Park Su Bin, Park Gwang Hun, Song Hun Min, Son Ho-Jun, Um Yurry, Kim Hyun-Seok, Jeong Jin Boo
Department of Medicinal Plant Resources, Andong National University, Andong, 36729, Republic of Korea.
Forest Medicinal Resources Research Center, National Institute of Forest Science, Yeongju, 36040, Republic of Korea.
BMC Complement Altern Med. 2017 Sep 5;17(1):445. doi: 10.1186/s12906-017-1954-2.
Although it has been reported to contain high polyphenols, the pharmacological studies of the calyx of Diospyros kaki Thunb (DKC) have not been elucidated in detail. In this study, we elucidated anti-cancer activity and potential molecular mechanism of DKC against human colorectal cancer cells.
Anti-cell proliferative effect of 70% ethanol extracts from the calyx of Diospyros kaki (DKC-E70) was evaluated by MTT assay. The effect of DKC-E70 on the expression of cyclin D1 in the protein and mRNA level was evaluated by Western blot and RT-PCR, respectively.
DKC-E70 suppressed the proliferation of human colorectal cancer cell lines such as HCT116, SW480, LoVo and HT-29. Although DKC-E70 decreased cyclin D1 expression in protein and mRNA level, decreased level of cyclin D1 protein by DKC-E70 occurred at the earlier time than that of cyclin D1 mRNA, which indicates that DKC-E70-mediated downregulation of cyclin D1 protein may be a consequence of the induction of degradation and transcriptional inhibition of cyclin D1. In cyclin D1 degradation, we found that cyclin D1 downregulation by DKC-E70 was attenuated in presence of MG132. In addition, DKC-E70 phosphorylated threonine-286 (T286) of cyclin D1 and T286A abolished cyclin D1 downregulation by DKC-E70. We also observed that DKC-E70-mediated T286 phosphorylation and subsequent cyclin D1 degradation was blocked in presence of the inhibitors of ERK1/2, p38 or GSK3β. In cyclin D1 transcriptional inhibition, DKC-E70 inhibited the expression of β-catenin and TCF4, and β-catenin/TCF-dependent luciferase activity.
Our results suggest that DKC-E70 may downregulate cyclin D1 as one of the potential anti-cancer targets through cyclin D1 degradation by T286 phosphorylation dependent on ERK1/2, p38 or GSK3β, and cyclin D1 transcriptional inhibition through Wnt signaling. From these findings, DKC-E70 has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.
尽管据报道柿蒂含有高含量的多酚，但柿蒂（DKC）的药理学研究尚未得到详细阐明。在本研究中，我们阐明了DKC对人结肠癌细胞的抗癌活性及潜在分子机制。
采用MTT法评估柿蒂70%乙醇提取物（DKC-E70）的抗细胞增殖作用。分别通过蛋白质印迹法和逆转录-聚合酶链反应（RT-PCR）评估DKC-E70对细胞周期蛋白D1在蛋白质和mRNA水平表达的影响。
DKC-E70抑制了人结肠癌细胞系如HCT116、SW480、LoVo和HT-29的增殖。尽管DKC-E70在蛋白质和mRNA水平均降低了细胞周期蛋白D1的表达，但DKC-E70使细胞周期蛋白D1蛋白质水平降低的时间早于其mRNA水平，这表明DKC-E70介导的细胞周期蛋白D1蛋白质下调可能是细胞周期蛋白D1降解诱导和转录抑制的结果。在细胞周期蛋白D1降解过程中，我们发现存在MG132时，DKC-E70对细胞周期蛋白D1的下调作用减弱。此外，DKC-E70使细胞周期蛋白D1的苏氨酸-286（T286）磷酸化，而T286A消除了DKC-E70对细胞周期蛋白D1的下调作用。我们还观察到，存在细胞外信号调节激酶1/2（ERK1/2）、p38或糖原合成酶激酶3β（GSK3β）抑制剂时，DKC-E70介导的T286磷酸化及随后的细胞周期蛋白D1降解被阻断。在细胞周期蛋白D1转录抑制方面，DKC-E70抑制了β-连环蛋白和TCF4的表达，以及β-连环蛋白/TCF依赖的荧光素酶活性。
我们的结果表明，DKC-E70可能通过依赖ERK1/2、p38或GSK3β的T286磷酸化导致细胞周期蛋白D1降解，以及通过Wnt信号通路抑制细胞周期蛋白D1转录，从而将细胞周期蛋白D1作为潜在的抗癌靶点之一进行下调。基于这些发现，DKC-E70有潜力成为人类结肠癌化学预防或治疗药物开发的候选物。
