Jou Yeong-Chin, Lin Guan-Ling, Lin Hon-Yi, Huang Wan-Hong, Chuang Yu-Ming, Lin Ru-Inn, Chen Pie-Che, Wu Shu-Fen, Shen Cheng-Huang, Chan Michael W Y
Department of Urology, Ditmanson Medical Foundation, Chiayi Christian Hospital, Chiayi, Taiwan.
Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan.
Cancer Cell Int. 2021 Apr 19;21(1):226. doi: 10.1186/s12935-021-01925-9.
Urothelial carcinoma (UC) is the second most common malignancy of the urinary system with high rate of recurrence, UC patients therefore needed to be treated with surgery followed by chemotherapy. Development of novel therapeutics with minimal side-effect is an urgent issue. Our previous study showed that cyproheptadine (CPH), an anti-histamine, exhibited antitumor activity in UC in vitro and in an xenograft model. However, the molecular mechanism of how CPH inhibits tumor progression is not fully understood.
Genes that were upregulated after treatment with CPH in UC cells, were examined by RNA-Seq. Real-time quantitative PCR (RT-qPCR) was employed to detect IRF6 expression while COBRA assay and bisulphite pyrosequencing were used to examine promoter methylation of IRF6. Enrichment of total H3K27 acetylation and H3K4 mono-methylation were detected by western blotting. Colony formation and flow cytometry were used to examine proliferation and apoptosis in UC cells overexpressed or depleted with IRF6. Nude mice xenograft model was used to examine the effect of IRF6 in UC.
Our result showed that several genes, including IRF6 were upregulated after treatment with CPH in BFTC905 UC cells. Further experiments found that treatment of CPH could restore the expression of IRF6 in several other UC cell lines, probably due to promoter hypomethylation and enrichment of H3K27 acetylation and H3K4 mono-methylation. These results may be due to the fact that CPH could alter the activity, but not the expression of epigenetic modifiers. Finally, re-expression of IRF6 in UC inhibited tumor growth in vitro and in an xenograft mouse model, by inducing apoptosis.
In conclusion, our results suggested that CPH may be an epigenetic modifier, modulating the expression of the potential tumor suppressor IRF6, in inhibiting tumor growth in UC.
尿路上皮癌（UC）是泌尿系统第二常见的恶性肿瘤，复发率高，因此UC患者需要接受手术治疗并辅以化疗。开发副作用最小的新型治疗方法是一个紧迫的问题。我们之前的研究表明，抗组胺药赛庚啶（CPH）在体外和异种移植模型中对UC具有抗肿瘤活性。然而，CPH抑制肿瘤进展的分子机制尚未完全明确。
通过RNA测序检测UC细胞经CPH处理后上调的基因。采用实时定量PCR（RT-qPCR）检测IRF6的表达，同时采用COBRA法和亚硫酸氢盐焦磷酸测序法检测IRF6的启动子甲基化。通过蛋白质印迹法检测总H3K27乙酰化和H3K4单甲基化的富集情况。采用集落形成和流式细胞术检测过表达或缺失IRF6的UC细胞的增殖和凋亡情况。利用裸鼠异种移植模型检测IRF6在UC中的作用。
我们的结果显示，在BFTC905 UC细胞中，经CPH处理后，包括IRF6在内的几个基因上调。进一步的实验发现，CPH处理可以恢复其他几种UC细胞系中IRF6的表达，这可能是由于启动子低甲基化以及H3K27乙酰化和H3K4单甲基化的富集。这些结果可能是因为CPH可以改变表观遗传修饰因子活性，但不改变其表达。最后，UC中IRF6的重新表达通过诱导凋亡在体外和异种移植小鼠模型中抑制肿瘤生长。
总之，我们的结果表明，CPH可能是一种表观遗传修饰因子，通过调节潜在肿瘤抑制因子IRF6的表达来抑制UC中的肿瘤生长。
