吉非替尼和順鉑聯合使用在非小細胞肺癌治療中的拮抗作用及其機制研究
發(fā)布時間：2018-07-18 21:47
【摘要】：背景與目的:支氣管肺癌是最常見的呼吸系統(tǒng)腫瘤,在全球范圍內發(fā)病率及死亡率均較高。非小細胞肺癌(Non-Small Cell Lung Cancer,NSCLC)占支氣管肺癌發(fā)病的大多數,目前手術仍然是治療NSCLC的主要手段。但是對于手術患者來說,兩年內的復發(fā)率約40%。故對于術后復發(fā)的NSCLC患者及無手術指征的晚期NSCLC患者,全身性化療及靶向治療仍為其主要的治療手段,但目前常見的NSCLC一線化療方案療效仍不理想,有效率僅為30%左右,因此尋找新的有價值治療手段和策略顯得十分必要。靶向治療與化療聯合能否提高化療療效,成為當前國內外研究的重要課題。含鉑類的聯合化療仍然是晚期NSCLC的標準一線化療方案,其中第一代鉑類藥物順鉑更是NSCLC化療的基石。然而單一化療并不能取得理想的治療效果,隨著生物制劑靶向治療藥物的出現,化療聯合靶向治療是否可以提高療效是一個值得研究的課題。吉非替尼是第一代表皮生長因子受體(Epidermal Growth Factor Receptor,EGFR)酪氨酸激酶抑制劑,該酶通常表達于上皮來源的實體瘤。作為第一代小分子酪氨酸激酶抑制劑,目前相關大型三期多中心臨床實驗證實在NSCLC的一線治療中,吉非替尼相對于化療來說,對于亞裔、腺癌和EGFR突變患者具有明顯的無進展生存期(Progress-Free Survival,PFS)優(yōu)勢,然而在能否與化療聯用方面,四個大型多中心臨床實驗均證實表皮生長因子受體酪氨酸激酶抑制劑(Tyrosine-Kinase Inhibitor TKI),例如吉非替尼和厄羅替尼都與與化療藥物聯用時對晚期非小細胞肺癌患者總生存期(Overall Survival,OS)無明顯生存獲益。提示吉非替尼與包括順鉑在內的化療藥物聯用時可能存在拮抗作用。雖然臨床試驗結果顯示順鉑和吉非替尼聯合使用存在拮抗作用,但導致這種拮抗作用的機制仍然不是十分清楚。闡明導致拮抗作用的機制可為臨床治療提供新的思路。本課題擬從自噬和外泌體兩個角度揭示吉非替尼與順鉑聯合使用時如何影響順鉑治療療效。研究一吉非替尼通過上調非小細胞肺癌細胞自噬水平誘導影響順鉑療效的作用及其機制研究方法(1)使用吉非替尼與不同濃度的順鉑聯合作用于人NSCLC細胞株PC9,采用MTT法測定不同濃度順鉑對PC9細胞增殖力的影響,同時計算吉非替尼和不同濃度順鉑之間的相互作用指數來分析吉非替尼對不同濃度順鉑的作用。Western-Blot方法分析吉非替尼與順鉑聯用與單獨使用順鉑作用于PC9細胞后自噬標記蛋白LC3-Ⅱ及P62的變化,電鏡觀察上述兩組自噬小體數量的變化,進而了解吉非替尼對順鉑誘導自噬水平的影響。(2)為進一步評估自噬強度,使用5μg/ml的溶酶體抑制劑氯喹(chloroquine,CQ),即抑制溶酶體降解的自噬抑制劑預處理細胞1小時,透射電子顯微鏡(Transmission Electron Microscope,TEM)用于確認CQ抑制自噬溶酶體的降解效果。然后加入吉非替尼和/或順鉑,比較無CQ時組間LC3表達的改變。從而判斷PC9細胞的自噬水平的改變是吉非替尼和/或順鉑誘導,而不是自噬降解缺陷所致。(3)為觀察抑制自噬后對吉非替尼和順鉑間拮抗效應的影響,我們使用MTT法分別檢測給予CQ處理后的吉非替尼和順鉑組PC9細胞,根據所測得OD值計算CDI,了解自噬在吉非替尼導致順鉑耐藥過程中的作用。(4)采用Annexin V-FITC/PI流式細胞技術檢測研究上述處理后各組細胞凋亡率,同時提取PC9細胞相應組別凋亡蛋白Bcl-2和Bax,以了解自噬水平改變后對PC9細胞凋亡的影響。同時我們采用流式細胞學技術(Flow cytometry,FCM)進行細胞周期分析,了解自噬對PC9細胞周期的影響。結果1吉非替尼聯合順鉑導致拮抗效應(1)采用MTT法,檢測不同濃度吉非替尼和順鉑對PC9細胞的增殖抑制作用,結果顯示,吉非替尼和順鉑對NSCLC細胞的抑制作用具有時間和濃度依賴性。相對于順鉑或吉非替尼單藥,其他各種聯合用藥組細胞增殖力并無明顯下降。(2)采用藥物相互作用系數(Coefficient of Drug Interaction,CDI)評估藥物作用性質,給予不同濃度吉非替尼和順鉑后CDI值為1.19±0.1(1.07-1.35),提示存在拮抗作用。2吉非替尼聯合順鉑誘導產生自噬(1)采用Western Blot法觀察吉非替尼和/或順鉑處理后的PC9細胞自噬標記物LC3及p62的表達情況,結果顯示,與對照未處理組相比,吉非替尼和順鉑可誘導上調LC3-II轉化,而吉非替尼合并順鉑與單獨吉非替尼用藥相比,LC3-II轉化明顯上升,但無統(tǒng)計學差異。同時吉非替尼和/或順鉑均顯著下調p62水平。相比單藥刺激,吉非替尼合并順鉑導致p62蛋白更加顯著的下調,提示吉非替尼合并順鉑相對單藥可誘導更高水平自噬。(2)采用透射電子顯微鏡(Transmission electron microscopy,TEM),觀察吉非替尼和/或順鉑處理后的PC9細胞自噬小體變化情況,結果顯示與單藥處理組相比,吉非替尼聯合順鉑相對單藥處理組自噬小體數目增多),并且吉非替尼合并順鉑相對單藥可誘導更高水平自噬。(3)采用Western Blot法觀察CQ預處理后,吉非替尼和/或順鉑處理后的PC9細胞自噬標記物LC3及p62的表達情況。該結果提示吉非替尼和/或順鉑可誘導PC9細胞自噬,而不是溶酶體降解所致。3抑制自噬可抵抗吉非替尼和順鉑間的拮抗效應(1)采用TEM檢測CQ預處理后吉非替尼和/或順鉑處理后的PC9細胞自噬溶酶體變化情況,結果顯示與吉非替尼和/或順鉑組相比,加用CQ后自噬溶酶體顯著增多,提示CQ可抑制自噬過程中自噬溶酶體的降解。(2)采用MTT檢測CQ預處理后,吉非替尼和/或順鉑處理后的PC9細胞增殖力,結果提示與無CQ組相比,給予CQ預處理時吉非替尼和順鉑顯著降低細胞增殖力。同時CQ預處理后吉非替尼和順鉑的協同作用相對增加。(3)采用Annexin V-FITC/PI凋亡檢測研究與無CQ預處理相比,CQ預處理可增加各組PC9凋亡細胞比例。CQ預處理時無論單藥還是兩藥聯合使用均可顯著增加凋亡細胞比,提示自噬在凋亡過程中扮演重要角色。4抑制自噬可促進凋亡(1)采用流式細胞技術(Flow Cytometry,FCM)進行對各組PC9細胞周期分析,結果提示吉非替尼和順鉑單藥均可導致G1期停滯,然而,吉非替尼和順鉑兩藥聯用僅稍微提高G1期PC9細胞比例。為進一步證實自噬與細胞周期停滯的關系,我們給吉非替尼和/或順鉑CQ(5μg/ml)預處理1小時,結果顯示CQ抑制自噬不能逆轉吉非替尼和/或順鉑誘導的細胞周期停滯。(2)采用Western Blot檢測是否CQ預處理可影響各組PC9細胞促凋亡蛋白和抗凋亡蛋白的表達水平。CQ處理后Bcl-2表達水平(經典抗凋亡家族蛋白)和Bax(促凋亡蛋白)表達分別顯著降低和升高。然而,與無CQ組相比,吉非替尼和/或順鉑加CQ預處理組的Bax/Bcl-2比例無顯著差異(分別為p=0.592,0.09及0.06)。上述結果提示CQ抑制自噬可通過上調Bax和下調Bcl-2表達來促進PC9細胞凋亡。研究二吉非替尼通過外泌體上調自噬拮抗順鉑誘導的非小細胞肺癌細胞凋亡的作用研究方法(1)采用Exo Quick沉淀液分離正常對照組PC9細胞或吉非替尼(1μM)/順鉑(7.5μM)給藥組的外泌體。外泌體從吉非替尼或順鉑作用的PC9細胞上清液提取,分為Exo-Con(Exosomes derived from negative Control),Exo-GF(Exosomes derived from gefitinib treated EGFR mutant lung cancer cells),Exo-DDP(Exosomes derived from cisplatin treated EGFR mutant lung cancer cells)。使用BCA蛋白檢測試劑盒對不同胞外體蛋白進行標準定量。(2)采用透射電子顯微鏡(TEM)觀察外泌體形成,鑒定是否提取成功。(3)采用CCK-8法檢測外泌體預處理后的各組細胞抑制率,我們使用一定濃度外泌體預孵育24小時后加1μM吉非替尼或7.5μM順鉑孵育24小時。使用CCK-8法計算其細胞增殖活力,同時計算其細胞抑制率。并計算兩種藥物間的藥物相互作用系數(Coefficient of Drug Interaction,CDI)。然后使用用外泌體抑制劑GW4869作用于各藥物處理組,了解抑制外泌體分泌對吉非替尼或順鉑對于PC9細胞效果的影響。(4)分別使用各組外泌體和藥物共同孵育細胞,Western Blot法檢測自噬標記蛋白LC3和p62的變化,以了解外泌體和自噬之間的相關性。同時對以上各組使用Annexin V-FITC/PI復染試劑及流式細胞技術(FCM)檢測細胞凋亡。并采用Western Blot法檢測凋亡蛋白Bcl-2和Bax,檢測外泌體對PC9細胞凋亡的影響。