顺铂对肿瘤间充质干细胞中IL-6表达的调控及p38MAPK通路参与机制
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摘要
目的探讨顺铂(CDDP)对肿瘤间充质干细胞(MSCs)中白细胞介素-6(IL-6)表达的调控作用与参与机制。方法采用CDDP分别处理小鼠Lewis肺癌(LLC)细胞和MSCs,聚合酶链反应阵列(PCR Array)检测、筛选其IL-6表达改变,并通过实时荧光定量聚合酶链反应(qRT-PCR)、酶联免疫吸附实验(ELISA)等方法进一步验证。Western blotting检测CDDP处理后MSCs中p38MAPK通路的表达改变;并通过该通路特异性抑制剂抑制其激活,ELISA检测IL-6的表达。结果 CDDP处理LLC细胞后,IL-6的表达未见有统计学意义的改变(P>0.05)。PCR Array结果显示,CDDP处理MCSs 24 h后,各白细胞介素因子有不同程度的表达改变,其中IL-6表达上升最为显著;进一步研究证实,CDDP作用于MCSs 3、6和24 h后,IL-6 mRNA和蛋白表达均升高,差异具有统计学意义(IL-6 mRNA:P=0.218;P=0.007;P<0.001;蛋白表达:P=0.001;P<0.001;P<0.001)。CDDP处理后MSCs中p38和pp38的表达均明显增强,且pp38/p38比值明显升高;采用SB203580抑制p38MAPK通路后,CDDP所致的IL-6上调表达被部分逆转。结论 CDDP对LLC细胞中IL-6的表达无统计学意义影响,但可显著上调MSCs中IL-6的表达。CDDP处理后,MSCs中p38MAPK通路表达上调,抑制该通路后可下调IL-6的表达,提示CDDP可能通过p38MAPK通路调控IL-6在MSCs的表达。
Objective To investigate the effect of cisplatin(CDDP) on interleukin(IL)-6 expression in tumor mesenchymal stem cells(MSCs) and explore its possible mechanisms. Methods Lewis lung carcinoma(LLC) cells or MSCs were treated with CDDP, and their IL gene expression was detected by polymerase chain reaction array(PCR Array) and further verified by quantitative real-time polymerase chain reaction(qRT-PCR) and enzyme-linked immunosorbent assay(ELISA). After CDDP treatment, the activation of p38 MAPK pathway in MSCs was analyzed by Western blotting. CDDP activated p38 MAPK pathway was inhibited by the specific inhibitor, and IL-6 expression in MSCs was measured by ELISA. Results CDDP treatment showed no significant effect on the expression of IL-6 in LLC cells(P>0.05). However, IL gene expression was changed in MSCs treated with CDDP for 24 h, and IL-6 expression was increased most significantly. Further studies confirmed the increase of IL-6 mRNA and protein in MSCs after CDDP treatment for 3 h, 6 h and 24 h(P<0.05). Higher expressions of p38 and pp38 were detected in MSCs after CDDP treatment. Moreover, the ratio of pp38/p38 was significantly increased. After the p38 MAPK pathway was inhibited by SB203580, the up-regulation of IL-6 caused by CDDP was partially reversed. Conclusion CDDP has no significant effect on IL-6 expression in LLC cells, but it significantly up-regulates the expression of IL-6 in MSCs. CDDP treatment activates the p38 MAPK pathway in MSCs, and inhibiting this pathway resultes in down-regulation of IL-6. These results indicate that CDDP might regulate the expression of IL-6 in MSCs via the p38 MAPK pathway.
Objective To investigate the effect of cisplatin(CDDP) on interleukin(IL)-6 expression in tumor mesenchymal stem cells(MSCs) and explore its possible mechanisms. Methods Lewis lung carcinoma(LLC) cells or MSCs were treated with CDDP, and their IL gene expression was detected by polymerase chain reaction array(PCR Array) and further verified by quantitative real-time polymerase chain reaction(qRT-PCR) and enzyme-linked immunosorbent assay(ELISA). After CDDP treatment, the activation of p38 MAPK pathway in MSCs was analyzed by Western blotting. CDDP activated p38 MAPK pathway was inhibited by the specific inhibitor, and IL-6 expression in MSCs was measured by ELISA. Results CDDP treatment showed no significant effect on the expression of IL-6 in LLC cells(P>0.05). However, IL gene expression was changed in MSCs treated with CDDP for 24 h, and IL-6 expression was increased most significantly. Further studies confirmed the increase of IL-6 mRNA and protein in MSCs after CDDP treatment for 3 h, 6 h and 24 h(P<0.05). Higher expressions of p38 and pp38 were detected in MSCs after CDDP treatment. Moreover, the ratio of pp38/p38 was significantly increased. After the p38 MAPK pathway was inhibited by SB203580, the up-regulation of IL-6 caused by CDDP was partially reversed. Conclusion CDDP has no significant effect on IL-6 expression in LLC cells, but it significantly up-regulates the expression of IL-6 in MSCs. CDDP treatment activates the p38 MAPK pathway in MSCs, and inhibiting this pathway resultes in down-regulation of IL-6. These results indicate that CDDP might regulate the expression of IL-6 in MSCs via the p38 MAPK pathway.
