We here evaluated the potential anti-colorectal cancer activity by erastin, a voltage-dependent anion channel (VDAC)-binding compound. colorectal cancer cells. On the other hand, over-expression of VDAC-1 augmented erastin-induced ROS production, mPTP opening, and colorectal cancer cell apoptosis. studies showed that PF-562271 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. Introduction The colorectal cancer is the major contributor of cancer-related mortality both in China [1] and around the world [2,3]. It is estimated that over 100,000 new cases of colorectal cancer are diagnosed each year, which cause over 50,000 deaths annually [4]. Chemotherapy has been widely-utilized for treatment of colorectal cancer, however drug resistance and/or off-target toxicity limit the efficiency of current chemo-drugs [5,6,7]. Thus, our group [8,9] and others [10,11] have been focusing on the development of novel and more efficient anti-colorectal cancer agents. Mitochondrial permeability transition pore (mPTP) is a multi-protein channel complex lying in the mitochondria, whose main function is to maintain the balance of mitochondrial respiratory chain [12]. mPTP PF-562271 is primarily composed of three proteins: including voltage-dependent anion channel (VDAC) in the out mitochondrial membrane (OMM), adenine nucleotide translocator 1 (ANT-1) in the inner mitochondrial membrane (IMM) and matrix locating cyclophilin-D (Cyp-D) [12]. It has been shown that multiple stimuli will induce ANT-1 and Cyp-D association and mPTP opening, thus leading to reactive oxygen species (ROS) production, PF-562271 ATP depletion and pro-apoptotic molecule (antitumor efficacy evaluation Tumor growth studies were performed in severe combined immunodeficient (SCID) mice xenograft model. All mice were purchased from the Animal Facility of Shanghai Jiao-tong University School of Medicine (Shanghai, China). Briefly, 2106 viable HT-29 cells in 100 L of growth medium (per mouse) were subcutaneously inoculated, and mice bearing ~100 mm3 tumors were randomly divided into three groups with 10 mice per group. Mice were treated daily with 10 or 30 mg/kg body weight of erastin (intraperitoneal injection, for 4 weeks) or vehicle control (Saline). Tumor volumes were calculated by the modified ellipsoid formula: ( / 6) AB2, where A is the longest and B is the shortest perpendicular axis of a tumor mass [22,23]. Mice body weights were also recorded every week. Humane endpoints were always utilized to minimize mice suffering. Animals were observed on daily bases. Signs such as significant-reduced locomotion, severe diarrhea, severe piloerection or a sudden weight loss (> 20%) were recorded. If animals reached these endpoints they were euthanized by exsanguination under 2,2,2-tribromoethanol anesthesia (4 mg/10 g body weight, Sigma). All injections were performed under the 2,2,2-tribromoethanol anesthesia method. The animal PF-562271 studies have been approved by the Shanghai Jiao-tong University School of Medicines Institutional Animal Care and Use Committee (IACUC) and Ethics committee (Contact person: Dr. Jun Wang, 2014126). 2.17. Statistical analysis All data were normalized to control values of each assay and were presented as mean standard deviation (SD). Data were analyzed by one-way ANOVA followed by a Scheffes f-test by using SPSS 16.0 software (SPSS Inc., Chicago, IL). Significance was chosen as p < 0.05. Results 3.1. Erastin exerts cytotoxic, but not cytostatic effects to cultured colorectal cancer cells To test erastins activity on colorectal cancer cell survival, HT-29 cells were treated with increasing concentrations of erastin (0.1C30 M). MTT assay was performed. As shown in Fig 1A, erastin potently inhibited HT-29 cell survival, which was evidenced by MTT OD reduction. Erastin showed a dose-dependent effect (Fig 1A), and 30 M of erastin displayed the most dramatic effect (Fig 1A). Erastin took at least 48 hours to exert significant cytotoxic effect in HT-29 cells (Fig 1A). The cytotoxic effect by erastin was also demonstrated by the trypan blue staining assay (Fig 1B) and colony formation assay (Fig 1C). Erastin (1C30 M) treatment significantly increased the number of trypan blue positive (dead) HT-29 cells (Fig 1B), whiling decreasing survival HT-29 colonies (Fig 1C). Fig 1 Erastin exerts cytotoxic, but not cytostatic, effects to cultured colorectal cancer cells. Interestingly, erastin (1C30 M) appeared ineffective in inhibiting HT-29 cell proliferation, and the BrdU incorporation was not changed in HT-29 cells after cytotoxic erastin (1C30 M) treatment (Fig 1D). Therefore, the cytotoxic effect by erastin is unlikely due to proliferation inhibition. MTT results in Fig 1E showed that erastin (1C30 M) was also cytotoxic to two other colorectal cancer cell lines: DLD-1 Rabbit polyclonal to ZNF768 and CaCo2. Yet, same erastin treatment was generally safe to the non-cancerous NCM460 colon epithelial cells (Fig 1E). Once.