Insert, knockdown of caspase 9 expression was confirmed by Western blot.
Figure 2
Figure 2. Autophagy is required for cell death induced by Chal-24 and cisplatin combination
(A) A549 cells were treated with cisplatin (10 μM), Chal-24 (1 μM) alone or in combination for the indicated times. The indicated proteins were detected by Western blot. β-tubulin was used as an input control. (B) the cells were preteated with chloroquine (CQ, 20 μM) for 30 min, and then treated with cisplatin and Chal-24 (24 h or 4 h for upper panel for p62 detection and lower panel for LC3 detection, respectively). The indicated proteins were detected by Western blot. GAPDH was detected as an input control. (C) A549 cells were pretreated with autophagy inhibitors (CQ, 20 μM; WTM, 1 μM; 3MA, 10 μM) for 30 min, followed by cisplatin (10 μM) and Chal-24 (1 μM) co-treatment for an additional 48 h, cell death was measured as described in Figure 1. (D) the cells were transfected with the indicated siRNA for 24 h, then the cells were treated with cisplatin (10 μM) and Chal-24 (1 μM) for 48 h, cell death was measured by LDH assay. *p < 0.05, **p < 0.01. Insert, knockdown of ATG7 expression was confirmed by Western blot.
Figure 3
Figure 3. Combination of cisplatin and Chal-24 activates JNK-and phosphorylation of Bcl-2
(A) A549 cells were treated with cisplatin (10 μM) and Chal-24 (1 μM) alone or in combination for indicated times. JNK1 and phospho-JNK were examined by Western blot. GAPDH was detected as an input control. (B) the cells were pretreated with SP600125 (10 μM) for 30 min, and then treated with cisplatin (10 μM) and Chal-24 (1 μM) for an additional 48 h, cell death were detected by LDH assay. **p < 0.01. (C) A549 cells were treated with cisplatin (10 μM) and Chal-24 (1 μM) alone or in combination for indicated times. The indicated proteins were detected by Western blot. GAPDH was used as an input control. (D, E) A549 cells were pretreated with CQ (20 μM) or MG132 (5 μM) for 30 min, respectively, then treated with cisplatin (10 μM), Chal-24 (1 μM) or in combination for an additional 8 h. The indicated proteins were detected by Western blot, GAPDH was detected as an input control.
Figure 4
Figure 4. Combination of cisplatin and Chal-24 induces ERK-mediated degradation of the IAP proteins
(A) A549 cells were treated with cisplatin (10 μM), Chal-24 (1 μM), or in combination for indicated times. The indicated proteins were detected by Western blot. β-actin was detected as an input control. (B) the cells were pretreated with MG132 (5 μM) for 30 min, and then treated with cisplatin (10 μM) and Chal-24 (1 μM) for an additional 24 h, the indicated proteins were detected by Western blot. β-tubulin was detected as an input control. (C), A549 cells were treated with cisplatin (10 μM), Chal-24 (1 μM), or in combination for indicated times. The indicated proteins were examined by Western blot. β-tubulin was detected as an input control. (D) the cells were pretreated with U0126 (10 μM) for 30 min, and then treated with cisplatin (10 μM) and Chal-24 (1 μM) for an additional 24 h, the indicated proteins were examined by Western blot. β-actin was detected as an input control. (E) the cells were pretreated with U0126 (10 μM) for 30 min, and then treated with cisplatin (10 μM) and Chal-24 (1 μM) for an additional 48 h, cell death was measured by LDH assay. (F) A549 cells were transfected with plasmids expressing the IAPs or the empty vector pcDNA with EGFP, and treated with cisplatin (10 μM) and Chal-24 (1 μM) for 40 h. Survival of EGFP-positive cells was quantified by counting live cells with green fluorescence. Data shown are mean ± SD. **p < 0.01. Upper, expression of transfected proteins was confirmed by Western blot.
Figure 5
Figure 5. Combination of cisplatin and Chal-24 triggers Ripoptosome formation and c-FLIPL degradation
(A) A549 cells were treated with cisplatin (10 μM), Chal-24 (1 μM), or in combination for 24 h, the indicated proteins were detected by Western blot after co-immunoprecipitation with an anti-RIP1 antibody. (B) the cells were transfected with the indicated siRNA for 24 h, then the cells were treated with (10 μM) and Chal-24 (1 μM) for 48 h, cell death was measured by LDH assay. *p < 0.05. Inset, knockdown of RIP1 expression was confirmed by Western blot. (C) the cells were pretreated with necrostatin-1 (NEC-1, 10 μM) for 30 min, and then treated with cisplatin (10 μM) and Chal-24 (1 μM) for an additional 48 h, cell death was measured by LDH assay. *p < 0.05. D, A549 cells were treated with cisplatin (10 μM), Chal-24 (1 μM) alone, or in combination for indicated times. The indicated proteins were examined by Western blot. β-actin was detected as an input control. (E) the cells were pretreated with MG132 (5 μM) for 30 min, and then treated with cisplatin (10 μM) and Chal-24 (1 μM) for an additional 24 h. The indicated proteins were examined by Western blot. GAPDH was detected as an input control. (F) A549 cells were transfected with V5-c-FLIPL or pcDNA with EGFP, and treated with cisplatin (10 μM) and Chal-24 (1 μM) for 40 h. Cell survival was quantified by counting cells with green fluorescence. Data shown are mean ± SD. *p < 0.05. Insert, expression of transfected protein was confirmed by Western blot.
Figure 6
Figure 6. A model for Chal-24 and cisplatin combination in induction of apoptotic cell death
Combination of Chal-24 and cisplatin kills cancer cells through apoptosis involving multiple pathways: the enhancement of Ripoptosome formation involving IAPs suppression and release the apoptosis brake involving c-FLIP degradation, autophagy involving JNK-mediated Bcl-2 phosphorylation, and cisplatin-induced DNA damage.
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