Honokiol Radiosensitizes Colorectal Cancer Cells:Enhanced Activity In Cells With Mismatch Repair Defects

Background:Both of the morbidity and mortality of colorectal cancer were in the third place among all kinds of malignant tumors in USA. Radiation therapy plays an important role in the complicated treatment of colorectal cancers, especially for those late-stage patients. However, high dose radiation will lead to some deadly side effects to those patients, some colorectal cancer cells are not sensitive to radiation therapy, and some cancer cells show the capability of radiation resistance. It is necessary to find an ideal radiosensitizer, which can not only inhibit the growth of colorectal cancer cells but also increase the sensitivity to radiation therapy. Mismatch repair is required for correcting any mismatches that are created during replication and recombination, and a defective mismatch repair system contributes to DNA damage-induced growth arrest. The colorectal cancer cell line HCT116is known to have a mutation in the hMLHl mismatch repair gene resulting in microsatellite instability and defective mismatch repair. Honokiol is a biphenolic compound that has been used in traditional Chinese medicine for treating various ailments including cancer.Objective:This study was designed to test the hypothesis that honokiol enhances the radiosensitivity of cancer cells with mismatch repair defect (HCT116) compared with those that are mismatch repair proficient (HCT116-CH3), and to explore the possible mechanism of hMLHl gene in adjusting rediosensitizing effect of honokiol in human colon cancer cells.Methods:Three human colon cancer cell lines HCT116cell, HCT116-CH2cell and HCT116-CH3cell were treated with the combination of different concentrations of honokiol(10umol/ml,20umol/ml,30umol/ml,40umol/ml,50umol/ml)and different dose of γ-irradiation(0,2.5,5.OGy) for different time points(24h,48h,72h), detecting honokiol's growth inhibition and radiosensitizing effects on human colon cancer cells. Cell colony experiement was used to test honokiol's long-time effects. With the same treatment, cells were collected and cell cycles were analyzed with Flow cytometry analysis. Caspase3,7assay kit help to detect the apoptosis happened the combination of honokiol and γ-irradiation. The protein and mRNA variation of apoptosis related gene, such as p53, Bax and Bcl-2, were detected with western blotting and RT-PCR.Results:First, Cell proliferation data indicated that the combination of honokiol and/or γ-irradiation treatment could lead to time-and dose-dependent inhibition of cell proliferation and colony formation in all three cell lines, the effect was more pronounced in hMLHl deficient HCT116and HCT116-CH2cells. Only treated with honokiol, the IC50of48hours is40umol/ml, and ID50the only treated with Y-irradiation was5Gy for72hours. While treated with the combination of honokiol and γ-irradiation, IC50were25umol/ml and2.5Gy for48hours, which indicated honokiol greatly increased the radiosensitivity of human colon cancer cells. With the same treatment, the proliferation inhibition of hMLHl deficient HCT116(41.55%) was more significant than that in hMLHl proficient HCT116-CH3cell (68.02%). Compared with HCT116-CH3cells, similarly, the combination of honokiol and Y irradiation treatment induced higher levels of apoptosis (caspase3activation, P-p53expression) in the HCT116cells. There were significantly lower amounts of phosphorylated p53in the HCT116-CH3cells, while there were much higher level of proapoptotic gene Bax and lower level of antiapoptotic gene Bcl-2expression. Cell cycle analyses revealed higher levels of dead cells in the HCT116cells than in the HCT116-CH3cell. The combination treatment reduced expression of cyclin B1and D1in both cell lines, which suggested that the high levels of hMLH1may reduce the radiosensitivity of human colon cancer cells. These data demonstrated that honokiol may be highly effective in radiosensitizing colorectal cancer cells, especially in those with a mismatch repair defect. hMLH1gene may reduce Honokiol's radiosensitizing effect by inhibit the activation of p53protein in colorectal cancer cells.Conclusion:1. Honokiol is an effective radiosensitizer for human colon cancer cells, especially for mismatch repair defect colon cancer cells.2. Honokiol can lead to the G0/G1phase arrest of human colon cancer cells.3. The combination of honokiol and γ-irradiation can increase the apoptosis of human colon cancer cells.4. hMLHl gene may reduce honokiol's radiosensitizing effect on human colon cancer cells by inhibiting the activity of P53.