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机制

黄英; 高媛媛; 赵艳姣; 李鹏

doi:10.13748/j.cnki.issn1007-7693.20250627

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机制

Mechanism of Lycium Barbarum Glycopeptide Inducing Ferroptosis in Triple-negative Breast Cancer Cells Through Akt/Nrf2/GPX4 Signaling Pathway

摘要

摘要:
目的探讨枸杞糖肽(Lycium barbarum glycopeptide，LbGp)对三阴性乳腺癌MDA-MB-231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+铁死亡抑制剂Ferrostatin-1(Fer-1)组(LbGp+Fer-1组)、LbGp+铁死亡抑制剂去铁酮(deferiprone，DFP)组(LbGp+DFP组)、LbGp+蛋白激酶B(protein kinase B，Akt)激动剂SC79组(LbGp+SC79组)。采用CCK-8法检测细胞活性；试剂盒检测乳酸脱氢酶(lactate dehydrogenase，LDH)释放率、还原型谷胱甘肽(glutathione，GSH)、超氧化物歧化酶(superoxide dismutase，SOD)和丙二醛(malondialdehyde，MDA)水平；免疫荧光检测活性氧(reactive oxygen species，ROS)水平和细胞内Fe²⁺含量；Western blotting检测转铁蛋白受体1(transferrin receptor 1，TFR1)、二价金属离子转运体1(divalent metal transporter 1，DMT1)、膜铁转运蛋白1(ferroportin 1，FPN1)、脂酰辅酶A合成酶长链家族成员4(acyl-CoA synthetase long-chain familymember 4，ACSL4)、溶质运载蛋白7家族成员11(solute carrier family 7 member 11，SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4，GPX4)、磷酸化蛋白激酶B(phosphorylated protein kinase B，p-Akt)、Akt、核因子E2相关因子2(nuclear factor erythroid 2-related factor-2，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组细胞的LDH释放率和ROS、Fe²⁺、MDA水平均明显增加(P<0.01)，TFR1、DMT1及ACSL4的蛋白表达明显上调(P<0.01)，而GSH、SOD水平及FPN1、SLC7A11、GPX4、p-Akt、Nrf2的蛋白表达均明显降低(P<0.01)。与LbGp组相比，LbGp+Fer-1组和LbGp+DFP组细胞的LDH释放率和ROS、Fe²⁺、MDA水平均明显降低(P<0.05或P<0.01)，TFR1、DMT1及ACSL4的蛋白表达明显下调(P<0.01)，而GSH、SOD水平及FPN1、SLC7A11、GPX4的蛋白表达均明显升高(P<0.01)。与 LbGp 组相比，LbGp+SC79 组p-Akt、Nrf2及GPX4的蛋白表达均明显增加(P<0.01)。
结论 LbGp通过诱导三阴性乳腺癌细胞铁死亡来抑制其增殖，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 of MDA-MB-231 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, 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+Fer-1 group), LbGp+ferroptosis inhibitor deferiprone(DFP) group(LbGp+DFP group), LbGp+protein kinase B(Akt) agonist(SC79) group(LbGp+SC79 group). CCK-8 method was used to detect cell viability. Commercial kits were used to detect the release rate of lactate dehydrogenase(LDH) and the levels of reduced glutathione(GSH), superoxide dismutase(SOD), and malondialdehyde(MDA). Immunofluorescence was used to detect intracellular Fe²⁺ content, reactive oxygen species(ROS) level. Western blotting was used to detect the protein expression of 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 nuclear factor erythroid-2 related factor 2(Nrf2).
RESULTS The viability of MDA-MB-231 cells decreased with the increase of LbGp concentration. Compared with the control group, the LbGp group showed significantly increased LDH release rate and levels of ROS, Fe²⁺ and MDA(P<0.01), as well as significantly upregulated protein expressions of TFR1, DMT1 and ACSL4(P<0.01), while the levels of GSH and SOD, and the protein expressions of FPN1, SLC7A11, GPX4, p-Akt, and Nrf2 were significantly decreased(P<0.01). Compared with the LbGp group, the LbGp+Fer-1 group and LbGp+DFP group showed significantly decreased LDH release rate and levels of ROS, Fe²⁺ and MDA(P<0.05 or P<0.01), as well as significantly downregulated protein expressions of TFR1, DMT1, and ACSL4(P<0.01), whereas the levels of GSH and SOD, and the protein expressions of FPN1, SLC7A11 and GPX4 were significantly increased(P<0.01). Compared with the LbGp group, the LbGp+SC79 group exhibited significantly increased protein expressions of p-Akt, Nrf2, and GPX4(P<0.01).
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.

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