抑制硬脂酰辅酶A去饱和酶1表达对乳腺癌细胞增殖和周期的影响
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摘要
目的检测硬脂酰辅酶A去饱和酶1(stearoyl-CoA desaturase 1,SCD1)在乳腺癌细胞中的表达,分析抑制SCD1对乳腺癌MCF-7细胞增殖和周期的影响及机制。方法采用蛋白质印迹法检测乳腺癌细胞株MCF-7和MDA-MB-231及正常人皮肤成纤维细胞株HSF中SCD1的表达。应用SCD1特异性抑制剂MF-438干预MCF-7细胞,采用MTS法测定细胞增殖的抑制率,计算IC_(50)值;采用PI染色流式细胞术分析细胞周期分布,蛋白质印迹法检测特异性周期蛋白Cyclin D1、Akt、pAkt、pAMPK、pACC蛋白的表达。结果乳腺癌MCF-7、MDA-MB-231细胞中SCD1的表达高于人皮肤成纤维细胞HSF细胞(P<0.05)。MF-438在100 nmol/L～100μmol/L浓度范围内,抑制低血清培养下的MCF-7细胞的增殖,并显示出显著的剂量依赖性,IC_(50)值为(3.9±0.45)μmol/L。5μmol/L MF-438干预MCF-7细胞后,处于细胞周期中S期和G_2/M期的细胞比例减少(P<0.01),G_0/G_1期细胞比例增加(P<0.01),Cyclin D1的表达水平降低(P<0.01);同时,pAkt及pAkt/Akt表达下降(P<0.05),pAMPK及pACC表达水平升高(P<0.05)。结论 SCD1在乳腺癌的发生和发展中发挥重要作用,抑制SCD1活性能通过下调Akt通路、活化AMPK通路,阻滞乳腺癌细胞周期进展,抑制细胞增殖。
Objective To investigate the expression of stearoyl-CoA desaturase-1(SCD1) in breast cancer cell lines. To analyze the effect of inhibiting SCD1 activity on the proliferation and cell cycle of MCF-7 breast cancer cell and its mechanism. Methods The expression of SCD1 protein were detected by Western blot techniques in breast cancer cell lines and humanskin fibroblasts.Cell viability of MCF-7 cells treated with MF-438 was measured using MTS assay and IC_(50) value was calculated.The distribution of cell cycle was determined by PI staining using flow cytometry.The expression of Cyclin D1 was detected by Western blot. The expression of Akt, pAkt, pAMPK and pACC were also detected by Western blot.Results The expression level of SCD1 in MCF-7 and MDA-MB-231 cells was significantly higher than that in HSF cells(P<0.05).MF-438 showed a significant dose-dependent proliferation inhibition effect on MCF-7 cells cultured in low serum at a concentration ranging from 100 nmol/L to 100 μmol/L with an IC_(50) value of(3.9±0.45) μmol/L. After intervention of 5 μmol/L MF-438 in MCF-7 cells, the proportion of cells in S phase and G_2/M phase was significantly decreased(P<0.01), the proportion of cells in G_0/G_1 phase increased(P<0.01), and the expression of Cyclin D1 was significantly decreased(P<0.05); Meanwhile,the expression of pAkt and pAkt/Akt value were significantly decreased(P<0.05) and the expression of pAMPK and pACC levels were significantly increased(P<0.05). Conclusion SCD1 plays an important role in the occurrence and development of breast cancer. Inhibition of SCD1 activity can inhibit cell cycle progression and impair cell proliferation by down-regulating the Akt pathway and activating the AMPK pathway. Further research on SCD1 is expected to provide a new target for molecular targeted therapy of breast cancer.
Objective To investigate the expression of stearoyl-CoA desaturase-1(SCD1) in breast cancer cell lines. To analyze the effect of inhibiting SCD1 activity on the proliferation and cell cycle of MCF-7 breast cancer cell and its mechanism. Methods The expression of SCD1 protein were detected by Western blot techniques in breast cancer cell lines and humanskin fibroblasts.Cell viability of MCF-7 cells treated with MF-438 was measured using MTS assay and IC_(50) value was calculated.The distribution of cell cycle was determined by PI staining using flow cytometry.The expression of Cyclin D1 was detected by Western blot. The expression of Akt, pAkt, pAMPK and pACC were also detected by Western blot.Results The expression level of SCD1 in MCF-7 and MDA-MB-231 cells was significantly higher than that in HSF cells(P<0.05).MF-438 showed a significant dose-dependent proliferation inhibition effect on MCF-7 cells cultured in low serum at a concentration ranging from 100 nmol/L to 100 μmol/L with an IC_(50) value of(3.9±0.45) μmol/L. After intervention of 5 μmol/L MF-438 in MCF-7 cells, the proportion of cells in S phase and G_2/M phase was significantly decreased(P<0.01), the proportion of cells in G_0/G_1 phase increased(P<0.01), and the expression of Cyclin D1 was significantly decreased(P<0.05); Meanwhile,the expression of pAkt and pAkt/Akt value were significantly decreased(P<0.05) and the expression of pAMPK and pACC levels were significantly increased(P<0.05). Conclusion SCD1 plays an important role in the occurrence and development of breast cancer. Inhibition of SCD1 activity can inhibit cell cycle progression and impair cell proliferation by down-regulating the Akt pathway and activating the AMPK pathway. Further research on SCD1 is expected to provide a new target for molecular targeted therapy of breast cancer.
