C6神经酰胺联合多西他赛抗乳腺癌作用的机制研究
发布时间：2018-07-25 10:19
【摘要】：研究背景转移性乳腺癌约占乳腺癌的30-40%,恶性程度高,5年生存率低,是人类难治的恶性肿瘤之一。它病程进展迅速但常无明显的临床表现,难以早期发现因而错过根治的机会。这些患者在进展期往往只有少数患者可以接受手术治疗,而化疗是其主要的治疗手段。以紫杉类为主的治疗是转移性乳腺癌的一线治疗方案,特别是在对蒽环类耐药的情况下。临床试验证实单药多西他赛与丝裂霉素联合长春新碱相比可以明显提高总生存率,提高缓解率,延长疾病进展期。另外,尽管紫杉醇和多西他赛两种紫杉类药物均用与转移性乳腺癌的治疗,在总生存率及疾病进展时间上多西他赛获益更多。Albain等人发现吉西他滨与紫杉醇联合用药疗效优于紫杉醇单药治疗。在我们的研究中,通过体外培养乳腺癌细胞来研究短链神经酰胺与多西他赛联合作用对乳腺癌的治疗疗效。神经酰胺,主要存在与细胞膜结构中,是脂类家族的结构蛋白。作为信使分子,神经酰胺也能引起凋亡。许多研究表明:神经酰胺与肿瘤细胞的凋亡有关,增加内源性神经酰胺的产生可以诱发凋亡。可溶性的短链神经酰胺在许多肿瘤细胞株中表现出抗肿瘤作用,例如黑色素瘤,软组织肉瘤,Jurkat白血病,头颈部鳞状细胞癌。我们课题研究要点是神经酰胺是如何增加化疗制剂的疗效。我们的前期实验发现C6神经酰胺联合多西他赛可以增加多西他赛的抗乳腺癌疗效,并且发现C6神经酰胺联合多西他赛引起乳腺癌细胞大量凋亡,并与AMPK通路的活化有关。它涉及的分子机制有待进一步深入的研究。研究方法和材料人乳腺癌细胞MCF-7,MDA-231,均从上海生命科学研究所(上海,中国)购买,乳腺癌原代细胞取自外科手术中乳腺癌患者的肿瘤组织,各细胞在RPMI/DMEM培养基中(Simga公司,圣路易斯,密苏里州),加入10%的FBS(Sigma公司,圣路易斯,密苏里州),青霉素/链霉素(1:100,Sigma),培养37℃CO2培养箱中。细胞给予C6神经酰胺和多西他赛处理,显微镜下观察细胞形态,运用MTT方法检测细胞存活率,同时对细胞进行台盼蓝染色,计算活细胞率;运用Annexin V试剂盒检测细胞凋亡,通过Western blot检测总的和磷酸化的AMPKα、ACC、信号水平的改变;运用程序性死亡抑制剂及凋亡抑制剂进一步验证C6神经酰胺和多西他赛诱导细胞死亡方式。建立AMPKα1-sh RNA及AMPK-α1显性失活(DN)突变(DN-AMPK-α1)c DNA稳转的肿瘤细胞,运用上述方法进一步验证信号通路的改变。为了了解线粒体通透性转换孔(m PTP)在协同用药对乳腺癌细胞的作用,通过JC-10染料测定细胞线粒体膜电位(MMP),FACS检测ROS生成,通过Western blot检测细胞色素C,C-caspase3,JNK,HER-1/2,Akt/Erk信号通路等,了解线粒体通路的改变,并通过MPTP抑制剂及ROS清除剂等方法来验证线粒体通路作用及相关信号通路改变。建立CYP-D-sh RNA稳转肿瘤,Cyp-D过表达乳腺癌细胞进一步验证C6神经酰胺和多西他赛诱导细胞死亡的信号通路改变。统计学处理在每个实验中,至少使用三个孔/平皿。每个实验重复至少三次,每次获得具有相似的结果。数据以均数±标准偏差(SD)。使用SPSS15.0软件进行分析统计,通过one-way ANOVA,Scheffe和Tukey检验。P0.05被认为有统计学意义。试剂的浓度和治疗持续时间基于发表文献和预实验的结果。研究结果1 C6神经酰胺显著增加多西他赛对乳腺癌细胞株的毒性作用无论是多西他赛(DTX,1.0μg/ml)或C6神经酰胺(C6,10μg/m L)单药都可以引起乳腺癌细胞死亡,当两者联用72h后,90%的乳腺癌细胞死亡,显著地抑制乳腺癌细胞生长并致死,在原代乳腺癌细胞也发生同样的现象。2 C6神经酰胺显著增加多西他赛引起的乳腺癌细胞凋亡单药多西他赛或单药神经酰胺均可引起乳腺癌细胞MCF-7,及MDA-231的凋亡,当两者联合使用时,乳腺癌细胞的凋亡显著增加。并用western-blot检测凋亡相关蛋白,在两药联合使用的MCF-7和MDA-231细胞中C-caspase-9表达上调的同时PARP下降。而且在使用一般凋亡抑制剂z VADfmk后,C6联合多西他赛引起的乳腺癌细胞MCF-7,MDA-231凋亡均被抑制。在原代乳腺癌细胞中,z VADfmk同样可以抑制C6联合多西他赛引起的细胞死亡。因此C6神经酰胺增加多西他赛引起的乳腺癌细胞死亡实质是通过引起凋亡。3 C6神经酰胺显著增加多西他赛对乳腺癌细胞的生长抑制作用细胞集落实验显示C6神经酰胺与多西他赛单药均可轻度抑制乳腺癌细胞MCF-7/MDA-231的增长,当两药联合使用后乳腺癌细胞集落明显下降。并且Cyclin D1的表达也明显下降。因此C6神经酰胺增加多西他赛对乳腺癌细胞的抑制,并引起的乳腺癌细胞细胞周期在G2M的停滞。4 C6神经酰胺与多西他赛联合用药通过活化AMPK抑制m TORC1的活性通过检测磷酸化的AMPKα1,及其下游磷酸化的ACC证实:在乳腺癌细胞C6神经酰胺与多西他赛协同活化AMPK,磷酸化AMPKα(Thr172)和ACC增加而总的AMPK和ACC是没有变化的。用sh RNA沉默AMPKα的表达,可以抑制C6联合多西他赛引起的对MDA-231乳腺癌细胞的毒性作用,在AMPK-负性突变的细胞株(DN,T172A)C6联合多西他赛的细胞毒作用也被抑制。这些结果表明,C6联合多西他赛对乳腺癌细胞的协同致死作用与AMPK活化有关。然后,我们检测了MDA-231细胞中m TORC1的活性。结果表明:MDA-231乳腺癌细胞在C6联合多西他赛的作用下,代表m TORC1功能的p S6活性被大大地抑制了。在用sh RNA沉默AMPKα表达的MDA-231乳腺癌细胞中,C6神经酰胺联合多西他赛对m TOR信号通路的抑制作用被阻断,p S6的生成增加,因此C6神经酰胺联合多西他赛对乳腺癌细胞m TORC1信号通路的阻断是通过AMPK信号通路的活化。5 C6神经酰胺联合多西他赛对乳腺癌细胞的毒性作用与JNK通路的活化有关C6神经酰胺联合多西他赛可以引起MDA-231乳腺癌细胞的JNK通路持续显著的活化,比单药使用C6神经酰胺或多西他赛作用均强。用JNK抑制剂SP600125和JNKi V预处理MDA231乳腺癌细胞后,C6神经酰胺与多西他赛联合用药引起的MDA231乳腺癌细胞的死亡及凋亡均被抑制。因此C6神经酰胺联合多西他赛对乳腺癌细胞的毒性作用与JNK通路的活化有关。6 C6神经酰胺与多西他赛联合用药引起的JNK/AMPK活化及细胞毒作用与线粒体通道(m PTP)开放,ROS生成有关我们检测了C6神经酰胺与多西他赛联合作用时乳腺癌细胞的线粒体膜电位的变化,以明确C6与多西他赛联合对癌细胞的毒性作用是否与线粒体通透性转换孔有关。结果表明:C6神经酰胺与多西他赛联合用药使乳腺癌细胞线粒体膜电位下降,并伴有ROS生成,细胞色素C增加及C-caspase-3增加。用m PTP抑制剂Sf A及ROS清除剂NAC预处理MDA-231后,C6神经酰胺联合多西他赛引起乳腺癌细胞的死亡被大大抑制,并且细胞色素C及C-caspase-3的生成均被抑制。根据这些结果我们认为C6神经酰胺与多西他赛联合引起的乳腺癌细胞的毒性作用与m PTP开放及ROS生成有关。为了进一步证实这一假说,我们用sh RNA沉默或抑制剂Cs A抑制m PTP主要结构蛋白Cyp-D的功能。结果表明:Cyp-D被抑制的MDA-231乳腺癌细胞,C6神经酰胺联合多西他赛引起的细胞毒作用明显减低。并且在Cyp-D过表达的乳腺癌细胞MDA-231对C6+多西他赛更加敏感。由于ROS可以激活JNK/AMPK的活性,我们检测了C6神经酰胺与多西他赛联合用药引起的JNK/AMPK活化是否与ROS有关。结果表明:ROS清除剂NAC抑制了联合用药引起的JNK/AMPK活化,并且过氧化氢(活性氧的一种)可以活化MDA-231乳腺癌细胞的AMPK和JNK通路。特别的是,用m PTP抑制剂Sf A预处理MDA-231后,联合用药引起的JNK/AMPK的活化也被抑制了。但是,单用Sf A或NAC作用,则不会抑制MDA-231的JNK/AMPK活性。因此:我们认为C6神经酰胺与多西他赛联合用药引起的乳腺癌细胞JNK/AMPK的活化与m PTP开放及ROS的生成有关。