Sriratanasak Nicharat, Chunhacha Preedakorn, Ei Zin Zin, Chanvorachote Pithi
Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand.
Center of Excellence in Cancer Cell and Molecular Biology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand.
Biomedicines. 2022 Oct 26;10(11):2703. doi: 10.3390/biomedicines10112703.
Cellular senescence is linked with chemotherapy resistance. Based on previous studies, GRP78 is a signal transducer in senescent cells. However, the association between GRP78 and stem cell phenotype remains unknown. Cisplatin treatment was clarified to induce cellular senescence leading to stemness induction via GRP78/Akt signal transduction. H460 cells were treated with 5 μM of cisplatin for 6 days to develop senescence. The colony formation assay and cell cycle analysis were performed. SA-β-galactosidase staining indicated senescence. Western blot analysis and RT-PCR were operated. Immunoprecipitation (IP) and immunocytochemistry assays (ICC) were also performed. Colony-forming activity was completely inhibited, and 87.07% of the cell population was arrested in the G2 phase of the cell cycle. mRNA of p21 and p53 increased approximately by 15.91- and 19.32-fold, respectively. The protein level of p21 and p53 was elevated by 9.57- and 5.9-fold, respectively. In addition, the c-Myc protein level was decreased by 0.2-fold when compared with the non-treatment control. Even though, the total of GRP78 protein was downregulated after cisplatin treatment, but the MTJ1 and downstream regulator, p-Akt/Akt ratio were upregulated by approximately 3.38 and 1.44-fold, respectively. GRP78 and MTJ1 were found at the cell surface membrane. Results showed that the GRP78/MTJ1 complex and stemness markers, including CD44, CD133, Nanog, Oct4, and Sox2, were concomitantly increased in senescent cells. MTJ1 anchored GRP78, facilitating the signal transduction of stem-like phenotypes. The strategy that could interrupt the binding between these crucial proteins or inhibit the translocation of GRP78 might beuseful for cancer therapy.
细胞衰老与化疗耐药性相关。基于先前的研究，GRP78是衰老细胞中的一种信号转导分子。然而，GRP78与干细胞表型之间的关联仍不清楚。已明确顺铂治疗可诱导细胞衰老，通过GRP78/Akt信号转导导致干性诱导。用5μM顺铂处理H460细胞6天以诱导衰老。进行了集落形成试验和细胞周期分析。SA-β-半乳糖苷酶染色表明细胞衰老。进行了蛋白质印迹分析和逆转录-聚合酶链反应。还进行了免疫沉淀（IP）和免疫细胞化学分析（ICC）。集落形成活性被完全抑制，87.07%的细胞群体停滞在细胞周期的G2期。p21和p53的mRNA分别增加了约15.91倍和19.32倍。p21和p53的蛋白质水平分别升高了9.57倍和5.9倍。此外，与未处理对照相比，c-Myc蛋白质水平降低了0.2倍。尽管顺铂处理后GRP78总蛋白水平下调，但MTJ1和下游调节因子p-Akt/Akt比值分别上调了约3.38倍和1.44倍。在细胞表面膜上发现了GRP78和MTJ1。结果表明，衰老细胞中GRP78/MTJ1复合物和干性标志物，包括CD44、CD133、Nanog、Oct4和Sox2同时增加。MTJ1锚定GRP78，促进干细胞样表型的信号转导。能够中断这些关键蛋白之间的结合或抑制GRP78易位的策略可能对癌症治疗有用。
