摘要
肺癌是当前世界上严重危害人类健康的恶性肿瘤之一,其发病率及死亡率很高,目前尚缺乏有效的治疗方法。近年国内外大量研究结果显示,他汀类药物具有多向性效应,除了降低血脂作用外,对多种肿瘤具有抑制细胞增殖、诱导细胞分化或凋亡、抑制血管生成、降低肿瘤细胞侵袭转移能力等作用,很可能成为一种具有应用前景的抗肿瘤药物。然而,有关氟伐他汀对小鼠Lewis肺癌抑制作用及其机制的研究尚未见报道。本研究应用移植性小鼠Lewis肺癌模型给予氟伐他汀处理后,观察对移植性肿瘤生长和肺转移的抑制作用;在体外观察氟伐他汀对小鼠Lewis肺癌细胞增殖和凋亡的影响。
实验结果表明,氟伐他汀对小鼠Lewis肺癌移植瘤的生长和肺转移有明显抑制作用,且呈剂量和时间依赖方式;氟伐他汀与环磷酰胺联合应用对Lewis肺癌移植瘤生长和肺转移的抑制效果,明显优于单独应用氟伐他汀或环磷酰胺。体外研究结果表明,氟伐他汀呈剂量和时间依赖方式抑制Lewis肺癌细胞增殖;在体外氟伐他汀能诱导Lewis肺癌细胞凋亡,这种诱导细胞凋亡的作用是通过甲羟戊酸途径介导的。
Lung carcinoma is one of the most common malignant tumors which severely harm the mankind health, both morbidity and mortality are high, and the effective therapeutic modalities remain insufficient.
Statins, 3-hydroxy-3-methylgluyaryl coenzyme A reductase inhibitors, are the most effective medication for lowering cholesterol, cardiovascular morbidity and mortality. Fluvastatin is one of statins. In the recent years, accumulating evidence suggests that statins have pleiotropic effects beyond reducing cholesterol synthesis. Statins have been shown to inhibit proliferation, induce differentiation and apoptosis, reduce angiogenesis and invasion capability in several tumor cells, whereas have no side effects on the normal cells, suggesting their potent applications as an antitumor reagent. The antitumor potential of statins and its mechanism have become one of research hot spots nowadays. However, the inhibitory effects of fluvastatin on the growth and apoptosis of murine Lewis lung carcinoma and their mechanisms have not been reported yet.
In the present work, the effect of Flu on the inhibition of tumor growth and pulmonary metastasis in a Lewis lung carcinoma murine model was studied. After Lewis lung carcinoma-bearing mice were administrated Flu at different doses for 15 days, the volume and mass weight of implanted tumors were measured, tumor weight inhibitory rates were calculated, lung weight and pulmonary metastasis nodules were counted, the inhibitory rates of pulmonary metastasis were calculated. The techniques of cytology, cytochemistry and flow cytometry were used to investigate the effects of fluvastatin on proliferation and apoptosis of Lewis lung carcinoma cells.
The main results of this study are as follows:
1. Flu showed significantly inhibitory effects on the growth of implanted tumors of Lewis lung carcinoma in mice. The inhibitory effects were increased with elevating dose of fluvastatin and extending action time, indicating a dose- and time-dependent manner, and the action of the high dose of fluvastatin was most obvious. As compared with treatment with fluvastatin or cyclophosphamide alone, combined treatment with both fluvastatin and cyclophosphamide resulted in more significant growth inhibition on implanted tumors.
2. The results revealed that fluvastatin exerted suppressive action on pulmonary metastasis of Lewis lung carcinoma model. As compared with control, the lung weight and pulmonary metastatic foci were obvioursly decreased, and inhibitory rates of pulmonary metastasis were markedly elevated in the groups at high and middling doses of fluvastatin, CYC and Flu+CYC. The inhibitory effects of combined treatment with both fluvastatin and cyclophosphamide were more significant.
3. After Lewis lung carcinoma cells were treated with 1,5, 10 and 20μmol/L of fluvastatin for 1, 2, 3, and 4 days in vitro, the cell growth activity was significantly suppressed. The inhibitory effects were increased with elevating concentration of fluvastatin and extending treated periods, suggesting a concentration- and time-dependent manner.
4. The results of CCK-8 assay indicated that Flu showed significantly suppressive effect on the proliferation of Lewis lung carcinoma cells. In the same time-point of treatment, the cell proliferation inhibitory rate was elevated with increasing concentrations of fluvastatin, displaying a concentration-dependent manner, among them, the effects were more obvious at 10 and 20μmol/L of fluvastatin. In the same concentration of fluvastatin, the cell proliferation inhibitory rate was elevated with periods treated with fluvastatin, indicating a time-dependent manner, the inhibitory effects were more potent in fluvastatin-treated for 48 and 72 hours.
