【摘要】：研究背景轉移性乳腺癌約占乳腺癌的30-40%,惡性程度高,5年生存率低,是人類難治的惡性腫瘤之一。它病程進展迅速但常無明顯的臨床表現(xiàn),難以早期發(fā)現(xiàn)因而錯過根治的機會。這些患者在進展期往往只有少數(shù)患者可以接受手術治療,而化療是其主要的治療手段。以紫杉類為主的治療是轉移性乳腺癌的一線治療方案,特別是在對蒽環(huán)類耐藥的情況下。臨床試驗證實單藥多西他賽與絲裂霉素聯(lián)合長春新堿相比可以明顯提高總生存率,提高緩解率,延長疾病進展期。另外,盡管紫杉醇和多西他賽兩種紫杉類藥物均用與轉移性乳腺癌的治療,在總生存率及疾病進展時間上多西他賽獲益更多。Albain等人發(fā)現(xiàn)吉西他濱與紫杉醇聯(lián)合用藥療效優(yōu)于紫杉醇單藥治療。在我們的研究中,通過體外培養(yǎng)乳腺癌細胞來研究短鏈神經(jīng)酰胺與多西他賽聯(lián)合作用對乳腺癌的治療療效。神經(jīng)酰胺,主要存在與細胞膜結構中,是脂類家族的結構蛋白。作為信使分子,神經(jīng)酰胺也能引起凋亡。許多研究表明:神經(jīng)酰胺與腫瘤細胞的凋亡有關,增加內(nèi)源性神經(jīng)酰胺的產(chǎn)生可以誘發(fā)凋亡�？扇苄缘亩替溕窠�(jīng)酰胺在許多腫瘤細胞株中表現(xiàn)出抗腫瘤作用,例如黑色素瘤,軟組織肉瘤,Jurkat白血病,頭頸部鱗狀細胞癌。我們課題研究要點是神經(jīng)酰胺是如何增加化療制劑的療效。我們的前期實驗發(fā)現(xiàn)C6神經(jīng)酰胺聯(lián)合多西他賽可以增加多西他賽的抗乳腺癌療效,并且發(fā)現(xiàn)C6神經(jīng)酰胺聯(lián)合多西他賽引起乳腺癌細胞大量凋亡,并與AMPK通路的活化有關。它涉及的分子機制有待進一步深入的研究。研究方法和材料人乳腺癌細胞MCF-7,MDA-231,均從上海生命科學研究所(上海,中國)購買,乳腺癌原代細胞取自外科手術中乳腺癌患者的腫瘤組織,各細胞在RPMI/DMEM培養(yǎng)基中(Simga公司,圣路易斯,密蘇里州),加入10%的FBS(Sigma公司,圣路易斯,密蘇里州),青霉素/鏈霉素(1:100,Sigma),培養(yǎng)37℃CO2培養(yǎng)箱中。細胞給予C6神經(jīng)酰胺和多西他賽處理,顯微鏡下觀察細胞形態(tài),運用MTT方法檢測細胞存活率,同時對細胞進行臺盼藍染色,計算活細胞率;運用Annexin V試劑盒檢測細胞凋亡,通過Western blot檢測總的和磷酸化的AMPKα、ACC、信號水平的改變;運用程序性死亡抑制劑及凋亡抑制劑進一步驗證C6神經(jīng)酰胺和多西他賽誘導細胞死亡方式。建立AMPKα1-sh RNA及AMPK-α1顯性失活(DN)突變(DN-AMPK-α1)c DNA穩(wěn)轉的腫瘤細胞,運用上述方法進一步驗證信號通路的改變。為了了解線粒體通透性轉換孔(m PTP)在協(xié)同用藥對乳腺癌細胞的作用,通過JC-10染料測定細胞線粒體膜電位(MMP),FACS檢測ROS生成,通過Western blot檢測細胞色素C,C-caspase3,JNK,HER-1/2,Akt/Erk信號通路等,了解線粒體通路的改變,并通過MPTP抑制劑及ROS清除劑等方法來驗證線粒體通路作用及相關信號通路改變。建立CYP-D-sh RNA穩(wěn)轉腫瘤,Cyp-D過表達乳腺癌細胞進一步驗證C6神經(jīng)酰胺和多西他賽誘導細胞死亡的信號通路改變。統(tǒng)計學處理在每個實驗中,至少使用三個孔/平皿。每個實驗重復至少三次,每次獲得具有相似的結果。數(shù)據(jù)以均數(shù)±標準偏差(SD)。使用SPSS15.0軟件進行分析統(tǒng)計,通過one-way ANOVA,Scheffe和Tukey檢驗。