藤梨根乙醇提取物通过AKT/PUMA通路诱导结直肠癌细胞凋亡

王晓戎; 黄禹舒; 陈仁杰; 陈欣德; 张田革; NISHIMWE Ange; 程峰; 吴志浩

doi:10.14148/j.issn.1672-0482.2023.0532

藤梨根乙醇提取物通过AKT/PUMA通路诱导结直肠癌细胞凋亡

doi: 10.14148/j.issn.1672-0482.2023.0532

1.
安徽中医药高等专科学校中医学系, 安徽芜湖 241000
2.
皖南医学院肿瘤微环境研究室, 安徽芜湖 241002

基金项目:

国家自然科学基金面上项目 81872371

安徽省自然科学基金面上项目 1708085MH203

分子肿瘤学国家重点实验室开放课题 SKL-KF-2019-11

安徽省高校学科(专业)拔尖人才学术资助项目 gxbjZD2020110

安徽省质量工程项目示范基层教学组织(中医教研室) 2020sjsfjxzz256

安徽高校自然科学研究项目 KJ2021A0831

详细信息

作者简介:
王晓戎, 女, 副教授，主治医师, E-mail: wxrxjp@163.com

通讯作者:
吴志浩, 男, 研究员, 主要从事肿瘤微环境研究, E-mail: zwu2ster@163.com

中图分类号: R285.5
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- 文章访问数: 157
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- 被引次数: 0
出版历程
- 收稿日期: 2022-12-06
- 网络出版日期: 2023-06-12
- 发布日期: 2023-06-10

Ethanol Extract of Tengligen Induces Apoptosis of Colorectal Cancer Cells through AKT/PUMA Pathway

1.
Department of Traditional Chinese Medicine, Anhui College of Traditional Chinese Medicine, Wuhu 241000, China
2.
Cancer Microenvironment Research Room, Wannan Medical College, Wuhu 241002, China

摘要

摘要: 目的研究藤梨根乙醇提取物(TLG)对结直肠癌细胞的作用及其抗肿瘤机制。方法利用MTT检测TLG对人结直肠腺癌细胞HCT-15、LoVo的毒性, 细胞平板克隆实验检测TLG对结直肠癌细胞HCT-15、LoVo增殖的影响, Hoechst-33258染色法和流式细胞术检测TLG对结直肠癌细胞凋亡的影响, Western blot实验检测TLG对HCT-15中相关蛋白Caspase-3、PARP、P65、PUMA、AKT、GSK-3β表达的影响, 双荧光素酶报告基因实验检测TLG对HCT-15中相关启动子活性的影响。结果结直肠癌细胞HCT-15、LoVo经TLG处理后, 与对照组相比, 细胞活力以时间及剂量依赖性下降(P < 0.01，P < 0.001), 转染AKT cDNA、PUMA siRNA或GSK-3β抑制剂处理后能够恢复增殖(P < 0.001);不同浓度TLG处理后, 细胞凋亡率提高(P < 0.01, P < 0.001), 转染AKT cDNA或GSK-3β抑制剂处理后能够遏制TLG诱导的凋亡(P < 0.01，P < 0.001);Western blot显示HCT-15细胞中的p-AKT/AKT、p-GSK-3β/GSK-3β蛋白表达量随TLG浓度上升而减少(P < 0.01), Cleaved-Caspase-3/Caspase-3、Cleaved-PARP/PARP、p65、PUMA蛋白表达量随TLG浓度上升而增加(P < 0.05, P < 0.01，P < 0.001), 同时转染AKT cDNA、PUMA siRNA或GSK-3β抑制剂处理后能够阻滞TLG对下游蛋白Cleaved-Caspase-3/Caspase-3、Cleaved-PARP/PARP、p65、PUMA的影响(P < 0.05, P < 0.01，P < 0.001);报告基因结果显示NF-κB、PUMA启动子活性随TLG浓度上升而增强(P < 0.05, P < 0.01), 同时转染AKT cDNA或GSK-3β抑制剂处理后恢复(P < 0.01, P < 0.001)。结论 TLG可以显著诱导结直肠癌细胞凋亡, 发挥较强的抑制肿瘤作用, 其机制可能与AKT/PUMA信号通路有关。
- 藤梨根乙醇提取物 /
- 结直肠癌 /
- 细胞凋亡 /
- AKT /
- GSK-3β /
- NF-κB /
- PUMA
Abstract: OBJECTIVE To investigate the effect and antitumor mechanism of ethanol extract of Tengligen (TLG) on colorectal cancer cells. METHODS The toxicity of TLG on human colorectal adenocarcinoma cells (HCT-15, LoVo) was detected by methyl thiazolyl tetrazolium (MTT). Cell plate cloning assay was used to detect the effect of TLG on colorectal cancer cell proliferation. Hoechst-33258 staining and flow cytometry were used to detect the effect of TLG on colorectal cancer cell apoptosis. Western blot assay was used to detect the effect of TLG on the related proteins Caspase-3, PARP, p65, PUMA, AKT, GSK-3β in HCT-15. Dual luciferase reporter assay was performed to detect the effect of TLG on the activity of related promoter in HCT-15. RESULTS Treatment of colorectal cancer cells (HCT-15, LoVo) with TLG resulted in a time- and dose-dependent decrease (P < 0.01, P < 0.001) in cell viability compared with control cells, and transfection with AKT cDNA, PUMA siRNA or GSK-3β inhibitor treatment was able to restore proliferation (P < 0.01). After treatment with different concentrations of TLG, the rate of apoptosis was increased (P < 0.01, P < 0.001), and cells transfected with AKT cDNA, PUMA siRNA, or GSK-3β inhibitor was able to suppress TLG induced apoptosis (P < 0.01, P < 0.001). Western blot showed that p-AKT/AKT and p-GSK-3β/GSK-3β protein expression in HCT-15 cells decreased (P < 0.001) while Cleaved-Caspase-3/Caspase-3, Cleaved PARP/PARP, p65 and PUMA protein levels increased with the increase of TLG concentrations (P < 0.05, P < 0.01, P < 0.001); simultaneous transfection of AKT cDNA, PUMA siRNA, or GSK-3β inhibitor was able to block the effects of TLG on downstream proteins Cleaved-Caspase-3/Caspase-3, Cleaved PARP/PARP, p65, PUMA (P < 0.05, P < 0.01, P < 0.001). Reporter gene results showed that NF-κB and PUMA promoter activities were enhanced (P < 0.05, P < 0.01) with the increase in TLG concentration, but recovered by simultaneous transfection of AKT cDNA or GSK-3β inhibitor treatment (P < 0.01, P < 0.001). CONCLUSION TLG can significantly induce apoptosis and exert strong tumor suppressive effects in colorectal cancer cells, and the mechanism may be related to the AKT/PUMA signaling pathway.
- ethanol extract of Tengligen /
- colorectal cancer /
- cell apoptosis /
- AKT /
- GSK-3β /
- NF-κB /
- PUMA

