Btk调控MDR1逆转急性淋巴细胞白血病耐药的研究
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摘要
目的:通过观察Btk抑制剂依鲁替尼作用于急性淋巴细胞白血病(ALL)细胞株(Sup-B15)后细胞中MDR1 mRNA、P-gp蛋白的表达及细胞对化疗药物敏感性的变化,探讨逆转ALL多药耐药的有效方法。方法:(1)利用依鲁替尼处理Sup-B15,PCR法检测处理后细胞中MDR1 mRNA水平变化;免疫印迹法检测P-gp等蛋白水平变化;(2)利用化疗药物作用于依鲁替尼处理后的Sup-B15,CCK-8法检测细胞增殖。结果:(1)在Sup-B15中,依鲁替尼作用后的MDR1 mRNA表达较对照组降低;细胞中P-gp等蛋白表达量较对照组降低。(2)依鲁替尼处理后的Sup-B15在相同Ara-C、ADR浓度作用下,其细胞增殖率较对照组降低(P <0. 05)。结论:(1)依鲁替尼可降低Sup-B15中MDR1 mRNA及P-gp蛋白的表达,其作用机制可能是通过抑制Btk、NFκB表达及生物学活性,从而抑制MDR1及P-gp的表达。(2)依鲁替尼增强ALL细胞对化疗药物的敏感性。
Objective: By targeting Btk in acute lymphoblastic leukemia(ALL) cells,to observe the expression of MDR1 and P-gp and the changes of cellular sensitivity to chemotherapy drugs of ALL cells,to further explore the role of Btk in the regulation of ALL multidrug resistance. Methods:(1) Sup-B15 were treated with ibrutinib,and the mRNA and the protein expression levels of Btk and MDR1(P-gp) were detected by PCR and Western blot,respectively.(2) Sup-B15 were first treated with ibrutinib,and then with Ara-C or ADR respectively,the cell proliferation was detected by CCK-8 method. Results:(1) After being treated with ibrutinib,the mRNA and protein expression levels of MDR1 and P-gp were decreased as compared with the control group.(2) When Sup-B15 were treated with ibrutinib and followed by Ara-C or ADR respectively,the cell proliferation rate was lower than that of the control group(P < 0. 05). Conclusion:(1) Ibrutinib can decrease the expression of MDR1 mRNA and P-gp protein by inhibiting the expression of Btk and NFκB in ALL cells.(2) Ibrutinib can enhance the sensitivity of Sup-B15 cells to Ara-C and ADR.
Objective: By targeting Btk in acute lymphoblastic leukemia(ALL) cells,to observe the expression of MDR1 and P-gp and the changes of cellular sensitivity to chemotherapy drugs of ALL cells,to further explore the role of Btk in the regulation of ALL multidrug resistance. Methods:(1) Sup-B15 were treated with ibrutinib,and the mRNA and the protein expression levels of Btk and MDR1(P-gp) were detected by PCR and Western blot,respectively.(2) Sup-B15 were first treated with ibrutinib,and then with Ara-C or ADR respectively,the cell proliferation was detected by CCK-8 method. Results:(1) After being treated with ibrutinib,the mRNA and protein expression levels of MDR1 and P-gp were decreased as compared with the control group.(2) When Sup-B15 were treated with ibrutinib and followed by Ara-C or ADR respectively,the cell proliferation rate was lower than that of the control group(P < 0. 05). Conclusion:(1) Ibrutinib can decrease the expression of MDR1 mRNA and P-gp protein by inhibiting the expression of Btk and NFκB in ALL cells.(2) Ibrutinib can enhance the sensitivity of Sup-B15 cells to Ara-C and ADR.
引文
[1]FYRBERG A,PETERSON C,KAGEDAL B,et al.Induction of fetal hemoglobin and ABCB1 gene expression in 9-beta-D-arabinofuranosylguanine-resistant MOLT-4 cells[J].Cancer Chemother Pharmacol,2011,68(3):583-591.