結果1吉非替尼合并順鉑治療產生拮抗效應采用CCK-8法,檢測吉非替尼和/或順鉑對PC9細胞增殖力的影響,結果顯示相比各濃度單藥吉非替尼(0.4-2μΜ)治療,聯合順鉑治療的吉非替尼抗增殖效應僅稍微增加。同時計算兩藥在PC9及A549細胞中的CDI值。結果提示在吉非替尼和順鉑聯用于EGFR突變及野生型NSCLC PC9細胞株時均存在相互拮抗作用。2 Exo-GF降低順鉑的抗腫瘤活性研究吉非替尼和順鉑間的拮抗效應由Exo-GF還是Exo-DDP介導。收集外泌體,加入一定濃度順鉑或吉非替尼共同作用于PC9細胞。CCK-8結果顯示Exo-GF可劑量依賴性對抗順鉑的抗腫瘤效應,然而Exo-Con對順鉑誘導的增殖抑制無顯著影響,同時Exo-DDP對吉非替尼幾乎無影響。采用CCK-8法檢測外泌體抑制劑GW4869預處理后,吉非替尼和/或順鉑作用于PC9細胞的抑制率,結果顯示加入GW4869后,單藥吉非替尼和順鉑對PC9細胞抑制率僅有輕微增加。但吉非替尼和順鉑聯合組使用GW4869預處理后抑制率顯著上升,同時兩藥CDI值在加入GW4869后顯著下降,提示抑制Exo-GF分泌可逆轉吉非替尼和順鉑間的拮抗作用。3 Exo-GF上調順鉑誘導的自噬水平采用Western blot檢測Exo-Con,Exo-GF及Exo-DDP作用于PC9細胞的LC3及p62表達。與對照未處理PC9細胞相比,Exo-Con,Exo-GF及Exo-DDP均可顯著上調自噬活性。相比單藥順鉑組,Exo-GF預處理順鉑組的自噬顯著增強。但Exo-DDP預處理幾乎對吉非替尼誘導的自噬無影響。4 Exo-GF下調順鉑誘導的細胞凋亡采用流式細胞技術(Flow Cytometry,FCM)檢測Exo-GF對于順鉑誘導的PC9細胞凋亡的影響,結果顯示相比使用Exo-Con預處理的順鉑組及順鉑單藥組,Exo-GF預處理加順鉑單藥組的凋亡細胞顯著減少。同CCK-8實驗結果一致,Exo-DDP對吉非替尼誘導凋亡無明顯影響。進一步采用Western Blot檢測上述處理各組PC9細胞Bcl-2及Bax的蛋白表達水平。與順鉑組或Exo-Con預處理加順鉑組相比,Exo-GF預處理加順鉑組PC9細胞Bcl-2表達顯著上調伴Bax下調。相比單純順鉑組,Exo-GF預處理加順鉑組Bcl-2/Bax比值顯著增高。但與單純吉非替尼組相比,Exo-DDP預處理加吉非替尼組PC9細胞凋亡水平無顯著差異。Exo-GF可顯著下調順鉑誘導的細胞凋亡。結論1.吉非替尼聯合順鉑作用于NSCLC細胞可產生拮抗效應;2.吉非替尼通過外泌體上調自噬拮抗順鉑誘導的NSCLC細胞凋亡;3.抑制外泌體分泌可通過抑制自噬逆轉吉非替尼和順鉑間的拮抗效應。
[Abstract]:Background and purpose: bronchial lung cancer is the most common respiratory system tumor, with high morbidity and mortality worldwide. Non small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) accounts for most of the incidence of bronchial lung cancer, and the operation is still the main means to treat NSCLC. The rate of hair is about 40%., so for patients with recurrent NSCLC and late NSCLC with no indication of operation, systemic chemotherapy and targeted therapy are still the main treatment methods. However, the common NSCLC first-line chemotherapy is still not ideal and the effective rate is only about 30%. Therefore, it is necessary to find new methods and Strategies of valuable treatment. The combination of targeted therapy and chemotherapy has become an important topic at home and abroad. Platinum group chemotherapy is still the standard first-line chemotherapy for advanced NSCLC. The first generation platinum group cisplatin is the cornerstone of NSCLC chemotherapy. However, single chemotherapy can not achieve the ideal therapeutic effect with the biological system. Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitor, which is the first representative of epidermal growth factor receptor (EGFR), is the first representative of the tyrosine kinase inhibitor, which is the first representative of the small molecule of casein. Acid kinase inhibitors, which are currently related to large three phase multicenter clinical validation of NSCLC first-line treatment, gefitinib has a distinct progression free survival (PFS) advantage for Asian, adenocarcinoma and EGFR mutations in patients with Asian, adenocarcinoma and EGFR mutations. However, in connection with chemotherapy, four large multicenter patients can be used in combination with chemotherapy. The bed test showed that the epidermal growth factor receptor tyrosine kinase inhibitor (Tyrosine-Kinase Inhibitor TKI), such as gefitinib and erlotinib, had no significant survival benefit for the total survival period of patients with advanced non-small cell lung cancer (Overall Survival, OS) when combined with chemotherapeutic drugs. There may be antagonism when combined with drugs. Although clinical trials have shown that the combination of cisplatin and gefitinib is antagonistic, the mechanism that causes this antagonism is still not very clear. Clarifying the mechanism that leads to antagonism can provide new ideas for clinical treatment. This topic is intended to be from two angles of autophagy and exocytosis. The effect of gefitinib and cisplatin on the therapeutic effect of cisplatin was revealed. The study of the effect and mechanism of gefitinib on the effect of autophagy induced autophagy in non small cell lung cancer cells (1) using gefitinib