西黄丸调节MEKK1/SEK1通路抑制小鼠乳腺癌生长机制研究
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摘要
目的研究西黄丸抑制4T1乳腺癌荷瘤小鼠肿瘤的生长及其可能的发生机制。方法建立4T1乳腺癌荷瘤小鼠模型,以西黄丸低、中、高剂量ig给药14 d,剥离肿瘤组织称重,TUNEL染色检测肿瘤细胞的凋亡,Real-time PCR法检测肿瘤组织中MEKK1和SEK1 mRNA表达,免疫荧光染色法和Western Blot法检测肿瘤组织中MEKK1和SEK1蛋白表达。结果与阴性对照组比较,给药组肿瘤体积及质量随西黄丸剂量的升高而降低;TUNEL染色显示凋亡的肿瘤细胞增加;Real-time PCR显示肿瘤组织中MEKK1和SEK1 mRNA表达升高;免疫荧光染色和Western Blot显示肿瘤组织中MEKK1和SEK1蛋白表达水平上调;以上结果均与西黄丸呈剂量依赖性,P <0.05具有统计学差异。结论西黄丸可能通过促进4T1乳腺癌荷瘤小鼠肿瘤细胞凋亡抑制肿瘤的生长,其机制可能与西黄丸上调4T1乳腺癌荷瘤小鼠肿瘤组织中MEKK1、SEK1 mRNA和蛋白表达有关。
Objective To study the effect of Xihuang Pill on the growth of 4T1 mouse breast cancer and its possible mechanism. Methods A mouse 4T1 breast cancer model has been established. Model mice are administered Xihuang Pill at low, medium and high dose for 2 weeks meanwhile tumor growth curve is drawn, and tumor tissues are then removed and weighed. Tumor cell apoptosis is detected by Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling staining(TUNEL), mRNA expression levels of Mitogen-activated protein kinase kinase kinase 1(MEKK1) and SAPK/Erk kinase 1(SEK1) in tumor tissue are detected by Real-time PCR and their protein expression levels is detected by immunofluorescence staining and Western Blot. ResultsComparing with the negative control group, tumor volumes and tumor weights in the Xihuang Pill groups decreas significantly with increasing doses of Xihuang Pill. TUNEL staining show that the number of apoptotic tumor cells increas with increasing doses of Xihuang Pill. Real-time PCR show that mRNA expression of MEKK1 and SEK1 in tumor tissue increase with increasing doses of Xihuang pill. Immunofluorescence staining and Western Blotting show that protein expression of MEKK1 and SEK1 in tumor tissue increase with increasing doses of Xihuang pill. Conclusion Xihuang Pill might promote tumor cell apoptosis and further inhibit the tumor growth of 4T1 mouse breast cancer. The mechanism of Xihuang Pill may be related to upregulation of mRNA and protein expression of MEKK1 and SEK1 in tumor tissue of 4T1 mouse breast cancer.
Objective To study the effect of Xihuang Pill on the growth of 4T1 mouse breast cancer and its possible mechanism. Methods A mouse 4T1 breast cancer model has been established. Model mice are administered Xihuang Pill at low, medium and high dose for 2 weeks meanwhile tumor growth curve is drawn, and tumor tissues are then removed and weighed. Tumor cell apoptosis is detected by Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling staining(TUNEL), mRNA expression levels of Mitogen-activated protein kinase kinase kinase 1(MEKK1) and SAPK/Erk kinase 1(SEK1) in tumor tissue are detected by Real-time PCR and their protein expression levels is detected by immunofluorescence staining and Western Blot. ResultsComparing with the negative control group, tumor volumes and tumor weights in the Xihuang Pill groups decreas significantly with increasing doses of Xihuang Pill. TUNEL staining show that the number of apoptotic tumor cells increas with increasing doses of Xihuang Pill. Real-time PCR show that mRNA expression of MEKK1 and SEK1 in tumor tissue increase with increasing doses of Xihuang pill. Immunofluorescence staining and Western Blotting show that protein expression of MEKK1 and SEK1 in tumor tissue increase with increasing doses of Xihuang pill. Conclusion Xihuang Pill might promote tumor cell apoptosis and further inhibit the tumor growth of 4T1 mouse breast cancer. The mechanism of Xihuang Pill may be related to upregulation of mRNA and protein expression of MEKK1 and SEK1 in tumor tissue of 4T1 mouse breast cancer.
引文
[1]Torre L A, Bray F, Siegel R L, et al. Global Cancer Statistic, 2012[J]. Ca A Cancer Journal for Clinicians, 2015, 65(2):87-108.
[2]Siegel R L, Miller K D, Jemal A. Cancer statistics, 2015[J]. Ca-A Cancer Journal for Clinicians, 2015, 65(1):5-29.
[3]Tao Z Q, Shi A, Lu C, et al. Breast Cancer:Epidemiology and Etiology[J]. Cell Biochemistry&Biophysics, 2015, 72(2):333-338.
[4]马杰,杨伟,关硕,等.西黄丸乙酸乙酯提取物抗肿瘤活性研究[J].医药导报, 2014, 33(3):303-306.
