Expressions of Sphingosine-1-phosphate (S1P) Receptors, Sphingosine Kinases in Malignant Bone and Soft Tissue Tumors, and The role of Sphingosine Kinase-1 in Growth of MFH Cell Lines

Shin-ichiro Kishimoto, Toshihiro Akisue, Kenta Kishimoto, Hitomi Hara, Masaya Imabori, Yoshiyuki Okada, Naomasa Fukase, Teruya Kawamoto, Ikuo Fujita, Takuya Fujimoto, Masahiro Kurosaka

.
Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kusunoki-cho, Chuo-ku, Kobe, JAPAN;.

DOI: 10.4236/jct.2011.22038                

Abstract

Sphingolipids are ubiquitous components of cell membranes. Their metabolites ceramide, sphingosine, and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. S1P is generated by phosphorylation of sphingosine catalyzed by sphingosine kinase-1 (SPHK1). The purpose of this study is to explore the roles of S1P, S1P receptors, and sphingosine kinases in malignant musculoskeletal tumors. Twenty-one tumor samples (7 liposarcomas, 3 chondrosarcomas, 6 osteosarcomas, 5 MFH) obtained at open biopsy, and four human MFH cell lines (Nara H, Nara F, TNMY1, GBS-1) were used. We examined the mRNA expression of S1P receptors by RT-PCR, and the expression levels of SPHK by Real-time PCR. We used 4 MFH cell lines to analyze SPHK1 proteins by Western blotting. SPHK1 siRNA was transfected into MFH cell lines by lipofection method. Cell proliferation (control and transfected with siRNA) was assayed using WST-8 (Cell Counting Kit-8) assay. All high grade malignant tumors expressed S1P1, S1P2, S1P3 receptors, whereas the expression of S1P1 receptor was detected in 50% of low-grade malignant tumors, S1P2 receptor in 30%, and S1P3 in 50%. No statistically significant difference was found in the expression level of SPHK1 between high-grade and low-grade malignant tumors by Real-time PCR. By results of Western blotting, proteins of SPHK1 were expressed in all MFH cell lines. In MFH cell lines, transfection with SPHK1 siRNA oligonucleotides resulted in approximately 50 to 80% suppression of SPHK1 mRNA expression as determined by real-time PCR. Down-regulation of SPHK1 with small interfering RNA significantly reduced SPHK1 protein levels by Western blotting. Knock down of SPHK1 expression significantly decreased cell proliferation of all MFH cells. These results suggest that the expression of S1P receptors may play an important role for cell proliferation and may correlate with histologic grade in malignant bone and soft tissue tumors, and that SPHK1 may be one of essential molecules for cell proliferation in MFH cell lines.

