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Ganoderma Tsugae Extracts Inhibit Colorectal Cancer Cell Growth

Ganoderma Tsugae Extracts Inhibit Colorectal Cancer Cell Growth

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Journal of Ethnopharmacology 120 (2008) 394–401

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Ganoderma tsugae extracts inhibit colorectal cancer cell growth via G2 /M cell cycle arrest
Shih-Chung Hsu a,b , Chien-Chih Ou c,d , Jhy-Wei Li e,f , Tzu-Chao Chuang g,h,∗∗ , Han-Pon Kuo i , Jah-Yao Liu c , Chin-Shiang Chen j , Song-Chow Lin k , Ching-Hua Su l , Ming-Ching Kao m,n,∗
a

Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan Kang-Ning Junior College of Medical Care and Management, Taipei, Taiwan Department of Obstetrics & Gynecology, Tri-Service General Hospital, Taipei, Taiwan d Department of Pharmacy, Keelung General Hospital, Keelung, Taiwan e Graduate Institute of Pathology, National Defense Medical Center, Taipei, Taiwan f Department of Pathology, Da-Chien General Hospital, Miaoli, Taiwan g Department of Chemistry, Tamkang University, Tamsui, Taipei, Taiwan h Graduate Institute of Life Sciences, Tamkang University, Tamsui, Taipei, Taiwan i Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan j Department of Horticulture and Biotechnology, Chinese Culture University, Taipei, Taiwan k Department & Institute of Pharmacology, Taipei Medical University, Taipei, Taiwan l Department of Microbiology and Immunology, Taipei Medical University, Taipei, Taiwan m Department of Biological Science and Technology, China Medical University, Taichung, Taiwan n Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan
b c

a r t i c l e

i n f o

a b s t r a c t
Ethnopharmacological relevance: Ganoderma, known as Lingzhi or Reishi, has been traditionally administered throughout Asia for centuries as a cancer treatment and for other medicinal purposes. Aim of the study: To investigate the inhibitory activity and explore the molecular mechanisms of antitumor effect on colorectal cancer cells in vitro and in vivo as well as to test the side effects of Ganoderma tsugae. Materials and methods: Methanol fraction was obtained from dried fruiting bodies of Ganoderma. TLC and HPLC were performed to differentiate and confirm the identification of different species as well as to quantify the bioactive molecules in methanol extracts of Ganoderma species. MTT and Trypan blue exclusion assay as well as tumorigenesis study were used to assess the anti-tumor effect in vitro and in vivo. Using flow cytometry and Western Blots, we examined further the molecular mechanisms of antitumor effect. Finally, biochemical and hematological profiles and pathological examinations were used to evaluate the safety. Results: The Ganoderma tsugae extracts inhibit colorectal cancer cell proliferation caused by accumulating cells in G2 /M phase, and it may be through downregulation of cyclin A and B1 and upregulation of p21 and p27. Tumorigenesis study in nude mice revealed the extracts caused tumor shrinkage. Additionally, safety assay showed Ganoderma tsugae extracts caused no significant side effects in an animal model. Conclusions: This study provides molecular evidence that Ganoderma tsugae extracts exert anti-tumor effects both in vitro and in vivo on colorectal adenocarcinoma cells by inducing G2 /M cell cycle arrest. More importantly, no significant physiological changes resulting from treatment with Ganoderma tsugae extracts were observed in the animal model. Therefore, these data provide new insights into the possible therapeutic use of Ganoderma tsugae for treating colorectal cancer. © 2008 Published by Elsevier Ireland Ltd.

