重组人骨形态发生蛋白2对乳腺癌MCF-7细胞增殖的影响
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摘要
背景:有研究发现重组人骨形态发生蛋白2的临床应用可增加恶性肿瘤的患病风险,但关于重组人骨形态发生蛋白2对乳腺癌MCF-7细胞增殖作用的影响及机制尚未被阐明。目的:探讨在体外及体内条件下重组人骨形态发生蛋白2对乳腺癌MCF-7细胞增殖能力的影响及其作用机制。方法:①体外实验:无血清条件下,不同浓度的重组人骨形态发生蛋白2处理MCF-7细胞,通过MTT检测重组人骨形态发生蛋白2对细胞增殖能力的影响。利用流式细胞分析技术检测细胞周期,通过Real-time PCR检测不同浓度的重组人骨形态发生蛋白2对p21、cyclinE表达的影响,应用Westernblot技术检测不同浓度的重组人骨形态发生蛋白2处理对p21、cyclinE蛋白水平及对PI3K/Akt磷酸化水平的影响;②体内实验:将14只雌性裸鼠随机等分为实验组和对照组,实验组成瘤方案为MCF-7细胞+重组人骨形态发生蛋白2,对照组成瘤方案为等量MCF-7细胞,分别于6周龄裸鼠皮下注射。结果与结论:①体外实验:无血清条件下,不同浓度的重组人骨形态发生蛋白2处理细胞,发现重组人骨形态发生蛋白2可显著抑制乳腺癌细胞系MCF-7的增殖能力。无血清条件下,不同浓度的重组人骨形态发生蛋白2处理细胞24 h,发现重组人骨形态发生蛋白2可增加G1期细胞所占比率。重组人骨形态发生蛋白2可显著促进p21的表达,显著抑制cyclinE的表达,并显著抑制PI3K/Akt信号通路的磷酸化过程;②体内实验:实验组裸鼠皮下瘤体体积均较对照组低,免疫组化检测显示重组人骨形态发生蛋白2处理可显著降低瘤体组织中ki-67的表达水平;③研究证实在体外及体内条件下重组人骨形态发生蛋白2通过影响PI3K/Akt信号通路对乳腺癌MCF-7细胞增殖能力发挥显著抑制作用,从基础水平进一步论证了重组人骨形态发生蛋白2的临床应用不能显著增加乳腺癌的患病风险,为脊柱融合及骨不连等骨科疾病的治疗中应用重组人骨形态发生蛋白2提供了一定的理论基础。
BACKGROUND: Clinical application of recombinant human bone morphogenetic protein-2(rhBMP-2) has been found to increase the risk for malignant tumors, but the effect of rhBMP-2 on the proliferation of breast cancer MCF-7 cells and the underlying mechanism remain unclear. OBJECTIVE: To explore the effects of rhBMP-2 on the proliferation of breast cancer MCF-7 cells and the underlying mechanism in vivo and in vitro. METHODS: In vitro study: cell proliferation of MCF-7 under different concentrations of rhBMP-2 was determined by MTT assay and flow cytometry in serum-free condition. The cell cycle was detected by flow cytometry. The expression levels of p21 and cyclin E were detected by real-time PCR. The protein levels of p21, cyclin E and phosphorylation of PI3 K/Akt were tested by western blot and real-time PCR. In vivo study: fourteen 6-week-old female nude mice were divided into two groups: experimental group(subcutaneous injection of rhBMP-2 plus MCF-7 cells), control group(subcutaneous injection of the same volume of MCF-7 cells). RESULTS AND CONCLUSION: In vitro study: rhBMP-2 markedly inhibited the proliferation of MCF-7 cells in serum-free condition. After 24-hour treatment, rhBMP-2 could increase the G1 cell ratio. rhBMP-2 could significantly increase the expression of p21 as well as significantly inhibit the expression of cyclin E and phosphorylation in the PI3 K/Akt signaling pathway. In vivo study: the volume of subcutaneous tumor in the experimental group was smaller than that in the control group. Immunohistochemistry revealed that rhBMP-2 could significantly decrease the expression level of ki-67 in tumor tissues. Our study suggests that rhBMP-2 has significantly suppressive effect on the proliferation of breast cancer MCF-7 cells via PI3 K/Akt pathway in vitro and in vivo. Therefore, we can provide the basic science data to avoid risks of breast cancer and to support the utilization of rhBMP-2 in the management of spinal fusion, treatment of bone ununion and other orthopedic disorders.
