M3R拮抗剂对人小细胞肺癌增殖、凋亡及粘附的影响
|
摘要
背景与目的研究表明毒蕈碱胆碱受体3(muscarinic receptor 3,M3R)在多种肿瘤的发生、发展中发挥重要的作用。本研究旨在探讨M3R在人小细胞肺癌(small cell lung cancer,SCLC)细胞株SBC3的表达,M3R拮抗剂对细胞增殖、凋亡及粘附的影响。方法体外培养SBC3细胞,RT-PCR和Western blot检测M3R的表达。MTT法及流式细胞法检测M3R拮抗剂(4-diphenylacetoxy-N-methylpiperidine methiodide,4-DAMP)对细胞增殖和凋亡的影响。流式细胞法检测细胞整合素的表达及碘化乙酰胆碱(acetylcholine iodide,Ach)和4-DAMP对整合素表达的影响。纤维结合蛋白(Fn)包被的96孔板用以研究Ach、4-DAMP及整合素抗体对细胞粘附的作用。结果 SBC3细胞表达M3R,4-DAMP浓度依懒性抑制细胞增殖。与对照组比较,10-4 M 4-DAMP能够明显地增加SBC3细胞凋亡。SBC3细胞表达αvβ1和α5β1整合素,10-4 M Ach刺激细胞粘附(P<0.01)的作用几乎被10-5M 4-DAMP、5μg/m L抗-β1抗体或抗-αv和α5抗体完全阻断(P<0.01),但Ach及4-DAMP不影响αv、α5和β1的表达水平。结论 SBC3细胞表达M3R,M3R拮抗剂能抑制细胞的增殖并促进凋亡。其抑制粘附的作用是通过抑制细胞含β1的整合素(αvβ1和α5β1)的功能实现的。
Background and objective Studies have shown that muscarinic receptors 3(M3R) plays key roles in regulating tumorigenesis and tumor growth. The aim of this study is to investigate the expression of M3R in human small cell lung cancer(SCLC) cell line SBC3 and to explore the effect of M3R antagonist on cell proliferation, apoptosis and adhesion. Methods SBC3 cells were cultured in vitro. RT-PCR and Western blot were used to detect the expression of M3R in SBC-3. MTT assay and flow cytometry were used to determine the effects of M3R antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide(4-DAMP) on the proliferation and apoptosis of SBC-3 cells. Flow cytometry was used to detect the integrin expression in SBC3 and alteration of integrin expression after treating the cells with cholinergic receptor agonist acetylcholine iodide(acetylcholine iodide, Ach) and 4-DAMP. Fibronectin(Fn) coated 96-well plates were used to detect the effect of Ach, 4-DAMP and anti-integrin antibody on cell adhesion. Results M3R was expressed in SBC3 cells. 4-DAMP significantly inhibited SBC3 cells proliferation in a dose-dependent manner. 4-DAMP promoted cell apoptosis at a concentration of 10-4 M of 4-DAMP compared with control. αvβ1 and α5β1 integrin were expressed in SBC3 cells. 10-4 M Ach stimulated cell adhesion toward Fn significantly(P<0.01). This effect was completely abrogated by 10~(-5) M 4-DAMP, 5 μg/m L anti-β1 integrin antibody or anti-αv and anti-α5 integrin antibodies(P<0.01) and partially abrogated by 5 μg/m L anti-αv or anti-α5 integrin antibody(P<0.05). But Ach and 4-DAMP did not alter Fn receptor(αvβ1 or α5β1 integrin) expression. Conclusion M3R is expressed in SBC3 cell. The M3R antagonist inhibits SBC3 cell proliferation, adhesion and enhances cell apoptosis. M3R antagonist in-hibiting SBC3 cell adhesion is presumably modulated by functional alteration of β1 containing integrins(αvβ1 and α5β1), but not by any variation in their expression.