5. Flow cytometry assays with Annexin V-FITC/PI double staining exhibited that compared with control, the apoptotic cells were increased after Lewis lung cancer cells were exposed to 20μmol/L of fluvastatin for 48 h (4.37% vs 12.83%, P<0.01), indicating fluvastatin induced apoptosis of Lewis lung carcinoma cells. Fluvastatin-induced cell apoptosis was reversed by coexposure to both fluvastatin and mevalonate (4.37% vs 5.89%, P>0.05), suggesting that the apoptosis inducing effect of fluvastatin was mediated through mevalonate pathway.
6. Electron microscopic study exhibited that after Lewis lung carcinoma cells were treated with fluvastatin (10μmol/L), the diminished cell body, condensed cytoplasm, dilated endoplasmic reticulum and vacuolization, as well as nuclear chromatin condensation with lumping and aggregating under the nuclear membrane and the apoptotic body were observed, showing the morphological changes of fluvastatin-induced apoptosis.
The conclusions of this study are as follows:
1. Fluvastatin exhibits significantly inhibitory effects on the growth and pulmonary metastasis of implanted tumors of Lewis lung carcinoma in mice in a dose- and time-dependent manner.
2. The combined treatment with both fluvastatin and cyclophosphamide can produce more significant inhibitory effects on growth and pulmonary metastasis of implanted tumors as compared with treatment with fluvastatin or cyclophosphamide alone.
3. Fluvastatin significantly inhibits proliferation of Lewis lung carcinoma cells in vitro, and the inhibitory effects are increased with elevating concentrations of fluvastatin and treated periods, suggesting a concentration- and time-dependent manner.
4. Fluvastatin treatment is able to induce apoptosis of Lewis lung carcinoma cells in vitro, the apoptosis inducing effect of fluvastatin was mediated through mevalonate pathway.
The creative points of the present study are: it is demonstrated that fluvastatin can suppress the growth and pulmonary metastasis of implanted tumors of Lewis lung carcinoma in mice, the combined treatment with both fluvastatin and cyclophosphamide can produce more significant inhibitory effects; fluvastatin inhibits proliferation of Lewis lung carcinoma cells in vitro in a concentration- and time-dependent manner, and induces apoptosis of Lewis lung carcinoma cells.
引文
1.Ferlay J,Bray F,Pisani P,et al.GLOBOCAN 2002:Cancer incidence,mortality and prevalence worldwide,version 2.0.IARC Cancer Base No.5 Lyon:IARC Press[M],2004.
2.Jakobisiak M,Golab J.Potential antitumor effects of statins[J].Int J Oncol,2003,23(4):1055-1065.
3.Oliveira KA,Zecchin KG,Alberici LC,et al.Simvastatin inducing PC3 prostate cancer cell necrosis mediated by calcineurin and mitochondrial dysfunction[J].J Bioenerg Biomembr,2008,40(4):307-314.
4.Kotamraju S,Willams CL,Kalyanaraman B.Statin-induced breast cancer cell death:role of inducible nitric oxide and arginase-dependent pathways[J].Cancer Res,2007,67(15):7386-7394.
5.Baulch-Brown C,Molloy TJ,Yeh SL,et al.Inhibitors of the mevalonate pathway as potential therapeutic agents in multiple myeloma[J].Leuk Res,2007,31(3):341-352.
6.Jones P,Kafonek S,Laurora I,et al.Comparative dose efficacy study of atorvastatin versus simvastatin,pravastatin,and fluvastatin in patients with hypercholesterolemia(the CURVES study)[J].Am J Cardiol,1998,81(5):582-587.
7.王怀良,常天辉.动脉粥样硬化的药物治疗[M].见:王怀良主编.临床药理学.北京:高等教育出版社,2004,277-287.
8.Calaro P,Yeh TH.The pleiotropic effects of statins[J].Curr Opin Cardiol,2005,20(6):541-546.
9.Amaud C,Burger F,Steffens S,e al.Statins reduce interleukin-6 induced C-reactive protein in human hepatocyces:new evidence for direct antiinflammatory effects of statins[J].Arterioscler Thromb Vasc Biol,2005,25(26):1231-12236.
10.Kataoka C,Egashira K,Inous S,et al.Important role of Rho kinase in the pathogenesis of cardiovascular inflammation and remodeling induced by long-term blockade of nitric oxide synthesis in rats[J].Hypertension,2002,39(2):245-250.
