Effects of Ginseng on the Proliferation of Human Lung Fibroblasts

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1 The American Journal of Chinese Medicine, Vol. 34, No. 1, World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine Effects of Ginseng on the Proliferation of Human Lung Fibroblasts Hye Hyun Yoo, *, Takako Yokozawa, * Akiko Satoh, * Ki Sung Kang * and Hyun Young Kim * Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama , Japan College of Pharmacy, Seoul National University, Seoul , Korea Abstract: In this study, we investigated the effects of methanolic extracts of white ginseng (Panax ginseng C.A. MEYER) and two kinds of heat-treated ginseng made by steaming fresh ginseng at 100 o C for 3 hours (HTG-100) or 120 o C for 3 hours (HTG-120) on the cell growth of human fibroblasts. All of the tested ginseng extracts stimulated cell growth, although the effect of HTG-120 was weaker than that of the other extracts. However, none of the ginseng extracts exhibited any effect on the growth of old cells with a population doubling level (PDL) of Flow cytometric analysis showed that ginseng extracts raised the population of cells in G 0 /G 1 phase after treatment for 24 hours, but did not exert any effect after treatment for 48 hours. These results suggest that ginsengs exert their cell growth-promoting action mainly on younger cells at an early stage of the cell cycle, and that this effect is closely associated with an increase in the population of cells in the G 0 /G 1 phase. Keywords: Panax ginseng C.A. MEYER; White Ginseng; Heat-Treated Ginseng; Cell Growth; Cell Cycle; Human Fibroblast. Introduction Korean ginseng (Panax ginseng C.A. MEYER; white ginseng, produced mainly in Korea and northeast China) is a herbal root that has been used extensively as a tonic food for more than 2000 years. The biochemical and pharmacological activities of ginseng are anti-aging, anti-diabetic, anti-carcinogenic, analgesic, anti-pyretic, anti-stress, antifatigue and tranquilizing activities, in addition to promotion of DNA, RNA and protein synthesis activities (Kumagai, 2000). In particular, there have been many reports that Correspondence to: Dr. Takako Yokozawa, Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama , Japan. Fax: (+81) , yokozawa@ms.toyamampu.ac.jp 137

2 138 H.H. YOO et al. ginseng possesses chemopreventive and therapeutic activities against cancers as well as improving general immune function (Ota et al., 1987 and 1991; Bhattacharya and Mitra, 1991; Baek et al., 1995; Lee et al., 1996 and 1997; Chen et al., 1998). Most studies on the chemopreventive or anticancer activities of ginseng have been conducted with cancer cells. However, the effects of ginsengs and their mechanisms of action in normal cells are rather less well understood. Generally, it is known that ginsengs have anti-proliferative activity against cancer cells (Kim et al., 2004). Contrary to their action on cancer cells, ginsengs exhibit proliferative activity on several types of normal cells. It enhances the proliferation of blood cells such as lymphocytes and hematopoietic progenitor cells related to immune function (Cho et al., 2002; Gai et al., 2003). Furthermore, it has been reported that ginsengs promote regeneration of excised liver tissue (Kwon and Jang, 2004), and stimulate the proliferation of epidermal cells (Choi, 2002) and vascular endothelial cells (Nakajima et al., 1998). However, the mechanism and active principles involved are not completely understood. Therefore, to help clarify the cell growth-stimulatory activity of ginsengs, we studied their effects on the cell proliferation of lung-originated human diploid fibroblasts (HDFs). HDFs are proliferative cells that display typical replicative senescence (Hayflick, 1976; Goldstein, 1990; Linskens et al., 1995; Campisi, 2000). In other words, after serial passage, they lose their ability to proliferate and become senescent, showing cellular changes related to the aging process. Thus it is possible to carry out experiments with these cells under conditions resembling those in normal human cells. In particular, lung fibroblasts are activated by inflammatory and immune cells, and play a role in the repair of endothelial or epithelial cell damage. The damaged cells may alter their immunological environment and promote the proliferation of immune cells such as lymphocytes, which in turn leads to proliferation of fibroblasts (Sheppard and Harrison, 1992; Sacco et al., 2004). In the present study, we investigated the effects of ginsengs on HDFs by measuring cell viability and cell cycle distribution employing flow cytometry. We also examined the changes in the effect of ginseng on cells as they undergo the aging process using cells at various population doubling levels (PDLs). In order to clarify whether the differences in processing affect the actions of ginsengs on cells, besides of using the methanolic extracts of white ginseng, we also used the extracts of heat-treated ginseng, which are made by heat-processing of white ginseng root. Materials and Methods Materials Basal Medium Eagle (BME) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Propidium iodide (PI) was purchased from Molecular Probes (Eugene, Oregon, USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Fetal bovine serum (FBS), trypsin solution and ribonuclease A (RNase) were purchased from Life Technologies Inc. (Grand Island, NY, USA), Nakarai (Kyoto, Japan) and Funakoshi (Tokyo, Japan),

