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Abstract：
BACKGROUND: Pancreatic cancer (PC) is associated with some of the worst prognoses of all major cancers. Thymoquinone (TQ) has a long history in traditional medical practice and is known for its anti-cancer, anti-inflammatory, anti-fibrosis and antioxidant pharmacological activities. Recent studies on hypoxia-inducible factor-1α (HIF-1α) and PC have shown that HIF-1α affects the occurrence and development of PC in many aspects. In addition, TQ could inhibit the development of renal cancer by decreasing the expression of HIF-1α. Therefore, we speculate whether TQ affects HIF-1α expression in PC cells and explore the mechanism.
OBJECTIVE: To elucidate the effect of TQ in PC cells and the regulatory mechanism of HIF-1α expression.
METHODS: Cell counting kit-8 assay, Transwell assay and flow cytometry were performed to detect the effects of TQ on the proliferative activity, migration and invasion ability and apoptosis of PANC-1 cells and normal pancreatic duct epithelial (hTERT-HPNE) cells. Quantitative real-time polymerase chain reaction and western blot assay were performed to detect the expression of HIF-1α mRNA and protein in PC cells. The effects of TQ on the HIF-1α protein initial expression pathway and ubiquitination degradation in PANC-1 cells were examined by western blot assay and co-immunoprecipitation.
RESULTS: TQ significantly inhibited proliferative activity, migration, and invasion ability and promoted apoptosis of PANC-1 cells; however, no significant effects on hTERT-HPNE cells were observed. TQ significantly reduced the mRNA and protein expression levels of HIF-1α in PANC-1, AsPC-1, and BxPC-3 cells. TQ significantly inhibited the expression of the HIF-1α initial expression pathway (PI3K/AKT/mTOR) related proteins, and promoted the ubiquitination degradation of the HIF-1α protein in PANC-1 cells. TQ had no effect on the hydroxylation and von Hippel Lindau protein mediated ubiquitination degradation of the HIF-1α protein but affected the stability of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90, thus promoting its ubiquitination degradation.
CONCLUSIONS: The regulatory mechanism of TQ on HIF-1α protein expression in PC cells was mainly to promote the ubiquitination degradation of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90; Secondly, TQ reduced the initial expression of HIF-1α protein by inhibiting the PI3K/AKT/mTOR pathway.
OBJECTIVE: To elucidate the effect of TQ in PC cells and the regulatory mechanism of HIF-1α expression.
METHODS: Cell counting kit-8 assay, Transwell assay and flow cytometry were performed to detect the effects of TQ on the proliferative activity, migration and invasion ability and apoptosis of PANC-1 cells and normal pancreatic duct epithelial (hTERT-HPNE) cells. Quantitative real-time polymerase chain reaction and western blot assay were performed to detect the expression of HIF-1α mRNA and protein in PC cells. The effects of TQ on the HIF-1α protein initial expression pathway and ubiquitination degradation in PANC-1 cells were examined by western blot assay and co-immunoprecipitation.
RESULTS: TQ significantly inhibited proliferative activity, migration, and invasion ability and promoted apoptosis of PANC-1 cells; however, no significant effects on hTERT-HPNE cells were observed. TQ significantly reduced the mRNA and protein expression levels of HIF-1α in PANC-1, AsPC-1, and BxPC-3 cells. TQ significantly inhibited the expression of the HIF-1α initial expression pathway (PI3K/AKT/mTOR) related proteins, and promoted the ubiquitination degradation of the HIF-1α protein in PANC-1 cells. TQ had no effect on the hydroxylation and von Hippel Lindau protein mediated ubiquitination degradation of the HIF-1α protein but affected the stability of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90, thus promoting its ubiquitination degradation.
CONCLUSIONS: The regulatory mechanism of TQ on HIF-1α protein expression in PC cells was mainly to promote the ubiquitination degradation of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90; Secondly, TQ reduced the initial expression of HIF-1α protein by inhibiting the PI3K/AKT/mTOR pathway.