Article history: Received 21 April 2008 Received in revised form 12 July 2008 Accepted 11 September 2008 Available online 2 October 2008 Keywords: Ganoderma tsugae Lingzhi (Reishi) Colo205 Colorectal cancer Cancer therapy

Abbreviations: G. tsugae, Ganoderma tsugae; TCM, traditional Chinese medicine; H&E stain, hematoxylin and eosin stain; TLC, thin layer chromatography; HPLC, high-performance liquid chromatography; IHC, immunohistochemistry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HPFs, high-power fields; CDKs, cyclin dependent kinases. ∗ Corresponding author at: Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan, ROC. Tel.: +886 4 22053366x2206; fax: +886 4 22070465. ∗∗ Corresponding author at: Department of Chemistry, and Graduate Institute of Life Sciences, Tamkang University, Tamsui, Taipei, Taiwan, ROC. Tel.: +886 2 26215656x2316; fax: +886 2 26209924. E-mail addresses: tcbc@mail.tku.edu.tw (T.-C. Chuang), mckao@mail.cmu.edu.tw (M.-C. Kao). 0378-8741/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2008.09.025

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1. Introduction Ganoderma (also known as Lingzhi or Reishi), a traditional Chinese medicine (TCM), has recently received considerable attention from the health care and cancer research communities in Taiwan. Colorectal cancer is of particular concern, due to the recent increases in prevalence and death from this disease; while early diagnosis and therapy improve the probability of colorectal cancer survival. In 2006, colorectal cancer was the second leading cause of cancer-related deaths in the United States (Wolpin et al., 2007) and the third in Taiwan. To investigate the possible application of Ganoderma in colon cancer therapy, we performed both in vitro and in vivo studies of Ganoderma activity. For centuries, Ganoderma has been used for medicinal purposes in Asian countries to treat many human diseases, including cancer. Ganoderma lucidum (G. lucidum) and Ganoderma tsugae (G. tsugae)

are the most widely cultivated species of the Ganoderma genus in Taiwan, and both have a long history of use in folk medicine in Asia. The biological activities of Ganoderma lucidum, especially its anti-tumor and immunomodulatory properties, have been welldocumented (Chen et al., 2004; Chien et al., 2004). Several reports have shown that the two major categories of bioactive ingredients that can be isolated from Ganoderma lucidum are polysaccharides and triterpenoids, both of which are potent inhibitors of in vitro and in vivo tumor growth (Miyazaki and Nishijima, 1981; Min et al., 2000; Kimura et al., 2002; Shiao, 2003; Lin and Zhang, 2004). Moreover, recent studies have demonstrated that Ganoderma lucidum suppresses cell motility, inhibits cell proliferation, induces apoptosis, and suppresses angiogenesis of highly invasive human breast and prostate cancer cells (Hu et al., 2002; Sliva et al., 2002; Jiang et al., 2004; Stanley et al., 2005). Although the anti-tumor activity of Ganoderma tsugae has been characterized (Wang et al., 1993), only few clinical or pharmacological studies of its efficacy have been pursued. Triterpenoids from Ganoderma tsugae can induce apoptosis and cell cycle arrest in human hepatoma cells (Gan et al., 1998), although the molecular mechanism of the anti-tumor effects of Ganoderma tsugae on human colorectal cancer cells has not been investigated. In this study, extracts from the three Ganoderma species (G. tsugae, G. lucidum, and G. formosanum) were examined by TLC and HPLC profiling to assess their quality prior to further experimentation. Because the Ganoderma tsugae extracts demonstrated the highest cytotoxicity in cancer cells as assessed by MTT assay, this study examined the anti-proliferative effects of Ganoderma tsugae as well as the possible mechanisms by which Ganoderma tsugae affects Colo205 human colorectal cancer cells. Furthermore, the safety of orally administered Ganoderma tsugae was evaluated in mouse studies. 2. Materials and methods 2.1. Cells and materials Human colorectal adenocarcinoma Colo205 cells were obtained from American Type Culture Collection and grown in RPMI 1640 medium supplemented with 10% FBS. Paclitaxel (Taxol) was purchased from Bristol-Myers Squibb (Wallingford, CT) and stored at −20 ◦ C before use. Paclitaxel was diluted in serum-free media at the required concentration before use. The Ganoderma tsugae extracts were directly added to cell cultures at the indicated concentrations. All the primary and secondary antibodies were purchased from Santa Cruz Biotechnology. 2.2. Sample extraction and preparation Ganoderma tsugae fruiting bodies were donated by the Luo Gui-Ying Fungi Agriculture Center (Taoyuan, Taiwan). Ganoderma lucidum and Ganoderma formosanum were purchased, in a dried form, at a traditional market in Taipei, Taiwan. The genus and species of the Ganoderma samples used in this study were identified and confirmed by Dr. C.H. Su (Graduate Institute of Medical Sciences, Taipei Medical University, Taiwan) as described (Su et al., 2001). All Ganoderma samples were dried and ground into fine powder, then extracted with methanol (MeOH) at room temperature. For MeOH extraction, 5 g of the powdered samples were mixed with 200 mL of methanol and then placed on a rotating shaker at 150 rpm for 24 h. Filtrate was collected twice by filter paper (Whatman No. 1) and MeOH was evaporated to dryness with a reduced pressure concentrator for approximately 3 h to obtain dry MeOH extracts. The extraction yield of the methanol extracts of Ganoderma tsugae is 30.3 ± 0.7%. All samples of Ganoderma extracts were dissolved in