3 GINSENG ON THE PROLIFERATION 139 respectively. Normal human lung diploid fibroblasts (TIG-1) were purchased from the Health Science Research Resources Bank (Osaka, Japan). Preparation of Methanol Extracts and Heat Treatment of White Ginseng White ginseng powder was produced by drying 100 g of fresh ginseng at 50 o C for 3 days and then ground to a fine powder. Each ginseng powder (50 g) was extracted under reflux with MeOH three times at 70 C for 2 hours. The solvent was evaporated in vacuo to give MeOH extract with a yield of about 20% of the original ginseng powder by weight. Heattreated ginseng was made by steaming fresh ginseng at 100 C for 3 hours (HTG-100) or at 120 C for 3 hours (HTG-120). For analysis of the constituents of each ginseng extract, analytical HPLC was conducted with a Hitachi L-7100 liquid chromatograph fitted with a C-18, reverse-phase column (5 µm, 25 cm 4 mm I.D.; YMC-Pack Pro) utilizing the solvent gradient system. The mobile phase consisted of water (solvent A) and AcCN (solvent B), and the flow rate was 1 ml/min. The detector was a SEDEX 55 ELSD (Sedere, France). Cell Experiments TIG-1 human diploid fibroblasts were cultivated in 10 mm culture dishes containing BME medium supplemented with 10% FBS at 37 C in a humidified atmosphere of 5% CO 2 in air. For experiments, cells were seeded at a density of 10 5 cells/ml in 6- or 96-well culture plates and incubated for 2 hours. Then the cells were treated with extracts of white or heattreated ginseng dissolved in media at final concentrations of 10, 50 and 100 µg/ml for the cell viability assay, and 10 and 100 µg/ml for measurement of the cell cycle. In each case, equivalent volumes of media were used as the control vehicle. The values of PDL of the fibroblasts were 30.5, 39.8 and 48.7, respectively. Cell viability and cell cycle distribution were measured after incubation for 24 and 48 hours, respectively. Cell Viability Cell viability was assessed using the MTT colorimetric assay (Mosmann, 1983). After treatment with each ginseng extract, the medium in each well was removed and 100 µl of MTT solution (1 mg/ml) was added. After incubation for 4 hours at 37 C, the medium containing MTT solution was removed and the incorporated formazan crystals in the viable cells were solubilized with 100 µl of dimethylsulfoxide. The absorbance of each well was read at 540 nm using a microplate reader (Model 3550-UV, Bio-Rad, Tokyo, Japan). Flow Cytometric Cell Cycle Analysis After treatment with each ginseng extract, the cells were collected by centrifugation, fixed for 1 hour at 4 C in 3 ml of 70% ice-cold ethanol, washed twice with PBS, incubated

4 140 H.H. YOO et al. with RNase solution for 30 minutes at 37 C and then treated with PI at 4 C for 30 minutes. The cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, CA, USA) with laser excitation at 488 nm using a 639 nm band, passed a filter to collect the red PI fluorescence. The percentages of cells at G 0 /G 1, S and G 2 /M phase were assessed using ModFit LT software (Verity Software House, Topsham, ME, USA) from the analyzed data. Statistics The values presented are means ± SE of five determinations for each of the independent experimental conditions. The significance of differences was tested using Dunnett s method. Differences at p < 0.05 were considered statistically significant. Figure 1. HPLC-ELSD chromatogram of ginseng extracts. White ginseng extract (A), HTG-100 extract (B), HTG- 120 extract (C). 1 = Re plus Rg 1, 2 = Rb 1, 3 = Rc, 4 = Rb 2, 5 = Rg 3 (S), 6 = Rg 3 (R), 7 = Rk 1 and 8 = Rg 5.