引文
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[15]Chang Q, Daly L, Bromberg J. The IL-6 feed-forward loop:a driver of tumorigenesis[J]. Semin Immunol, 2014, 26(1):48-53.
[16]Nguyen DP, Li JY, Tewari AK. Inflammation and prostate cancer:the role of interleukin 6 (IL-6)[J]. BJU Int, 2014, 113(6):986-992.
[17]Yao X, Huang JQ, Zhong HH, et al. Targeting interleukin-6 in inflammatory autoimmune diseases and cancers[J]. Pharmacol Ther, 2014, 141(2):125-139.
[18]Rossi JF, Lu ZY, Jourdan M, et al. Interleukin-6 as a therapeutic target[J]. Clin Cancer Res, 2015, 21(6):1248-1257.
[19]Davies R, Choy E. Clinical experience of IL-6 blockade in rheumatic diseases - implications on IL-6 biology and disease pathogenesis[J]. Semin Immunol, 2014, 26(1):97-104.
[20]Inoue N, Shimizu M, Tsunoda S, et al. Cytokine profile in adult-onset Still's disease:Comparison with systemic juvenile idiopathic arthritis[J]. Clin Immunol, 2016, 169:8-13. doi:10.1016/j.clim.2016.05.010.
[21]Melzer C, von der Ohe J, Hass R. Concise review:crosstalk of mesenchymal Stroma/Stem-like cells with cancer cells provides therapeutic potential[J]. Stem Cells, 2018, 36(7):951-968.
[22]Jones BE, Maerz MD, Buckner JH. IL-6:a cytokine at the crossroads of autoimmunity[J]. Curr Opin Immunol, 2018, 55:9-14. doi:10.1016/j.coi.2018.09.002.
[23]Jiang XY, Zhu XS, Liu N, et al. Diallyl trisulfide inhibits growth of NCI-H460 in Vitro and in Vivo, and ameliorates cisplatin-induced oxidative injury in the treatment of lung carcinoma in xenograft mice[J]. Int J Biol Sci, 2017, 13(2):167-178.
[24]Cuadrado A, Lafarga V, Cheung PC, et al. A new p38 MAP kinase-regulated transcriptional coactivator that stimulates p53-dependent apoptosis[J]. EMBO J, 2007, 26(8):2115-2126.
[25]Park GH, Park JH, Eo HJ, et al. The induction of activating transcription factor 3 (ATF3) contributes to anti-cancer activity of Abeliophyllum distichum Nakai in human colorectal cancer cells[J]. BMC Complement Altern Med, 2014, 14:487. doi:10.1186/1472-6882-14-487.
[2]王善容, 丁月云, 朱宗平, 等. SPECT/CT融合骨显像诊断肺癌单发骨转移的价值[J]. 山东大学学报(医学版), 2018, 56(2):41-46. WANG Shanrong, DING Yueyun, ZHU Zongping, et al. Validity of SPECT/CT bone fusing imaging in the diagnosis of single bone metastasis of lung cancer[J]. Journal of Shandong University (Health Science), 2018, 56(2):41-46.
[3]Rossi A, Di Maio M. Platinum-based chemotherapy in advanced non-small-cell lung cancer:optimal number of treatment cycles[J]. Expert Rev Anticancer Ther, 2016, 16(6):653-660.
[4]费成明, 常春康. 骨髓间充质干细胞介导恶性血液病耐药的研究进展[J]. 中华血液学杂志, 2018, 39(8):697-700. FEI Chengming, CHANG Chunkang. Bone marrow mesenchymal stromal cells mediated drug resistance in hematological malignancies[J]. Chinese Journal of Hematology, 2018, 39(8):697-700.
[5]Sun Y. Tumor microenvironment and cancer therapy resistance[J]. Cancer Lett, 2016, 380(1):205-215.