Fig. 1. Analysis of Ganoderma species by TLC and HPLC profiling. (A) Identification of Ganoderma species by TLC. Lanes 1 and 2, Ganoderma lucidium; lanes 3 and 4, Ganoderma formosanum; lanes 5–7, G. tsugae. (B) Quantification of the methanol extracts from G. tsugae (GT1, GT2 and GT3) and Ganoderma lucidium (GL1 and GL2) by HPLC.

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MeOH as a stock solution (300 mg/ml) for tests in cells. For in vivo studies of tumorigenesis, dry Ganoderma tsugae extracts were dissolved in ethanol (EtOH, 100 mg/ml) and diluted to the indicated concentrations (10 mg/ml) with suspension solution (74.5% corn oil, 16% PEG-400, 4% Tween-80, 4% Cremophor EL, and 1.5% EtOH, v/v). 2.3. TLC and HPLC analysis TLC and HPLC analytical procedures were done according to the report of Su et al., 2001. 2.4. Drug sensitivity analysis by MTT assay The MTT assay was performed as described (Chuang et al., 2003). The GI50 (drug concentration causing 50% growth inhibition) was

obtained by plotting the drug concentration against the percentage of growth inhibition. 2.5. Cell counting and Trypan blue exclusion assay Cells (2.5 × 106 ) were seeded onto a 100-mm Petri dish to measure cell proliferation. Cell growth rate and viability were determined using a hemacytometer to count the number of cells as a function of time following Trypan blue staining. 2.6. Cell cycle analysis by flow cytometry and Western blotting To determine the cell cycle phase distribution and the molecular markers associated with each phase, flow cytometry and Western blot analysis were performed, respectively, as described (Chuang et al., 2003).

Fig. 2. Effect of Ganoderma extracts on human colorectal cancer Colo205 cells. (A) Inhibition of cell growth was demonstrated in Colo205 cells by MTT assay. Cells were treated with three methanol extracts of Ganoderma tsugae, GT1 (a), GT2 (b) and GT3 (c); one methanol extracts of Ganoderma formosanum, GF1 (e); and two methanol extracts of Ganoderma lucidium, GL1 (f) and GL2 (g) as well as one water extracts of GT2 (d) at the indicated concentrations for 3 days. The Colo205 cells treated with Taxol were used as positive control (h). Data were expressed as the percentage of positive cells compared to the vehicle-treated control group. (B) Colo205 cells were treated with various doses of Ganoderma tsugae extracts (0.3, 0.75, 1.5, 2.25 and 3.0 mg/ml) for 72 h. Cell viability was measured by MTT assay and the results were presented as the calculated cell survival rate. Values are presented as mean ± S.E. of three independent experiments. * P < 0.01 and ** P < 0.001 versus the vehicle-treated control group.

