Anti-TUMOUR activity of Ganoderma lucidum (Ling Zhi) and its mechanism
Zhi-Bin Lin, Department of Pharmacology, School of Basic Medical Sciences, Beijing University, Beijing 100083, China
Abstract Ganoderma lucidum (Leyss. ex fr.) Karst. (Lingzhi) is a medicinal fungus with a long history in China as a tonic and remedy. Modern pharmacological and clinical investigations have demonstrated that Lingzhi has anti-tumour activities. The anti-tumour effects of G. lucidum extract (GLE) and Ganoderma polysaccharides B (GL-B) and its mechanism were investigated in this laboratory. The studies have demonstrated that both GLE and GL-B significantly inhibited the growth of implanted sarcoma (S)-180 in mice. Co-administration of GL-B potentiated the anti-tumour activity of cyclophosphamide in mice. The addition of either GLE or GL-B to culture medium neither suppressed the proliferation of S-180 and HL-60 tumour cells nor induced apoptosis of both tumour cells in vitro. However, the serum from GLE and GL-B treated mice could suppress the proliferation of S-180 and HL-60 cells and induced its apoptosis in vitro. In addition, splenocyte conditioned medium with GL-B (GL-B-S-CM) and peritoneal macrophages conditioned medium with GL-B (GL-B-PM-CM) also significantly inhibited HL-60 proliferation and induced its apoptosis in vitro. Further study indicated that GLE and GL-B could induce tumour necrosis factor (TNF) production in murine peritoneal macrophages and interferon production in murine spleen cells in vitro. GLE and GL-B could also induce TNF-a mRNA expression in murine peritoneal macrophages and IFN-g mRNA expression in murine spleen cells. These results suggest that the anti-tumour effects of G. lucidum are associated with the activation of macrophages and spleen cells, the induction of cytokines such as TNF-a and IFN-g at mRNA and protein levels.
Key words: Apoptosis, Ganoderma lucidum, Tumour necrosis factor-a, Interferon-g, Polysaccharides.
Ganoderma lucidum (Leyss.ex fr.) Karst. (Lingzhi) is a medicinal fungus with long history in China as a tonic and remedy. Lingzhi was highly ranked as an herbal medicine in Shen Nong Materia Medica (Shen Nung Ben Cao Jing) which was published in the second century B.C. Shi-Zhen Li, a well known ancient Chinese physician, also described the efficacy and medical uses of Lingzhi in the world renown classic Compendium of Materia (Ben Cao Gang Mu) in the 16th century. Ancient Chinese medical scholars suggested that Lingzhi could strengthen body resistance and consolidate the constitution of patients i.e., “Fuzheng Guben” which is one of the major principles in the therapeutics of traditional Chinese medicine.
Lingzhi has a widespread use as a herbal medicine in clinical settings. Many Chinese physicians have co-administered Lingzhi in cancer patients receiving conventional chemotherapy and/or radiation treatment, to build up immune resistance and decrease toxicity. It has been reported that the extract and polysaccharides isolated from G. lucidum significantly inhibits the growth of implanted Sarcoma (S)-180 and Lewis lung-carcinoma in animal models in vivo (Lin, 1996). Although its anti-tumour activity is beyond doubt, the detailed mechanisms remain unclear. Some researchers have suggested that its anti-tumour activity may be mediated to some extent by activation of host immune functions. In according to establishment of this hypothesis, we have investigated the anti-tumour activity of Ganoderma extract (GLE) and Ganoderma polysaccharides B (GL-B) isolated from G. lucidum and its possible mechanisms at cellular and molecular level both in vivo and in vitro.
METHODS AND RESULTS
Anti-tumour activity of GLE and GL-B on implanted Sarcoma 180 in Vivo
GLE or GL-B was administered by stomach tube once a day for 10 days in mice. The study indicated that GLE at 5, 10, and 20 g (crude materials)/kg inhibited the growth of S-180 in a dose-dependent manner, with an inhibitory rates of 22.8, 41.6, and 60.9%, respectively (Zhang et al., 2000). Similarly, GL-B at 50, 100, 200 mg/kg inhibited the growth of S-180, with an inhibitory rate of 27.7, 55.8, and 66.7% respectively. Co-administration of GL-B potentiated the anti-tumour activity of cyclophosphamide in mice, resulting in an inhibitory rate of 81.0, 45.5, and 75.95% respectively, which were significantly higher than those in the groups treated with GL-B or cyclophosphamide alone (Zhang and Lin, 1999c). Recently, we found that the polysaccharides isolated from mycelia of G. lucidum at 50, 100 mg/kg inhibited the growth of S-180 in Balb/c mice and Kun-ming mice, with an inhibitory rates of 37.8 – 78.1% (Hu and Lin, 1999b). These results indicate that either G. lucidum or its active component, Ganoderma polysaccharides, has anti-tumour activity in mice in vivo, and Ganoderma polysaccharides have synergic effect on the anti-tumour activity of cytotoxic drug such as cyclophosphamide.
