ATPR对人肺癌细胞分化和增殖的影响及其作用机制的初步探讨
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摘要
目的:观察4-氨基-2-三氟甲基苯基维甲酸酯(ATPR)对人肺腺癌细胞A549和鳞癌细胞L78生长抑制、分化的影响。
方法:将两株细胞分为4-氨基-2-三氟甲基苯基维甲酸酯(ATPR)组、空白对照组及全反式维甲酸(ATRA)组.前组分别以0.01umol/L、0.1 umolL、1 umol/L、5 umol/L、10 umol/L的ATPR与肺腺癌细胞A549和鳞癌细胞L78作用24h,48h,72h,96h。运用MTT法检测细胞吸光度并计算细胞的生长抑制率,流式细胞仪检测细胞周期。0.1 umolL、1 umol/L、5 umol/L的ATPR与肺腺癌细胞A549和鳞癌细胞L78作用72h,苏木精—伊红(HE)染色观察肺癌细胞形态变化。1 umol/L的ATPR作用肺癌细胞72h后, RT-PCR方法检测细胞核维甲酸α受体(RARα)、表皮生长因子受体(EGFR)表达的变化。
结果:ATPR具有抑制A549、L78细胞生长的作用,生长抑制率有剂量和时间依赖关系。流式细胞仪分析结果显示A549、L78细胞被阻滞于G0—G1期。A549、L78细胞在ATPR的作用下,发生了细胞胞体变小、分裂状态的细胞数减少等形态学变化。通过对肺癌细胞的RT-PCR法检测,均发现EGFR和RARα的特异性条带。空白对照组L78细胞高表达EGFR;A549细胞低表达EGFR,EGFR的表达自24h开始逐渐下降并呈时间依赖。空白对照组L78细胞和A549细胞低表达RARα,RARα的mRNA表达逐渐增加,并呈时间依赖。
结论:1.从MTT结果证实不同浓度的ATPR能够抑制细胞的增值,随着浓度的增加细胞活力依赖性下降,即抑制率逐渐增加。
2.流式细胞仪结果示ATPR使细胞被阻滞于G0-G1期。
3.从细胞形态学观察结果证实ATPR使肺癌细胞出现明显的分化征象。
4.从mRNA水平证实肺癌细胞株A549、L78表达EGFR、RARα。
5. ATPR以时间依赖的方式下调肺癌细胞中EGFR的mRNA表达并上调RARα的mRNA表达。
综上所述,ATPR可明显抑制A549、L78细胞增值并诱导其分化以及显著降低肺癌细胞EGFR的表达同时增加RARα的表达。
Objective: to investigate the effects and mechanisms of ATPR on differentiation and proliferation of adenocarcinoma and squamous-celled carcinoma of the human lung cell lines.( A549 and L78).
Methods: Dividing A549 and L78 lung cancer cells into three groups. ATPR group, ATRA group and control group. In ATPR group ,cells were cultured with ATPR (0.01umol/L,0.1 umol/L ,1 umol/L,5 umol/L,10 umol/L) for 24h,48,72h,96h, The absorbance and growth inhibitory effect of ATPR on ( A549 and L78) was observed by MTT method. the cell cycle and was assayed by flow cytometry. cells were cultured with ATPR (1 umol/L,5 umol/L,10 umol/L) for 72h, HE coloretur and observe morphologic change of lung cancer cells. cells were cultured with ATPR with 1 umol/L for 72h, RT-PCR was used to analyze the expression changes of EGFR and RARα.
Results : ATPR obviously inhibited the proliferation of (A549 and L78) cells ,and the cell cycle was arrested in G0/G1 phase.We observed the specificity EGFR and RARαstrap,through detecting A549 and L78 cells . The expression of EGFR in L78 cell of control group was on a high level .After incubation with ATPR,the expression of EGFR mRNA in L78 cell started to decrease after 24h;The expression of EGFR o in A549 cell was on a low level . the expression of EGFR mRNA in A549 cell was decreased in a time dependent manner. the expression of RARαin A549 and L78 cells were on a low level and the expressions were increased in a time dependent manner.