5 GINSENG ON THE PROLIFERATION 141 Results HPLC Chromatogram The representative ginsenoside contents of each ginseng extract were as follows: Re + Rg 1 (5.1%), Rb 1 (5.2%), Rc (2.6%) and Rb 2 (2%) in white ginseng extract; Re + Rg 1 (7.3%), Rb 1 (5.4%), Rc (3.0%) and Rb 2 (2.0%) in HTG-100 extract; Re + Rg 1 (2.0%), Rb 1 (4.1%), Rg 3 (S) (2.6%), Rg 3 (R) (1.5%), Rk 1 (3%) and Rg 5 (3.5%) in HTG-120 extract, as shown in Fig. 1. Cell Viability As shown in Table 1, the results from the MTT assays using human fibroblasts with a PDL of 30.5 showed that cell viability increased after treatment with the ginseng extracts. On the whole, more stimulation of cell growth was observed in the extracts of white ginseng and HTG-100 than in the extract of HTG-120, and cell viability was also increased to a greater degree after incubation for 48 hours than after 24 hours. When cells were treated for 24 hours with the extracts of white ginseng, HTG-100 and HTG-120 at concentrations ranging from 10 to 100 µg/ml, cell viability increased to 125.6%, 123.6% and 112.1% of the control value, respectively. When treated for 48 hours, the respective increases were 136.8%, 140.2% and 129.6%. Meanwhile, as the PDL of the cells increased, the activity Table 1. Effect of Ginseng Extract on Cell Viability of Human Fibroblast with a PDL Value of 30.5 Incubation Time Groups Concentration Cell Viability (Hours) (µg/ml) (% of the Control) 24 Control ± 1.4 White ginseng ± ± ± 1.6 HTG ± 1.7 * ± ± 1.3 HTG ± 1.9 * ± 1.5 * ± 2.1 * 48 Control ± 3.0 White ginseng ± ± ± 3.0 HTG ± ± ± 2.9 HTG ± ± 4.4 * ± 3.3 Statistical significance: * p < 0.01, p < versus control values.

6 142 H.H. YOO et al. Table 2. Effect of Ginseng Extract on Cell Viability of Human Fibroblast with Various PDL Value Incubated for 24 Hours Groups Concentration Cell Viability (% of the Control) (µg/ml) PDL = 30.5 PDL = 39.8 PDL = 48.7 Control ± ± ± 3.5 White ginseng ± ± ± ± ± ± ± ± ± 2.9 * HTG ± 1.7 * ± 1.4 * 97.1 ± ± ± ± ± ± ± 2.0 HTG ± 1.9 * ± ± ± 1.5 * ± ± ± 2.1 * ± ± 3.1 Statistical significance: * p < 0.05, p < versus control values. of the ginsengs on cell growth was reduced (Table 2). The effects of ginsengs on cells with a PDL of 39.8 remained unchanged from those on cells with a PDL of However, on cells with a PDL of 48.7, the activity of ginsengs was reduced significantly and there was no difference in cell viability between ginseng-treated and untreated cells, although the activity of white ginseng was still evident at a concentration of 100 µg/ml. Cell Cycle Analysis In order to investigate the mechanism of cell growth stimulation by the ginseng extracts, TIG-1 cells with a PDL of 30.5 on which the effect of ginsengs was most potent were treated with each ginseng extract for 24 and 48 hours and analyzed for cell cycle distribution by flow cytometry. It was found that the ginseng extracts altered the cell cycle distribution of the asynchronously growing TIG-1 cells. Among cells incubated for 24 hours, ginseng extracts mainly raised the G 0 /G 1 phase population of cells and consequently reduced the S phase population, when compared with untreated cells (Table 3). HTG-120 in particular produced a marked elevation of the G 0 /G 1 phase population from 46.1% to 64.4% at a concentration of 100 µg/ml. When cells were incubated for 48 hours, as they came to confluence, the G 0 /G 1 phase population increased to about 70%. In the case of the cells treated with the ginseng extracts and incubated for 48 hours, the cell cycle distribution showed hardly any difference from that of the untreated control cells, but the G 0 /G 1 phase population of cells treated with HTG-120 was slightly higher than that of the cells treated with the other ginseng extracts. Discussion The present results indicated that all of the tested ginseng extracts generally stimulated the growth of human fibroblasts, although each had a distinctive type of action. The effects

