| Objective The nasopharyngeal carcinoma is one of the most common malignant tumors and threatens the people’s health seriously. Now the main methods of curing with nasopharyngeal carcinoma include radiotherapy and chemotherapy, both of them have severe side effect and making the influence of further therapy. So to search new drug of anti-nasopharyngeal carcinoma is a top topic. Recently, some studies have shown that matrine can inhibit tumor growth, induce and kill apoptosis of tumor cell, and getting widely attention. But the dose of inhibit tumor growth of matrine is too high, and it is not a high efficient drug of anti-cancer. To make the higher effect of anti-cancer, we synthesized novel20matrine derivants that based on matrine as the lead compound by chemical derivant to transform its structure, and after carrying out the screening of anti-nasopharyngeal carcinoma, obtained dirivant X—14-thienyl methylene matrine as an optimal anti-tumor compound that have more higher effect of anti-nasopharyngeal carcinoma than matrine in prophase research. In order to further research the effects and its mechanism of14-thienyl methylene matrine on anti-nasopharyngeal carcinoma, NPC cell atrains, CNE1and CNE2will be used in these experimental researchs on its effects in vitro and in vivo so as to find a higher effective drug of anti-nasopharyngeal carcinoma.Research contentsPart oneStudying on the inhibiting anti-nasopharyngeal carcinoma effect of14-thieny1methylene matrine in vitroMethods:In order to research the effects and its mechanism of14-thienyl methylene matrine on anti-nasopharyngeal carcinoma, interfering NPC cell CNE1and CNE2with the14-thienyl methylene matrine and matrine. Use MTT to detect the grouth-inhibitory curves of cells. Observe the change of the cell shape by inverted microscope. Effect of mechanism of dirivant X of the Matrine on ultrastructures of CNE1cells was observed by transmission electron microscope. The cell apoptosis rates and cell cycle were analyzed by flow cytometry. The correlated apoptotic protein expression was examined by western blotting.Results:Matrine and its dirivant X can inhibit the proliferation of CNE1and CNE2cells in dose dependent manners in vitro by MTT. Dirivant X which based on matrine for the lead compound through chemical synthesis to transform its structure, have more higher effect of CNE1and CNE2cells than matrine. The IC50(1-5days) of derivant X were lower than matrine’s.After incubation of CNE1and CNE2cells with derivant X of the Matrine for48h, cells alter to rounded and smaller. Characteristic morphology of apoptosis, such as shrinkage, heterochromatin significantly increased in nuclear, increased gap of periphery in nucleus uncertainly, and apoptotic bodies, etc were found under the transmission electron microscope.Flow cytometry analysis revealed the apoptosis rate in different dose of derivant X, the dose of IC50of matrine and DDP group which showed the significant difference compared with the control group both in CNE1and CNE2cells(P<0.05). The apoptosis rate show that derivant X has stronger depressant effect on CNE1than on CNE2.The detect of flow cytometry reveal, for CNE1cells, the cycle blockage happen at the stage of G1both in different dose of derivant X and matrine groups. The cell proportion of G1period is higher than the control group (p<0.01). The cycle blockage of the DDP group take place at the stage of S (P<0.01). Fellow the dose of derivant X increasing, the blockage effect of derivant X were increasing. For the CNE2, the cell proportion in different dose of derivant X of S period is higher than the control group. Compared with control group, high dose of derivant X can significant increase the cell proportion of S (p<0.01). The cycle blockage of the matrine and DDP group take place at the stage of G1(P<0.01).After incubation of CNE1cells with derivant X of the Matrine for48h, Western blotting showed that the expression of Bax and p53protein were significantly increased by derivant X in dose dependent(P<0.05, P<0.01), at the same time, the expression of Caspase-3,-8and-9were increased in a dose dependent manner(P<0.05, P<0.01). Compared with the control group, the expression of Caspase-3,-8and-9were significantly increased in the middle and high dose of derivant X of the Matrine group (P<0.05, P<0.01); The expression of Bax, Caspase-3,-8,-9protein were significantly increased by matrine (P<0.05). In matrine group, the expression of p53was also increased but no significantly different. In DDP group, the expression of Bax, p53, Caspase-3,-8and-9were significantly increased compared with control group (P<0.05, P<0.01). The expression of Bcl-2were significantly decreased in matrine, derivant X and DDP group(P<0.05, P<0.01).For the CNE2cell, the expression of Bax and Caspase-9protein were significantly increased by derivant X by derivant X of the Matrine in middle and high dose for48h(P<0.05, P<0.01). The expression of Caspase-3were increased in a dose dependent manner by matrine(P<0.05, P<0.01). At the same time, Bcl-2was significantly decreased by derivant X (P<0.05, P<0.01); The expression proteins of DDP group were similar with matrine gtoup. In matrine group, the expression of Bax and Caspase-9were also increased but no significantly difference. Caspase-3was significantly decreased and Bcl-2was significantly decreased by matrine (P<0.05).Part oneStudying on the inhibiting anti-nasopharyngeal carcinoma effect of14-thienyl methylene matrine in vivoMethods:The CNE1and CNE2cells were grafted in the null mice, when the tumor growed to a certain volume, the null divided into martrine group, derivant X group, DDP group and control group at random. The weight of the null mice and the volume of tumor were measured every day. The tumor was taken from mice and the weight of the tumor was measured after treatment of15days. The tumor issue was investigated by HE staining. Ultra microstructure changes of cells were observed by transmission electron microscope. TUNEL stain was used to detect the apoptosis of CNE1and CNE2cells. The expression of Bcl-2, Bax, p53, Caspase-3and Caspase-9were detected by immunohistochemistry.Results:The volume and the weight of tumor were inhibited by14-thienyl methylene matrine. HE staining of tumor cell indicated that the number of cancer cell was reduced, the infiltration of a great amount of inflammatory cells were found, and the tumor cell was encapsulated by fiber tissue.Electron microscopy indicated the apoptosis of tumor cells with nuclei condensation and nuclei fragmentation, and apoptotic bodies were observed. The proportion of apoptotic cell of drug groups was higher than the control group. The expression of Bax、p53、Caspase-3and Caspase-9were increased and the expression of of the Bcl-2were decreased than control group.Conclusion:14-thienyl methylene matrine can inhibit the growth of CNE1and CNE2cells both in vitro and in vivo. The effective dose is lower than matrine’. The possible mechanism is related with inducing apoptosis of cell, arresting the cell cycle, and increasing of the apoptotic protein Bax、 p53、Caspase-3、-8and-9expression level, decreasing of the anti-apoptotic protein Bcl-2protein expression level. This may indicate that the caspase-dependent apoptotic pathway were involved. |
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