Zaric Milan, Mitrovic Marina, Nikolic Ivana, Baskic Dejan, Popovic Suzana, Djurdjevic Predrag, Milosavljevic Zoran, Zelen Ivanka
Department of Biochemistry, Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovica 69, 34000 Kragujevac, Serbia.
Anticancer Agents Med Chem. 2015;15(2):189-95. doi: 10.2174/1871520614666140924123116.
Chronic lymphocytic leukemia (CLL) develops due to an imbalance between apoptosis and proliferation of B lymphocytes. Chrysin induced apoptosis in leukemia cell lines such as U937, MO7e, THP-1 and HL-60, but there has not yet been data demonstrating the apoptotic effect of chrysin on CLL cells. Therefore, in our investigation we examined the cytotoxicity of chrysin against two leukemia cell lines, MOLT-4 and JVM-13, peripheral blood lymphocytes isolated from B-CLL patients and peripheral blood mononuclear cells (PBMC) from healthy individuals in vitro. The effect of chrysin on viability of MOLT-4 and JVM-13 cell lines, B-CLL cells derived from 28 patients and PBMC from 16 healthy subjects was determined by MTT assay. The type of cell death induced by chrysin was verified by Annexin V/7AAD assay and acridine orange and ethidium bromide (AO/EB) staining assay. Intracellular localisation and endogenic expression of apoptotic proteins including Bax, Bcl-2, cytochrome c and caspase-3 were determined by flow cytometry and fluorescent microscopy. Our results demonstrated that exposure of MOLT-4, JVM-13 cell lines and B-CLL cells to the concentration of chrysin of 10μM and higher selectively decreased viability of cells in this cell population, but not in the PBMC derived from healthy subjects; LC50 values of chrysin for B-CLL cells were 51μM for 24 hours and 32μM for 48 hours of incubation, respectively. Our findings demonstrated that chrysin induces the activation of proapoptotic Bax and decreases the expression of antiapoptotic Bcl-2 protein, releases cytochrome c from mitochondria into cytosol and cleavages/activates caspase-3, subsequently leading to the activation of apoptosis of B-CLL cells. Together, these findings suggest that chrysin selectively induces apoptosis of peripheral blood lymphocytes isolated from human chronic lymphocytic leukemia patients via mitochondrial pathway in vitro and that it might have a promising role as a potential future antileukemic remedy.
慢性淋巴细胞白血病（CLL）是由于B淋巴细胞凋亡与增殖失衡所致。白杨素可诱导U937、MO7e、THP-1和HL-60等白血病细胞系凋亡，但尚无数据表明白杨素对CLL细胞的凋亡作用。因此，在我们的研究中，我们检测了白杨素对两种白血病细胞系MOLT-4和JVM-13、从B-CLL患者分离的外周血淋巴细胞以及健康个体的外周血单个核细胞（PBMC）的体外细胞毒性。通过MTT法测定白杨素对MOLT-4和JVM-13细胞系、28例患者来源的B-CLL细胞以及16例健康受试者的PBMC活力的影响。通过膜联蛋白V/7-氨基放线菌素D（Annexin V/7AAD）检测和吖啶橙与溴化乙锭（AO/EB）染色检测来验证白杨素诱导的细胞死亡类型。通过流式细胞术和荧光显微镜测定包括Bax、Bcl-2、细胞色素c和半胱天冬酶-3在内的凋亡蛋白的细胞内定位和内源性表达。我们的结果表明，将MOLT-4、JVM-13细胞系和B-CLL细胞暴露于浓度为10μM及以上的白杨素中，可选择性降低该细胞群体中细胞的活力，但对健康受试者来源的PBMC无此作用；白杨素作用于B-CLL细胞24小时和48小时的半数致死浓度（LC50）分别为51μM和32μM。我们的研究结果表明，白杨素可诱导促凋亡蛋白Bax的激活，降低抗凋亡蛋白Bcl-2的表达，使细胞色素c从线粒体释放到细胞质中，并切割/激活半胱天冬酶-3，随后导致B-CLL细胞凋亡的激活。总之，这些发现表明，白杨素在体外通过线粒体途径选择性诱导从人类慢性淋巴细胞白血病患者分离的外周血淋巴细胞凋亡，并且它可能作为一种潜在的未来抗白血病药物具有广阔的应用前景。
