Akiyama Hidehiko, Endo Masahiro, Matsui Taei, Katsuda Itsurou, Emi Nobuhiko, Kawamoto Yasuko, Koike Takaaki, Beppu Hidehiko
Department of Clinical Hematology, Fujita Health University School of Health Sciences, Toyoake, Aichi 470-1192, Japan.
Biochim Biophys Acta. 2011 May;1810(5):519-25. doi: 10.1016/j.bbagen.2011.02.010. Epub 2011 Mar 5.
Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine.
Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells.
Nuclear damage, DNA fragmentation, was observed by Wright-Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10μg/mL of agaritine for 48h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment.
These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria.
This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients.
姬松茸已被证明具有免疫刺激和抗癌活性；然而，其作用机制尚不清楚。我们最近发现，姬松茸热水提取物的可扩散部分对白血病细胞具有抗肿瘤活性，并将其鉴定为含肼化合物伞菌氨酸。在本研究中，我们检测了伞菌氨酸对U937细胞的形态学和细胞化学作用，以阐明伞菌氨酸的杀肿瘤机制。
使用U937细胞检测伞菌氨酸处理前后磷脂酰丝氨酸的表面表达（通过膜联蛋白V结合评估）、Fas抗原、TUNEL染色检测DNA裂解、半胱天冬酶活性变化和细胞色素c释放。
当U937细胞与10μg/mL伞菌氨酸孵育48小时后，通过瑞氏-吉姆萨染色、TUNEL染色和琼脂糖凝胶电泳观察到核损伤和DNA片段化。流式细胞术分析表明，伞菌氨酸增加了膜联蛋白V阳性U937细胞的比例，而Fas抗原表达无显著变化。加入伞菌氨酸后，半胱天冬酶-3、-8和-9的活性逐渐增加。在存在半胱天冬酶-3或颗粒酶B抑制剂的情况下，除了半胱天冬酶-8抑制剂外，膜联蛋白V表达显著降低，这表明主要是半胱天冬酶-3和-9参与凋亡途径。此外，伞菌氨酸处理后通过蛋白质印迹分析检测到细胞色素c释放。
这些结果强烈表明，姬松茸成分伞菌氨酸通过线粒体释放细胞色素c激活半胱天冬酶，适度诱导U937白血病细胞凋亡。
这是首次报道表明伞菌氨酸的抗肿瘤作用是通过凋亡介导的。目前的结果可能为针对白血病患者的抗肿瘤药物设计提供有益的建议。