BACKGROUND: Clinical application of recombinant human bone morphogenetic protein-2(rhBMP-2) has been found to increase the risk for malignant tumors, but the effect of rhBMP-2 on the proliferation of breast cancer MCF-7 cells and the underlying mechanism remain unclear. OBJECTIVE: To explore the effects of rhBMP-2 on the proliferation of breast cancer MCF-7 cells and the underlying mechanism in vivo and in vitro. METHODS: In vitro study: cell proliferation of MCF-7 under different concentrations of rhBMP-2 was determined by MTT assay and flow cytometry in serum-free condition. The cell cycle was detected by flow cytometry. The expression levels of p21 and cyclin E were detected by real-time PCR. The protein levels of p21, cyclin E and phosphorylation of PI3 K/Akt were tested by western blot and real-time PCR. In vivo study: fourteen 6-week-old female nude mice were divided into two groups: experimental group(subcutaneous injection of rhBMP-2 plus MCF-7 cells), control group(subcutaneous injection of the same volume of MCF-7 cells). RESULTS AND CONCLUSION: In vitro study: rhBMP-2 markedly inhibited the proliferation of MCF-7 cells in serum-free condition. After 24-hour treatment, rhBMP-2 could increase the G1 cell ratio. rhBMP-2 could significantly increase the expression of p21 as well as significantly inhibit the expression of cyclin E and phosphorylation in the PI3 K/Akt signaling pathway. In vivo study: the volume of subcutaneous tumor in the experimental group was smaller than that in the control group. Immunohistochemistry revealed that rhBMP-2 could significantly decrease the expression level of ki-67 in tumor tissues. Our study suggests that rhBMP-2 has significantly suppressive effect on the proliferation of breast cancer MCF-7 cells via PI3 K/Akt pathway in vitro and in vivo. Therefore, we can provide the basic science data to avoid risks of breast cancer and to support the utilization of rhBMP-2 in the management of spinal fusion, treatment of bone ununion and other orthopedic disorders.
引文
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[16]Malham GM, Giles GG, Milne RL, et al. Bone Morphogenetic Proteins in Spinal Surgery:What Is the Fusion Rate and Do They Cause Cancer? Spine(Phila Pa 1976). 2015;40(22):1737-1742.
[17]Skovrlj B, Koehler SM, Anderson PA, et al. Association Between BMP-2 and Carcinogenicity. Spine(Phila Pa 1976). 2015;40(23):1862-1871.
[18]Wozney JM, Rosen V. Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair.Clin Orthop Relat Res. 1998;(346):26-37.
[19]Wang Y, He T, Liu J, et al. Synergistic effects of overexpression of BMP-2 and TGF-β3 on osteogenic differentiation of bone marrow mesenchymal stem cells. Mol Med Rep. 2016;14(6):5514-5520.
[20]Zheng Y, Wang X, Wang H, et al. Bone morphogenetic protein 2inhibits hepatocellular carcinoma growth and migration through downregulation of the PI3K/AKT pathway. Tumour Biol. 2014;35(6):5189-5198.
[21]Zhao X, Wang Q, Yang S, et al. Quercetin inhibits angiogenesis by targeting calcineurin in the xenograft model of human breast cancer. Eur J Pharmacol. 2016;781:60-68.
[22]No?l A, De Pauw-Gillet MC, Purnell G, et al. Enhancement of tumorigenicity of human breast adenocarcinoma cells in nude mice by matrigel and fibroblasts. Br J Cancer. 1993;68(5):909-915.
[23]Chen W, Zheng R, Baade PD, et al. Cancer statistics in China,2015. CA Cancer J Clin. 2016;66(2):115-132.
[24]Fontanella C, Fanotto V, Rihawi K, et al. Skeletal metastases from breast cancer:pathogenesis of bone tropism and treatment strategy. Clin Exp Metastasis. 2015;32(8):819-833.
[25]Sciubba DM, Goodwin CR, Yurter A, et al. A Systematic Review of Clinical Outcomes and Prognostic Factors for Patients Undergoing Surgery for Spinal Metastases Secondary to Breast Cancer.Global Spine J. 2016;6(5):482-496.
[2]Salazar VS, Gamer LW, Rosen V. BMP signalling in skeletal development, disease and repair. Nat Rev Endocrinol. 2016;12(4):203-221.
[3]Kang MH, Kang HN, Kim JL, et al. Inhibition of PI3 kinase/Akt pathway is required for BMP2-induced EMT and invasion. Oncol Rep. 2009;22(3):525-534.
[4]Lao L, Shen J, Tian H, et al. Secreted phosphoprotein 24kD(Spp24)inhibits growth of hepatocellular carcinoma in vivo.Environ Toxicol Pharmacol. 2017;51:51-55.