Conclusion:1. ATPR minght inhibit proliferation and induce differentiation of (A549 and L78) .2. HE coloretur and observe morphologic change of lung cancer cells.3. EGFR and RARαmRNA were detected in A549 and L78 cells.
4. ATPR upregulated RARαmRNA expression and down-regulated EGFR mRNA expression in a time dependent manner.
方法:将两株细胞分为4-氨基-2-三氟甲基苯基维甲酸酯(ATPR)组、空白对照组及全反式维甲酸(ATRA)组.前组分别以0.01umol/L、0.1 umolL、1 umol/L、5 umol/L、10 umol/L的ATPR与肺腺癌细胞A549和鳞癌细胞L78作用24h,48h,72h,96h。运用MTT法检测细胞吸光度并计算细胞的生长抑制率,流式细胞仪检测细胞周期。0.1 umolL、1 umol/L、5 umol/L的ATPR与肺腺癌细胞A549和鳞癌细胞L78作用72h,苏木精—伊红(HE)染色观察肺癌细胞形态变化。1 umol/L的ATPR作用肺癌细胞72h后, RT-PCR方法检测细胞核维甲酸α受体(RARα)、表皮生长因子受体(EGFR)表达的变化。
结果:ATPR具有抑制A549、L78细胞生长的作用,生长抑制率有剂量和时间依赖关系。流式细胞仪分析结果显示A549、L78细胞被阻滞于G0—G1期。A549、L78细胞在ATPR的作用下,发生了细胞胞体变小、分裂状态的细胞数减少等形态学变化。通过对肺癌细胞的RT-PCR法检测,均发现EGFR和RARα的特异性条带。空白对照组L78细胞高表达EGFR;A549细胞低表达EGFR,EGFR的表达自24h开始逐渐下降并呈时间依赖。空白对照组L78细胞和A549细胞低表达RARα,RARα的mRNA表达逐渐增加,并呈时间依赖。
结论:1.从MTT结果证实不同浓度的ATPR能够抑制细胞的增值,随着浓度的增加细胞活力依赖性下降,即抑制率逐渐增加。
2.流式细胞仪结果示ATPR使细胞被阻滞于G0-G1期。
3.从细胞形态学观察结果证实ATPR使肺癌细胞出现明显的分化征象。
4.从mRNA水平证实肺癌细胞株A549、L78表达EGFR、RARα。
5. ATPR以时间依赖的方式下调肺癌细胞中EGFR的mRNA表达并上调RARα的mRNA表达。
综上所述,ATPR可明显抑制A549、L78细胞增值并诱导其分化以及显著降低肺癌细胞EGFR的表达同时增加RARα的表达。
Objective: to investigate the effects and mechanisms of ATPR on differentiation and proliferation of adenocarcinoma and squamous-celled carcinoma of the human lung cell lines.( A549 and L78).
Methods: Dividing A549 and L78 lung cancer cells into three groups. ATPR group, ATRA group and control group. In ATPR group ,cells were cultured with ATPR (0.01umol/L,0.1 umol/L ,1 umol/L,5 umol/L,10 umol/L) for 24h,48,72h,96h, The absorbance and growth inhibitory effect of ATPR on ( A549 and L78) was observed by MTT method. the cell cycle and was assayed by flow cytometry. cells were cultured with ATPR (1 umol/L,5 umol/L,10 umol/L) for 72h, HE coloretur and observe morphologic change of lung cancer cells. cells were cultured with ATPR with 1 umol/L for 72h, RT-PCR was used to analyze the expression changes of EGFR and RARα.
Results : ATPR obviously inhibited the proliferation of (A549 and L78) cells ,and the cell cycle was arrested in G0/G1 phase.We observed the specificity EGFR and RARαstrap,through detecting A549 and L78 cells . The expression of EGFR in L78 cell of control group was on a high level .After incubation with ATPR,the expression of EGFR mRNA in L78 cell started to decrease after 24h;The expression of EGFR o in A549 cell was on a low level . the expression of EGFR mRNA in A549 cell was decreased in a time dependent manner. the expression of RARαin A549 and L78 cells were on a low level and the expressions were increased in a time dependent manner.
