木犀草素通过增加ROS的产生和下调AKT/mTOR通路及HO-1蛋白表达抑制肺癌A549细胞增殖

doi:10.12122/j.issn.1673-4254.2024.12.12

摘要/Abstract

摘要：

目的探究木犀草素（Lut）对肺癌A549细胞增殖的抑制作用及其内在机制。方法用不同浓度的Lut处理A549细胞48 h，通过MTT法检测细胞活性，通过平板克隆和EdU染色检测细胞增殖，通过DCFH-DA法检测细胞活性氧（ROS）水平，通过Hoechst33258 染色法检测细胞凋亡水平，通过MDC染色法检测细胞自噬水平，通过Western blotting实验检测细胞凋亡相关蛋白Bax、Bcl-2、Cleaved caspase-9，自噬相关蛋白LC3B、Beclin1、P62，AKT/mTOR通路蛋白以及HO-1蛋白的表达。结果 Lut剂量依赖性的抑制A549细胞的活力和增殖能力（P<0.05），引发细胞内ROS水平增加（P<0.05），上调凋亡相关蛋白Bax、Cleaved caspase-9和自噬相关蛋白Beclin1的表达，增加LC3B-II/LC3B-I的比值，下调抗凋亡蛋白Bcl-2和自噬相关蛋白P62的表达，诱导细胞凋亡和自噬（P<0.001）。此外，Lut可显著抑制AKT和mTOR的磷酸化，下调HO-1蛋白的表达（P<0.05）。结论 Lut通过增加细胞内ROS的产生，抑制AKT/mTOR通路以及下调HO-1蛋白水平诱导A549细胞的凋亡和自噬。

关键词: 木犀草素, 活性氧, 凋亡, 自噬, AKT/mTOR通路, HO-1

Abstract:

Objective To investigate the mechanism of luteolin for inhibiting proliferation of lung cancer A549 cells. Methods A549 cells treated with different concentrations of luteolin for 48 h were evaluated for changes in cell viability, proliferation, reactive oxygen species (ROS) production and apoptosis using MTT assay, plate cloning assay, EdU staining, DCFH-DA assay and Hoechst33258 staining. The changes in cell autophagy were examined with MDC staining, and the expressions of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-9), autophagy-related proteins (LC3B, Beclin 1, and P62), AKT/mTOR pathway proteins, and HO-1 protein were detected using Western blotting. Results Treatment with luteolin dose-dependently inhibited the viability and proliferation of A549 cells, increased intracellular ROS levels, up-regulated the expressions of Bax, cleaved caspase-9, and Beclin 1, increased the LC3B-II/LC3B-I ratio, down-regulated the expressions of Bcl-2 and P62, and induced cell apoptosis and autophagy. Luteolin also significantly inhibited the phosphorylation of AKT and mTOR and down-regulated the expression of HO-1 protein in the cells. Conclusion Luteolin induces apoptosis and autophagy to inhibit proliferation of A549 cells by increasing ROS production, inhibiting the AKT/mTOR pathway and down-regulating HO-1 protein expression.

Key words: luteolin, reactive oxygen species, apoptosis, autophagy, AKT/mTOR signaling pathway, HO-1

李欢, 邱紫欣, 徐文洁, 陈雪, 魏典典, 王允. 木犀草素通过增加ROS的产生和下调AKT/mTOR通路及HO-1蛋白表达抑制肺癌A549细胞增殖[J]. 南方医科大学学报, 2024, 44(12): 2367-2374.

Huan LI, Zixin QIU, Wenjie XU, Xue CHEN, Diandian WEI, Yun WANG. Luteolin inhibits proliferation of lung cancer A549 cells by increasing ROS production and inhibiting the AKT/mTOR signaling pathway and HO-1 expression[J]. Journal of Southern Medical University, 2024, 44(12): 2367-2374.