7 GINSENG ON THE PROLIFERATION 143 Table 3. Effect of Ginseng Extract on Cell Cycle of Human Fibroblast with a PDL Value of 30.5 Incubation Time Groups Concentration % of Cells in Each Phase of Cell Cycle (Hours) (µg/ml) G 0 /G 1 S G 2 /M 24 Control 46.1 ± ± ± 2.6 White ginseng ± ± ± ± ± 0.1 * 22.1 ± 0.4 HTG ± 1.3 * 23.2 ± ± ± 1.4 * 21.4 ± ± 2.6 HTG ± ± 2.8 * 17.1 ± ± ± 2.9 * 17.3 ± Control 70.0 ± ± ± 0.8 White ginseng ± ± ± ± ± ± 0.3 HTG ± ± ± ± 1.1 * 18.6 ± 0.4 * 7.0 ± 0.7 HTG ± 1.8 * 18.1 ± 1.3 * 6.2 ± ± 0.3 * 20.5 ± ± 0.3 Statistical significance: * p < 0.05, p < 0.01 versus control values. of white ginseng and HTG-100 on cell growth were similar, but that of HTG-120 was generally weaker than that of the others. This was considered to be due to the differences in their constituents. HTG-100 and HTG-120 are made by heat-processing of white ginseng at 100 C and 120 C, respectively. This heat processing gives rise to chemical transformation of the components of ginsengs, and consequently may bring about alteration of their bioactivities (Park, 1996; Kim et al., 2000). There are, in fact, differences in the content and composition of ginsenosides, the main bioactive principles of ginsengs, among the ginseng extracts. Particularly, in the case of HTG-120, the conditions of processing are more severe than those for HTG-100, so more drastic changes in the chemical constituents are generated by chemical reactions such as hydrolysis of glycosidic bonds or dehydroxylation (Park et al., 1998). From the HPLC chart in Fig. 1, it is evident that in HTG-120 polar ginsenosides such as Re, Rg 1 and Rb 1 were decreased, whereas relatively non-polar ginsenosides such as Rg 3, Rk 1 and Rg 5 were increased or newly formed. Although it is still unclear which ginsenosides exert effects on cell growth, this finding may at least partly explain why ginsenosides stimulate the growth of human fibroblasts. The major active components might be ginsenosides such as Re, Rg 1 and Rb 1, which are abundant in white ginseng and HTG-100. There are reports that ginsenoside-rg 1 and -Rb 1 stimulate the proliferation of human marrow granulocyte-macrophage progenitor cells (Niu et al., 2001), and Rb 1 and Re are known to enhance lymphocyte proliferation (Cho et al., 2002). Rb 2 has been found to have an epidermis-proliferative effect (Choi, 2002). However, there is also a report that ginseng extract contains a basic fibroblast growth factor-like molecule, probably a nonsaponin substance, which has a mitogenic effect on BALB/c3T3 fibroblasts (Takei et al., 1996). Thus, we cannot exclude the possibility that the proliferation of fibroblasts treated with ginseng is due to a non-ginsenoside substance.

