內(nèi)質(zhì)網(wǎng)應(yīng)激和自噬在順鉑誘導(dǎo)肺癌細(xì)胞凋亡中作用的研究
發(fā)布時間：2018-08-02 12:48
【摘要】：肺癌是一種常見的,嚴(yán)重威脅人群健康和生命的肺部惡性腫瘤,非小細(xì)胞肺癌約占肺癌細(xì)胞的80%。近50年來許多國家都報道肺癌的發(fā)病率和死亡率都明顯增高,肺癌的病因目前還不是完全清楚,但越來越多的文獻(xiàn)表明肺癌的常見病因包括吸煙、電離輻射、大氣污染和和既往肺部慢性感染等。順鉑(cis-diamminedichloroplatinum II,CDDP)是臨床上治療肺癌最常用的化療藥物。順鉑作用位點(diǎn)在癌細(xì)胞DNA的嘌呤和嘧啶堿基,它通過干預(yù)DNA損傷修復(fù)機(jī)制和抑制DNA復(fù)制誘導(dǎo)癌細(xì)胞凋亡。目前,順鉑在臨床上廣泛應(yīng)用于腫瘤治療過程中,并取得了良好的效果。但順鉑在使用過程中機(jī)體容易對其產(chǎn)生耐受性,同時順鉑所具有的耳毒性、神經(jīng)毒性和腎毒性等毒副作用也限制了它的臨床應(yīng)用。因此,明確順鉑誘導(dǎo)細(xì)胞凋亡的機(jī)制尤為重要,越來越多的文獻(xiàn)表明順鉑在誘導(dǎo)癌細(xì)胞凋亡過程中伴隨著內(nèi)質(zhì)網(wǎng)應(yīng)激和自噬的發(fā)生。內(nèi)質(zhì)網(wǎng)(Endoplasmic Reticulum,ER)是真核細(xì)胞內(nèi)的一種膜結(jié)合細(xì)胞器,它的主要功能是對膜蛋白和分泌蛋白進(jìn)行正確折疊和翻譯后修飾。此外,內(nèi)質(zhì)網(wǎng)在脂類生物合成,能量代謝,細(xì)胞內(nèi)Ca~（2+）穩(wěn)態(tài)和氧化還原平衡方面也發(fā)揮重要的作用。內(nèi)質(zhì)網(wǎng)中蛋白質(zhì)的折疊功能對細(xì)胞外和細(xì)胞內(nèi)的刺激都非常敏感,包括缺血再灌注,炎癥,糖基化和Ca~（2+）平衡失調(diào)等。內(nèi)質(zhì)網(wǎng)管腔中錯誤折疊或未折疊蛋白的聚集會誘導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic Reticulum stress,ER stress),進(jìn)而會激活未折疊蛋白反應(yīng)(unfolded protein response,UPR)。UPR可以增加內(nèi)質(zhì)網(wǎng)對蛋白質(zhì)的折疊和修飾的能力,減弱m RNA的翻譯,通過內(nèi)質(zhì)網(wǎng)相關(guān)蛋白降解(ER-associated degradation,ERAD)和自噬降解未折疊和錯誤折疊蛋白。在內(nèi)質(zhì)網(wǎng)應(yīng)激的早期階段,內(nèi)質(zhì)網(wǎng)內(nèi)的蛋白質(zhì)合成會減少,調(diào)控蛋白質(zhì)翻譯和正確折疊的相關(guān)基因會被激活,這都有利于維持細(xì)胞的正常生理學(xué)功能,從而促進(jìn)了細(xì)胞存活。然而,當(dāng)ER stress持續(xù)時間過久或UPR功能受到損傷時,內(nèi)質(zhì)網(wǎng)內(nèi)就會聚集大量的錯誤折疊或未折疊蛋白。這時,促凋亡信號就會被激活。目前,有研究表明心肌細(xì)胞、胰腺細(xì)胞的內(nèi)質(zhì)網(wǎng)功能失調(diào)可能是心腦組織缺血阻塞、糖尿病的主要發(fā)病機(jī)制。因此,ER stress既可以作為維持細(xì)胞存活的生存手段,也可以誘導(dǎo)細(xì)胞凋亡的重要機(jī)制。在大部分真核細(xì)胞內(nèi),自噬是一種廣泛存在的生命現(xiàn)象,是一種依賴于溶酶體途徑對細(xì)胞內(nèi)代謝產(chǎn)物和細(xì)胞器進(jìn)行降解并具有高度保守性的過程。自噬具有降解和回收代謝物以及細(xì)胞器的功能,在維持細(xì)胞內(nèi)環(huán)境穩(wěn)態(tài)方面起到至關(guān)重要的作用。自噬在腫瘤細(xì)胞中的作用具有雙重性:一方面,自噬可以通過清除有缺陷的蛋白質(zhì)或受損的細(xì)胞器,最終通過維持細(xì)胞內(nèi)環(huán)境的穩(wěn)態(tài)促進(jìn)癌細(xì)胞存活;另一方面,過度活躍的自噬通過誘導(dǎo)細(xì)胞器的損傷和細(xì)胞內(nèi)環(huán)境穩(wěn)態(tài)失調(diào),激活自噬性程序性細(xì)胞死亡。越來越多的研究表明自噬在腫瘤中所發(fā)揮的具體作用依賴于腫瘤發(fā)展的特定階段和特殊腫瘤類型。因此,探討自噬在順鉑誘導(dǎo)肺癌細(xì)胞凋亡中的作用具有很重要的意義。本文以人肺癌A549和H460細(xì)胞為研究對象,探討了內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬反應(yīng)在順鉑誘導(dǎo)肺癌A549和H460細(xì)胞凋亡中的作用及機(jī)制,為臨床治療肺癌提供新的思路。方法:(1)分別用不同濃度的順鉑(5μM、10μM、20μM、40μM和80μM)處理肺癌A549和H460細(xì)胞24 h。MTT法檢測不同濃度的順鉑對肺癌A549和H460細(xì)胞生存率的影響,LDH試劑盒檢測A549和H460細(xì)胞LDH漏出率。(2)分別用不同濃度的順鉑(10μM、20μM和40μM)處理肺癌A549和H460細(xì)胞24 h。通過流式細(xì)胞儀和熒光分光計分別檢測細(xì)胞凋亡和線粒體膜電位的變化,Western blot方法檢測cleaved caspase3、cleaved PARP、胞漿Cyt c、Grp78、PERK、IRE1的變化。(3)順鉑聯(lián)合內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑4-PBA和TUDC。MTT檢測聯(lián)合內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑對A549和H460細(xì)胞生存率的變化,Western blot檢測cleaved caspase3和Cyt c的變化。(4)觀察自噬在順鉑誘導(dǎo)A549和H460細(xì)胞凋亡過程中的變化。Western blot檢測LC3、Beclin 1蛋白的變化。(5)順鉑聯(lián)合自噬抑制劑3-MA和CQ。MTT檢測聯(lián)合自噬抑制劑對A549和H460細(xì)胞生存率的變化,Western blot檢測cleaved caspase3和Cyt c的變化。結(jié)果:(1)順鉑以劑量依賴性的方式抑制肺癌A549和H460細(xì)胞生長,同樣地,順鉑也能以劑量依賴性的方式誘導(dǎo)了LDH釋放率升高。(2)順鉑誘導(dǎo)了A549和H460細(xì)胞凋亡并降低其線粒體膜電位,順鉑誘導(dǎo)cleaved caspase3和cleaved PARP的活化,胞漿Cyt c、Grp78、PERK和IRE1表達(dá)上調(diào)。