背景:黄酮类化合物具有多种生物活性,如抗炎,抗肿瘤,抗血栓形成等。莫鲁辛醇,作为一种从桑树根皮中提取的新型异戊二烯类黄酮,在以前的研究中具有抗动脉血栓形成和抗炎的作用。然而,莫鲁辛醇的抗癌机制尚不清楚.
目的:在本研究中,我们主要研究了莫鲁辛醇的抗肿瘤作用及其在黑色素瘤中的作用方式。
方法:用MTT法评价莫鲁辛醇对黑色素瘤的抗癌作用,EdU,平板克隆形成和软琼脂测定。流式细胞术检测细胞周期和细胞凋亡。采用γ-H2AX免疫荧光法和碱性彗星法检测DNA损伤,采用Western印迹法检测DNA损伤相关蛋白的表达。还通过使用免疫沉淀测定法检测了CHK1的泛素化和周转。在体内使用细胞系衍生的异种移植(CDX)小鼠模型来评估莫鲁辛醇对致瘤性的影响。
结果:我们证明了莫鲁辛醇不仅具有抑制细胞增殖的能力,但也诱导细胞周期停滞在G0/G1期,人黑色素瘤细胞中caspase依赖性凋亡和DNA损伤。此外,莫来素在体内有效抑制黑色素瘤异种移植物的生长。更引人注目的是,CHK1在维持细胞周期的完整性方面发挥了重要作用,基因组稳定性和细胞活力,在莫来素治疗后,以剂量和时间依赖性方式下调。进一步的研究表明,CHK1被泛素-蛋白酶体途径降解。之后,莫鲁辛醇诱导的细胞周期停滞,在黑色素瘤细胞系中过度表达CHK1可以部分挽救细胞凋亡和DNA损伤。在这里,进一步的实验表明,莫鲁司醇在体外和体内增加了达卡巴嗪(DTIC)对黑色素瘤化疗的敏感性。
结论:莫鲁辛醇通过泛素-蛋白酶体途径诱导CHK1降解,从而诱导细胞周期停滞,黑色素瘤细胞凋亡和DNA损伤反应。本研究首次为莫鲁辛醇成为临床治疗肿瘤的候选药物提供了理论依据,比如黑色素瘤,单独或与达卡巴嗪结合。
BACKGROUND: Flavonoids have a variety of biological activities, such as anti-inflammation, anti-tumor, anti-thrombosis and so on. Morusinol, as a novel isoprene flavonoid extracted from Morus alba root barks, has the effects of anti-arterial thrombosis and anti-inflammatory in previous studies. However, the anti-cancer mechanism of morusinol remains unclear.
OBJECTIVE: In present study, we mainly studied the anti-tumor effect of morusinol and its mode of action in melanoma.
METHODS: The anti-cancer effect of morusinol on melanoma were evaluated by using the MTT, EdU, plate clone formation and soft agar assay. Flow cytometry was used for detecting cell cycle and apoptosis. The ɣ-H2AX immunofluorescence and the alkaline comet assay were used to detect DNA damage and the Western blotting analysis was used to investigate the expressions of DNA-damage related proteins. Ubiquitination and turnover of
CHK1 were also detected by using the immunoprecipitation assay. The cell line-derived xenograft (CDX) mouse models were used in vivo to evaluate the effect of morusinol on tumorigenicity.
RESULTS: We demonstrated that morusinol not only had the ability to inhibit cell proliferation, but also induced cell cycle arrest at G0/G1 phase, caspase-dependent apoptosis and DNA damage in human melanoma cells. In addition, morusinol effectively inhibited the growth of melanoma xenografts in vivo. More strikingly,
CHK1, which played an important role in maintaining the integrity of cell cycle, genomic stability and cell viability, was down-regulated in a dose- and time-dependent manner after morusinol treatment. Further research showed that
CHK1 was degraded by the ubiquitin-proteasome pathway. Whereafter, morusinol-induced cell cycle arrest, apoptosis and DNA damage were partially salvaged by overexpressing
CHK1 in melanoma cell lines. Herein, further experiments demonstrated that morusinol increased the sensitivity of dacarbazine (DTIC) to chemotherapy for melanoma in vitro and in vivo.
CONCLUSIONS: Morusinol induces
CHK1 degradation through the ubiquitin-proteasome pathway, thereby inducing cell cycle arrest, apoptosis and DNA damage response in melanoma. Our study firstly provided a theoretical basis for morusinol to be a candidate drug for clinical treatment of cancer, such as melanoma, alone or combinated with dacarbazine.