7 C6神经酰胺与多西他赛联合用药引起乳腺癌细胞HER-1/2的下调及Akt/Er K通路的抑制在MDA-231乳腺癌细胞中,我们发现C6神经酰胺联合多西他赛可以协同下调HER-1/2的表达,并抑制Akt和Erk1/2的表达。值得注意的是,HER-1/2的下调及p-Akt和p-Erk表达下降均开始于联合用药6-12h时,提示乳腺癌细胞Akt/Erk通路的抑制可能与HER-1/2的下调有关。AG1478,HER的抑制剂,在MDA-231及MCF-7乳腺癌细胞均能抑制Akt和Erk的磷酸化,同样也抑制乳腺癌细胞的细胞活性。因此:C6神经酰胺与多西他赛联合用药引起乳腺癌细胞HER-1/2的下调及Akt/Er K通路的抑制。结论1.C6神经酰胺显著增加多西他赛对乳腺癌细胞株的毒性作用2.C6神经酰胺显著增加多西他赛引起的乳腺癌细胞凋亡3.C6神经酰胺显著增加多西他赛对乳腺癌细胞的生长抑制作用4.C6神经酰胺增加多西他赛的抗乳腺癌作用通过活化AMPK并抑制m TORC1的活性5.C6神经酰胺联合多西他赛对乳腺癌细胞的毒性作用与JNK通路的活化有关6.C6神经酰胺与多西他赛联合用药引起的JNK/AMPK活化及细胞毒作用与线粒体通道(m PTP)开放,ROS生成有关7.C6神经酰胺与多西他赛联合用药引起乳腺癌细胞HER-1/2的下调及Akt/Er K通路的抑制
[Abstract]:Background metastatic breast cancer accounts for about 30-40% of breast cancer, with high malignancy and low 5 year survival rate. It is one of the most difficult human malignant tumors. Chemotherapy is the main treatment. Treatment based on paclitaxel is a first-line treatment for metastatic breast cancer, especially in the case of anthracycline resistance. Clinical trials have proved that the single drug docetaxel can significantly improve the total survival rate, improve the remission rate, and prolong the progression of the disease. Although two paclitaxel and docetaxel drugs are used for the treatment of metastatic breast cancer, more.Albain and others have benefited from the total survival rate and the time of disease progression. The combination of gemcitabine and taxol has been found to be more effective than paclitaxel. In our study, breast cancer cells were cultured in vitro. Study the therapeutic effect of short chain ceramide and docetaxel in the treatment of breast cancer. Ceramide, mainly in the membrane structure, is the structural protein of the lipid family. As a messenger, ceramide can also cause apoptosis. Many studies have shown that ceramide is associated with the apoptosis of tumor cells and increases endogenous ceramide. Production can induce apoptosis. Soluble short chain ceramide shows antitumor effects in many tumor cell lines, such as melanoma, soft tissue sarcoma, Jurkat leukemia, and head and neck squamous cell carcinoma. Our main point is how ceramide increases the efficacy of chemotherapeutic agents. Our early experiments found C6 neuroacyl. Amines combined with docetaxel can increase the efficacy of docetaxel against breast cancer, and it is found that C6 ceramide combined with docetaxel causes large number of apoptosis in breast cancer cells and is related to the activation of the AMPK pathway. Its molecular mechanisms need to be further studied. Methods and materials for human breast cancer cells, MCF-7, MDA-231, are all from Shanghai. The Life Science Institute (Shanghai, China) purchased the primary mammary cancer cells from the tumor tissue of the breast cancer patients in surgery, each cell in the RPMI/DMEM medium (Simga, Saint Louis, Missouri), and 10% of FBS (Sigma, Saint Louis, Missouri), penicillin / streptomycin (1:100, Sigma), culture, and culture of CO2 culture. In the box, the cells were treated with C6 ceramide and docetaxel, the cell morphology was observed under the microscope, the cell survival rate was detected by the MTT method, and the cells were stained with trypan blue to calculate the living cell rate; the apoptosis was detected by Annexin V kit, and the changes of the total and phosphorylated AMPK a, ACC, and signal levels were detected by Western blot. Use programmed death inhibitors and apoptosis inhibitors to further validate C6 ceramide and docetaxel induced cell death. To establish AMPK alpha 1-sh