和厚朴酚通过UbcH8诱导AML1-ETO蛋白降解
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摘要
目的:研究和厚朴酚诱导AML1-ETO蛋白降解的可能机制。方法:用10、20、40 μmol/L的和厚朴酚处理Kasumi-1细胞,分别在24 h和48 h后qRT-PCR检测AML1-ETO、UbcH8 mRNA的表达;Western blot法检测蛋白表达;40 μmol/L和厚朴酚处理24 h,基因芯片分析寻找靶基因;构建UbcH8过表达质粒,通过反转录病毒转染Kasumi-1细胞,检测AML1-ETO蛋白;通过短发夹RNA(sh RNA)敲除Ubc H8的表达,40 μmol/L和厚朴酚处理48 h,同时检测AML1-ETO和UbcH8蛋白;构建异种移植小鼠模型,和厚朴酚处理,检测肿瘤的成瘤性和相关蛋白表达。结果:和厚朴酚减少Kasumi-1细胞中AML1-ETO蛋白的表达,并具有浓度和时间依赖性,但没有影响AML1-ETO mRNA的表达;和厚朴酚处理后的AML1-ETO蛋白稳定性降低;基因芯片分析发现,和厚朴酚将Ubc H8 m RNA的表达提高了约4倍;和厚朴酚处理后,Kasumi-1细胞中的Ubc H8 mRNA和蛋白的表达量均升高;过表达UbcH8的Kasumi-1细胞中,AML1-ETO蛋白表达量减少;用和厚朴酚处理Ubc H8敲除后的Kasumi-1细胞,AML1-ETO的降解被阻断;和厚朴酚抑制了异种移植小鼠模型的肿瘤生长,肿瘤中的AML1-ETO蛋白显著降低,而UBCH8表达升高。结论:和厚朴酚能通过UbcH8降解白血病细胞中的AML1-ETO蛋白。
Objective: To investigate the possible mechanism of the degradation of AML1-ETO protein induced by honokiol. Methods: Kasumi-1 cells were treated with 10, 20 and 40 μmol/L honokiol for 24 h and 48 h.The mRNA expression of AML1-ETO and UbcH8 and the protein expression were detected by qRT-PCR and Western blot respectively. Microarry analysis was used to select the target gene. To explore the role of UbcH8 in the degradation of AML1-ETO protein induced by honokiol, the expression of UbcH8 was knocked down by small hairpin RNAs(shRNAs) and overexpressed by MSCV-based vector to detect the related indicators. A xenograft mice model was treated by honokiol to detect tumorigenicity and related protein expression. Results:Honokiol decreased the protein expression of AML1-ETO in a time-and concentration-dependent manner, and reduced the stability of AML1-ETO protein, but did not affect the mRNA expression of AML1-ETO in kasumi-1 cell. We identified UbcH8 as the target gene by honokiol through microarray analysis. Honokiol obviously increased the expression of UbcH8 by 4-fold. Furthermore, overexpression of UbcH8 decreased the expression of AML1-ETO and knockdown of UbcH8 by shRNAs prevented honokiol-induced degradation of AML-ETO,suggesting that UbcH8 plays a critical role in the degradation of AML1-ETO by honokiol. Finally, honokiol decreased xenograft tumor size in a xenograft leukemia mouse model. Meanwhile, the protein of AML1-ETO was significantly reduced, while UBCH8 expression was elevated. Conclusion: Honokiol can degrade AML1-ETO oncoprotein through upregulation of UbcH8 in acute myeloid leukemia.
Objective: To investigate the possible mechanism of the degradation of AML1-ETO protein induced by honokiol. Methods: Kasumi-1 cells were treated with 10, 20 and 40 μmol/L honokiol for 24 h and 48 h.The mRNA expression of AML1-ETO and UbcH8 and the protein expression were detected by qRT-PCR and Western blot respectively. Microarry analysis was used to select the target gene. To explore the role of UbcH8 in the degradation of AML1-ETO protein induced by honokiol, the expression of UbcH8 was knocked down by small hairpin RNAs(shRNAs) and overexpressed by MSCV-based vector to detect the related indicators. A xenograft mice model was treated by honokiol to detect tumorigenicity and related protein expression. Results:Honokiol decreased the protein expression of AML1-ETO in a time-and concentration-dependent manner, and reduced the stability of AML1-ETO protein, but did not affect the mRNA expression of AML1-ETO in kasumi-1 cell. We identified UbcH8 as the target gene by honokiol through microarray analysis. Honokiol obviously increased the expression of UbcH8 by 4-fold. Furthermore, overexpression of UbcH8 decreased the expression of AML1-ETO and knockdown of UbcH8 by shRNAs prevented honokiol-induced degradation of AML-ETO,suggesting that UbcH8 plays a critical role in the degradation of AML1-ETO by honokiol. Finally, honokiol decreased xenograft tumor size in a xenograft leukemia mouse model. Meanwhile, the protein of AML1-ETO was significantly reduced, while UBCH8 expression was elevated. Conclusion: Honokiol can degrade AML1-ETO oncoprotein through upregulation of UbcH8 in acute myeloid leukemia.
