Novel smac mimetic APG-1387 elicits ovarian cancer cell killing through TNF-alpha, Ripoptosome and autophagy mediated cell death pathway

Abstract

Background: Ovarian cancer is a deadly disease. Inhibitors of apoptosis proteins (IAPs) are key regulators of apoptosis and are frequently dysregulated in ovarian cancer. Overexpression of IAPs proteins has been correlated with tumorigenesis, treatment resistance and poor prognosis. Reinstalling functional cell death machinery by pharmacological inhibition of IAPs proteins may represent an attractive therapeutic strategy for treatment of ovarian cancer.

Methods: CCK-8 and colony formation assay was performed to examine cytotoxic activity. Apoptosis was analyzed by fluorescence microscopy, flow cytometry and TUNEL assay. Elisa assay was used to determine TNFα protein. Caspase activity assay was used for caspase activation evaluation. Immunoprecipitation and siRNA interference were carried out for functional analysis. Western blotting analysis were carried out to test protein expression. Ovarian cancer cell xenograft nude mice model was used for in vivo efficacy evaluation.

Results: APG-1387 demonstrated potent inhibitory effect on ovarian cancer cell growth and clonogenic cell survival. APG-1387 induced RIP1- and TNFα-dependent apoptotic cell death in ovarian cancer through downregulation of IAPs proteins and induction of caspase-8/FADD/RIP1 complex, which drives caspase-8 activation. NF-κB signaling pathway was activated upon APG-1387 treatment and RIP1 contributed to NF-κB activation. APG-1387 induced cytoprotective autophagy while triggering apoptosis in ovarian cancer cells and inhibition of autophagy enhanced APG-1387-induced apoptotic cell death. APG-1387 exhibited potent antitumor activity against established human ovarian cancer xenografts.

Conclusions: Our results demonstrate that APG-1387 targets IAPs proteins to potently elicit apoptotic cell death in vitro and in vivo, and provide mechanistic and applicable rationale for future clinical evaluation of APG-1387 in ovarian cancer.

Keywords: APG-1387; Apoptosis; Autophagy; Ovarian cancer.

Fig. 1

Effects of APG-1387 on cell viability in ovarian cancer. a, b APG-1387 inhibited the proliferation of SKOV3 and OVCAR3 cell lines. They were treated with the indicated concentrations of APG-1387 for 72 h and cell viability was determined by the CCK-8 assay. c Colony-forming test results. SKOV3 cells were incubated with APG-1387 (0, 3 nM) for 7 days. d Statistical analysis of the percentage of clone numbers. Columns, mean (n = 3); bars, SD. *P < 0.05 vs. untreated group

Fig. 2

Effects of APG-1387 on apoptosis in ovarian cancer. a. APG-1387 inhibited the proliferation of SKOV3 cell line. They were treated with the indicated concentrations of APG-1387 for 24, 48, 72 h. Cell viability was determined by the CCK-8 assay. b Morphology of SKOV3 cells exposed to APG-1387(0, 10 nM) photographed under a fluorescence microscope (original magnification× 10). c APG-1387-induced apoptosis in SKOV3 cells was assessed by Hoechst33258 staining. Morphology of SKOV3 cells exposed to APG-1387 at different concentrations photographed under a fluorescence microscope (original magnification × 10). Condensated and fragmented nuclears were the mean ± SEM of 5 randomized areas. P < 0.01. d SKOV3 cells were treated with 10 nM APG-1387 for the indicated times. The cells were stained for phosphorylated H₂AX and DAPI, then were analyzed by fluorescence microscopy (original magnification × 200). γ-H₂AX positive spots were the mean ± SEM of 5 randomized areas. P < 0.01. e, f SKOV3 and OVCAR3 cells were exposed to various concentrations of APG-1387 (0, 10, 30 nM) for 24 h followed cell apoptosis analysis by flow cytometry. g Western blot analysis of caspase-3/PARP SKOV3 cells were treated withAPG-1387 (0, 3, 10, 30, 100, 300 nM) for 24 h. The data shown are representative of three different experiments. h SKOV3 cells were stimulated with APG-1387 for indicated periods of concentrations, caspase activation were tested by caspase activity assay

Fig. 3

APG-1387-induced apoptosis in caspase dependent manner. a Cells with or without addition of Z-VAD-FMK. Morphology of cells exposed to different treatment groups photographed under a fluorescence microscope (original magnification × 10). b APG-1387 was coadministered with or without addition of caspase inhibitor (Z-VAD-FMK). Cell viability was determined by the CCK-8 assay. c Western blot analysis of the effect of APG-1387 with or without addition of Z-VAD-FMK on caspase-3/PARP expression level in SKOV3 cells. d Western blot analysis of the expression levels of IAPs at different concentrations of APG-1387 in SKOV3 cells. e Cells were treated with different time points, and the effect of APG-1387 on IAP family members expression level was determined by western blot. Data represent one of three experiments yielding similar results

