【作者】 朱琳；

【导师】 周玉冰；

【作者基本信息】 郑州大学 ， 药学（专业学位）， 2025， 硕士

【摘要】 目的细胞周期蛋白依赖性激酶4/6(Cyclin-dependent kinases 4/6,CDK4/6)抑制剂被纳入激素受体呈阳性且人表皮生长因子受体2呈阴性(HR+/HER2-)乳腺癌的临床治疗指南,但其临床疗效仍存在局限性。最近的研究表明,二甲双胍能增强传统癌症疗法的抗肿瘤效果。将二甲双胍纳入乳腺癌治疗方案,对提高患者的临床疗效具有重要的研究潜力。本研究旨在探讨二甲双胍联合CDK4/6抑制剂哌柏西利应用于HR+/HER2-乳腺癌细胞的作用及其潜在机制,为二甲双胍作为乳腺癌新型治疗药物及策略的应用提供理论基础和实践指导。方法1.二甲双胍和哌柏西利对HR+/HER2-乳腺癌细胞活力的影响:首先利用CCK-8法检测哌柏西利和二甲双胍分别对两种HR+/HER2-乳腺癌细胞MCF7和T47D细胞活力的影响并计算IC50;利用IC10或IC20的二甲双胍联合哌柏西利共同处理细胞检测其细胞活性;利用Compusyn软件分析药物组合浓度对应的药物组合指数(Combination index,CI),评估二甲双胍联合哌柏西利的协同抑制效果。2.二甲双胍协同哌柏西利对HR+/HER2-乳腺癌细胞增殖、迁移和侵袭能力的影响:利用克隆形成实验、CCK-8法和EdU增殖实验检测二甲双胍、哌柏西利或联合处理HR+/HER2-乳腺癌细胞增殖的变化;利用免疫荧光检测二甲双胍、哌柏西利或联合处理细胞中增殖标志物KI67蛋白的表达;利用伤口愈合实验和Transwell实验检测二甲双胍、哌柏西利或联合处理HR+/HER2-乳腺癌细胞迁移和侵袭的变化。3.二甲双胍协同哌柏西利对HR+/HER2-乳腺癌细胞凋亡和周期的影响:利用流式细胞术检测二甲双胍、哌柏西利或联合处理HR+/HER2-乳腺癌细胞凋亡数量的变化;利用Western blot检测二甲双胍、哌柏西利或联合处理细胞中抗凋亡蛋白BCL-2和MCL-1的表达;利用流式细胞术检测二甲双胍、哌柏西利或联合处理HR+/HER2-乳腺癌细胞周期参数。4.二甲双胍联合哌柏西利协同作用的潜在机制:通过mRNA转录组测序分析利用哌柏西利和二甲双胍联合哌柏西利作用之间的差异基因和富集通路;Western blot检测二甲双胍、哌柏西利或联合处理HR+/HER2-乳腺癌细胞中PI3K/AKT/mTOR通路及下游通路相关蛋白;在联合用药的基础上加入PI3K激动剂740Y-P,分别检测740Y-P单独处理、二甲双胍联合哌柏西利处理以及740Y-P联合二甲双胍和哌柏西利处理细胞后,PI3K/AKT/mTOR信号通路相关蛋白的表达水平变化;利用CCK-8法、集落形成实验和EdU实验评估加入740Y-P后联合用药对细胞增殖能力的影响;利用伤口愈合和Transwell实验观察加入740Y-P后联合用药对细胞迁移和侵袭能力的作用。5.体内验证二甲双胍协同哌柏西利对HR+/HER2-乳腺癌小鼠模型中肿瘤的影响:构建荧光素酶表达的鼠源HR+/HER2-乳腺癌细胞EMT6细胞;将Luc-EMT6在小鼠皮下种瘤以构建HR+/HER2-乳腺癌小鼠模型;分别二甲双胍、哌柏西利或联合给药小鼠14天,利用小动物活体成像观察肿瘤体积并记录肿瘤生长曲线;给药结束后称量并测量解剖后小鼠肿瘤的重量和大小;利用免疫组化分析小鼠肿瘤组织中Ki67、p-AKT和p-mTOR的表达水平。结果1.CCK-8测定显示哌柏西利和二甲双胍分别对两种HR+/HER2-乳腺癌细胞MCF7和T47D具有显著的剂量依赖性和时间依赖性抑制增殖作用。哌柏西利处理MCF7和T47D细胞48小时的IC50分别为9.71±0.23 μM和8.55±0.22μM,72小时的IC50分别为5.09±0.37 μM和4.70±0.10 μM。二甲双胍处理的MCF7 和T47D细胞 48 小时的IC50 为 30.37±1.51 mM和 20.21±1.32 mM,72小时的IC50为 13.80±1.06 mM和 7.92±0.38 mM。IC10和IC20的二甲双胍联合梯度浓度的哌柏西利处理MCF7和T47D细胞产生协同抑制细胞活性作用。2.与单药处理组相比,二甲双胍联合哌柏西利处理组的MCF7和T47D细胞克隆形成能力、时间依赖性增殖能力、EdU细胞增殖能力显著降低,Ki67蛋白表达强度显著下降以及伤口愈合能力、细胞迁移和侵袭能力显著抑制,说明二甲双胍协同哌柏西利抑制HR+/HER2-乳腺癌细胞增殖、迁移和侵袭能力。3.在二甲双胍联合哌柏西利处理组中,MCF7和T47D细胞的凋亡率较单药组显著提高,同时抗凋亡蛋白BCL-2和MCL-1的表达水平显著降低。这说明二甲双胍协同哌柏西利促进HR+/HER2-乳腺癌细胞凋亡,并且与抑制BCL-2和MCL-1蛋白表达相关。4.哌柏西利显著促进MCF7和T47D细胞周期停滞能力,诱导细胞在G0/G1期停留。但相比于哌柏西利处理组,二甲双胍联合哌柏西利处理组的细胞周期参数未有显著差异,说明二甲双胍并未显著增强哌柏西利诱导的细胞周期阻滞能力。5.转录组测序结果分析PI3K/AKT通路在二甲双胍联合哌柏西利作用中发挥关键调控作用。二甲双胍联合哌柏西利抑制MCF7和T47D细胞内的PI3K、p-AKT和p-mTOR蛋白表达。二甲双胍下调CDK4和Cyclin D1蛋白表达。二甲双胍联合哌柏西利抑制Rb蛋白磷酸化和E2F蛋白表达。在二甲双胍与哌柏西利联合处理的基础上添加740Y-P后,p-AKT和p-mTOR蛋白的表达水平显著上调。740Y-P逆转二甲双胍联合哌柏西利导致的HR+/HER2-乳腺癌细胞致癌功能抑制,进一步说明二甲双胍联合哌柏西利对HR+/HER2-乳腺癌细胞的协同抑制作用与抑制PI3K/AKT/mTOR通路相关。6.与单一用药组相比,二甲双胍联合哌柏西利给药组的HR+/HER2-乳腺癌小鼠模型表现出更为显著的肿瘤生长抑制效果。二甲双胍联合哌柏西利给药组小鼠的肿瘤体积和肿瘤明显低于单药处理组。在二甲双胍与哌柏西利联合用药组中,小鼠肿瘤组织的Ki67、p-AKT和p-mTOR表达水平较单一用药组显著降低。这说明二甲双胍协同哌柏西利抑制HR+/HER2-乳腺癌小鼠体内肿瘤生长,且与抑制PI3K/AKT/mTOR通路相关。结论1.二甲双胍协同哌柏西利抑制HR+/HER2-乳腺癌细胞增殖、迁移和侵袭能力。2.二甲双胍协同哌柏西利促进HR+/HER2-乳腺癌细胞凋亡,其作用机制与抑制BCL-2和MCL-1蛋白表达相关。3.二甲双胍联合哌柏西利通过抑制PI3K/AKT/mTOR通路发挥协同抗HR+/HER2-乳腺癌作用。4.二甲双胍协同哌柏西利抑制HR+/HER2-乳腺癌小鼠体内肿瘤生长。更多还原

