绿原酸通过抑制PI3K-Akt信号通路诱导肺癌A549细胞线粒体功能障碍

doi:10.3760/cma.j.cn371439-20230906-00002

摘要/Abstract

摘要： 目的探讨绿原酸能否通过作用于PI3K-Akt信号通路造成线粒体功能障碍，从而抑制肺癌A549细胞增殖、迁移、侵袭和促进其凋亡。方法采用梯度浓度（0、25、50、100、150、200 μg/ml）的绿原酸干预A549细胞48 h，CCK-8实验检测细胞增殖率并计算半抑制浓度（IC₅₀）。将A549细胞分成空白组、绿原酸组（IC₅₀）和绿原酸+740YP组（IC₅₀绿原酸+50 μg/ml 740YP），干预48 h后使用细胞划痕实验检测细胞迁移距离，细胞侵袭实验检测细胞侵袭能力，流式细胞术检测细胞周期、细胞凋亡水平与线粒体膜电位变化情况，酶联免疫吸附试验检测细胞上清丙二醛（MDA）含量，蛋白质印迹法检测细胞中p-PI3K、p-Akt和Caspase3蛋白表达情况。结果绿原酸对A549细胞的IC₅₀为57.45 μg/ml。细胞划痕实验结果显示，空白组、绿原酸组和绿原酸+740YP组细胞48 h迁移距离分别为（424.80±14.43）、（289.67±18.93）和（402.22±17.99）μm，细胞侵袭实验结果显示，3组细胞48 h侵袭细胞数量分别为（96.00±6.24）、（35.33±7.64）和（83.00±2.00）个，流式细胞术结果显示，3组细胞48 h凋亡率分别为（6.15±0.17）%、（54.63±0.72）%和（17.27±0.39）%，差异均具有统计学意义（F=105.98，P<0.001；F=90.62，P<0.001；F=8 321.99，P<0.001）；与空白组相比，绿原酸组和绿原酸+740YP组细胞迁移距离和侵袭数量均减少（均P<0.05），细胞凋亡率显著升高（均P<0.001）；与绿原酸组相比，绿原酸+740YP组细胞迁移距离增加（P<0.001），细胞侵袭数量增多（P<0.001），细胞凋亡率降低（P<0.001）。流式细胞术结果显示，空白组、绿原酸组和绿原酸+740YP组细胞G₀/G₁期比例分别为（65.75±0.58）%、（55.84±0.78）%和（55.24±1.37）%，G₂/M期比例分别为（11.21±1.03）%、（20.23±0.62）%和（9.96±0.33）%，S期比例分别为（23.04±0.49）%、（23.92±1.36）%和（34.80±1.15）%，差异均具有统计学意义（F=111.02，P<0.001；F=181.26，P<0.001；F=113.05，P<0.001）；与空白组相比，绿原酸组和绿原酸+740YP组细胞G₀/G₁期比例减少（均P<0.001），绿原酸组G₂/M期比例增加（P<0.001），绿原酸+740YP组细胞S期比例增加（P<0.001）；与绿原酸组相比，绿原酸+740YP组细胞G₂/M期比例减少、S期比例增加（均P<0.001）。线粒体膜电位检测结果显示，空白组、绿原酸组和绿原酸+740YP组线粒体JC-1荧光强度分别为39.51±1.32、10.05±0.19和21.85±1.45，差异具有统计学意义（F=508.82，P<0.001）；与空白组相比，绿原酸组和绿原酸+740YP组荧光强度均降低（均P<0.001）；与绿原酸组相比，绿原酸+740YP组荧光强度升高（P<0.001）。酶联免疫吸附试验结果显示，空白组、绿原酸组和绿原酸+740YP组细胞MDA含量分别为（0.47±0.01）、（0.61±0.01）和（0.56±0.01）nmol/ml，差异具有统计学意义（F=162.30，P<0.001）；与空白组相比，绿原酸组和绿原酸+740YP组细胞MDA含量均增加（均P<0.001）；与绿原酸组相比，绿原酸+740YP组细胞MDA含量减少（P=0.001）。蛋白质印迹法结果显示，空白组、绿原酸组和绿原酸+740YP组细胞p-PI3K蛋白相对表达水平分别为1.01±0.33、0.28±0.14和0.34±0.20，p-Akt蛋白相对表达水平分别为1.00±0.16、0.43±0.05和0.95±0.14，Caspase3蛋白相对表达水平分别为1.00±0.04、1.41±0.05和0.70±0.13，差异均具有统计学意义（F=8.48，P=0.018；F=19.11，P=0.002；F=57.50，P<0.001）；与空白组相比，绿原酸组细胞p-PI3K、p-Akt蛋白表达均降低、Caspase3蛋白表达升高（均P<0.05），绿原酸+740YP组p-PI3K、Caspase3蛋白表达均降低（均P<0.05）；与绿原酸组相比，绿原酸+740YP组细胞p-Akt蛋白表达升高、Caspase3蛋白表达降低（均P<0.05）。结论绿原酸可能通过减少PI3K与Akt蛋白磷酸化抑制PI3K-Akt信号通路，造成线粒体功能受损，引起MDA累积，最终导致肺癌A549细胞功能受损与活性降低，促进细胞凋亡。