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2.7. Laboratory animals and tumorigenicity assay Four to five week-old nude mice were obtained and bred in the Animal Center of NDMC, Taipei, Taiwan. Care and use of the animals was in accordance with institutional guidelines (Confirmed by AAALAC, USA). Animal experiments were performed as described (Wang et al., 2007). Briefly, 5 × 106 viable Colo205 cancer cells were subcutaneously inoculated into a dorsal site in nude mice. When tumors grew to approximately 100–200 mm3 in size, the mice were sorted into experimental groups of eight animals each for further testing. Ganoderma tsugae extracts were orally administered in 10 mg/ml of suspension solution to the nude mice. Either the vehicle or extract was given p.o. once/day for three weeks at a dose of 0.1 ml/10 g of body weight. Tumor growth was monitored daily for three weeks, at which point the animals were sacrificed. Biochemical and hematological measurements were evaluated for the safety of the drug as described by Xing et al. (1997). 2.8. Histochemical and immunohistochemical (IHC) stains Xenografted Colo205 tumors and the surrounding mouse tissues were excised, fixed in neutral formalin, embedded in paraffin, and sliced for hematoxylin and eosin (H&E) staining to measure the extent of necrosis and mitotic figures. Proliferation was also measured by Ki-67 IHC analysis. The preparation of samples for H&E staining and IHC was performed as previously described (Li et al., 2005). Five high-power fields (5 HPFs, 400×) of H&E-stained slides were counted using the image selection function of Adobe Photoshop, Version 7.0 (Adobe Systems, CA). The ratio of necrosis was defined as the area of the pale-stained, necrotic region of the tumor divided by the total tumor area on the section. The number of mitotic figures was counted in 5 HPFs of H&E-stained, necrosis-free areas. The anti-human Ki-67 antibody (Clone MIB-1, Dakocytomation, mouse mAb; 1:150 dilution) was used as the primary antibody. Any nuclear staining, regardless of intensity, was considered positive for Ki-67. The Ki-67 labeling index (LI) was expressed as the percentage of tumor cells with an immunoreactive nucleus in a sample size of one thousand tumor cells. 2.9. Statistical analysis All data represent mean ± standard error (mean ± S.E.) from three independent experiments. Statistical analysis was performed by Student’s t test. Differences between treatment groups were analyzed for significance by multiple comparisons using the analysis of variance. A P value of <0.01 was considered statistically significant. * P < 0.01 and ** P < 0.001 versus the vehicle-treated control group. 3. Results 3.1. Analysis of Ganoderma sp. by TLC and HPLC Of the numerous species of Ganoderma, this study classified seven samples of Ganoderma originating from different places in Taiwan by the morphological characteristics of the fruiting body and TLC chromatograms of their triterpenoids (Su et al., 2001). As Fig. 1A shows, the seven samples could be grouped into the following three species: Ganoderma lucidum, Ganoderma formosanum and Ganoderma tsugae. These three species are among the most common Ganoderma species used in East Asian folk medicine, but the morphology of Ganoderma tsugae is more easily confused. Therefore, HPLC analysis of the triterpenoids was performed, not only

to differentiate and confirm the identification of different species (Fig. 1B), but also to quantify the bioactive molecules in methanol extracts of Ganoderma lucidum and Ganoderma tsugae (Su et al., 2001). The analytical results showed that the small molecule content of Ganoderma tsugae methanol extracts is greater than for Ganoderma lucidum.

3.2. The Ganoderma extracts inhibit colorectal cancer cell proliferation Fig. 2 shows the results of experiments in which the anti-tumor effects of the methanol extracts from three species of Ganoderma were examined in human colorectal cancer cells by MTT assay. The methanol extracts from both Ganoderma lucidum and Ganoderma tsugae significantly inhibited the growth of colorectal cancer cells as measured by MTT analysis in Colo205 cells at 72 h compared to vehicle-treated controls (Fig. 2A, a–c and f–g). For growth inhibition, the effective concentration of methanol extracts from both Ganoderma lucidum and Ganoderma tsugae ranged from 1 to 3 mg/ml with a GI50 of approximately 1.8 mg/ml. In contrast, exposure of cells to water extracts of Ganoderma tsugae (>1000 mg/ml) (Fig. 2A, d) and methanol extracts of Ganoderma formosanum (>3 mg/ml) (Fig. 2A, e) had no effect on Colo205 colorectal cancer cells in culture. Cells were also treated with Taxol for experimental reference (Fig. 2A, h). Furthermore, colorectal cancer cells were incubated with various doses of Ganoderma tsugae extracts (GT3), and cell viability was determined. Our results revealed that cell viability decreased in response to dose-dependent treatment with 0.3–3 mg/ml of Ganoderma tsugae extracts (Fig. 2B). We next sought to evaluate whether the observed inhibition of cell growth in cancer cells treated with Ganoderma tsugae extracts was due to cell death or cell cycle arrest. To do this, we performed a Trypan blue exclusion assay to assess the viability of the Colo205 cells. As Fig. 3 shows, a significant difference in cell number was noted between vehicle-treated and extract-treated cells. Additionally, the Trypan blue exclusion assay revealed the extract-treated cells (3.0 mg/ml) maintained a stable number of cells for up to 48 h after treatment, suggesting that the growth inhibition observed in Colo205 cells was not due to cytotoxicity. Taking these findings together, we postulated that G. tsugae extracts caused the observed inhibition of Colo205 cell growth by arresting the cell cycle.

