Calebin-A誘導人類腸癌細胞週期停滯並抑制腫瘤細胞生長之體外及體內試驗研究

Abstract

Calebin-A 是從薑黃中分離出的類薑黃素化合物。先前研究指出其具有抑制人類胃腺癌細胞生長及誘導人類肝癌細胞凋亡的功效，然而在腸癌細胞中的抗癌活性機制尚不明確，因此本篇目的為探討calebin-A之抗腸癌功效與其相關分子機制，此外，本實驗更進一步使用異種移植動物模式探討calebin-A對腫瘤生長之影響。結果顯示，calebin-A具有抑制人類腸癌細胞HCT116生長之活性，流式細胞儀分析指出calebin-A誘導細胞停滯於S和G2/M期，在分子機制上，流式細胞儀與彗星電泳結果指出，經calebin-A處理使胞內活性氧分子 (Reactive oxygen species, ROS) 含量增加與引起DNA損傷，隨後上調r-H2AX、磷酸化chk1、chk2、p53與p53、p21之蛋白質表現量、降低cdc25A、 cyclinB、 cyclinA與cdc2表現量，經自由基清除劑N-乙醯基半胱氨酸 (N-acetyl-L-cysteine, NAC) 預處理後，能回復細胞週期的分布，表示ROS可能參與calebin-A所誘導的細胞週期停滯之途徑。在異種移植模式中，評估calebin-A對於HCT116體內異種移植之抗腫瘤活性，以腹腔注射方式給予裸calebin-A (25 mg/kg) 能顯著降低腫瘤體積，組織染色分析指出，calebin-A能減小腫瘤細胞及降低細胞增生指標 (Proliferating cell nuclear antigen, PCNA) 蛋白質表現量。綜合上述實驗結果，calebin-A在體外及體內模式中皆呈現抗人類腸癌細胞之潛力，據此可作為腸癌化學預防劑開發之重要依據。

Calebin-A is a curcuminoids compound isolated from turmeric. Previous studies indicated that calebin-A inhibits cell growth and induces apoptosis in drug-resistant human gastric cancer cells and also restrain the proliferation of human hepatoma cell line HepG2 cells; however, the molecular mechanism of anti-cancer activity in colon cancer remains unclear. Therefore, the objectives of research are to study the anti-cancer efficacy of calebin-A and its underlying molecular mechanism. Furthermore, we use xenograft animal model to further investigate the anti-tumor effect of calebin-A. The results showed calebin-A was able to inhibit colon cancer cells growth and induced cell cycle to arrest in S and G2/M phase. At the molecular levels, calebin-A exhibited the anti-cancer ability by increasing reactive oxygen species (ROS) level and triggering DNA damage response as evidenced by flow cytometry and comet assay. Western blot analysis showed calebin-A up-regulation of phosphorylation of H2AX, chk1, chk2, p53 and p53, p21 protein levels, decreased cdc25A, cyclinB, cyclinA and cdc2 protein levels. Moreover, pretreatment of N-acetyl-L-cysteine (NAC) reversed the cell cycle population, indicated ROS might involve in the cell cycle arrest signaling pathway caused by the toxic of calebin-A. In the xenograft model, we evaluated the anti-tumor ability of calebin-A on HCT116 tumor xenografts in vivo. Intraperitoneal injection of calebin-A (25 mg/kg) to nude mice significantly decreased tumor volume as well as reduced tumor size and proliferating cell nuclear antigen (PCNA) protein by Hematoxylin & Eosin (HE) and immunohistochemistry (IHC) stain, respectively. These results suggested calebin-A with potent anti-cancer effect in vitro and in vivo, could provided a molecular basis for the development of colon cancer chemopreventive agent.