HAPLN1通过IKK/p65信号通路诱导大肠癌HT-29细胞产生MTX耐药
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摘要
目的:研究甲氨蝶呤(methotrexate,MTX)耐药前后人大肠癌(结直肠癌)HT-29细胞中透明质酸蛋白聚糖连结蛋白1(hyaluronan and proteoglycan link protein 1,HAPLN1)表达的变化及其对MTX耐药性的影响,并探究可能的分子机制。方法:采用浓度梯度递增法构建耐药细胞株HT-29/MTX;RT-PCR检测HAPLN1和多药耐药相关蛋白2(multidrug resistance-associated protein 2,MRP2)的mRNA表达水平;采用脂质体介导法将人HAPLN1和MRP2基因的干扰质粒转染HT-29/MTX细胞并筛选出稳定表达的细胞系;采用CCK-8法检测细胞活力;流式细胞术检测细胞凋亡;采用Western blot检测HAPLN1、MRP2、IκB激酶(IκB kinase,IKK)α/β、p-IKKα/β(Ser176/Ser177)、p65和p-p65(Ser536)的蛋白水平。结果:HT-29/MTX细胞中HAPLN1和MRP2的mRNA和蛋白表达水平都显著高于其亲代HT-29细胞(P<0.05),耐药倍数高达463.756。抑制HAPLN1和MRP2基因表达使MTX对HT-29/MTX细胞的IC_(50)从15.304μmol/L分别降至6.119μmol/L和7.801μmol/L,逆转倍数分别为2.501和1.962,并增强MTX诱导的细胞凋亡(P<0.05)。敲减HAPLN1基因表达和使用IKK抑制剂IKK16均可下调IKKα/β和p65蛋白磷酸化水平以及MRP2蛋白表达水平(P<0.05),但IKK16未对HAPLN1蛋白表达产生影响(P>0.05)。结论:敲减HAPLN1基因表达可在体外增强人大肠癌耐药细胞株HT-29/MTX对MTX的敏感性,这可能与其抑制IKK/p65信号通路活化,继而下调MRP2基因表达有关。
AIM: To investigate the changes of hyaluronan and proteoglycan link protein 1(HAPLN1) expression before and after resistance to methotrexate(MTX) in human colorectal cancer HT-29 cells and its effect on this drug resistance, and to explore the molecular mechanism in the process. METHODS: The drug-resistant HT-29/MTX cells were established by stepwise exposure of the cells to MTX, and then the HT-29/MTX cells were stably transfected with specific shRNA interference plasmid vectors targeting HAPLN1 and multidrug resistance-associated protein 2(MRP2). The mRNA expression levels of HAPLN1 and MRP2 were measured by RT-PCR. CCK-8 assay was used to detect the viability of HT-29/MTX cells. The apoptosis rate was analyzed by flow cytometry. The protein levels of HAPLN1, MRP2, IκB kinase(IKK) α/β, p-IKKα/β(Ser176/Ser177), p65 and p-p65(Ser536) were determined by Western blot. RESULTS: The HT-29/MTX cells had significantly higher mRNA and protein levels of HAPLN1 and MRP2 than HT-29 cells(P<0.05) with resistant factor of 463.756. HAPLN1 and MRP2 gene silencing significantly increased the cytotoxicity and apoptosis of HT-29/MTX cells induced by MTX(P<0.05). The IC_(50) value was decreased from 15.304 μmol/L to 6.119 μmol/L and 7.801 μmol/L, respectively, and their reversal folds were 2.501 and 1.962, respectively. Silencing of HAPLN1 and IKK inhibitor IKK16 inhibited the phosphorylation of IKKα/β and p65(P<0.05), and down-regulated the protein level of MRP2 in the HT-29/MTX cells(P<0.05). However, IKK16 did not affect the protein level of HAPLN1 in the HT-29/MTX cells.CONCLUSION: Knock-down of HAPLN1 gene expression reverses the resistance to MTX in human colorectal cancer HT-29/MTX cells possibly by blocking the IKK/p65 signaling pathway and thus down-regulating the expression of MRP2.
