摘要
卵巢癌作为一类常见的女性癌症,是目前世界上第5大高发癌症,严重威胁着女性的健康和生命,而且80 %以上的患者是死于癌症的转移。因此,预防癌细胞的恶性侵袭和转移是抗击卵巢癌的行之有效的治疗手段。
在肿瘤的发生和发展中,炎症被认为是重要因素之一。研究证明长期存在于局部的炎症通过DNA损伤、抵抗凋亡等机制诱导肿瘤的发生,并能够进一步募集大量免疫细胞侵润,从而形成肿瘤微环境继续促进肿瘤发展。
没食子儿茶素没食子酸酯EGCG作为绿茶的主要成分,具有较广泛的抗肿瘤及抗炎活性。EGCG的抗肿瘤机制尚未被人们系统明确的了解,还有待进一步研究。因此本研究选取低转移卵巢癌细胞株HO-8910和从HO-8910分化而来的高转移卵巢癌细胞株HO-8910PM为研究对象,首先给予卵巢癌细胞系不同剂量的EGCG处理,筛选出抑制癌细胞生长作用效果显著的50μM作为最终的EGCG工作浓度。
然后,我们分别用50μM EGCG给予卵巢癌细胞预处理,通过肿瘤细胞的软琼脂克隆形成试验、创口愈合实验和基质胶侵染实验分别检测转移能力不同的两株卵巢癌细胞对照组和实验组中癌细胞转移表型的变化,实验结果证明与HO-8910相比HO-8910PM表现出更强的增殖及转移活力;50μM EGCG能显著抑制两株卵巢癌细胞的非锚定生长能力、迁移能力以及侵袭转移能力,且对高转移株HO-8910PM的作用效果尤为显著。
最后,再用50μM EGCG预处理卵巢癌细胞后,利用荧光定量PCR技术在mRNA水平检测炎症基因IL-8、TNF-α及基因MMP9、VEGF表达的变化,利用ELISA实验技术检测细胞因子IL-8和TNF-α的分泌水平。实验结果证明50μM EGCG使高转移株HO-8910PM的IL-8、TNF-α的表达水平显著下调,而低转移株虽有下调趋势,但作用效果并不显著。50μM EGCG处理后,两株卵巢癌细胞MMP9和VEGF基因的表达水平均没有受到影响。
综上,我们认为EGCG能降低癌细胞的生长能力,而低剂量的EGCG能针对性地降低恶性肿瘤的侵袭转移生物学特性。EGCG的这种作用效果极有可能是通过干预炎症因子IL-8及TNF-α的表达减缓炎症损伤进而削弱肿瘤细胞的恶性表型。关注EGCG抗肿瘤与抗炎活性的关联性,将为进一步探究其抗肿瘤应用的新靶点创新思路。
Ovarian cancer, as a class of common cancers of the female and the fifth high incidence cancer, is a serious threat to women's health and more than 80% of patients were died from the metastasis of cancer at present. Therefore, the prevention of malignant invasion and metastasis of cancer cells is an effective treatment against ovarian cancer.
Inflammation has been considered to be an important factor in the development and progression of tumors. Studies have shown that local existing inflammation, induced tumors by DNA damage, apoptosis and so on, could raise a lot of immune cells invasion, thus form the microenvironment to promote the development of tumor.
Inflammation has been regarded as an important component of carcinogenesis and cancer progression. It is increasingly clear that the inflammatory tumor environment promote the neoplasia process, cancer cell proliferation, survival and migration.
Epigallocatechin gallate EGCG as the main component of green tea, have a wider anti-tumor and anti-inflammatory activity. The anti-tumor mechanism of EGCG has not been understood systematically and clearly, and remains to be further studied. Therefore, we selected the low-metastatic ovarian carcinoma cell lines HO-8910 and the highly-metastatic ovarian carcinoma cell lines HO-8910PM differentiated from HO-8910 as materials. We treated cells with different doses of EGCG, and selected 50μM EGCG which inhibited the growth of cancer cell significantly as a final working concentration.
Then, we pretreated the ovarian cancer cells with 50μM EGCG, and detected the phenotypic changes in the control and experimental group of the two ovarian cancer cells by the experiment of tumor cell colony formation in soft agar, wound healing and matrigel infection assays. The results proved that HO-8910PM showed greater proliferation and transfer activity compared with HO-8910; 50μM EGCG could significantly inhibit the growth capacity of non-anchor, migration, invasion and metastasis of two ovarian cancer cells; the effect on the high transfer cell lines HO-8910PM was particularly significant.
Finally, we pretreated the ovarian cancer cells with 50μM EGCG again, detected the expression changes of gene IL-8, TNF-αand gene MMP9, VEGF by real-time quantitative PCR on mRNA level, and detected the level of inflammatory cytokine IL-8 and TNF-αby ELISA. The results showed that 50μM EGCG reduced the expression of inflammatory gene IL-8 and TNF-αin highly-metastatic line HO-8910PM significantly, while the down regulation effect on low-metastatic line HO-8910 was not significant; 50μM EGCG had no influence on the expression of gene MMP9 and VEGF.
In summary, we believed that EGCG could reduce the growth capacity of cancer cells, while low doses of EGCG could reduce the biological characteristics of invasion and metastasis of malignant tumors contrarily. This effect of EGCG was likely to slow down the inflammatory damage to further weak the malignant phenotype of tumor cells by intervening the expression of inflammatory factor IL-8 and TNF-α. Concerning the relevance of anti-tumor and anti-inflammatory activity of EGCG will further explore the application of its new target for anti-tumor innovative ideas.
引文
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