Dukel Muzaffer, Kalabalik Zumre Ayca
Molecular Biology and Genetics Department, Faculty of Art and Science, Mehmet Akif Ersoy University, Burdur, 15100, Turkey.
Mol Biol Rep. 2025 Jun 3;52(1):534. doi: 10.1007/s11033-025-10612-1.
The protein kinase CβII (PKCβII) is a conventional PKC isoform that exerts both over-expression and downregulation in tumors. Roles of PKCβII in tumor formation and progression remains poorly understood. In the present study, we aimed to investigate the hypothesis that PKCβII overexpression promotes anoikis resistance in colon cancer cells.
Colon cancer cells were grown in suspension, and their susceptibility to anoikis was assessed using cell viability assay and number of apoptotic cells were measured with flow cytometry. We analyzed expression of PKC isoforms by western blot and qRT-PCR. Our findings demonstrated that PKCβII was overexpressed by ~ threefold in LoVo and T84 compared to CCD-18Co and SW480 cells. Anoikis resistant cells were treated with PKC inhibitors to assess requirement for PKCβII activity in the growth of these cells. To further elucidate the role of PKCβII in regulating growth and anoikis resistance, we used both Crispr/Cas9 to knock out PKCβII and shRNA approaches for partial PKCβII knockdown. Furthermore, we overexpressed PKCβII in anoikis-sensitive cells and assessed its impact on cell survival under suspension conditions. Our in vitro experiments revealed a correlation between PKCβII expression levels and anoikis resistance in T84 and LoVo cell lines. At the molecular level, reduced PKCβII expression or activity in suspended T84 and LoVo cells resulted in induce apoptosis. Treatment with the PKC inhibitor staurosporine promoted anoikis in LoVo and T84 cells, while PKC activator induced anoikis resistance in sensitive cells, suggesting that PKCβII might regulate anoikis in suspended cells. Furthermore, overexpression of PKCβII in CCD-18Co and SW480 cells restored cells resistance to anoikis.
Taken together, our findings demonstrate that PKCβII selectively promotes anoikis resistance of human colon cancer cells and targeting PKCβII may provide new strategies for colon cancer therapy.
蛋白激酶CβII（PKCβII）是一种传统的蛋白激酶C亚型，在肿瘤中既表现为过表达，也存在下调情况。PKCβII在肿瘤形成和进展中的作用仍知之甚少。在本研究中，我们旨在探讨PKCβII过表达促进结肠癌细胞失巢凋亡抗性这一假说。
将结肠癌细胞悬浮培养，使用细胞活力测定法评估其对失巢凋亡的敏感性，并通过流式细胞术检测凋亡细胞数量。我们通过蛋白质免疫印迹法和定量逆转录聚合酶链反应分析蛋白激酶C亚型的表达情况。我们的研究结果表明，与CCD - 18Co和SW480细胞相比，LoVo和T84细胞中PKCβII的表达量大约高出三倍。对失巢凋亡抗性细胞用蛋白激酶C抑制剂进行处理，以评估这些细胞生长过程中对PKCβII活性的需求。为了进一步阐明PKCβII在调节细胞生长和失巢凋亡抗性中的作用，我们使用CRISPR/Cas9敲除PKCβII，并采用短发夹RNA方法部分敲低PKCβII。此外，我们在对失巢凋亡敏感的细胞中过表达PKCβII，并评估其在悬浮条件下对细胞存活的影响。我们的体外实验揭示了T84和LoVo细胞系中PKCβII表达水平与失巢凋亡抗性之间的相关性。在分子水平上，悬浮的T84和LoVo细胞中PKCβII表达或活性降低会导致细胞凋亡。用蛋白激酶C抑制剂星形孢菌素处理可促进LoVo和T84细胞的失巢凋亡，而蛋白激酶C激活剂可诱导敏感细胞产生失巢凋亡抗性，这表明PKCβII可能调节悬浮细胞中的失巢凋亡。此外，在CCD - 18Co和SW480细胞中过表达PKCβII可恢复细胞对失巢凋亡的抗性。
综上所述，我们的研究结果表明PKCβII选择性地促进人结肠癌细胞的失巢凋亡抗性，靶向PKCβII可能为结肠癌治疗提供新策略。