Conclusion:1. ATPR minght inhibit proliferation and induce differentiation of (A549 and L78) .2. HE coloretur and observe morphologic change of lung cancer cells.3. EGFR and RARαmRNA were detected in A549 and L78 cells.
4. ATPR upregulated RARαmRNA expression and down-regulated EGFR mRNA expression in a time dependent manner.
引文
[1]赵先文,韩存芝,荆洁等.肺癌患者血清肿瘤标志物联合检测及5678[J].肿瘤研究与临床,2005,17(3):170- 172.
[2] Testi A M,Biondi A, Lo Coco F, et al GIMEMA-AIEOPAIDA protocol for the treatment of newly diagnosed acute promyelocytic len-kemia(APL)in children [J].Blood,2005,106(2):447-53.
[3] Chang J,Lin C, Smith D E,et al Restoration of retinoic acid concentration suppresse sethanol-enhanced expression and hepatocyte proliferation in rat liver[J].Carcinogenesis,2001,22(8):1213-9.
[4] Spaulding D C, Spaulding B O. Epidermal growth factor receptor expression and measurement in solid tumors[J]. Semin Oncol, 2002 Oct; 29(5 Suppl 14): 45- 54.
[5] Saski H, Yukiue H, Mizumo K, et al. Elevated serum epidermal growth factor receptor level is correlated with lymph node metastasis in lung cancer [J]. Int J Clin Oncol, 2003Apr; 8(2): 79- 82.
[6] Clared L E, Leitzel K, Smith J, et al. Epidermal growth factor receptor mRNA in peripheral blood of patients with pancreatic, lung,and colon carcinomas detected by RT- PCR[J]. Int J Oncol, 2003 Feb, 22(2):425- 430.
[7] Gonzalez- Larriba JL, Giaccone G, van Oosterom AT, et al. Combination therapy with gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, gemcitabine and cisplatin in patients with advanced solid tumors [J]. Ann Oncol, 2004 May, 15(5): 831-838.
[8] Fukuodk M, Yano S, Giaccone G, et al. Final results from a phase trial of ZD 1839(' lressa' )[J]. Proc Am Soc Clin Oncol, 2002, 21(11): 298a(All88).
[9] Dancey J E, Freidlin B. Targeting epidermal growth factor receptor are we missingthe mark?[J]. Lancet, 2003, 362(9377): 62- 64.
[10]李榕,韩宝惠。EGFR检测在肺癌中的临床意义[J].临床肿瘤学杂志, 2004,9(4):420- 424.
[11] Ohsaki Y, Tanno S , Fujita Y, et al . Epidermal growth factor receptor expression correlates with poor prognosis in non-small cell lung cancer patients with p53 overexpression[J]. Oncol Rep ,2000 ,7 (3) :603-607.
[12] Chandrika J , Piyat hilake CJ , Andra R , et al . Differential Expression of Growt h Factors in Squamous Cell Carcinoma and Pre-cancerous Lesions of t he lung[J]. Clin Cancer Res ,2002 ,8 (3) :734-744.
[13] Maxwell S A.Retinoblastoma protein in non-small lung carcinoma,cells arrested for growth by retinoic acid[J]. Anticancer Res,1994,14(4A):1535-1540.
[14] Polosa R , Prosperini G,Leir SH , et al . Expression of c-erbB receptor and ligands in human bronchial mucosa[J]. Am J Respir Cell Molbiol ,1999 ,20(5) :914-923.
[15] Nicholson RI , Gee JM , Harper ME. EGFR and cancer prognosis[J]. Eur J Cancer,2001 ,37 (4) :9-14.
[16] Brabender J , Danenberg K D , Metzger R , et al . Epidermal growt h factor receptor and HER2-neu mRNA expression in non-small cell lung cancer is correlated with survival[J] . Clin Cancer Res ,2001 ,7 (7) :1850-1855.
[17] Cox G,Jones JL ,Byrne KJ . Mat rixmetallo proteinase and t he epidermal growt h factor signal pat hway in operable non-small cell lung cancer[J] . Clin Cancer Res ,2000 ,6 (6) :2349-2355.
[18] Eccles SA. Cell biology of lymphatic metastasis. The potential role of cerb2B oncogene signaling[J]. Recent Result s Cancer Res ,2000 ,157 (10) :41-54.