P0.05被認為有統(tǒng)計學意義。試劑的濃度和治療持續(xù)時間基于發(fā)表文獻和預實驗的結果。研究結果1 C6神經(jīng)酰胺顯著增加多西他賽對乳腺癌細胞株的毒性作用無論是多西他賽(DTX,1.0μg/ml)或C6神經(jīng)酰胺(C6,10μg/m L)單藥都可以引起乳腺癌細胞死亡,當兩者聯(lián)用72h后,90%的乳腺癌細胞死亡,顯著地抑制乳腺癌細胞生長并致死,在原代乳腺癌細胞也發(fā)生同樣的現(xiàn)象。2 C6神經(jīng)酰胺顯著增加多西他賽引起的乳腺癌細胞凋亡單藥多西他賽或單藥神經(jīng)酰胺均可引起乳腺癌細胞MCF-7,及MDA-231的凋亡,當兩者聯(lián)合使用時,乳腺癌細胞的凋亡顯著增加。并用western-blot檢測凋亡相關蛋白,在兩藥聯(lián)合使用的MCF-7和MDA-231細胞中C-caspase-9表達上調(diào)的同時PARP下降。而且在使用一般凋亡抑制劑z VADfmk后,C6聯(lián)合多西他賽引起的乳腺癌細胞MCF-7,MDA-231凋亡均被抑制。在原代乳腺癌細胞中,z VADfmk同樣可以抑制C6聯(lián)合多西他賽引起的細胞死亡。因此C6神經(jīng)酰胺增加多西他賽引起的乳腺癌細胞死亡實質(zhì)是通過引起凋亡。3 C6神經(jīng)酰胺顯著增加多西他賽對乳腺癌細胞的生長抑制作用細胞集落實驗顯示C6神經(jīng)酰胺與多西他賽單藥均可輕度抑制乳腺癌細胞MCF-7/MDA-231的增長,當兩藥聯(lián)合使用后乳腺癌細胞集落明顯下降。并且Cyclin D1的表達也明顯下降。因此C6神經(jīng)酰胺增加多西他賽對乳腺癌細胞的抑制,并引起的乳腺癌細胞細胞周期在G2M的停滯。4 C6神經(jīng)酰胺與多西他賽聯(lián)合用藥通過活化AMPK抑制m TORC1的活性通過檢測磷酸化的AMPKα1,及其下游磷酸化的ACC證實:在乳腺癌細胞C6神經(jīng)酰胺與多西他賽協(xié)同活化AMPK,磷酸化AMPKα(Thr172)和ACC增加而總的AMPK和ACC是沒有變化的。用sh RNA沉默AMPKα的表達,可以抑制C6聯(lián)合多西他賽引起的對MDA-231乳腺癌細胞的毒性作用,在AMPK-負性突變的細胞株(DN,T172A)C6聯(lián)合多西他賽的細胞毒作用也被抑制。這些結果表明,C6聯(lián)合多西他賽對乳腺癌細胞的協(xié)同致死作用與AMPK活化有關。然后,我們檢測了MDA-231細胞中m TORC1的活性。結果表明:MDA-231乳腺癌細胞在C6聯(lián)合多西他賽的作用下,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表達的MDA-231乳腺癌細胞中,C6神經(jīng)酰胺聯(lián)合多西他賽對m TOR信號通路的抑制作用被阻斷,p S6的生成增加,因此C6神經(jīng)酰胺聯(lián)合多西他賽對乳腺癌細胞m TORC1信號通路的阻斷是通過AMPK信號通路的活化。5 C6神經(jīng)酰胺聯(lián)合多西他賽對乳腺癌細胞的毒性作用與JNK通路的活化有關C6神經(jīng)酰胺聯(lián)合多西他賽可以引起MDA-231乳腺癌細胞的JNK通路持續(xù)顯著的活化,比單藥使用C6神經(jīng)酰胺或多西他賽作用均強。用JNK抑制劑SP600125和JNKi V預處理MDA231乳腺癌細胞后,C6神經(jīng)酰胺與多西他賽聯(lián)合用藥引起的MDA231乳腺癌細胞的死亡及凋亡均被抑制。因此C6神經(jīng)酰胺聯(lián)合多西他賽對乳腺癌細胞的毒性作用與JNK通路的活化有關。