关键词: 癌，非小细胞肺, 绿原酸, 磷酸肌醇3-激酶类, 细胞凋亡, 线粒体

Abstract: Objective To investigate whether chlorogenic acid can inhibit the proliferation， migration， invasion and promote apoptosis of lung cancer A549 cells by causing mitochondrial dysfunction through PI3K-Akt pathway. Methods A549 cells were treated with chlorogenic acid at concentrations of 0， 25， 50， 100， 150， and 200 μg/ml for 48 h. CCK-8 assay was used to detect the cell proliferation rate and calculate the half maximal inhibitory concentration （IC₅₀）. A549 cells were divided into three groups： control group， chlorogenic acid group （IC₅₀） and chlorogenic acid + 740-YP group （IC₅₀ chlorogenic acid +50 μg/ml 740YP）. After 48 h of intervention， the cell migration distance was detected by cell scratch assay. Cell invasion assay was used to detect cell invasion ability. Cell cycle， apoptosis and mitochondrial membrane potential were detected by flow cytometry. The content of malondialdehyde （MDA） in cell supernatant was detected by enzyme-linked immunosorbent assay （ELISA）. Western blotting was used to detect the protein expression of p-PI3K， p-Akt and Caspase3. Results The IC₅₀ of chlorogenic acid to A549 cells was 57.45 μg/ml. The results of cell scratch assay showed that the 48 h migration distances of the control group， chlorogenic acid group and chlorogenic acid + 740YP group were （424.80±14.43），（289.67±18.93） and （402.22±17.99） μm， respectively. The results of cell invasion assay showed that the numbers of invasive cells after 48 h were 96.00±6.24， 35.33±7.64 and 83.00±2.00， and the results of flow cytometry showed that the 48 h apoptosis rates were （6.15±0.17）%，（54.63±0.72）% and （17.27±0.39）%， respectively， among the three groups with statistically significant differences （F=105.98， P<0.001； F=90.62， P<0.001； F=8 321.99， P<0.001）. Compared with the control group， the cell migration distances and invasive numbers of chlorogenic acid group and chlorogenic acid + 740YP group were decreased （all P<0.05）， while the apoptosis rates were significantly increased （both P<0.001）. Compared with chlorogenic acid group， the cell migration distance of chlorogenic acid + 740YP group increased （P<0.001）， the number of cell invasion increased （P<0.001）， and the apoptosis rate decreased （P<0.001）. The results of flow cytometry showed that the proportions of cells in G₀/G₁ phase in the control group， chlorogenic acid group and chlorogenic acid + 740YP group were （65.75±0.58）%，（55.84±0.78）% and （55.24±1.37）%， respectively. The proportions of G₂/M phase were （11.21±1.03）%，（20.23±0.62）% and （9.96±0.33）%， and the proportions of S phase were （23.04±0.49）%，（23.92±1.36）% and （34.80±1.15）%， respectively， with statistically significant differences （F=111.02， P<0.001； F=181.26， P<0.001； F=113.05， P<0.001）. Compared with the control group， the proportions of G₀/G₁ phase cells in chlorogenic acid group and chlorogenic acid + 740YP group decreased （both P<0.001）， and the proportion of G₂/M phase in chlorogenic acid group increased （P<0.001）， and the proportion of S phase cells in chlorogenic