combined with different concentrations of cisplatin in human NSCLC cell line PC9, the MTT assay was used to determine the effect of gefitinib The effect of different concentrations of cisplatin on the proliferation of PC9 cells and the interaction index between gefitinib and cisplatin at different concentrations to analyze the effect of gefitinib on different concentrations of cisplatin by.Western-Blot method analysis gefitinib and cisplatin combined with cisplatin and the action of cisplatin alone on PC9 cell autophagic protein LC3- II and P62 Change and electron microscopy to observe the changes in the number of autophagic corpuscles in these two groups, and then understand the effect of gefitinib on the autophagy induced autophagy. (2) to further evaluate autophagy, the use of chloroquine (CQ), a lysosome inhibitor (CQ), which is a lysosome inhibitor, is used to pretreat cells for 1 hours and transmission electron microscopy. Transmission Electron Microscope (TEM) was used to confirm the degradation effect of CQ on autophagic lysosomes. Then we added gefitinib and / or cisplatin to compare the changes in LC3 expression between groups without CQ. Thus, the changes in the autophagy level of PC9 cells were determined by gefitinib and / or cisplatin, rather than autophagic degradation defects. (3) observation of inhibition (3) The effect of autophagy on the antagonism of gefitinib and cisplatin, we used MTT to detect PC9 cells in gefitinib and cisplatin group after CQ treatment, and calculated the CDI according to the measured OD value. (4) the study of Annexin V-FITC/PI flow cytometry was used to investigate the role of autophagy in the process of cisplatin resistance. The apoptosis rate of each cell after treatment was described, and the apoptosis protein Bcl-2 and Bax of the corresponding group of PC9 cells were extracted to understand the effect of the autophagy level on the apoptosis of PC9 cells. At the same time, we used the flow cytometry (Flow cytometry, FCM) to carry out the cell cycle analysis to understand the effect of autophagy on the cycle of PC9 cells. Results 1 combined with gefitinib. The antagonistic effect of cisplatin (1) was used to detect the inhibitory effect of gefitinib and cisplatin on the proliferation of PC9 cells by MTT. The results showed that the inhibition of gefitinib and cisplatin on NSCLC cells was time and concentration dependent. (2) the drug interaction coefficient (Coefficient of Drug Interaction, CDI) was used to evaluate the properties of the drug. The CDI value of gefitinib and cisplatin at different concentrations was 1.19 + 0.1 (1.07-1.35), suggesting the existence of the antagonistic effect of.2 gefitinib and cisplatin induced autophagy (1) using Western Blot to observe gefitinib and / or cisplatin. The expression of autophagic markers LC3 and p62 after treatment showed that gefitinib and cisplatin could induce the up-regulation of LC3-II transformation compared with the control group, while gefitinib and cisplatin were significantly increased in LC3-II transformation compared with the single gefitinib, but no statistical difference was found. Both gefitinib and / or cisplatin were significantly different. Lower p62 level. Compared with single drug stimulation, gefitinib and cisplatin resulted in a more significant downregulation of p62 protein, suggesting that gefitinib and cisplatin could induce higher level autophagy relative to the single drug. (2) a transmission electron microscope (Transmission electron microscopy, TEM) was used to observe the autophagic corpuscle of PC9 cells treated with gefitinib and / or cisplatin. The results showed that the number of autophagic corpuscles in gefitinib combined with cisplatin was increased in comparison with the single drug treatment group, and a higher level of autophagy could be induced by gefitinib and cisplatin relative to the single drug. (3) the Western Blot method was used to observe the autophagic marker LC of PC9 cells treated with gefitinib and / or cisplatin. 