6 C6神經(jīng)酰胺與多西他賽聯(lián)合用藥引起的JNK/AMPK活化及細胞毒作用與線粒體通道(m PTP)開放,ROS生成有關我們檢測了C6神經(jīng)酰胺與多西他賽聯(lián)合作用時乳腺癌細胞的線粒體膜電位的變化,以明確C6與多西他賽聯(lián)合對癌細胞的毒性作用是否與線粒體通透性轉換孔有關。結果表明:C6神經(jīng)酰胺與多西他賽聯(lián)合用藥使乳腺癌細胞線粒體膜電位下降,并伴有ROS生成,細胞色素C增加及C-caspase-3增加。用m PTP抑制劑Sf A及ROS清除劑NAC預處理MDA-231后,C6神經(jīng)酰胺聯(lián)合多西他賽引起乳腺癌細胞的死亡被大大抑制,并且細胞色素C及C-caspase-3的生成均被抑制。根據(jù)這些結果我們認為C6神經(jīng)酰胺與多西他賽聯(lián)合引起的乳腺癌細胞的毒性作用與m PTP開放及ROS生成有關。為了進一步證實這一假說,我們用sh RNA沉默或抑制劑Cs A抑制m PTP主要結構蛋白Cyp-D的功能。結果表明:Cyp-D被抑制的MDA-231乳腺癌細胞,C6神經(jīng)酰胺聯(lián)合多西他賽引起的細胞毒作用明顯減低。并且在Cyp-D過表達的乳腺癌細胞MDA-231對C6+多西他賽更加敏感。由于ROS可以激活JNK/AMPK的活性,我們檢測了C6神經(jīng)酰胺與多西他賽聯(lián)合用藥引起的JNK/AMPK活化是否與ROS有關。結果表明:ROS清除劑NAC抑制了聯(lián)合用藥引起的JNK/AMPK活化,并且過氧化氫(活性氧的一種)可以活化MDA-231乳腺癌細胞的AMPK和JNK通路。特別的是,用m PTP抑制劑Sf A預處理MDA-231后,聯(lián)合用藥引起的JNK/AMPK的活化也被抑制了。但是,單用Sf A或NAC作用,則不會抑制MDA-231的JNK/AMPK活性。因此:我們認為C6神經(jīng)酰胺與多西他賽聯(lián)合用藥引起的乳腺癌細胞JNK/AMPK的活化與m PTP開放及ROS的生成有關。7 C6神經(jīng)酰胺與多西他賽聯(lián)合用藥引起乳腺癌細胞HER-1/2的下調(diào)及Akt/Er K通路的抑制在MDA-231乳腺癌細胞中,我們發(fā)現(xiàn)C6神經(jīng)酰胺聯(lián)合多西他賽可以協(xié)同下調(diào)HER-1/2的表達,并抑制Akt和Erk1/2的表達。值得注意的是,HER-1/2的下調(diào)及p-Akt和p-Erk表達下降均開始于聯(lián)合用藥6-12h時,提示乳腺癌細胞Akt/Erk通路的抑制可能與HER-1/2的下調(diào)有關。AG1478,HER的抑制劑,在MDA-231及MCF-7乳腺癌細胞均能抑制Akt和Erk的磷酸化,同樣也抑制乳腺癌細胞的細胞活性。因此:C6神經(jīng)酰胺與多西他賽聯(lián)合用藥引起乳腺癌細胞HER-1/2的下調(diào)及Akt/Er K通路的抑制。結論1.C6神經(jīng)酰胺顯著增加多西他賽對乳腺癌細胞株的毒性作用2.C6神經(jīng)酰胺顯著增加多西他賽引起的乳腺癌細胞凋亡3.C6神經(jīng)酰胺顯著增加多西他賽對乳腺癌細胞的生長抑制作用4.C6神經(jīng)酰胺增加多西他賽的抗乳腺癌作用通過活化AMPK并抑制m TORC1的活性5.C6神經(jīng)酰胺聯(lián)合多西他賽對乳腺癌細胞的毒性作用與JNK通路的活化有關6.C6神經(jīng)酰胺與多西他賽聯(lián)合用藥引起的JNK/AMPK活化及細胞毒作用與線粒體通道(m PTP)開放,ROS生成有關7.C6神經(jīng)酰胺與多西他賽聯(lián)合用藥引起乳腺癌細胞HER-1/2的下調(diào)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
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.9
，

本文編號：2143512