应激对小鼠乳腺癌生长转移的作用及机制
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摘要
目的探讨应激对小鼠乳腺癌生长转移的作用及机制。方法 6～8周龄BALB/c小鼠接种4T1细胞制备乳腺癌移植瘤模型,分为对照组、慢性应激组、Iso[异丙肾上腺素,10 mg/(kg.d)]组、慢性应激+DMSO(二甲基亚砜)组和慢性应激+Rapa[雷帕霉素,15 mg/(kg.d)]组。记录癌组织体积变化,并于造模第21天处死小鼠,检测瘤内Beclin1、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)和p62表达变化;同时进行肺墨汁染色,计数小鼠肺内转移结节。采用去甲肾上腺素(NE)处理4T1细胞,观察自噬相关分子Beclin 1、LC3-Ⅱ、p62 mRNA和蛋白表达的变化。结果与对照组[(1359.7±173.9) mm3]相比,应激刺激[(2119.7±130.0) mm3]和Iso注射[(1947.0±102.8) mm3]可促进乳腺瘤体的生长(P<0.05),同时,肺内转移结节数也呈现同样的变化趋势[慢性应激组(10.3±1.1)个,Iso组(8.8±0.5)个vs.对照组(4.3±0.3)个,P<0.05];慢性应激组瘤内Beclin 1、LC3-Ⅱ表达减少,而自噬的抑制性分子标志物p62表达增加(P<0.05);自噬诱导剂Rapa可逆转应激的促瘤效果[(2275.477±187.397) mm3vs.(1360.097±213.938) mm3,P<0.05]。与生理盐水组相比,NE处理后4T1细胞内Beclin 1表达减少约60%(100%vs. 39.8%±2.0%,P<0.05),绿色荧光蛋白(GFP)-LC3的点状聚集也明显减少(P<0.05)。生物信息学分析显示高表达Beclin1的乳腺癌患者生存预后更好,高表达p62者则正相反(P<0.001)。结论应激通过抑制细胞自噬促进乳腺癌的生长和转移。
Objective To investigate the potential mechanisms of chronic stress-induced breast cancer progression.Methods Mouse breast cancer xenograft model was established by injecting 4 T1 cells into 6-week-old BALB/c mice, followed by randomized into the control group(no induced stress or drug treatment), chronic stress group, Iso injection group [10 mg/(kg.d),served as positive control], chronic stress + DMSO group(served as control for drug treatment), and chronic stress +Rapa group[15 mg/(kg.d)]. The tumor size was monitored up to 21 days. The intratumor expression levels of Beclin1, LC3-Ⅱ, and p62 were detected. The pulmonary metastatic nodules were visualized and counted using lung ink staining. The expression of autophagyrelated molecules in 4 T1 cells after NE treatment was also examined in vitro. Results Compared with the control group[(1359.7±173.9) mm3], chronic stress [(2119.7±130.0) mm3], and Iso [(1947.0±102.8) mm3] promoted the growth of breast cancer cells(P<0.05). Consistently, the lung nodules numbers were significantly increased in the chronic stress group(10.3±1.1)and the Iso group(8.8±0.5), compared to control group(4.3±0.3, P<0.05). In addition, compared to the control group, Beclin1 expression from samples of the stress group were decreased while p62 expression increased(P<0.05). Interestingly, the autophagy inducer Rapa reversed the pro-tumorigenic effect of chronic stress [(2275.477±187.397) mm3 vs.(1360.097±213.938) mm3,P<0.05]. We further confirmed that 4 T1 cells treated with NE resulted in 60% decreased of Beclin1 expression in 4 T1(100% vs.39.8%±2.0%, P<0.05) the fluorescence intensity of LC3 decreased as well(P<0.05). Bioinformatics analysis showed that breast cancer patients with high expression of Beclin1 had better survival prognosis, while those with high expression of p62 showed worse outcome(P<0.001). Conclusion Stress promotes the growth and metastasis of breast cancer through suppressing cell autophagy.
