一種新穎化合物CR-108經由含氧自由基與粒線體損傷路徑誘發人類乳癌細胞的凋亡

Abstract

維生素K衍生物已被證實具有抗癌活性，在本論文我們研究一種新穎的維生素K衍生物，稱為CR-108，探討其誘發人類乳癌細胞凋亡的分子機轉。在非HER-2過度表現的MCF-7與過度表現HER-2的BT-474人類乳癌細胞中，處理2-8 μM的 CR-108 經24小時後，隨著藥物濃度增加，癌細胞的存活率隨之降低。利用自由基的螢光染劑H2DCF-DA及流式細胞儀與共軛焦顯微鏡的分析，發現CR-108大量誘發MCF-7乳癌細胞中含氧自由基的產生。以JC-1及 DiOC6 的膜電位螢光染劑分析，發現CR-108會顯著抑制粒線體膜電位(ΔΨm)。CR-108會促使cytochrome c從粒線體釋放到細胞質，並造成PARP蛋白的切割及細胞凋亡產生。處理5 mM含氧自由基的清除者NAC 24小時後，完全阻斷CR-108所誘發的含氧自由基、粒線體損傷及細胞凋亡。CR-108同時也增加p38磷酸化蛋白激酶及p53 (Ser-15) 磷酸化蛋白的表現，卻抑制survivin蛋白的表現。處理NAC可有效降低磷酸化p38蛋白激酶及p53蛋白的表現，並回復survivin蛋白的表達。處理p38蛋白激酶的專一性抑制劑SB202190能回復CR-108所抑制的survivin蛋白表現，並降低CR-108所造成的細胞毒性。然而，處理p53專一性抑制劑pifithrin-α會加強CR-108所誘發的細胞死亡。此外，CR-108會誘發乳癌細胞的細胞週期停滯，並在處理NAC後回復CR-108所造成的細胞週期停滯。綜合以上結果，我們推測CR-108是經由含氧自由基與粒線體損傷路徑誘發癌細胞凋亡，而p38蛋白激酶、p53及survivin為參與調控人類乳癌細胞凋亡的重要角色。

Vitamin K derivatives have been shown to exert anticancer activities. In this report, we found a novel vitamin K derivative, CR-108, which studied its molecular mechanism on apoptosis induction in the human breast cancer cells. Treatment with 2-8 μM CR-108 for 24 h reduced the cell viability via a concentration-dependent manner in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. Interestingly, CR-108 highly elicited the intracellular reactive oxygen species (ROS) level by staining with H2DCF-DA and detected using flow cytometer and confocal microscope in MCF-7 cells. CR-108 markedly induced the loss of mitochondrial membrane potential (ΔΨm) analysed by fluorescence intensities of JC-1 and DiOC6. CR-108 induced cytochrome c release from mitochondria to cytosol, elevated the levels of cleaved PARP proteins, and increased apoptotic induction. Treatment with a ROS scavenger NAC (5 mM for 24 h) completely blocked the CR-108-induced ROS generation, mitochondrial damage, and apoptosis. Meantime, CR-108 elicited the phosphorylations of p38 mitogen-activated protein (MAP) kinase and p53 (Ser-15) but conversely inhibited survivin protein expression. Treatment with NAC reduced the phosphorylated p38 MAP kinase and p53 (Ser-15) proteins and restored the survivin protein level. SB202190, a specific p38 MAP kinase inhibitor, restored the survivin protein level and attenuated CR-108-induced cytotoxicity. However, treatment with a specific p53 inhibitor, pifithrin-α, enhanced the CR-108-induced cancer cell death. In addition, CR-108 induced cell cycle arrest in MCF-7 cells and NAC restored the CR-108-induced cell cycle arrest. Taken together, we suggest that CR-108 induces apoptosis through ROS generation and mitochondrial damage pathway, and p38 MAP kinase, p53 and survivin play important roles in regulating apoptosis in the human breast cancer cells.