AIM: To investigate the changes of hyaluronan and proteoglycan link protein 1(HAPLN1) expression before and after resistance to methotrexate(MTX) in human colorectal cancer HT-29 cells and its effect on this drug resistance, and to explore the molecular mechanism in the process. METHODS: The drug-resistant HT-29/MTX cells were established by stepwise exposure of the cells to MTX, and then the HT-29/MTX cells were stably transfected with specific shRNA interference plasmid vectors targeting HAPLN1 and multidrug resistance-associated protein 2(MRP2). The mRNA expression levels of HAPLN1 and MRP2 were measured by RT-PCR. CCK-8 assay was used to detect the viability of HT-29/MTX cells. The apoptosis rate was analyzed by flow cytometry. The protein levels of HAPLN1, MRP2, IκB kinase(IKK) α/β, p-IKKα/β(Ser176/Ser177), p65 and p-p65(Ser536) were determined by Western blot. RESULTS: The HT-29/MTX cells had significantly higher mRNA and protein levels of HAPLN1 and MRP2 than HT-29 cells(P<0.05) with resistant factor of 463.756. HAPLN1 and MRP2 gene silencing significantly increased the cytotoxicity and apoptosis of HT-29/MTX cells induced by MTX(P<0.05). The IC_(50) value was decreased from 15.304 μmol/L to 6.119 μmol/L and 7.801 μmol/L, respectively, and their reversal folds were 2.501 and 1.962, respectively. Silencing of HAPLN1 and IKK inhibitor IKK16 inhibited the phosphorylation of IKKα/β and p65(P<0.05), and down-regulated the protein level of MRP2 in the HT-29/MTX cells(P<0.05). However, IKK16 did not affect the protein level of HAPLN1 in the HT-29/MTX cells.CONCLUSION: Knock-down of HAPLN1 gene expression reverses the resistance to MTX in human colorectal cancer HT-29/MTX cells possibly by blocking the IKK/p65 signaling pathway and thus down-regulating the expression of MRP2.
引文
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[3] 李聪. 结直肠癌筛查现状[J]. 广东医学, 2016, 37(22):3328-3330.
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[9] Scheibner KA, Lutz MA, Boodoo S, et al. Hyaluronan fragments act as an endogenous danger signal by engaging TLR2[J]. J Immunol, 2006, 177(2):1272-1281.
[10] Mebarki S, Désert R, Sulpice L, et al. De novo HAPLN1 expression hallmarks Wnt-induced stem cell and fibrogenic networks leading to aggressive human hepatocellular carcinomas[J]. Oncotarget, 2016, 7(26):39026-39043.
[11] Huynh M, Pak C, Markovina S, et al. Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resis-tance in multiple myeloma[J]. J Biol Chem, 2018, 293(7):2452-2465.
[12] DeSouza LV, Matta A, Karim Z, et al. Role of moesin in hyaluronan induced cell migration in glioblastoma multiforme[J]. Mol Cancer, 2013, 12:74.
[13] Bourguignon LY, Wong G, Earle CA, et al. Interaction of low molecular weight hyaluronan with CD44 and toll-like receptors promotes the actin filament-associated protein 110-actin binding and MyD88-NFκB signaling leading to proinflammatory cytokine/chemokine production and breast tumor invasion[J]. Cytoskeleton (Hoboken), 2011, 68(12):671-693.
[14] Ween MP, Oehler MK, Ricciardelli C. Role of versican, hyaluronan and CD44 in ovarian cancer metastasis[J]. Int J Mol Sci, 2011,12(2):1009-1029.
[15] Slevin M, Krupinski J, Gaffney J, et al. Hyaluronan-mediated angiogenesis in vascular disease: uncovering RHAMM and CD44 receptor signaling pathways[J]. Matrix Biol, 2007, 26(1):58-68.