图/表 5

图1 Lut抑制A549细胞的活力、增殖和克隆形成能力

Fig.1 Luteolin (Lut) inhibits viability, proliferation, and clone-forming ability of A549 cells. A: MTT assay of A549 cells treated with different concentrations of luteolin for 48 h. B: MTT assay of A549 and Beas-2B cells treated with 20 and 40 μmol/L luteolin for 48 h. C, D: EdU assay of A549 cells treated with 20 and 40 μmol/L luteolin for 48 h (Original magnification: ×100). E, F: Clone formation assay of A549 cells treated with 20 and 40 μmol/L luteolin for 48 h. *P<0.05, **P<0.01, ***P<0.001 vs control.

图2 Lut诱导A549细胞氧化应激

Fig.2 Luteolin induces oxidative stress in A549 cells. A, B: Detection of intracellular ROS levels in luteolin-treated A549 cells by flow cytometry. C: MTT assay of A549 cells treated with luteolin in the presence or absence of NAC for 48 h. *P<0.05, ***P<0.001vs control; ##P<0.01,###P<0.001 vs NAC- group.

图3 Lut诱导A549细胞凋亡

Fig.3 Luteolin induces apoptosis of A549 cells. A: Hoechst 33258 staining of luteolin-treated A549 cells (×100). B-D: Western blotting for detecting expression levels of Bax, Bcl-2, cleaved-caspase-9 in luteolin-treated A549 cells. ***P<0.001 vs control.

图4 Lut诱导A549细胞自噬

Fig.4 Luteolin induces autophagy in A549 cells. A, B: MDC staining for detecting autophagy in luteolin-treated A549 cells (×100). C-F: Western blotting for detecting expression levels of autophagy-related proteins Beclin 1, P62, and LC3B in luteolin-treated A549 cells. *P<0.05, ***P<0.001 vs control.

图5 Lut对A549细胞AKT/mTOR通路、HO-1蛋白水平的改变

Fig.5 Luteolin-induced changes in AKT/mTOR signaling and HO-1 protein levels in A549 cells. A-D: Western blotting for detecting expression levels of AKT, P-AKT, mTOR, P-mTOR, and HO-1 proteins in luteolin-treated A549 cells. E, F: Western blotting for detecting HO-1 protein expression levels in A549 cells treated with luteolin in the presence or absence of LY294002 for 48 h. G-I: Western blotting of P-AKT and P-mTOR protein expression levels in A549 cells treated with luteolin in the presence or absence of NAC for 48 h. ***P<0.001 vs control; #P<0.05, ###P<0.001 vs Lut group.