Effect of GLE and GL-B on the proliferation and apoptosis of tumour cells in vitro
The addition of either GLE or GL-B (both at 50, 100, 200 mg/ml) to the cultures of S-180 and HL-60 tumour cells had no inhibition against the proliferation of tumour cells, even at the very high concentration such as GL-B 400 mg/ml (Hu and Lin, 1999b; Zhang et al., 2000).
A number of anti-tumour drugs, including alkylating drugs, antimetabolites, and antihormones can induce the apoptosis of tumour cells, contributing to their anti-tumour activities (Makin and Hickman, 2000). We investigated the effects of GLE and GL-B on apoptosis of tumour cells using the agarose-gel-electrophoresis and analysis of propidinmiodide fluorescence of individual nuclei by FAC Scan flow cytometer. The study indicated that GLE at 50, 100, 200 mg (crude materials)/ml could not induce S-180 cell apoptosis; the proportions of apoptotic cells were 0.64 * 0.30, 0.75 * 0.41, and 0.70 * 0.36% respectively. The same result found that GL-B 50, 100, 200 mg/ml also cannot induce HL-60 cell apoptosis; the proportions of apoptotic cells were 0.87 * 0.24 0.65 * 0.31, and 0.63 * 0.45%, respectively (Hu and Lin, 1999b). Etopeside, used as a positive control, significantly induced apoptosis of S-180 cell and HL-60 cell; apoptotic cells were 69.6 * 8.0 and 67.7 * 4.0% respectively (Zhang et al., 2000).
Further evidence also indicated that the polysaccharides isolated from mycelia of G. lucidum neither inhibited HL-60 cell proliferation nor induced HL-60 cell apoptosis in vitro even at the very high concentration (750 mg/ml).
These results, taken together with that G. lucidum and its polysaccharides neither inhibited tumour cell proliferation nor induced their apoptosis in vitro, i.e. they have not cytotoxic effect, suggest that mechanisms other than direct cytotoxicity may be involved in the anti-tumour activity of G. lucidum.
Using the method of serologic pharmacology to study the anti-tumour effect of GLE and GL-B
Serologic pharmacology means to make in vitro experiment using an animal serum with drug when the animal has been taken this drug. The drug treated-serum may contain drug, metabolites of the drug, or endogenous active substances induced by drug in vivo, thus the serum with drug produces pharmacological effect. In recent years, serologic pharmacological method is widely used to investigate traditional Chinese medicine.
At the dose of 10, 20 g (crude materials)/kg, GLE were administered by stomach tube, once a day for 10 days. The mice were killed 10 days later, and serums were taken and stored at -20oC for use. We added GLE-treated serum to the in vitro S-180 culture media. Surprisingly, GLE-treated serum could inhibit proliferation of S-180 cells and induced their apoptosis in vitro (Zhang et al., 2000). Similarly, GL-B-treated serum also inhibited proliferation of HL-60 cells and induced these cells apoptosis in vitro (Zhang and Lin, 1999c). These results suggest that GLE-treated serum and GL-B-treated serum may have the substances with anti-tumour activity.
What active substances could contain in the serums? TNF-a and IFN-g are known to play important roles in suppressing tumour cell growth and inducing apoptosis of many different kinds of tumour cells. Many studies have showed that TNF-a and IFN-g work together in inducing tumour cell apoptosis. Therefore, above-mentioned results with GLE-treated serum and GL-B-treated serum may be associated with these two cytokines, they are endogenous active products by stimulating effect of GLE or GL-B on immune system in vivo. We used bioassay method and ELISA kit to detect the tumour necrosis factor (TNF)-a activity and interferon (IFN)-g level in serum. The results showed that the TNF-a and IFN-g levels were increased in GLE-treated serum and GL-B treated serum (Zhang and Lin, 1999c; Zhang et al., 2000).
Effects of GL-B-M-CM and GL-B-T-CM on the proliferation and apoptosis of HL-60 cells in vitro
To further study the effect of GL-B on stimulating cytokine production by T lymphocytes and macrophages, and the effect of GL-B-conditioned medium (GL-B-M-CM) with T lymphocytes and macrophages on proliferation and apoptosis of tumour cells, a pure population of macrophages or T lymphocytes was incubated separately in an RPMI 1640 medium containing 10% NBS with or without various concentrations of GL-B at 37oC for 12 – 72 h, which were called macrophage culture medium with GL-B (GL-B-M-CM) and T lymphocyte culture medium with GL-B (GL-B-T-CM), were then collected, filtered, and stored at -70oC, for determination of cells proliferation and analysis of apoptosis in vitro.