[6]Hui LL, Chen Y. Tumor microenvironment:Sanctuary of the devil[J]. Cancer Lett, 2015, 368(1):7-13.
[7]Melzer C, Yang YY, Hass R. Interaction of MSC with tumor cells[J]. Cell Commun Signal, 2016, 14(1):20. doi:10.1186/s12964-016-0143-0.
[8]Kraakman MJ, Kammoun HL, Allen TL, et al. Blocking IL-6 trans-signaling prevents high-fat diet-induced adipose tissue macrophage recruitment but does not improve insulin resistance[J]. Cell Metab, 2015, 21(3):403-416.
[9]Alspach E, Flanagan KC, Luo XM, et al. p38MAPK plays a crucial role in stromal-mediated tumorigenesis[J]. Cancer Discov, 2014, 4(6):716-729.
[10]Gupta J, del Barco Barrantes I, Igea A, et al. Dual function of p38α MAPK in colon cancer:suppression of colitis-associated tumor initiation but requirement for cancer cell survival[J]. Cancer Cell, 2014, 25(4):484-500.
[11]Ling WF, Zhang JM, Yuan ZR, et al. Mesenchymal stem cells use IDO to regulate immunity in tumor microenvironment[J]. Cancer Res, 2014, 74(5):1576-1587.
[12]Ren GW, Zhang LY, Zhao X, et al. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide[J]. Cell Stem Cell, 2008, 2(2):141-150.
[13]Zimmermann S, Dziadziuszko R, Peters S. Indications and limitations of chemotherapy and targeted agents in non-small cell lung cancer brain metastases[J]. Cancer Treat Rev, 2014, 40(6):716-722.
[14]Wu T, Dai Y. Tumor microenvironment and therapeutic response[J]. Cancer Lett, 2017, 387:61-68. doi:10.1016/j.canlet.2016.01.043.
[15]Chang Q, Daly L, Bromberg J. The IL-6 feed-forward loop:a driver of tumorigenesis[J]. Semin Immunol, 2014, 26(1):48-53.
[16]Nguyen DP, Li JY, Tewari AK. Inflammation and prostate cancer:the role of interleukin 6 (IL-6)[J]. BJU Int, 2014, 113(6):986-992.
[17]Yao X, Huang JQ, Zhong HH, et al. Targeting interleukin-6 in inflammatory autoimmune diseases and cancers[J]. Pharmacol Ther, 2014, 141(2):125-139.
[18]Rossi JF, Lu ZY, Jourdan M, et al. Interleukin-6 as a therapeutic target[J]. Clin Cancer Res, 2015, 21(6):1248-1257.
[19]Davies R, Choy E. Clinical experience of IL-6 blockade in rheumatic diseases - implications on IL-6 biology and disease pathogenesis[J]. Semin Immunol, 2014, 26(1):97-104.
[20]Inoue N, Shimizu M, Tsunoda S, et al. Cytokine profile in adult-onset Still's disease:Comparison with systemic juvenile idiopathic arthritis[J]. Clin Immunol, 2016, 169:8-13. doi:10.1016/j.clim.2016.05.010.
[21]Melzer C, von der Ohe J, Hass R. Concise review:crosstalk of mesenchymal Stroma/Stem-like cells with cancer cells provides therapeutic potential[J]. Stem Cells, 2018, 36(7):951-968.
[22]Jones BE, Maerz MD, Buckner JH. IL-6:a cytokine at the crossroads of autoimmunity[J]. Curr Opin Immunol, 2018, 55:9-14. doi:10.1016/j.coi.2018.09.002.
[23]Jiang XY, Zhu XS, Liu N, et al. Diallyl trisulfide inhibits growth of NCI-H460 in Vitro and in Vivo, and ameliorates cisplatin-induced oxidative injury in the treatment of lung carcinoma in xenograft mice[J]. Int J Biol Sci, 2017, 13(2):167-178.
[24]Cuadrado A, Lafarga V, Cheung PC, et al. A new p38 MAP kinase-regulated transcriptional coactivator that stimulates p53-dependent apoptosis[J]. EMBO J, 2007, 26(8):2115-2126.
[25]Park GH, Park JH, Eo HJ, et al. The induction of activating transcription factor 3 (ATF3) contributes to anti-cancer activity of Abeliophyllum distichum Nakai in human colorectal cancer cells[J]. BMC Complement Altern Med, 2014, 14:487. doi:10.1186/1472-6882-14-487.