肺癌異基因的改變與14-3-3sigma蛋白在肺癌細胞株中cisplatin-quercetin-所誘導之細胞增生與凋亡

於人類肺癌中因不正常促進子(promoter)處的甲基化而導致抑癌基因不表現常發生，而且此機制似乎在這些癌症的增生扮演著舉足輕重的角色。而EDNRB(endothelin reporter B)基因其promoter區在一些人類癌症中，發現有高度甲基化的現象，但其在肺癌的形成仍不是很清楚。本篇研究，利用methylation-specific PCR(MSP)分析肺癌患者EDNRB promoter region之甲基化狀態。結果發現，有32.9%的肺癌患者，其EDNRB有不正常的甲基化現象。之後再以去甲基化藥物5’-aza-2’-deoxycytidine處理H1355(人類肺癌細胞株)，以進一步去佐證於promoter區之甲基化與EDNRB基因表現與否兩者間的關係為何。在本論文的結果顯示：因epigenetic alternation而造成EDNRB基因不表現在肺癌生成扮演著重要的角色。在7種14-3-3家族異構物(isoform)中，以14-3-3σ和癌症有最直接的關係，它可視為一個抑癌基因。Cisplatin為治療癌症上常使用的抗癌藥物。研究顯示，當天然食材中所富含的Quercetin與Cisplatin一起使用時，可以提高細胞對Cisplatin細胞毒素的敏感度，而提高了屠殺癌細胞的作用。本篇我們利用MTT assay、DNA fragmentation、Western blot與flow cytometry分析，在處理了兩種抗癌藥物後，有或無overexpressed 14-3-3σ的細胞，其生理反應之差益異為何。在MTT assay與flow cytometry的結果中發現，有14-3-3σ存在時，當細胞受到傷害時，細胞會引發細胞凋亡，並停止於細胞週期的G2/M時期。

Epigenetic inactivation of tumor suppressor genes by aberrant promoter methylation is frequently observed in lung carcinomas and seems so play an important role in the pathogenesis of this tumor type. Hypermethylation has been shown in the promoter region of the endothelin receptor B (EDNRB) gene in several human tumors, but its role in lung cancer formation is unclear. In this study, genomic DNA from lung cancer patients was subjected to methylation-specific PCR to determine the methylation status of the EDNRB gene in lung cancer. Aberrant methylation of the EDNRB gene was detected in 32.9% (26 of 79) lung cancer patients. Treatment H1355 (human lung cancer cell line) with 5’-aza-2’-deoxycytidine (demethylation reagent) to determine the relationship between hypermethylation of the promoter region and the activation of EDNRB gene. Our results suggest that inactivation of the EDNRB gene through epigenetic alteration may play an important role in lung cancer tumorigenesis. Of the 7 14-3-3 isoforms, 14-3-3σ has been linked to cancer most directly, and its acts as a tumor suppressor gene. Cisplatin is widely used anti-cancer agents in the treatment of various tumors. The natural product Quercetin has been shown to sensitize cells to the cytotoxic potential of Cisplatin. In this study, we use MTT assay, DNA fragmentation, Western blotting and flow cytometry to analyze the cellular response of overexpressed 14-3-3σ that treat with Cisplatin and Quercetin alone or combination. The rusult of MTT assay and flow cytometry showed that after treated with DNA damage reagent overexpressed 14-3-3σ induct cell apoptosis and promote cell cycle arrest at G2/M phase.