RNA and AMPK- alpha 1 dominant inactivation (DN) mutation (DN) mutation (DN-AMPK- alpha 1) C DNA stable transformation of the tumor cells, using the above methods to further verify the signal transduction pathway changes. The effect of M PTP on the breast cancer cells in synergistic drug use. The cell mitochondrial membrane potential (MMP) was measured by JC-10 dye, and FACS was used to detect the formation of ROS. The changes of cytochrome C, C-caspase3, JNK, HER-1/2, and signal pathway were detected by Western blot. The role of mitochondrial pathway and related signaling pathway change. Establish CYP-D-sh RNA stable tumor, Cyp-D overexpression of breast cancer cells further verify the signal pathway change of C6 ceramide and docetaxel induced cell death. Statistical treatment in each experiment, at least three holes / Petri dishes. Each experiment repeats at least three times, each time obtained. Similar results. Data with mean number + standard deviation (SD). Using SPSS15.0 software for analysis and statistics,.P0.05 was considered statistically significant through one-way ANOVA, Scheffe and Tukey test. The concentration of reagents and duration of treatment were based on published literature and pre experiment results. Results 1 C6 ceramide significantly increased docetaxel. The toxic effects on breast cancer cell lines, either DTX, 1 mu g/ml or C6 ceramide (C6,10 mu g/m L), can cause death of breast cancer cells. When both of them are combined with 72h, 90% of the breast cancer cells die, significantly inhibiting the growth and death of breast cancer cells, and the same phenomenon in the primary breast cancer cells,.2 C6 God, is also occurring in the primary breast cancer cells. The apoptosis of breast cancer cells, docetaxel or single drug ceramide, can cause MCF-7 and MDA-231 apoptosis in breast cancer cells. The apoptosis of breast cancer cells was significantly increased when both were used together. The apoptosis related proteins were detected by Western-blot, and MCF-7 and MDA-231 in combination of two drugs were used. In the cells, the expression of C-caspase-9 is up and PARP decreases. And after the use of the general apoptosis inhibitor Z VADfmk, C6 combined with docetaxel induced MCF-7, MDA-231 apoptosis is suppressed. In primary breast cancer cells, Z VADfmk also inhibits the cell death caused by C6 combined with docetaxel. Therefore C6 ceramide increases. The death of breast cancer cells caused by docetaxel is essentially by inducing apoptotic.3 C6 ceramide to significantly increase the inhibitory effect of docetaxel on the growth of breast cancer cells, which shows that C6 ceramide and docetaxel can slightly inhibit the growth of MCF-7/MDA-231 in breast cancer cells, when two drugs are combined with breast cancer. The cell colonies decreased significantly and the expression of Cyclin D1 decreased significantly. Therefore, C6 ceramide increased the inhibition of docetaxel to breast cancer cells, and the cell cycle of breast cancer cells at the stagnation of G2M,.4 C6 ceramide combined with docetaxel by activating AMPK to inhibit m TORC1 activity by detecting phosphorylated AMPK alpha 1, ACC and its downstream phosphorylation showed that the total AMPK and ACC were not changed in CO activation of AMPK, phosphorylated AMPK alpha (Thr172) and ACC, and the expression of SH RNA silenced AMPK alpha could inhibit the toxic effect of C6 combined docetaxel on breast cancer cells in breast cancer cells, and the expression of SH RNA could inhibit the