11.刘永铭,严祥,张钲,等.他汀类调脂药物对高脂血症患者黏附分子的影响[J].中华心血管病杂志,2001,29(3):140-143.
12.Halcox JP,Deanfield JE.Beyond the laboratory:clinical implications for statin pleiotropy[J].Circulation,2004,109(Suppl.Ⅱ):Ⅱ42-Ⅱ48.
13.Sever PS,Dahl(o|¨)f B,Poulter NR,et al.Prevention of coronary and stroke events with atorvastatin in hypertensive patients who have average or lower-than-average cholesterol concentrations,in the Anglo-Scandinavian Cardiac Outcome Trial-Lipid Lowering Arm(ASCOT-LLA):a multicenter randomized controlled trial[J].Lancet,2003,361(9364):1149-1158.
14.Hindler K,Cleeland CS,Rivera E,et al.The role of statins in cancer therapy[J].The Oncologist,2006,11(3):306-315.
15.Graaf MR,Richel DJ,van Noorden CJ,et al.Effects of statins and farnesyltransferase inhibitors on the development and progression of cancer[J].Cancer Treat Rev,2004,30(32):609-641.
16.Mo H,Elson CE.Studies of the isoprenoid-mediated inhibition of mevalonate synthesis applied to cancer chemotherapy and chemoprevention[J].Exp Biol Med,2004,229(6):567-585.
17.周永,麇漫天,朱俊东,等.洛伐他汀对MCF-7细胞增殖分化功能及间隙连接细胞通讯的影响[J].癌症,2003,22(3):257-261.
18.Florio T,Thellung S,Arena S,et al.Somatostatin and its analog lanreotide inhibit the proliferation of dispersed human non-functioning pituitary adenoma cells in vitro[J].Eur J Endocrinol,1999,141(4):396-408.
19.Holstein SA,Hohl RJ.Interaction of cytosine arabinoside and lovastatin in haman leukemia cells[J].Leuk Res,2001,25(8):651-660.
20.Zuckerbraum BS,Shapiro RA,Billiar TR,et al.Rho A influences the nuclear localization of extracellular signal-regulated kinases to modulate p21Waf/Cipl expression[J]. Circulation, 2003, 108(7): 876-881.
21. Liao JK, Lanfs U. Pleiotropic effects of statins[J]. Annu Rev Pharmacol Toxicol, 2005, 45(12): 89-118.
22. Xia Z, Tan MM, Wong WW, et al. Blocking protein geranylgeranylation is essential for lovastatin-induced apoptosis of human acute myeloid leukemia cells[J]. Leukemia, 2001, 15(4): 1398-1407.
23. Blaneo-Colio LM, Justo P, Daehn I, et al. Bcl-xL overexpression protects from apoptosis induced by HMG-CoA reductase inhibitors in murine tubular cells[J]. Kidney Int, 2003, 64(2): 181-191.
24. Lin R, Liu J, Peng N, et al. Lovastatin reduces nuclear factor kappa B activation induced by C-reactive protein in human vascular endothelial cells[J]. Biol Pharm Bull, 2005, 28(9): 1630-1634.
25. Gnad R, Aktories K, Kaina B, et al. Inhibition of protein isoprenylation impairs Rho-regulated early cellular response to genotoxic stress[J]. Mol Pharmacol, 2000, 58(6): 1389-1397.
26. Fromigue O, Hamidouche Marie PJ. Blockade of the Rho-JNK-C-Jun- MMP2 cascade by atorvastatin reduces osteosarcoma cell invasion[J]. J Biol Chem, 2008, 283(45): 30549-30556.
27. Shannon J, Tewoderos S, Grazotto M, et al.Statins and prostate cancer risk: a case-control study[J]. Am J Epidemiol, 2005,162(4): 318-325.
28. van der Spek E, Bloem AC, Lokhorst HM, et al. Inhibition of the mevalonate pathway potentiates the effect of lenalidomide in myeloma[J]. Leuk Res, 2009, 33(1): 100-108.
29. Sassano A, Katsoulidis E, Antico G, et al. Suppressive effects of statins on acute promyelocytic leukemia cells[J]. Cancer Re, 2007, 67(9): 4524-4532.
30. Katz MS. Therapy insight: potential of statins for cancer chemoprevention and therapy[J]. Nat Clin Pract Oncol, 2005, 2(2): 82-89.
