Objective: Alpha tocopherol, other than its anti-oxidant activity, also has a non anti-oxidant activity that correlates with inhibition of PKC-alpha isozyme activity. Several studies had confirmed its role in inhibition of vascular smooth muscle proliferation. Inhibition of stromal cell proliferation is an alternative method of prevention in BPH development. The aim of this in vitro study is to confirm that alpha-tocopherol, by its ability to inhibit PKC-alpha activity, has the capability to inhibit human prostatic stromal cell proliferation rate. Materials & Method: Primary cultures of human prostatic stromal muscle cells were obtained from 4 patients with bladder outlet obstruction caused by benign prostatic enlargement. The effects of various concentrations of alpha-tocopherol (20, 30, 40, 50, and 60 microM) were examined by cell proliferation and PKC-alpha isozyme activity assay. Cell proliferation study was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method. Caspase-3 activity assay was also performed to examine apoptotic mechanism. Results: MTT method, which is used to count viable cells, showed that exposures to various doses of alpha-tocopherol resulted in decreased proliferation rate. The decrement of proliferation rate is linear with the increment of alpha-tocopherol dose. Immunocytochemistry staining with anti caspase-3 antibody showed that no cells developed apoptosis. Alpha-tocopherol also inhibits PKC-alpha activity. Isozyme activity is reduced along with the increment of the dose (dose-dependent). There is a correlation between PKC-alpha activity with cell proliferation rate after exposure to alpha-tocopherol (R2=0,9279; p=0,000). Conclusion: Alpha-tocopherol at different concentrations reduces prostatic stromal cell proliferation rate, without effect on apoptosis. This antiproliferative property correlates with decreased activity of PKC-alpha isozyme.
protein kinase C-alpha, primary culture, human prostatic stromal cells, antiproliferative effect
Berry SJ, Coffey DS, Walsh PC, Ewing LL. The development of human benign prostatic hyperplasia with age. J Urol. 1984; 132: 474-9.
Deering RE, Bigler SA, King J, Choongkit taworn M, Aramburu E, Brawer MK. Morpho-metric quantitation of stroma in human benign prostatic hyperplasia.Urology1994; 44: 64-70.
Claus S, Berges R, Senge T, Schulze H. Cell Kinetic in Epithelium and Stroma of Benign Prostatic Hyperplasia. J Urol 1997; 158: 217-21.
Newton AC. Protein Kinase C: Structure, Function, and Regulation. J Biol Chem 1995; 270: 28495–98.
Nishizuka Y. Protein Kinase C Isotypes and Their Specific Functions. Discovery and Prospect of Protein Kinase C Research: Epilogue. J. Biochem. 2003; 133: 155–8.
Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA, Messing RO. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 2000; 279: L429-L 438.
Kazanietz MG & Blumberg PM. Protein kinase C and signal transduction in normal and neoplastic cells. In Sirica (ed) Cellular and molecular pathogenesis. Lippincott-Raven, Philadelphia, 1996: 389-402.
Ron D, Kazanietz MG. New insights into the regulation of protein kinase C and novel phorbol ester receptors. FASEB J. 1999; 13: 1658–76.
Egger T, Schuligoi R, Wintersperger A, Amann R, Malle E, Sattler W. Vitamin E (α-tocopherol) attenuates cyclo-oxygenase 2 transcription and synthesis in immortalized murine BV-2 microglia. Biochem J. 2003; 370: 459-67.
Boscoboinik D, Szewczyk A, Hensey C and Azzi A. Inhibition of cell proliferation by alpha-tocopherol. Role of protein kinase C. J. Biol. Chem.1991; 266: 6188-94.
Tasinato A, Boscoboinik D, Bartoli GM, Maroni P, Azzi A. d-α-Tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentration, correlates with protein kinase C inhibition, and is independent of its antioxidant properties. Proc. Nas. Acad. Sci. USA 1995; 92: 12190-94.
Ricciarelli R, Zingg JM, Azzi A. Vitamin E: protective role of a Janus molecule. FASEB J. 2001; 15: 2314–25.
Venkateswaran V, Fleshner NE, Klotz LH. Modulation of cell proliferation and cell cycle regulators by vitamin E in human prostate carcinoma cell lines. J Urol 2002; 169: 1578-82
Zhang Y, Ni J, Messing EM, Chang E, Yang CR, Yeh S. Vitamin E succinate inhibits the function of androgen receptor and the expression of prostate-specific antigen in prostate cancer cells. PNAS 2002; 99: 7408-13.
Jiang Q, Wong J, Fyrst H, Saba JD, Ames BN. γ–Tocopherol or combination of vitamin E forms induce cell death in human prostate cancer cells by interupting sphingolipid synthesis. PNAS 2004; 101: 17825-30
Helzlsouer KJ, Huang H-Y, Alberg AJ, Hoffman S, Burke A, Norkus EP, Morris JS, Comstock GW. Association Between α-Tocopherol, γ-Tocopherol, Selenium, and Subsequent Prostate Cancer. J Natl Cancer Inst 2000; 92:2018–23
Chan JM, Stampfer MJ, Jing Ma, Rimm EB, Willett WC, Giovannucci EL. Supplemental Vitamin E Intake and Prostate Cancer Risk in a Large Cohort of Men in the United States. Cancer Epidemiology, Biomarkers & Prevention 1999; 8: 893–9.
