The Effect And Mechanism Of Hematoporphyrin Monomethyl Ether-SDT Combined With Doxorubicin On The Proliferation Of QBC Cell Lines

Purpose:As a traditional anti-cancer chemotherapy drug, doxorubicin has been used until 1970s, but the heart toxicity and other side effect have seriously affected its clinical effects. Studying the effect and mechanism of HMME-SDT combined with DOX in QBC cell line in this study to reduce the toxicity and increased the effect. And as we know that both DOX and HMME-SDT can induce cell apoptosis, but the mechanism about the combination of DOX and HMME-SDT is unknown.Methods:Human cholangiocarcinoma QBC cells we used for the experiment, there are 12 experimental groups:the control group, the ultrasound control group, the DOX treated group, the DOX dealt with ultrasound, HMME low dose group, HMME low dose dealt with ultrasound group, HMME high dose group, HMME high dose dealt with ultrasound group, HMME low dose combine with DOX group, HMME low dose combine with DOX and ultrasound group, HMME high dose combine with DOX group, HMME high dose combine with DOX and ultrasound group. After study, cell were sonicated by 1.2 MHZ pulsed ultrasound at intensities of 1.0 W/cm2 for 120 s; The cells were incubated for 24 h, and treated with drugs and ultrasound, the results were analyzed by MTT assay; The levels of apoptosis and cell cycle arrest was evaluated using Annexin V-FITC/Propidium iodide flow cytometry analysis; The nuclear morphology of cells was studied by using the cell-permeable DNA dye Hoechst 33258;Western blot were employed to examine the p53,Fas, Bax, Bcl-2, activated caspase-3 protein alterations in response to drug and ultrasound. The DNA damage was observed by single cell gel electrophoresis and DNA fragmentation (DNA ladder); Western blot were employed to examine the p53, y-H2A.X and MDM2 protein alterations in response to drug and ultrasound. We measured the antitumor effect by the methods witch used the expression of human cholangiocarcinoma cells in nude mice and injection to study the effect of ultrasound combined with HMME and DOX in vivo.Results:The data of MTT assay indicated that HMME high dose combine with DOX dealt with ultrasound group was better than the other group in QBC Cell proliferation inhibition rate, in the 24h results, the cell viability was only 36.2±0.58%; The results from flow cytometer showed that the early and late apoptotic cell populations of QBC cell significantly increased after treat with HMME-SDT combine with DOX, and HMME-SDT combine with DOX groups induced G2/M phase cell cycle arrest; Similarly, the Hoechst staining showed that the nuclear morphology of cells were significantly change when dealt with HMME-SDT combine with DOX; The expression of protein p53, Fas, Bax, caspase-3 were up-regulated and Bcl-2 protein was down-regulated in the experimental groups with treatment. The results of Single cell gel electrophoresis and DNA fragmentation indicated the effect of DNA damage of HMME-SDT combine with DOX groups were better than the other; The expression of p53,γ-H2A.X protein were up-regulated and MDM2 protein was down-regulated in the experimental groups with treatment whatever after 12h and 24h. After the experiment of nude mice, we found that the tumor in nude mice treated with ultrasound combined with HMME and DOX was significantly inhibited.Conclusion:All data showed that the combination of HMME-SDT and DOX not only decreased the toxicity but also increased the effect, in other words, the effect of HMME-SDT combined with DOX is Synergistic effect, and the mechanism of that may increased DNA damage.