(3)用4-PBA和TUDC抑制內(nèi)質(zhì)網(wǎng)應(yīng)激,可以增加順鉑對細(xì)胞的生長抑制作用,增加順鉑誘導(dǎo)的凋亡,增加順鉑誘導(dǎo)的cleaved caspase3和Cyt c的表達(dá)。(4)順鉑誘導(dǎo)了A549和H460細(xì)胞內(nèi)自噬相關(guān)蛋白LC3II/LC3I、Beclin 1表達(dá)上調(diào)。(5)用3-MA和CQ抑制自噬,可以增加順鉑對細(xì)胞的生長抑制作用,增加順鉑誘導(dǎo)的凋亡,以及增加順鉑誘導(dǎo)的cleaved caspase3和Cyt c的表達(dá)。結(jié)論:我們的實(shí)驗(yàn)數(shù)據(jù)表明順鉑能誘導(dǎo)人非小細(xì)胞肺癌A549和H460細(xì)胞凋亡。一方面,通過線粒體途徑介導(dǎo)的細(xì)胞凋亡;一方面,通過內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)凋亡途徑。減輕內(nèi)質(zhì)網(wǎng)應(yīng)激可以顯著地增加順鉑誘導(dǎo)的細(xì)胞凋亡。順鉑在誘導(dǎo)細(xì)胞凋亡的同時也激活了自噬。自噬在順鉑誘導(dǎo)的細(xì)胞凋亡過程中發(fā)揮著促存活的作用,抑制自噬增加了肺癌細(xì)胞對順鉑的敏感性。本研究探討了內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬反應(yīng)在順鉑誘導(dǎo)肺癌A549和H460細(xì)胞凋亡中的作用及機(jī)制,進(jìn)一步揭示順鉑的作用機(jī)制,為臨床治療肺癌提供了可行的理論依據(jù)。
[Abstract]:Lung cancer is a common type of malignant tumor that seriously threatens the health and life of the population. Non small cell lung cancer accounts for approximately 50 years of lung cancer cells. In recent 50 years, many countries have reported that the incidence and mortality of lung cancer are obviously increased. The cause of lung cancer is not completely clear, but more and more literature shows the common cause of lung cancer. Including smoking, ionizing radiation, air pollution and chronic pulmonary chronic infection. Cis-diamminedichloroplatinum II (CDDP) is the most commonly used chemotherapeutic drug for the treatment of lung cancer. The cisplatin action site is in the purine and pyrimidine bases of the cancer cell DNA. It can induce the repair mechanism of DNA damage and inhibit DNA replication to induce the apoptosis of cancer cells. Before, cisplatin is widely used in the clinical treatment of cancer and has achieved good results. However, cisplatin is easily tolerated during the use of cisplatin. At the same time, the toxic side effects of cisplatin, such as ototoxicity, neurotoxicity and nephrotoxicity, also restrict its clinical application. Therefore, the mechanism of cisplatin induced apoptosis is clearly defined. In particular, more and more literature shows that cisplatin is accompanied by endoplasmic reticulum stress and autophagy during the induction of apoptosis in cancer cells. Endoplasmic Reticulum (ER) is a membrane binding organelle in eukaryotic cells. Its main function is to fold and post translational modifications to membrane proteins and secretory proteins. The mass network also plays an important role in lipid biosynthesis, energy metabolism, intracellular Ca~ (2+) homeostasis and redox balance. The folding function of protein in the endoplasmic reticulum is very sensitive to both extracellular and intracellular stimuli, including ischemia reperfusion, inflammation, glycosylation and Ca~ (2+) imbalance. The aggregation of unfolded proteins induces endoplasmic reticulum stress (Endoplasmic Reticulum stress, ER stress), and then activates the unfolded protein reaction (unfolded protein response, UPR).UPR that can increase the ability of the endoplasmic reticulum to fold and modify proteins, weaken the translation of M RNA, and degrade through endoplasmic reticulum related proteins. On, ERAD) and autophagy degrade unfolded and erroneous folding proteins. In the early stages of endoplasmic