acid + 740YP group increased （P<0.001）. Compared with chlorogenic acid group， the proportion of G₂/M phase cells decreased and the proportion of S phase cells increased in chlorogenic acid + 740YP group （both P<0.001）. The results of mitochondrial membrane potential detection showed that the JC-1 fluorescence intensity of mitochondria in the control group， chlorogenic acid group and chlorogenic acid + 740YP group were 39.51±1.32， 10.05±0.19 and 21.85±1.45， respectively， with a statistically significant difference （F=508.82， P<0.001）. Compared with the control group， the fluorescence intensity of chlorogenic acid group and chlorogenic acid + 740YP group decreased （both P<0.001）. Compared with chlorogenic acid group， the fluorescence intensity of chlorogenic acid + 740YP group increased （P<0.001）. ELISA results showed that the MDA contents of the control group， chlorogenic acid group and chlorogenic acid + 740YP group were （0.47±0.01），（0.61±0.01） and （0.56±0.01） nmol/ml， respectively， with a statistically significant difference （F=162.30， P<0.001）. Compared with the control group， MDA contents in chlorogenic acid group and chlorogenic acid + 740YP group increased （both P<0.001）. Compared with chlorogenic acid group， MDA content in chlorogenic acid + 740YP group decreased （P=0.001）. Western blotting results showed that the relative protein expression levels of p-PI3K in the control group， chlorogenic acid group and chlorogenic acid + 740YP group were 1.01±0.33， 0.28±0.14 and 0.34±0.20， respectively. The relative protein expression levels of p-Akt were 1.00±0.16， 0.43±0.05 and 0.95±0.14， and the relative protein expression levels of Caspase3 were 1.00±0.04， 1.41±0.05 and 0.70±0.13， respectively， and there were statistically significant differences （F=8.48， P=0.018； F=19.11， P=0.002； F=57.50， P<0.001）. Compared with the control group， the expressions of p-PI3K and p-Akt protein in chlorogenic acid group decreased， and the expression of Caspase3 protein increased （all P<0.05）. The expressions of p-PI3K and Caspase3 protein in chlorogenic acid + 740YP group decreased （both P<0.05）. Compared with chlorogenic acid group， the expression of p-Akt protein in chlorogenic acid + 740YP group increased， and the expression of Caspase3 protein decreased （both P<0.05）. Conclusion Chlorogenic acid may inhibit the PI3K-Akt pathway by reducing the phosphorylation of PI3K and Akt proteins， resulting in the damage of mitochondrial function and the accumulation of MDA， which eventually leads to the damage of lung cancer A549 cells function and the reduction of cells activity， and then promotes cells apoptosis.

Key words: Carcinoma， non-small-cell lung, Chlorogenic acid, Phosphatidylinositol 3-kinases, Apoptosis, Mitochondria

张科平, 赵永生, 杨娟, 付茂勇. 绿原酸通过抑制PI3K-Akt信号通路诱导肺癌A549细胞线粒体功能障碍[J]. 国际肿瘤学杂志, 2024, 51(1): 21-28.

Zhang Keping, Zhao Yongsheng, Yang Juan, Fu Maoyong. Chlorogenic acid induces mitochondrial dysfunction in lung cancer A549 cells by inhibiting the PI3K-Akt pathway[J]. Journal of International Oncology, 2024, 51(1): 21-28.