【Abstract】 ObjectiveCDK4/6 inhibitors are recommended as the first-line treatment for HR+/HER2breast cancer,but their efficacy is limited.Metformin has recently shown to enhance the antitumor effects of conventional cancer therapies.The integration of metformin into the therapeutic regimen for breast cancer holds significant research potential for enhancing the clinical outcomes in patients.In this study,we investigated the role and potential mechanisms of metformin combined with the CDK4/6 inhibitor palbociclib in HR+/HER2-breast cancer cells,aiming to provide an experimental basis and scientific evidence for the use of metformin as a novel therapeutic agent and strategy for breast cancer.Methods1.Effects of Metformin and Palbociclib on HR+/HER2-Breast Cancer Cell Viability:The impact of palbociclib and metformin on the viability of MCF7 and T47D cells,which are HR+/HER2-breast cancer cell models,was assessed using the CCK-8 assay,and the IC50 values were calculated.Cell activity was further evaluated by treating MCF7 and T47D cells with metformin and palbociclib in combination at IC10 or IC20 concentrations.The synergistic inhibitory effect of the drug combination was quantified by calculating the CI using Compusyn software.2.Effects of Metformin and Palbociclib on Cellular Proliferation,Migratory Capacity,and Invasive Potential in HR+/HER2-Breast Cancer Cell Models:Proliferation changes in HR+/HER2-breast cancer cells treated with metformin,palbociclib,or their combination were measured using clone formation,CCK-8,and EdU proliferation assays.The expression of the proliferation marker KI67 was assessed by immunofluorescence in cells treated with metformin,palbociclib,or their combination.Additionally,cell migration and invasion were evaluated using wound healing and Transwell assays.3.Effects of Metformin Combined with Palbociclib on Apoptosis and Cell Cycle of HR+/HER2-Breast Cancer Cells:The number of apoptotic cells in HR+/HER2-breast cancer cells treated with metformin,palbociclib,or their combination was performed through flow cytometric assessment.The expression levels of anti-apoptotic proteins BCL-2 and MCL-1 were analyzed by Western blot.Changes in the cell cycle parameters were also performed through flow cytometry in cells treated with metformin,palbociclib,or their combination.4.Mechanisms of the Synergistic Inhibitory Effect of Metformin Combined with Palbociclib:Through mRNA transcriptome sequencing analysis,the differential genes and enriched pathways between the effects of palbociclib alone and the combination of palbociclib with metformin were investigated.The expression levels of proteins related to the PI3K/AKT/mTOR pathway and its downstream pathways were analyzed in HR+/HER2-breast cancer cells using Western blot following treatment with metformin,palbociclib,or their combination.To further