3 and p62 expression. The results suggest that gefitinib and / or cisplatin can induce autophagy in PC9 cells, instead of lysosomal degradation induced by.3 inhibition of autophagy against gefitinib and cisplatin (1) changes in the autophagic lysosomes of PC9 cells treated with gefitinib and / or cisplatin after CQ preconditioning. The results show that the changes in the autophagy of the autophagic lysosomes after the treatment of gefitinib and / or cisplatin Compared with gefitinib and / or cisplatin group, autophagic lysosomes increased significantly after adding CQ, suggesting that CQ could inhibit the degradation of autophagic lysosomes during autophagy. (2) the proliferation of PC9 cells treated with gefitinib and / or cisplatin after CQ preconditioning was detected by MTT. The results suggested that gefitinib and cisplatin were given to CQ pretreatment than in the CQ group. The synergism of gefitinib and cisplatin was increased by CQ pretreatment. (3) compared with Annexin V-FITC/PI apoptosis, CQ preconditioning could increase the proportion of apoptotic cells in each group of PC9 cells to increase the ratio of apoptotic cells, regardless of the combination of single or two drugs. Autophagy plays an important role in the process of apoptosis,.4 inhibits autophagy and promotes apoptosis (1) using flow cytometry (Flow Cytometry, FCM) to analyze the cycle of PC9 cells in each group. The results suggest that gefitinib and cisplatin can lead to the stagnation of G1 phase, however, the combined use of gefitinib and cisplatin only slightly increases the proportion of PC9 cells in G1 phase. To further confirm the relationship between autophagy and cell cycle stagnation, we pretreated gefitinib and / or cisplatin CQ (5 g/ml) for 1 hours. The results showed that the inhibition of autophagy by CQ could not reverse the cell cycle stagnation induced by gefitinib and / or cisplatin. (2) the use of Western Blot to detect CQ preconditioning could affect the apoptotic protein and resistance of PC9 cells in each group. The expression level of Bcl-2 (classic anti apoptotic family protein) and Bax (pro apoptotic protein) was significantly reduced and elevated after.CQ treatment. However, there was no significant difference in the proportion of Bax/Bcl-2 in gefitinib and / or cisplatin plus CQ preconditioning group (p=0.592,0.09 and 0.06 respectively). The results suggested CQ inhibition. Autophagy can promote the apoptosis of PC9 cells by up regulation of Bax and down regulation of Bcl-2. Study the effect of two gefitinib on the inhibition of autophagy against cisplatin induced non small cell lung cancer cell apoptosis (1) the separation of PC9 cells from normal control group or gefitinib (1 M) / cisplatin (7.5 mu M) by Exo Quick precipitate PC9 cell supernatant from gefitinib or cisplatin is extracted from the supernatant of PC9 cells, which is divided into Exo-Con (Exosomes derived from negative Control). The white detection kit carried out a standard quantitative determination of different extracellular body proteins. (2) a transmission electron microscope (TEM) was used to observe the formation of exosbodies and whether the extraction was successful. (3) the cell inhibition rate was detected by CCK-8 method. We used a certain concentration of exote for 24 hours to add 1 mu M to gefitinib or 7.5 M cisplatin. The cell proliferation activity was calculated by CCK-8 method and the cell inhibition rate was calculated by CCK-8 method. The drug interaction coefficient (Coefficient of Drug Interaction, CDI) between the two drugs was calculated. Then the exocrine inhibitor GW4869 was used in the drug treatment group to understand the inhibition of exocrine secretion to gefitinib or cisplatin. The influence