Objective To investigate the potential mechanisms of chronic stress-induced breast cancer progression.Methods Mouse breast cancer xenograft model was established by injecting 4 T1 cells into 6-week-old BALB/c mice, followed by randomized into the control group(no induced stress or drug treatment), chronic stress group, Iso injection group [10 mg/(kg.d),served as positive control], chronic stress + DMSO group(served as control for drug treatment), and chronic stress +Rapa group[15 mg/(kg.d)]. The tumor size was monitored up to 21 days. The intratumor expression levels of Beclin1, LC3-Ⅱ, and p62 were detected. The pulmonary metastatic nodules were visualized and counted using lung ink staining. The expression of autophagyrelated molecules in 4 T1 cells after NE treatment was also examined in vitro. Results Compared with the control group[(1359.7±173.9) mm3], chronic stress [(2119.7±130.0) mm3], and Iso [(1947.0±102.8) mm3] promoted the growth of breast cancer cells(P<0.05). Consistently, the lung nodules numbers were significantly increased in the chronic stress group(10.3±1.1)and the Iso group(8.8±0.5), compared to control group(4.3±0.3, P<0.05). In addition, compared to the control group, Beclin1 expression from samples of the stress group were decreased while p62 expression increased(P<0.05). Interestingly, the autophagy inducer Rapa reversed the pro-tumorigenic effect of chronic stress [(2275.477±187.397) mm3 vs.(1360.097±213.938) mm3,P<0.05]. We further confirmed that 4 T1 cells treated with NE resulted in 60% decreased of Beclin1 expression in 4 T1(100% vs.39.8%±2.0%, P<0.05) the fluorescence intensity of LC3 decreased as well(P<0.05). Bioinformatics analysis showed that breast cancer patients with high expression of Beclin1 had better survival prognosis, while those with high expression of p62 showed worse outcome(P<0.001). Conclusion Stress promotes the growth and metastasis of breast cancer through suppressing cell autophagy.
引文
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[2]Xiao B,Liu RR,Liu BT,et al.Induction effect of TTF1-NPon human hepatoma cell apoptosis through ERS-mediated pathway[J].J Jilin Univ(Med Ed),2015,41(6):1118-1123,12.[肖斌,刘荣荣,刘炳彤,等.TTF1-NP诱导人肝癌HepG-2细胞凋亡的内质网应激作用[J].吉林大学学报(医学版),2015,41(6):1118-1123,12.]
[3]Cole SW.Nervous system regulation of the cancer genome[J].Brain Behav Immun,2013,30(Suppl):S10-S18.
[4]Rosengren A,Hawken S,Ounpuu S,et al.Association of psychosocial risk factors with risk of acute myocardial infarction in 11119 cases and 13648 controls from 52countries(the INTERHEART study):case-control study[J].Lancet,2004,364(9438):953-962.
[5]Krizanova O,Babula P,Pacak K.Stress,catecholaminergic system and cancer[J].Stress,2016,19(4):419-428.
[6]Sloan EK,Capitanio JP,Tarara RP,et al.Social stress enhances sympathetic innervation of primate lymph nodes:mechanisms and implications for viral pathogenesis[J].J Neurosci,2007,27(33):8857-8865.
[7]Shan MH,Qin JF,Jin FJ,et al.Autophagy suppresses isoprenaline-induced M2 macrophage polarization via the ROS/ERK and mTOR signaling pathway[J].Free Radical Bio Med,2017,110(9):432-443.
[8]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):939-944.
[9]Qin JF,Jin FJ,Li N,et al.Adrenergic receptorβ2 activation by stress promotes breast cancer progression through macrophages M2 polarization in tumor microenvironment[J].BMB Rep,2015,48(5):295-300.
[10]Cordes MC,Scherwath A,Ahmad T,et al.Distress,anxiety and depression in patients with brain metastases before and after radiotherapy[J].BMC Cancer,2014,14:731-741.
[11]Fitzgerald PJ.Is norepinephrine an etiological factor in some types of cancer?[J].Int J Cancer,2009,124(2):257-263.