引文
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[13]LAFIURA K M,EDWARDS H,TAUB J W,et al.Identification and characterization of novel AML1-ETO fusion transcripts in pediatrict(8;21)acute myeloid leukemia:a report from the Children’s Oncology Group[J].Oncogene,2008,27(36):4933-4942.
[14]邢冲云,高申孟,梁彬,等.实时荧光定量PCR检测急性髓系白血病患者WT1基因表达及其临床意义[J].温州医学院学报,2011,41(6):527-530.
[15]NISHIDA S,HOSEN N,SHIRAKATA T,et al.AML1-ETOrapidly induces acute myeloblastic leukemia in cooperation with the Wilms tumor gene,WT1[J].Blood,2006,107(8):3303-3312.
[16]ZHAO S,ZHANG Y,SHA K,et al.KRAS(G12D)Cooperates with AML1/ETO to initiate a mouse model mimicking human acute myeloid leukemia[J].Cell Physiol Biochem,2014,33(1):78-87.
[17]GAO X N,YAN F,LIN J,et al.AML1/ETO cooperates with HIF1αto promote leukemogenesis through DNMT3a transactivation[J].Leukemia,2015,29(8):1730-1740.
[18]WANG Z,ZHANG X.Chemopreventive activity of honokiol against 7,12-dimethylbenz[a]anthracene-induced mammary cancer in female sprague dawley rats[J].Front Pharmacol,2017,8:320.
[19]SENGUPTA S,NAGALINGAM A,MUNIRAJ N,et al.Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3[J].Oncogene,2017,36(41):5709-5721.
[20]PAN J,LEE Y,ZHANG Q,et al.Honokiol decreases lung cancer metastasis through inhibition of the STAT3 signaling pathway[J].Cancer Prev Res(Phila),2017,10(2):133-141.
[21]LU C H,CHEN S H,CHANG Y S,et al.Honokiol,a potential therapeutic agent,induces cell cycle arrest and program cell death in vitro and in vivo in human thyroid cancer cells[J].Pharmacol Res,2017,115:288-298.
[22]CHO J H,JEON Y J,PARK S M,et al.Multifunctional effects of honokiol as an anti-inflammatory and anti-cancer drug in human oral squamous cancer cells and xenograft[J].Biomaterials,2015,53:274-284.
[23]KRAMER O H,MULLER S,BUCHWALD M,et al.Mechanism for ubiquitylation of the leukemia fusion proteins AML1-ETO and PML-RARalpha[J].FASEB J,2008,22(5):1369-1379.
[24]TOPALY J,ZELLER W J,FRUEHAUF S.Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells[J].Leukemia,2001,15(3):342-347.
[25]HUANG M E,YE Y C,CHEN S R,et al.Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia[J].Blood,1988,72(2):567-572.
[26]陈月苗,俞康,郑翠苹,等.CD56阳性对急性早幼粒细胞白血病患者预后的影响[J].温州医科大学学报,2019,49(2):96-99.
[2]PABST T,MUELLER B U,HARAKAWA N,et al.AML1-ETO downregulates the granulocytic differentiation factor C/EBPalpha in t(8;21)myeloid leukemia[J].Nat Med,2001,7(4):444-451.
[3]WANG Y Y,ZHAO L J,WU C F,et al.C-KIT mutation cooperates with full-length AML1-ETO to induce acute myeloid leukemia in mice[J].Proc Natl Acad Sci U S A,2011,108(6):2450-2455.
[4]GRISOLANO J L,ONEAL J,CAIN J,et al.An activated receptor tyrosine kinase,TEL/PDGFbetaR,cooperates with AML1/ETO to induce acute myeloid leukemia in mice[J].Proc Natl Acad Sci U S A,2003,100(16):9506-9511.
[5]CHEN G,IZZO J,DEMIZU Y,et al.Different redox states in malignant and nonmalignant esophageal epithelial cells and differential cytotoxic responses to bile acid and honokiol[J].Antioxid Redox Signal,2009,11(5):1083-1095.