of PC9 cell effect. (4) the changes of autophagic marker protein LC3 and p62 were detected by Western Blot method, and the correlation between autophagy and autophagy was detected by using both exocrine and drug incubating cells. The apoptosis of the cells was detected by Annexin V-FITC/PI reagents and flow cytometry (FCM). The effects of Bcl-2 and Bax on apoptosis of PC9 cells were detected by Western Blot. Results the antagonistic effect of 1 gefitinib combined with cisplatin was detected by CCK-8, and the effects of gefitinib and / or cisplatin on the proliferation of PC9 cells were detected. The results showed that compared with the concentration of the single drug gefitinib (0.4-2 mu), the combination of cisplatin and cisplatin was compared. The antiproliferative effect of gefitinib was only slightly increased. The CDI values of the two drugs in PC9 and A549 cells were calculated. The results suggest that the antagonistic effect of.2 Exo-GF on the antitumor activity of cisplatin and the antagonism of gefitinib and cisplatin in the combination of gefitinib and cisplatin in the EGFR mutation and the wild type NSCLC PC9 cell lines It should be mediated by Exo-GF or Exo-DDP. Collecting exosecrete, adding a certain concentration of cisplatin or gefitinib to the PC9 cell.CCK-8 results showed that Exo-GF was dose-dependent against the antitumor effect of cisplatin. However, Exo-Con had no significant effect on cisplatin induced proliferation inhibition, while Exo-DDP had little effect on gefitinib. CCK-8 The inhibition rates of gefitinib and / or cisplatin on PC9 cells were detected by GW4869 pretreatment with exo secreting inhibitors. The results showed that the inhibition rate of PC9 cells was only slightly increased after the addition of GW4869 to GW4869, but the inhibition rate of GW4869 in the combination group of gefitinib and cisplatin increased significantly, and the CDI value of the two drugs was at the same time. After adding GW4869, the inhibition of Exo-GF secretion could reverse the antagonism of gefitinib and cisplatin..3 Exo-GF up-regulated the level of autophagy induced by cisplatin. Western blot was used to detect Exo-Con, Exo-GF and Exo-DDP were used in LC3 and p62 expressions in PC9 cells. Compared with cisplatin group, Exo-GF pretreated cisplatin group significantly increased autophagy, but Exo-DDP pretreatment almost caused gefitinib induced autophagy.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2
本文編號：2132875
[Abstract]:Background and purpose: bronchial lung cancer is the most common respiratory system tumor, with high morbidity and mortality worldwide. Non small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) accounts for most of the incidence of bronchial lung cancer, and the operation is still the main means to treat NSCLC. The rate of hair is about 40%., so for patients with recurrent NSCLC and late NSCLC with no indication of operation, systemic chemotherapy and targeted therapy are still the main treatment methods. However, the common NSCLC first-line chemotherapy is still not ideal and the effective rate is only about 30%. Therefore, it is necessary to find new methods and Strategies of valuable treatment. The combination of targeted therapy and chemotherapy has become an important topic at home and abroad. Platinum group chemotherapy is still the standard first-line chemotherapy for advanced NSCLC. The first generation platinum group cisplatin is the cornerstone of NSCLC chemotherapy. However, single chemotherapy can not achieve the ideal therapeutic effect with the biological system. Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitor, which is the first representative of epidermal growth factor receptor (EGFR), is the first representative of the tyrosine kinase inhibitor, which is the first representative of the small molecule of casein. Acid kinase inhibitors, which are currently related to large three phase multicenter clinical validation of NSCLC first-line treatment, gefitinib has a distinct progression free survival (PFS) advantage for Asian, adenocarcinoma and EGFR mutations in patients with Asian, adenocarcinoma and EGFR mutations. However, in connection with chemotherapy, four large multicenter patients can be used in combination with chemotherapy. The bed test showed that the epidermal growth factor receptor tyrosine kinase inhibitor (Tyrosine-Kinase Inhibitor TKI), such as gefitinib and erlotinib, had no significant survival benefit for the total survival period of patients with advanced non-small cell lung cancer (Overall Survival, OS) when combined with chemotherapeutic drugs. There may be antagonism when combined with drugs. Although clinical trials have shown that the combination of cisplatin and gefitinib is antagonistic, the mechanism that causes this antagonism is still not very clear. Clarifying the mechanism that leads to antagonism can provide new ideas for clinical treatment. This topic is intended to be from two angles of autophagy and exocytosis. The effect of gefitinib and cisplatin on the therapeutic effect of cisplatin was revealed. The study of the effect and mechanism of gefitinib on the effect of autophagy induced autophagy in non small cell lung cancer cells (1) using gefitinib combined with different concentrations of cisplatin in human NSCLC cell line PC9, the MTT assay was used to determine the effect of gefitinib The effect of different concentrations of cisplatin on the proliferation of PC9 cells and the interaction index between gefitinib and cisplatin at different concentrations to analyze the effect of gefitinib on different concentrations of cisplatin by.Western-Blot method analysis gefitinib and cisplatin combined with cisplatin and the action of cisplatin alone on PC9 cell autophagic protein LC3- II and P62 Change and electron microscopy to observe the changes in the number of autophagic corpuscles in these two groups, and then understand the effect of gefitinib on the autophagy induced autophagy. (2) to further evaluate autophagy, the use of chloroquine (CQ), a lysosome inhibitor (CQ), which is a lysosome inhibitor, is used to pretreat cells for 1 hours and transmission electron microscopy. Transmission Electron Microscope (TEM) was used to confirm the degradation effect of CQ on autophagic lysosomes. Then we added gefitinib and / or cisplatin to compare the changes in LC3 expression between groups without CQ. Thus, the changes in the autophagy level of PC9 cells were determined by gefitinib and / or cisplatin, rather than autophagic degradation defects. (3) observation of inhibition (3) The effect of autophagy on the antagonism of gefitinib and cisplatin, we used MTT to detect PC9 cells in gefitinib and cisplatin group after CQ treatment, and calculated the CDI according to the measured OD value. (4) the study of Annexin V-FITC/PI flow cytometry was used to investigate the role of autophagy in the process of cisplatin resistance. The apoptosis rate of each cell after treatment was described, and the apoptosis protein Bcl-2 and Bax of the corresponding group of PC9 cells were extracted to understand the effect of the autophagy level on the apoptosis of PC9 cells. At the same time, we used the flow cytometry (Flow cytometry, FCM) to carry out the cell cycle analysis to understand the effect of autophagy on the cycle of PC9 cells. Results 1 combined with gefitinib. The antagonistic effect of cisplatin (1) was used to detect the inhibitory effect of gefitinib and cisplatin on the proliferation of PC9 cells by MTT. The results showed that the inhibition of gefitinib and cisplatin on NSCLC cells was time and concentration dependent. (2) the drug interaction coefficient (Coefficient of Drug Interaction, CDI) was used to evaluate the properties of the