Genistein inhibits cell invasion and motility by inducing cell differentiation in murine osteosarcoma cell line LM8

BMC Cell Biology

Hiroshi Masuno¹  Hiromasa Miura²  Yoshiaki Norimatsu¹  Tomoyo Takata¹  Teruki Kidani²  Kenshi Sakayama³  Junichi Aizawa²  Atsushi Nakamura¹ 

[1] Department of Medical Technology, Faculty of Health Sciences, Ehime Prefectural University of Health Sciences, Takooda, Tobe-cho, Iyo-gun, Ehime, 791-2101, Japan;Department of Bone and Joint Surgery, Ehime University Graduate School of Medicine, Toon, Ehime, 791-0295, Japan;Musculoskeletal Tumor Surgery Center, Minami Matsuyama Hospital, Matsuyama, Ehime, 790-8534, Japan

关键词: Cell differentiation;    β-catenin;    Matrix metalloproteinase-2;    Cell invasion;    LM8;    Genistein;   

Others  :  856760
DOI  :  10.1186/1471-2121-13-24

 received in 2012-06-13, accepted in 2012-09-20,  发布年份 2012

【 摘 要 】

Background

One of the problems associated with osteosarcoma is the frequent formation of micrometastases in the lung prior to diagnosis because the development of metastatic lesions often causes a fatal outcome. Therefore, the prevention of pulmonary metastases during the early stage of tumor development is critical for the improvement of the prognosis of osteosarcoma patients. In Japan, soy is consumed in a wide variety of forms, such as miso soup and soy sauce. The purpose of this study is to investigate the effect of genistein, an isoflavone found in soy, on the invasive and motile potential of osteosarcoma cells.

Methods

LM8 cells were treated for 3 days with various concentrations of genistein. The effect of genistein on cell proliferation was determined by DNA measurement in the cultures and 5-bromo-2’-deoxyuridine (BrdU) incorporation study. The assays of cell invasion and motility were performed using the cell culture inserts with either matrigel-coated membranes or uncoated membranes in the invasion chambers. The expression and secretion of MMP-2 were determined by immunohistochemistry and gelatin zymography. The subcellular localization and cellular level of β-catenin were determined by immunofluorescence and Western blot. For examining cell morphology, the ethanol-fixed cells were stained with hematoxylin-eosin (H&E). The expressionof osteocalcin mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR).

Results

Genistein dose-dependently inhibits cell proliferation. Genistein-treated cells were less invasive and less motile than untreated cells. The expression and secretion of MMP-2 were lower in the genistein-treated cultures than in the untreated cultures. β-Catenin in untreated cells was located in the cytoplasm and/or nucleus, while in genistein-treated cells it was translocated near to the plasma membrane. The level of β-catenin was higher in genistein-treated cells than in untreated cells. Treatment of LM8 cells with genistein induced morphological changes, markedly decreased the formation of multilayer masses of cells, and markedly increased the expression of osteocalcin mRNA.

Conclusions

Genistein decreased invasive and motile potential by inducing cell differentiation in LM8 cells. Genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects.