Keywords

Sarcoma, Sphingosine-1-Phosphate, S1p Receptor, Sphingosine Kinase, MIB-1, MFH

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	T. Hla, “Signaling and Biological Actions of Sphingosine 1-phosphate,” Pharmacological Research, Vol. 47, No. 5, 2003, pp. 401-407. doi:10.1016/S1043-6618(03)00046-X
[2]	S. E. Alvarez, S. Milstien and S. Spiegel, “Autocrine and Paracrine Roles of Sphingosine-1-phosphate,” Trends in Endocrinology & Metabolism, Vol. 18, No. 8, 2007, pp. 300-307. doi:10.1016/j.tem.2007.07.005
[3]	Y. A. Hannun and L. M. Obeid, “Principles of Bioactive Lipid Signalling: Lessons from Sphingolipids,” Nature Reviews Molecular Cell Biology, Vol. 9, No. 2, 2008, pp.139-150. doi:10.1038/nrm2329
[4]	T. C. Pham, J. I. Sr. Fells, D. A. Osborne, E. J. North, M. M. Naor and A. L. Parrill, “Molecular Recognition in the Sphingosine-1-phosphate Receptor Family,” Journal of Molecular Graphics and Modeling, Vol. 26, No. 8, 2008, pp. 1189-1201. doi:10.1016/j.jmgm.2007.11.001
[5]	T. A. Taha, K. M. Argraves and L. M. Obeid, “Sphingosine-1-phosphate Receptors: Receptor Specificity Versus Functional Redundancy,” Biochimica et Biophysica Acta, Vol. 1682, No. 1-3, 2004, pp. 48-55.
[6]	V. Brinkmann, “Sphingosine-1-phosphate Receptors in Health and Disease: Mechanistic Insights from Gene Deletion Studies and Reverse Pharmacology,” Pharmacology & Therapeutics, Vol. 115, No. 1, 2007, pp. 84-105. doi:10.1016/j.pharmthera.2007.04.006
[7]	E. J. Goetzl, H. Dolezalova, Y. Kong and L. Zeng, “Dual Mechanisms for Lysophospholipid Induction of Proliferation of Human Breast Carcinoma Cells,” Cancer Research, Vol. 59, No. 18, 1999, pp. 4732-4737.
[8]	J. R. Van Brocklyn, N. Young and R. Roof, “Sphingosine-1-phosphate Stimulates Motility and Invasiveness of Human Glioblastoma Multiforme Cell,” Cancer Letters, Vol. 199, No. 1, 2003, pp. 53-60. doi:10.1016/S0304-3835(03)00334-3
[9]	H. Yamaguchi, J. Kitayama, N. Takuwa, K. Arikawa, I. Inoki, K. Takehara, H. Nagawa and Y. Takuwa, “Sphingosine-1-phosphate Receptor Subtype-Specific Positive and Negative Regulation of Rac and Haematogenous Metastasis of Melanoma Cells,” Biochemical Journal, Vol. 374, No. 3, 2003, pp. 715-722. doi:10.1042/BJ20030381
[10]	N. C. Hait, C. A. Oskeritzian, S. W. Paugh, S. Milstien and S. Spiegel, “Sphingosine Kinases, Sphingosine- 1-phosphate, Apoptosis and Diseases,” Biochimica et Biophysica Acta, Vol. 1758, No. 12, 2006, pp. 2016- 2026. doi:10.1016/j.bbamem.2006.08.007
[11]	D. R. Gude, S. E. Alvarez, S. W. Paugh, P. Mitra P, J. Yu, R. Griffiths, S. E. Barbour, S. Milstien and S. Spiegel, “Apoptosis Induces Expression of Sphingosine Kinase 1 to Release Sphingosine-1-phosphate as a ‘Come-and- Get-Me’ Signal,” The FASEB Journal, Vol. 22, No. 8, 2008, pp. 2629-2638. doi:10.1096/fj.08-107169
[12]	S. Pyne, S. C. Lee, J. Long and N. J. Pyne, “Role of Sphingosine Kinases and Lipid Phosphate Phosphatases in Regulating Spatial Sphingosine-1-phosphate Signalling in Health and Disease,” Cell Signal, Vol. 21, No. 1, 2009, pp. 14-21. doi:10.1016/j.cellsig.2008.08.008
[13]	A. J. Melendez, “Sphingosine Kinase Signalling in Immune Cells: Potential as Novel Therapeutic Targets,” Biochimica et Biophysica Acta, Vol. 1784, No. 1, 2008, pp. 66-75.