参考文献 40

1	He SY, Xia CF, Li H, et al. Cancer profiles in China and comparisons with the USA: a comprehensive analysis in the incidence, mortality, survival, staging, and attribution to risk factors[J]. Sci China Life Sci, 2024, 67(1): 122-31.
2	Varghese R, Efferth T, Ramamoorthy S. Carotenoids for lung cancer chemoprevention and chemotherapy: promises and controversies[J]. Phytomedicine, 2023, 116: 154850.
3	Klaunig JE, Kamendulis LM, Hocevar BA. Oxidative stress and oxidative damage in carcinogenesis[J]. Toxicol Pathol, 2010, 38(1): 96-109.
4	Panieri E, Santoro MM. ROS homeostasis and metabolism: a dangerous liason in cancer cells[J]. Cell Death Dis, 2016, 7(6): e2253.
5	Huang R, Chen H, Liang JY, et al. Dual role of reactive oxygen species and their application in cancer therapy[J]. J Cancer, 2021, 12(18): 5543-61.
6	Zhang HB, Ma L, Kim E, et al. Rhein induces oral cancer cell apoptosis and ROS via suppresse AKT/mTOR signaling pathway in vitro and in vivo [J]. Int J Mol Sci, 2023, 24(10): 8507.
7	Liou YF, Chen PN, Chu SC, et al. Thymoquinone suppresses the proliferation of renal cell carcinoma cells via reactive oxygen species-induced apoptosis and reduces cell stemness[J]. Environ Toxicol, 2019, 34(11): 1208-20.
8	Gong XM, Smith JR, Swanson HM, et al. Carotenoid lutein selectively inhibits breast cancer cell growth and potentiates the effect of chemotherapeutic agents through ROS-mediated mechanisms[J]. Molecules, 2018, 23(4): 905.
9	Fan JJ, Ren DM, Wang JX, et al. Bruceine D induces lung cancer cell apoptosis and autophagy via the ROS/MAPK signaling pathway in vitro and in vivo [J]. Cell Death Dis, 2020, 11(2): 126.
10	NavaneethaKrishnan S, Rosales JL, Lee KY. ROS-mediated cancer cell killing through dietary phytochemicals[J]. Oxid Med Cell Longev, 2019, 2019: 9051542.
11	Yao WS, Lin Z, Shi PY, et al. Delicaflavone induces ROS-mediated apoptosis and inhibits PI3K/AKT/mTOR and Ras/MEK/Erk signaling pathways in colorectal cancer cells[J]. Biochem Pharmacol, 2020, 171: 113680.
12	Liu YH, Shi CJ, He Z, et al. Inhibition of PI3K/AKT signaling via ROS regulation is involved in Rhein-induced apoptosis and enhancement of oxaliplatin sensitivity in pancreatic cancer cells[J]. Int J Biol Sci, 2021, 17(2): 589-602.
13	Imran M, Rauf A, Abu-Izneid T, et al. Luteolin, a flavonoid, as an anticancer agent: a review[J]. Biomed Pharmacother, 2019, 112: 108612.
14	You YJ, Wang R, Shao NY, et al. Luteolin suppresses tumor proliferation through inducing apoptosis and autophagy via MAPK activation in glioma[J]. Onco Targets Ther, 2019, 12: 2383-96.
15	Wu L, Lin YD, Gao SY, et al. Luteolin inhibits triple-negative breast cancer by inducing apoptosis and autophagy through SGK1-FOXO3a-BNIP3 signaling[J]. Front Pharmacol, 2023, 14: 1200843.
16	Yang HB, Zhao YY, Song W, et al. The inhibition of β‑catenin activity by luteolin isolated from Paulownia flowers leads to growth arrest and apoptosis in cholangiocarcinoma[J]. Int J Biol Macromol, 2024, 254(Pt 1): 127627.
17	Geng AZ, Luo L, Ren FY, et al. MiR-29a-3p inhibits endometrial cancer cell proliferation, migration and invasion by targeting VEGFA/CD C42/PAK1[J]. BMC Cancer, 2021, 21(1): 843.
18	Zhou XJ, Chen Y, Wang FF, et al. Artesunate induces autophagy dependent apoptosis through upregulating ROS and activating AMPK-mTOR-ULK1 axis in human bladder cancer cells[J]. Chem Biol Interact, 2020, 331: 109273.
19	Liu SJ, Bu QQ, Tong JS, et al. MiR-486 responds to apoptosis and autophagy by repressing SRSF3 expression in ovarian granulosa cells of dairy goats[J]. Int J Mol Sci, 2023, 24(10): 8751.
20	Zhao YJ, Guo WY, Gu XL, et al. Repression of deoxynivalenol-triggered cytotoxicity and apoptosis by mannan/β‑glucans from yeast cell wall: involvement of autophagy and PI3K-AKT-mTOR signaling pathway[J]. Int J Biol Macromol, 2020, 164: 1413-21.