[12]McGregor BA,Antoni MH.Psychological intervention and health outcomes among women treated for breast cancer:a review of stress pathways and biological mediators[J].Brain Behav Immun,2009,23(2):159-166.
[13]Khan S,Tisdale MJ.Catabolism of adipose tissue by a tumourproduced lipid-mobilising factor[J].Int J Cancer,1999,80(3):444-447.
[14]Drell TL,Joseph J,Lang K,et al.Effects of neurotransmitters on the chemokinesis and chemotaxis of MDA-MB-468 human breast carcinoma cells[J].Breast Cancer Res Treat,2003,80(1):63-70.
[15]Yao H,Duan Z,Wang M,etal.Adrenalineinduces chemoresistance in HT-29 colon adenocarcinoma cells[J].Cancer Genet Cytogen,2009,190(2):81-87.
[16]Deng GH,Liu J,Zhang J,et al.Exogenous norepinephrine attenuates the efficacy of sunitinib in a mouse cancer model[J].JExp Clin Cancer Res,2014,33(1):21-32.
[17]Cecconi F,Levine B.The role of autophagy in mammalian development:cell makeover rather than cell death[J].Dev Cell,2008,15(3):344-357.
[18]Qin JF,Liu H,Li YF,et al.Effect of hormones from adrenal gland on breast cancer metastasis in mice[J].Cancer Res Prev Treat,2016,43(12):1013-1017.[秦君芳,刘华,李云飞,等.肾上腺激素水平对小鼠乳腺癌转移的影响[J].肿瘤防治研究,2016,43(12):1013-1017.]
[19]Li HM,Wu J,Wu S,et al.Effects of saturated and unsaturated fatty acids on proliferation and autophagy of lung cancer cells[J].Med J Chin PLA,2017,42(8):668-673.[李慧敏,伍俊,吴尚,等.饱和脂肪酸和不饱和脂肪酸对肺癌细胞增殖与自噬的影响[J].解放军医学杂志,2017,42(8):668-673.]
[20]Wang DD,Chen XS.Cell autophagy and its progress in breast cancer[J].J Mod Oncol,2017,25(22):3704-3707.[王丹丹,陈雪松.细胞自噬及其在乳腺癌中的研究进展[J].现代肿瘤医学,2017,25(22):3704-3707.]
[21]Liang XH,Jackson S,Seaman M,et al.Induction of autophagy and inhibition of tumorigenesis by beclin 1[J].Nature,1999,402(6762):672-676.
[22]Gozuacik D,Kimchi A.Autophagy as a cell death and tumor suppressor mechanism[J].Oncogene,2004,23(16):2891-2906.
[23]Liu HB,Hong J,Cui C.Meta-analysis of the expression of p62protein and survival in breast cancer[J].Clin J Med Officer,2018,46(9):1027-1029,1032.[刘会彬,洪健,崔琮.自噬相关蛋白p62表达与乳腺癌预后关系Meta分析[J].临床军医杂志,2018,46(9):1027-1029,1032.]
[24]Wang X,Li L,Niu X,et al.mTOR enhances foam cell formation by suppressing the autophagy pathway[J].DNA Cell Biol,2014,33:198-204.
[25]Qi Y,Zhang M,Li H,et al.Autophagy inhibition by sustained overproduction of IL6 contributes to arsenic carcinogenesis[J].Cancer Res,2014,74(14):3740-3752.
[26]Ichimura Y,Kumanomidou T,Sou YS,et al.Structural basis for sorting mechanism of p62 in selective autophagy[J].J Biol Chem,2008,283(33):22847-22857.
[27]Wu FQ.ADRB2 signaling pathway promotes the tumorigenesis and development o f hepatocel l u lar carcinoma and i t s mechanisms[D].Shanghai:Second Military Medical University,2015.[邬福全.ADRB2信号通路调控肝癌发生及发展的机制研究[D].上海:第二军医大学,2015.]