reticulum stress, protein synthesis in the endoplasmic reticulum will be reduced, and genes related to protein translation and correct folding are activated, which all contribute to the maintenance of normal physiological functions of the cells and thus promote cell survival. However, when ER stress continues, it continues. In the endoplasmic reticulum, a large number of misfolded or unfolded proteins are aggregated in the endoplasmic reticulum when the time is too long or the UPR function is damaged. At this time, the apoptosis signal can be activated. At present, there is a study showing that the dysfunction of the endoplasmic reticulum in the pancreatic cells may be the main pathogenesis of ischemia and diabetes. Therefore, ER stress is the main mechanism of the disease. In most eukaryotic cells, autophagy is a widespread life phenomenon in most eukaryotic cells. It is a process that relies on lysosome pathway to degrade intracellular metabolites and organelles and is highly conserved. Autophagy degrades and returns. The function of metabolites and organelles plays a vital role in maintaining the homeostasis of the cell. Autophagy plays a dual role in tumor cells: on the one hand, autophagy can promote the survival of cancer cells by maintaining the homeostasis of the intracellular environment by eliminating defective proteins or damaged organelles; On the other hand, hyperactive autophagy activates autophagic programmed cell death by inducing cellular organelle damage and homeostasis of intracellular homeostasis. More and more studies have shown that the specific role of autophagy in tumors depends on the specific stage and specific tumor type of tumor development. Therefore, the study of autophagy induces lung cancer cells in cisplatin. The role of apoptosis is of great significance. In this paper, the role and mechanism of endoplasmic reticulum stress and autophagy in the apoptosis of lung cancer A549 and H460 cells induced by cisplatin in human lung cancer A549 and H460 cells were studied. Methods: (1) different concentrations of cisplatin (5 M, 10 u M, 20 mu M, 40 mu) were used respectively. The effects of different concentrations of cisplatin on the survival rate of A549 and H460 cells in lung cancer were detected by M and 80 M H460 cells. The LDH kit was used to detect the LDH leakage of A549 and H460 cells. (2) different concentrations of cisplatin (10 micron, 20 mu and 40 micron) were used to treat lung cancer and 24 cells through flow cytometer and fluorescent spectrometer. The changes in apoptosis and mitochondrial membrane potential were detected respectively. Western blot method was used to detect cleaved Caspase3, cleaved PARP, cytoplasmic Cyt C, Grp78, PERK, IRE1. (3) cisplatin combined with endoplasmic reticulum stress inhibitors and the changes in the survival rate of endoplasmic reticulum stress inhibitors Changes in ED Caspase3 and Cyt C. (4) observe the changes of autophagy during the apoptosis of A549 and H460 cells induced by cisplatin and.Western blot to detect the changes of LC3, Beclin 1 protein. (5) the changes of cisplatin combined with autophagy inhibitor, 3-MA and CQ.MTT detection combined autophagic