探討致癌基因KRAS在薑黃素合併FDA核准標靶用藥對人類大腸直腸癌細胞的角色

Abstract

薑黃素為天然多酚類化合物，許多研究證實具有抗癌功效。大腸直腸癌在臺灣癌症死因中排名第三位，全世界約有35-45%的大腸直腸癌病患會有致癌基因KRAS突變的問題，KRAS突變也與治療產生阻抗及預後較差有關。Regorafenib為小分子多激酶抑制劑，無論病患KRAS基因狀態皆可使用，是目前治療大腸直腸癌的最後一線用藥 。本研究使用薑黃素合併Regorafenib進行實驗，使用MTT assay發現Curcumin合併Regorafenib在HCT 116（KRAS mutant）細胞的生長抑制效果顯著優於parental HT-29（KRAS wild type）細胞，以流式細胞儀分析細胞凋亡及細胞自噬結果發現HCT 116細胞的sub-G1（細胞凋亡）及AVOs（細胞自噬）的比例顯著高於parental HT-29細胞。使用HT-29 KRAS mutant inducible細胞檢測發現在HT-29 induced KRAS mutant細胞的生長抑制效果較HT-29 KRAS mutant細胞好。最後利用U0126處理HCT 116、parental HT-29細胞，發現Curcumin合併Regorafenib在KRAS mutant細胞中可能藉由標靶RAF及MEK蛋白引發合成致死作用，增加細胞死亡數量。

Curcumin, a polyphenol mainly from Asian foods, has been reported to exhibit anti-cancer activity. Colorectal cancer is third most common cause of cancer-related death in Taiwan. KRAS is the most frequently mutated RAS isoform, having been shown to be mutated in 35-45% of colorectal cancers (CRC) worldwide. KRAS mutation failed to targeted therapies and led to poor prognosis. Regorafenib is a FDA-approved multi-kinase inhibitor used as the last-line drug for CRC patients despite the RAS genotype. To determine whether Curcumin can enhance Regorafenib-induced cytotoxicity of CRC with KRAS mutation, CRC HCT 116 (KRAS mutant), parental HT-29 (KRAS wild-type) and KRAS mutant HT-29 (with inducible KRAS mutant gene) cells were used. MTT analysis indicated that Curcumin in the presence of Regorafenib had a higher growth inhibition on HCT 116 cells than parental HT-29 cells. Addition of Curcumin not only significantly enhanced Regorafenib-induced elevation in the percentage of cells at the sub-G1 phase (induction of apoptosis) and cells with acidic vesicular organelles (induction of autophagic flux) on HCT 116 cells, compared to that on parental HT-29 cells using flow cytometry. MTT analysis also indicated that Curcumin in the presence of Regorafenib had a higher growth inhibition on HT-29 induced KRAS mutant cells than HT-29 KRAS mutant cells. The use of MEK inhibitor U0126 increased the cytoxicity, percentage of cells at the sub-G1 phase and AVOs on HCT 116 and parental HT-29 cells.Taken together, these data suggest that Curcumin plus Regorafenib may target one more gene other than mutant KRAS and enhanced the cytotoxicity (Synthetic Lethality) on CRC, indicating a critical role of Curcumin on KRAS mutant CRC treated with Regorafenib.