8 144 H.H. YOO et al. Our results suggested that ginseng s effect on cell growth became weaker as cells underwent the aging process. Although ginseng s cell growth-stimulatory effect was maintained on cells with a PDL of 39.8, the activity was significantly reduced and was barely evident on cells with a PDL of This indicates that young cells at the early passage stage are more susceptible than older cells to ginseng s growth-stimulatory action. Cell cycle analysis revealed that ginseng s effect on cell growth might be partly related to the G 0 /G 1 phase population. There have been several reports about the effects of ginseng on the cell cycle of cancer cells. Ginsenoside-Rh 2 is known to block the cell cycle of human hepatoma cells at the G 1 /S boundary by selectively inducing the expression of p27 KiP1 protein (Lee et al., 1996). Ginsenoside-Rs 4 is known to arrest the cell cycle at the G 1 /S boundary and consequently induce apoptosis by down-regulating the activities of both cyclin E- and A-associated kinases and elevating the levels of p53 and p21 WAF1 proteins in human hepatoma cells (Kim et al., 1999). In addition, panaxydol, a cytotoxic polyacetylenic compound of ginseng, is reported to induce G 1 cell cycle arrest to inhibit the proliferation of human melanoma cells by inducing p27 KIP1 expression and reducing the activity of cyclin-dependent kinase 2 (Cdk2) (Moon et al., 2000). Thus, ginsengs work on cancer cells mainly through G 1 phase arrest of the cell cycle to induce apoptosis and inhibit cell proliferation. However, our results showed that ginseng extracts elevated the population of cells in G 0 /G 1 phase, but stimulated the proliferation of human fibroblasts. When cells reached confluence and the G 0 /G 1 phase population increased to 70%, ginseng did not exert any significant effect on the cell cycle of fibroblasts. The present results do not indicate the pathway by which ginseng induces the G 0 /G 1 phase increase, and it is still unclear whether the cell proliferation induced by ginseng is directly related to elevation of the G 0 /G 1 phase population. However, our findings suggest that the effects of ginseng on the cell cycle, primarily at the early stage of cell growth, might regulate cell proliferation. In summary, ginseng extracts have been shown to stimulate the proliferation of human fibroblasts. Their effects are exerted mainly on young cells at an early stage of the cell cycle. This cell growth-stimulatory activity was reduced somewhat when ginseng was heatprocessed at 120ºC. Further studies should be done to elucidate the active principle(s) and the molecular mechanism(s) responsible for the cell growth-stimulatory activity of ginseng and to clarify the relationship between this effect and cell cycle regulation. Although it is still unclear whether this effect of ginseng can be extrapolated to normal cells in vivo, our findings may at least partly explain the effect of ginseng a popular tonic food for promotion of health on cell proliferation. References Baek, N.I., D.S. Kim, Y.H. Lee, J.D. Park, C.B. Lee and S.I. Kim. Cytotoxicities of ginseng saponins and their degradation products against some cancer cell lines. Arch. Pharm. Res. 18: , Bhattacharya, S.K. and S.M. Mitra. Anxiolytic activity of Panax ginseng roots: an experimental study. J. Ethnopharmacol. 34: 87 92, 1991.