toxicity of C6 combined with docetaxel on breast cancer cells. The cytotoxic effects of DN (T172A) C6 combined with docetaxel were also suppressed. These results showed that the synergistic lethal effect of C6 combined with docetaxel on breast cancer cells was associated with AMPK activation. Then, we detected the activity of M TORC1 in MDA-231 cells. The results showed that the effect of MDA-231 breast cancer cells in C6 combined with docetaxel. The activity of P S6 representing the function of M TORC1 was greatly suppressed. In MDA-231 mammary cancer cells expressed in AMPK alpha with SH RNA, C6 ceramide combined with docetaxel's inhibition of M TOR signaling pathway, and the generation of P glands increased, thus blocking ceramide combined with docetaxel to block the signaling pathway of breast cancer cells. The activation of.5 C6 neuroamide through the AMPK signaling pathway combined with docetaxel's toxicity to breast cancer cells and activation of the JNK pathway associated with C6 neuroamide combined with docetaxel can cause a sustained and significant activation of the JNK pathway of MDA-231 breast cancer cells, stronger than the use of C6 neuroamide or docetaxel by single drug. After the pretreatment of MDA231 breast cancer cells with SP600125 and JNKi V, the death and apoptosis of MDA231 breast cancer cells caused by the combination of C6 ceramide and docetaxel are suppressed. Therefore, the toxicity of C6 ceramide combined with docetaxel on the breast cancer cells and the activation of JNK pathway is associated with the combination of.6 C6 ceramide and docetaxel. The activation and cytotoxicity of JNK/AMPK and cytotoxicity are related to the opening of mitochondrial channel (m PTP) and ROS generation. We detected the changes in the mitochondrial membrane potential of breast cancer cells when C6 ceramide combined with docetaxel to determine whether the toxic effects of C6 and docetaxel on the cancer cells were related to the mitochondrial permeability transition pore. The results showed that the combination of C6 ceramide and docetaxel reduced the mitochondrial membrane potential of breast cancer cells, accompanied by ROS formation, increased cytochrome C and increased C-caspase-3. After M PTP inhibitor Sf A and ROS scavenger NAC preprocessing MDA-231, the death of mammary cancer cells induced by C6 ceramide combined with docetaxel was greatly suppressed and fine. The formation of cytochrome C and C-caspase-3 were suppressed. According to these results, we believe that the toxicity of C6 ceramide and docetaxel combined with the opening of M PTP and the formation of ROS. In order to further confirm this hypothesis, we use sh RNA silencing or inhibitor Cs A to inhibit the function of M PTP. The results showed that the cytotoxic effect of C6 ceramide combined with docetaxel was significantly reduced in Cyp-D suppressed MDA-231 breast cancer cells. And the Cyp-D overexpressed breast cancer cell MDA-231 was more sensitive to C6+ docetaxel. As ROS activates the activity of JNK/AMPK, we detected a combination of C6 ceramide and docetaxel. Whether the activation of JNK/AMPK is associated with ROS. The results show that the ROS scavenger NAC inhibits the activation of JNK/AMPK caused by the combination of drugs, and hydrogen peroxide (one of the reactive oxygen species) can activate the AMPK and JNK pathway of MDA-231 breast cancer cells. It is inhibited. However, the action of Sf A or NAC alone does not inhibit the JNK/AMPK activity of MDA-231. Therefore, we