[2]LIN J,XING H Y,GONG Y P,et al.Multidrug resistant gene MDR1 contributes to development of imatinib-resistance in Ph(+)acute lymphoblastic leukemia cell line SUP-BS15RI[J].Journal of Sichuan University(Medical Science Edition),2012,43(5):657-660,665.
[3]RAHGOZAR S,MOAFI A,ABEDI M,et al.m RNA expression profile of multidrug-resistant genes in acute lymphoblastic leukemia of children,a prognostic value for ABCA3 and ABCA2[J].Cancer Biol Ther,2014,15(1):35-41.
[4]PONGSTAPOM W,PAKAKASAM S,CHAKSANGCHAI-CHOTE P,et al.MDR1 C3435T and C1236T polymorphisms:association with high-risk childhood acute lymphoblastic leukemia[J].Asian Pac J Cancer Prev,2015,16(7):2839-2843.
[5]ANSARII M,SAUTY G,LABUDA M,et al.Polymorphism in multidrug resistance-associated protein gene 3 is associated with outcomes in childhood acute lymphoblastic leukemia[J].Pharmacogenomics J,2012,12(5):386-394.
[6]SU Y,LEE S H,SINKO P J.Inhibition of efflux transporter ABCG2/BCRP does not restore mitoxantrone sensitivity in irinotecan-selected human leukemia CPT-K5 cells:evidence for multifactorial multidrug resistance[J].Eur J Pharm Sci,2006,29(2):102-110.
[7]HUANG F F,WU D S,ZHANG L,et al.Inactivation of PTENincreases ABCG2 expression and the side population through the PI3K/Akt pathway in adult acute leukemia[J].Cancer Lett,2013,336(1):96-105.
[8]江雪杰.成人急性淋巴细胞白血病中乳腺癌耐药蛋白基因表达及其临床意义[J].中国实验血液学杂志,2008,16(1):31-34.
[9]SHAH D S,KUMAR R.Steroid resistance in leukemia[J].World J Exp Med,2013,3(2):21-25.
[10]TAO S D,WANG C L,CHEN Y,et al.Btk and NFκB as prognostic biomarkers and potential therapeutic targets in Bcell acute lymphoblastic leukemia[J].Int J Clin Exp Pathol,2016,9(8):7995-8005.
[11]DAVOUDI Z,AKBARZADEH A,RAHMATIYAMCHI M,et al.Molecular target therapy of AKT and NF-k B signaling pathways and multidrug resistance by specific cell penetrating inhibitor peptides in HL-60 cells[J].Asian Pac J Cancer Prev,2014,15(10):4353-4358.
[12]SOH S X,LIM J Y,HUANG J W,et al.Multi-agent chemotherapy overcomes glucocorticoid resistance conferred by a BIM deletion polymorphism in pediatric acute lymphoblastic leukemia[J].PLo S One,2014,9(8):e103435.
[13]VASSILEV A,OZER Z,NAVARA C,et al.Bruton's tyrosine kinase as an inhibitor of the Fas/CD95 death-inducing signaling complex[J].J Biol Chem,1999,274(3):1646-1656.
[14]LIN K,GLENN M A,HARRIS R J,et al.c-Abl expression in chronic lymphocytic leukemia cells:clinical and therapeutic implications[J].Cancer Res,2006,66(15):7801-7809.
[15]陶善东,邓媛,何正梅,等.Btk及NFκB在急性髓系白血病细胞中的表达及其意义[J].中国实验血液学杂志,2013,21(1):25-28.
[16]朱文艳,王志清,华海应.急性淋巴细胞性白血病13个月后初次缓解1例并文献复习[J].现代医学,2015,43(9):1163-1164.
[17]邓媛,陶善东,张欣,等.PCI-32765和Bortezomib对B细胞肿瘤细胞系细胞增殖、凋亡的影响及其机制的研究[J].中国实验血液学杂志,2013,21:1178-1182.