[5]洪日,吴永强,吴越.西黄丸辅助治疗中晚期乳腺癌的疗效观察[J].中国中药杂志, 2014, 39(6):1120-1123.
[6]李德辉,范焕芳,孙春霞.西黄丸在乳腺癌中的应用研究进展[J].时珍国医国药, 2016, 27(9):2247-2248.
[7]芦琴,项景芳,张秉.西黄丸/胶囊辅助治疗乳腺癌有效性和安全性Meta分析[J].中国老年学杂志, 2015, 35(24):7092-7094.
[8]岳雁鸿,曾燕,郑华芳,等. TAC方案联合西黄丸对Ⅲ期乳腺癌患者P53, HER-2及TOPⅡ水平的影响[J].现代生物医学进展, 2017, 17(8):1505-1508.
[9]吴国玉.西黄丸辅助治疗乳腺癌的疗效及对患者免疫功能的影响[J].山西医药杂志, 2016, 45(21):2477-2479.
[10] Guo Q, Lin J, Liu R, et al. Review on the Applications and Molecular Mechanisms of Xihuang Pill in Tumor Treatment[J]. Evidence-Based Complementray and Alternative Medicine, 2015(2015-6-10), 2015, 2015(4):1-10.
[11] Zheng W, Han S, Jiang S, et al. Multiple effects of Xihuang pill aqueous extract on the Hs578T triple-negative breast cancer cell line[J]. Biomed Rep,2016, 5(5):559-566.
[12]沈雁鸣.西黄丸联合西医治疗乳腺癌随机平行对照研究[J].实用中医内科杂志, 2014, 28(3):127-128.
[13] Lu H, Ning X, Tao X, et al. MEKK1 Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage[J]. Neurochemical Research, 2016,41(12):1-14.
[14]鲁明骞,孔庆志,许新华,等. MKK4基因启动子区-1304T/G多态性与散发性鼻咽癌易感性的关系[J].临床耳鼻咽喉头颈外科杂志, 2016,30(4):287-290.
[15] Lan Z, Mi Z, Wang Y, et al. miR-92a inhibits vascular smooth muscle cell apoptosis:role of the MKK4-JNK pathway[J]. Apoptosis An International Journal on Programmed Cell Death, 2014, 19(6):975-983.
[16] Dunnill C J, Ibraheem K, Mohamed A, et al. A redox state-dictated signalling pathway deciphers the malignant cell specificity of CD40-mediated apoptosis:[J]. Oncogene, 2017, 36(18):2515-2528.
[17]王一尧,任征远,焦战,等.西黄丸对肿瘤迁移微环境的影响[J].中药药理与临床, 2014, 30(4):11-13.
[18]杨洋博君,陈蓉,李舒,等.复方大七气汤对裸鼠卵巢癌皮下移植瘤生长及凋亡的影响[J].中国医院药学杂志, 2014, 34(24):2102-2107.
[19] Pham T T, Angus S P, Johnson G L. MAP3K1:Genomic Alterations in Cancer and Function in Promoting Cell Survival or Apoptosis[J]. Genes&Cancer,2013, 4(12):419-26.
[20] Owen G R, Achilonu I, Dirr H W. High yield purification of JNK1β1 and activation by in vitro reconstitution of the MEKK1→MKK4→JNK MAPK phosphorylation cascade.[J]. Protein Expression&Purification, 2013, 87(2):87-99.
[21]万强琨,汪庚明,江浩. MKK4基因的抑癌及致癌作用[J].医学综述,2014, 20(17):3117-3119.
[22] Zheng J, Liu B, Zhang L, et al. The protective role of polymorphism MKK4-1304 T> G in nasopharyngeal carcinoma is modulated by Epstein–Barr virus'infection status[J]. International Journal of Cancer Journal International Du Cancer, 2012, 130(9):1981-1990.
[23]鲁明骞,孔庆志,许新华,等.鼻咽癌MKK4蛋白表达与-1044A/T多态性的相关性分析[J].中国免疫学杂志, 2016, 32(8):1137-1140.
[24] Thuong P T, Khoi N M, Ohta S, et al. ent-kaurane diterpenoids from Croton tonkinensis induce apoptosis in colorectal cancer cells through the phosphorylation of JNK mediated by reactive oxygen species and dualspecificity JNK kinase MKK4[J]. Anti-Cancer Agents in Medicinal Chemistry(Formerly Current Medicinal Chemistry-Anti-Cancer Agents), 2014, 14(7):1051-1061.
[25] Davies C, Tournier C. Exploring the function of the JNK(c-Jun N-terminal kinase)signalling pathway in physiological and pathological processes to design novel therapeutic strategies[J]. Biochemical Society Transactions,2012, 40(1):85-89.
[2]Siegel R L, Miller K D, Jemal A. Cancer statistics, 2015[J]. Ca-A Cancer Journal for Clinicians, 2015, 65(1):5-29.