[14]	M. Maceyka, H. Sankala, N. C. Hait, H. Le Stunff, H. Liu, R. Toman, C. Collier, M. Zhang, L. S. Satin, A. H. Mer- rill Jr., S. Milstien and S. Spiegel, “SPHK1 and SPHK2, Sphingosine Kinase Isoenzymes with Opposing Functions in Sphingolipid Metabolism,” The Journal of Biological Chemistry, Vol. 280, No. 44, 2005, pp. 37118-37129. doi:10.1074/jbc.M502207200
[15]	M. Vadas, P. Xia, G. McCaughan and J. Gamble, “The Role of Sphingosine Kinase 1 in Cancer: Oncogene or Non-Oncogene Addiction?” Biochimica et Biophysica Acta, Vol. 1781, No. 9, 2008, pp. 442-447.
[16]	J. R. van Brocklyn, C. A. Jackson, D. K. Pearl, M. S. Kotur, P. J. Snyder and T. W. Prior, “Sphingosine Kinase-1 Expression Correlates with Poor Survival of Patients with Glioblastoma Multiforme: Roles of Sphingosine Kinase Isoforms in Growth of Glioblastoma Cell Lines,” Journal of Neuropathology & Experimental Neurology, Vol. 64, No. 8, 2005, pp. 695- 705. doi:10.1097/01.jnen.0000175329.59092.2c
[17]	R. Alemany, C. J. van Koppen, K. Danneberg, M. Ter Braak and D. Meyer zu Heringdorf, “Regulation and Functional Roles of Sphingosine Kinases,” Naunyn-Schmiedeberg’s Archives of Pharmacology, Vol. 374, No. 5-6, 2007, pp. 413-428. doi:10.1007/s00210-007-0132-3
[18]	C. Ricci, F. Onida, F. Servida, F. Radaelli, G. Saporiti, K. Todoerti, G. L. Deliliers and R. Ghidoni, “In Vitro Anti-Leukaemia Activity of Sphingosine Kinase Inhibitor,” British Journal of Haematology, Vol. 144, No. 3, 2009, pp. 350-357. doi:10.1111/j.1365-2141.2008.07474.x
[19]	Y. Kiyozuka, H. Nakagawa, Y. Uemura, H. Senzaki, A. Yamamoto, T. Noguchi, H. Mizuta, K. Nakanishi, S. Nakano and A. Tsubura, “Novel Cell Lines Established from a Human Myxoid Malignant Fibrous Histiocytoma Arising in the Uterus,” Cancer Genetics and Cytogenetics, Vol. 127, No. 1, 2001, pp. 7-15. doi:10.1016/S0165-4608(00)00413-1
[20]	T. Nakatani, T. Marui, T. Yamamoto, M. Kurosaka, T. Akisue and K. Matsumoto, “Establishment and Characterization of Cell Line TNMY1 Derived from Human Malignant Fibrous Histiocytoma,” Pathology International, Vol. 51, No. 8, 2001, pp. 595-602. doi:10.1046/j.1440-1827.2001.01253.x
[21]	Z. Fang, H. Mukai, K. Nomura, K. Shinomiya, S. Matsumoto, N. Kawaguchi, T. Kitagawa and H. Kanda, “Establishment and Characterization of a Cell Line from a Malignant Fibrous Histiocytoma of Bone Developing in a Patient with Multiple Fibrous Dysplasia,” Journal of Cancer Research and Clinical Oncology, Vol. 128, No. 1, 2002, pp. 45-49. doi:10.1007/s00432-001-0295-0
[22]	C. Hornuss, R. Hammermann, M. Fuhrmann, U. R. Juergens and K. Racké, “Human and Rat Alveolar Macrophages Express Multiple EDG Receptors,” European Journal of Pharmacology, Vol. 429, No. 1-3, 2001, pp. 303-308. doi:10.1016/S0014-2999(01)01329-2
[23]	X. Pi, S. Y. Tan, M. Hayes, L. Xiao, J. A. Shayman, S. Ling and J. Holoshitz, “Sphingosine Kinase 1-Mediated Inhibition of Fas Death Signaling in Rheumatoid Arthritis B Lymphoblastoid Cells,” Arthritis & Rheumatism, Vol. 54, No. 3, 2006, pp. 754-764. doi:10.1002/art.21635
[24]	S. Pyne and N. Pyne, “Sphingosine-1-phosphate Signalling via the Endothelial Differentiation Gene Family of G-Protein-Coupled Receptors,” Pharmacology & Therapeutics, Vol. 88, No. 2, 2000, pp. 115-131. doi:10.1016/S0163-7258(00)00084-X
