PO.ET01.05 · 实验与分子治疗
选择性非典型蛋白激酶 C 抑制剂降低恶性子宫内膜癌细胞的增殖并促进其凋亡
Selective atypical protein kinase C inhibitor reduces proliferation and promotes apoptosis in malignant endometrial cancer cells
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摘要 Abstract
中文摘要
子宫内膜癌是一种子宫癌。它起始于子宫的内层，即子宫内膜。子宫内膜癌是美国最常见的影响妇科器官的癌症类型。在全球范围内，子宫内膜癌每年约有 420,000 例新发病例和近 98,000 例死亡（截至 2022 年），使其成为全球最常见的妇科癌症之一。子宫内膜癌主要影响绝经后妇女，发病高峰在 60 至 70 岁之间。总体而言，它仍然是一种主要与年长的绝经后妇女相关的疾病。在蛋白激酶 C（PKC）家族中，由蛋白激酶 C-iota（PKC-ι）和蛋白激酶 C-zeta（PKC-ζ）组成的非典型 PKC（aPKC）亚组已被认为与癌细胞的增殖和存活有关。尤其是 PKC-ι，在包括子宫内膜癌在内的多种恶性肿瘤中经常过表达。5-amino-1-((1R, 2S, 3S, 4R)-2, 3-dihydroxy-4-methyl cyclopentyl)-1H-imidazole-4-carboxamide（ICA-1S）是一种对 PKC-ι 具有选择性的抑制剂，已在胶质母细胞瘤、前列腺癌、卵巢癌和肺癌等多种肿瘤类型中显示出抗癌活性。在我们的研究中，将 50,000 个 Hec1A 子宫内膜癌细胞接种于六孔板中，并用不同浓度的 ICA-1S 处理。观察到细胞增殖呈浓度依赖性下降，在 1 μM、5 μM、10 μM、20 μM 和 50 μM ICA-1S 下分别降低了 17%、22%、52%、38% 和 58%。选择产生显著抑制作用的 10 μM 浓度用于后续实验。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）随后进行 Western Blot 分析显示，PKC-ι 的表达和磷酸化显著降低。同样，PKC-ζ 和磷酸化 PKC-ζ 的水平也显著降低。此外，与未处理的对照相比，ICA-1S 处理的细胞中关键凋亡标志物如 survivin、caspase-3 和多聚（ADP-核糖）聚合酶（PARP）的表达显著降低，提示凋亡的诱导。未来的实验将着重于通过更多的 Western Blotting、免疫荧光、免疫共沉淀和流式细胞术来确定受 ICA-1S 处理影响的信号通路，以进一步阐明 Hec1A 细胞在 PKC-ι 抑制后观察到的增殖降低和凋亡增强背后的分子机制。
查看英文原文 English abstract
Endometrial cancer is a type of uterine cancer. It starts in the inner lining of the uterus, known as the endometrium. Endometrial cancer is the most commonly diagnosed type of cancer that affects the gynecologic organs in the United States. Globally, endometrial cancer accounts for about 420,000 new cases and nearly 98,000 deaths annually (as of 2022), making it one of the most common gynecologic cancers worldwide. Endometrial cancer primarily affects postmenopausal women, with peak incidence between 60 and 70 years of age. Overall, it remains a disease largely associated with older, postmenopausal women. Among the Protein Kinase C (PKC) family, the atypical PKC (aPKC) subgroup, consisting of Protein Kinase C-iota (PKC-ι) and Protein Kinase C-zeta (PKC-ζ), has been implicated in cancer cell proliferation and survival. PKC-ι, in particular, is frequently overexpressed in various malignancies, including endometrial cancer. 5-amino-1-((1R, 2S, 3S, 4R)-2, 3-dihydroxy-4-methyl cyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S), an inhibitor selective for PKC-ι, has demonstrated anticancer activity in multiple tumor types such as glioblastoma, prostate, ovarian, and lung cancers. In our study, 50,000 Hec1A endometrial cancer cells were seeded in six-well plates and treated with various concentrations of ICA-1S. A concentration-dependent decrease in cell proliferation was observed, with reductions of 17%, 22%, 52%, 38%, and 58% at 1 µM, 5 µM, 10 µM, 20 µM, and 50 µM ICA-1S, respectively. The 10 µM concentration, which produced a significant inhibitory effect, was selected for subsequent experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western Blot analysis showed a marked reduction in the expression and phosphorylation of PKC-ι. Similarly, there was a significant reduction in the levels of PKC-ζ and phosphorylated PKC-ζ. Additionally, expression of key apoptotic markers such as survivin, caspase-3, and poly (ADP-ribose) polymerase (PARP) was significantly decreased in ICA-1S-treated cells compared to untreated controls, suggesting induction of apoptosis. Future experiments will focus on identifying the signaling pathways affected by ICA-1S treatment through more Western Blotting, Immunofluorescence, Co-Immunoprecipitation, and Flow Cytometry to elucidate further the molecular mechanisms underlying the reduced proliferation and enhanced apoptosis observed in Hec1A cells following PKC-ι inhibition.
利益披露 Disclosure
G. R. Khanal, None.