PO.ET01.05 · 实验与分子治疗
ICA-1S 靶向蛋白激酶 C-iota 以抑制肝细胞癌中的 WNT/beta-catenin 信号通路
ICA-1S targets protein kinase C-iota to inhibit the WNT/beta-catenin signaling in hepatocellular carcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
肝癌是美国癌症相关死亡的主要原因之一，也是公共健康的重大威胁。尽管世界卫生组织预测到 2030 年全球肝癌死亡人数将超过 100 万，但在肿瘤药物研发中，肝癌仍然受到的关注有限。WNT/beta-catenin 信号通路的激活是肝细胞癌（HCC）的关键分子事件，约三分之一的 HCC 病例中经典通路被激活。然而，由于特异性方面的挑战，针对该通路的治疗一直受到限制。在本研究中，我们探讨了非典型蛋白激酶 C-iota（PKC-ι）及其特异性抑制剂 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide（ICA-1S）在调节 beta-catenin 稳定性以及抑制其在肝癌细胞中的信号传导方面的作用。我们通过细胞增殖实验、Western blotting、免疫共沉淀（Co-immunoprecipitation）和 siRNA 敲低来评估 PKC-ι 抑制所影响的通路。HepG2 肝癌细胞以每孔 8×10⁴ 接种，并用 ICA-1S（10-50 μM）处理 72 小时。未处理孔作为对照。作为比较，我们使用基于聚合物的转染试剂 siTran 2.0 以 80 nM siRNA 实现了 PKC-ι 基因敲低。随后我们通过 Western blotting 评估蛋白表达，并以 beta-actin 进行归一化。ICA-1S 的剂量-反应曲线显示，在 10 μM、20 μM、30 μM、40 μM 和 50 μM 浓度下经三天处理后，增殖分别减少了 15%、35%、53%、55% 和 63%。此外，用 20 μM ICA-1S 处理使细胞色素 C（cytochrome C）增加 39%、Bcl-2 相互作用的细胞死亡介导因子（BIM）增加 25%、切割型 Caspase-3 增加 24%、切割型多聚（ADP-核糖）聚合酶（PARP）增加 24%，同时总 PARP 减少 35%，表明存在内源性凋亡反应。ICA-1S 降低了 beta-catenin 的稳定性，提示 PKC-ι 维持了抗 beta-catenin 降解复合物。我们还观察了 WNT/beta-catenin 信号通路下游效应因子对 20 μM ICA-1S 的反应，结果显示 Axin-1 上调 53%、蓬乱同源节段极性蛋白-3（DVL3）下调 37%，以及低密度脂蛋白受体相关蛋白-6（LRP6）和磷酸化 LRP6 水平分别下调 55% 和 63%。上皮钙黏蛋白（Epithelial cadherin）和肿瘤蛋白 p53 分别上调了 41% 和 13%。本研究表明，ICA-1S 可通过破坏 beta-catenin 的稳定性并扰乱 WNT/beta-catenin 信号通路，抑制肝癌细胞的增殖并诱导凋亡。我们的发现将 PKC-ι 确定为一个有前景的治疗靶点，支持进一步开发 ICA-1S 或相关化合物作为潜在的抗 HCC 药物，以克服肝癌治疗中的通路特异性挑战。
查看英文原文 English abstract
Liver cancer is one of the leading causes of cancer-related mortality in the United States and a significant threat to public health. Despite the projection of the World Health Organization that the global deaths from liver cancer would exceed one million by 2030, liver cancer continues to receive limited attention in oncology drug development. The activation of the WNT/beta-catenin signaling pathway is a key molecular event in hepatocellular carcinoma (HCC), and the canonical pathway is activated in approximately one-third of HCC cases. However, therapeutic targeting of this pathway has been limited by challenges in specificity. In this study, we investigated the role of atypical protein kinase C-iota (PKC-ι) and its specific inhibitor, 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S), in regulating beta-catenin stability and suppressing its signaling in liver cancer cells. We assessed the pathways affected by PKC-ι inhibition by cell proliferation assays, Western blotting, Co-immunoprecipitation, and siRNA knockdowns. HepG2 liver cancer cells were seeded 8×10⁴ per well and treated with ICA-1S (10-50 µM) for 72 hours. Untreated wells served as controls. In comparison, we achieved PKC-ι gene knockdown with 80 nM siRNA using polymer-based transfection reagent siTran 2.0. We then assessed protein expression by Western blotting and normalized it to beta-actin. The dose-response curve of ICA-1S showed a 15%, 35%, 53%, 55%, and 63% reduction in proliferation at concentrations of 10 µM, 20 µM, 30 µM, 40 µM, and 50 µM, respectively, after three days of treatment. Moreover, treatment with 20 µM ICA-1S increased cytochrome C (39%), Bcl-2 Interacting Mediator of cell death (BIM) (25%), cleaved Caspase-3 (24%), and cleaved Poly (ADP-ribose) polymerase (PARP) (24%), while reducing total PARP (35%), indicating an intrinsic apoptotic response. ICA-1S reduced beta-catenin stability, suggesting that PKC-ι maintains the anti-beta-catenin degradation complex. We also observed the downstream effectors of the WNT/beta-catenin signaling pathway in response to 20 µM ICA-1S, which resulted in 53% upregulation of Axin-1, 37% downregulation of disheveled segment polarity protein-3 (DVL3), and 55% and 63% downregulation of Low-density lipoprotein receptor-related protein-6 (LRP6) and phospho-LRP6 levels, respectively. Epithelial cadherin and tumor protein p53 were upregulated by 41% and 13%. We have shown in this study that ICA-1S could inhibit proliferation and induce apoptosis in liver cancer cells by destabilizing beta-catenin and disrupting the WNT/beta-catenin signaling pathway. Our findings identify PKC-ι as a promising therapeutic target, supporting the further development of ICA-1S or related compounds as potential anti-HCC agents that can overcome pathway-specificity challenges in liver cancer therapy.
利益披露 Disclosure
A. O. Olatunji, None..
N. N. Oishee, None..
A. H. Shourav, None..
G. Teodoro, None..
M. Acevedo-Duncan, None.