Background and objective Studies have shown that muscarinic receptors 3(M3R) plays key roles in regulating tumorigenesis and tumor growth. The aim of this study is to investigate the expression of M3R in human small cell lung cancer(SCLC) cell line SBC3 and to explore the effect of M3R antagonist on cell proliferation, apoptosis and adhesion. Methods SBC3 cells were cultured in vitro. RT-PCR and Western blot were used to detect the expression of M3R in SBC-3. MTT assay and flow cytometry were used to determine the effects of M3R antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide(4-DAMP) on the proliferation and apoptosis of SBC-3 cells. Flow cytometry was used to detect the integrin expression in SBC3 and alteration of integrin expression after treating the cells with cholinergic receptor agonist acetylcholine iodide(acetylcholine iodide, Ach) and 4-DAMP. Fibronectin(Fn) coated 96-well plates were used to detect the effect of Ach, 4-DAMP and anti-integrin antibody on cell adhesion. Results M3R was expressed in SBC3 cells. 4-DAMP significantly inhibited SBC3 cells proliferation in a dose-dependent manner. 4-DAMP promoted cell apoptosis at a concentration of 10-4 M of 4-DAMP compared with control. αvβ1 and α5β1 integrin were expressed in SBC3 cells. 10-4 M Ach stimulated cell adhesion toward Fn significantly(P<0.01). This effect was completely abrogated by 10~(-5) M 4-DAMP, 5 μg/m L anti-β1 integrin antibody or anti-αv and anti-α5 integrin antibodies(P<0.01) and partially abrogated by 5 μg/m L anti-αv or anti-α5 integrin antibody(P<0.05). But Ach and 4-DAMP did not alter Fn receptor(αvβ1 or α5β1 integrin) expression. Conclusion M3R is expressed in SBC3 cell. The M3R antagonist inhibits SBC3 cell proliferation, adhesion and enhances cell apoptosis. M3R antagonist in-hibiting SBC3 cell adhesion is presumably modulated by functional alteration of β1 containing integrins(αvβ1 and α5β1), but not by any variation in their expression.
引文
1 Shah N,Khurana S,Raufman JP.Muscarnic receptor and ligands in cancer.Am J Physiol Cell Physiol.2009,296(2):C221-C232.
2 Jiang MY,Chen J,Wang J,et al.Nitric oxide modulates cardiovascular function in the rat by activating adenosine A2A receptors and inhibiting acetylcholine release in the rostral ventrolateral medulla.Clin Exp Pharmacol Physiol,2011,38(6):380-386.
3 Jiang M,Wang Q,Karasawa T,et al.Sodium-glucose transporter-2(SGLT2;SLC5A2)enhances cellular uptake of aminoglycosides.PLo S One,2014,9 (9):e108941.
4 Wessler I,Kirk Patrick C J,RackéK.Non-neuronal acetylcholine,a locally acting molecule,widely distributed in biological systems:expression and function in humans.Pharmacol Ther,1998,77(1):59-79.
5 Williams CL.Muscarinic signaling in carcinoma cells.Life Sci,2003,72(18-19):2173-2182.
6 Song P,Sekhon HS,Lu A,et al.M3 muscarinic receptor antagonists inhibit small cell lung carcinoma growth and mitogen-activated protein kinase phosphorylation induced by acetylcholine secretion.Cancer Res,2007,67(8):3936-3944.
7 Espanol AJ,Jacob G,Dmytrenko G,et al.Muscarinic activation enhances the anti-proliferative effect of Paclitaxel in murine breast tumor cells.Anticancer Agents Med chem,2013,13(8):1273-1279.
8 Nicke B,Detjen K,Logston CD,et al.Muscarinic cholinergic receptors activate both inhibitory and stimulatory growth mechanisms in NIH3T3cells.Biol chem,1999,274(31):21701-21706.
9 De Sarno P,Shestopal SA,King TD,et al.Muscarinic receptor activation protects cells from apoptotic effects of DNA damage,oxidative stress,and mitochondrial inhibition.Biol Chem,2003,278(13):11086-11093.
10 Budd DC,Spragg EJ,Ridd K,et al.Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway.Biol Chem,2004,381(pt 1):43-49.
11 Profita M,Riccobono L,Montalbano AM,et al.In vitro anticholinergic drugs affect CD8+peripheral blood T-cells apoptosis in COPD.Immunobiology,2012,217(3):345-353.
12 Quigley RL,Shafer SH,Williams CL.Regulation of integrin-mediated adhesion by muscarinic acetylcholine receptors and protein kinase C in small cell lung carcinoma.Chest,1998,114(3):839-846.
13 Humphries MJ.Integrin Structure.Biochem Soc Trans,2000,28(4):311-339.
14 Pankov R,Yamada KM.Fibronectin at a glance.Cell Sci,2002,115(Pt 20):3861-3863.
15 Vogel BE,Tarone G,Giancotti FG,et al.A novel fibronectin receptor with an unexpected subunit composition(αvβ1).Biol Chem,1990,265(11):5934-5937.
