| Background:Nasopharyngeai carcinoma (NPC) is originated from the epithelial lining of the nasopharynx with remarkable ethnic and geographic distribution. Nasopharyngeai car cinoma is common in southern China and south-east Asia. The estimated age adjus ted incidence is20per100,000, whereas it is only one in100,000in other parts of the wo rld. Each year,80,000new cases of NPC are diagnosed worldwide, and50,000ind-ividuals die of this disease. According to the statistics of the World Health organizati-on, about80%patients of NPC are diagnosed in our country, and the rate of incidence rises gradually year by year rencently. Both its cause and pathogenesis are not clearily.Many studies show that NPC presents as a complex disease that is caused by an interaction among genetic,EBV chronic infection and environmental factors.At present,radical radiotherapy in combination with chemotherapy is the treatment of early or local nasopharyngeai carcinoma (NPC) standard solution.but the relapse rate still as high as50%after radiotherapy combined with chemotherapy,5-year survival rate only is34%—53%, the prognosis for metastatic NPC even poorer. Resistance to adiotherapy and chemotherapy has been an important reason for the failure of comprehensive treatment of nasopharyngeal carcinoma (NPC). Numerous studies show that many chemotherapy drugs play a role through inducing apoptosis, so the apoptosis of block have close relationship with tumor resistance to chemotherapy.There are many overexpressed oncogenes in NPC, such as antiapoptotic Bcl-2family members, c-Myc and LMP1, may cause the lack of long term efficacy of conventional therapies. The Bcl-2is considered to be a kind of generalized antiapoptotic genes, encoding a type of carcinoma protein with relative molecular weight of25×1000u, the Bcl-2protein is an important antiapoptotic protein in the Bcl-2family of cell apoptosis related proteins. It can inhibit the program of tumor cell apoptosis or autophagy, resulting in the occurrence and development of tumor, and it is considered to be associated with the resistance and relapse of cancer.Lu detected that the Bcl-2were highly expressed in80%nasopharyngeal carcinoma specimens by FISH and immunohistochemical method. In addition, most of the studies show that its high expression can block the apoptosis of tumor cells, and may closely related to the occurrence, development and metastasis of nasopharyngeal carcinoma.Reed was first reported that they use antisense Bcl-2gene therapy inhibit the growth of leukemia cells in vitro in1990. In some vivo studies at home and aborad, show that antisense Bcl-2gene therapy can enhance the sensitivity to chemotherapy in leukemia patients. So the Bcl-2May become a target for targeted treatment of nasoph aryngeal carcinoma (NPC). Because of the lack of long term efficacy of conventional therapies, antisense Bcl-2gene therapy may open up new prospects for treatment of nasopharyngeal carcinoma, but there still are many difficultise for gene therapy.With the development of relationship between Bcl-2family and tumor, the anticancer drugs have been developed depended on the mechanism of action.The biological treatment and advantages of peptides, small organic molecules and other drugs have been used for some appropriate anti-apoptotic protein based on Bcl-2. The small organicmolecules derivatives of Bcl-2inhibitor are the important direction of development.Gossypol is a yellow polyphenolic compound high contented in the Malvaceae Gossypium cotton plant seeds, leaves, stems and roots and other organs. It is a small molecule inhibitor of Bcl-2. Studies showed that L-gossypol is the active ingredient of gossypol, and play a major role in the Bcl-2/Bcl-XL gene,and prevent its ro-apoptotic genes such as Bak, Bax, Bad, etc. to form heterodimers,induce apoptosis of tumor cells. The vivo and vitro experiments have demonstrated that gossypol has the effective anti-tumor activity. However,it was onsidered that the aldehyde in the ends of the molecular structure of L-gossypol are related with toxicity, although the effect is weak, but limits the body’s maximum tolerance dose. In addition, this mixture into the body is easily combined with other proteins, which weaked the effectiveness of drug treatment,increased the toxicity. Apogossyplone (ApoG2), removed the two aldehyde groups, is a new derivative of gossypol. Its cytotoxicity tests showed that the inhibition of tumor cell growth is more than the L-gossypol, and showing the better prospects.Paclitaxel is a typical taxanes chemotherapy drugs extracted from the Pacific yew tree bark. Paclitaxel induces cell apoptosis by inhibiting the formation of the mitotic spindlethe