甘草查尔酮B诱导小鼠黑色素瘤细胞凋亡作用

Apoptosis-inducing Effect of Licochalcone B on Mouse Melanoma and its Mechanism

摘要:

目的: 研究甘草查尔酮B(licochalcone B)诱导小鼠黑色素瘤细胞(B16F0)凋亡作用并初步探讨其机制。 方法: 取对数生长期B16F0细胞按5 000个/孔接种于 96 孔板中,MTT染色法检测甘草查尔酮B(5,7.5,10,12.5,15 mg·L^-1)作用24 h后,对B16F0细胞的增殖抑制作用;取对数生长期B16F0细胞按1×10⁵个/孔接种于6 孔板中,甘草查尔酮B(7.5, 15 mg·L^-1)作用24 h后,Hoechst 33258染色法观察细胞凋亡形态;取对数生长期B16F0细胞按5×10⁵个/瓶接种于细胞培养瓶中,甘草查尔酮B(5,7.5,10,12.5 mg·L^-1)作用24 h后,流式细胞仪检测细胞凋亡率;RT-PCR法检测与凋亡相关基因Bcl-2相关X蛋白(Bax),B淋巴细胞瘤-2(Bcl-2) mRNA表达;荧光法检测半胱氨酸蛋白酶蛋白9(Caspase-9)和半胱胺酸蛋白酶蛋白3(Caspase-3)相对活性。 结果: 甘草查尔酮B能有效抑制B16F0细胞增殖,作用24 h后对细胞的生长有明显的抑制作用,IC₅₀ 11.3 mg·L^-1,在5~15 mg·L^-1表现出明显的剂量依赖,细胞出现明显凋亡特征,随甘草查尔酮B浓度的增加凋亡率逐渐升高;甘草查尔酮B(10,12.5 mg·L^-1)能明显下调Bcl-2/Bax,上调Caspase-3,Caspase-9的表达,其中7.5 mg·L^-1甘草查尔酮B引起Caspase-9,Caspase-3的活化程度较12.5 mg·L^-1高。 结论: 甘草查尔酮B可能通过激活线粒体调控的凋亡通路诱导B16F0细胞凋亡。

Abstract:

Objective: To evaluate the mechanism of licochalcone B induce mouse melanoma B16F0 cells apoptosis. Method: Effect of licochalcone B on proliferation of B16F0 cells was determined by MTT. Hoechst 33258 fluorescent staining were used to observe the morphological changes of apoptotic cell. Apoptosis was determined by staining cells with annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling.The mRNA expression levels of B cell lymphoma/lewkmia-2(Bcl-2) associated X protein(Bax) and Bcl-2 were detected using RT-PCR analysis. Changes of Caspase-9 and Caspase-3 were measured with fluorescence assay. Result: Licochalcone B(5-15 mg·L^-1) can effectively inhibit B16F0 cell proliferation in a dose-dependent manner. IC₅₀ was 11.3 mg·L^-1, and cell obviously appeared apoptosis characteristics, with the concentration of licochalcone B increased, the apoptosis rate was increased, licochalcone B(10, 12.5 mg·L^-1) significantly lowered the mRNA expression levels of Bcl-2/Bax, and raised the expression of caspase-9 and caspase-3, licochaleone B(7.5 mg·L^-1) cause the activation degree of Caspase-9 and Caspase-3 higher than 12.5 mg·L^-1. Conclusion: Licochalcone B induce B16F0 cell apoptosis through mitochondria-dependent pathway.