Study On The Radiosensitizing Effects Of Tanshinone Compounds

Objective:Salvia is a clinical medicine in blood circulation. Many studies about modern medicine have reported that Danshen displayed a variety of biological activities, such as prevention of myocardial ischemia, myocardial infarction and other cardiovascular diseases.Meanwhile,it can also treat liver cancer, leukemia, etc. Tanshinone compounds played anti-tumor effects mainly by inhibiting proliferation and promoting apoptosis of tumor cells. In this paper, the cytotoxicity and radiosensitizing effects of tanshinone compounds (TanshinoneⅡA,TanshinoneⅠ, Dihydrotanshinone, Cryptotanshinone) on human cervical cancer Hela cell were investigated. The varieties of structure that associated with cytotoxicity and relative mechanism were investigated. The modification of TanshinoneⅡA and the cytotoxicity of its derivatives were also explored. These work will contribute to the radiosensitizing drugs with low toxicity and high efficiency.Methods:The MTT assay was used to detect the growth inhibition rates of Hela cell at 24h,48h,72h which cultured with tanshinone compounds in different concentrations. With multi-target single-hit model, the radiosensitization effect was investigated by clone formation assay through sensitizing enhancement ratio (SER).The cell cycle distribution and apoptosis were measured by flow cytometry. The protein expressions of E6,p53 and p21 were studied by Western blot. The mouse of hepatoma H22 model was established, the growth of tumor's conditions after irradiated was observated. Mices were killed after 22 days, the inhibition rate and enhancement factor (EF) were calculated. TanshinoneⅡA, with weak cytotoxicity, was modificated by Mannich reaction.The cytotoxic activity of its derivative was detected.Results:The different concentrations (0.5-16μg/ml) of TanⅡA, TanⅠ, dihydrotanshinone and cryptotanshinone had the cytotoxicity on Hela cell, which were clearly toxic dose and time-dependent. The IC50 of 24h:24.1μg/ml,20.1μg/ml,8.4μg/ml, 17.8μg/ml; The IC50 of 48h:7.97μg/ml,11.1μg/ml,2.2μg/ml,8.2μg/ml; The IC50 of 72h: 4.6μg/ml,3.5μg/ml,1.5μg/ml,8.6μg/ml,respectively. The radiosensitization of Crypto-tanshinone and Dihydrotanshinone were proved on Hela cell line. Further clone formation assay indicated that the sensitization enhancement ratio of IC20 was 1.4 and 1.35, respectively. Hela cells were treated with drugs which had significant effects on cell cycle and induced apoptosis. Western blot showed that cryptotanshinone decreased expression of HPV E6 and increased expression of p53 and p21 protein. Drug (high, medium and low) groups inhibited the tumor growth better than the contral groups, and drug (medium) groups had the significant radiosensitizing effects. The inhibition of derivatives on Hela cell was closely related to concentration and time of administration. And the strongest inhibition of cell proliferation was detected on the concentration of 1μg/ml at 24 and 48h.Conclusion:Tanshinone compounds had proliferating inhibition effects on Hela cell line. Dihydrotanshinone had the strongest cytotoxicity on the Hela cell line.The disparity between A ring and D ring,with aromatic and furan ring structure, may make the difference on growth inhibition in Hela cell obviously. Cryptotanshinone and Dihydrotanshinone had significant cytotoxicity and radiosensitization effects on cervical cancer Hela cell. In addition, the capacity of delaying tumor growth in nude mice transplanted with hepatoma cell was evaluated. One of the mechanism maybe that it can make significant G0/G1 phase arrest and induce apoptosis, decrease the proportion of cells in S phase, also decrease expression of HPV E6 and restore the function of p53 to induce apoptosis.So cells were more sensitive to radiation in human cervical cancer. TanshinoneⅡA was modificated by Mannich reaction, after introducted the sodium salt of glycine. The derivative was stronger in inhibiting proliferation of Hela cell than that of tanshinoneⅡA on low dose of concentration (below 1μg/ml) owing to improvement of solubility.