investigate the role of the PI3K pathway,the PI3K agonist 740Y-P was added to the co-treatment regimen.Subsequent evaluation of PI3K/AKT/mTOR signaling pathway components was conducted in cells treated with 740Y-P,metformin+palbociclib,or the combination of 740Y-P,metformin,and palbociclib.The effects of 740Y-P+metformin+palbociclib on cell proliferation were evaluated including CCK-8 viability testing,colony formation analysis,and EdU incorporation assay.Additionally,the impact of 740Y-P+metformin+palbociclib on cell migration and invasion was examined using wound healing and Transwell assays.5.In Vivo Validation of the Effect of Metformin Combined with Palbociclib on Tumor Growth in HR+/HER2-Breast Cancer Mice:Luciferase-expressing HR+/HER2-breast cancer cells(Luc-EMT6)were constructed and implanted subcutaneously in mice to establish a HR+/HER2breast cancer model.Mice were treated with metformin,palbociclib,or their combination for 14 days.Tumor volume was monitored and tumor growth curves were recorded using small-animal live imaging.At the end of the treatment period,the tumors were excised,and their weight and size were measured.The effects of metformin in combination with palbociclib on tumor growth were assessed using immunoassays.Immunohistochemical analysis was subsequently performed to evaluate the tissue expression profiles of proliferation marker Ki67 and phosphorylated signaling molecules p-AKT and p-mTOR in tumor specimens.Results1.The CCK-8 assay revealed significant dose-dependent and time-dependent inhibitory effects of palbociclib and metformin on the proliferation of MCF7 and T47D.For palbociclib,the IC50 values of MCF7 and T47D cells were 9.71±0.23μM and 8.55±0.22 μM at 48 hours,and 5.09±0.37 μM and 4.70±0.10 μM at 72 hours.For metformin,the IC50 values at 48 hours were 30.37±1.51 mM for MCF7 and 20.21±1.32 mM for T47D,and at 72 hours,these values decreased to 13.80±1.06 mM and 7.92±0.38 mM,respectively.IC10 or IC20 concentrations of metformin synergized with gradient concentrations of palbociclib to inhibit proliferation of HR+/HER2-breast cancer cells.2.Compared to single-agent treatments,the combination of metformin and palbociclib significantly reduced the clone formation ability,time-dependent proliferation,and EdU-labeled proliferating cell populations in MCF7 and T47D cells.Additionally,there was a notable decrease in Ki67 protein expression intensity in the combination treatment group.Furthermore,metformin synergized with palbociclib to inhibit the migration and invasion of MCF7 and T47D cells.These findings indicate that metformin synergistically inhibits cellular proliferation,migratory capacity,and invasive potential in HR+/HER2-breast cancer cell models.3.Furthermore,the combination of metformin and palbociclib significantly increased apoptosis in MCF7 and T47D cells