[5]Li CS, Tian H, Zou M, et al. Secreted phosphoprotein 24?k D(Spp24)inhibits growth of human pancreatic cancer cells caused by BMP-2.Biochem Biophys Res Commun. 2015;466(2):167-172.
[6]Yu JS, Cui W. Proliferation, survival and metabolism:the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination. Development. 2016;143(17):3050-3060.
[7]Faundez A, Tournier C, Garcia M, et al. Bone morphogenetic protein use in spine surgery-complications and outcomes:a systematic review. Int Orthop. 2016;40(6):1309-1319.
[8]James AW, LaChaud G, Shen J, et al. A Review of the Clinical Side Effects of Bone Morphogenetic Protein-2. Tissue Eng Part B Rev. 2016;22(4):284-297.
[9]Bodalia PN, Balaji V, Kaila R, et al. Effectiveness and safety of recombinant human bone morphogenetic protein-2 for adults with lumbar spine pseudarthrosis following spinal fusion surgery:A systematic review. Bone Joint Res. 2016;5(4):145-152.
[10]Ghosh-Choudhury N, Woodruff K, Qi W, et al. Bone morphogenetic protein-2 blocks MDA MB 231 human breast cancer cell proliferation by inhibiting cyclin-dependent kinase-mediated retinoblastoma protein phosphorylation.Biochem Biophys Res Commun. 2000;272(3):705-711.
[11]Katsuno Y, Hanyu A, Kanda H, et al. Bone morphogenetic protein signaling enhances invasion and bone metastasis of breast cancer cells through Smad pathway. Oncogene. 2008;27(49):6322-6333.
[12]Ye S, Park BH, Song KJ, et al. In vivo inhibition of bone morphogenetic protein-2 on breast cancer cell growth. Spine(Phila Pa 1976). 2013;38(3):E143-150.
[13]Vavken J, Mameghani A, Vavken P, et al. Complications and cancer rates in spine fusion with recombinant human bone morphogenetic protein-2(rhBMP-2). Eur Spine J. 2016;25(12):3979-3989.
[14]Tannoury CA, An HS2. Complications with the use of bone morphogenetic protein 2(BMP-2)in spine surgery. Spine J.2014;14(3):552-559.
[15]Young A, Mirarchi A. Soft Tissue Swelling Associated with the Use of Recombinant Human Bone Morphogenetic Protein-2 in Long Bone Non-unions. J Orthop Case Rep. 2015;5(3):18-21.
[16]Malham GM, Giles GG, Milne RL, et al. Bone Morphogenetic Proteins in Spinal Surgery:What Is the Fusion Rate and Do They Cause Cancer? Spine(Phila Pa 1976). 2015;40(22):1737-1742.
[17]Skovrlj B, Koehler SM, Anderson PA, et al. Association Between BMP-2 and Carcinogenicity. Spine(Phila Pa 1976). 2015;40(23):1862-1871.
[18]Wozney JM, Rosen V. Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair.Clin Orthop Relat Res. 1998;(346):26-37.
[19]Wang Y, He T, Liu J, et al. Synergistic effects of overexpression of BMP-2 and TGF-β3 on osteogenic differentiation of bone marrow mesenchymal stem cells. Mol Med Rep. 2016;14(6):5514-5520.
[20]Zheng Y, Wang X, Wang H, et al. Bone morphogenetic protein 2inhibits hepatocellular carcinoma growth and migration through downregulation of the PI3K/AKT pathway. Tumour Biol. 2014;35(6):5189-5198.
[21]Zhao X, Wang Q, Yang S, et al. Quercetin inhibits angiogenesis by targeting calcineurin in the xenograft model of human breast cancer. Eur J Pharmacol. 2016;781:60-68.
[22]No?l A, De Pauw-Gillet MC, Purnell G, et al. Enhancement of tumorigenicity of human breast adenocarcinoma cells in nude mice by matrigel and fibroblasts. Br J Cancer. 1993;68(5):909-915.
[23]Chen W, Zheng R, Baade PD, et al. Cancer statistics in China,2015. CA Cancer J Clin. 2016;66(2):115-132.
[24]Fontanella C, Fanotto V, Rihawi K, et al. Skeletal metastases from breast cancer:pathogenesis of bone tropism and treatment strategy. Clin Exp Metastasis. 2015;32(8):819-833.
[25]Sciubba DM, Goodwin CR, Yurter A, et al. A Systematic Review of Clinical Outcomes and Prognostic Factors for Patients Undergoing Surgery for Spinal Metastases Secondary to Breast Cancer.Global Spine J. 2016;6(5):482-496.
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