The Inhibitory Effect Of Methyl Jasmonate On Adenoid Cystic Carcinoma Of Salivary Glands

Background:Salivary adenoid cystic carcinoma (SACC) is one of the most common malignant tumor of salivary glands, accounting for about11%of salivary epithelial tumors, and27%of all salivary gland malignant carcinomas. The majority of these tumors occur in the minor salivary glands, and minority of the large salivary glands. SACC can occur at any age, and the majority of the tumors occur in patients between40to60years old. There is no significant gender differences. Its clinical course is charactered by a slow but locally aggressive growth pattern along nerves and blood vessels irreversibly. Radical surgical resection approaches frequently fail to attain clean surgical margins, which is metastases in lung, liver and the skeletal system. Therefore, it is important to find new treatment methods and high effective anti-cancer drugs to SACC.Plant hormones in plants, play very important roles in all aspects as chemical information communication in metabolism, cell growth, morphogenesis, physiological activities of plants. At present, the anti-tumor effect of the plant hormones have achieved remarkable results. Jasmonic acid (JA) and its methyl ester (Methyl Jasmonate, Me-Ja) are a class of endogenous signal molecule, which regulate higher plant development, response to external stimuli (especially trauma), and gene expression. It widespread in the plant kingdom, the16th International Conference on Plant growth had accepted it as a new class of plant hormones. Recent studies have shown that, JA and Me-Ja can induce Molt-4lymphoblastic leukemia cell death, inhibition of human breast cancer MCF-7cells, melanoma SK-28cells, prostate cancer LNcaP cells proliferation, and methyl jasmonate could lead to all of the above cells death. Me-Ja can also inhibit oral squamous carcinoma cells,, liver cancer cells, human neuroblastoma cells growth, induce those cells apoptosis. In view of different cells exist differences in the sensitivity to Me-Ja, we chose human salivary adenoid cystic carcinoma cell ACC-2stem in the experiment, to observe the effects of Me-Ja on ACC-2cells, in order to provide a theoretical basis for the treatment of SACC.Objective:By experiment of methyl jasmonate on salivary adenoid cystic carcinoma ACC-2cell stem, we study the mechanism of inhibition of proliferation and apoptosis of ACC-2cells after dosed by methyl jasmonate, further explore the mechanism of salivary adenoid cystic carcinoma and to find a new drug who has low toxicity and highly effective for SACC.Methods:1. Cultivation ACC-2cells in vitro, testing an effective concentration of methyl jasmonate to ACC-2cells and testing the inhibitory rate of each group ACC-2cells by CCK-8method; observing the changes in shape of the cells by inverted microscope; and observing the cellular ultrastructure by transmission electron microscope; detecting the cell cycles and apoptosis by flow cytometry.2. Detecting the Bcl-2and Bax mRNA and protein expressions By RT-PCR and immunocytochemistry method.Results:1. Me-Ja has the inhibitory effect on ACC-2cells growth, and this effect shows in the dose/time dependent manner.2. The50%inhibitory concentration (IC50) of Me-Ja to ACC-2cells was9.20μmol/L, we choose1/2IC50as the working concentration of Me-Ja for the later experiments.3. Morphology observing of Me-Ja acting on ACC-2cells growth inhibition: The cells in control group grow well in adherence. In the Me-Ja and5-Fu group, part of ACC-2cells ablate, and adherent cells decrease obviously.while in the5-Fu+Me-Ja group:adherent cells decrease obviously.4. The inhibition rate of ACC-2cell proliferation results:5-Fu Group is44.22%, Me-Ja group is44.92%, Me-Ja+5-Fu Group is49.3%.5-Fu group compared with Me-Ja group, P>0.05, joint action group compared with the monotherapy Group P<0.05. The results instructions there is no significant difference in the inhibitory rate of5-Fu and Me-Ja separately effected on ACC-2, combined medication has more effective than medication alone.5. The changes of each group ACC-2cells ultramicrostructure:In control group, the ACC-2cells are in low differentiation, which is the typical morphologic character of malignant tumor cells. In Me-Ja and5-Fu group, cell junction and microvillus disappeared, apoptotic body appeared in part of ACC-2cells. In Me-Ja+5-Fu group: some mitochondria swell, microvillus disappeared and apoptotic body appeared.6. The changes of cell cycles of each ACC-2cells:In control group:the ratio of cells at stage G1was40.8%,24.6%was at stage S. In Me-Ja group:the ratio of cells at stage G1was62.1%, and17.3%was at stage S, the cell cycle has been blocked to stage G1; apoptosis may happen to some cells because of peak of diploid appeared. In5-Fu group:the ratio of cells at stage G1was55.9%; the ratio at stage S was17.2%, the cell cycle has been obviously blocked to stage G1:In Me-Ja+5-Fu group:the radio of cells at stage G1was61.2%, ratio at stage S was7.1%, most cells have been blocked to stage G1, typical peak of apoptosis diploid appears.7. RT-PCR tested Bcl-2and Bax mRNA expression:the Bcl-2mRNA expression of Me-Ja,5-Fu and Me-Ja+5-Fu group of ACC-2cells were significantly decreased (P<0.05), while Bax mRNA expression levels were significantly higher than the control group(P<0.05).8. Bcl-2and Bax immunohistochemical results:Bcl-2protein in the cytoplasm of expression of ACC-2cells in Me-Ja,5-Fu and Me-Ja+5-Fu group were significantly lower than control group (P<0.05); while Bax protein levels were more significant increase than control group (P<0.05). Conclusion:Methyl jasmonate can inhibit ACC-2cells growth obviously; Me-Ja could block ACC-2cellular cycle, and promote ACC-2cell apoptosis; So it has the effect of anti-adenoid cystic carcinoma. Methyl jasmonate induced apoptosis of ACC-2cell through the way of lowering Bcl-2mRNA and protein expression levels, and raising Bax mRNA and protein expression levels.