think that the activation of JNK/AMPK in breast cancer cells caused by the combination of C6 ceramide and docetaxel is associated with m PTP opening and the formation of ROS, and that.7 C6 ceramide and docetaxel are associated with breast cancer cells. The inhibition of Akt/Er K pathway in MDA-231 breast cancer cells, we found that C6 ceramide combined with docetaxel can down regulate the expression of HER-1/2 and inhibit the expression of Akt and Erk1/2. It is worth noting that the downregulation of HER-1/2 and the decline of p-Akt and p-Erk expressions begin at the 6-12h of the combined drug use, suggesting the Akt/Erk pathway of breast cancer cells. Inhibition may be associated with the downregulation of HER-1/2,.AG1478, inhibitors of HER, both in MDA-231 and MCF-7 breast cancer cells can inhibit the phosphorylation of Akt and Erk, and also inhibit the cell activity of breast cancer cells. Therefore, the combination of C6 ceramide and docetaxel induces the downregulation of HER-1/2 in breast cancer cells and the inhibition of Akt/Er K pathway. Conclusion 1.C6 Effect of ceramide on the toxic effect of docetaxel on breast cancer cell strain 2.C6 ceramide significantly increased the apoptosis of breast cancer cells induced by docetaxel, 3.C6 ceramide significantly increased the inhibitory effect of docetaxel on the growth of breast cancer cells, 4.C6 ceramide increased the anti breast cancer effect of docetaxel by activating AMPK and inhibiting the action of breast cancer. M TORC1 active 5.C6 ceramide combined with docetaxel's toxicity to breast cancer cells and JNK pathway activation related to JNK/AMPK activation and cytotoxicity of 6.C6 ceramide and docetaxel combined with mitochondrial pathway (m PTP) opening, ROS formation associated with 7.C6 ceramide and docetaxel combined use of the mammary gland Downregulation of HER-1/2 and inhibition of Akt/Er K pathway in cancer cells
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9
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本文编号：2143512
[Abstract]:Background metastatic breast cancer accounts for about 30-40% of breast cancer, with high malignancy and low 5 year survival rate. It is one of the most difficult human malignant tumors. Chemotherapy is the main treatment. Treatment based on paclitaxel is a first-line treatment for metastatic breast cancer, especially in the case of anthracycline resistance. Clinical trials have proved that the single drug docetaxel can significantly improve the total survival rate, improve the remission rate, and prolong the progression of the disease. Although two paclitaxel and docetaxel drugs are used for the treatment of metastatic breast cancer, more.Albain and others have benefited from the total survival rate and the time of disease progression. The combination of gemcitabine and taxol has been found to be more effective than paclitaxel. In our study, breast cancer cells were cultured in vitro. Study the therapeutic effect of short chain ceramide and docetaxel in the treatment of breast cancer. Ceramide, mainly in the membrane structure, is the structural protein of the lipid family. As a messenger, ceramide can also cause apoptosis. Many studies have shown that ceramide is associated with the apoptosis of tumor cells and increases endogenous ceramide. Production can induce apoptosis. Soluble short chain ceramide shows antitumor effects in many tumor cell lines, such as melanoma, soft tissue sarcoma, Jurkat leukemia, and head and neck squamous cell carcinoma. Our