-
焦虑是一种使人内心混乱的、不愉悦的精神状态[1]。有研究[2-3]发现,焦虑症状在乳腺癌病人中发生率较高且影响抗肿瘤治疗效果,并且与乳腺癌病人总生存时间存在显著相关性[4-5]。有报道显示,Akt通路作为一种特异性的丝氨酸/苏氨酸蛋白激酶,对于许多细胞生理过程的调控起着关键的作用[6-7]。本研究拟用焦虑模型以及去甲肾上腺素处理的乳腺癌细胞,探究焦虑状态下乳腺癌发展的可能分子机制。现作报道。
-
小鼠高架十字迷宫实验结果显示,造模组小鼠进入开放臂次数比例为13.75%±4.16%,明显低于对照组的40.68%±9.87%(t=7.95,P < 0.01)。ELISA实验结果显示,造模组小鼠血清中的去甲肾上腺素的水平为(195.64±17.53)pg/mL, 低于对照组的(65.69±16.41)pg/ mL(t=17.11,P < 0.01)。造模组小鼠的去甲肾上腺素水平与焦虑程度呈显著正相关关系(r=0.78,P < 0.01)。造模组小鼠的肿瘤体积(2 830.13±188.68)mm3, 大于对照组的(2 155.33±212.43)mm3(t=7.51,P < 0.05)。
-
Western blotting结果显示,β1-AR和β2-AR均可在MDA-MB-231细胞系中表达,相对表达分别为1.63%±0.09%、1.72%±0.23%。免疫荧光实验支持这一结果(见图 1、2)。
-
去甲肾上腺素对MDA-MB-231的增殖有明显的促进作用,且具有时间和剂量依赖性, 各实验组与对照组间相比有统计学意义(P < 0.05)(见表 1)。与对照组相比,去甲肾上腺素组细胞的迁移指数有明显增加(P < 0.01),经PRO预处理后,细胞迁移能力较单加去甲肾上腺素明显减弱(P < 0.05)。去甲肾上腺素处理组的跨膜细胞数量相比对照组有明显增加(P < 0.01),而经PRO预处理组的细胞跨膜数量比单用去甲肾上腺素组减少(P < 0.05)。去甲肾上腺素上调Akt表达量(P<0.01),PRO组Akt信号通路蛋白相对表达量比NE组明显降低(P<0.01)(见表 2)。
分组 0 h 24 h 48 h 72 h 对照组 0.125±0.005 0.233±0.063 0.36±0.024 0.448±0.028 0.1 μmol/L 0.123±0.004 0.228±0.027 0.40±0.019 0.413±0.011 1 μmol/L 0.124±0.004 0.349±0.039 0.471±0.010 0.482±0.013 10 μmol/L 0.124±0.003 0.428±0.020* 0.562±0.036** 0.567±0.026** 100 μmol/L 0.127±0.004 0.517±0.029* 0.555±0.022** 0.586±0.041** F 0.74 10.45 43.64 21.95 P > 0.05 < 0.05 < 0.01 < 0.01 MS组内 0.001 0.010 0.010 0.010 q检验:与对照组比较*P<0.05,**P<0.01 表 1 CCK-8实验结果OD值比较(ni=3;x±s)
分组 迁移指数 跨膜细胞数量 相对表达量 对照组 0.22±0.05 38.33±5.13 1.00±0.02 NE组 0.68±0.16** 86.67±17.16** 3.18±0.65** PRO+NE组 0.23±0.11# 54.33±5.69# 2.44±0.31## PRO组 0.18±0.06 20.33±4.16** 0.30±0.09** F 15.91 25.70 33.33 P < 0.01 < 0.01 < 0.01 MS组内 0.010 92.580 0.214 q检验:与对照组比较**P<0.01;与NE组比较# P<0.05,##P < 0.01 表 2 各组细胞迁移指数、跨膜细胞数量及相对表达量比较(ni=3;x±s)
-
流式细胞仪结果显示,去甲肾上腺素作用后,S期细胞比例相较对照组明显增加(P<0.01),G1期细胞的比例明显减少(P<0.01)。而如果用PRO预处理,则上述作用明显减弱(P<0.01)(见表 3)。
分组 G1期 S期 G2期 对照组 67.93±2.12 23.55±1.05 8.04±0.59 NE组 47.64±1.63** 44.08±1.66** 7.95±0.64 PRO+NE组 56.75±1.35## 35.11±0.67## 7.98±0.31 PRO组 73.93±3.24 18.73±1.21** 8.01±0.44 F 84.32 270.60 0.05 P < 0.01 < 0.01 > 0.05 MS组内 4.870 1.450 0.260 q检验:与对照组比较**P<0.01;与NE组相比##P<0.01 表 3 细胞周期检测实验各期细胞数量百分比比较(ni=3;x±s)
-
模型组小鼠肿瘤体积大于对照组肿瘤体积(P<0.05)。PRO组肿瘤体积与模型组比较明显缩小(P<0.05)(见表 4)。
分组 肿瘤体积/mm3 F P MS组内 对照组 2 149.51±145.97 模型组 2 910.74±433.01* 8.96 < 0.05 86 699.900 PRO组 1 945.13±226.33# q检验:与对照组比较*P<0.05;与模型组比较#P<0.05 表 4 对照组、模型组及PRO组小鼠肿瘤体积比较(ni=10;x±s)
焦虑状态下去甲肾上腺素激活Akt信号通路对乳腺癌作用研究
Study on norepinephrine activating Akt signaling pathway in breast cancer under anxiety state
-
摘要:
目的探讨去甲肾上腺素在焦虑状态下对乳腺癌的作用及机制。 方法将20只C57BL/6小鼠分为对照组及模型组,通过接种肿瘤细胞和实验刺激建立焦虑荷瘤小鼠模型,造模结束后检测血清中去甲肾上腺素水平并测量肿瘤体积。人乳腺癌细胞系MDA-MB-231细胞在去甲肾上腺素处理后检测其增殖、迁移、侵袭、细胞周期分布及Akt信号通路的激活状态。 结果模型组小鼠肿瘤体积大于对照组(P < 0.05),血清中去甲肾上腺素含量明显升高(P < 0.01),且同焦虑程度呈现相关关系(r=0.78)。去甲肾上腺素提高了MDA-MB-231细胞的增殖、迁移和侵袭能力,并且促进DNA复制以及Akt信号通路的激活。 结论去甲肾上腺素在焦虑状态下激活AKt信号通路,促进乳腺癌的发展。 