【 授权许可】

   
2012 Nakamura et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

【 参考文献 】

• [1]Dome JS, Schwartz CL: Osteosarcoma. Cancer Treat Res 1997, 92:215-251.
• [2]Bacci G, Longhi A, Versari M, Mercuri M, Briccoli A, Picci P: Prognostic factors for osteosarcoma of the extremity treated with neoadjuvant chemotherapy: 15-year experience in 789 patients treated at a single institution. Cancer 2006, 106:1154-1161.
• [3]Ferguson WS, Goorin AM: Current treatment of osteosarcoma. Cancer Invest 2001, 19:292-315.
• [4]Saeter G, Høie J, Stenwig AE, Johansson AK, Hannisdal E, Solheim OP: Systemic relapse of patients with osteogenic sarcoma: Prognostic factors for long term survival. Cancer 1995, 75:1084-1093.
• [5]Bacci G, Briccoli A, Ferrari S, Saeter G, Donati D, Longhi A, Manfrini M, Bertoni F, Rimondini S, Monti C, Forni C: Neoadjuvant chemotherapy for osteosarcoma of the extremities with synchronous lung metastases: treatment with cisplatin, adriamycin and high dose of methotrexate and ifosfamide. Oncol Rep 2000, 7:339-346.
• [6]Asai T, Ueda T, Itoh K, Yoshioka K, Aoki Y, Mori S, Yoshikawa H: Establishment and characterization of a murine osteosarcoma cell line (LM8) with high metastatic potential to the lung. Int J Cancer 1998, 76:418-422.
• [7]Kamei S, Sakayama K, Tamashiro S, Aizawa J, Miyawaki J, Miyazaki T, Yamamoto H, Norimatsu Y, Masuno H: Ketoprofen in topical formulation decreases the matrix metalloproteinase-2 expression and pulmonary metastatic incidence in nude mice with osteosarcoma. J Orthop Res 2009, 27:909-915.
• [8]Aizawa J, Sakayama K, Kamei S, Kidani T, Yamamoto H, Norimatsu Y, Masuno H: Effect of troglitazone on tumor growth and pulmonary metastasis development of the mouse osteosarcoma cell line LM8. BMC Cancer 2010, 10:51. BioMed Central Full Text
• [9]Stetler-Stevenson WG, Aznavoorian S, Liotta LA: Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu Rev Cell Biol 1993, 9:541-573.
• [10]Kaya M, Wada T, Akatsuka T, Kawaguchi S, Nagoya S, Shindoh M, Higashino F, Mezawa F, Okada F, Ishii S: Vascular endothelial growth factor expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis. Clin Cancer Res 2000, 6:572-577.
• [11]Yamamoto S, Sobue T, Kobayashi M, Sasaki S, Tsugane S: Soy, isoflavones, and breast cancer risk in Japan. J Natl Cancer Inst 2003, 95:906-913.
• [12]Record IR, Broadbent JL, King RA, Dreosti IE, Head RJ, Tonkin AL: Genistein inhibits growth of B16 melanoma cells in vivo and in vitro and promotes differentiation in vitro. Int J Cancer 1997, 72:860-864.
• [13]Morris C, Thorpe J, Ambrosio L, Santin M: The soybean isoflavone genistein induces differentiation of MG63 human osteosarcoma osteoblasts. J Nutr 2006, 136:1166-1170.
• [14]Singh AV, Franke AA, Blackburn GL, Zhou J-R: Soy phytochemicals prevent orthotopic growth and metastasis of bladder cancer in mice by alterations of cancer cell proliferation and apoptosis and tumor angiogenesis. Cancer Res 2006, 66:1851-1858.
• [15]Lin S-Y, Xia W, Wang JC, Kwong KY, Spohn B, Wen Y, Pestell RG, Hung MC: β-Catenin, a novel prognostic marker for breast cancer: Its roles in cyclin D1 expression and cancer progression. Proc Natl Acad Sci USA 2000, 97:4262-4266.
• [16]Iwaya K, Ogawa H, Kuroda M, Izumi M, Ishida T, Mukai K: Cytoplasmic and/or nuclear staining of beta-catenin is associated with lung metastasis. Clin Exp Metastasis 2003, 20:525-529.
• [17]Yoshizumi T, Ohta T, Ninomiya I, Terada I, Fushida S, Fujimura T, Nishimura G, Shimizu K, Yi S, Miwa K: Thiazolidinedione, a peroxisome proliferator-activated receptor-γ ligand, inhibits growth and metastasis of HT-29 human colon cancer cells through differentiation-promoting effects. Int J Oncol 2004, 25:631-639.
• [18]Aronow MA, Gerstenfeld LC, Owen TA, Tassinari MS, Stein GS, Lian JB: Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells. J Cell Physiol 1990, 143:213-221.
• [19]van Belzen N, Dinjens WN, Diesveld MP, Groen NA, van der Made AC, Nozawa Y, Vlietstra R, Trapman J, Bosman FT: A novel gene which is up-regulated during colon epithelial cell differentiation and down-regulated in colorectal neoplasms. Lab Invest 1997, 77:85-92.