in cells and then blocking cell in G2/M phase. Paclitaxel combine with cisplatin and5-fluorouracil have a better effect for the treatment of head and neck cancer, this strategy is also used for nasopharyngeal carcinoma. Chemotherapy is more and more important for nasopharyngeal carcinoma in recent years, especially for locally advanced nasopharyngeal carcinoma, compared with radiation, chemo-radiotherapy can significantly gain curative effect in the near future and long-term survival, chemotherapy plays an increasing radiation sensitivity and control distant small metastases. But chemotherapy has bigger toxicity, and prone to resistance and other shortcomings.Autophagy and apoptosis are two completely different ways of programmed cell death, are the important mechanism of the self balance of cell, they play an important role in biological tissue differentiation, growth, development, self-renewal, disease, and tumor formation. Chemotherapy drugs kill cancer cells mainly through induction of cell apoptosis, and autophagy has long been regarded as a mechanism for cell survival. Autophagy gradually become a research hotspot in recent years. Research shows that autophagy is another important way of tumor cell death for certain chemo-therapeutic drugs. It finds that Beclin1and LC3are two important autophagy genes. Beclinl was the first one associated with autophagy, Beclinl gene as a tumor supp-ressor gene reach a consensus. There is alleles deficiency in the human chromosome17q21loci in40%～75%spontaneous breast cancer, ovarian cancer and nasopharyn geal carcinoma.To improve NPC survival rates, In our study, we choose the human Nasopharyngeai carcinoma cell line CNE-2as the research object, discuss its possible mechanism for treatment of ApoG2in CNE-2cells, and invegestgate whether ApoG2can enhance the sensitivity of paclitaxel to CNE-2cells chemotherapy in vitro and in vivo, provide some new research directions of Nasopharyngeai carcinoma.Objective:To invegestgate the inhibitory effect and induced apotosis and autophagy effects of apogossypolone(ApoG2) combined with Paclitaxel on Human NasopharyngealCareinoma cells in Vitro and in Vivo.Methods:1.The inhibitory effect of different concentrations of ApoG2,paclitaxel alone or combination of ApoG2and paclitaxel on CNE-2cells were measured by MTT assay. and the CDI value was calculated.2.CNE-2cells of the control group, the ApoG2group, the Paclitaxel group and the combination group were stained by acridine orange and Hoechst33258to respectively observe the morphology of apoptotic cells and acidic vesicular organelles (AVOs).3.The apotosis rate and fluorescence intensity of autophagy were determined by flow cytometry.4. Western blot analysis was used to detect the expression of apotosis related proteins Bcl-2in CNE-2cells treated with ApoG2.5.The effect of ApoG2combined with Paclitaxel on human nasopharyngealcarcinoma were studied be establishing the xenografts of human nasopharyngealcarcinoma in nude mice.6.Using SP staining of immunohistochemistry detected the expression of apotosis related proteins Bcl-2and autophagy related proteins Beclinl.Results:1.MTT assay showed that CNE-2cells were sensitive to paclitaxel and ApoG2in a dose-dependent manner. MTT assay also showed ApoG2combined with paclitaxel had synergistic effect.(CDI<1).2.Hoechst33258staining showed more appearance of apoptosis.nuclear condensation and fragmentation, chromatin condensation, and apoptotic body formation in the combination group compared with other groups. AO fluorescent staining showed that the cytoplasm or nucleus can be seen bright red fluorescent,which meaned acidic autophagic vacuoles. The autophagy is more obvious in combined group compared with that in single treatment group.3.FCM assay indicated increased apotosis rate in combination group, which was statistically significant difference(P<0.01).Autophagy fluorescence intensity of combined group was more significant than that in single treatment groups (P<0.01).4.Western blot showed that the ApoG2group could obviously inhibit the expression of Bcl-2protein compared to the control group.(p<0.01).5.The tumor volume and the rate of tum-or suppression of the combined group reached85%in vivo experiment, which was significant compared to the separate group(P<0.05).6.1mmunohistochemistry showed that the combined group could obviously inhibit the expression of Bcl-2protein and induced the expression of Beclinl protein(p<0.01).Conclusions:ApoG2effectively enhances the sensitivity of paclitaxel to CNE-2cells chemotherapy in vitro and in vivo, and its mechanism was related with induced apotosis and autophagy. |
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