16 Sethi T,Rintoul RC,Moore SM,et al.Extracellular matrix proteins protect small cell lung cancer cells against apoptosis:A mechanism for small cell lung cancer growth and drug resistance in vivo.Nat Med,1999,5(6):662-668.
17 Koivisto L,Grenman R,Heino J,et al.Itegrins alpha5beta1,alphavbeta1,and alphavbeta6 collaborate in squamous carcinoma cell spreading and migration on fibronectin.Exp Cell Res,2000,255(1):10-17.
18 He H,Ma AL,Zhang SX.Expression of muscarinic cholinergic receptor3 in small cell lung cancer and its clinical significance.Lin Chuang Yu Shi Yan Bing Li Xue Za Zhi,2014,30(11):1237-1241.[何花,马爱玲,张淑香.小细胞肺癌中M3R的表达及意义.临床与实验病理学杂志,2014,30(11):1237-1241.]
2 Jiang MY,Chen J,Wang J,et al.Nitric oxide modulates cardiovascular function in the rat by activating adenosine A2A receptors and inhibiting acetylcholine release in the rostral ventrolateral medulla.Clin Exp Pharmacol Physiol,2011,38(6):380-386.
3 Jiang M,Wang Q,Karasawa T,et al.Sodium-glucose transporter-2(SGLT2;SLC5A2)enhances cellular uptake of aminoglycosides.PLo S One,2014,9 (9):e108941.
4 Wessler I,Kirk Patrick C J,RackéK.Non-neuronal acetylcholine,a locally acting molecule,widely distributed in biological systems:expression and function in humans.Pharmacol Ther,1998,77(1):59-79.
5 Williams CL.Muscarinic signaling in carcinoma cells.Life Sci,2003,72(18-19):2173-2182.
6 Song P,Sekhon HS,Lu A,et al.M3 muscarinic receptor antagonists inhibit small cell lung carcinoma growth and mitogen-activated protein kinase phosphorylation induced by acetylcholine secretion.Cancer Res,2007,67(8):3936-3944.
7 Espanol AJ,Jacob G,Dmytrenko G,et al.Muscarinic activation enhances the anti-proliferative effect of Paclitaxel in murine breast tumor cells.Anticancer Agents Med chem,2013,13(8):1273-1279.
8 Nicke B,Detjen K,Logston CD,et al.Muscarinic cholinergic receptors activate both inhibitory and stimulatory growth mechanisms in NIH3T3cells.Biol chem,1999,274(31):21701-21706.
9 De Sarno P,Shestopal SA,King TD,et al.Muscarinic receptor activation protects cells from apoptotic effects of DNA damage,oxidative stress,and mitochondrial inhibition.Biol Chem,2003,278(13):11086-11093.
10 Budd DC,Spragg EJ,Ridd K,et al.Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway.Biol Chem,2004,381(pt 1):43-49.
11 Profita M,Riccobono L,Montalbano AM,et al.In vitro anticholinergic drugs affect CD8+peripheral blood T-cells apoptosis in COPD.Immunobiology,2012,217(3):345-353.
12 Quigley RL,Shafer SH,Williams CL.Regulation of integrin-mediated adhesion by muscarinic acetylcholine receptors and protein kinase C in small cell lung carcinoma.Chest,1998,114(3):839-846.
13 Humphries MJ.Integrin Structure.Biochem Soc Trans,2000,28(4):311-339.
14 Pankov R,Yamada KM.Fibronectin at a glance.Cell Sci,2002,115(Pt 20):3861-3863.
15 Vogel BE,Tarone G,Giancotti FG,et al.A novel fibronectin receptor with an unexpected subunit composition(αvβ1).Biol Chem,1990,265(11):5934-5937.
16 Sethi T,Rintoul RC,Moore SM,et al.Extracellular matrix proteins protect small cell lung cancer cells against apoptosis:A mechanism for small cell lung cancer growth and drug resistance in vivo.Nat Med,1999,5(6):662-668.
17 Koivisto L,Grenman R,Heino J,et al.Itegrins alpha5beta1,alphavbeta1,and alphavbeta6 collaborate in squamous carcinoma cell spreading and migration on fibronectin.Exp Cell Res,2000,255(1):10-17.
18 He H,Ma AL,Zhang SX.Expression of muscarinic cholinergic receptor3 in small cell lung cancer and its clinical significance.Lin Chuang Yu Shi Yan Bing Li Xue Za Zhi,2014,30(11):1237-1241.[何花,马爱玲,张淑香.小细胞肺癌中M3R的表达及意义.临床与实验病理学杂志,2014,30(11):1237-1241.]