Huang Yajing, Wu Hao, Li Xingrui
Department of Breast and Thyroid Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China.
Department of General Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei China.
Cancer Cell Int. 2020 Oct 12;20:501. doi: 10.1186/s12935-020-01597-x. eCollection 2020.
Triple-negative breast cancer (TNBC) is a highly aggressive malignancy that lacks sensitivity to chemotherapy, endocrine therapy or targeted therapy. CDK4/6 inhibitors, combined with endocrine therapy, have been shown to be effective in postmenopausal women with HR-positive, HER2-negative advanced or metastatic breast cancer. Therefore, we investigated whether the CDK4/6 inhibitor palbociclib (PD) could enhance the effects of cisplatin (CDDP) on TNBC.
The effects of different drug regimens consisting of PD and CDDP on MDA-MB-231 and RB-knockdown MDA-MB-231 (sh-MDA-MB-231) cells were assessed in vitro and in vivo. MDA-MB-468 and RB-overexpressing MDA-MB-468 cells were used to assess the effect of the PD-CDDP regimens in vitro. Immunoblotting illustrated the role of the cyclin D1/RB/E2F axis signalling pathway.
PD induced G1 phase cell cycle arrest in the MDA-MB-231 cell line. However, synchronous treatment with PD and CDDP for 24 h, treatment with PD for 24 h followed by CDDP and treatment with CDDP for 24 h followed by PD had no influence on MDA-MB-231 cell apoptosis. We further investigated the effect of PD or CDDP withdrawal on the effects of sequential treatment and found that PD treatment for 48 h followed by withdrawal for 48 h and subsequent CDDP treatment (PD-CDDP) significantly increased apoptosis and inhibited the cell viability and colony formation of MDA-MB-231 cells, while with other regimens, PD and CDDP had an additive or antagonistic response. The preferential use of PD increased DNA damage induced by CDDP, as measured through γH2AX immunofluorescence. These findings were not observed in sh-MDA-MB-231 cells, and experiments to assess cell function in MDA-MB-468 and RB-overexpressing MDA-MB-468 cells yielded similar results, which indicated that PD enhanced the sensitivity of TNBC cells to CDDP in an RB-dependent manner. In vivo, compared with single drug treatment, combination treatment inhibited tumour growth and Ki-67 expression in MDA-MB-231 xenograft models. Western blot analysis revealed that PD enhanced sensitivity to CDDP through the CDK4/6-cyclin D1-RB-E2F pathway.
Pre-treatment with PD synchronized the tumour cell cycle through the CDK4/6-cyclin D1-RB-E2F pathway, which increased the antitumour effect of CDDP. Thus, PD-CDDP might be an effective treatment for RB-proficient TNBC patients.
三阴性乳腺癌（TNBC）是一种侵袭性很强的恶性肿瘤，对化疗、内分泌治疗或靶向治疗均不敏感。已证实，细胞周期蛋白依赖性激酶4/6（CDK4/6）抑制剂联合内分泌治疗对激素受体（HR）阳性、人表皮生长因子受体2（HER2）阴性的绝经后晚期或转移性乳腺癌患者有效。因此，我们研究了CDK4/6抑制剂哌柏西利（PD）是否能增强顺铂（CDDP）对TNBC的治疗效果。
在体外和体内评估由PD和CDDP组成的不同药物方案对MDA-MB-231细胞和视网膜母细胞瘤抑制蛋白（RB）基因敲除的MDA-MB-231细胞（sh-MDA-MB-231）的作用。使用MDA-MB-468细胞和RB过表达的MDA-MB-468细胞在体外评估PD-CDDP方案的效果。免疫印迹法阐明了细胞周期蛋白D1/RB/E2F轴信号通路的作用。
PD可诱导MDA-MB-231细胞系的G1期细胞周期阻滞。然而，PD与CDDP同步处理24小时、先使用PD处理24小时后使用CDDP以及先使用CDDP处理24小时后使用PD，均对MDA-MB-231细胞凋亡无影响。我们进一步研究了撤用PD或CDDP对序贯治疗效果的影响，发现先使用PD处理48小时，撤药48小时后再使用CDDP（PD-CDDP）可显著增加MDA-MB-231细胞的凋亡，并抑制其细胞活力和集落形成，而在其他方案中，PD和CDDP具有相加或拮抗作用。通过γH2AX免疫荧光检测发现，优先使用PD可增加CDDP诱导的DNA损伤。在sh-MDA-MB-231细胞中未观察到这些结果，评估MDA-MB-468细胞和RB过表达的MDA-MB-468细胞功能的实验也得到了类似结果，这表明PD以RB依赖的方式增强了TNBC细胞对CDDP的敏感性。在体内，与单药治疗相比，联合治疗可抑制MDA-MB-231异种移植模型中的肿瘤生长和Ki-67表达。蛋白质免疫印迹分析显示，PD通过CDK4/6-细胞周期蛋白D1-RB-E2F途径增强了对CDDP的敏感性。
PD预处理通过CDK4/6-细胞周期蛋白D1-RB-E2F途径使肿瘤细胞周期同步，从而增强了CDDP的抗肿瘤作用。因此，PD-CDDP可能是RB功能正常的TNBC患者的一种有效治疗方法。