HTML全文

图 1 TLG对结直肠癌细胞增殖能力的影响

注：A.MTT实验检测不同浓度TLG分别处理HCT-15、LoVo细胞24、36、48 h的细胞活力情况; B.平板克隆实验检测不同浓度TLG处理HCT-15、LoVo细胞4 h, 培养1周后细胞克隆集落数量; 与溶媒对照组比较, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.000 1。x±s, n=3。

Figure 1. The effect of TLG on the proliferation ability of colorectal cancer cells

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图 2 TLG对结直肠癌细胞凋亡的影响

注：A.Annexin V-FITC/PI双染细胞流式术检测TLG对HCT-15、LoVo细胞诱导凋亡的情况; B.Hoechst-33258染色, 观察HCT-15细胞中核固缩、白染的细胞数量；与溶媒对照组比较, ^**P < 0.01，^***P < 0.001。x±s，n=3。

Figure 2. TLG promoted apoptosis of colorectal cancer cell line HCT-15

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图 3 TLG对HCT-15细胞凋亡相关蛋白Cleaved-Caspase-3/Caspase-3、Cleaved-PARP/PARP的影响

注：与溶媒对照组(0 μg·mL^-1)比较, ^**P < 0.01, ^***P < 0.001。x±s，n=3。

Figure 3. Effects of TLG on apoptosis related proteins Cleaved-Caspase-3/Caspase-3, Cleaved-PARP/PARP in HCT-15 cells

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图 4 TLG诱导结直肠癌细胞HCT-15细胞系凋亡与NF-κB/PUMA通路有关

注：A.Western blot实验检测不同浓度TLG对HCT-15细胞中p65、PUMA蛋白表达水平调控的情况; B~C.双荧光素酶报告基因实验检测不同浓度TLG对HCT-15细胞中NF-κB、PUMA启动子的影响; D.Western blot实验检测HCT-15细胞在利用PUMA siRNA敲低PUMA蛋白表达后, 对下游Cleaved-Caspase-3/Caspase-3、Cleaved-PARP/PARP的表达影响; E.MTT实验检测HCT-15细胞在利用PUMA siRNA敲低PUMA蛋白表达后, TLG对HCT-15细胞的抑制效果改变情况。与溶媒对照组比较, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.000 1；与50 μg·mL^-1 TLG组比较，^###P < 0.001。x±s，n=3。

Figure 4. TLG induced apoptosis in human colorectal cancer cell line HCT-15 by NF-κB/PUMA pathway

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图 5 TLG可能通过AKT/GSK-3β信号通路影响NF-κB/PUMA通路

注：A.Western blot实验检测不同浓度TLG对HCT-15细胞中p-AKT/AKT、p-GSK-3β/GSK-3β蛋白表达水平调控的情况; B~C.双荧光素酶报告基因实验检测HCT-15细胞过表达AKT蛋白(GSK-3β抑制剂)同时TLG加药处理对NF-κB、PUMA启动子的影响; D~E.Western blot实验检测HCT-15细胞过表达AKT蛋白(GSK-3β抑制剂)同时TLG加药处理对下游蛋白的表达影响; 与溶媒对照组比较, ^**P < 0.01, ^***P < 0.001；与50 μg·mL^-1 TLG组比较，^##P < 0.01，^###P < 0.001。x±s，n=3。

Figure 5. TLG may affect NF-κB/PUMA pathway by AKT/GSK-3β signaling pathway

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图 6 GSK-3β抑制剂和AKT-cDNA能够有效遏制TLG对HCT-15的杀伤效果

注: A~B.MTT实验检测HCT-15细胞在利用AKT cDNA过表达及GSK-3β抑制剂处理后, TLG对HCT-15细胞活力的抑制效果改变情况; C.平板克隆实验检测HCT-15细胞在利用AKT cDNA过表达及GSK-3β抑制剂处理后, TLG对HCT-15细胞克隆集落数量的抑制效果改变情况; D.Hoechst-33258染色实验检测HCT-15细胞在利用AKT cDNA过表达及GSK-3β抑制剂处理后, TLG对HCT-15细胞凋亡的诱导效果改变情况; 与溶媒对照组比较, ^**P < 0.01, ^***P < 0.001；与50 μg·mL^-1 TLG组比较，^##P < 0.01，^###P < 0.001。

Figure 6. Utilization of GSK-3β inhibitor and overexpression of AKT cDNA to effectively suppress the killing effect of TLG on HCT-15

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