31. Poynter JN, Gruber SB, Higgins PD, et al. Statins and the risk of colorectal cancer[J]. N Engl J Med, 2005, 352(21): 2184-2192
32. Platz EA, Leitzmann MF, Visvanathan K, Statin drugs and risk of advanced prostate cancer[J]. J Natl Cancer Inst, 2006, 98(24): 1819-1825.
33 . Bonovas S, Filioussi K, Tsavaris N, et al. Statins and cancer risk: a literature-based meta-analysis and meta-regression analysis of 35 randomized controlled trials[J]. J Clin Oncol, 2006, 24(30): 4808-4817.
34. Dale KM, Coleman CI, Henyan NN, et al. Statins and cancer risk: a meta -analysis[J]. JAMA, 2006, 295(1): 74-80.
35. Browning DR, Martin RM. Statins and risk of cancer: a systematic review and metaanalysis[J]. Int J Cancer, 2007, 120(4): 833-843.
36. Farwell WR, Scranton RE, Lawler EV, et al. The association between statins and cancer incidence in a veterans population[J]. J Natl Cancer Inst, 2008, 100(2): 134-139.
37. Karp I, Behlouli H, Lelorier J, et al. Statins and cancer risk[J]. Am J Med, 2008, 121(4): 302-309.
38. Paragh G, Kertai P, Kovacs P, et al. HMG CoA reductase inhibitor fluvastatin arrests the development of implanted hepatocarcinoma in rats[J]. Anticancer Res, 2003, 23(5A): 3949-3954.
39. Fujiwara K, Tsubaki M, Yamazoe Y, et al. Fluvastatin induces apoptosis on human tongue carcinoma cell line HSC-3[J]. Yakugaku Zasshi, 2008, 128(1): 153-158.
40. Budman DR, Tai J, Calabro A. Fluvastatin enhancement of trastuzumab and classical cytotoxic agents in defined breast cancer cell lines in vitro[J]. Breast Cancer Res Treat, 2007, 104(1): 93-101.
41. Issat T, Nowis D, Legat M, et al.Potentiated antitumor effects of the combination treatment with statins and pamidronate in vitro and in vivo[J]. Int J Oncol, 2007, 30(6): 1413-1425.
42. .van der Weide K, de Jonge-Peeters SD, Kuipers F, et al. Combining simvastatin with the famesyl transferase inhibitor tipifarnib results in an enhanced cytotoxic effect i a subset of primary CD34~+ acute myeloid leukemia samples[J]. Clin Cancer Res, 2009, 15(9): 3076-3083.
43.Wali VB, Bachawal SV, Sylvester PW. Suppression in mevalonate synthesis mediates antitumor effects of combined statin and gamm-tocotrienol treatment[J].Lipids, Epub 2009 Sep 24.
44. Horiguchi A, Sumitomo M, Asakuma J, et al. 3-hydroxy-3- methylglutaryl -coenzyme a reductase inhibitor, fluvastatin, as a novel agent for prophylaxis of renal cancer metastasis[J]. Clin Cancer Res, 2004, 10(24): 8648-8655.
44. Kamat AM, Nelkin GM. Atorvastatin: a potential chemopreventive agent in bladder cancer[J]. Urology, 2005,6,6(6): 1209-1212.
46. Saito A, Saito N, Mol W, et al. Simvastatin inhibits growth via apoptosis and the induction of cell cycle arrest in human melanoma cells[J]. Melanoma Res, 2008,18(2): 85-94.
47. Zhang G, Gurtu V, Kain SR, et al. Early detection of apoptosis using a fluorescent conjugate of annexin V[J]. Biotechques, 1997, 23(3): 525-531.
48. Cafforio P, Dammacco F, Gernone A, et al. Statins activate the mitochondrial pathway of apoptosis in human lymphoblasts and myeloma cells[J]. Carcinogenesis, 2005, 26(5): 883-891.
49. Hoque A, Chen H, Xu XC. Statin induces apoptosis and cell growth arrest in prostate cancer cells[J]. Cancer Epidemiol Biomarkers Prev, 2008, 17(1): 88-94.
50. Buhaescu I, Izzedine H. Mevalonate pathway: a review of clinical and therapeutical implications[J]. Clin Biochem, 2007,40(9-10): 575-584.
51. Cordle A, Koenigsknecht-Talboo J, Wilkinson B, et al. Mechanisms of statin-mediated inhibition of small G-protein function[J]. J Biol Chem, 2005,280(40): 34202-34209.
52 . Swanson KM, Hohl RJ. Anti-cancer therapy: targeting the mevalonate pathway[J]. Curr Cancer Drug Targets, 2006, 6(1): 15-37.