drug. The CDI value of gefitinib and cisplatin at different concentrations was 1.19 + 0.1 (1.07-1.35), suggesting the existence of the antagonistic effect of.2 gefitinib and cisplatin induced autophagy (1) using Western Blot to observe gefitinib and / or cisplatin. The expression of autophagic markers LC3 and p62 after treatment showed that gefitinib and cisplatin could induce the up-regulation of LC3-II transformation compared with the control group, while gefitinib and cisplatin were significantly increased in LC3-II transformation compared with the single gefitinib, but no statistical difference was found. Both gefitinib and / or cisplatin were significantly different. Lower p62 level. Compared with single drug stimulation, gefitinib and cisplatin resulted in a more significant downregulation of p62 protein, suggesting that gefitinib and cisplatin could induce higher level autophagy relative to the single drug. (2) a transmission electron microscope (Transmission electron microscopy, TEM) was used to observe the autophagic corpuscle of PC9 cells treated with gefitinib and / or cisplatin. The results showed that the number of autophagic corpuscles in gefitinib combined with cisplatin was increased in comparison with the single drug treatment group, and a higher level of autophagy could be induced by gefitinib and cisplatin relative to the single drug. (3) the Western Blot method was used to observe the autophagic marker LC of PC9 cells treated with gefitinib and / or cisplatin. 3 and p62 expression. The results suggest that gefitinib and / or cisplatin can induce autophagy in PC9 cells, instead of lysosomal degradation induced by.3 inhibition of autophagy against gefitinib and cisplatin (1) changes in the autophagic lysosomes of PC9 cells treated with gefitinib and / or cisplatin after CQ preconditioning. The results show that the changes in the autophagy of the autophagic lysosomes after the treatment of gefitinib and / or cisplatin Compared with gefitinib and / or cisplatin group, autophagic lysosomes increased significantly after adding CQ, suggesting that CQ could inhibit the degradation of autophagic lysosomes during autophagy. (2) the proliferation of PC9 cells treated with gefitinib and / or cisplatin after CQ preconditioning was detected by MTT. The results suggested that gefitinib and cisplatin were given to CQ pretreatment than in the CQ group. The synergism of gefitinib and cisplatin was increased by CQ pretreatment. (3) compared with Annexin V-FITC/PI apoptosis, CQ preconditioning could increase the proportion of apoptotic cells in each group of PC9 cells to increase the ratio of apoptotic cells, regardless of the combination of single or two drugs. Autophagy plays an important role in the process of apoptosis,.4 inhibits autophagy and promotes apoptosis (1) using flow cytometry (Flow Cytometry, FCM) to analyze the cycle of PC9 cells in each group. The results suggest that gefitinib and cisplatin can lead to the stagnation of G1 phase, however, the combined use of gefitinib and cisplatin only slightly increases the proportion of PC9 cells in G1 phase. To further confirm the relationship between autophagy and cell cycle stagnation, we pretreated gefitinib and / or cisplatin CQ (5 g/ml) for 1 hours. The results showed that the inhibition of autophagy by CQ could not reverse the cell cycle stagnation induced by gefitinib and / or cisplatin. (2) the use of Western Blot to detect CQ preconditioning could affect the apoptotic protein and resistance of PC9 cells in each group. The expression level of Bcl-2 (classic anti apoptotic family protein) and Bax (pro apoptotic protein) was significantly reduced and elevated after.CQ treatment. However, there was no significant difference in the proportion of Bax/Bcl-2 in gefitinib and / or cisplatin plus CQ preconditioning group (p=0.592,0.09 and 0.06 respectively). The