奥希替尼联合槲皮素对H1975细胞增殖的协同抑制作用
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摘要
目的 探索奥希替尼联合槲皮素对非小细胞肺癌(NSCLC)H1975细胞的增殖抑制作用以及潜在的作用机制。方法 采用MTT法检测2.625、5.25、10.5、21、42、84 nmol/L奥希替尼和15.625、31.25、62.5、125、250、500μmol/L槲皮素对细胞增殖的抑制效应(设置单一药物组及药物联合组),根据检测的结果计算得出奥希替尼和槲皮素对细胞的半数抑制浓度(IC_(50))以及联合指数(CI)。单药组、两药联合组及空白对照组的细胞凋亡率采用流式细胞仪进行检测。Western blot检测各组细胞中Akt/p-Akt、Bcl-2及Bax蛋白的表达水平变化。结果 不同浓度的奥希替尼和槲皮素均可抑制H1975细胞的增殖(P<0.01),IC_(50)值分别为(21.54±1.05) nmol/L和(109.7±1.08)μmol/L;各浓度奥希替尼与槲皮素联合时的CI值均<1,表现为协同抑制效应。两药联合作用后表现出更强的诱导细胞凋亡作用。奥希替尼联合槲皮素组相对两药单药组可观察到p-Akt及Bcl-2蛋白的表达明显下调(P<0.01),而两药联合组Bax蛋白的表达水平显著高于单药组(P<0.01)。结论 奥希替尼联合槲皮素表现出的协同效应使H1975细胞增殖受到明显抑制,其机制可能与药物作用使p-Akt蛋白的表达下调以及促细胞凋亡有关。
Objective To explore the effect of osimertinib combined with quercetin inhibiting non-small cell lung cancer(NSCLC) H1975 cells proliferation and potential mechanism. Methods MTT method was used to detect the effects of osimertinib(2.625, 5.25, 10.5, 21, 42 and 84 nmol/L) and quercetin(15.625, 31.25, 62.5, 125, 250 and 500 μmol/L) as single drug and the combination of the two drugs on proliferation of H1975 cells. The half inhibitory concentration(IC_(50)) and the combination index(CI) values of osimertinib and quercetin on H1975 cells were calculated.The apoptotic rate of each group of H1975 cells was detected by flow cytometry.The changes of Akt/p-Akt, bcl-2 and bax protein expression levels in cells of each group were tested by Western blot. Results Osimertinib and quercetin at different concentrations could inhibit the proliferation of H1975 cells(both P<0.01). The IC_(50) values were(21.54±1.05)nmol/L and(109.7±1.08)μmol/L, respectively.CI values were < 1 when osimertinib combined with quercetin at all concentrations, showing a synergistic inhibitory effect.The combination of the two drugs showed a stronger effect of inducing apoptosis.The expressions of p-Akt and bcl-2 proteins in the osimertinib combined with quercetin group were significantly down-regulated compared with the single drug groups(both P<0.01), while the bax protein expression level in the combination group was significantly higher than that in the single drug groups(P<0.01). Conclusion Osimertinib and quercetin can exert synergistic action,which can significantly inhibit H1975 cell proliferation.The mechanism may be related to the down-regulation of p-Akt protein expression and the promotion of apoptosis.
