Inhibition of Human Smooth Muscle Cell Proliferation by Gamigeonsim-Tang through the Transcriptional Regulation of Cell Cycle-Controlling Genes

American Journal of Chinese Medicine,  Wntr, 2000  by Seong-Gyu Ko,  Kyung-Sup Lee,  Ki-Ho Cho,  Young-Suk Kim,  Hyung-Sup Bae,  Sang-Kwan Moon

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Abstract: The effects of Gamigeonsim-tang (GGT) on cellular proliferation and expression of cell cycle-related genes were investigated in human smooth muscle cell HISM. HISM cells were treated with an aqueous extract of GGT. Cellular proliferation was investigated by an immunocytometric analysis of PCNA expression and a flow cytometric analysis of the cell cycle progression. Reduced expression of PCNA and a significant accumulation of [G.sub.1] phase cells were observed following treatment, indicating that GGT inhibits cellular proliferation of human smooth muscle cells. To explore whether GGT affects the transcription of cell cycle-regulating genes, we evaluated mRNA expression of p53, [p21.sup.Waf1], PCNA, Cyclin D1, Cdc2, Histone H3, c-Myc , and c-Fos using a quantitative RT-PCR analysis. While increased expressions of two negative cell cycle regulators, p53 and [p21.sup.Waf1] were found, reduced expressions of cell cycle stimulators, PCNA, c-Fos, and c-Myc, were identified following treatment. Taken together, our study demonstrates that GGT inhibits cellular proliferation of human smooth muscle cell through the up- and down-regulation of growth-inhibiting and growth-promoting genes, respectively.

Smooth muscle cells are one of the main cellular components of the walls of blood vessels and they play an important role in vascular pathology (Simionescu, 1992). Smooth muscle cells are capable of many functions including vasoconstriction and dilatation in response to normal or pharmacologic stimuli; synthesis of various types of collagen, elastin, and proteoglycan; elaboration of growth factors and cytokines; and migration and proliferation (Casscells, 1992). Normally the predominant cellular element of the vascular media, smooth muscle cells are an important element of vascular reparative processes and proliferative diseases. It is well known that smooth muscle cell proliferation and production of extracellular matrix components contribute to atherosclerotic plaque (Kocher, 1991). Thus, the structural and functional integrity of smooth muscle cells is a fundamental requirement for maintenance of vessel wall homeostasis and circulatory function (Pober et al., 1990). There is considerable experimental support for smooth muscle dysfunction as a major factor in several vascular diseases including atherosclerosis (Ross, 1993)

The migratory and proliferative activity of smooth muscle cells is physiologically regulated by both growth promoters and inhibitors. Promoters include platelet-derived growth factor (PDGF) derived from not only platelets but also endothelial cells and macrophages, basic fibroblast growth factor (bFGF), and IL-1 (Grainger, 1993). Inhibitors inelude heparan sulfates, nitric oxide (NO)/endothelial-derived relaxing factor (EDRF), interferon-gamma, and transforming growth factor-beta (TGF-beta). Some promoters and inhibitors are endogenous to the vascular wall (PDGF, heparan sulfates, and TGF-beta); others, such as IL-l, are not. In essence, vascular injury stimulates smooth muscle cell growth by disrupting the physiologic balance between smooth muscle cell growth inhibition and growth stimulation (Nachman, 1992).

Despite the fact that mortality due to atherosclerosis has been increased and several Korean traditional formulas have been proved to have valuable clinical effects for patients, no molecular biological characterization for the effects of these Korean traditional formulas has been described. In this study we examined the effect of GGT, which has been frequently used for vascular diseases including atherosclerosis and ischemic heart diseases.

p53 is a tumor suppressor gene which functions in cellular proliferation, differentiation, and maintenance of genomic stability (Levine, 1992; Lane, 1992). The growth inhibitory functions of p53 are thought to result from its ability to modulate transcription of several cell cycle-related genes such as [p21.sup.Waf1] or MDM2 by binding to p53 response elements (Zambetti et al., 1992; Weintraub et al., 1991; El-Deiry et al., 1993). Accumulation of p53 is associated with an arrest of the cell cycle and induction of apoptosis, p53-induced [G.sub.1] arrest of the cell cycle is believed to allow for DNA repair or apoptosis, thus preventing an accumulation of mutations (Yonish-Rouach et al., 1991; Kastan et al., 1992). [p21.sup.Waf1], a cyclin-dependent kinase inhibitor, is induced by p53 and is a downstream effector in the cascade that results in cell cycle arrest (El-Deiry et al., 1993; Harper et al., 1993). The role of [p21.sup.Waf1] in cell cycle regulation was demonstrated by strong induction of [p21.sup.Waf1] in wild-type p53 cells following exposure to DNA-damaging agents and subsequent p53-associated [G.sub.1] cell cycle arrest or apoptosis (El-Deiry et al., 1994).

In the present study, we demonstrate that GGT inhibits the transition of [G.sub.1] to S phase of the cell cycle. The growth inhibition of human smooth muscle cells by GGT is accompanied by the transcriptional up-regulation of cell cycle inhibitors, p53 and [p21.sup.Waf1], and down-regulation of cell cycle promoters, c-Myc and c-Fos.