Abstract:ObjectiveTo explore the effects and mechanism of norepinephrine in breast cancer under anxiety state. MethodsTwenty C57BL/6 mice were divided into the control group and model group.The mice models with anxiety tumor-bearing were established using inoculation of tumor cells and experimental stimulation.The serum level of norepinephrine was detected, and the tumor volume was measured after modeling.The proliferation, migration, invasion, cell cycle distribution and activation of Akt signaling pathway of MDA-MB-231 breast cancer cells treated with norepinephrine were detected. ResultsThe tumor volume in model group was significantly larger than that in control group (P < 0.05), and the serum concentration of norepinephrine significantly increased (P < 0.01), which was correlated with the anxiety degree (r=0.78).Norepinephrine enhanced the proliferation, migration and invasion of MDA-MB-231 cells, promoted DNA replication, and activated the Akt signaling pathway. ConclusionsNorepinephrine activates the Akt signaling pathway to promote the development of breast cancer under state anxiety. -
Key words:
- breast neoplasms /
- anxiety /
- norepinephrine /
- Akt signaling pathway
-
表 1 CCK-8实验结果OD值比较(ni=3;x±s)
分组 0 h 24 h 48 h 72 h 对照组 0.125±0.005 0.233±0.063 0.36±0.024 0.448±0.028 0.1 μmol/L 0.123±0.004 0.228±0.027 0.40±0.019 0.413±0.011 1 μmol/L 0.124±0.004 0.349±0.039 0.471±0.010 0.482±0.013 10 μmol/L 0.124±0.003 0.428±0.020* 0.562±0.036** 0.567±0.026** 100 μmol/L 0.127±0.004 0.517±0.029* 0.555±0.022** 0.586±0.041** F 0.74 10.45 43.64 21.95 P > 0.05 < 0.05 < 0.01 < 0.01 MS组内 0.001 0.010 0.010 0.010 q检验:与对照组比较*P<0.05,**P<0.01 表 2 各组细胞迁移指数、跨膜细胞数量及相对表达量比较(ni=3;x±s)
分组 迁移指数 跨膜细胞数量 相对表达量 对照组 0.22±0.05 38.33±5.13 1.00±0.02 NE组 0.68±0.16** 86.67±17.16** 3.18±0.65** PRO+NE组 0.23±0.11# 54.33±5.69# 2.44±0.31## PRO组 0.18±0.06 20.33±4.16** 0.30±0.09** F 15.91 25.70 33.33 P < 0.01 < 0.01 < 0.01 MS组内 0.010 92.580 0.214 q检验:与对照组比较**P<0.01;与NE组比较# P<0.05,##P < 0.01 表 3 细胞周期检测实验各期细胞数量百分比比较(ni=3;x±s)
分组 G1期 S期 G2期 对照组 67.93±2.12 23.55±1.05 8.04±0.59 NE组 47.64±1.63** 44.08±1.66** 7.95±0.64 PRO+NE组 56.75±1.35## 35.11±0.67## 7.98±0.31 PRO组 73.93±3.24 18.73±1.21** 8.01±0.44 F 84.32 270.60 0.05 P < 0.01 < 0.01 > 0.05 MS组内 4.870 1.450 0.260 q检验:与对照组比较**P<0.01;与NE组相比##P<0.01 表 4 对照组、模型组及PRO组小鼠肿瘤体积比较(ni=10;x±s)
分组 肿瘤体积/mm3 F P MS组内 对照组 2 149.51±145.97 模型组 2 910.74±433.01* 8.96 < 0.05 86 699.900 PRO组 1 945.13±226.33# q检验:与对照组比较*P<0.05;与模型组比较#P<0.05 -
[1] ROSENHAN DL, SELIGMAN ME.Abnormal psychology[M].New York:WW Norton & Co., 1995. [2] YI JC, SYRJALA KL.Anxiety and Depression in Cancer Survivors[J].Med Clin North Am, 2017, 101(6):1099. doi: 10.1016/j.mcna.2017.06.005 [3] MCGUIRE S.World Cancer Report 2014.Geneva, Switzerland:World Health Organization, International Agency for Research on Cancer, WHO Press, 2015[J].Adv Nutr, 2016, 7(2):418. [4] ZHAO W, WU Z, CHEN J, et al.Survival prediction of anxious emotion in advanced cancer patients receiving palliative care[J].Psychooncology, 2017, 26(10):1463. doi: 10.1002/pon.4314 [5] 阎子海, 肖永红, 张文杰, 等.心理因素与女性乳腺癌关系的Meta分析[J].中国公共卫生, 2006, 22(6):748. doi: 10.3321/j.issn:1001-0580.2006.06.060 [6] FRANKE TF, KAPLAN DR, CANTLEY LC, et al.Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3, 4-bisphosphate[J].Science, 1997, 275(5300):665. doi: 10.1126/science.275.5300.665 [7] PALMIERI M, PAL R, NELVAGAL HR, et al.mTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases[J].Nat Commun, 2017, 8:14338. doi: 10.1038/ncomms14338 [8] 翟亚南, 陈柏安, 郭萌, 等.束缚应激的研究进展[J].实验动物科学, 2013, 30(4):56. doi: 10.3969/j.issn.1006-6179.2013.04.015 [9] SHAO F, LIN W, WANG W, et al.The effect of emotional stress on the primary humoral immunity of rats[J].J Psychopharmacol, 2003, 17(2):179. doi: 10.1177/0269881103017002005 [10] 谢贵林, 陈璐艳, 陈新华, 等.纳秒脉冲电场消融乳腺癌的实验研究[J].中国医药导报, 2015, 12(16):4. [11] OUYANG X, ZHU Z.Epinephrine increases malignancy of breast cancer through p38 MAPK signaling pathway in depressive disorders[J].Int J Clin Exp Pathol, 2019, 12(46):1932 [12] INHESTERN L, BEIERLEIN V, BULTMANN J C, et al.Anxiety and depression in working-age cancer survivors:a register-based study[J].BMC Cancer, 2017, 17(1):347. doi: 10.1186/s12885-017-3347-9 [13] PYTER LM, SUAREZ-KELLY LP, CARSON WE, et al.Novel rodent model of breast cancer survival with persistent anxiety-like behavior and inflammation[J].Behav Brain Res, 2017, 330(108):17. [14] DHABHAR FS, SAUL AN, HOLMES TH, et al.High-anxious individuals show increased chronic stress burden, decreased protective immunity, and increased cancer progression in a mouse model of squamous cell carcinoma[J].PLoS One, 2012, 7(4):e33069. doi: 10.1371/journal.pone.0033069 [15] LUTGENDORF S K, DEGEEST K, DAHMOUSH L, et al.Social isolation is associated with elevated tumor norepinephrine in ovarian carcinoma patients[J].Brain Behav Immun, 2011, 25(2):250. doi: 10.1016/j.bbi.2010.10.012 [16] YASUNARI K, MATSUI T, MAEDA K, et al.Anxiety-induced plasma norepinephrine augmentation increases reactive oxygen species formation by monocytes in essential hypertension[J].Am J Hypertens, 2006, 19(6):573. doi: 10.1016/j.amjhyper.2005.10.027 [17] REEDER A, ATTAR M, NAZARIO L, et al.Stress hormones reduce the efficacy of paclitaxel in triple negative breast cancer through induction of DNA damage[J].Br J Cancer, 2015, 112(9):1461. doi: 10.1038/bjc.2015.133 [18] THAKER PH, HAN LY, KAMAT AA, et al.Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma[J].Nat Med, 2006, 12(8):939. doi: 10.1038/nm1447 [19] DETHLEFSEN C, HANSEN LS, LILLELUND C, et al.Exercise-Induced Catecholamines Activate the Hippo Tumor Suppressor Pathway to Reduce Risks of Breast Cancer Development[J].Cancer Res, 2017, 77(18):4894. doi: 10.1158/0008-5472.CAN-16-3125 [20] DETHLEFSEN C, LILLELUND C, MIDTGAARD J, et al.Exercise regulates breast cancer cell viability:systemic training adaptations versus acute exercise responses[J].Breast Cancer Res Treat, 2016, 159(3):469. doi: 10.1007/s10549-016-3970-1 [21] CUI B, LUO Y, TIAN P, et al.Stress-induced epinephrine enhances lactate dehydrogenase A and promotes breast cancer stem-like cells[J].2019, 129(3): 1030. [22] KAMIYA A, HAYAMA Y, KATO S, et al.Genetic manipulation of autonomic nerve fiber innervation and activity and its effect on breast cancer progression[J].Nat Neurosci, 2019, 22(8):1289. doi: 10.1038/s41593-019-0430-3