Fig. 3. Inhibition of Colo205 cell growth by Ganoderma tsugae extracts. Cell numbers were counted via Trypan blue stain after treatment for 0.5, 2, 4, 6, 8, 16, 24 and 48 h. Cell growth was inhibited after treatment for 24- and 48-h by 3 mg/ml of Ganoderma tsugae extracts but not by 0.3 mg/ml.

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3.3. Ganoderma tsugae extracts accumulate Colo205 cells in G2 /M phase To determine the effects of Ganoderma tsugae extracts on the cell cycle, cell cycle phase distributions were examined by flow cytometry. As Fig. 4A shows, the proportion of cells in G2 /M phase was markedly increased after treatment with increasing concentrations of Ganoderma tsugae extracts. Similarly, using increasing concentrations of Taxol to treat Colo205 cells also increases the percentage of cells in G2 /M phase. Since the progression of the cell cycle is largely controlled by cyclins, we examined these proteins as molecular markers of the cell cycle by immunoblotting. Using these markers, our aim was to validate the effects of Ganoderma tsugae extracts on the cell cycle

in Colo205 cells. Cyclin D and cyclin E are known to drive the progression of a cell through G1 into S phase. The cyclin A is important for G2 phase. Lastly, cyclin B1 interacts with CDK1 to form an active complex necessary for entry into mitosis. The expression of cyclin B1 starts in late S phase and remains elevated until its degradation in anaphase of mitosis (Akli and Keyomarsi, 2003; Muraoka-Cook et al., 2006). As Fig. 4B illustrates, treatment of Colo205 with Ganoderma tsugae extracts decreased cyclin A and B1, but not cyclin D and E. We also examined the involvement of cyclin dependent kinases (CDKs), which promote cell cycle progression, and the CDK inhibitors, p21 and p27. Increased expression of p21 and p27 was noted in Colo205 cells treated with Ganoderma tsugae extracts for 24 h (Fig. 4B). Taken together, these results demonstrate that treatment with Ganoderma

Fig. 4. Effect of Ganoderma tsugae extracts on cell cycle transition in Colo205 cells. (A) Different concentrations of Ganoderma tsugae extracts (0.3, 1.5 and 3.0 mg/ml) (upper panel) or Taxol (1, 10 and 100 ng/ml) (lower panel) were added and incubated for 24 h, and cellular DNA content was measured by FACS analysis. Both treatment of Ganoderma tsugae extracts and Taxol caused cell growth arrest at G2 /M phase of Colo205 cells. (B) Effect of Ganoderma tsugae extracts on cyclin and CDK inhibitor protein levels. The value below the figures represents the change in protein expression normalized against actin.

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Fig. 5. Inhibition of Colo205-induced tumor growth by Ganoderma tsugae extracts in nude mice. Tumor volumes were estimated from caliper measurements of three dimensions of the tumor. Estimated tumor weight was calculated as L × W2 × 0.5, where L is the major axis, and W is tumor width. Data are represented as mean ± S.E. (n = 8).