[25]	H. Yamashita, J. Kitayama, D. Shida, H. Yamaguchi, K. Mori, M. Osada, S. Aoki, Y. Yatomi, Y. Takuwa and H. Nagawa, “Sphingosine-1-phosphate Receptor Expression Profile in Human Gastric Cancer Cells: Differential Regulation on the Migration and Proliferation,” Journal of Surgical Research, Vol. 130, No. 1, 2006, pp. 80-87. doi:10.1016/j.jss.2005.08.004
[26]	M. Murph and G. B. Mills, “Targeting the Lipids LPA and S1P and Their Signalling Pathways to Inhibit Tumour Progression,” Expert Reviews in Molecular Medicine, Vol. 9, No. 28, 2007, pp. 1-18. doi:10.1017/S1462399407000476
[27]	S. Sarkar, M. Maceyka, N. C. Hait, S. W. Paugh, H. Sankala, S. Milstien and S. Spiegel, “Sphingosine Kinase 1 is Required for Migration, Proliferation and Survival of MCF-7 Human Breast Cancer Cells,” FEBS Letters, Vol. 579, No. 24, 2005, pp. 5313-5317. doi:10.1016/j.febslet.2005.08.055
[28]	S. W. Weiss and J. R. Goldblum, “Enzinger & Weiss’s Soft Tissue Tumors,” Mosby Inc., Louisville, 2008. doi:10.1158/1078-0432.CCR-08-1158
[29]	W. Li, C. P. Yu, J. T. Xia, L. Zhang, G. X. Weng, H. Q. Zheng, Q. L. Kong, L. J. Hu, M. S. Zeng, Y. X. Zeng, M. Li, J. Li and L. B. Song, “Sphingosine Kinase 1 is Associated with Gastric Cancer Progression and Poor Survival of Patients,” Clinical Cancer Research, Vol. 15, No. 4, 2009, pp. 1393-1399.
[30]	T. Kawamori, T. Kaneshiro, M. Okumura, S. Maalouf, A. Uflacker, J. Bielawski, Y. A. Hannun and L. M. Obeid, “Role for Sphingosine Kinase 1 in Colon Carcinogenesis,” The FASEB Journal, Vol. 23, No. 2, 2009, pp. 405-414. doi:10.1096/fj.08-117572
[31]	J. Li, H. Y. Guan, L. Y. Gong, L. B. Song, N. Zhang, J. Wu, J. Yuan, Y. J. Zheng, Z. S. Huang and M. Li, “Clinical Significance of Sphingosine Kinase-1 Expression in Human Astrocytomas Progression and Overall Patient Survival,” Clinical Cancer Research, Vol. 14, No. 21, 2008, pp. 6996-7003. doi:10.1158/1078-0432.CCR-08-0754
[32]	K. J. French, J. J. Upson, S. N. Keller, Y. Zhuang, J. K. Yun and C. D. Smith, “Antitumor Activity of Sphingosine Kinase Inhibitors,” Journal of Neuropathology & Experimental Neurology, Vol. 318, No. 2, 2006, pp. 596-603.
[33]	K. J. French, R. S. Schrecengost, B. D. Lee, Y. Zhuang, S. N. Smith, J. L. Eberly, J. K. Yun and C. D. Smith, “Discovery and Evaluation of Inhibitors of Human Sphingosine Kinase,” Cancer Research, Vol. 63, No. 18, 2003, pp. 5962-5969.
[34]	M. E. Leroux, E. Auzenne, R. Evans, N. Jr. Hail, W. Spohn, S. C. Ghosh, D. Farquhar, T. McDonnell and J. Klostergaard, “Sphingolipids and the Sphingosine Kinase Inhibitor, SKI II, Induce BCL-2-Independent Apoptosis in Human Prostatic Adenocarcinoma Cells,” Prostate, Vol. 67, No. 15, 2007, pp. 1699-1717. doi:10.1002/pros.20645
[35]	S. W. Paugh, B. S. Paugh, M. Rahmani, D. Kapitonov, J. A. Almenara, T. Kordula, S. Milstien, J. K. Adams, R. E. Zipkin, S. Grant and S. Spiegel, “A Selective Sphingosine Kinase 1 Inhibitor Integrates Multiple Molecular Therapeutic Targets in Human Leukemia,” Blood, Vol. 112, No. 4, 2008, pp. 1382-1391. doi:10.1182/blood-2008-02-138958
[36]	D. Kapitonov, J. C. Allegood, C. Mitchell, N. C. Hait, J. A. Almenara, J. K. Adams, R. E. Zipkin, P. Dent, T. Kordula, S. Milstien and S. Spiegel, “Targeting Sphingosine Kinase 1 Inhibits Akt Signaling, Induces Apoptosis, and Suppresses Growth of Human Glioblastoma Cells and Xenografts,” Cancer Research, Vol. 69, No. 17, 2009, pp. 6915-6923. doi:10.1158/0008-5472.CAN-09-0664