摘要
肝癌是我国常见的恶性肿瘤之一,恶性程度高,病情进展快,治疗难度大,疗效差。化疗是传统的肝癌治疗手段,但肝癌对各种化疗药物均高度耐药,因此,急需进一步探讨全新的肝癌化疗药物。
肝癌的发生发展是一个多因素协同作用的过程,包括病毒感染、致癌物作用、癌基因激活和抑癌基因失活、肝细胞凋亡和增殖调节失调等多个因素多个环节,其中肝细胞增殖调节失控在肝癌的形成过程中起着非常关键的作用。随着肝癌发生发展过程中新的肿瘤分子机制的不断阐明,全新的肝癌治疗策略应用而生。
FOX(forkhead/winged helix transcription factor)被称为叉头框基因家族,该家族包括50多个转录因子,以共有一段由100多个氨基酸组成的同源保守性翼状螺旋DNA结构域为特征,该结构域具有DNA结合、转录活化和转录抑制等功能。FOX蛋白家族成员主要参与细胞的生长、增殖、分化、寿命决定和转化等过程。其中,FOX基因家族的成员之一FOXM1基因,是一类增殖特异性基因,与胚胎发育、衰老、再生和肿瘤等许多病理生理过程关系密切。大量研究表明,FOXM1癌性转录因子可通过对细胞周期不同阶段的影响而促进细胞增殖,如可通过对其下游SKP2、CyclinD1、CDK2和CDK4等靶基因的调节而促进细胞周期G1期向S期、G2期向M期过渡和诱导有丝分裂,由此提示:FOXM1基因可能通过参与细胞增殖调节而在肝癌的发生发展过程中发挥重要作用。同时,已有大量研究发现,FOXM1基因在不同组织起源的人类肿瘤,包括肝细胞肝癌、乳腺癌、肺癌、前列腺癌、胃癌、胰腺癌等肿瘤组织中均普遍上调。并且,最新的研究表明,FOXM1可使乳腺癌细胞对顺铂这一化疗药物产生获得性耐药。鉴于FOXM1癌性转录因子在细胞周期调控、肿瘤发生和化疗耐药等方面的重要作用,已成为一种潜在的全新肿瘤治疗靶点。
最新的研究表明,FOXM1上调与大鼠对肝细胞肝癌(hepatocellular carcinoma,HCC)的易感性密切相关,而另一方面,siRNA和p19ARF介导的FOXM1表达或功能缺失可致啮齿类动物肝细胞增殖受阻,并对肝癌发生产生抵抗性。而FOXM1在HCC临床标本中的表达及其临床意义尚未见报道。因此,本研究旨在证实临床HCC标本高表达FOXM1蛋白的基础上进一步探讨靶向性抑制FOXM1在肝癌治疗中的潜在作用。
硫链丝菌素(thiostrepton,TST)是一种自然循环寡肽抗生素,可从数株青链霉菌中分离得到。在动物医学中,TST已被用于由革兰阴性菌感染引起的乳腺炎和皮肤科疾病的治疗。TST也可抵抗革兰阳性菌感染。最近,TST被报道可通过靶向作用于FOXM1转录因子而选择性抑制乳腺癌细胞增殖。另有研究表明,TST对FOXM1转录因子有特异性抑制作用,而对其它转录因子的转录活性无影响。此外,用此类抗菌素处理不同来源的人肿瘤细胞株,可在下调FOXM1基因表达的同时诱导细胞凋亡。
本课题以38例HCC临床标本以及HepG2和HepG2.2.15两株人肝癌细胞为研究对象,在采用免疫组织化学法验证HCC临床标本和HCC细胞株高表达FOXM1蛋白的前提下,采用MTT法检测TST药物处理前后细胞增殖变化情况,使用流式细胞技术测定TST药物处理前后细胞周期的变化,同时收集HepG2和HepG2.2.15细胞培养上清液,定量检测TST药物处理前后肝癌细胞AFP分泌量的改变。同时,选取HepG2和HepG2.2.15两株人肝癌细胞为研究对象,通过半定量RT-PCR、实时荧光定量PCR和Western Blot技术,检测了FOXM1本身及其下游与细胞增殖相关的靶基因,如CyclinD1、CDK2、CDK4和SKP2等蛋白的表达变化,以及FOXM1上游FOXO3a转录因子的表达情况,对TST抑制肝癌细胞增殖的分子机制进行了初步探讨。
研究结果显示:FOXM1蛋白高表达于65.8%(25/38)的HCC临床组织标本以及HepG2和HepG2.2.15等HCC细胞株中,TST处理HepG2、HepG2.2.15细胞24h、48h后,可明显抑制细胞增殖,诱导细胞周期发生G1/S期阻滞,同时下调细胞的AFP蛋白分泌量。分子机制研究显示,TST显著抑制人肝癌细胞中FOXM1基因mRNA和蛋白的表达(p<0.05),且其表达水平的变化与其下游调控的与细胞增殖相关的CyclinD1、CDK2、CDK4和SKP2等靶基因的表达变化呈一定相似性;而其上游FOXO3a基因的表达变化则显著上调(P<0.05)。以上结果提示:FOXM1是良好的肝癌治疗靶点,TST作为全新的FOXM1特异性抑制剂,可成为肝癌治疗的全新策略。
Liver cancer is one of the most common malignancies in China. Being highly malignant with rapid progression, the treatment for human liver cancer is difficult and the therapeutic effect is usually poor. Chemotherapy is the traditional choice for unresectable or metastatic disease; however, HCC is highly resistant to almost all kinds of chemotherapeutic drugs. Thus, there is an urgent need to develop novel therapeutic agents for the treatment of HCC.