mice. Mice were first given oral doses of Ganoderma tsugae extracts for 21 days to determine maximum tolerable dose (data not shown). After the tumor volume reached approximately 100–200 mm3 , the mice were treated for three weeks p.o. with either Ganoderma tsugae extracts (dissolved in EtOH) at 100 mg/kg daily, or vehicle only. Fig. 5 shows that mice treated with Ganoderma tsugae extracts exhibited a marked suppression in Colo205 tumor growth relative to vehicle-treated controls. These results demonstrate that Ganoderma tsugae extracts can suppress tumor growth in vivo (Fig. 5). Tumor xenografts were stained with H&E, which revealed markedly more advanced necrosis (white zones) in extract-treated tumors than controls (Fig. 6, A1 vs. A2). Additionally, both mitotic counts and proliferation (as measured by Ki67-LI) were decreased after Ganoderma tsugae treatment (Fig. 6, B1 vs. B2; C1 vs. C2), indicating that Ganoderma tsugae extracts both induce cell death and inhibit cell proliferation in human colorectal cancer cells in vivo. To investigate the safety of Ganoderma tsugae extracts, pathologic examinations, clinical chemistry, and hematological analyses were performed. Several organs were examined by histopathology for visceral congestion, inflammation, hemorrhage or hyperplasia: brain, heart, liver, spleen, lung, kidney, stomach, adrenal glands, and intestine. No significant microscopic aberrations were noted comparing to vehicle-treated controls (data not shown). Moreover, peripheral blood samples were assayed by both clinical chemistry and hematological analyses. Importantly, the nude mice apparently tolerated repeated treatment with Ganoderma tsugae extracts,

tsugae extracts leads to the downregulation of cyclin A and B1 and upregulation of p21 and p27, causing G2/M arrest in Colo205 cells. 3.4. Ganoderma tsugae extracts cause regression of Colo205 xenografted tumors To analyze the in vivo anti-tumor effect of Ganoderma tsugae extracts, xenografts of Colo205 cells were implanted into nude

Fig. 6. H&E staining and IHC analysis of representative histological sections taken from tumors of mice inoculated with Colo205 cells. Ganoderma tsugae extract-treated mice had more extensive necrosis (white zones, necrotic ratio: 57% in A2) and a larger tumor mass than controls (necrotic ratio: 3% in A1). Sections from control mice showed more mitotic figures (B1, 99/5HPFs) than Ganoderma tsugae extract-treated mice (B2, 37/5 HPFs). The proliferation index was higher in control mice (C1, positive brown-red nuclei count, Ki67-LI: 2.4) than in Ganoderma tsugae extract-treated mice (C2, Ki67-LI: 1.6). White lines and arrows indicate scale bars and mitotic figures, respectively.

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Table 1 Biochemical and hematological profiles in mice following twenty-one daily doses of the Ganoderma tsugae extracts by oral administration. Itema BUN (mg/dl) Creatinine (mg/dl) sGOT (IU/l) sGPT (IU/l) Sodium (mmol/l) Potassium (mmol/l) Chloride (mmol/l) Magnesium (mg/dl) WBC (mm3 ) Neutro-Seg % Neutro-Seg (mm3 ) Lymph % Lymph (mm3 ) Mono % Mono (mm3 ) RBC (106 /mm3 ) Hb (g/dl) Hct (%) MCV ( m6 ) MCH (pg) MCHC (%) Platelet (103 /mm3 ) Vehicleb 14.5 0.2 359.0 45.4 151.8 9.8 132.4 2.2 2650.0 65.6 1744.9 31.4 825.9 3.1 82.9 6.0 10.2 30.3 50.8 17.0 33.5 260.1 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.7 0.0 42.2 7.0 1.3 0.5 1.7 0.1 174.4 0.5 122.3 0.6 47.3 0.2 9.3 0.2 0.4 1.1 0.4 0.1 0.2 26.2 Ganoderma tsugaeb 17.6 0.2 270.8 38.9 154.9 9.0 136.6 2.0 1821.4 62.7 1158.1 34.9 619.6 2.4 43.7 5.5 9.2 28.0 50.8 16.7 32.9 274.0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2.0 0.0 37.1 5.6 0.9 0.5 1.0 0.1 271.4 0.8 189.4 0.8 77.8 0.1 5.8 0.2 0.3 1.0 0.4 0.1 0.2 41.4

a BUN, blood urea nitrogen; sGOT, serum glutamic-oxaloacetic transaminase; sGPT, serum glutamic-pyruvic transaminase; WBC, white blood cell; Neutro-Seg, segmented neutrophil; Lymph, lymphocyte; Mono, monocyte; RBC, red blood cell; Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration. b Values represented as mean ± S.E.