inhibitors Results: (1) cisplatin inhibited the growth of lung cancer A549 and H460 cells in a dose-dependent manner. Similarly, cisplatin also induced the increase of LDH release rate in a dose-dependent manner. (2) cisplatin induced apoptosis of A549 and H460 cells and reduced the mitochondrial membrane potential. Cisplatin induced the activation of cleaved Caspase3 and cleaved PARP, cytoplasmic Cy. T C, Grp78, PERK and IRE1 are up-regulated. (3) inhibition of endoplasmic reticulum stress by 4-PBA and TUDC can increase the inhibition of cisplatin on cell growth, increase cisplatin induced apoptosis, increase the expression of cisplatin induced cleaved Caspase3 and Cyt C. (4) cisplatin induced A549 and intracellular autophagy related protein, up regulation of 1. (5) Inhibition of autophagy with 3-MA and CQ can increase cisplatin's inhibitory effect on cell growth, increase cisplatin induced apoptosis, and increase cisplatin induced cleaved Caspase3 and Cyt C expression. Conclusion: our experimental data suggest that cisplatin can induce apoptosis of A549 and H460 in human non-small cell lung cancer. On the one hand, it is mediated by mitochondrial pathway. Apoptosis, on the one hand, through endoplasmic reticulum stress related apoptosis pathway. Reduction of endoplasmic reticulum stress can significantly increase cisplatin induced apoptosis. Cisplatin also activates autophagy while inducing apoptosis. Autophagy plays a role in promoting survival during cisplatin induced apoptosis, and inhibition of autophagy increases lung cancer cells. This study explored the role and mechanism of endoplasmic reticulum stress and autophagy in the apoptosis of lung cancer A549 and H460 cells induced by cisplatin, and further revealed the mechanism of cisplatin, which provides a feasible theoretical basis for the clinical treatment of lung cancer.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R734.2
本文編號：2159490
[Abstract]:Lung cancer is a common type of malignant tumor that seriously threatens the health and life of the population. Non small cell lung cancer accounts for approximately 50 years of lung cancer cells. In recent 50 years, many countries have reported that the incidence and mortality of lung cancer are obviously increased. The cause of lung cancer is not completely clear, but more and more literature shows the common cause of lung cancer. Including smoking, ionizing radiation, air pollution and chronic pulmonary chronic infection. Cis-diamminedichloroplatinum II (CDDP) is the most commonly used chemotherapeutic drug for the treatment of lung cancer. The cisplatin action site is in the purine and pyrimidine bases of the cancer cell DNA. It can induce the repair mechanism of DNA damage and inhibit DNA replication to induce the apoptosis of cancer cells. Before, cisplatin is widely used in the clinical treatment of cancer and has achieved good results. However, cisplatin is easily tolerated during the use of cisplatin. At the same time, the toxic side effects of cisplatin, such as ototoxicity, neurotoxicity and nephrotoxicity, also restrict its clinical application. Therefore, the mechanism of cisplatin induced apoptosis is clearly