main point is how ceramide increases the efficacy of chemotherapeutic agents. Our early experiments found C6 neuroacyl. Amines combined with docetaxel can increase the efficacy of docetaxel against breast cancer, and it is found that C6 ceramide combined with docetaxel causes large number of apoptosis in breast cancer cells and is related to the activation of the AMPK pathway. Its molecular mechanisms need to be further studied. Methods and materials for human breast cancer cells, MCF-7, MDA-231, are all from Shanghai. The Life Science Institute (Shanghai, China) purchased the primary mammary cancer cells from the tumor tissue of the breast cancer patients in surgery, each cell in the RPMI/DMEM medium (Simga, Saint Louis, Missouri), and 10% of FBS (Sigma, Saint Louis, Missouri), penicillin / streptomycin (1:100, Sigma), culture, and culture of CO2 culture. In the box, the cells were treated with C6 ceramide and docetaxel, the cell morphology was observed under the microscope, the cell survival rate was detected by the MTT method, and the cells were stained with trypan blue to calculate the living cell rate; the apoptosis was detected by Annexin V kit, and the changes of the total and phosphorylated AMPK a, ACC, and signal levels were detected by Western blot. Use programmed death inhibitors and apoptosis inhibitors to further validate C6 ceramide and docetaxel induced cell death. To establish AMPK alpha 1-sh RNA and AMPK- alpha 1 dominant inactivation (DN) mutation (DN) mutation (DN-AMPK- alpha 1) C DNA stable transformation of the tumor cells, using the above methods to further verify the signal transduction pathway changes. The effect of M PTP on the breast cancer cells in synergistic drug use. The cell mitochondrial membrane potential (MMP) was measured by JC-10 dye, and FACS was used to detect the formation of ROS. The changes of cytochrome C, C-caspase3, JNK, HER-1/2, and signal pathway were detected by Western blot. The role of mitochondrial pathway and related signaling pathway change. Establish CYP-D-sh RNA stable tumor, Cyp-D overexpression of breast cancer cells further verify the signal pathway change of C6 ceramide and docetaxel induced cell death. Statistical treatment in each experiment, at least three holes / Petri dishes. Each experiment repeats at least three times, each time obtained. Similar results. Data with mean number + standard deviation (SD). Using SPSS15.0 software for analysis and statistics,.P0.05 was considered statistically significant through one-way ANOVA, Scheffe and Tukey test. The concentration of reagents and duration of treatment were based on published literature and pre experiment results. Results 1 C6 ceramide significantly increased docetaxel. The toxic effects on breast cancer cell lines, either DTX, 1 mu g/ml or C6 ceramide (C6,10 mu g/m L), can cause death of breast cancer cells. When both of them are combined with 72h, 90% of the breast cancer cells die, significantly inhibiting the growth and death of breast cancer cells, and the same phenomenon in the primary breast cancer cells,.2 C6 God, is also occurring in the primary breast cancer cells. The apoptosis of breast cancer cells, docetaxel or single drug ceramide, can cause MCF-7 and MDA-231 apoptosis in breast cancer cells. The apoptosis of breast cancer cells was significantly increased when both were used together. The apoptosis related proteins were detected by Western-blot, and MCF-7 and MDA-231 in combination of two drugs were used. In the cells, the