Mangiferin联合硼替佐米对Burkitt淋巴瘤恶性生物学行为的调控机制及对CXCR表达的影响

Regulatory Mechanism of Mangiferin Combined with Bortezomib on Malignant Biological Behavior of Burkitt Lymphoma and Its Effect on Expression of CXC Chemokine Receptors

目的:分析芒果苷(mangiferin)联合硼替佐米对人Burkitt淋巴瘤Raji细胞增殖、侵袭、凋亡和自噬的作用及对CXC趋化因子受体家族(CXCR)表达的影响,并探究其间存在的分子机制,为Burkitt淋巴瘤基础研究与临床提供科学依据。方法:采用不同浓度Mangiferin、硼替佐米单药或联合干预Raji细胞,利用CCK-8法检测细胞增殖,Transwell小室法检测细胞侵袭能力,Annexin V/PI双染流式细胞术检测细胞凋亡,Western blot检测凋亡、自噬及Akt/m TOR通路蛋白表达情况,实时荧光定量PCR检测CXCR家族的表达变化。结果:不同浓度Mangiferin干预Raji细胞不同时间后,可抑制Raji细胞活力,且呈浓度及时间依赖性(r=-0.682,r=-0.836);与单药组相比,Mangiferin联合硼替佐米干预Raji细胞时,细胞增殖与侵袭能力显著下降、凋亡水平显著升高(P<0.01)。Mangiferin联合硼替佐米干预Raji细胞后,可上调促凋亡蛋白Bax表达并显著下调抗凋亡蛋白Bcl-2的表达,同时亦使Caspase-3水解活化,继而诱导Raji细胞发生凋亡。Mangiferin联合硼替佐米干预Raji细胞后可上调LC3Ⅱ蛋白的表达,且细胞中LC3Ⅱ/LC3Ⅰ比值较单药或对照组显著上调(P<0.01)。Mangiferin联合硼替佐米可显著抑制Akt和m TOR的磷酸化水平,通过抑制Akt/m TOR通路来使Raji细胞增殖及侵袭受抑,并诱导细胞发生自噬与凋亡。Mangiferin及硼替佐米单药干预Raji细胞后,可下调CXCR4、CXCR7 m RNA的表达,当两药联合时CXCR4、CXCR7 m RNA表达下调更为显著(P<0.01)。Mangiferin单药或联合硼替佐米干预Raji细胞后CXCR5 m RNA表达无显著变化(P>0.05),但两药联合时可使CXCR3表达下调(P<0.05)。结论:Mangiferin联合硼替佐米能协同抑制Raji细胞增殖、侵袭,并诱导其发生自噬与凋亡,机制可能与抑制Akt/m TOR信号通路并通过下调抗凋亡蛋白Bcl-2和上调促凋亡蛋白Bax以及使CXCR家族表达受抑等有关。

Objective:To analyze the effects of mangiferin combined with bortezomib on the proliferation,invasion,apoptosis and autophagy of human Burkitt lymphoma Raji cells,as well as the expression of CXC chemokine receptors(CXCRs)family,and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma.Methods:Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination,then cell proliferation was detected by CCK-8 assay,cell invasion ability was detected by Transwell chamber method,cell apoptosis was detected by Annexin V/PI double-staining flow cytometry,apoptosis,autophagy and Akt/mTOR pathway protein expression were detected by Western blot,and the expression changes of CXCR family was detected by real-time quantitative PCR(RT-qPCR).Results:Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration-and time-dependent manner(r=-0.682,r=-0.836).When Raji cells were intervened by combination of mangiferin and bortezomib,compared with single drug group,the proliferation and invasion abilities were significantly decreased,while the apoptosis level was significantly increased(P<0.01).Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells.Caspase-3 was also hydrolyzed and activated,and then induced the apoptosis of Raji cells.Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱprotein in Raji cells,and the ratio of LC3Ⅱ/LC3Ⅰin cells was significantly up-regulated compared with single drug or control group(P<0.01).Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR,inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway,and induce cell autophagy and apoptosis.Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells,and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined(P<0.01).Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells(P>0.05),while the combination of the two drugs could down-regulate the expression of CXCR3(P<0.05).Conclusion:Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells,and induce autophagy and apoptosis.The mechanism may be related to the inhibition of Akt/mTOR signaling pathway,down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax,and the inhibition of the expression of CXCR family.