21	Chu N, Zhang X, Chen S, et al. Luteolin has a significant protective effect against cadmium-induced injury in lung epithelial Beas-2B cells[J]. J South Med Univ, 2021, 41(5): 729-35.
22	Hseu YC, Lee MS, Wu CR, et al. The chalcone flavokawain B induces G2/M cell-cycle arrest and apoptosis in human oral carcinoma HSC-3 cells through the intracellular ROS generation and downregulation of the Akt/p38 MAPK signaling pathway[J]. J Agric Food Chem, 2012, 60(9): 2385-97.
23	Amawi H, Ashby CR Jr, Tiwari AK. Cancer chemoprevention through dietary flavonoids: what's limiting[J]? Chin J Cancer, 2017, 36(1): 50.
24	Holmström KM, Finkel T. Cellular mechanisms and physiological consequences of redox-dependent signalling[J]. Nat Rev Mol Cell Biol, 2014, 15(6): 411-21.
25	Li XM, Hou YN, Zhao JT, et al. Combination of chemotherapy and oxidative stress to enhance cancer cell apoptosis[J]. Chem Sci, 2020, 11(12): 3215-22.
26	Ullah A, Razzaq A, Alfaifi MY, et al. Sanguinarine attenuates lung cancer progression via oxidative stress-induced cell apoptosis[J]. Curr Mol Pharmacol, 2024, 17: e18761429269383.
27	Wang T, Wu X, Al Rudaisat M, et al. Curcumin induces G2/M arrest and triggers autophagy, ROS generation and cell senescence in cervical cancer cells[J]. J Cancer, 2020, 11(22): 6704-15.
28	Wang Q, Wang HD, Jia Y, et al. Luteolin induces apoptosis by ROS/ER stress and mitochondrial dysfunction in gliomablastoma[J]. Cancer Chemother Pharmacol, 2017, 79(5): 1031-41.
29	Zhou CC, Qian WK, Li J, et al. High glucose microenvironment accelerates tumor growth via SREBP1-autophagy axis in pancreatic cancer[J]. J Exp Clin Cancer Res, 2019, 38(1): 302.
30	Zhang N, Xue ML, Wang Q, et al. Inhibition of fucoidan on breast cancer cells and potential enhancement of their sensitivity to chemotherapy by regulating autophagy[J]. Phytother Res, 2021, 35(12): 6904-17.
31	Sun Y, Qiao YN, Liu Y, et al. Ent-Kaurane diterpenoids induce apoptosis and ferroptosis through targeting redox resetting to overcome cisplatin resistance[J]. Redox Biol, 2021, 43: 101977.
32	Pistritto G, Trisciuoglio D, Ceci C, et al. Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies[J]. Aging, 2016, 8(4): 603-19.
33	Sahoo G, Samal D, Khandayataray P, et al. A review on caspases: key regulators of biological activities and apoptosis[J]. Mol Neurobiol, 2023, 60(10): 5805-37.
34	Bortnik S, Gorski SM. Clinical applications of autophagy proteins in cancer: from potential targets to biomarkers[J]. Int J Mol Sci, 2017, 18(7): 1496.
35	Jovanović L, Nikolić A, Dragičević S, et al. Prognostic relevance of autophagy-related markers p62, LC3, and Beclin1 in ovarian cancer[J]. Croat Med J, 2022, 63(5): 453-60.
36	Li W, Cai ZN, Mehmood S, et al. Polysaccharide FMP-1 from Morchella esculenta attenuates cellular oxidative damage in human alveolar epithelial A549 cells through PI3K/AKT/Nrf2/HO-1 pathway[J]. Int J Biol Macromol, 2018, 120(Pt A): 865-75.
37	Li H, Song F, Duan LR, et al. Paeonol and danshensu combination attenuates apoptosis in myocardial infarcted rats by inhibiting oxidative stress: roles of Nrf2/HO-1 and PI3K/Akt pathway[J]. Sci Rep, 2016, 6: 23693.
38	Hu YW, Liu Y, Guo EY, et al. Naphtho-γ-pyrone dimers from an endozoic Aspergillus niger and the effects of coisolated monomers in combination with cisplatin on a cisplatin-resistant A549 cell line[J]. J Nat Prod, 2021, 84(7): 1889-97.
39	Kim YC, Guan KL. mTOR: a pharmacologic target for autophagy regulation[J]. J Clin Invest, 2015, 125(1): 25-32.
40	Yang JL, Pi CC, Wang GH. Inhibition of PI3K/Akt/mTOR pathway by apigenin induces apoptosis and autophagy in hepatocellular carcinoma cells[J]. Biomed Pharmacother, 2018, 103: 699-707.

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