Mechanism Of Growth Inhibitory Effects Of6R On Human Colon Cancer Cells

BackgroundsCancer remains a serious threat to mankind. Currently, chemotherapy is still the major treatment for cancer. Besides, targeted drugs have been focused in recent years, which will lead the development trends of chemotherapy in future. Colorectal cancer is one of the main causes of death with high incidence rates. Thus, to find a targeted and effective therapeutic agent is quite necessary. The physiological function of phosphatase and tensin homologue (PTEN) is a novel tumor suppressor gene. PTEN’s expression and activity has great implications with tumorigenesis and progress of cancer, but its exact mechanism and signaling pathways are still unclear. Studies have showed that PTEN played a key role in tumorigenesis and progress of human colon cancer.5-[2,3-Dichloro-4-(2-methylene-l-oxobutyl) phenoxymethyl]-3-methyl-1,2,4-ox-adiazole (6r) is a new ethacrynic acid (EA) oxadiazole containing analogues. It has been demonstrated that6r inhibited the proliferation of a variety of cancer cells in vitro and in vivo and could induce apoptosis, such as human prostate cancer, human myeloid leukemia, human cervical cancer, human promyelocytic leukaemia and human colon cancer, which hints good antitumor activity of6r.MethodsHuman colon cancer cells HT29, SW620, HCT116and Lovo were used to observe the antitumor effcts of6r. The anti-proliferative activity was determined by the MTT assay. Western blotting analysis was employed to examine the expression level of PTEN protein in the presence or absence of6r.The effects of6r on PTEN-dependent pathway were investigated. FITC-AnnexinV/PI double staining assay was carried out to examine the apoptosis induction of HT29and HCT116cells. PI staining assay was performed to measure the cell cycle arrest of HT29and HCT116cells after6r treatment. The scratch assay was employed to investigate the effects of6r on the migration ability of HT29cells. Western blotting analysis was employed to determine the expression of PI3K, P-AKT, MDM2and P53in HT29and HCT116cells.To further confirm the effects of6r on PTEN and its down-stream signal pathway, VO-OHpic was used to inhibit PTEN-dependent pathway. In the presence of VO-OHpic, the anti-proliferative effects of6r against HT29and HCT116cells were determined by the MTT assay. Cytotoxicity of6r against HT29and HCT116cells was determined by trypan blue dye exclusion method. Western blotting analysis was employed to determine the expression of P-AKT and P53in HT29and HCT116cells.Results1The antitumor effects of6r on human colon cancer cells were related to PTEN levelThe inhibitory effects of6r on human colon cancer cells growth was evaluated by MTT assay. HCT116, HT29and SW620cell lines were highly sensitive to6r, while Lovo cell line was less sensitive to6r. Western blotting analysis showed that the expression of PTEN was gradually reduced in HCT116, HT29, SW620and Lovo cells.6r could significantly increase the expression of PTEN in HCT116, HT29, SW620and Lovo cells in a concentration-dependent manner.26r activated PTEN-dependent pathwayFITC-AnnexinV/PI double staining showed that the apoptosis rate was increased after treatment with2,4and6μM of6r for48h in HT29and HCT116cells. PI staining results showed that6r could induce cancer cell cycle arrest at S phase in HT29and HCT116cells. Incubation with cells for12,24,36and48h,4,6μM6r inhibited the migration of HT29cell at36h and48h, as estimated by scratch assay. Western blotting showed that6r significantly reduced the expressioin of pro-casepase-3and pro-casepase-9and increased the ratio of Bax/Bcl-2in HT29and HCT116cells.6r could also reduce the levels of PI3K, P-AKT and MDM2and increase the levels of P53in HCT116、HT29and SW620cells.3VO-OHpic attenuated antitumor effects of6r by inhibiting PTEN-dependent pathwayIn the presence of VO-OHpic, the anti-proliferation of6r on HT29and HCT116cells was inhibited significantly in comparison with6r treatment alone. Moreover,6r decreased the protein level of P-AKT and increased the protein level of P53. However, the regulation of6r on the proteins expression was inhibited by VO-Ohpic.ConclusionThe inhibitory strength of colon cancer by6r is closely related to the expression level of PTEN in colon cancer cells.6r can induce apoptosis in colon cancer cells by up-regulating the expression of PTEN and activating PTEN-dependent pathways, resulting in strong anti-tumor effects on colon cancer cells.