阻断STAT3信号通路对恶性淋巴瘤细胞凋亡的影响
|
摘要
目的探讨阻断信号转导与转录激活因子3(STAT3)信号通路对恶性淋巴瘤细胞凋亡的影响。方法用0、200、400、800、1600 nmol/L的STAT3信号通路特异性阻断剂JSI-124作用于恶性淋巴瘤细胞株Raji,噻唑蓝(MTT)法检测细胞增殖水平,计算半数抑制浓度。用半数抑制浓度的JSI-124作用于Raji细胞,流式细胞术检测细胞凋亡和细胞周期,Western blot法检测细胞周期蛋白D1(cyclin D1)、STAT3、磷酸化的STAT3(p-STAT3)、活化的半胱氨酸天冬氨酸蛋白水解酶3(cleaved caspase 3)的蛋白相对表达水平。结果随着JSI-124作用浓度的升高,细胞存活率逐渐下降。计算半数抑制浓度为(615.62±52.98)nmol/L,故后续选用600 nm/L的JSI-124作用于恶性淋巴瘤细胞。600 nm/L作用组细胞凋亡率明显高于0 nm/L作用组(P﹤0.01)。600 nm/L作用组G0/G1期细胞所占比例明显低于0 nm/L作用组,G2/M期细胞所占比例明显高于0 nm/L作用组,差异均有统计学意义(P﹤0.01);两组S期细胞所占比例比较,差异无统计学意义(P﹥0.05)。600 nm/L作用组p-STAT3、cyclin D1蛋白相对表达水平明显低于0 nm/L作用组,cleaved caspase 3蛋白相对表达水平明显高于0 nm/L作用组,差异均有统计学意义(P﹤0.01)。结论阻断STAT3信号通路可促进恶性淋巴瘤细胞凋亡,抑制细胞增殖,将细胞周期阻滞在G2/M期。
Objective To investigate the effect of signal transducer and activator of transcription 3(STAT3) signaling pathway on the apoptosis of malignant lymphoma cells. Method Raji, a malignant lymphoma cell line, was treated with JSI-124, a specific blocker of STAT3 signaling pathway, at the concentration of 0, 200, 400, 800 and 1600 nmol/L. MTT assay was used to detect cell proliferation. The half inhibitory concentration was calculated. Raji cells were treated with JSI-124 at half inhibitory concentration. Cell apoptosis and cell cycle were detected by flow cytometry, besides, the relative expression levels of cyclin D1, STAT3, phosphorylated STAT3(p-STAT3) and cleaved caspase 3 were determined by Western blot. Result With the increase of JSI-124 concentration, cell viability declined gradually, the half inhibitory concentration was calculated as(615.62 ± 52.98) nmol/L, which therefore was used to treat the malignant lymphoma cells.The apoptosis rate in 600 nm/L group was significantly higher than that in 0 nm/L group(P<0.01). The percentage of G0/G1 phase cells in 600 nm/L group was significantly lower than that in 0 nm/L group, while the proportion of G2/M phase cells was significantly higher in 600 nm/L group than that of 0 nm/L group(P<0.01), there was no significant difference in regard to the proportion of S phase cells(P>0.05). The relative expression levels of p-STAT3 and cyclin D1 protein in 600 nm/L group were significantly lower than those in 0 nm/L group(P<0.01), and the cleaved caspase 3 protein was significantly higher than that of 0 nm/L group, the difference was statistically significant(P<0.01). Conclusion Blocking STAT3 signaling pathway can promote the apoptosis of malignant lymphoma cells, inhibit cell proliferation and block cell cycle in G2/M phase.
Objective To investigate the effect of signal transducer and activator of transcription 3(STAT3) signaling pathway on the apoptosis of malignant lymphoma cells. Method Raji, a malignant lymphoma cell line, was treated with JSI-124, a specific blocker of STAT3 signaling pathway, at the concentration of 0, 200, 400, 800 and 1600 nmol/L. MTT assay was used to detect cell proliferation. The half inhibitory concentration was calculated. Raji cells were treated with JSI-124 at half inhibitory concentration. Cell apoptosis and cell cycle were detected by flow cytometry, besides, the relative expression levels of cyclin D1, STAT3, phosphorylated STAT3(p-STAT3) and cleaved caspase 3 were determined by Western blot. Result With the increase of JSI-124 concentration, cell viability declined gradually, the half inhibitory concentration was calculated as(615.62 ± 52.98) nmol/L, which therefore was used to treat the malignant lymphoma cells.The apoptosis rate in 600 nm/L group was significantly higher than that in 0 nm/L group(P<0.01). The percentage of G0/G1 phase cells in 600 nm/L group was significantly lower than that in 0 nm/L group, while the proportion of G2/M phase cells was significantly higher in 600 nm/L group than that of 0 nm/L group(P<0.01), there was no significant difference in regard to the proportion of S phase cells(P>0.05). The relative expression levels of p-STAT3 and cyclin D1 protein in 600 nm/L group were significantly lower than those in 0 nm/L group(P<0.01), and the cleaved caspase 3 protein was significantly higher than that of 0 nm/L group, the difference was statistically significant(P<0.01). Conclusion Blocking STAT3 signaling pathway can promote the apoptosis of malignant lymphoma cells, inhibit cell proliferation and block cell cycle in G2/M phase.