[16] Wang Z, Sun X, Feng Y, et al. Dihydromyricetin reverses MRP2-mediated MDR and enhances anticancer activity induced by oxaliplatin in colorectal cancer cells[J]. Anticancer Drugs, 2017, 28(3):281-288.
[17] Woillard JB, Debord J, Benz-de-Bretagne I, et al. A time-dependent model describes methotrexate elimination and supports dynamic modification of MRP2/ABCC2 acti-vity[J]. Ther Drug Monit, 2017, 39(2):145-156.
[18] Liu Y, Yin Y, Sheng Q, et al. Association of ABCC2 -24C>T polymorphism with high-dose methotrexate plasma concentrations and toxicities in childhood acute lymphoblastic leukemia[J]. PLoS One, 2014, 9(1):e82681.
[19] Zgheib NK, Akra-Ismail M, Aridi C, et al. Genetic polymorphisms in candidate genes predict increased toxicity with methotrexate therapy in Lebanese children with acute lymphoblastic leukemia[J]. Pharmacogenet Genomics, 2014, 24(8):387-396.
[20] Sharifi MJ, Bahoush G, Zaker F, et al. Association of -24CT, 1249GA, and 3972CT ABCC2 gene polymorphisms with methotrexate serum levels and toxic side effects in children with acute lymphoblastic leukemia[J]. Pediatr Hematol Oncol, 2014, 31(2):169-177.
[21] Ke SZ, Ni XY, Zhang YH, et al. Camptothecin and cis-platin upregulate ABCG2 and MRP2 expression by activating the ATM/NF-κB pathway in lung cancer cells[J]. Int J Oncol, 2013, 42(4):1289-1296.
[22] Zhang J, Zhang M, Sun B, et al. Hyperammonemia enhances the function and expression of P-glycoprotein and Mrp2 at the blood-brain barrier through NF-κB[J]. J Neurochem, 2014, 131(6):791-802.
[23] Balasubramaniyan N, Ananthanarayanan M, Suchy FJ. Nuclear factor-κB regulates the expression of multiple genes encoding liver transport proteins[J]. Am J Physiol Gastrointest Liver Physiol, 2016, 310(8):G618-G628.
[24] 赵京山, 孙佳欢, 于琨, 等. Roscovitine通过影响核因子κB活化抑制大鼠颈动脉内膜损伤导致的炎性增生[J]. 中国病理生理杂志, 2017, 33(2):233-238.
[2] Chen W, Zheng R, Zhang S, et al. Cancer incidence and mortality in China, 2013[J]. Cancer Lett, 2017, 401:63-71.
[3] 李聪. 结直肠癌筛查现状[J]. 广东医学, 2016, 37(22):3328-3330.
[4] Kotelevets L, Chastre E, Desma?le D, et al. Nanotechnologies for the treatment of colon cancer: from old drugs to new hope[J]. Int J Pharm, 2016, 514(1):24-40.
[5] Du J, He Y, Li P, et al. IL-8 regulates the doxorubicin resistance of colorectal cancer cells via modulation of multidrug resistance 1 (MDR1)[J]. Cancer Chemother Pharmacol, 2018, 81(6):1111-1119.
[6] Chung YH, Kim D. Enhanced TLR4 expression on colon cancer cells after chemotherapy promotes cell survival and epithelial-mesenchymal transition through phosphorylation of GSK3β[J]. Anticancer Res, 2016, 36(7):3383-3394.
[7] Sui H, Zhou LH, Zhang YL, et al. Evodiamine suppresses ABCG2 mediated drug resistance by inhibiting p50/p65 NF-κB pathway in colorectal cancer[J]. J Cell Biochem, 2016, 117(6):1471-1481.
[8] Misra S, Hascall VC, Markwald RR, et al. Interactions between hyaluronan and its receptors (CD44, RHAMM) regulate the activities of inflammation and cancer[J]. Front Immunol, 2015, 6:201.
[9] Scheibner KA, Lutz MA, Boodoo S, et al. Hyaluronan fragments act as an endogenous danger signal by engaging TLR2[J]. J Immunol, 2006, 177(2):1272-1281.