9 GINSENG ON THE PROLIFERATION 145 Campisi, J. Cancer, aging and cellular senescence. In Vivo 14: , Chen, X., H. Liu, X. Lei, Z. Fu, Y. Li, L. Tao and R. Han. Cancer chemopreventive and therapeutic activities of red ginseng. J. Ethnopharmacol. 60: 71 78, Cho, J.Y., A.R. Kim, E.S. Yoo, K.U. Baik and M.H. Park. Ginsenosides from Panax ginseng differentially regulate lymphocyte proliferation. Planta Med. 68: , Choi, S. Epidermis proliferative effect of the Panax ginseng ginsenoside Rb 2. Arch. Pharm. Res. 25: 71 76, Gai, Y., R.L. Gao and Y.P. Niu. Effect of Panax notoginsenosides on the proliferation of hematopoietic progenitor cells in mice with immune-mediated aplastic anemia. Zhongguo Zhong Xi Yi Jie He Za Zhi 23: , Goldstein, S. Replicative senescence: the human fibroblast comes of age. Science 249: , Hayflick, L. The cell biology of human aging. N. Engl. J. Med. 295: , Kim, H.S., E.H. Lee, S.R. Ko, K.J. Choi, J.H. Park and D.S. Im. Effects of ginsenosides Rg 3 and Rh 2 on the proliferation of prostate cancer cells. Arch. Pharm. Res. 27: , Kim, S.E., Y.H. Lee, J.H. Park and S.K. Lee. Ginsenoside-Rs 4, a new type of ginseng saponin concurrently induces apoptosis and selectively elevates protein levels of p53 and p21 WAF1 in human hepatoma SK-HEP-1 cells. Eur. J. Cancer 35: , Kim, W.Y., J.M. Kim, S.B. Han, S.K. Lee, N.D. Kim, M.K. Park, C.K. Kim and J.H. Park. Steaming of ginseng at high temperature enhances biological activity. J. Nat. Prod. 63: , Kumagai, A. Ginseng Kyoritsu Shuppan Co., Tokyo, 2000, pp Kwon, Y.S. and K.H. Jang. The effect of Korean red ginseng on liver regeneration after 70% hepatectomy in rats. J. Vet. Med. Sci. 66: , Lee, K.Y., Y.H. Lee, S.I. Kim, J.H. Park and S.K. Lee. Ginsenoside-Rg 5 suppresses cyclin E-dependent protein kinase activity via up-regulating p21 Cip/WAF1 and down-regulating cyclin E in SK- HEP-1 cells. Anticancer Res. 17: , Lee, K.Y., J.A. Park, E. Chung, Y.H. Lee, S.I. Kim and S.K. Lee. Ginsenoside-Rh 2 blocks the cell cycle of SK-HEP-1 cells at the G 1 /S boundary by selectively inducing the protein expression of p27 kip1. Cancer Lett. 110: , Linskens, M.H., C.B. Harley, M.D. West, J. Campisi and L. Hayflick. Replicative senescence and cell death. Science 267: 17, Moon, J., S.J. Yu, H.S. Kim and J. Sohn. Induction of G 1 cell cycle arrest and p27 KIP1 increase by panaxydol isolated from Panax ginseng. Biochem. Pharmacol. 59: , Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55 63, Nakajima, S., Y. Uchiyama, K. Yoshida, H. Mizukawa and E. Haruki. The effects of ginseng radix rubra on human vascular endothelial cells. Am. J. Chin. Med. 26: , Niu, Y.P., J.M. Jin, R.L. Gao, G.L. Xie and X.H. Chen. Effects of ginsenosides Rg 1 and Rb 1 on proliferation of human marrow granulocyte-macrophage progenitor cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi 9: , Ota, T., K. Fujikawa-Yamamoto, Z.P. Zong, M. Yamazaki, S. Odashima, I. Kitagawa, H. Abe and S. Arichi. Plant-glycoside modulation of cell surface related to control of differentiation in cultured B16 melanoma cells. Cancer Res. 47: , Ota, T., M. Maeda and S. Odashima. Mechanism of action of ginsenoside Rh 2 ; uptake and metabolism of ginsenoside Rh 2 by cultured B16 melanoma cells. J. Pharm. Sci. 80: , Park, J.D. Recent studies on the chemical constituents of Korean ginseng (Panax ginseng C. A. Meyer). Kor. J. Ginseng Sci. 20: , 1996.

10 146 H.H. YOO et al. Park, J.H., J.M. Kim, S.B. Han, N.Y. Kim, Y.J. Surh, S.K. Lee, N.D. Kim and M.K. Park. A new processed ginseng with fortified activity. In: H. Hur, K.J. Choi and Y.C. Kim (eds.) Advances in Ginseng Research (Proceedings of the 7th International Symposium on Ginseng). Korean Society of Ginseng, Seoul, 1998, pp Sacco, O., M. Silvestri, F. Sabatini, R. Sale, A.C. Defilippi and G.A. Rossi. Epithelial cells and fibroblasts: structural repair and remodeling in the airways. Paediatr. Respir. Rev. 5(Suppl. A): S35 S40, Sheppard, M.N. and N.K. Harrison. New perspectives on basic mechanisms in lung disease. 1. Lung injury, inflammatory mediators, and fibroblast activation in fibrosing alveolitis. Thorax 47: , Takei, Y., T. Yamamoto, H. Higashira and K. Hayashi. Identification of basic fibroblast growth factorlike immunoreactivity in Panax ginseng extract: investigation of its molecular properties. Biosci. Biotechnol. Biochem. 60: , 1996.

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