引文
[1] Ladetto M, Buske C, Hutchings M, et al. ESMO consensusconference on malignant lymphoma:general perspectivesand recommendations for prognostic tools in mature B-celllymphomas and chronic lymphocytic leukaemia[J]. AnnOncol, 2016, 27(12):2149-2160.
[2] McCarthy CL, Uchihara Y, Vlychou M, et al. Developmentof malignant lymphoma after metal-on-metal hip replace-ment:a case report and review of the literature[J]. SkeletalRadiol, 2017, 46(6):831-836.
[3] Horlad H, Ma C, Yano H, et al. An IL-27/Stat3 axis inducesexpression of programmed cell death 1 ligands(PD-L1/2)on infiltrating macrophages in lymphoma[J]. Cancer Sci,2016, 107(11):1696-1704.
[4] McEachron TA, Kirov I, Wungwattana M, et al. Successfultreatment of genetically profiled pediatric extranodal NK/T-Cell lymphoma targeting oncogenic STAT3 mutation[J]. Pe-diatr Blood Cancer, 2016, 63(4):727-730.
[5]李凤萍,杨孟雪,李显文,等. 2型糖尿病大血管病变患者血清诱导的人脐静脉内皮细胞中JAK2/STAT3信号通路的表达[J].第二军医大学学报, 2016, 37(4):452-457.
[6] Cha YJ, Lee JH, Han HH, et al. MicroRNA alteration andputative target genes in high-grade prostatic intraepithelialneoplasia and prostate cancer:STAT3 and ZEB1 are upregu-lated during prostate carcinogenesis[J]. Prostate, 2016, 76(10):937-947.
[7]廖明媚,王成志,杨满意,等. JAK2-STAT3信号通路在肝细胞癌中的研究进展[J].中国普通外科杂志, 2017, 26(1):102-108.
[8]杨谋广,徐岩,邹传德,等.抑制LIF/Stat3信号通路促进小鼠胚胎干细胞向心肌细胞分化[J].安徽医科大学学报,2017, 52(5):645-650.
[9] Guo X, Qiu J, Tu T, et al. Induction of innate lymphoid cell-derived interleukin-22 by the transcription factor STAT3mediates protection against intestinal infection[J]. Immuni-ty, 2014, 40(1):25-39.
[10] Wilson RP, Ives ML, Rao G, et al. STAT3 is a critical cell-intrinsic regulator of human unconventional T cell num-bers and function[J]. J Exp Med, 2015, 212(6):855-864.
[11] Lee HJ, Zhuang G, Cao Y, et al. Drug resistance via feed-back activation of Stat3 in oncogene-addicted cancer cells[J]. Cancer Cell, 2014, 26(2):207-221.
[12] McFarland BC, Gray GK, Nozell SE, et al. Activation ofthe NF-κB pathway by the STAT3 inhibitor JSI-124 in hu-man glioblastoma cells[J]. Mol Cancer Res, 2013, 11(5):494-505.
[13]杨卫富,吴红艳,朱新国. JSI-124阻断STAT3信号转导通路对人胆囊癌细胞生长的影响[J].江苏医药, 2014, 40(12):1381-1384.
[14]吴思思,朱国念,倪银芸,等.葫芦素Ⅰ(JSI-124)通过p53信号通路诱导HepG2细胞凋亡[J].细胞与分子免疫学杂志, 2017, 33(1):33-38.
[15] Ishdorj G, Johnston JB, Gibson SB. Inhibition of constitu-tive activation of STAT3 by curcurbitacin-I(JSI-124)sen-sitized human B-leukemia cells to apoptosis[J]. Mol Can-cer Ther, 2010, 9(12):3302-3314.
[16]万英明.重组人乳铁蛋白对口腔癌Tca8113细胞周期及周期调控因子cyclinD1、p19和p27基因表达的影响[J].中国老年学杂志, 2015, 35(5):1323-1325.
[17]李萱,刘颖群,方兆武. P16、Ki67和cyclinD1在宫颈癌中的表达及TCT与HPV-DNA检查在宫颈癌早期诊断中的研究[J].中国妇幼保健, 2016, 31(4):829-831.
[18]牛德方,崔岩(综述),陈辉(审校). CyclinD1表达与泌尿系统肿瘤关系的研究进展[J].实用肿瘤学杂志, 2016, 30(6):547-550.
[19]贺骏. Pin1和CyclinD1在肿瘤中的表达及其意义研究[D].武汉:华中科技大学, 2005.
[20] Zheng LC, Yang MD, Kuo CL, et al. Norcantharidin-in-duced apoptosis of ags human gastric cancer cells throughreactive oxygen species production, and caspase-and mi-tochondria-dependent signaling pathways[J]. AnticancerRes, 2016, 36(11):6031-6042.