defined. In particular, more and more literature shows that cisplatin is accompanied by endoplasmic reticulum stress and autophagy during the induction of apoptosis in cancer cells. Endoplasmic Reticulum (ER) is a membrane binding organelle in eukaryotic cells. Its main function is to fold and post translational modifications to membrane proteins and secretory proteins. The mass network also plays an important role in lipid biosynthesis, energy metabolism, intracellular Ca~ (2+) homeostasis and redox balance. The folding function of protein in the endoplasmic reticulum is very sensitive to both extracellular and intracellular stimuli, including ischemia reperfusion, inflammation, glycosylation and Ca~ (2+) imbalance. The aggregation of unfolded proteins induces endoplasmic reticulum stress (Endoplasmic Reticulum stress, ER stress), and then activates the unfolded protein reaction (unfolded protein response, UPR).UPR that can increase the ability of the endoplasmic reticulum to fold and modify proteins, weaken the translation of M RNA, and degrade through endoplasmic reticulum related proteins. On, ERAD) and autophagy degrade unfolded and erroneous folding proteins. In the early stages of endoplasmic reticulum stress, protein synthesis in the endoplasmic reticulum will be reduced, and genes related to protein translation and correct folding are activated, which all contribute to the maintenance of normal physiological functions of the cells and thus promote cell survival. However, when ER stress continues, it continues. In the endoplasmic reticulum, a large number of misfolded or unfolded proteins are aggregated in the endoplasmic reticulum when the time is too long or the UPR function is damaged. At this time, the apoptosis signal can be activated. At present, there is a study showing that the dysfunction of the endoplasmic reticulum in the pancreatic cells may be the main pathogenesis of ischemia and diabetes. Therefore, ER stress is the main mechanism of the disease. In most eukaryotic cells, autophagy is a widespread life phenomenon in most eukaryotic cells. It is a process that relies on lysosome pathway to degrade intracellular metabolites and organelles and is highly conserved. Autophagy degrades and returns. The function of metabolites and organelles plays a vital role in maintaining the homeostasis of the cell. Autophagy plays a dual role in tumor cells: on the one hand, autophagy can promote the survival of cancer cells by maintaining the homeostasis of the intracellular environment by eliminating defective proteins or damaged organelles; On the other hand, hyperactive autophagy activates autophagic programmed cell death by inducing cellular organelle damage and homeostasis of intracellular homeostasis. More and more studies have shown that the specific role of autophagy in tumors depends on the specific stage and specific tumor type of tumor development. Therefore, the study of autophagy induces lung cancer cells in cisplatin. The role of apoptosis is of great significance. In this paper, the role and mechanism of endoplasmic reticulum stress and autophagy in the apoptosis of lung cancer A549 and H460 cells induced by cisplatin