expression of C-caspase-9 is up and PARP decreases. And after the use of the general apoptosis inhibitor Z VADfmk, C6 combined with docetaxel induced MCF-7, MDA-231 apoptosis is suppressed. In primary breast cancer cells, Z VADfmk also inhibits the cell death caused by C6 combined with docetaxel. Therefore C6 ceramide increases. The death of breast cancer cells caused by docetaxel is essentially by inducing apoptotic.3 C6 ceramide to significantly increase the inhibitory effect of docetaxel on the growth of breast cancer cells, which shows that C6 ceramide and docetaxel can slightly inhibit the growth of MCF-7/MDA-231 in breast cancer cells, when two drugs are combined with breast cancer. The cell colonies decreased significantly and the expression of Cyclin D1 decreased significantly. Therefore, C6 ceramide increased the inhibition of docetaxel to breast cancer cells, and the cell cycle of breast cancer cells at the stagnation of G2M,.4 C6 ceramide combined with docetaxel by activating AMPK to inhibit m TORC1 activity by detecting phosphorylated AMPK alpha 1, ACC and its downstream phosphorylation showed that the total AMPK and ACC were not changed in CO activation of AMPK, phosphorylated AMPK alpha (Thr172) and ACC, and the expression of SH RNA silenced AMPK alpha could inhibit the toxic effect of C6 combined docetaxel on breast cancer cells in breast cancer cells, and the expression of SH RNA could inhibit the toxicity of C6 combined with docetaxel on breast cancer cells. The cytotoxic effects of DN (T172A) C6 combined with docetaxel were also suppressed. These results showed that the synergistic lethal effect of C6 combined with docetaxel on breast cancer cells was associated with AMPK activation. Then, we detected the activity of M TORC1 in MDA-231 cells. The results showed that the effect of MDA-231 breast cancer cells in C6 combined with docetaxel. The activity of P S6 representing the function of M TORC1 was greatly suppressed. In MDA-231 mammary cancer cells expressed in AMPK alpha with SH RNA, C6 ceramide combined with docetaxel's inhibition of M TOR signaling pathway, and the generation of P glands increased, thus blocking ceramide combined with docetaxel to block the signaling pathway of breast cancer cells. The activation of.5 C6 neuroamide through the AMPK signaling pathway combined with docetaxel's toxicity to breast cancer cells and activation of the JNK pathway associated with C6 neuroamide combined with docetaxel can cause a sustained and significant activation of the JNK pathway of MDA-231 breast cancer cells, stronger than the use of C6 neuroamide or docetaxel by single drug. After the pretreatment of MDA231 breast cancer cells with SP600125 and JNKi V, the death and apoptosis of MDA231 breast cancer cells caused by the combination of C6 ceramide and docetaxel are suppressed. Therefore, the toxicity of C6 ceramide combined with docetaxel on the breast cancer cells and the activation of JNK pathway is associated with the combination of.6 C6 ceramide and docetaxel. The activation and cytotoxicity of JNK/AMPK and cytotoxicity are related to the opening of mitochondrial channel (m PTP) and ROS generation. We detected the changes in the mitochondrial membrane potential of breast cancer cells when C6 ceramide combined with docetaxel to determine whether the toxic effects of C6 and docetaxel on the cancer cells were related to the mitochondrial permeability transition pore. The results showed that the combination of C6 ceramide and docetaxel reduced