Dewi N. Karakterisasi ekstrak kasar enzim Protein Kinase C-α hasil isolasi dari serum darah penderita pre-ekalmsia. Skripsi, Unibraw 2000
Guh JH, Hwang TL, Ko FN, Chueh SC, Lai MK, Teng CM. Antiproliferative effect in human prostatic smooth muscle cells by nitric oxide donor. Mol Pharm, 1998; 53: 467-74.
Keaney JF, Guo Y, Cunningham D, Shwaery GT, Xu A, Vita JA. Vascular incorporation of α-tocopherol prevents endothelial dysfunction due to oxidized LDL by inhibiting protein kinase C stimulation. J. Clin. Invest.1996; 98: 386–94
Kunisaki M, Bursell SE, Umeda F, Nawata H, King GL. Normalization of diacylglycerol-protein kinase C activation by vitamin E in aorta of diabetic rats and cultured rat smooth muscle cells exposed to elevated glucose levels. Diabetes. 1994; 43:1372-77.
Kunisaki M, Bursell SE, Clermont AC, Ishii H, Ballas LM, Jirousek MR, Umeda F, Nawata H, King GL.Vitamin E prevents diabetes-induced abnormal retinal blood flow via the diacylglycerol-protein kinase C pathway. Am J Physiol. 1995; 269: E239-46.
Ganz MB & Seftel A. Glucose-induced changes in protein kinase C and nitric oxide are prevented by vitamin E. Am J Physiol. Endocrinol. Metab. 2000; 278: E 146- E 152.
Boudreau RTM, Garduno R, Lin T-J. Protein phosphatase 2A and protein kinase Cα are physically associated and are involved in Pseudomonas aeruginosa-induced interleukin 6 production by mast cells. J. Biol. Chem. 2002; 227: 5322-29
Lue TF. Physiology of penile erection and pathophysiology of erectile dysfunction and priapism. In: Campbell’s Urology, edisi ke 7. editor: Walsh PC, Retik AB, Vaughan ED, dan Wein AJ. Philadelphia: WB Saunders Co., 2002; 1591-618.
Chueh S-C, Guh J-H, Chen J, Lai M-K, Teng C-M. Dual effects of ouabain on the regulation of proliferation and apoptosis in human prostatic smooth muscle cells. J Urol 2001; 166: 347-53.
Albert B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Cell communication. Dalam Molecular biology of the cell,edisi ke-4, Garland Publishing, Inc, NewYork, 2002:831-906.
DeWolf WC & Gaston SM. The cell cycle and its relevance to the urologist. Jurol 2004; 171: 1674-81.
Braz TC, Bueno OF, DEWindt LJ, Molkentin JD. PKCα regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase 1 / 2 (ERK 1 / 2 ). J. Cell Biol. 2002; 5: 905-19.
Mauro A, Ciccarelli C, Cesaris PD, Scoglio A, Bouche M, Molinaro M et al. PKCα-mediated ERK, JNK, and p38 activation regulates the myogenic program in human rhabdomyosarco-ma cells. JourCellScience 2002; 115: 3587-99.
Marcus R & Coulston AM. Fat soluble vitamins. Vitamin A, D, and E. In: Hardman JG, Limbird LE, and GilmanAG (editor). Goodman & Gilman’s The Pharmacolocical basis of therapeutics (edisi ke-10), 2001: 1733-91.
Ricciarelli, R., Maroni, P., Ozer, N., Zingg, J. M., and Azzi, A. Age-dependent increase of collagenase expression can be reduced by alpha-tocopherol via protein kinase C inhibition. Free Radic. Biol. Med. 1999; 27: 729–37.
Brigelius-Flohe R, Traber MG. Vitamin E: function and metabolism. FASEB J. 1999; 13: 1145–55.
Aratri, E., Spycher, S. E., Breyer, I., and Azzi, A. Modulation of alpha-tropomyosin expression by alpha-tocopherol in rat vascular smooth muscle cells. FEBS Lett. 1999; 447: 91–4.
McIntyre BS, Briski KP, Gapor A, Sylvester PW. Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells (44544). P.S.E.B.M 2000; 224: 292-301
Neuzil J, Weber T, Schroder A, Lu M, Ostermann G, Gellert N, Mayne GC et al. Induction of cancer cell apoptosis by α-tocopheryl succinate: molecular pathways and structural. FASEB J. 2001; 15: 403–15
Neuzil J, Zhao M, Ostermann G, Sticha M, Gellert N, Weber C, Eaton JW, Brunk UT. a-Tocopheryl succinate, an agent with in vivo anti-tumour activity, inducesapoptosis by causing lysosomal instability. Biochem J 2002; 362: 709-15
Maroni PD, Koul S, Meacham RB, Koul HK. Mitogen Activated Protein kinase signal transduction pathways in the prostate. Cell Communication and Signaling 2004, diakses dari: http://www.biosignaling.com/content/2/1/5
Price DT, Della Roca G, Guo C, Ballo MS, Schwinn DA, Luttrell LM. Activation of extracellular signal-regulated kinase in human prostate cancer. J Urol 1999; 162: 1537-42.