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Medicina

探究红景天苷抑制MCF-7细胞增殖和迁移的药理作用及分子机制

Published: June 09, 2023
doi:
10.3791/65634
, , , , , , ,
1Pathology Department,Suining Central Hospital, 2General Surgery Day Ward, Department of General Surgery,the Third People’s Hospital of Chengdu & the Affiliated Hospital of Southwest Jiaotong University

Summary

本方案描述了一种综合策略，用于评估红景天苷在抑制MCF-7细胞增殖和迁移中的药理作用和机制。

Abstract

红景天苷（Sal）具有抗癌、抗缺氧和抗炎药理活性。然而，其潜在的抗乳腺癌机制尚未完全阐明。因此，该协议旨在解码Sal在调节人乳腺癌MCF-7细胞恶性增殖中PI3K-AKT-HIF-1α-FoxO1通路的潜力。首先，通过CCK-8和细胞划痕试验评估Sal对MCF-7的药理活性。此外，通过迁移和基质胶侵袭试验测量MCF-7细胞的抗性。对于细胞凋亡和周期测定，分别使用膜联蛋白 V-FITC/PI 和细胞周期染色检测试剂盒分步处理 MCF-7 细胞进行流式细胞术分析。DCFH-DA和Fluo-4 AM免疫荧光染色检测活性氧（ROS）和Ca2+水平。使用相应的商业试剂盒测定Na+-K+-ATPase和Ca2+-ATPase的活性。分别采用Western blot和qRT-PCR分析进一步测定细胞凋亡和PI3K-AKT-HIF-1α-FoxO1通路中的蛋白和基因表达水平。我们发现Sal治疗显着限制了MCF-7细胞的增殖、迁移和侵袭，并具有剂量依赖性效应。同时，Sal 给药还显着迫使 MCF-7 细胞发生凋亡和细胞周期停滞。免疫荧光试验显示，Sal可观察到刺激MCF-7细胞中ROS和Ca2+的产生。进一步数据证实，Sal促进了促凋亡蛋白Bax、Bim、裂解caspase-9/7/3及其相应基因的表达水平。一致地，Sal 干预显着降低了 Bcl-2、p-PI3K/PI3K、p-AKT/AKT、mTOR、HIF-1α 和 FoxO1 蛋白及其相应基因的表达。总之，Sal 可作为治疗乳腺癌的潜在草药衍生化合物，因为它可以通过抑制 PI3K-AKT-HIF-1α-FoxO1 通路来减少 MCF-7 细胞的恶性增殖、迁移和侵袭。

Introduction

作为最常见的癌症和最常见的恶性肿瘤之一，最新统计数据显示，截至2020年，全球出现了230万例乳腺癌病例，占所有癌症病例的11.7%1。乳腺癌的常见症状包括乳房压痛和刺痛、乳房肿块和疼痛、溢液、皮肤糜烂或凹陷以及腋窝淋巴结肿大 1,2。更令人震惊的是，乳腺癌的新病例数和总体发病率每年继续以惊人的速度增长，占癌症相关死亡人数的6.9%1。目前，乳腺癌干预仍主要涉及化疗、手术、放疗和综合治疗。虽然治疗可以有效降低患者的复发率和死亡率，但长期应用治疗往往会引起产生多重耐药性、大面积脱发、恶心呕吐，以及严重的精神和心理负担2,3。值得注意的是，乳腺癌多器官转移的潜在风险也迫使人们寻求新的草药来源的药物治疗4,5。

磷酸肌醇 3 激酶 （PI3K） 介导的信号转导通过影响多个基因表达的剪接与乳腺癌的生长、增殖和存活有关6.作为 PI3K 的下游信号感应蛋白，大量证据表明蛋白激酶 B （AKT） 可以与哺乳动物雷帕霉素靶标 （mTOR） 蛋白偶联，以进一步增加乳腺癌 7,8,9。此外，PI3K/AKT/mTOR 信号转导的失活也被认为是抑制乳腺癌恶性增殖和刺激细胞凋亡的药物的关键成分10,11,12。众所周知，肿瘤微环境中的极度缺氧迫使缺氧诱导因子 1 α （HIF-1α） 大量激增，这进一步恶化了乳腺癌的进展13,14,15。同时，AKT 刺激也会导致 HIF-1α 的过度积累，从而限制乳腺癌样本中的细胞凋亡16,17。有趣的是，PI3K-AKT-HIF-1α 信号转导的激活已被证实参与多种癌症的病理进展和转移，包括肺癌18、结直肠癌19、卵巢癌20 和前列腺癌21。除了由 HIF-1α 协调外，AKT 信号刺激还触发分叉头转录因子 1 （FoxO1） 过表达，从而促进乳腺癌细胞的周期停滞和细胞凋亡抑制 22,23。综上所述，上述确凿证据表明，抑制PI3K-AKT-HIF-1α-FoxO1信号转导的级联反应可能是乳腺癌药物治疗的潜在新靶点。

红景天苷 （Sal） 已被广泛证明具有抗癌 24,25、抗缺氧26、27、28、29 和增强免疫力的药理活性30。它是一种浅棕色或棕色粉末，易溶于水，是苯乙烷苷的一种，化学式为C14H 20 O7，分子量为300.331,32。现代药理学研究表明，Sal可以通过抑制PI3K-AKT-mTOR信号传导来促进胃癌细胞的凋亡24。进一步的证据还表明，Sal 治疗对 PI3K-AKT-HIF-1α 信号传导的抑制可能通过增强癌细胞对化疗药物的敏感性来促进癌细胞的凋亡25。证据还表明，Sal 通过促进人乳腺癌 MCF-7 细胞的凋亡来限制细胞迁移和侵袭并导致周期停滞33,34。然而，Sal能否调控PI3K-AKT-HIF-1α-FoxO1信号传导并抑制MCF-7细胞的恶性增殖还有待观察。因此，该方案旨在通过PI3K-AKT-HIF-1α-FoxO1途径探索Sal对MCF-7细胞迁移，侵袭，细胞周期和细胞凋亡的影响。采用常规、低成本、自主实验的综合研究策略，如细胞迁移和侵袭评估、流式细胞术细胞凋亡和细胞周期检测、活性氧（ROS）和Ca2+荧光测定等，可为传统草药抗癌研究的实验整体设计提供参考。本研究的实验过程如图1所示。