[6]AVTANSKI D B,NAGALINGAM A,KUPPUSAMY P,et al.Honokiol abrogates leptin-induced tumor progression by inhibiting Wnt1-MTA1-beta-catenin signaling axis in a microRNA-34a dependent manner[J].Oncotargets,2015,6(18):16396-16410.
[7]PANDEY M K,SUNG B,AGGARWAL B B.Betulinic acid suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase SHP-1 in human multiple myeloma cells[J].Int J Cancer,2010,127(2):282-292.
[8]BI L,YU Z,WU J,et al.Honokiol inhibits constitutive and inducible STAT3 signaling via PU.1-induced SHP1 expression in acute myeloid leukemia cells[J].Tohoku J Exp Med,2015,237(3):163-172.
[9]SINGH T,GUPTA N A,XU S,et al.Honokiol inhibits the growth of head and neck squamous cell carcinoma by targeting epidermal growth factor receptor[J].Oncotargets,2015,6(25):21268-21282.
[10]LI H Y,YE H G,CHEN C Q,et al.Honokiol induces cell cycle arrest and apoptosis via inhibiting class I histone deacetylases in acute myeloid leukemia[J].J Cell Biochem,2015,116(2):287-298.
[11]SERNIWKA S A,SHAW G S.The structure of the UbcH8-ubiquitin complex shows a unique ubiquitin interaction site[J].Biochemistry,2009,48(51):12169-12179.
[12]MAN N,TAN Y,SUN X J,et al.Caspase-3 controls AML1-ETO-driven leukemogenesis via autophagy modulation in a ULK1 dependent manner[J].Blood,2017,129(20):2782-2792.
[13]LAFIURA K M,EDWARDS H,TAUB J W,et al.Identification and characterization of novel AML1-ETO fusion transcripts in pediatrict(8;21)acute myeloid leukemia:a report from the Children’s Oncology Group[J].Oncogene,2008,27(36):4933-4942.
[14]邢冲云,高申孟,梁彬,等.实时荧光定量PCR检测急性髓系白血病患者WT1基因表达及其临床意义[J].温州医学院学报,2011,41(6):527-530.
[15]NISHIDA S,HOSEN N,SHIRAKATA T,et al.AML1-ETOrapidly induces acute myeloblastic leukemia in cooperation with the Wilms tumor gene,WT1[J].Blood,2006,107(8):3303-3312.
[16]ZHAO S,ZHANG Y,SHA K,et al.KRAS(G12D)Cooperates with AML1/ETO to initiate a mouse model mimicking human acute myeloid leukemia[J].Cell Physiol Biochem,2014,33(1):78-87.
[17]GAO X N,YAN F,LIN J,et al.AML1/ETO cooperates with HIF1αto promote leukemogenesis through DNMT3a transactivation[J].Leukemia,2015,29(8):1730-1740.
[18]WANG Z,ZHANG X.Chemopreventive activity of honokiol against 7,12-dimethylbenz[a]anthracene-induced mammary cancer in female sprague dawley rats[J].Front Pharmacol,2017,8:320.
[19]SENGUPTA S,NAGALINGAM A,MUNIRAJ N,et al.Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3[J].Oncogene,2017,36(41):5709-5721.
[20]PAN J,LEE Y,ZHANG Q,et al.Honokiol decreases lung cancer metastasis through inhibition of the STAT3 signaling pathway[J].Cancer Prev Res(Phila),2017,10(2):133-141.
[21]LU C H,CHEN S H,CHANG Y S,et al.Honokiol,a potential therapeutic agent,induces cell cycle arrest and program cell death in vitro and in vivo in human thyroid cancer cells[J].Pharmacol Res,2017,115:288-298.
[22]CHO J H,JEON Y J,PARK S M,et al.Multifunctional effects of honokiol as an anti-inflammatory and anti-cancer drug in human oral squamous cancer cells and xenograft[J].Biomaterials,2015,53:274-284.
[23]KRAMER O H,MULLER S,BUCHWALD M,et al.Mechanism for ubiquitylation of the leukemia fusion proteins AML1-ETO and PML-RARalpha[J].FASEB J,2008,22(5):1369-1379.
[24]TOPALY J,ZELLER W J,FRUEHAUF S.Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells[J].Leukemia,2001,15(3):342-347.
[25]HUANG M E,YE Y C,CHEN S R,et al.Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia[J].Blood,1988,72(2):567-572.
[26]陈月苗,俞康,郑翠苹,等.CD56阳性对急性早幼粒细胞白血病患者预后的影响[J].温州医科大学学报,2019,49(2):96-99.