in human lung cancer A549 and H460 cells were studied. Methods: (1) different concentrations of cisplatin (5 M, 10 u M, 20 mu M, 40 mu) were used respectively. The effects of different concentrations of cisplatin on the survival rate of A549 and H460 cells in lung cancer were detected by M and 80 M H460 cells. The LDH kit was used to detect the LDH leakage of A549 and H460 cells. (2) different concentrations of cisplatin (10 micron, 20 mu and 40 micron) were used to treat lung cancer and 24 cells through flow cytometer and fluorescent spectrometer. The changes in apoptosis and mitochondrial membrane potential were detected respectively. Western blot method was used to detect cleaved Caspase3, cleaved PARP, cytoplasmic Cyt C, Grp78, PERK, IRE1. (3) cisplatin combined with endoplasmic reticulum stress inhibitors and the changes in the survival rate of endoplasmic reticulum stress inhibitors Changes in ED Caspase3 and Cyt C. (4) observe the changes of autophagy during the apoptosis of A549 and H460 cells induced by cisplatin and.Western blot to detect the changes of LC3, Beclin 1 protein. (5) the changes of cisplatin combined with autophagy inhibitor, 3-MA and CQ.MTT detection combined autophagic inhibitors Results: (1) cisplatin inhibited the growth of lung cancer A549 and H460 cells in a dose-dependent manner. Similarly, cisplatin also induced the increase of LDH release rate in a dose-dependent manner. (2) cisplatin induced apoptosis of A549 and H460 cells and reduced the mitochondrial membrane potential. Cisplatin induced the activation of cleaved Caspase3 and cleaved PARP, cytoplasmic Cy. T C, Grp78, PERK and IRE1 are up-regulated. (3) inhibition of endoplasmic reticulum stress by 4-PBA and TUDC can increase the inhibition of cisplatin on cell growth, increase cisplatin induced apoptosis, increase the expression of cisplatin induced cleaved Caspase3 and Cyt C. (4) cisplatin induced A549 and intracellular autophagy related protein, up regulation of 1. (5) Inhibition of autophagy with 3-MA and CQ can increase cisplatin's inhibitory effect on cell growth, increase cisplatin induced apoptosis, and increase cisplatin induced cleaved Caspase3 and Cyt C expression. Conclusion: our experimental data suggest that cisplatin can induce apoptosis of A549 and H460 in human non-small cell lung cancer. On the one hand, it is mediated by mitochondrial pathway. Apoptosis, on the one hand, through endoplasmic reticulum stress related apoptosis pathway. Reduction of endoplasmic reticulum stress can significantly increase cisplatin induced apoptosis. Cisplatin also activates autophagy while inducing apoptosis. Autophagy plays a role in promoting survival during cisplatin induced apoptosis, and inhibition of autophagy increases lung cancer cells. This study explored the role and mechanism of endoplasmic reticulum stress and autophagy in the apoptosis of lung cancer A549 and H460 cells induced by cisplatin, and further revealed the mechanism of cisplatin, which provides a feasible theoretical basis for the clinical treatment of lung cancer.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R734.2
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