[19] koo J S, Jetten A M,Kim Y D ,et al.Role of retinoid receptors in the regulation of mucin gene expression by retinoic acid in human tracheobronchial epithelial cells [J] Biochem J,1999(2):351-7.
[20] Chambon P .A decade of molecular biology of retinoic acid receptors [J]. FASEB J,1996,10(9):940-954.
[21] Wartz J M.A canonical structure for the ligand binding domain of nuclear receptors [J].Nat Structure Biol,1996,3(1):87-94.
[22] Naumorski L,Cleary ML,Michacl L.Bcl-2 inhibits apoptosis associated with terminal differentiation of HL-60 myeloid leukemia cells [J]. Bloods,1994,83(8):2261-2267.
[23] Bhatia U,Traganos F,Darzykiewicz Z. Induction of cell differentiation potintiates apoptosis triggered by prior exposure to DNA damaging drugs [J]. Cell Growth Differ,1995,6(8):937-944.
[1]王振义,陈竺.肿瘤的诱导分化和凋亡疗法【M】.上海:上海科学技术出版社.1998.65-66.
[2] Yumi W,Takeshi W,Masatoshi K,et al. pRb phosphorylation is regulated differentially by cyclin dependent kinase(cdk)2 and cdk4 in retinoic acid - induced neuronal differentiation of P19 cells[J]. Brain Res,1999,8(42):342 - 350.
[3] Brambilla E,Moro D,Gazzeri S,et al. Alterations of expression of Rb,P16(INK4A)and cyclin D1 in non - small cell lung carcinoma and their clinical significance[J]. J Pathol,1999,188(4):351 - 360.
[4] Malusecka E,Zborek A,Krzyzowska - Gruca S. Changes in expression of pRb,P16,and cyclin D1 in non - small cell lung cancer :an immunohistochemical study [J]. Folia Histochem Cytobiol,1999,37(1):19 - 24.
[5] Lonardo F,Dragnev KH,Freemantle SJ,et al. Evidence for the epidermal growth factor receptor as a target for lung cancer[J]. Clin Cancer Res. 2002 ,8(1):54-60.
[6]任杰桂,刘志方,丁磊等. 9 -顺-维甲酸对肺腺癌细胞cyclin D1 cdk4转录的影响[J].中国病理生理杂志, 2004,20(3):359– 362.
[7]游庆军,胡国强,于雪艳等. 9 -顺-维甲酸对肺鳞、腺癌细胞株c - erbB - 2和Rb表达的影响[J].山东医科大学学报,2000,38(4):339– 341.
[8]游庆军,沈振亚,金小寅等. 9-顺-维A酸诱导肺鳞、腺癌细胞株凋亡与CyclinD1和Rb基因表达的关系[J].中华实验外科杂志,2003,20(1):251-253.
[9] Panigone S,Debermardi S.pRb and Cdk regulation by N-(4-hydroxypheny)retinamide.On cogene,2000,19(35):4035-4041.
[10] Hayashi Y.Mechanism of tumor igenesis caused by tumor suppressor gene.Nipp on Rinsho,2000,58(6):1231-1236.
[11]于雪艳,田克立,张世国等.9一顺一维甲酸对人肺鳞癌细胞分化和凋亡的影响[J].山东医科大学学报,2001,39(4)289-291. [12 ] Sengupta S, Ray S, Chattopadhyay N, et al. Effect of retinoic acid on integrin receptors of B16F10 melanoma cells[J ]. J Exp Clin Cancer Res,2000,19(1):81-87. [13 ]张元雪,王萍,刘国柱.维甲酸联合化疗治疗晚期非小细胞肺癌的临床研究[J]。临沂医学专科学校学报.2003,25(5):341-343.
[14]黄贵华.维甲酸联合化疗治疗肺腺癌30例临床分析[J] . Journal of Practical Oncology ,2004, 19(6): 538-539.
[15] Zouboulis CC. Retinoids-which dermatological indicationsWill benefit in the near future?[ J].S kin Pharmacol ApplSkin Physiol, 2001,14(5):303-315.
