Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Changan-ku, Chunchun-dong, Kyunggi-do, Suwon 440-746, Korea and
1 Department of Pharmacology, Faculty of Medicine, University of Ottawa, Ontario, Canada
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Abstract |
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Abbreviations: 8-OH-dG, 8-hydroxydeoxyguanosine; APS, Aloe barbadensis Miller polysaccharide; CPS, Coriolus versicolor polysaccharide; GPS, Ganoderma lucidum polysaccharide; GST, glutathione S-transferase; LPS, Lentinus edodes polysaccharide; ODC, ornithine decarboxylase; PMA, phorbol myristic acetate; TK, tyrosine kinase.
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Introduction |
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Plant polysaccharides have traditionally been used around the world as a folk remedy for various diseases due to their multiple biological properties including anti-inflammation, wound healing, antihepatitis, anti-ulcer and anti-neoplastic effects (911). Aloe polysaccharide (APS) has recently been shown to exert an inhibitory effect on benzo[a]pyrene (B[a]P)DNA adduct formation in vitro and in vivo (12). A number of other plant polysaccharides from Basidiomycetes, such as Lentinus edodes (LPS), Ganoderma lucidum (GPS), and Coriolus versicolor (CPS), have also been shown to produce anti-tumor effects, thus being potentially useful in cancer therapy (1315). However, the mechanisms responsible for chemopreventive effects of plant polysaccharides are poorly understood.
In the present study, several structurally similar plant polysaccharides (APS, LPS, GPS and CPS) were screened for their chemopreventive effects using biomarkers involved in chemical carcinogenesis. Biomarkers used for initiation stage of cancer were: (i) DNA adduct formation (e.g. B[a]PDNA adducts); (ii) 8-hydroxydeoxyguanosine (8-OH-dG), representing oxidative DNA damage; and (iii) induction of glutathione S-transferase (GST) activity. Biomarkers for promotion stage of cancer were (i) phorbol myristic acetate (PMA)-induced tyrosine kinase (TK) activity increase in human leukemia cells [HL-60; American Tissue Culture Collection (ATCC), Manassas, VA]; (ii) PMA-induced ornithine decarboxylase (ODC) activity elevation in Balb/3T3 cells (ATCC); and (iii) free radical formation in PMA-induced HL-60 cells (ATCC). LPS, GPS and CPS were prepared as described previously (15) and APS provided by ALOECORP (Harlingen, TX).
The total polysaccharide content was measured by the phenolsulphuric acid method (16) and protein content was determined by the Bradford method (17). The sugar composition of crude polysaccharides was analyzed by HPLC (18). The purified polysaccharides were white to off-white fluffy powders with <16% protein and >67% carbohydrates (Table I). APS consists of ~0.5% protein and 85.1% polysaccharides (73% being mannose). Carbohydrate analysis indicated that glucose was the major sugar in GPS and CPS. However, mannose and arabinose were the major monosaccharides in LPS. A B[a]PDNA binding assay was carried out with mouse normal liver NCTC clone-1469 cells (1x107) plated in culture flasks with NCTC-clone 115 medium (Gibco BRL, Grand Island, NY) supplemented with penicillin, streptomycin and 10% fetal bovine serum (FBS; Gibco BRL). The cells were maintained for 6 h, treated with 3H-labeled B[a]P (4 nmol/ml, 52 Ci/nmol) (Amersham, Arlington Heights, IL) in the absence or presence of polysaccharides (6, 20, 60 or 180 µg/ml) and incubated for 12 h. After incubation, whole cells were harvested and DNA was isolated from the cells by addition of DNAzol (MRC, Cincinnati, OH). Concentration-dependent inhibition of 3H-B[a]PDNA adduct formation was determined as described previously (19) and was observed only with APS treatment (Table II
). APS at concentrations of 20180 µg/ml significantly inhibited 3H-B[a]PDNA adduct formation by 2050% of control (8.95 ± 2.13 pmol/mg DNA). Except for LPS, GPS and CPS at all concentrations produced moderate effects on DNA adduct formation. Using our experimental conditions, no cytotoxic effect on the 1469 cells was observed at 180 µg/ml concentration. Effects of APS, GPS, LPS and CPS on 8-OH-dG and superoxide anion formation are shown in Figure 1
. The baseline levels of 8-OH-dG as measured by the method of Kasai et al. (20) in NCTC-clone 1469 cells were 2.47 ± 0.76 residues/105 dG (data not shown) whereas a significant increase in 8-OH-dG formation was observed in cells treated with B[a]P (4.47 ± 0.76 residues/105 dG). APS and CPS at concentrations of 180 µg/ml significantly inhibited 8-OH-dG formation, but LPS and CPS produced moderate effects on B[a]P-induced 8-OH-dG formation (Figure 1A
). Effects of APS, LPS, GPS and CPS on free radical formation were investigated in PMA-induced HL-60 cells using a superoxide dismutase-sensitive cytochrome c reduction system as described by Pick and Mizel (21). In 96-well plates suspended in Hank's balanced salts solution (Gibco BRL), a time-dependent increase in cellular production of free radicals was observed in HL-60 cells (1x106 cells) treated with PMA (data not shown). As shown in Figure 1B
, APS and CPS at 180 µg/ml concentration significantly inhibited the free radical formation in PMA-induced HL-60 cells. However, GPS and LPS produced moderate effects on PMA-induced inhibition of free radical formation. GST activity was measured in NCTC-clone cells (1x106 cells/dish, supplemented with 10% FBS) treated with or without plant polysaccharides as described previously (22). GPS significantly increased GST activities by 2030%, but APS and CPS did not, and LPS moderately increased GST activities (Table II