results suggested CQ inhibition. Autophagy can promote the apoptosis of PC9 cells by up regulation of Bax and down regulation of Bcl-2. Study the effect of two gefitinib on the inhibition of autophagy against cisplatin induced non small cell lung cancer cell apoptosis (1) the separation of PC9 cells from normal control group or gefitinib (1 M) / cisplatin (7.5 mu M) by Exo Quick precipitate PC9 cell supernatant from gefitinib or cisplatin is extracted from the supernatant of PC9 cells, which is divided into Exo-Con (Exosomes derived from negative Control). The white detection kit carried out a standard quantitative determination of different extracellular body proteins. (2) a transmission electron microscope (TEM) was used to observe the formation of exosbodies and whether the extraction was successful. (3) the cell inhibition rate was detected by CCK-8 method. We used a certain concentration of exote for 24 hours to add 1 mu M to gefitinib or 7.5 M cisplatin. The cell proliferation activity was calculated by CCK-8 method and the cell inhibition rate was calculated by CCK-8 method. The drug interaction coefficient (Coefficient of Drug Interaction, CDI) between the two drugs was calculated. Then the exocrine inhibitor GW4869 was used in the drug treatment group to understand the inhibition of exocrine secretion to gefitinib or cisplatin. The influence of PC9 cell effect. (4) the changes of autophagic marker protein LC3 and p62 were detected by Western Blot method, and the correlation between autophagy and autophagy was detected by using both exocrine and drug incubating cells. The apoptosis of the cells was detected by Annexin V-FITC/PI reagents and flow cytometry (FCM). The effects of Bcl-2 and Bax on apoptosis of PC9 cells were detected by Western Blot. Results the antagonistic effect of 1 gefitinib combined with cisplatin was detected by CCK-8, and the effects of gefitinib and / or cisplatin on the proliferation of PC9 cells were detected. The results showed that compared with the concentration of the single drug gefitinib (0.4-2 mu), the combination of cisplatin and cisplatin was compared. The antiproliferative effect of gefitinib was only slightly increased. The CDI values of the two drugs in PC9 and A549 cells were calculated. The results suggest that the antagonistic effect of.2 Exo-GF on the antitumor activity of cisplatin and the antagonism of gefitinib and cisplatin in the combination of gefitinib and cisplatin in the EGFR mutation and the wild type NSCLC PC9 cell lines It should be mediated by Exo-GF or Exo-DDP. Collecting exosecrete, adding a certain concentration of cisplatin or gefitinib to the PC9 cell.CCK-8 results showed that Exo-GF was dose-dependent against the antitumor effect of cisplatin. However, Exo-Con had no significant effect on cisplatin induced proliferation inhibition, while Exo-DDP had little effect on gefitinib. CCK-8 The inhibition rates of gefitinib and / or cisplatin on PC9 cells were detected by GW4869 pretreatment with exo secreting inhibitors. The results showed that the inhibition rate of PC9 cells was only slightly increased after the addition of GW4869 to GW4869, but the inhibition rate of GW4869 in the combination group of gefitinib and cisplatin increased significantly, and the CDI value of the two drugs was at the same time. After adding GW4869, the inhibition of Exo-GF secretion could reverse the antagonism of gefitinib and cisplatin..3 Exo-GF up-regulated the level of autophagy induced by cisplatin. Western blot was used to detect Exo-Con, Exo-GF and Exo-DDP were used in LC3 and p62 expressions in PC9 cells. Compared with cisplatin group, Exo-GF pretreated cisplatin group significantly increased autophagy, but Exo-DDP pretreatment almost caused gefitinib induced autophagy.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2
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