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参考文献 25

[1]	Horvath L, Lang C, Boettiger K, et al. Potential subtype-specific therapeutic approaches in small cell lung cancer[J]. Curr Opin Oncol, 2024, 36(1): 51-56. DOI: 10.1097/CCO.0000000000001005.
[2]	Cai C, Yao S, Zou Y, et al. KRAS^G12C mutation-induced TOPK overexpression contributes to tumour progression in non-small cell lung cancer[J]. J Cell Mol Med, 2023, 27(12): 1637-1652. DOI: 10.1111/jcmm.17640.
[3]	Luo Y, Ma J, Lu W. The significance of mitochondrial dysfunction in cancer[J]. Int J Mol Sci, 2020, 21(16): 5598. DOI: 10.3390/ijms21165598.
[4]	Xie Y, Shi X, Sheng K, et al. PI3K/Akt signaling transduction pathway, erythropoiesis and glycolysis in hypoxia (review)[J]. Mol Med Rep, 2019, 19(2): 783-791. DOI: 10.3892/mmr.2018.9713. pmid: 30535469
[5]	刘嘉欣, 黎同明, 黄慧贤, 等. 基于PI3K/Akt信号通路探讨姜黄素诱导A549细胞凋亡的机制[J]. 中国临床药理学杂志, 2023, 39(17): 2487-2491. DOI: 10.13699/j.cnki.1001-6821.2023.17.012.
[6]	张亚玲, 刘单, 邓述恺. 贝伐珠单抗通过调控PI3K/AKT信号通路下调肺腺癌H1299细胞PD-L1表达[J]. 中国免疫学杂志, 2023, 39(1): 104-108. DOI: 10.3969/j.issn.1000-484X.2023.01.018.
[7]	Miao M, Xiang L. Pharmacological action and potential targets of chlorogenic acid[J]. Adv Pharmacol, 2020, 87: 71-88. DOI: 10. 1016/bs.apha.2019.12.002.
[8]	Singh SS, Rai SN, Birla H, et al. Effect of chlorogenic acid supplementation in MPTP-intoxicated mouse[J]. Front Pharmacol, 2018, 9: 757. DOI: 10.3389/fphar.2018.00757. pmid: 30127737
[9]	Yang H, Said AM, Huang H, et al. Chlorogenic acid depresses cellular bioenergetics to suppress pancreatic carcinoma through modulating c-Myc-TFR1 axis[J]. Phytother Res, 2021, 35(4): 2200-2210. DOI: 10.1002/ptr.6971. pmid: 33258205
[10]	Wang X, Liu J, Xie Z, et al. Chlorogenic acid inhibits proliferation and induces apoptosis in A498 human kidney cancer cells via inactivating PI3K/Akt/mTOR signalling pathway[J]. J Pharm Pharmacol, 2019, 71(7): 1100-1109. DOI: 10.1111/jphp.13095. pmid: 30989669
[11]	Rossi A, La Salvia A, Di Maio M. Chemotherapy and intercalated gefitinib or erlotinib in the treatment of advanced non-small-cell lung cancer[J]. Expert Rev Respir Med, 2017, 11(3): 171-180. DOI: 10.1080/17476348.2017.1290526.
[12]	Li J, Kwok HF. Current strategies for treating NSCLC: from biological mechanisms to clinical treatment[J]. Cancers (Basel), 2020, 12(6): 1587. DOI: 10.3390/cancers12061587.
[13]	Upadhyay R, Mohan Rao LJ. An outlook on chlorogenic acids-occurrence, chemistry, technology, and biological activities[J]. Crit Rev Food Sci Nutr, 2013, 53(9): 968-984. DOI: 10.1080/10408398.2011.576319. pmid: 23768188
