人参皂苷Rh_2(S)抑制HDAC6促白血病细胞凋亡作用研究
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摘要
目的研究人参皂苷Rh2(S)促白血病K562和KG1a细胞凋亡的机制。方法 CCK-8法检测人参皂苷Rh2(S)对细胞增殖的影响;镜下观察细胞的生长情况;流式细胞术(FCM)检测细胞周期和凋亡;Western blotting法检测周期和凋亡相关蛋白以及组蛋白去乙酰化酶6(HDAC6)、热休克蛋白90(HSP90)、蛋白激酶B(Akt)、糖原合成酶激酶-3(GSK-3β)和β-连环蛋白(β-catenin)的表达。结果 CCK-8结果显示人参皂苷Rh2(S)对K562和KG1a细胞具有较明显的增殖抑制作用;FCM结果显示人参皂苷Rh2(S)能够将K562和KG1a细胞周期阻滞在G0/G1期,同时促进细胞凋亡;Westernblotting结果显示人参皂苷Rh2(S)能够抑制Bcl-2、Cyclin D1、HDAC6、HSP90、p-Akt和β-catenin蛋白的表达,促进Bax、Ac-α-tubulin和GSK-3β蛋白的表达。结论 Rh2(S)通过抑制HDAC6的表达导致HSP90的表达下降,进一步抑制Akt的活性,激活GSK-3β,最后抑制Wnt/β-catenin信号通路,促进白血病细胞凋亡。
Objective To explore the specific mechanism of ginsenoside Rh2(S) inducing the apoptosis of leukemia cells. Methods The effect of Rh2(S) on proliferation of leukemia cells K562 and KG1 a was measured by cell counting kit-8 assay(CCK-8 assay). The growth states of cells were observed under the inverted phase microscope, and cell cycle distribution, and apoptosis were determined by flow cytometry(FCM). The expression levels of HDAC6, HSP90, Akt, GSK-3β, β-catenin, and cell cycle apoptosis related proteins were ascertained by Western blotting. Results The results of CCK-8 showed that Rh2(S) had the most obvious inhibitory effect on the proliferation of leukemia cells. Rh2(S) significantly induced apoptosis and led to cell cycle arrest at G0/G1 phase of leukemia cells by FCM. While the microscope observation showed that the number of cells was decreased and normal cell morphology changed. Rh2(S) decreased the expression of Bcl-2, Cyclin D1, HDAC6, HSP90, p-Akt, and β-catenin and increased the expression of Bax, Ac-α-tubulin, and GSK-3β by Western blotting. Conclusion Rh2(S) can effectively inhibit the proliferation and promote the apoptosis of K562 and KG1 a cells, its specific mechanism may relate to inhibitting the expression of HDAC6, resulting in a decline in the expression of HSP90, so as to further inhibit the activity of Akt activation of GSK-3, and finally inhibit Wnt/β-catenin pathway.
Objective To explore the specific mechanism of ginsenoside Rh2(S) inducing the apoptosis of leukemia cells. Methods The effect of Rh2(S) on proliferation of leukemia cells K562 and KG1 a was measured by cell counting kit-8 assay(CCK-8 assay). The growth states of cells were observed under the inverted phase microscope, and cell cycle distribution, and apoptosis were determined by flow cytometry(FCM). The expression levels of HDAC6, HSP90, Akt, GSK-3β, β-catenin, and cell cycle apoptosis related proteins were ascertained by Western blotting. Results The results of CCK-8 showed that Rh2(S) had the most obvious inhibitory effect on the proliferation of leukemia cells. Rh2(S) significantly induced apoptosis and led to cell cycle arrest at G0/G1 phase of leukemia cells by FCM. While the microscope observation showed that the number of cells was decreased and normal cell morphology changed. Rh2(S) decreased the expression of Bcl-2, Cyclin D1, HDAC6, HSP90, p-Akt, and β-catenin and increased the expression of Bax, Ac-α-tubulin, and GSK-3β by Western blotting. Conclusion Rh2(S) can effectively inhibit the proliferation and promote the apoptosis of K562 and KG1 a cells, its specific mechanism may relate to inhibitting the expression of HDAC6, resulting in a decline in the expression of HSP90, so as to further inhibit the activity of Akt activation of GSK-3, and finally inhibit Wnt/β-catenin pathway.
引文
[1]何道同,王兵,陈珺明.人参皂苷药理作用研究进展[J].辽宁中医药大学学报,2012,14(7):118-121.
[2]Xu Z.Modernization:One step at a time[J].Nature,2011,480(7378):90-92.
[3]权恺,刘群,李萍,等.人参皂苷抗癌活性的最新研究进展[J].医学研究生学报,2015,12(4):427-431.