Protocol

用于本研究的 MCF-7 细胞是从商业来源获得的（参见 材料表）。 1. 细胞培养 在37°C的湿润5%CO2 气氛中用含有10S和1%青霉素（10,000U / mL）/链霉素（10,000μg/ mL）的DMEM培养MCF-7细胞（参见 材料表）。注：覆盖培养皿底部90%的细胞用于实验，并分为以下组：对照组，盐酸多柔比星组（DXR，5μM）和Sal组（20μM，40μM和80μM），?…

Representative Results

Sal 对抑制 MCF-7 细胞过度增殖和延缓伤口愈合的影响为了探究 Sal 对抗乳腺癌的潜力，我们首先使用细胞增殖毒性和人乳腺癌 MCF-7 细胞系的划痕测定测试了其抗癌特性。将这些细胞与浓度系列的Sal（5-320μM）共同孵育24小时，并使用CCK-8测定法评估细胞增殖。观察到Sal对细胞增殖的剂量依赖性抑制作用，在40μM时细胞活力下降50%（图2A）。然后选择 20 μM、40 μM ?…

Discussion

乳腺癌影响所有年龄段的人，并造成不可估量的身心负担和巨大的经济压力1.乳腺癌的发病率和死亡率逐年增加，在寻求传统治疗之外的有效草药复合疗法方面也引起了全世界的关注4,5。有希望的是，大量证据揭示了 Sal24,25,38 的抗癌作用。不幸的是，Sal在乳腺癌中?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了四川省卫生健康委员会（120025）的支持。

Materials

1% penicillin/streptomycin HyClone SV30010
AKT antibody ImmunoWay Biotechnology Company YT0185
Annexin V-FITC/PI kit MultiSciences Biotech Co., Ltd. AP101
Automatic microplate reader Molecular Devices SpectraMax iD5
Bax antibody Cell Signaling Technology, Inc. #5023
BCA kit Biosharp Life Sciences BL521A
Bcl-2 antibody Cell Signaling Technology, Inc. #15071
Bim antibody Cell Signaling Technology, Inc. #2933
Ca2+–ATPase assay kit Nanjing Jiancheng Bioengineering Institute A070-4-2
Cell counting kit-8 Biosharp Life Sciences BS350B
Cell cycle staining kit MultiSciences Biotech Co., Ltd. CCS012
cleaved caspase-3 Cell Signaling Technology, Inc. #9661
cleaved caspase-7 Cell Signaling Technology, Inc. #8438
cleaved caspase-9 Cell Signaling Technology, Inc. #20750
Crystal violet solution Beyotime Biotechnology C0121
DMEM high glucose culture medium Servicebio Technology Co., Ltd. G4510
Doxorubicin hydrochloride MedChemExpress HY-15142
ECL chemiluminescent solution Biosharp Life Sciences BL520B
Fetal bovine serum Procell Life Science & Technology Co., Ltd. 164210
Flow cytometer BD FACSCanto Equation 1
Fluo-4 AM Beyotime Biotechnology S1060
FoxO1 antibody ImmunoWay Biotechnology Company YT1758
Goat anti-rabbit IgG secondary antibody MultiSciences Biotech Co., Ltd. 70-GAR0072
GraphPad Prism software La Jolla Version 6.0
HIF-1α antibody Affinity Biosciences BF8002
Human breast cancer cell line MCF-7 Procell Life Science & Technology Co., Ltd. CL-0149
Loading buffer Biosharp Life Sciences BL502B
LY294002 MedChemExpress HY-10108
Matrigel Thermo  356234
mTOR antibody Servicebio Technology Co., Ltd. GB11405
Na+–K+–ATPase assay kit Nanjing Jiancheng Bioengineering Institute A070-2-2
Optical microscope Olympus IX71PH
p-AKT antibody ImmunoWay Biotechnology Company YP0006
PI3K antibody Servicebio Technology Co., Ltd. GB11525
p-PI3K antibody Affinity Biosciences AF3241
Quantitative western blot imaging system Touch Image Pro eBlot
Reverse transcription first strand cDNA synthesis kit Servicebio Technology Co., Ltd. G3330-100
ROS assay kit Beyotime Biotechnology S0033S DCFH-DA fluorescence probe is included here
Salidroside Chengdu Herbpurify Co., Ltd. H-040
SDS-PAGE kit Servicebio Technology Co., Ltd. G2003-50T
Total RNA isolation kit Foregene RE-03014
Trypsin HyClone SH30042.01
β-actin Affinity Biosciences AF7018
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Tags

Keywords: SalidrosideMCF-7 CellsBreast CancerPI3K/AKT/HIF-1α/FoxO1 SignalingProliferationMigrationInvasionApoptosisCell CycleROSCa2+Na+-K+-ATPaseCa2+-ATPase
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Cui, L., Ye, C., Luo, T., Jiang, H., Lai, B., Wang, H., Chen, Z., Li, Y. Exploring the Pharmacological Action and Molecular Mechanism of Salidroside in Inhibiting MCF-7 Cell Proliferation and Migration. J. Vis. Exp. (196), e65634, doi:10.3791/65634 (2023).
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