[16] Ong C, Sullivan J, Hertzberg M, et al. Stage 1V CD30+anaplastic large cell lymphoma: response to acitretin andinterferon- alpha[ J]. AustralasJ Dermatol, 2002,43(3):207- 210 .
[2] Testi A M,Biondi A, Lo Coco F, et al GIMEMA-AIEOPAIDA protocol for the treatment of newly diagnosed acute promyelocytic len-kemia(APL)in children [J].Blood,2005,106(2):447-53.
[3] Chang J,Lin C, Smith D E,et al Restoration of retinoic acid concentration suppresse sethanol-enhanced expression and hepatocyte proliferation in rat liver[J].Carcinogenesis,2001,22(8):1213-9.
[4] Spaulding D C, Spaulding B O. Epidermal growth factor receptor expression and measurement in solid tumors[J]. Semin Oncol, 2002 Oct; 29(5 Suppl 14): 45- 54.
[5] Saski H, Yukiue H, Mizumo K, et al. Elevated serum epidermal growth factor receptor level is correlated with lymph node metastasis in lung cancer [J]. Int J Clin Oncol, 2003Apr; 8(2): 79- 82.
[6] Clared L E, Leitzel K, Smith J, et al. Epidermal growth factor receptor mRNA in peripheral blood of patients with pancreatic, lung,and colon carcinomas detected by RT- PCR[J]. Int J Oncol, 2003 Feb, 22(2):425- 430.
[7] Gonzalez- Larriba JL, Giaccone G, van Oosterom AT, et al. Combination therapy with gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, gemcitabine and cisplatin in patients with advanced solid tumors [J]. Ann Oncol, 2004 May, 15(5): 831-838.
[8] Fukuodk M, Yano S, Giaccone G, et al. Final results from a phase trial of ZD 1839(' lressa' )[J]. Proc Am Soc Clin Oncol, 2002, 21(11): 298a(All88).
[9] Dancey J E, Freidlin B. Targeting epidermal growth factor receptor are we missingthe mark?[J]. Lancet, 2003, 362(9377): 62- 64.
[10]李榕,韩宝惠。EGFR检测在肺癌中的临床意义[J].临床肿瘤学杂志, 2004,9(4):420- 424.
[11] Ohsaki Y, Tanno S , Fujita Y, et al . Epidermal growth factor receptor expression correlates with poor prognosis in non-small cell lung cancer patients with p53 overexpression[J]. Oncol Rep ,2000 ,7 (3) :603-607.
[12] Chandrika J , Piyat hilake CJ , Andra R , et al . Differential Expression of Growt h Factors in Squamous Cell Carcinoma and Pre-cancerous Lesions of t he lung[J]. Clin Cancer Res ,2002 ,8 (3) :734-744.
[13] Maxwell S A.Retinoblastoma protein in non-small lung carcinoma,cells arrested for growth by retinoic acid[J]. Anticancer Res,1994,14(4A):1535-1540.
[14] Polosa R , Prosperini G,Leir SH , et al . Expression of c-erbB receptor and ligands in human bronchial mucosa[J]. Am J Respir Cell Molbiol ,1999 ,20(5) :914-923.
[15] Nicholson RI , Gee JM , Harper ME. EGFR and cancer prognosis[J]. Eur J Cancer,2001 ,37 (4) :9-14.
[16] Brabender J , Danenberg K D , Metzger R , et al . Epidermal growt h factor receptor and HER2-neu mRNA expression in non-small cell lung cancer is correlated with survival[J] . Clin Cancer Res ,2001 ,7 (7) :1850-1855.
[17] Cox G,Jones JL ,Byrne KJ . Mat rixmetallo proteinase and t he epidermal growt h factor signal pat hway in operable non-small cell lung cancer[J] . Clin Cancer Res ,2000 ,6 (6) :2349-2355.
[18] Eccles SA. Cell biology of lymphatic metastasis. The potential role of cerb2B oncogene signaling[J]. Recent Result s Cancer Res ,2000 ,157 (10) :41-54.
[19] koo J S, Jetten A M,Kim Y D ,et al.Role of retinoid receptors in the regulation of mucin gene expression by retinoic acid in human tracheobronchial epithelial cells [J] Biochem J,1999(2):351-7.