[4]Jia W D,Sun H C,Zhang J B,et al.A novel peptide that selectively binds highly metastatic hepatocellular carcinoma cell surface is related to invasion and metastasis[J].Cancer Lett,2007,247(2):234-242.
[5]Wang S,Wu X,Tan M,et al.Fighting fire with fire:Poisonous Chinese herbal medicine for cancer therapy[J].J Ethnopharmacol,2012,140(1):33-45.
[6]Chung K S,Cho S H,Shin J S,et al.Ginsenoside Rh2induces cell cycle arrest and differentiation in human leukemia cells by upregulating TGF-beta expression[J].Carcinogenesis,2013,34(2):331-340.
[7]Arrowsmith C H,Bountra C,Fish P V,et al.Epigenetic protein families:A new frontier for drug discovery[J].Nat Rev Drug Discov,2012,11(5):384-400.
[8]刘仁帅,方浩.组蛋白去乙酰化酶6的结构、功能及选择性抑制剂的研究进展[J].药学学报,2015,7(1):7-14.
[9]Liu Z H,Li J,Xia J,et al.Ginsenoside 20(S)-Rh2 as potent natural histone deacetylase inhibitors suppressing the growth of human leukemia cells[J].Chem-Biol Interact,2015,24(2):227-234.
[10]Kovacs J J,Murphy P J,Gaillard S,et al.HDAC6regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor[J].Mol Cell,2005,18(5):601-607.
[11]Bali P,Pranpat M,Bradner J,et al.Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90:A novel basis for antileukemia activity of histone deacetylase inhibitors[J].J Biol Chem,2005,280(29):26729-26734.
[12]Iaconelli J,Lalonde J,Watmuff B,et al.Lysine deacetylation by HDAC6 regulates the kinase activity of Akt in human neural progenitor cells[J].ACS Chem Biol,2017,12(8):39-48.
[13]Seo S B,Hur J G,Kim M J,et al.Trail sensitize MDRcells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3beta pathway and activation of caspases[J].Mol Cancer,2010,9(12):199-205.
[14]Sun C,Luan S,Zhang G,et al.Cebpa-mediated upregulation of the lncRNA PLIN2 promotes the development of chronic myelogenous leukemia via the GSK3 and Wnt/beta-catenin signaling pathways[J].Am JCancer Res,2017,7(5):1054-1067.
[2]Xu Z.Modernization:One step at a time[J].Nature,2011,480(7378):90-92.
[3]权恺,刘群,李萍,等.人参皂苷抗癌活性的最新研究进展[J].医学研究生学报,2015,12(4):427-431.
[4]Jia W D,Sun H C,Zhang J B,et al.A novel peptide that selectively binds highly metastatic hepatocellular carcinoma cell surface is related to invasion and metastasis[J].Cancer Lett,2007,247(2):234-242.
[5]Wang S,Wu X,Tan M,et al.Fighting fire with fire:Poisonous Chinese herbal medicine for cancer therapy[J].J Ethnopharmacol,2012,140(1):33-45.
[6]Chung K S,Cho S H,Shin J S,et al.Ginsenoside Rh2induces cell cycle arrest and differentiation in human leukemia cells by upregulating TGF-beta expression[J].Carcinogenesis,2013,34(2):331-340.
[7]Arrowsmith C H,Bountra C,Fish P V,et al.Epigenetic protein families:A new frontier for drug discovery[J].Nat Rev Drug Discov,2012,11(5):384-400.
[8]刘仁帅,方浩.组蛋白去乙酰化酶6的结构、功能及选择性抑制剂的研究进展[J].药学学报,2015,7(1):7-14.
[9]Liu Z H,Li J,Xia J,et al.Ginsenoside 20(S)-Rh2 as potent natural histone deacetylase inhibitors suppressing the growth of human leukemia cells[J].Chem-Biol Interact,2015,24(2):227-234.
[10]Kovacs J J,Murphy P J,Gaillard S,et al.HDAC6regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor[J].Mol Cell,2005,18(5):601-607.
[11]Bali P,Pranpat M,Bradner J,et al.Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90:A novel basis for antileukemia activity of histone deacetylase inhibitors[J].J Biol Chem,2005,280(29):26729-26734.
[12]Iaconelli J,Lalonde J,Watmuff B,et al.Lysine deacetylation by HDAC6 regulates the kinase activity of Akt in human neural progenitor cells[J].ACS Chem Biol,2017,12(8):39-48.
[13]Seo S B,Hur J G,Kim M J,et al.Trail sensitize MDRcells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3beta pathway and activation of caspases[J].Mol Cancer,2010,9(12):199-205.
[14]Sun C,Luan S,Zhang G,et al.Cebpa-mediated upregulation of the lncRNA PLIN2 promotes the development of chronic myelogenous leukemia via the GSK3 and Wnt/beta-catenin signaling pathways[J].Am JCancer Res,2017,7(5):1054-1067.