A Research on the Mechanism of TH-302 on Hypoxic Condition and TH-302 Combined with Radiation Applied in Gastrointestinal Tumors

Abstract

Tumors often contain hypoxic regions resistant to chemo- and radiotherapy. TH-302 is an investigational hypoxia-activated prodrug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. TH-302 mainly acts on hypoxic condition instead of normal oxygen condition. There have been some phase II clinical trials of TH-302 in soft tissue sarcoma and pancreatic cancer, it shows that TH-302 has good effect on these tumors. Radiation is less sensitive to tumor cells on hypoxic conditon. This study is to investigate the effect of TH-302 combined with radiation in gastrointestinal tumors and the mechanism of TH-302 on hypoxic condition. HCT116 cells, HT29 cells (both are intestinal cancer cells) and MKN28 cells (gastric cancer cells) are chosen for the following experiments. Firstly, 48h and 72h IC50s of TH-302 both on normal oxygen condition (O2%=21%) and hypoxic condition (O2%=1.3%) are obtained through drug cytotoxicity tests. Then tumor cells on hypoxic condition are dealt with TH-302 and radiation, CCK8 of the cells are tested. What’s more, cells are treated with TH302 (24h) on normal oxygen condition (O2%=21%) and hypoxic condition (O2%=1.3%) respectively. Cell apoptosis is examined using Flow Cytometry. At last, expression of HIF-1α protein related to hypoxia mechanism is examined through Western Blot and expression of HIF-1α gene is examined through qPCR. TH-302 is sensitive to tumor cells on hypoxic condition and has lower IC50.The 48h IC50 of TH-302 in HCT116 cells on normal oxygen and hypoxic condition is 2.8*10ˆ8nmol/L and 2,074nmol/L respectively.In HT29 and MKN28 cells the data is 118,400 nmol/L vs 40,790 nmol/L and 29,143 nmol/L vs 1,473 nmol/L.TH-302 combined with radiation on hypoxic condition has more effect than radiation and TH-302 alone. The figure will be shown in the final paper. Tumor cells dealt with TH-302 have more apoptosis on hypoxic condition than normal oxygen condition. In HCT116 +/+, HCT116 -/-, MKN28 cells, the apoptosis ratio is 11.3% vs 0.9%, 17.0% vs 4.3% and 73.8% vs 16.5%. There is not much difference of HIF-1 gene expression between hypoxia and normal oxygen condition. ON hypoxic condition, tumor cells have higher HIF-1 protein expression level. TH-302 is sensitive to tumor cells on hypoxic condition. TH-302 can be used as a radiosensitizer in gastrointestinal tumors. The mechanism of how TH-302 works on hypoxic condition has relationship with apoptosis.