Erastin Disrupts Mitochondrial Permeability Transition Pore (mPTP) and Induces Apoptotic Death of Colorectal Cancer Cells

Abstract

We here evaluated the potential anti-colorectal cancer activity by erastin, a voltage-dependent anion channel (VDAC)-binding compound. Our in vitro studies showed that erastin exerted potent cytotoxic effects against multiple human colorectal cancer cell lines, possibly via inducing oxidative stress and caspase-9 dependent cell apoptosis. Further, mitochondrial permeability transition pore (mPTP) opening was observed in erastin-treated cancer cells, which was evidenced by VDAC-1 and cyclophilin-D (Cyp-D) association, mitochondrial depolarization, and cytochrome C release. Caspase inhibitors, the ROS scavenger MnTBAP, and mPTP blockers (sanglifehrin A, cyclosporin A and bongkrekic acid), as well as shRNA-mediated knockdown of VDAC-1, all significantly attenuated erastin-induced cytotoxicity and apoptosis in colorectal cancer cells. On the other hand, over-expression of VDAC-1 augmented erastin-induced ROS production, mPTP opening, and colorectal cancer cell apoptosis. In vivo studies showed that intraperitoneal injection of erastin at well-tolerated doses dramatically inhibited HT-29 xenograft growth in severe combined immunodeficient (SCID) mice. Together, these results demonstrate that erastin is cytotoxic and pro-apoptotic to colorectal cancer cells. Erastin may be further investigated as a novel anti-colorectal cancer agent.

Fig 1. Erastin exerts cytotoxic, but not cytostatic, effects to cultured colorectal cancer cells.

Colorectal cancer cells (HT-29, DLD-1 and Caco-2 lines) or NCM460 colon epithelial cells were treated with vehicle control (0.1% DMSO, “Ctrl”) or indicated concentrations of erastin for applied time, cell survival was tested by MTT assay (A and E) and colony formation assay (C); The percentage of trypan blue positive (“dead” cells) was recorded (B); Cell proliferation was tested by BrdU incorporation assay (D and F). For each assay, n = 5. The data presented were mean ± SD. Experiments were repeated three times with similar results obtained. * p < 0.05 vs. group of “Ctrl”.

Fig 2. Erastin induces ROS production and caspase-dependent apoptosis in cultured colorectal cancer cells.

Colorectal cancer cells (HT-29, DLD-1 and Caco-2 lines) or NCM460 colon epithelial cells were treated with vehicle control (0.1% DMSO, “Ctrl”) or indicated concentrations of erastin for applied time, cell apoptosis was examined by listed assays (A-C, G and H); ROS production was also examined (D and I). HT-29 cells were pre-treated with z-DEVD-fmk (“zDEVD”, 50 μM), z-LEHD-fmk (“zLEHD”, 50 μM) or MnTBAP (10 μM) for 1 hour prior to applied erastin stimulation, cell survival and cell death were tested by MTT assay (E) and trypan blue assay (F), respectively. For each assay, n = 5. The data presented were mean ± SD. Experiments were repeated three times with similar results obtained. * p < 0.05 vs. group of “Ctrl”. ^# p < 0.05 vs. group of erastin only (E and F).

Fig 3. Erastin induces mPTP opening in cultured colorectal cancer cells.

HT-29 cells were treated with applied erastin for indicated time, mPTP opening was evidenced by mitochondrial VDAC-1-ANT-1 association (A), cytochrome C (“Cyto-C”) release (B) and JC-10 intensity increase (C). HT-29 cells were pre-treated with sanglifehrin A (SfA, 2.5 μM), cyclosporin A (CsA, 0.5 μM) or bongkrekic acid (BA, 5 μM) prior to erastin (10 μM) treatment, cell survival (D) and apoptosis (E) were analyzed afterwards. Stably HT-29 cells expressing VDAC-1 shRNA-1/-2 or scramble control shRNA (“scr shRNA”) were treated with erastin (10 μM), VDAC-1 expression (F), cell survival (G) and apoptosis (H) were tested. ANT-1-assocaited Cyp-D (A), cytosol cytochrome C expression (B) and VDAC-1 expression (F) were quantified. For each assay, n = 4. The data presented were mean ± SD. Experiments were repeated three times with similar results obtained. * p < 0.05 vs. group of “Ctrl”. ^# p < 0.05 vs. group of erastin only (D and E) or the “scr shRNA” group (G and H). “Trans” stands for transfection control (F-H).

Fig 4. VDAC-1 over-expression potentiates erastin’s cytotoxicity.

Stably HT-29 cells or NCM460 colon epithelial cells expressing empty vector (pSuper-puro, “Vec”) or VDAC-1 cDNA (“VDAC-1”) were treated with designed erastin for applied time, VDAC-1 expression, cell survival and apoptosis were tested by Western blotting assay (A and F), MTT assay (B and G) and histone DNA ELISA assay (C), respectively; ROS production (D) and JC-10 intensity (E) were also analyzed. For each assay, n = 4. The data presented were mean ± SD. Experiments were repeated three times with similar results obtained. VDAC-1 expression was quantified (F).* p < 0.05 vs. Ctrl group of “Vec” cells (B-E). ^# p < 0.05 vs. erastin group of “Vec” cells (B-E). “Trans” stands for transfection control (D and E).

Fig 5. Erastin administration inhibits HT-29 xenograft growth in SCID mice.

HT-29 tumor bearing SCID mice were intraperitoneally administrated with erastin (10/30 mg/kg body weight or “bw”, daily, for 4 weeks) or vehicle (Saline) control, tumor volumes (A) and mice body weights (B) were recorded weekly for five weeks; Daily tumor growth was also calculated (C). At the termination of experiments, all the xenografts were isolated and weighted (D). For each assay, n = 10. The data presented were mean ± SD. Experiments were repeated twice with similar results obtained. * p < 0.05 vs. group of “Vehicle”. ^# p < 0.05 vs. group of erastin at 10 mg/kg of body weight.