[18]CHIEN W W,LE B C,RACHINEL N,et al.Differential mechanisms of asparaginase resistance in B-type acute lymphoblastic leukemia and malignant natural killer cell lines[J].Sci Rep,2015,5:8068.
[19]BUCHNER M,PARK E,GENG H,et al.Identification of FOXM1 as a therapeutic target in B-cell lineage acute lymphoblastic leukaemia[J].Nat Commun,2015,6:6471.
[20]ZHOU L,BAI H,WANG C,et al.micro RNA125b promotes leukemia cell resistance to daunorubicin by inhibiting apoptosis[J].Mol Med Rep,2014,9(5):1909-1916.
[21]de OLIVEIRA J C,BRASSESCO M S,SCRIDELI C A,et al.MicroRNA expression and activity in pediatric acute lymphoblastic leukemia(ALL)[J].Pediatr Blood Cancer,2012,59(4):599-604.
[22]LIAO W,LIU X,PENG H,et al.The expression and functional study of mi R-181a in pediatric acute lymphoblastic leukemia[J].Chinese Journal of Hematology,2015,36(1):53-57.
[23]NISHIOKA C,IKEZOE T,YANG J,et al.Downregulation of mi R-217 Correlates with resistance of Ph(+)leukemia cells to ABL tyrosine kinase inhibitors[J].Cancer Sci,2014,105(3):297-307.
[24]FERNANDO T R,RODRIGUEZ-MALAVE N I,WATERS EV,et al.Lnc RNA expression discriminates karyotype and predicts survival in b-lymphoblastic leukemia[J].Mol Cancer Res,2015,13(5):839-851.
[25]SHISHIDO S,BONIG H,KIM Y M.Role of integrin alpha4 in drug resistance of leukemia[J].Front Oncol,2014,4:99.
[26]SHENG X,MITTELMAN S D.The role of adipose tissue and obesity in causing treatment resistance of acute lymphoblastic leukemia[J].Front Pediatr,2014,2:53.
[27]SAMUELS A L,BEESLEY A H,YADAV B D,et al.A preclinical model of resistance to induction therapy in pediatric acute lymphoblastic leukemia[J].Blood Cancer J,2014,4:e232.
[28]YEN C Y,CHIANG W F,LIU S Y,et al.Long-term stimulation of areca nut components results in increased chemoresistance through elevated autophagic activity[J].J Oral Pathol Med,2014,43(2):91-96.
[29]王婷,陈芳源,韩洁英,等.CYP3A5参与急性白血病耐药机制的研究[J].中华血液学杂志,2003,24(6):286-289.
[30]KHARABI M B,GENG H,HURTZ C,et al.Mechanistic rationale for targeting the unfolded protein response in pre-B acute lymphoblastic leukemia[J].Proc Natl Acad Sci U S A,2014,111(21):E2219-E2228.
[31]COOLS J,MENTENS N,FURET P,et al.Prediction of resistance to small molecule FLT3 inhibitors:implications for molecularly targeted therapy of acute leukemia[J].Cancer Res,2004,64(18):6385-6389.
[32]夏君燕.我国210例伊马替尼耐药慢性髓细胞白血病和Ph阳性急性淋巴细胞白血病ABL1基因突变特征[J].中华检验医学杂志,2012,35(1):17-22.
[33]TAMAI H,MIYAKE K,YAMAGUCHI H,et al.Resistance of MLL-AFF1-positive acute lymphoblastic leukemia to tumor necrosis factor-alpha is mediated by S100A6 upregulation[J].Blood Cancer J,2011,1:e38.
[34]BURKE M J,LAMBA J K,POUNDS S,et al.A therapeutic trial of decitabine and vorinostat in combination with chemotherapy for relapsed/refractory acute lymphoblastic leukemia[J].Am J Hematol,2014,89(9):889-895.
