| BackgroundBreast cancer is one common tumor to life and health of women recently. China is more growing incidence of breast cancer,Chinese Anti-Cancer Association released statistics showed that:in recent years,the incidence of breast cancer speeded at an annual rate of3%,and became the faster growing cancer in cities.In addition, the age of patient is more and more younger.Despite we had made great improvement in treating breast cancer in recent years, there were still many patients developed metastasis or recurrence during or post-treaments. Therefore, we need to continue studing the pathogenesis of breast cancer, and looking for more and better approaches of treatment for breast cancer.Chemotherapy is an important way to treat breast cancer, but its side effects is not be ignored,such as bone marrow suppression, gastrointestinal reactions, decreased immune function, decreased resistance to infection, skin and mucous membrane damage, nervous system damage.when the patient can not tolerate, it will seriously affect the treatment effect. For radiotherapy, preoperative radiotherapy or postoperative radiotherapy can reduce local recurrence rates of tumor and improve overall survival rate. In the course of treatment of breast cancer, radiation therapy has become an important tool. But in the clinical application, a variety of factors may affect the efficacy of radiation therapy.including the tumor cells themselves radiosensitivity and tumor radiation resistance, and normal tissues limits the radiation dose, which may have a serious impact on the efficacy of radiation treatment.how to overcome the radioresistance of the tumor, change the radiation sensitivity of tumors has become the constant attentional and explorative subject of research and clinical treatment. Currently, using radiosensitizers to enhance the effect of radiotherapy has become a hot topic. The radio sensitizer means through the application of chemical or biological means to increase the sensitivity of tumor cells to radiation therapy. However, not to add a significant amount of normal tissue radiosensitivity, an ideal radiation sensitizer, should meet the following six requirements:(1) It can increase the sensitivity of tumor tissue to radiation but not increase the sensitivity of normal tissue cells to radiation;(2)non-toxic to normal tissues or its toxicity is low;(3) ideal lipid-water distribution system, can penetrate into the tumor cells and the hypoxic zone, distribution or less distribution to normal tissue where easily cause the toxicity;(4)The relatively stable chemical properties, appropriate biological half-life;(5) It can play the entire cell cycle;(6)in the conventional therapy, lower drug doses can have a radiosensitizing effect.Taxol is an anticancer drug, which is extracted from the bark of paclitaxel (Taxus), is a complex secondary metabolites in the genus Taxus, is currently understood only one that can promote microtubule polymerization andthe stability of the polymerized microtubule drugs. Isotope tracing showed that paclitaxel only bind to the polymerization of microtubules, not reaction to polymerization of tubulin dimer. When cells are exposured to paclitaxel, a large number of microtubules accumulate in the intracellular, microtubule accumulation interferes with the various functions of the cell, especially make cell division stop in mitosis, block the normal cell division. Taxol is mainly applied to ovarian and breast cancer, lung cancer, colorectal cancer, melanoma, head and neck cancer, lymphoma, brain tumors have a certain effect. The main adverse effects include allergic reactions, bone marrow suppression, neurotoxicity, cardiovascular toxicity, gastrointestinal reactions, liver toxicity, hair loss and intravenous infusion of drugs and drug extravasation of local inflammation. The clinical application has some limitations. 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.It can prevent its pro-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 effective anti-tumor activity. Apogossyplone (ApoG2), is a new derivative of gossypol, which has low toxicity and side effects,but has strong tumor-killing effect.Its cytotoxicity tests showed that the inhibition of tumor cell growth is more than the L-gossypol, and showing the better prospects. In our study, we choose the human breast cancer cell line MCF-7as the research object to explore whether or not ApoG2combined with radiotherapy or chemotherapy have synergistic effects, to explore the possible mechanism, which may provide some new research directions for the treatment of breast cancer.ObjectiveThis experiment is to make use of high expression of Bcl-2protein in breast cancer cell line MCF-7as the research object, to study radiosensitizing effect on brest cancer cell Line by ApoG2and synergistic effect by ApoG2combined with paclitaxel and to explore its possible mechanism.which may Provide experimental evidence and theoretical basis for further research of ApoG2in clinical application.Methods1.Using MTT proliferation studies research the growth inhibition of ApoG2irradiation and ApoG2combined with irradiation act on the breast cancer cell line MCF-7for72h in vitro,calculating the q value.2.Using MTT proliferation studies research the growth inhibition of ApoG2paclitaxel and ApoG2combined with paclitaxel on the breast cancer cell line MCF-7for48h in vitro,calculating the CDI(drug interaction index) value.3.Using colony forming assay to claculate cell survival fraction(SF), using "multi-target mathematical model" to fit the cell survival curve in order to observe tumor-killing effect of (2,4,6,8Gy) radiation group and (2.5,5μmol/L)ApoG2+(2,4,6,8Gy) group on MCF-7cell line. 4.Group1:Cells were divided into control group,5μmol/LApoG2group,8Gy irradiation group,5μmol/LApoG2+8Gy irradiation group, group2:Cells were divided into control group10μmol/LApoG2,0.25μmol/paclitaxel group,10μmol/LApoG2+0.25μmol/paclitaxel group. Using Hoechst33258fluorescent staining and AO fluorescence