[14]	Wang H, Chu W, Ye C, et al. Chlorogenic acid attenuates virulence factors and pathogenicity of Pseudomonas aeruginosa by regulating quorum sensing[J]. Appl Microbiol Biotechnol, 2019, 103(2): 903-915. DOI: 10.1007/s00253-018-9482-7. pmid: 30421108
[15]	Wang L, Du H, Chen P. Chlorogenic acid inhibits the proliferation of human lung cancer A549 cell lines by targeting annexin A2 in vitro and in vivo[J]. Biomed Pharmacother, 2020, 131: 110673. DOI: 10.1016/j.biopha.2020.110673. pmid: 32882585
[16]	Sun Z, Sun L, Tu L. GABAB receptor-mediated PI3K/Akt signa-ling pathway alleviates oxidative stress and neuronal cell injury in a rat model of Alzheimer's disease[J]. J Alzheimers Dis, 2020, 76(4): 1513-1526. DOI: 10.3233/JAD-191032.
[17]	Buskaran K, Hussein MZ, Moklas MAM, et al. Graphene oxide loaded with protocatechuic acid and chlorogenic acid dual drug nanodelivery system for human hepatocellular carcinoma therapeutic application[J]. Int J Mol Sci, 2021, 22(11): 5786. DOI: 10.3390/ijms22115786.
[18]	崔营营, 李诗扬, 赵雅男, 等. 磷脂酰肌醇蛋白聚糖1表达对肺腺癌A549细胞增殖、凋亡及周期的影响[J]. 中国生物制品学杂志, 2021, 34(10): 1191-1196. DOI: 10.13200/j.cnki.cjb.003443.
[19]	Chen Z, Tang WJ, Zhou YH, et al. Andrographolide inhibits non-small cell lung cancer cell proliferation through the activation of the mitochondrial apoptosis pathway and by reprogramming host glucose metabolism[J]. Ann Transl Med, 2021, 9(22): 1701. DOI: 10.21037/atm-21-5975. pmid: 34988210
[20]	Gao J, Qiu X, Xi G, et al. Downregulation of GSDMD attenuates tumor proliferation via the intrinsic mitochondrial apoptotic pathway and inhibition of EGFR/Akt signaling and predicts a good prognosis in non‑small cell lung cancer[J]. Oncol Rep, 2018, 40(4): 1971-1984. DOI: 10.3892/or.2018.6634. pmid: 30106450
[21]	Chen CJ, Shih YL, Yeh MY, et al. Ursolic acid induces apoptotic cell death through AIF and Endo G release through a mitochondria-dependent pathway in NCI-H292 human lung cancer cells in vitro[J]. In Vivo, 2019, 33(2): 383-391. DOI: 10.21873/invivo.11485.
[22]	Li W, Ping Z, Xuemei G, et al. Chlorogenic acid regulates the proliferation and migration of high-grade serous ovarian cancer cells through modulating the miR199a5p/DDR1 axis[J]. Acta Biochim Pol, 2022, 69(4): 855-864. DOI: 10.18388/abp.2020_6381. pmid: 36508480
[23]	Tewari D, Patni P, Bishayee A, et al. Natural products targeting the PI3K-Akt-mTOR signaling pathway in cancer: a novel therapeutic strategy[J]. Semin Cancer Biol, 2022, 80: 1-17. DOI: 10.1016/j.semcancer.2019.12.008.
[24]	熊刚, 邹华, 胡承莲, 等. 绿原酸基于PI3K/Akt信号通路对结肠癌细胞增殖和凋亡的调控作用[J]. 西部中医药, 2020, 33(5): 71-74. DOI: 10.12174/j.issn.1004-6852.2020.05.20.
[25]	刘铭, 王俊, 李丽, 等. 杜仲绿原酸通过PI3K/AKT/Nrf-2通路增强视网膜母细胞瘤细胞系HXO-Rb44放射敏感性[J]. 中国临床解剖学杂志, 2019, 37(6): 644-649, 655. DOI: 10.13418/j.issn.1001-165x.2019.06.008.