[10] Mebarki S, Désert R, Sulpice L, et al. De novo HAPLN1 expression hallmarks Wnt-induced stem cell and fibrogenic networks leading to aggressive human hepatocellular carcinomas[J]. Oncotarget, 2016, 7(26):39026-39043.
[11] Huynh M, Pak C, Markovina S, et al. Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resis-tance in multiple myeloma[J]. J Biol Chem, 2018, 293(7):2452-2465.
[12] DeSouza LV, Matta A, Karim Z, et al. Role of moesin in hyaluronan induced cell migration in glioblastoma multiforme[J]. Mol Cancer, 2013, 12:74.
[13] Bourguignon LY, Wong G, Earle CA, et al. Interaction of low molecular weight hyaluronan with CD44 and toll-like receptors promotes the actin filament-associated protein 110-actin binding and MyD88-NFκB signaling leading to proinflammatory cytokine/chemokine production and breast tumor invasion[J]. Cytoskeleton (Hoboken), 2011, 68(12):671-693.
[14] Ween MP, Oehler MK, Ricciardelli C. Role of versican, hyaluronan and CD44 in ovarian cancer metastasis[J]. Int J Mol Sci, 2011,12(2):1009-1029.
[15] Slevin M, Krupinski J, Gaffney J, et al. Hyaluronan-mediated angiogenesis in vascular disease: uncovering RHAMM and CD44 receptor signaling pathways[J]. Matrix Biol, 2007, 26(1):58-68.
[16] Wang Z, Sun X, Feng Y, et al. Dihydromyricetin reverses MRP2-mediated MDR and enhances anticancer activity induced by oxaliplatin in colorectal cancer cells[J]. Anticancer Drugs, 2017, 28(3):281-288.
[17] Woillard JB, Debord J, Benz-de-Bretagne I, et al. A time-dependent model describes methotrexate elimination and supports dynamic modification of MRP2/ABCC2 acti-vity[J]. Ther Drug Monit, 2017, 39(2):145-156.
[18] Liu Y, Yin Y, Sheng Q, et al. Association of ABCC2 -24C>T polymorphism with high-dose methotrexate plasma concentrations and toxicities in childhood acute lymphoblastic leukemia[J]. PLoS One, 2014, 9(1):e82681.
[19] Zgheib NK, Akra-Ismail M, Aridi C, et al. Genetic polymorphisms in candidate genes predict increased toxicity with methotrexate therapy in Lebanese children with acute lymphoblastic leukemia[J]. Pharmacogenet Genomics, 2014, 24(8):387-396.
[20] Sharifi MJ, Bahoush G, Zaker F, et al. Association of -24CT, 1249GA, and 3972CT ABCC2 gene polymorphisms with methotrexate serum levels and toxic side effects in children with acute lymphoblastic leukemia[J]. Pediatr Hematol Oncol, 2014, 31(2):169-177.
[21] Ke SZ, Ni XY, Zhang YH, et al. Camptothecin and cis-platin upregulate ABCG2 and MRP2 expression by activating the ATM/NF-κB pathway in lung cancer cells[J]. Int J Oncol, 2013, 42(4):1289-1296.
[22] Zhang J, Zhang M, Sun B, et al. Hyperammonemia enhances the function and expression of P-glycoprotein and Mrp2 at the blood-brain barrier through NF-κB[J]. J Neurochem, 2014, 131(6):791-802.
[23] Balasubramaniyan N, Ananthanarayanan M, Suchy FJ. Nuclear factor-κB regulates the expression of multiple genes encoding liver transport proteins[J]. Am J Physiol Gastrointest Liver Physiol, 2016, 310(8):G618-G628.
[24] 赵京山, 孙佳欢, 于琨, 等. Roscovitine通过影响核因子κB活化抑制大鼠颈动脉内膜损伤导致的炎性增生[J]. 中国病理生理杂志, 2017, 33(2):233-238.