苦瓜凝集素诱导肝癌细胞凋亡的分子机制

Utilizing active ingredient of Chinese herbal medicine to tumor treatment has been currently attracting more and more interest. Lately, we found that Momordica Charantia Lectin (MCL), a component purified from traditional Chinese herb bitter gourd, significantly inhibited cell growth. Further study revealed that MCL exposure in hepatocellular carcinoma (HCC) resulted in Fas upregulation, decline of mitochondrial membrane potential, and activation of H2AX and caspase 8/9.These findings indicated that MCL induced DNA damage and apoptosis in HCC. However, the detailed mechanism remains elusive. In this study, we aim to, in vitro and in vivo, elucidate the mechanism through which MCL induces growth inhibition, to quest for the molecular target of MCL, to disclose the mechanism of MCL-mediated DNA damage response, and to investigate the mechanism via which MCL activates the Fas-dependent and mitochondria-dependent apoptotic pathways, using a series of biomolecular and immunological experiments in Bak-/- and/or Bid-/- cell and animal models. Likely, our study will provide not only solid evidence that MCL is capable of inhibiting cell growth in HCC, but also a scientific basis for HCC chemotherapeutic treatment mediated by active ingredient extracted from traditional Chinese Medicine.

寻找抗肿瘤中药有效成分及其作用机制是目前研究热点和难点之一。我们前期发现苦瓜凝集素（MCL），一种自行从中药苦瓜子中提纯的单一成分，显著抑制肝癌细胞生长。进一步实验表明MCL上调Fas，降低线粒体跨膜电位，并激活H2AX和 caspase 8/9，提示MCL引起DNA损伤及细胞凋亡，但其具体分子机制尚未清楚。本项目拟采用一系列分子细胞生物学及免疫学实验技术，利用Bak和/或Bid缺失细胞模型和裸鼠模型，在蛋白表达调控及细胞功能上阐明MCL抑制肝癌细胞生长的分子机制，寻找MCL的作用靶点，揭示MCL引起DNA损伤的作用及其机理，探讨MCL激活Fas和线粒体凋亡信号转导通路的分子机制，明确MCL诱导的DNA损伤与细胞凋亡的相互影响，从而获得MCL抑制肝癌细胞生长的可靠证据，为MCL治疗肝癌的临床研究奠定坚实基础，并为诠释中草药有效成分治疗肿瘤提供充分的科学依据。

肝细胞癌在全球范围的发病率和死亡率长期居高不下。从天然植物中筛选抗肿瘤药物是治疗肝癌的其中一种可行方法。本研究从传统中药苦瓜子中提取、纯化并鉴定出苦瓜凝集素(momordica charantia lectin, MCL)，并检测其在抗肝癌中的作用。我们发现MCL在体内外能显著抑制肝癌细胞增殖，而低浓度(< 5 μmol/L)的MCL并不影响正常肝细胞株的生长。MCL上调p21导致细胞阻滞在G2/M期，激活LC3-II引起细胞自噬。进一步研究发现MCL诱导DNA片段化，线粒体损伤和细胞凋亡，并伴随caspase 8，caspase 9和MAPK通路的激活。Z-VAD-FMK (caspase蛋白抑制剂), SB 203580 (p38 MAPK抑制剂) 和SP 600125 (JNK抑制剂)显著抑制MCL介导的细胞凋亡。体内外实验发现MCL激活Bid；统计学分析提示Bid表达水平与MCL的IC50呈负性相关。Bid siRNA, BI-6C9 (Bid抑制剂) and Z-IETD-FMK (caspase 8抑制剂)阻断MCL介导的细胞增殖抑制、caspase 3活化、线粒体跨膜电位去极化和细胞凋亡。此外，MCL联合化疗药物Sorafenib显著增强抗肿瘤效果。综上所述，我们的研究表明天然药物成分MCL有效抑制肝癌细胞生长，为利用MCL辅助治疗肝癌提供可靠的科学依据。