摘要：
背景：胰腺癌（PC）与所有主要癌症中一些最差的预后有关。胸醌（TQ）在传统医学实践中有着悠久的历史，以其抗癌作用而闻名，抗炎,抗纤维化和抗氧化药理活性。最近对缺氧诱导因子-1α(HIF-1α)和PC的研究表明,HIF-1α在许多方面影响PC的发生和发展。此外,TQ可能通过降低HIF-1α的表达来抑制肾癌的发生发展。因此,我们推测TQ是否影响PC细胞中HIF-1α的表达并探讨其机制。
目的：阐明TQ在PC细胞中的作用及其对HIF-1α表达的调控机制。
方法：细胞计数试剂盒-8测定，进行Transwell分析和流式细胞术检测TQ对增殖活性的影响,PANC-1细胞和正常胰管上皮（hTERT-HPNE）细胞的迁移和侵袭能力以及凋亡。实时定量聚合酶链反应和免疫印迹法检测PC细胞中HIF-1αmRNA和蛋白的表达。通过Westernblot分析和免疫共沉淀检测TQ对PANC-1细胞中HIF-1α蛋白初始表达途径和泛素化降解的影响。
结果：TQ显著抑制增殖活性，迁移,和侵袭能力，促进PANC-1细胞凋亡；然而，未观察到对hTERT-HPNE细胞的显著影响。TQ显著降低PANC-1、AsPC-1和BxPC-3细胞中HIF-1α的mRNA和蛋白表达水平。TQ显著抑制HIF-1α初始表达通路(PI3K/AKT/mTOR)相关蛋白的表达,并促进PANC-1细胞中HIF-1α蛋白的泛素化降解。TQ对HIF-1α蛋白的羟基化和vonHippelLindau蛋白介导的泛素化降解没有影响，但通过抑制HIF-1α与HSP90之间的相互作用影响HIF-1α蛋白的稳定性，从而促进其泛素化降解。
结论：TQ对PC细胞HIF-1α蛋白表达的调控机制主要是通过抑制HIF-1α与HSP90的相互作用，促进HIF-1α蛋白的泛素化降解；其次，TQ通过抑制PI3K/AKT/mTOR途径降低HIF-1α蛋白的初始表达。
目的：阐明TQ在PC细胞中的作用及其对HIF-1α表达的调控机制。
方法：细胞计数试剂盒-8测定，进行Transwell分析和流式细胞术检测TQ对增殖活性的影响,PANC-1细胞和正常胰管上皮（hTERT-HPNE）细胞的迁移和侵袭能力以及凋亡。实时定量聚合酶链反应和免疫印迹法检测PC细胞中HIF-1αmRNA和蛋白的表达。通过Westernblot分析和免疫共沉淀检测TQ对PANC-1细胞中HIF-1α蛋白初始表达途径和泛素化降解的影响。
结果：TQ显著抑制增殖活性，迁移,和侵袭能力，促进PANC-1细胞凋亡；然而，未观察到对hTERT-HPNE细胞的显著影响。TQ显著降低PANC-1、AsPC-1和BxPC-3细胞中HIF-1α的mRNA和蛋白表达水平。TQ显著抑制HIF-1α初始表达通路(PI3K/AKT/mTOR)相关蛋白的表达,并促进PANC-1细胞中HIF-1α蛋白的泛素化降解。TQ对HIF-1α蛋白的羟基化和vonHippelLindau蛋白介导的泛素化降解没有影响，但通过抑制HIF-1α与HSP90之间的相互作用影响HIF-1α蛋白的稳定性，从而促进其泛素化降解。
结论：TQ对PC细胞HIF-1α蛋白表达的调控机制主要是通过抑制HIF-1α与HSP90的相互作用，促进HIF-1α蛋白的泛素化降解；其次，TQ通过抑制PI3K/AKT/mTOR途径降低HIF-1α蛋白的初始表达。