[2]LIN J,XING H Y,GONG Y P,et al.Multidrug resistant gene MDR1 contributes to development of imatinib-resistance in Ph(+)acute lymphoblastic leukemia cell line SUP-BS15RI[J].Journal of Sichuan University(Medical Science Edition),2012,43(5):657-660,665.
[3]RAHGOZAR S,MOAFI A,ABEDI M,et al.m RNA expression profile of multidrug-resistant genes in acute lymphoblastic leukemia of children,a prognostic value for ABCA3 and ABCA2[J].Cancer Biol Ther,2014,15(1):35-41.
[4]PONGSTAPOM W,PAKAKASAM S,CHAKSANGCHAI-CHOTE P,et al.MDR1 C3435T and C1236T polymorphisms:association with high-risk childhood acute lymphoblastic leukemia[J].Asian Pac J Cancer Prev,2015,16(7):2839-2843.
[5]ANSARII M,SAUTY G,LABUDA M,et al.Polymorphism in multidrug resistance-associated protein gene 3 is associated with outcomes in childhood acute lymphoblastic leukemia[J].Pharmacogenomics J,2012,12(5):386-394.
[6]SU Y,LEE S H,SINKO P J.Inhibition of efflux transporter ABCG2/BCRP does not restore mitoxantrone sensitivity in irinotecan-selected human leukemia CPT-K5 cells:evidence for multifactorial multidrug resistance[J].Eur J Pharm Sci,2006,29(2):102-110.
[7]HUANG F F,WU D S,ZHANG L,et al.Inactivation of PTENincreases ABCG2 expression and the side population through the PI3K/Akt pathway in adult acute leukemia[J].Cancer Lett,2013,336(1):96-105.
[8]江雪杰.成人急性淋巴细胞白血病中乳腺癌耐药蛋白基因表达及其临床意义[J].中国实验血液学杂志,2008,16(1):31-34.
[9]SHAH D S,KUMAR R.Steroid resistance in leukemia[J].World J Exp Med,2013,3(2):21-25.
[10]TAO S D,WANG C L,CHEN Y,et al.Btk and NFκB as prognostic biomarkers and potential therapeutic targets in Bcell acute lymphoblastic leukemia[J].Int J Clin Exp Pathol,2016,9(8):7995-8005.
[11]DAVOUDI Z,AKBARZADEH A,RAHMATIYAMCHI M,et al.Molecular target therapy of AKT and NF-k B signaling pathways and multidrug resistance by specific cell penetrating inhibitor peptides in HL-60 cells[J].Asian Pac J Cancer Prev,2014,15(10):4353-4358.
[12]SOH S X,LIM J Y,HUANG J W,et al.Multi-agent chemotherapy overcomes glucocorticoid resistance conferred by a BIM deletion polymorphism in pediatric acute lymphoblastic leukemia[J].PLo S One,2014,9(8):e103435.
[13]VASSILEV A,OZER Z,NAVARA C,et al.Bruton's tyrosine kinase as an inhibitor of the Fas/CD95 death-inducing signaling complex[J].J Biol Chem,1999,274(3):1646-1656.
[14]LIN K,GLENN M A,HARRIS R J,et al.c-Abl expression in chronic lymphocytic leukemia cells:clinical and therapeutic implications[J].Cancer Res,2006,66(15):7801-7809.
[15]陶善东,邓媛,何正梅,等.Btk及NFκB在急性髓系白血病细胞中的表达及其意义[J].中国实验血液学杂志,2013,21(1):25-28.
[16]朱文艳,王志清,华海应.急性淋巴细胞性白血病13个月后初次缓解1例并文献复习[J].现代医学,2015,43(9):1163-1164.
[17]邓媛,陶善东,张欣,等.PCI-32765和Bortezomib对B细胞肿瘤细胞系细胞增殖、凋亡的影响及其机制的研究[J].中国实验血液学杂志,2013,21:1178-1182.