compared to single-agent treatments.The expression of anti-apoptotic proteins BCL-2 and MCL-1 was also significantly suppressed in the combination treatment group.This suggests that the synergistic effect of metformin and palbociclib promotes apoptosis in HR+/HER2-breast cancer cells,potentially through the inhibition of BCL-2 and MCL-1 expression.4.Palbociclib-mediated cell cycle modulation resulted in marked G0/G1 phase accumulation in MCF7 and T47D cells,demonstrating its specific inhibitory effect on cell cycle progression.However,there was no significant difference in cell cycle parameters between the palbociclib-treated group and the metformin-palbociclib combination group.This indicates that metformin did not significantly augment the cell cycle arrest induced by palbociclib.5.Transcriptome sequencing analysis revealed that the PI3K/AKT pathway plays a critical regulatory role in the combined treatment of metformin and palbociclib.Western blot analysis revealed that the combination of metformin and palbociclib inhibited the expression of PI3K,p-AKT,and p-mTOR proteins in MCF7 and T47D cells.Metformin alone downregulated the expression of CDK4,Cyclin D1,E2F and Rb phosphorylated protein.The addition of the PI3K agonist 740Y-P to the metformin-palbociclib treatment led to an upregulation of p-AKT and p-mTOR expression.740Y-P reversed the functional impairment of HR+/HER2breast cancer cells induced by the combination treatment,suggesting that the synergistic inhibitory effect of metformin and palbociclib on HR+/HER2-breast cancer cells is mediated through the inhibition of the PI3K/AKT/mTOR pathway.6.In the HR+/HER2-breast cancer mice model,the combination of metformin and palbociclib resulted in more significant tumor growth inhibition compared to single-agent treatments.Both tumor volume and weight were significantly lower in the combination group than in the single-drug groups.The combinatorial treatment regimen demonstrated significant downregulation of Ki67,p-AKT and p-mTOR in neoplastic tissues compared to monotherapy groups.These findings suggest that Metformin synergized with palbociclib to inhibit tumor growth in HR+/HER2-breast cancer mice,and this effect is associated with the inhibition of the PI3K/AKT/mTOR pathway.Conclusions1.Metformin synergized with palbociclib to inhibit the proliferation,migration and invasion of HR+/HER2-breast cancer cells.2.Metformin synergized with palbociclib promoted apoptosis in HR+/HER2-breast cancer cells,and its mechanism of action was related to the inhibition of the expression of anti-apoptotic proteins BCL-2 and MCL-1.3.Metformin in combination with palbociclib exerts synergistic anti-HR+/HER2-breast cancer effects by inhibiting the PI3K/AKT/mTOR pathway.4.Metformin synergized with palbociclib to inhibit tumor growth in HR+/HER2-breast cancer mice.更多还原