Fig. 4

APG-1387 is RIP1-dependent in ovarian cancer induced apoptosis. a Western blot analysis of the expression levels of caspases-8 and RIP1 at different concentrations of APG-1387 in cells. b Cells were treated with APG-1387 (10 nM) for the indicated times. The indicated proteins were detected by western blot after co-Immunoprecipitation with an antibody for RIP1, GADPH was detected as a control for specificity of co-Immunoprecipitation. c Western blot analysis of the effect of APG-1387 with or without addition of Z-VAD-FMK on RIP1 expression level in the SKOV3 cells. d Cells were transfected with caspases-8 siRNAs, cell viability was determined by the CCK-8 assay. e Cells were transfected with RIP1 siRNAs, cell viability was determined by the CCK-8 assay. f APG-1387 was coadministered with or without Necrostatin-1, cell viability was determined by the CCK-8 assay. g Cells were transfected with RIP1 siRNAs. Western blot analysis of the expression levels of caspases-3 and PARP. h The protein levels of cleaved-caspases-3/8 and PARP in cells by western blot after APG-1387 was coadministered with or without Necrostatin-1

Fig. 5

APG-1387 induces cell death partly through TNF-alpha signaling pathway. a ELISA analysis of the secretion of TNF-alpha after different time points of APG-1387 on SKOV3 cells streatment. b Cells were treated with different time points of APG-1387, and the secretion of TNF-alpha were analyzed by ELISA. c Cells were transfected with TNFR1 siRNAs, cell viability was determined by the CCK-8 assay. d Cells were transfected with TNF-alpha siRNAs, Cell viability was determined by the CCK-8 assay. e APG-1387 was coadministered with or without TNF-alpha neutralizing antibody, cell viability was determined by the CCK-8 assay. f Cells were treated with different concentrations of APG-1387 on cells after 24 h treatment and the expression levels of NF-κB1/p50 and NF-κB2/p52 were determined by western blot. g The protein levels of NF-κB1/p50 and NF-κB2/p52 in cells by western blot after APG-1387 was coadministered with or without Necrostatin-1

Fig. 6

APG-1387 induces autophagy in ovarian cancer cells. a The expression of LC3, Beclin1 and P62 was measured by western blot. Cells were treated with APG-1387(0, 3, 10, 30, 100, 300nM) for 24 h. b Cells were transfected with GFP-LC3 plasmids, and then maintained in media with or without 3 nM APG-1387 for 24 h. The cells were then stained with DAPI and analyzed by fluorescence microscopy. c Statistical analysis of the percentage of LC3 puncta per cell. Columns, mean (n=3); bars, SD. *P<0.01 vs. untreated group. LC3 puncta per cell were quantified. d Cells were transfected with Beclin1 siRNAs. Western blot was used to detect the expression of Beclin1. e Cells were transfected with ATG7 siRNAs. Western blot was used to detect the expression of ATG7. f Cells were transfected with Beclin1 siRNAs. After 24 h treatment with or without 3 nM APG-1387, western blot analysis was performed for indicated proteins. g Cells were transfected with ATG7 siRNAs. After 24 h treatment with or without 3 nM APG-1387, western blot analysis was performed for indicated proteins. h Western blot analysis was performed for indicated proteins in cells transfected with siBeclin-1 and treated with 10 nM APG-1387. i Western blot analysis was performed for indicated proteins in cells transfected with siATG7-1 and treated with 10 nM APG-1387

Fig. 7

APG-1387-induced autophagy promotes cell survival in SKOV3 cells. a, b, c The cell death was clearly evident in all cell lines by phase-contrast microscopy, Cells were incubated for 2 h in the presence or absence of 3-MA (20 mM), Baf A1 (100 nM) and CQ (10 μM), then, APG-1387 combined with or without 3-MA (20 mM), Baf A1 (100 nM) and CQ (10 μM) for 24 h. d, e, f Cells were incubated for 2 h in the presence or absence of 3-MA, Baf A1 and CQ, then, Cell viability were tested after APG-1387 combined with or without 3-MA, Baf A1 and CQ treatment 24 h was determined by the CCK-8 assay. g, h, i The expression of caspase-3 was measured by western blot. Cells were incubated for 2 h in the presence or absence of 3-MA, Baf A1 and CQ, then, APG-1387 combined with or without 3-MA, Baf A1 and CQ for 24 h. The data shown are representative of three different experiments

Fig. 8

APG-1387 inhibits tumor growth in xenografted ovarian models. a, b Changes in tumor volume and tumor growth between APG-1387 (1, 3, 10 mg/kg) group and vehicle group of SKOV3 tumor-bearing mice. Data are shown as mean tumor volume ± SD (eight mice/group). c, e, d, f Mice bearing SKOV3 xenograft tumor were treated with a single intravenous dose of APG-1387 and tumors were collected at the indicated time points for western blot analysis (Group 1: c, e and Group 1: d, f) or TUNEL assay (g, h). For western blot analysis, tumor lysates were assigned into two groups (Group 1: c, e and Group 1: d, f) and analyzed for the indicated proteins. g, h IHC staining assay (testing TUNEL-positive cells). All values were expressed as mean ± SD. *p < 0.05 vs. group of “control”. Bar ¼ 100 mm