Rattanapornsompong Khanti, Khattiya Janya, Phannasil Phatchariya, Phaonakrop Narumon, Roytrakul Sittiruk, Jitrapakdee Sarawut, Akekawatchai Chareeporn
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand.
Biochem Biophys Rep. 2021 Jan 12;25:100903. doi: 10.1016/j.bbrep.2020.100903. eCollection 2021 Mar.
Previous studies showed that suppression of pyruvate carboxylase (PC) expression in highly invasive breast cancer cell line, MDA-MB-231 inhibits cell growth as a consequence of the impaired cellular biosynthesis. However, the precise cellular mechanism underlying this growth restriction is unknown.
We generated the PC knockdown (PCKD) MDA-MB-231 cells and assessed their phenotypic changes by fluorescence microscopy, proliferation, apoptotic, cell cycle assays and proteomics.
PC knockdown MDA-MB-231 cells had a low percentage of cell viability in association with accumulation of abnormal cells with large or multi-nuclei. Flow cytometric analysis of annexin V-7-AAD positive cells showed that depletion of PC expression triggers apoptosis with the highest rate at day 4. The increased rate of apoptosis is consistent with increased cleavage of procaspase 3 and poly (ADP-Ribose) polymerase. Cell cycle analysis showed that the apoptotic cell death was associated with G2/M arrest, in parallel with marked reduction of cyclin B levels. Proteomic analysis of PCKD cells identified 9 proteins whose expression changes were correlated with the degree of apoptosis and G2/M cell cycle arrest in the PCKD cells. STITCH analysis indicated 3 of 9 candidate proteins, CCT3, CABIN1 and HECTD3, that form interactions with apoptotic and cell cycle signaling networks linking to PC via MgATP.
Suppression of PC in MDA-MB-231 cells induces G2/M arrest, leading to apoptosis. Proteomic analysis supports the potential involvement of PC expression in the aberrant cell cycle and apoptosis, and identifies candidate proteins responsible for the PC-mediated cell cycle arrest and apoptosis in breast cancer cells.
Our results highlight the possibility of the use of PC as an anti-cancer drug target.
先前的研究表明，在高侵袭性乳腺癌细胞系MDA-MB-231中抑制丙酮酸羧化酶（PC）的表达会因细胞生物合成受损而抑制细胞生长。然而，这种生长限制背后的确切细胞机制尚不清楚。
我们构建了PC基因敲低（PCKD）的MDA-MB-231细胞，并通过荧光显微镜、增殖、凋亡、细胞周期分析和蛋白质组学评估其表型变化。
PC基因敲低的MDA-MB-231细胞活力百分比低，伴有大量或多核异常细胞的积累。膜联蛋白V-7-AAD阳性细胞的流式细胞术分析表明，PC表达的缺失在第4天触发凋亡，凋亡率最高。凋亡率的增加与procaspase 3和聚（ADP-核糖）聚合酶的切割增加一致。细胞周期分析表明，凋亡性细胞死亡与G2/M期阻滞相关，同时细胞周期蛋白B水平显著降低。对PCKD细胞的蛋白质组学分析鉴定出9种蛋白质，其表达变化与PCKD细胞中的凋亡程度和G2/M细胞周期阻滞相关。STITCH分析表明，9种候选蛋白中的3种，即CCT3、CABIN1和HECTD3，通过MgATP与凋亡和细胞周期信号网络形成相互作用，这些网络与PC相关。
在MDA-MB-231细胞中抑制PC会诱导G2/M期阻滞，导致凋亡。蛋白质组学分析支持PC表达可能参与异常细胞周期和凋亡，并鉴定出负责PC介导的乳腺癌细胞周期阻滞和凋亡的候选蛋白。
我们的结果突出了将PC用作抗癌药物靶点的可能性。
