Department of Pharmaceutical Sciences, Washington State University, Spokane, WA, 99202, United States.
Department of Pharmaceutical Sciences, Washington State University, Spokane, WA, 99202, United States.
Toxicon. 2024 Oct;249:108072. doi: 10.1016/j.toxicon.2024.108072. Epub 2024 Aug 20.
Microcystin-LR (MCLR) exposure has been associated with development of hepatocellular carcinoma (HCC). Many of the carcinogenic mechanisms for MCLR have been attributed to the induction of cell survival and proliferation through altered protein phosphorylation pathways by inhibition of protein phosphatases 1 (PP1) and PP2A. The current study determined MCLR effects on the phosphoproteome in human HepaRG cells. Differentiated HepaRG cells were treated with either vehicle or MCLR followed by phosphoproteomic analysis and Western blotting of MAPK-activated proteins. MCLR decreased cell viability at 24 h at doses as low as 0.03 μM. MCLR also caused a dose-dependent increase in phosphorylation of signaling and stress kinases. The number of decreased phosphosites by 0.1 μM MCLR was similar between the 2 h (212) and 24 h (154) timepoints. In contrast, a greater number of phosphosites were increased at 24 h (567) versus the 2 h timepoint (136), indicating the hyperphosphorylation state caused by MCLR-mediated inhibition of PPs is time-dependent. A kinase perturbation analysis predicted that MCLR exposure at both 2 h and 24 h increased the function of aurora kinase B (AURKB), checkpoint kinase 1 (CHEK1), and serum and glucocorticoid-regulated kinase 1 (SGK1). STRING database analysis of the phosphosites altered by MCLR exposure revealed pathways associated with cell proliferation and survival, including ribosomal protein S6 kinase (RSK), and vascular endothelial growth factor receptor (VEGFR2)-mediated vascular permeability. In addition, several cancer-related KEGG pathways were enriched at both 2 h and 24 h timepoints, and multiple cancer-related disease-gene associations were identified at the 24 h timepoint. Many of the kinases and pathways described above play crucial roles in the development of HCC by affecting processes such as invasion and metastasis. Overall, our data indicate that MCLR-mediated changes in protein phosphorylation involve biological pathways related to carcinogenesis that may contribute to the development of HCC.
微囊藻毒素-LR（MCLR）暴露与肝细胞癌（HCC）的发展有关。MCLR 的许多致癌机制归因于通过抑制蛋白磷酸酶 1（PP1）和 PP2A 改变蛋白磷酸化途径来诱导细胞存活和增殖。本研究确定了 MCLR 对人 HepaRG 细胞磷酸蛋白质组的影响。分化的 HepaRG 细胞用载体或 MCLR 处理，然后进行磷酸蛋白质组分析和 MAPK 激活蛋白的 Western blot 分析。MCLR 在低至 0.03μM 的剂量下在 24 小时时降低细胞活力。MCLR 还导致信号和应激激酶的磷酸化呈剂量依赖性增加。0.1μM MCLR 引起的磷酸化减少的磷酸化位点在 2 小时（212）和 24 小时（154）时间点之间相似。相比之下，在 24 小时（567）与 2 小时时间点（136）相比，更多的磷酸化位点增加，表明 MCLR 介导的 PP 抑制引起的过度磷酸化状态是时间依赖性的。激酶扰动分析预测，MCLR 暴露在 2 小时和 24 小时都会增加 Aurora 激酶 B（AURKB）、检查点激酶 1（CHEK1）和血清和糖皮质激素调节激酶 1（SGK1）的功能。MCLR 暴露改变的磷酸化位点的 STRING 数据库分析显示与细胞增殖和存活相关的途径，包括核糖体蛋白 S6 激酶（RSK）和血管内皮生长因子受体（VEGFR2）介导的血管通透性。此外，在 2 小时和 24 小时时间点均富集了多个与癌症相关的 KEGG 途径，并且在 24 小时时间点确定了多个与癌症相关的疾病基因关联。上述许多激酶和途径通过影响侵袭和转移等过程在 HCC 的发展中起着至关重要的作用。总的来说，我们的数据表明，MCLR 介导的蛋白质磷酸化变化涉及与致癌作用相关的生物学途径，这些途径可能有助于 HCC 的发展。