Objective To explore the effect of osimertinib combined with quercetin inhibiting non-small cell lung cancer(NSCLC) H1975 cells proliferation and potential mechanism. Methods MTT method was used to detect the effects of osimertinib(2.625, 5.25, 10.5, 21, 42 and 84 nmol/L) and quercetin(15.625, 31.25, 62.5, 125, 250 and 500 μmol/L) as single drug and the combination of the two drugs on proliferation of H1975 cells. The half inhibitory concentration(IC_(50)) and the combination index(CI) values of osimertinib and quercetin on H1975 cells were calculated.The apoptotic rate of each group of H1975 cells was detected by flow cytometry.The changes of Akt/p-Akt, bcl-2 and bax protein expression levels in cells of each group were tested by Western blot. Results Osimertinib and quercetin at different concentrations could inhibit the proliferation of H1975 cells(both P<0.01). The IC_(50) values were(21.54±1.05)nmol/L and(109.7±1.08)μmol/L, respectively.CI values were < 1 when osimertinib combined with quercetin at all concentrations, showing a synergistic inhibitory effect.The combination of the two drugs showed a stronger effect of inducing apoptosis.The expressions of p-Akt and bcl-2 proteins in the osimertinib combined with quercetin group were significantly down-regulated compared with the single drug groups(both P<0.01), while the bax protein expression level in the combination group was significantly higher than that in the single drug groups(P<0.01). Conclusion Osimertinib and quercetin can exert synergistic action,which can significantly inhibit H1975 cell proliferation.The mechanism may be related to the down-regulation of p-Akt protein expression and the promotion of apoptosis.
引文
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[2] Chen W,Zheng R,Zhang S,et al.Annual report on status of cancer in China,2010[J].Chin J Cancer Res,2014,26(1):48-58.
[3] Saad N,Poudel A,Basnet A,et al.Epidermal growth factor receptor T790M mutation-positive metastatic non-small-cell lung cancer:focus on osimertinib (AZD9291)[J].Onco Targets Ther,2017,10:1757-66.
[4] Wu M,Yuan Y,Pan Y Y,et al.Combined gefitinib and pemetrexed overcome the acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer[J].Mol Med Rep,2014,10(2):931-8.
[5] Cross D A,Ashton S E,Ghiorghiu S,et al.AZD9291,an irreversible EGFR TKI,overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer[J].Cancer Discov,2014,4(9):1046-61.
[6] Lee S H,Lee E J,Min K H,et al.Quercetin enhances chemosensitivity to gemcitabine in lung cancer cells by inhibiting heat shock protein 70 expression[J].Clin Lung Cancer,2015,16(6):e235-43.
[7] 王静,袁媛,潘跃银,等.吉非替尼联合槲皮素对非小细胞肺癌PC9/GR细胞增殖的协同抑制作用[J].肿瘤,2018,38(3):182-8.
[8] Midha A,Dearden S,Mccormack R.EGFR mutation incidence in non-small cell lung cancer of adenocarcinoma histology:a systematic review and global map by ethnicity (mutmapII)[J].Am J Cancer Res,2015,5(9):2892-911.
[9] Martin M J,Eberlein C,Taylor M,et al.Inhibition of oxidative phosphorylation suppresses the development of osimertinib resistance in a preclinical model of EGFR-driven lung adenocarcinoma[J].Oncotarget,2016,7(52):86313-25.
[10] J?nne P A,Yang J C,Kim D W,et al.AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer[J].N Engl J Med,2015,372(18):1689-99.
[11] Mukherjee A,Khuda-Bukhsh A R.Quercetin down-regulates IL-6/STAT-3 signals to induce mitochondrial-mediated apoptosis in a nonsmall-cell lung-cancer cell line,A549[J].J Pharmacopuncture,2015,18(1):19-26.
[12] Song B Q,Chi Y,Li X,et al.Inhibition of notch signaling promotes the adipogenic differentiation of mesenchymal stem cells through autophagy activation and PTEN-PI3K/AKT/mTOR pathway[J].Cell Physiol Biochem,2015,36(5):1991-2002.
[13] Wang H J,Yang Z X,Dai X T,et al.Bisdemethoxycurcumin sensitizes cisplatin-resistant lung cancer cells to chemotherapy by inhibition of CA916798 and PI3K/AKT signaling[J].Apoptosis,2017,22(9):1157-68.
[14] 赵楠,王红,穆春青,等.雷公藤红素抑制A549人肺癌细胞的增殖并增加其凋亡[J].细胞与分子免疫学杂志,2018,34(12):1111-5.