). PMA-induced TK activities were measured in HL-60 cells treated with APS, GPS, LPS and CPS (Table II
). HL-60 cells were cultured in RPMI-1640 media (Gibco BRL) supplemented with 10% FBS and allowed to reach a maximum density of 1x106 cells/ml. The cells were then plated at a density of 5x105 cells in six-well plates, treated with PMA alone (0.1 µM in 0.001% dimethyl sulfoxide; Sigma, St Louis, MO) to induce TK activity, and immediately followed by the addition of genistein (103 M), as a positive control or with various concentrations of polysaccharides for 24 h at 37°C in an incubator containing 5% CO2. TK activity was measured using poly(GluTyr) (4.1:0.9; Sigma) as a substrate based on a previous method (23). In cells treated with PMA alone for 6 h, TK activity was significantly increased by 50% compared with control. Genistein, as a positive control, almost completely inhibited TK activity (data not shown). APS produced concentration-dependent inhibition of TK activity and was significantly inhibited by 30% at the highest concentration. In contrast, other polysaccharides induced moderate effects on TK activity. ODC activities were measured by the method described previously (24). When Balb/3T3 cells were treated with PMA alone, ODC activity was significantly increased by 50% compared with control. Among plant polysaccharides tested, APS induced concentration-dependent inhibition of ODC activity and significantly decreased it by 2030% of control at 180 µg/ml (Table II
). However, LPS, GPS and CPS produced moderate effects on ODC activity.
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It has been also suggested that the chemopreventive mechanisms of some compounds are related to the inhibition of carcinogen activation systems or to the induction of detoxification enzymes (25). In our study, GPS significantly increased GST activity by 2030%. Although the induction of GST activity was low, it might be possible that plant polysaccharides could be effective in the induction of other detoxification enzymes. The mode of action of polysaccharides on GST activity is not clearly understood, but the induction of GST activity by GPS appears to be related to the amount of protein present in polysaccharideprotein complexes. Singh et al. (26) demonstrated that chlorophyllin also significantly elevated GST activity and glutathione levels in the liver and skin tissue.
Mechanisms of the inhibition of tumor promotion by polysaccharides toward different types of promoters remain unclear. However, tumor formation can be prevented by certain agents including retinoids, difluoromethylornithine (DFMO) and indomethacin, that block induction of ODC (27,28). Therefore, it is possible that ODC inhibition may be useful for the screening of chemopreventive agents. Moreover, it has been demonstrated that protein phosphorylation by TK is closely associated with cellular proliferation and differentiation. The inhibition of tyrosine TK may lead to suppression or reversal of carcinogenic processes (29). In our study, ODC and TK activities were significantly decreased by APS as compared with control in a concentration-dependent manner.
There are various naturally occurring products and synthetic compounds having both blocking and suppressing properties. The effect of certain fruits and vegetables containing high levels of natural antioxidants has been associated with scavenging of free radicals (30,31). Hence, the decrement in the rate of oxidative DNA damage induced by fruits and vegetables may serve as a basis for chemopreventive mechanisms. Liu et al. (32) suggested that mushroom polysaccharide extracts possessed superoxide and hydroxyl radical scavenging activities. However, the mechanism of free radical scavenging by polysaccharides is still not fully understood. It is possible that the protein content of polysaccharide extracts may be directly effective in free radical scavenging activity. It is known that phenolic compounds may react with superoxide radicals by a one-electron transfer mechanism (33). APS and CPS also significantly inhibited free radical formation in PMA-induced HL-60 cells, but it is not clear whether the mechanism of superoxide and hydroxyl radical scavenging by plant polysaccharides examined in this study is similar to that of phenolic compounds. In general, plant polysaccharides showed moderate or significant effects on the modulation of in vitro biomarkers associated with the carcinogenesis process. This effect might be related to the composition of sugars in polysaccharides as well as protein content.
In conclusion, the chemopreventive potential of polysaccharides was evaluated using biomarkers for carcinogenesis. Among polysaccharides tested, APS was found to be the most effective in anti-genotoxic and anti-promoting activities. GPS was also effective in GST activity induction and CPS was effective in inhibition of both 8-OH-dG formation and superoxide anion formation. LPS was the least effective and showed only moderate effects on some biomarkers such as 8-OH-dG, GST, TK and superoxide anion formation. These results suggest that some plant polysaccharides might be useful for chemoprevention application.
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