• [20]Harmon AW, Harp JB: Differential effects of flavonoids on 3T3-L1 adipogenesis and lipolysis. Am J Physiol Cell Physiol 2001, 280:C807-C813.
• [21]Sandoval MJ, Cutini PH, Rauschemberger MB, Massheimer VL: The soyabean isoflavone genistein modulates endothelial cell behaviour. Br J Nutr 2010, 104:171-179.
• [22]Kashima T, Kawaguchi J, Takeshita S, Kuroda M, Takanashi M, Horiuchi H, Imamura T, Ishikawa Y, Ishida T, Mori S, Machinami R, Kudo A: Anomalous cadherin expression in osteosarcoma. Possible relationships to metastasis and morphogenesis. Am J Pathol 1999, 155:1549-1555.
• [23]Hugh TJ, Dillon SA, O’Dowd G, Getty B, Pignatelli M, Poston GJ, Kinsella AR: β-Catenin expression in primary and metastatic colorectal carcinoma. Int J Cancer 1999, 82:504-511.
• [24]Su Y, Simmen RCM: Soy isoflavone genistein upregulates epithelial adhesion molecule E-cadherin expression and attenuates β-catenin signaling in mammary epithelial cells. Carcinogenesis 2009, 30:331-339.
• [25]Su Y, Simmen FA, Xiao R, Simmen RCM: Expression profiling of rat mammary epithelial cells reveals candidate signaling pathways in dietary protection from mammary tumors. Physiol Genomics 2007, 30:8-16.
• [26]He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW: Identification of c-MYC as a target of the APC pathway. Science 1998, 281:1509-1512.
• [27]Miyazawa K, Iwaya K, Kuroda M, Harada M, Serizawa H, Koyanagi Y, Sato Y, Mizokami Y, Matsuoka T, Mukai K: Nuclear accumulation of beta-catenin in intestinal-type gastric carcinoma: correlation with early tumor invasion. Virchows Arch 2000, 437:508-513.
• [28]Abercrombie M: Contact inhibition and malignancy. Nature 1979, 281:259-262.
• [29]Kurdistani SK, Arizti P, Reimer CL, Sugrue MM, Aaronson SA, Lee SW: Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage. Cancer Res 1998, 58:4439-4444.
• [30]Guan RJ, Ford HL, Fu Y, Li Y, Shaw LM, Pardee AB: Drg-1 as a differentiation-related, putative metastatic suppressor gene in human colon cancer. Cancer Res 2000, 60:749-755.
• [31]Chua M-S, Sun H, Cheung ST, Mason V, Higgins J, Ross DT, Fan ST, So S: Overexpression of NDRG1 is an indicator of poor prognosis in hepatocellular carcinoma. Mod Pathol 2007, 20:76-83.
• [32]Bandyopadhyay S, Wang Y, Zhan R, Pai SK, Watabe M, Iiizumi M, Furuta E, Mohinta S, Liu W, Hirota S, Hosobe S, Tsukada T, Miura K, Takano Y, Saito K, Commes T, Piquemal D, Hai T, Watabe K: The tumor metastasis suppressor gene Drg-1 down-regulates the expression of activating transcription factor 3 in prostate cancer. Cancer Res 2006, 66:11983-11990.
• [33]Matsugaki T, Zenmyo M, Hiraoka K, Fukushima N, Shoda T, Komiya S, Ono M, Kuwano M, Nagata K: N-myc downstream-regulated gene 1/Cap43 expression promotes cell differentiation of human osteosarcoma cells. Oncol Rep 2010, 24:721-725.
• [34]Wu AH, Wan P, Hankin J, Tseng C-C, Yu MC, Pike MC: Adolescent and adult soy intake and risk of breast cancer in Asian-Americans. Carcinogenesis 2002, 23:1491-1496.
• [35]Horn-Ross PL, John EM, Lee M, Stewart SL, Koo J, Sakoda LC, Shiau AC, Goldstein J, Davis P, Perez-Stable EJ: Phytoestrogen consumption and breast cancer risk in a multiethnic population: The Bay Area breast cancer study. Am J Epidemiol 2001, 154:434-441.
• [36]Lakshman M, Xu L, Ananthanarayanan V, Cooper J, Takimoto CH, Helenowski I, Pelling JC, Bergan RC: Dietary genistein inhibits metastasis of human prostate cancer in mice. Cancer Res 2008, 68:2024-2032.
• [37]Hinegardner RT: An improved fluorometric assay for DNA. Anal Biochem 1971, 39:197-201.
• [38]Zamurovic N, Cappellen D, Rohner D, Susa M: Coordinated activation of Notch, Wnt, and transforming growth factor-β signaling pathways in bone morphogenic protein 2-induced osteogenesis. J Biol Chem 2004, 279:37704-37715.
• [39]Song C, Guo Z, Ma Q, Chen Z, Liu Z, Jia H, Dang G: Simvastatin induces osteoblastic differentiationand inhibits adipocytic differentiationin mouse bone marrowstromal cells. Biochem Biophys Res Commun 2003, 308:458-462.