引文
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[11] PLAS DR,THOMPSON CB.Akt-dependent transformation:there is more to growth than just surviving.Oncogene,2005,24(50):7435-7442.
[12] PORSTMANN T,GRIFFITHS B,CHUNG YL,et al.PKB/Akt induces transcription of enzymes involved in cholesterol and fatty acid biosynthesis via activation of SREBP.OncogeneJ,2005,24(43) 6465-6481.
[13] KAHN BB,ALQUIER T,CARLING D,et al.AMP-activated protein kinase:ancient energy gauge provides clues to modern understanding of metabolism.Cell Metabolism,2005,1(1):15-25.
[14] FORBES-HERNáNDEZ TY,GIAMPIERI F,GASPARRINI M,et al.Lipid accumulation in HepG2 cells is attenuated by strawberry extract through AMPK activation.Nutrients,2017,9(6).pii:E621.
[15] ELSTROM RL,BAUER DE,BUZZAI M,et al.Akt stimulates aerobic glycolysis in cancer cells.Cancer Res,2004,64(11):3892-3899.
[16] ALTOMARE DA,TESTA JR.Perturbations of the AKT signaling pathway in human cancer.Oncogene,2005,24(50):7455-7464.
[17] RICOULT SJ,YECIES JL,BENSAHRA I,et al.Oncogenic PI3K and K-Ras stimulate de novo lipid synthesis through mTORC1 and SREBP.Oncogene,2015,35(10):1250-1260.
[18] GALDIERI L,GATLA H,VANCUROVA I,et al.Activation of AMP-activated protein kinase by metformin induces protein acetylation in prostate and ovarian cancer cells.J Biol Chem,2016,291(48):25154-25166.
[2] NOTO A,RAFFA S,DE VC,et al.Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells.Cell Death Dis,2013,4:e947[2018-10-08].https://www.nature.com/articles/cddis2013444.doi:10.1038/cddis.2013.444.
[3] VON ROEMELING CA,MARLOW LA,WEI JJ,et al.Stearoyl-CoA desaturase 1 is a novel molecular therapeutic target for clear cell renal cell carcinoma.Clin Cancer Res,2013,19(9):2368-2380.
[4] FERLAY J,SOERJOMATARAM I,DIKSHIT R,et al.Cancer incidence and mortality worldwide:sources,methods and major patterns in GLOBOCAN 2012.Int J Cancer,2015,136(5):E359-E386.
[5] ALEGRE MM,KNOWLES MH,ROBISON RA,et al.Mechanics behind breast cancer prevention - focus on obesity,exercise and dietary fat.Asian Pac J Cancer Prev,2013,14(4):2207-2212.
[6] CHAJèS V,ASSI N,BIESSY C,et al.A prospective evaluation of plasma phospholipid fatty acids and breast cancer risk in the EPIC study.Ann Oncol,2017,28(11):2836-2842.
[7] ZHAO J,ZHI Z,WANG C,et al.Exogenous lipids promote the growth of breast cancer cells via CD36.Oncol Rep,2017,38(4):2105-2115.
[8] JAIN R,STRICKLER HD,FINE E,et al.Clinical studies examining the impact of obesity on breast cancer risk and prognosis.J Mammary Gland Biol Neoplasia,2013,18(3/4):257-266.
[9] IWAI T,KUME S,CHIN-KANASAKI M,et al.Stearoyl-CoA eesaturase-1 protects cells against lipotoxicity-mediated apoptosis in proximal tubular cells.Int J Mol Sci,2016,17.pii.E1868.
[10] BAXTER AA,HULETT MD,IKH P.The phospholipid code:a key component of dying cell recognition,tumor progression and host-microbe interactions.Cell Death Differ,2015,22(12):1893-1905.
[11] PLAS DR,THOMPSON CB.Akt-dependent transformation:there is more to growth than just surviving.Oncogene,2005,24(50):7435-7442.
[12] PORSTMANN T,GRIFFITHS B,CHUNG YL,et al.PKB/Akt induces transcription of enzymes involved in cholesterol and fatty acid biosynthesis via activation of SREBP.OncogeneJ,2005,24(43) 6465-6481.
[13] KAHN BB,ALQUIER T,CARLING D,et al.AMP-activated protein kinase:ancient energy gauge provides clues to modern understanding of metabolism.Cell Metabolism,2005,1(1):15-25.
[14] FORBES-HERNáNDEZ TY,GIAMPIERI F,GASPARRINI M,et al.Lipid accumulation in HepG2 cells is attenuated by strawberry extract through AMPK activation.Nutrients,2017,9(6).pii:E621.
[15] ELSTROM RL,BAUER DE,BUZZAI M,et al.Akt stimulates aerobic glycolysis in cancer cells.Cancer Res,2004,64(11):3892-3899.
[16] ALTOMARE DA,TESTA JR.Perturbations of the AKT signaling pathway in human cancer.Oncogene,2005,24(50):7455-7464.
[17] RICOULT SJ,YECIES JL,BENSAHRA I,et al.Oncogenic PI3K and K-Ras stimulate de novo lipid synthesis through mTORC1 and SREBP.Oncogene,2015,35(10):1250-1260.
[18] GALDIERI L,GATLA H,VANCUROVA I,et al.Activation of AMP-activated protein kinase by metformin induces protein acetylation in prostate and ovarian cancer cells.J Biol Chem,2016,291(48):25154-25166.
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