also revealed that the Ganoderma tsugae methanol extracts induced Colo205 cancer cell arrest at G2 /M phase (Fig. 4), which is similar to the previous report that the Ganoderma lucidum ethanol extracts caused G2 /M phase arrest in hepatoma cells (Lin et al., 2003). Cell cycle arrest in the G2 phase can be triggered by stress from several different stimuli. A recent report demonstrated that microtubule-interacting agents (i.e., Taxol) influence the expression of several important regulators of the G2 /M transition. For example, p34cdc2 and topoisomerase II-alpha (TOP2A) were both found to be downregulated by agents targeting microtubules, while the CDK inhibitor, p21 was upregulated (Chen et al., 2003; Bani et al., 2004; Skladanowski et al., 2005). Our Western blotting results demonstrate that Ganoderma tsugae extracts upregulate p21 and p27 and downregulate cyclin A and cyclin B1 (Fig. 4B). However, further investigation is required to better understand the molecular mechanisms by which Ganoderma tsugae extracts affect these cell cycle regulators in Colo205 human colorectal adenocarcinoma cells. In summary, this study provides molecular evidence that Ganoderma tsugae extracts exert anti-tumor effects both in vitro and in vivo on colorectal adenocarcinoma cells by inducing G2 /M cell cycle arrest. More importantly, based on the biochemical, hematological (Table 1) and pathological examinations, no significant physiological toxicities resulting from treatment with Ganoderma tsugae extracts were observed in the in vivo animal model. Therefore, Ganoderma tsugae extracts may prove clinically useful as an anticancer treatment for human colorectal adenocarcinoma.

Acknowledgements showing no adverse effects on renal, hepatic, and hematological parameters (Table 1). 4. Discussion Some chemopreventive extracts of herbs or plants, including Ganoderma (Lingzhi), are known to be anti-tumorigenic. Among them, Ganoderma lucidum and Ganoderma tsugae are the most widely used Ganoderma species for folk remedies in Asia. The antitumor effects of Ganoderma are apparently mediated by numerous biologically active compounds, such as polysaccharides, triterpenes and immunomodulatory proteins (Sliva et al., 2002). Because of variations in the composition of extracts, as well as differences in culturing methods and climate, individual Ganoderma species may differ in their bioactivity. Use of TLC and HPLC (Su et al., 2001) in this study enabled the easy identification and classification of species from the Ganoderma genus (Fig. 1). Furthermore, as Fig. 2A shows, the antitumor bioactivity of Ganoderma in Colo205 colorectal cancer cells was observed in Ganoderma lucidum and Ganoderma tsugae, but not in Ganoderma formosanum. It has been widely reported that Ganoderma lucidum extracts interfere with the cell cycle to act as anticancer agents. Ganoderma lucidum alcohol extracts were found to arrest the cell cycle in G1 /S phase in cervical tumor HeLa cells and breast tumor MCF-7 cells (Zhu et al., 2000; Hu et al., 2002); similarly, Ganoderma lucidum hot water extracts and triterpene-enriched, soluble ethanol fractions caused G2 /M phase arrest in hepatoma cells (Lin et al., 2003). The differential effects may be due to variations in the composition of the extracts (Tang et al., 2006). However, little is known about the anti-tumor properties and mechanisms of action of methanol extracts of Ganoderma tsugae. This study demonstrated that treating human colorectal Colo205 tumor cells with Ganoderma tsugae extracts induced necrosis and inhibited tumor cell growth in a dose-dependent manner, both in vitro (Figs. 2 and 3) and in vivo in a xenograft model (Figs. 5 and 6). In addition, our experiments This work was supported by the Department of Health (DOH93TD-F-113-030 & DOH94-TD-F-113-023), Taiwan, ROC, and the China Medical University (CMU95-296), Taichung, Taiwan, granted to M.C.K.

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