[3]Tao Z Q, Shi A, Lu C, et al. Breast Cancer:Epidemiology and Etiology[J]. Cell Biochemistry&Biophysics, 2015, 72(2):333-338.
[4]马杰,杨伟,关硕,等.西黄丸乙酸乙酯提取物抗肿瘤活性研究[J].医药导报, 2014, 33(3):303-306.
[5]洪日,吴永强,吴越.西黄丸辅助治疗中晚期乳腺癌的疗效观察[J].中国中药杂志, 2014, 39(6):1120-1123.
[6]李德辉,范焕芳,孙春霞.西黄丸在乳腺癌中的应用研究进展[J].时珍国医国药, 2016, 27(9):2247-2248.
[7]芦琴,项景芳,张秉.西黄丸/胶囊辅助治疗乳腺癌有效性和安全性Meta分析[J].中国老年学杂志, 2015, 35(24):7092-7094.
[8]岳雁鸿,曾燕,郑华芳,等. TAC方案联合西黄丸对Ⅲ期乳腺癌患者P53, HER-2及TOPⅡ水平的影响[J].现代生物医学进展, 2017, 17(8):1505-1508.
[9]吴国玉.西黄丸辅助治疗乳腺癌的疗效及对患者免疫功能的影响[J].山西医药杂志, 2016, 45(21):2477-2479.
[10] Guo Q, Lin J, Liu R, et al. Review on the Applications and Molecular Mechanisms of Xihuang Pill in Tumor Treatment[J]. Evidence-Based Complementray and Alternative Medicine, 2015(2015-6-10), 2015, 2015(4):1-10.
[11] Zheng W, Han S, Jiang S, et al. Multiple effects of Xihuang pill aqueous extract on the Hs578T triple-negative breast cancer cell line[J]. Biomed Rep,2016, 5(5):559-566.
[12]沈雁鸣.西黄丸联合西医治疗乳腺癌随机平行对照研究[J].实用中医内科杂志, 2014, 28(3):127-128.
[13] Lu H, Ning X, Tao X, et al. MEKK1 Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage[J]. Neurochemical Research, 2016,41(12):1-14.
[14]鲁明骞,孔庆志,许新华,等. MKK4基因启动子区-1304T/G多态性与散发性鼻咽癌易感性的关系[J].临床耳鼻咽喉头颈外科杂志, 2016,30(4):287-290.
[15] Lan Z, Mi Z, Wang Y, et al. miR-92a inhibits vascular smooth muscle cell apoptosis:role of the MKK4-JNK pathway[J]. Apoptosis An International Journal on Programmed Cell Death, 2014, 19(6):975-983.
[16] Dunnill C J, Ibraheem K, Mohamed A, et al. A redox state-dictated signalling pathway deciphers the malignant cell specificity of CD40-mediated apoptosis:[J]. Oncogene, 2017, 36(18):2515-2528.
[17]王一尧,任征远,焦战,等.西黄丸对肿瘤迁移微环境的影响[J].中药药理与临床, 2014, 30(4):11-13.
[18]杨洋博君,陈蓉,李舒,等.复方大七气汤对裸鼠卵巢癌皮下移植瘤生长及凋亡的影响[J].中国医院药学杂志, 2014, 34(24):2102-2107.
[19] Pham T T, Angus S P, Johnson G L. MAP3K1:Genomic Alterations in Cancer and Function in Promoting Cell Survival or Apoptosis[J]. Genes&Cancer,2013, 4(12):419-26.
[20] Owen G R, Achilonu I, Dirr H W. High yield purification of JNK1β1 and activation by in vitro reconstitution of the MEKK1→MKK4→JNK MAPK phosphorylation cascade.[J]. Protein Expression&Purification, 2013, 87(2):87-99.
[21]万强琨,汪庚明,江浩. MKK4基因的抑癌及致癌作用[J].医学综述,2014, 20(17):3117-3119.
[22] Zheng J, Liu B, Zhang L, et al. The protective role of polymorphism MKK4-1304 T> G in nasopharyngeal carcinoma is modulated by Epstein–Barr virus'infection status[J]. International Journal of Cancer Journal International Du Cancer, 2012, 130(9):1981-1990.
[23]鲁明骞,孔庆志,许新华,等.鼻咽癌MKK4蛋白表达与-1044A/T多态性的相关性分析[J].中国免疫学杂志, 2016, 32(8):1137-1140.
[24] Thuong P T, Khoi N M, Ohta S, et al. ent-kaurane diterpenoids from Croton tonkinensis induce apoptosis in colorectal cancer cells through the phosphorylation of JNK mediated by reactive oxygen species and dualspecificity JNK kinase MKK4[J]. Anti-Cancer Agents in Medicinal Chemistry(Formerly Current Medicinal Chemistry-Anti-Cancer Agents), 2014, 14(7):1051-1061.
[25] Davies C, Tournier C. Exploring the function of the JNK(c-Jun N-terminal kinase)signalling pathway in physiological and pathological processes to design novel therapeutic strategies[J]. Biochemical Society Transactions,2012, 40(1):85-89.