枸杞糖肽通过Akt/Nrf2/GPX4信号通路诱导三阴性乳腺癌细胞铁死亡的作用机制

Functional mechanism of Lycium barbarum glycopeptide inducing ferroptosis in triple-negative breast cancer cells through Akt/Nrf2/GPX4 signaling pathway

关键词:  枸杞糖肽  三阴性乳腺癌  铁死亡  GPX4  Nrf2

Key words:  Lycium barbarum glycopeptide  triple-negative breast cancer  ferroptosis  GPX4  Nrf2

摘要:

目的? 探讨枸杞糖肽(Lycium barbarum glycopeptide,LbGp)对三阴性乳腺癌细胞铁死亡的影响及作用机制。方法? CCK-8法检测不同浓度LbGp(0、0.4、0.8、1.6、2.4、3.2、6.4 mg·mL-1)处理后三阴性乳腺癌MDA-MB-231细胞增殖率,筛选合适的LbGp作用浓度。体外培养的MDA-MB-231细胞随机分为对照组、LbGp组、LbGp+铁死抑制剂Ferrostatin-1(Fer-1)组、LbGp+铁死亡抑制剂去铁酮(DFP)组、LbGp+Akt激动剂(SC79)组。采用CCK-8法检测细胞活性；试剂盒检测乳酸脱氢酶(LDH)释放、还原型谷胱甘肽(GSH)、超氧化物歧化酶(SOD)和丙二醛(MDA)水平；免疫荧光检测ROS水平和细胞内Fe2+含量；Western blot检测TFR1、DMT1、FPN1、ACSL4、SLC7A11、GPX4、p-Akt、Akt、Nrf2蛋白的表达。结果? MDA-MB-231细胞活力随LbGp浓度的升高而降低。与对照组相比,LbGp组LDH释放、ROS、Fe2+含量、MDA水平、TFR1、DMT1及ACSL4蛋白表达量均增加(P<0.05),GSH、SOD水平、FPN1、SLC7A11、GPX4、p-Akt及Nrf2蛋白表达量均降低(P<0.05)。使用SC79后,Akt/Nrf2/GPX4通路被激活(P<0.01)。结论 LbGp通过诱导三阴性乳腺癌细胞铁死亡进而抑制其增殖,其机制可能与Akt/Nrf2/GPX4信号通路的调控有关。

Abstract:

OBJECTIVE To investigate the effect and mechanism of Lycium barbarum glycopeptide (LbGp) on ferroptosis in triple-negative breast cancer cells. METHODS The proliferation rate of MDA-MB-231 cells after treatment with different concentrations of LbGp (0, 0.4, 0.8, 1.6, 2.4, 3.2, and 6.4 mg·mL-1) was detected by CCK-8 method, and the appropriate concentration of LbGp was screened out. The MDA-MB-231 cells cultured in vitro were randomly separated into control group, LbGp group, LbGp+ferroptosis inhibitor Ferrostatin-1 (Fer-1) group, LbGp+ferroptosis inhibitor deferiprone (DFP) group and LbGp+Akt agonist (SC79) group. CCK-8 method was used to detect cell viability in each group; commercial kits were used to detect the release of lactate dehydrogenase (LDH), reduced glutathione (GSH), superoxide?dismutase (SOD) and malondialdehyde (MDA) level; immunofluorescence was used to detect intracellular Fe2+ content and reactive oxygen species?(ROS) level; Western blot was used to detect transferrin receptor 1 (TFR1), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), acyl-CoA synthetase long-chain family member 4 (ACSL4), solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), phosphorylated protein kinase B (p-Akt), Akt and regulating nuclear factor erythroid-2 related factor 2 (Nrf2) protein expression. RESULTS The viability of MDA-MB-231 cells decreased with the increase of LbGp concentration. Compared with the control group, the release of LDH, ROS, intracellular Fe2+ content, MDA level, TFR1, DMT1 and ACSL4 protein expressions were increased, and GSH, SOD level, FPN1, SLC7A11, GPX4, p-Akt and Nrf2 protein expressions were decreased in the LbGp group. The application of SC79 obviously caused significant activation of the Akt/Nrf2/GPX4 pathway. CONCLUSION LbGp inhibits the proliferation of triple-negative breast cancer cells by inducing ferroptosis, its mechanism may be related to the regulation of Akt/Nrf2/GPX4 signaling pathway.