The development of liver cancer is a multi-factor synergized process. A variety of elements inclucing virus infection, carcinogens, oncogene activation, inactivation of tumor suppressor genes, dysregulation of apoptosis and proliferation acted together, among which dysregulation of proliferation plays a key role during hepatocarcinogenesis. Novel therapeutic strategies derived from increased knowledge of molecular oncology underlying hepatocarcinogenesis are constantly being developed to cure this disease.
FOX (forkhead/winged helix) transcription factor, known as the forkhead box gene family with more than 50 transcription factors, is defined by a homologous conserved winged helix DNA binding domain with more than 100 amino acids, which demonstrated its functions in DNA binding, transcriptional activation and transcriptional repression. Fox protein family members play important roles in cell growth, proliferation, differentiation, longevity determination and cellular transformation. FOXM1 transcription factor, an important member of the FOX gene family and a proliferation-specific gene, is involved in a variety of patho-physiological processes such as embryonic development, aging, tissue regeneration and cancinogenesis. Numerous studies have shown that the FOXM1 oncogenic transcription factor can promote cell proliferation through its effect on different phases of cell cycle progression. For instance, an increase in FOXM1 activity may promote tumorigenesis through driving mitosis and cell cycle progression at both G1-S and G2-M transitions by regulating downstream targets such as SKP2, CyclinD1, CDK2 and CDK4. These results indicated that: the FOXM1 transcription factor may play an important role in hepatocarcinogenesis through its regulation on cell proliferation. Meanwhile, over-expressions of FOXM1 oncogenic transcription factor have been identified in many human malignancies including HCC, breast cancer, lung cancer, prostate cancer, gastric cancer, pancreatic cancer and so on. Moreover, FOXM1 was also demonstrated to confer an acquired cisplatin resistance in breast cancer cells. Because of their important roles in mitotic control, tumorigenesis and drug resistance, FOXM1 has attracted much attention as potential cancer therapeutic targets.
Recently, FOXM1 up-regulation has been demonstrated to be associated with the acquisition of a susceptible HCC phenotype in rats; on the other hand, both siRNA- and p19ARF- mediated FOXM1 depletions led to proliferation inhibition and resistance to hepatocarcinogenesis in rodents. However, FOXM1 expression in clinical HCC tissues and its clinical significance have not been well investigated. The present study was therefore initiated to (1) demonstrate the frequent over-expression of FOXM1 in clinical HCC samples, and (2) to investigate the potential effect of targeted inhibition of FOXM1 in the treatment of liver cancer.
Thiostrepton is a natural cyclic oligopeptide antibiotic, which can be derived from several strains of strepromycetes. Among which Thiostrepton has been used in veterinary medicine in mastitis caused by gram-negative organisms and in dermatologic disorders. It is also active against gram-positive bacteria. Recently, Thiostrepton has been reported to selectively target breast cancer cells through inhibition of FOXM1 expression. Other research also shows that thiostrepton specifically inhibited FOXM1 transcription factor with no effect on other transcription factors. In addition, Thiostrepton was shown to induce apoptosis after its inhibition on FOXM1 expression in a variety of human tumor cell lines with different origines.
38 HCC clinical samples and 2 human liver cancers cells, HepG2 and HepG2.2.15 were included into the present study. After demonstrating that FOXM1 was immunochemically over-expressed in both clinical HCC tissues and HCC cell lines, MTT assay was employed for the detection of cellular proliferation before and after Thiostrepton treatment; cell cycle profiles were assessed by flow cytometry; and the cell culture supernatants from both HepG2 and HepG2.2.15 cells were collected and AFP was quantified before and after Thiostrepton treatment; and the possible molecular targets were explored through semi-quantitative RT-PCR, real time quantitative RT-PCR and Western blot, focusing on Thiostrepton-mediated FOXM1 inhibition and its downstream proliferation-associated targets such as CyclinD1, CDK2, CDK4 and SKP2, and its induction of the FOXM1 up-stream FOXO3a transcription factor.
The results showed that at 24h and 48h after treatment,TST induced inhibition of cellular proliferation and cell cycle block at G1/S transition in both HepG2 and HepG2.2.15 HCC cells,concomitant with down-regulated AFP secretions. Exploration of the molecular mechanisms revealed that Thiostrepton effectively inhibited FOXM1 expression at both mRNA and protein level(p<0.05), furthermore, its expression dynamics were similar to those of its downstream proliferation-associated targets including CyclinD1, CDK2, CDK4 and SKP2; while on the other hand, the expression of its up-stream FOXO3a was significantly up-regulated(P<0.05). All these results suggested that FOXM1 is an attractive therapeutic target in the fight against HCC. Thiostrepton may specifically target FOXM1 and could be potentially developed as a novel anticancer drug against HCC.
引文
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