[18]CHIEN W W,LE B C,RACHINEL N,et al.Differential mechanisms of asparaginase resistance in B-type acute lymphoblastic leukemia and malignant natural killer cell lines[J].Sci Rep,2015,5:8068.
[19]BUCHNER M,PARK E,GENG H,et al.Identification of FOXM1 as a therapeutic target in B-cell lineage acute lymphoblastic leukaemia[J].Nat Commun,2015,6:6471.
[20]ZHOU L,BAI H,WANG C,et al.micro RNA125b promotes leukemia cell resistance to daunorubicin by inhibiting apoptosis[J].Mol Med Rep,2014,9(5):1909-1916.
[21]de OLIVEIRA J C,BRASSESCO M S,SCRIDELI C A,et al.MicroRNA expression and activity in pediatric acute lymphoblastic leukemia(ALL)[J].Pediatr Blood Cancer,2012,59(4):599-604.
[22]LIAO W,LIU X,PENG H,et al.The expression and functional study of mi R-181a in pediatric acute lymphoblastic leukemia[J].Chinese Journal of Hematology,2015,36(1):53-57.
[23]NISHIOKA C,IKEZOE T,YANG J,et al.Downregulation of mi R-217 Correlates with resistance of Ph(+)leukemia cells to ABL tyrosine kinase inhibitors[J].Cancer Sci,2014,105(3):297-307.
[24]FERNANDO T R,RODRIGUEZ-MALAVE N I,WATERS EV,et al.Lnc RNA expression discriminates karyotype and predicts survival in b-lymphoblastic leukemia[J].Mol Cancer Res,2015,13(5):839-851.
[25]SHISHIDO S,BONIG H,KIM Y M.Role of integrin alpha4 in drug resistance of leukemia[J].Front Oncol,2014,4:99.
[26]SHENG X,MITTELMAN S D.The role of adipose tissue and obesity in causing treatment resistance of acute lymphoblastic leukemia[J].Front Pediatr,2014,2:53.
[27]SAMUELS A L,BEESLEY A H,YADAV B D,et al.A preclinical model of resistance to induction therapy in pediatric acute lymphoblastic leukemia[J].Blood Cancer J,2014,4:e232.
[28]YEN C Y,CHIANG W F,LIU S Y,et al.Long-term stimulation of areca nut components results in increased chemoresistance through elevated autophagic activity[J].J Oral Pathol Med,2014,43(2):91-96.
[29]王婷,陈芳源,韩洁英,等.CYP3A5参与急性白血病耐药机制的研究[J].中华血液学杂志,2003,24(6):286-289.
[30]KHARABI M B,GENG H,HURTZ C,et al.Mechanistic rationale for targeting the unfolded protein response in pre-B acute lymphoblastic leukemia[J].Proc Natl Acad Sci U S A,2014,111(21):E2219-E2228.
[31]COOLS J,MENTENS N,FURET P,et al.Prediction of resistance to small molecule FLT3 inhibitors:implications for molecularly targeted therapy of acute leukemia[J].Cancer Res,2004,64(18):6385-6389.
[32]夏君燕.我国210例伊马替尼耐药慢性髓细胞白血病和Ph阳性急性淋巴细胞白血病ABL1基因突变特征[J].中华检验医学杂志,2012,35(1):17-22.
[33]TAMAI H,MIYAKE K,YAMAGUCHI H,et al.Resistance of MLL-AFF1-positive acute lymphoblastic leukemia to tumor necrosis factor-alpha is mediated by S100A6 upregulation[J].Blood Cancer J,2011,1:e38.
[34]BURKE M J,LAMBA J K,POUNDS S,et al.A therapeutic trial of decitabine and vorinostat in combination with chemotherapy for relapsed/refractory acute lymphoblastic leukemia[J].Am J Hematol,2014,89(9):889-895.