[20] Chambon P .A decade of molecular biology of retinoic acid receptors [J]. FASEB J,1996,10(9):940-954.
[21] Wartz J M.A canonical structure for the ligand binding domain of nuclear receptors [J].Nat Structure Biol,1996,3(1):87-94.
[22] Naumorski L,Cleary ML,Michacl L.Bcl-2 inhibits apoptosis associated with terminal differentiation of HL-60 myeloid leukemia cells [J]. Bloods,1994,83(8):2261-2267.
[23] Bhatia U,Traganos F,Darzykiewicz Z. Induction of cell differentiation potintiates apoptosis triggered by prior exposure to DNA damaging drugs [J]. Cell Growth Differ,1995,6(8):937-944.
[1]王振义,陈竺.肿瘤的诱导分化和凋亡疗法【M】.上海:上海科学技术出版社.1998.65-66.
[2] Yumi W,Takeshi W,Masatoshi K,et al. pRb phosphorylation is regulated differentially by cyclin dependent kinase(cdk)2 and cdk4 in retinoic acid - induced neuronal differentiation of P19 cells[J]. Brain Res,1999,8(42):342 - 350.
[3] Brambilla E,Moro D,Gazzeri S,et al. Alterations of expression of Rb,P16(INK4A)and cyclin D1 in non - small cell lung carcinoma and their clinical significance[J]. J Pathol,1999,188(4):351 - 360.
[4] Malusecka E,Zborek A,Krzyzowska - Gruca S. Changes in expression of pRb,P16,and cyclin D1 in non - small cell lung cancer :an immunohistochemical study [J]. Folia Histochem Cytobiol,1999,37(1):19 - 24.
[5] Lonardo F,Dragnev KH,Freemantle SJ,et al. Evidence for the epidermal growth factor receptor as a target for lung cancer[J]. Clin Cancer Res. 2002 ,8(1):54-60.
[6]任杰桂,刘志方,丁磊等. 9 -顺-维甲酸对肺腺癌细胞cyclin D1 cdk4转录的影响[J].中国病理生理杂志, 2004,20(3):359– 362.
[7]游庆军,胡国强,于雪艳等. 9 -顺-维甲酸对肺鳞、腺癌细胞株c - erbB - 2和Rb表达的影响[J].山东医科大学学报,2000,38(4):339– 341.
[8]游庆军,沈振亚,金小寅等. 9-顺-维A酸诱导肺鳞、腺癌细胞株凋亡与CyclinD1和Rb基因表达的关系[J].中华实验外科杂志,2003,20(1):251-253.
[9] Panigone S,Debermardi S.pRb and Cdk regulation by N-(4-hydroxypheny)retinamide.On cogene,2000,19(35):4035-4041.
[10] Hayashi Y.Mechanism of tumor igenesis caused by tumor suppressor gene.Nipp on Rinsho,2000,58(6):1231-1236.
[11]于雪艳,田克立,张世国等.9一顺一维甲酸对人肺鳞癌细胞分化和凋亡的影响[J].山东医科大学学报,2001,39(4)289-291. [12 ] Sengupta S, Ray S, Chattopadhyay N, et al. Effect of retinoic acid on integrin receptors of B16F10 melanoma cells[J ]. J Exp Clin Cancer Res,2000,19(1):81-87. [13 ]张元雪,王萍,刘国柱.维甲酸联合化疗治疗晚期非小细胞肺癌的临床研究[J]。临沂医学专科学校学报.2003,25(5):341-343.
[14]黄贵华.维甲酸联合化疗治疗肺腺癌30例临床分析[J] . Journal of Practical Oncology ,2004, 19(6): 538-539.
[15] Zouboulis CC. Retinoids-which dermatological indicationsWill benefit in the near future?[ J].S kin Pharmacol ApplSkin Physiol, 2001,14(5):303-315.
[16] Ong C, Sullivan J, Hertzberg M, et al. Stage 1V CD30+anaplastic large cell lymphoma: response to acitretin andinterferon- alpha[ J]. AustralasJ Dermatol, 2002,43(3):207- 210 .