香菇多糖的抗肿瘤构效关系及诱导肿瘤细胞凋亡的分子机制研究

Lentinan has been used for auxiliary treatment of various malignant tumor in clinical, as an immunomodulatory agent. However,the precise antitumor mechanisms of lentinan are unknown. In previous study ,we found that lentinan could inhibit the growth of cancer cells significantly by the induction of apoptosis. Marked morphological and biochemical changes of apoptosis were observed clearly after the treatment of lentinan ,which indicated that lentinan can suppress the growth and even kill cancer cells directly via multiple pathways besides the immune system. To our knowledge ,it is an innovative report that lentinan could directly inhibit the growth of cancer cells. Morever, it is a new field in the pharmacological study of lentinan as well. But the molecular mechanism remains unclear. This subject is to extract lentinan which has antitumor effect from Lentinus edodes,with the separation and purification according to different molecular weights. Examine the effects of lentinan on inhibiting cancer cell proliferation both in vitro used in many kinds of tumor cells and in vivo which adopted carcinoma nude mouse subcutaneous transplantation model. And study the molecular mechanisms of inducing tumor cell apoptosis in regulating cancer cell cycle,activating or inhibiting signaling pathways and increasing the level of reactive oxygen species. Besides, we decide to discuss its qualitative and quantitative structure-activity relationship from the main chain structure, molecular weight, branching degree, spiral structure, water-solubility and computer-aided drug design. This subject will make contributions to the further development of new pharmacological effects and mechanisms of polysaccharides, and provide a new thought and approach to search new compounds with higher antitumor activities and lower toxicity.

香菇多糖作为免疫调节剂，在临床上已用于各种恶性肿瘤的辅助治疗，但具体作用机制尚未定论。本课题组在前期研究中发现，肿瘤细胞在香菇多糖作用下出现一系列凋亡细胞所特有的形态学和生化特征，即香菇多糖能够直接诱导肿瘤细胞凋亡，推测香菇多糖可能通过其他非免疫途径直接抑制肿瘤细胞增殖。这是香菇多糖药理作用研究中从未涉及的新领域，但其具体分子机制尚不明确。本课题拟提取具有抗肿瘤作用的香菇多糖，并对其按照不同分子量段进行分离纯化。体内试验采用人癌裸小鼠皮下移植模型，体外试验采用多种肿瘤细胞模型，从调控肿瘤细胞周期、激活或抑制信号通路及升高细胞内活性氧水平等方面研究香菇多糖诱导肿瘤细胞凋亡的作用机制。并从主链结构、分子量、分支度、螺旋结构、水溶性和计算机辅助药物设计等方面探讨其定性定量构效关系。为发现和研究多糖类药物新的药理作用和作用机理作贡献，也为寻找高效低毒天然抗肿瘤药物提供新的思路和途径。

研究报道植物多糖可通过多种非免疫途径直接杀伤肿瘤细胞发挥抗肿瘤作用，诱导肿瘤细胞凋亡是当前国内外肿瘤防治的重要策略。本课题以香菇子实体为原材料，经水提、碱提醇沉和H2O2脱色，超滤分级分离得5种不同分子量精制香菇多糖，LST1，LST2，LJT1，LJT2和LJT3。分子量分别为524kDa、65.5kDa、697kDa、200kDa、 98.4kDa。结构研究表明香菇多糖均由葡萄糖组成，其4种类型的糖残基所占比例（1-6和末端：1-3和1-3-6）依次约为1:1.6,1:1,1:3,1:2和1:2，且具有三螺旋结构。体外抗肿瘤活性实验结果表明五种多糖对H22、S180和HepG2肿瘤细胞均具有一定的直接抑制作用，且表现出良好的量效依赖关系。采用皮下注射H22和MCF-7细胞建立荷瘤小鼠模型，给药香菇多糖治疗。结果表明水提香菇多糖低、中、高剂量对H22实体瘤的抑制率分别为23.31%、39.85% 和65.41%，对MCF-7裸鼠肿瘤抑制率分别为26%，39%和46%，总体呈剂量依赖性。碱提香菇多糖对H22抑制率则分别为38.17%，61.07%和29.01%。两种香菇多糖组分均可显著提高H22荷瘤小鼠脾指数及血清中IL-2和TNF-α水平。构效关系研究发现，β-(1→3)-D-葡聚糖主链结构，同一提取方法中大分子量多糖，分支度较小的香菇多糖抗肿瘤活性较好。此外，螺旋结构和水溶性与抗肿瘤活性也有关系。采用HE、AO/EB、Hoechst 33258 和Annexin V-FITC/PI 染色，结果均表明香菇多糖有诱导肿瘤细胞凋亡的作用。香菇多糖低、中、高剂量对H22、HepG2和MCF-7细胞的凋亡率分别为14.9%、15.1%、24.0%，9.96%、18.00%、24.73% 和7.20%、10.69%、17.49%。流式细胞仪和western blot检测结果表明，香菇多糖可阻滞H22肿瘤细胞周期于G2/M期，且可升高H22和HT-29细胞内ROS的水平，进而发挥抗肿瘤作用。机制研究表明香菇多糖可通过激活JNK，抑制Akt、p38MAPK通路，使肿瘤组织中Bax、p53和cleaved caspase-3表达增多，Bcl-2、cyclinB1表达下降，从而触发细胞凋亡信号通路诱导肿瘤细胞发生凋亡。此研究为最大限度发挥香菇多糖的临床作用提供了新的理论和实验基础。