The study indicates that GL-B-M-CM and GL-B-T-CM at either concentration significantly (P < 0.05) inhibited the proliferation of HL-60 cells in vitro, compared with RPMI 1640 and macrophage culture medium with normal saline (N-M-CM) or T lymphocyte culture medium with normal saline (N-T-CM) groups. At 50, 100, 200 mg/ml, GL-B-M-CM significantly (P < 0.01) induced HL-60 cells apoptosis in vitro; the proportions of apoptotic cells were 18.8 * 0.93, 21.0 * 1.6, and 23.0 * 0.6% respectively, compared with N-M-CM group (7.44 * 1.07%). At these concentrations, GL-B-T-CM also significantly (P < 0.01) induced HL-60 cell apoptosis in vitro, the proportions of apoptotic cells were 19.4 * 1.1, 21.9 * 0.8, and 22.9 * 1.5% respectively, compared with N-T-CM group (7.8 * 1.1%) (Zhang and Lin, 1999c). Similar results were observed with the conditioned medium from the polysaccharides isolated from mycelia of G. lucidum-activated splenocytes or macrophages markedly induced HL-60 apoptosis (Hu and Lin, 1999b; Zhang et al., 2000).
To further reveal the mechanism of anti-tumour effect of GL-BM-CM and GL-B-T-CM in vitro, the levels of TNF in GL-B-M-CM and IFN in GL-B-T-CM were assayed by using bioassay and ELISA, respectively.
Effect of GL-B on TNF-a production of macrophage and IFN-g production of T lymphocytes in vitro
Our experimental results showed that the TNF-a level in the supernatant of 12.5-400 mg/ml GL-B cultured with macrophages was rose during 24 h as the dose increased. Similarly, the IFN-g level in the supernatant of 12.5 – 200 mg/ml GL-B cultured with T lymphocytes was increased during 24 h as the dose was increased to 400 mg/ml. Moreover, our results have indicated that there was a positive correlation between the level of TNF-a in GL-B M-CM and IFN-g in GL-B-T-CM and the anti-tumour effect of GL-B-M-CM and GL-B-T-CM (Zhang and Lin, 1999b). It was also found that at the dose of 12.5, 50, and 200 mg/ml, the macrophage culture medium with polysaccharides isolated from mycelia of G. lucidum inhibited proliferation of HL-60 cells and induced its apoptosis significantly; with an increased TNF level in the cultured supernatant (Hu et al., 1999a). These results together with those from other laboratories suggest that all of these cytokines may be involved in the anti-tumour effect of Ganoderma polysaccharides in vivo.
Effects of GLE and GL-B on TNF and IFN mRNA expression
In order to further investigate the mechanism of GLE or GL-B on the production of cytokines, we observed the effect of GL-B on TNF mRNA expression from macrophages and IFN mRNA expression from T lymphocytes using RT-PCR method in vitro. The study showed that the addition of GL-B (50 – 200 mg/ml) to the in vitro macrophages or T-lymphocytes culture media, resulted in an significantly increased TNF-a and IFN-g mRNA expression in a concentration- dependent manner (Zhang and Lin, 1999a; Zhang and Lin, 1999b). Following the administration of GLE at 5, 10 20 g (crude material)/kg by forced stomach tube feeding, we found that TNF-a and IFN-g mRNA expression was increased markedly (Zhang and Lin, 1999c). These results indicate that GLE and GL-B could induce TNF and IFN mRNA expression in vitro and in vivo.
Taking these results together, several conclusions may be draw. Firstly, GLE and GL-B could inhibit the growth of implanted S-180 in vivo. Secondly, GLE or GL-B added to culture medium neither induced S-180 or HL-60 apoptosis nor restrained their proliferation in vitro, whereas GLE- or GL-B-treated serum could induce tumour cells apoptosis and inhibit their proliferation in vitro. Thirdly, the macrophage or T lymphocyte culture medium treated with GL-B significantly induced HL-60 apoptosis and inhibited tumour cell proliferation. Simultaneously, GL-B significantly increased TNF and IFN release in a dose-dependent mode. Finally, both GLE and GL-B could induce TNF and IFN expression, the latter are known to play an important role in suppressing tumour cells growth and inducing apoptosis of tumour cells, suggesting that the anti-tumour activity of GLE and GL-B may be related to the induction of TNF from macrophages and IFN from T lymphocytes.
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