staining to observe apoptosis and autophagy morphological changes of cells treated in ways above for48h5.Flow cytometry (FCM) was used to measure the apoptosis and autophagy fluorescence intensity change in breast cancer MCF-7cells treated in ways above for48h6.Western blot analysis was used to detect Bcl-2protein and Beclinl protein in breast cancer MCF-7cells treated in ways above for48hResults1.MTT assay showed that ApoG2、irradiation can respectively inhibit the growth of MCF-7cells(FApoG2=197.338, P=0.000; F射线=647.817, P=0.000),when the cells were treated with ApoG2combined with irradiation for72h, with drug concentration and radiation dose increasing, the inhibition rate was enhanced significantly, Between ApoG2and irradiation had a interaction effect (F=15.200, P=0.000). and the q value>1.15, the two had a synergistic effect. MTT assay also showed that ApoG2、 paclitaxel can respectively inhibit the growth of MCF-7cells (FApoG2=156.916, P=0.000; Fpaclitaxel=129.904, P=0.000). when the cells were treated with ApoG2combined with paclitaxel for48h, with drug concentration of ApoG2and paclitaxel increasing, the inhibition rate was increased significantly, Between ApoG2and paclitaxel had a interaction effect (F=12.402, P=0.000). and the CDI value<1, the two had a synergistic effect.2.Cloning formation experiments showed that the cells treated with (2,4,6,8Gy) irradiation,(2.5,5μmol/L)ApoG2+(2,4,6,8Gy) irradiation for14days, with irradiation dose increasing, cell viability of irradiation group,2.5μmol/L+irradiation group, and5μmol/L+irradiation group gradually reduced, the difference was statistically significant (F单纯照射=264.308, P单纯照射=0.000; F2.5μmol/L+照射 =180.204, P2.5μmol/L+照射=0.000; F5μmol/L+照射=214.737, P5μmol/L+照射=0.000).Using the multi-target model to calculate Do, Dq, SER(Do)and SER (Dq)value,which were respectively1.925Gy (Do) and3.564Gy(Dq) in control group. In2.5μmol/L ApoG2+irradiation group, the value of Do, Dq, SER(Do)and SER (Dq) were respectively1.688Gy,2.123Gy,1.140, and1.679, in contrast with1.317Gy,1.675Gy,1.462,2.128in5μmol/L ApoG2+irradiation group. Compared with control group, the Do,Dq value of2.5μmol/L ApoG2+irradiation group and5μmol/L ApoG2+irradiation were lower, the amount of area "shoulder area" of experimental curve of the low dose group was narrowed significantly,and the survival fraction of each dose point were lower than control group. It suggested that sub-lethal radiation damage repair ability of cells was reduced.3.Group1:Cells were divided into control group,5μmol/LApoG2group,8Gy irradiation group,5μmol/LApoG2+8Gy irradiation group, group2:Cells were divided into control group10μmol/LApoG2,0.25μmol/L paclitaxel group, lOumol/LApoG2+0.25μmol/L paclitaxel group.when cells were treated in ways above for48h, Hoechst33258fluorescence staining showing a clear nuclear condensation and fragmentation, chromatin condensation, apoptotic body formation of apoptosis in experiment gpoup,and control cells are presented uniform dispersion, the normal light blue nucleus. The apoptotic phenomenon is more obvious in ApoG2combined with paclitaxel group compared with that in single treatment group. While in ApoG2combined with irradiation group, the apoptotic phenomenon did not increase significantly compared with that in single treatment group.AO (acridine orange) fluorescent staining showed dyed bright green fluorescence in cytoplasm or nucleus of the cells in the control group, in the experiment group,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.4. Flow cytometry showed that when cells were treated with5μmol/LApoG2,8Gy irradiation and5μmol/LApoG2,+8Gy irradiation for48h, Apoptosis rate in different treatment groups was statistically significant difference (F=35.074, P=0.000), autophagy fluorescence intensity in different treatment groups was statistically significant difference (F=470.809, P=0.000). Autophagy fluorescence intensity of combined group was more significant than that in single treatment groups (P<0.05), compared with separate treatment groups, apoptosis rate of combined group was not statistically significant. when cells were treated with10μmol/LApoG2,0.25μmol/L paclitaxel and10μmol/LApoG2+0.25μmol/Lpaclitaxel for48h Apoptosis rate in different treatment groups was statistically significant difference (F-571.563, P=0.000), autophagy fluorescence intensity in different treatment groups was statistically significant difference (F=204.422, P=0.000). apoptosis rate and autophagy fluorescence intensity of combined group were more significant than that in single treatment groups (P<0.05).5. Western blot showed that when cells were treated with5μmol/LApoG2,8Gy irradiation and5μmol/LApoG2,+8Gy irradiation for48h, Beclinl and Bcl-2protein expression in different treatment groups were statistically significant difference (FBeclin1=176.562, PBeclinl=0.000; FBcl-2=470.809,PBcl-2=0.000), Beclinl protein expression in combined group was more than that in single treatment groups (P <0.05), Bcl-2protein expression in combined group was less than that in single treatment groups (P<0.05). when cells were treated with10μmol/LApoG2,0.25μmol/L paclitaxel and10μmol/LApoG2+0.25μmol/Lpaclitaxel for48h, Beclinl and Bcl-2protein expression in different treatment groups were statistically significant difference (FBeclinl=57.266, PBeclinl=0.000; FBcl-2=138.495, PBcl-2=0.000).Beclinl protein expression in combined group was higher than that in single treatment groups (P<0.05), Bcl-2protein expression in combined group was lower than that in single treatment groups (P<0.05).Conclusion:ApoG2can increase radiosensitivity and chemosensitivity (paclitaxel) of breast cancer MCF7cell line.The mechanism may be related to induced apoptosis and autophagy, In radiotherapy sensitivity experiments, autophagy may play an important role in inhibiting cell proliferation. In chemotherapy sensitivity tests, apoptosis and autophagy may have a synergistic effect on tumor-killing. |
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