[21] Duan WR, Garner DS, Williams SD, et al. Comparison ofimmunohistochemistry for activated caspase-3 and cleavedcytokeratin 18 with the TUNEL method for quantificationof apoptosis in histological sections of PC-3 subcutaneousxenografts[J]. J Pathol, 2003, 199(2):221-228.
[2] McCarthy CL, Uchihara Y, Vlychou M, et al. Developmentof malignant lymphoma after metal-on-metal hip replace-ment:a case report and review of the literature[J]. SkeletalRadiol, 2017, 46(6):831-836.
[3] Horlad H, Ma C, Yano H, et al. An IL-27/Stat3 axis inducesexpression of programmed cell death 1 ligands(PD-L1/2)on infiltrating macrophages in lymphoma[J]. Cancer Sci,2016, 107(11):1696-1704.
[4] McEachron TA, Kirov I, Wungwattana M, et al. Successfultreatment of genetically profiled pediatric extranodal NK/T-Cell lymphoma targeting oncogenic STAT3 mutation[J]. Pe-diatr Blood Cancer, 2016, 63(4):727-730.
[5]李凤萍,杨孟雪,李显文,等. 2型糖尿病大血管病变患者血清诱导的人脐静脉内皮细胞中JAK2/STAT3信号通路的表达[J].第二军医大学学报, 2016, 37(4):452-457.
[6] Cha YJ, Lee JH, Han HH, et al. MicroRNA alteration andputative target genes in high-grade prostatic intraepithelialneoplasia and prostate cancer:STAT3 and ZEB1 are upregu-lated during prostate carcinogenesis[J]. Prostate, 2016, 76(10):937-947.
[7]廖明媚,王成志,杨满意,等. JAK2-STAT3信号通路在肝细胞癌中的研究进展[J].中国普通外科杂志, 2017, 26(1):102-108.
[8]杨谋广,徐岩,邹传德,等.抑制LIF/Stat3信号通路促进小鼠胚胎干细胞向心肌细胞分化[J].安徽医科大学学报,2017, 52(5):645-650.
[9] Guo X, Qiu J, Tu T, et al. Induction of innate lymphoid cell-derived interleukin-22 by the transcription factor STAT3mediates protection against intestinal infection[J]. Immuni-ty, 2014, 40(1):25-39.
[10] Wilson RP, Ives ML, Rao G, et al. STAT3 is a critical cell-intrinsic regulator of human unconventional T cell num-bers and function[J]. J Exp Med, 2015, 212(6):855-864.
[11] Lee HJ, Zhuang G, Cao Y, et al. Drug resistance via feed-back activation of Stat3 in oncogene-addicted cancer cells[J]. Cancer Cell, 2014, 26(2):207-221.
[12] McFarland BC, Gray GK, Nozell SE, et al. Activation ofthe NF-κB pathway by the STAT3 inhibitor JSI-124 in hu-man glioblastoma cells[J]. Mol Cancer Res, 2013, 11(5):494-505.
[13]杨卫富,吴红艳,朱新国. JSI-124阻断STAT3信号转导通路对人胆囊癌细胞生长的影响[J].江苏医药, 2014, 40(12):1381-1384.
[14]吴思思,朱国念,倪银芸,等.葫芦素Ⅰ(JSI-124)通过p53信号通路诱导HepG2细胞凋亡[J].细胞与分子免疫学杂志, 2017, 33(1):33-38.
[15] Ishdorj G, Johnston JB, Gibson SB. Inhibition of constitu-tive activation of STAT3 by curcurbitacin-I(JSI-124)sen-sitized human B-leukemia cells to apoptosis[J]. Mol Can-cer Ther, 2010, 9(12):3302-3314.
[16]万英明.重组人乳铁蛋白对口腔癌Tca8113细胞周期及周期调控因子cyclinD1、p19和p27基因表达的影响[J].中国老年学杂志, 2015, 35(5):1323-1325.
[17]李萱,刘颖群,方兆武. P16、Ki67和cyclinD1在宫颈癌中的表达及TCT与HPV-DNA检查在宫颈癌早期诊断中的研究[J].中国妇幼保健, 2016, 31(4):829-831.
[18]牛德方,崔岩(综述),陈辉(审校). CyclinD1表达与泌尿系统肿瘤关系的研究进展[J].实用肿瘤学杂志, 2016, 30(6):547-550.
[19]贺骏. Pin1和CyclinD1在肿瘤中的表达及其意义研究[D].武汉:华中科技大学, 2005.
[20] Zheng LC, Yang MD, Kuo CL, et al. Norcantharidin-in-duced apoptosis of ags human gastric cancer cells throughreactive oxygen species production, and caspase-and mi-tochondria-dependent signaling pathways[J]. AnticancerRes, 2016, 36(11):6031-6042.
[21] Duan WR, Garner DS, Williams SD, et al. Comparison ofimmunohistochemistry for activated caspase-3 and cleavedcytokeratin 18 with the TUNEL method for quantificationof apoptosis in histological sections of PC-3 subcutaneousxenografts[J]. J Pathol, 2003, 199(2):221-228.