the mitochondrial membrane potential of breast cancer cells, accompanied by ROS formation, increased cytochrome C and increased C-caspase-3. After M PTP inhibitor Sf A and ROS scavenger NAC preprocessing MDA-231, the death of mammary cancer cells induced by C6 ceramide combined with docetaxel was greatly suppressed and fine. The formation of cytochrome C and C-caspase-3 were suppressed. According to these results, we believe that the toxicity of C6 ceramide and docetaxel combined with the opening of M PTP and the formation of ROS. In order to further confirm this hypothesis, we use sh RNA silencing or inhibitor Cs A to inhibit the function of M PTP. The results showed that the cytotoxic effect of C6 ceramide combined with docetaxel was significantly reduced in Cyp-D suppressed MDA-231 breast cancer cells. And the Cyp-D overexpressed breast cancer cell MDA-231 was more sensitive to C6+ docetaxel. As ROS activates the activity of JNK/AMPK, we detected a combination of C6 ceramide and docetaxel. Whether the activation of JNK/AMPK is associated with ROS. The results show that the ROS scavenger NAC inhibits the activation of JNK/AMPK caused by the combination of drugs, and hydrogen peroxide (one of the reactive oxygen species) can activate the AMPK and JNK pathway of MDA-231 breast cancer cells. It is inhibited. However, the action of Sf A or NAC alone does not inhibit the JNK/AMPK activity of MDA-231. Therefore, we think that the activation of JNK/AMPK in breast cancer cells caused by the combination of C6 ceramide and docetaxel is associated with m PTP opening and the formation of ROS, and that.7 C6 ceramide and docetaxel are associated with breast cancer cells. The inhibition of Akt/Er K pathway in MDA-231 breast cancer cells, we found that C6 ceramide combined with docetaxel can down regulate the expression of HER-1/2 and inhibit the expression of Akt and Erk1/2. It is worth noting that the downregulation of HER-1/2 and the decline of p-Akt and p-Erk expressions begin at the 6-12h of the combined drug use, suggesting the Akt/Erk pathway of breast cancer cells. Inhibition may be associated with the downregulation of HER-1/2,.AG1478, inhibitors of HER, both in MDA-231 and MCF-7 breast cancer cells can inhibit the phosphorylation of Akt and Erk, and also inhibit the cell activity of breast cancer cells. Therefore, the combination of C6 ceramide and docetaxel induces the downregulation of HER-1/2 in breast cancer cells and the inhibition of Akt/Er K pathway. Conclusion 1.C6 Effect of ceramide on the toxic effect of docetaxel on breast cancer cell strain 2.C6 ceramide significantly increased the apoptosis of breast cancer cells induced by docetaxel, 3.C6 ceramide significantly increased the inhibitory effect of docetaxel on the growth of breast cancer cells, 4.C6 ceramide increased the anti breast cancer effect of docetaxel by activating AMPK and inhibiting the action of breast cancer. M TORC1 active 5.C6 ceramide combined with docetaxel's toxicity to breast cancer cells and JNK pathway activation related to JNK/AMPK activation and cytotoxicity of 6.C6 ceramide and docetaxel combined with mitochondrial pathway (m PTP) opening, ROS formation associated with 7.C6 ceramide and docetaxel combined use of the mammary gland Downregulation of HER-1/2 and inhibition of Akt/Er K pathway in cancer cells
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9
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本文编号：2143512
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