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Nutrition and Cancer

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Triterpenes From Ganoderma Lucidum Induce Autophagy in Colon Cancer Through the Inhibition of p38 Mitogen-Activated Kinase (p38 MAPK)
Anita Thyagarajan , Andrej Jedinak , Hai Nguyen , Colin Terry , Lee Ann Baldridge , Jiahua Jiang & Daniel Sliva
a b a a b a a a a b

Methodist Research Institute, Indianapolis, Indiana, USA Indiana University School of Medicine, Indianapolis, USA

Available online: 22 Jun 2010

To cite this article: Anita Thyagarajan, Andrej Jedinak, Hai Nguyen, Colin Terry, Lee Ann Baldridge, Jiahua Jiang & Daniel Sliva (2010): Triterpenes From Ganoderma Lucidum Induce Autophagy in Colon Cancer Through the Inhibition of p38 MitogenActivated Kinase (p38 MAPK), Nutrition and Cancer, 62:5, 630-640 To link to this article: http://dx.doi.org/10.1080/01635580903532390

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Nutrition and Cancer, 62(5), 630640 Copyright C 2010, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635580903532390

Triterpenes From Ganoderma Lucidum Induce Autophagy in Colon Cancer Through the Inhibition of p38 Mitogen-Activated Kinase (p38 MAPK)
Anita Thyagarajan, Andrej Jedinak, Hai Nguyen, and Colin Terry
Methodist Research Institute, Indianapolis, Indiana, USA

Lee Ann Baldridge

Indiana University School of Medicine, Indianapolis, USA

Jiahua Jiang
Downloaded by [University of Windsor] at 13:24 14 February 2012
Methodist Research Institute, Indianapolis, Indiana, USA

Daniel Sliva
Methodist Research Institute, Indianapolis, Indiana, and Indiana University School of Medicine, Indianapolis, Indiana, USA

Medicinal mushroom Ganoderma lucidum is one of the most esteemed natural products that have been used in the traditional Chinese medicine. In this article, we demonstrate that G. lucidum triterpene extract (GLT) suppresses proliferation of human colon cancer cells HT-29 and inhibits tumor growth in a xenograft model of colon cancer. These effects of GLT are associated with the cell cycle arrest at G0/G1 and the induction of the programmed cell death Type IIautophagy in colon cancer cells. Here, we show that GLT induces formation of autophagic vacuoles and upregulates expression of Beclin-1 (1.3-fold increase) and LC-3 (7.3-fold increase) proteins in colon cancer cells and in tumors in a xenograft model (Beclin-1, 3.9-fold increase; LC-3, 1.9-fold increase). Autophagy is mediated through the inhibition of p38 mitogen-activated protein kinase (p38 MAPK) because p38 MAPK inhibitor, SB202190, induces autophagy and expression of Beclin-1 (1.2-fold increase) and LC-3 (7.4-fold increase), and GLT suppresses phosphorylation of p38 MAPK (60% inhibition) in colon cancer cells. Taken together, our data demonstrate a novel mechanism responsible for the inhibition of colon cancer cells by G. lucidum and suggest GLT as natural product for the treatment of colon cancer.

INTRODUCTION Colon cancer is a leading contributor to cancer prevalence and mortality in the Western world, and in the United States, it is the third most common diagnosed cancer with an estiSubmitted 20 March 2009; accepted in nal form 26 October 2009. Address correspondence to Daniel Sliva, Cancer Research Laboratory, Methodist Research Institute, 1800 N. Capitol Ave., E504, Indianapolis, IN 46202. Phone: 317-962-5731. Fax: 317-962-9369. E-mail: dsliva@clarian.org

mated 50,000 deaths in 2009 (1). The association among diet, colon cancer risk, and mortality is well established (2), and an inverse correlation between mushroom intake and the risk of gastric cancer has been demonstrated (3). Ganoderma lucidum is one of the important Asian fungi that were recognized in China and Korea as Ling Zhi (mushroom of immortality) and in Japan as reishi mushroom or mannentake (10,000 yr mushroom) more than 4,000 yr ago (4). Although G. lucidum was originally used to improve health and promote longevity, its potential therapeutic effects were recognized in traditional Chinese medicine for the treatment of a variety of diseases. Two major groups of biologically active compounds isolated from G. lucidum are polysaccharides (mainly glucans and glycoproteins) and lanostane-type triterpenes (ganoderic acids, ganoderic alcohols, and their derivatives) (5). Most of the anticancer effects of G. lucidum polysaccharides have been attributed to the modulation of the immune system through the activation of macrophages, neutrophils, dendritic cells, natural killer cells, and T- and B-lymphocytes (6,7). Mechanistically, G. lucidum polysaccharides (GLP) induced cytokine expression via Tolllike receptors (TLR)-4 in macrophages and dendritic cells; whereas immunoglobulin production was mediated through TLR-4/TLR-2 in B-lymphocytes (8,9). The anticancer activities of G. lucidum triterpenes were originally demonstrated by their cytotoxic/killing effects on hepatoma (10), naso-pharynx carcinoma (11), lung carcinoma and sarcoma (12), breast cancer (13), and leukemia cells (14). Mechanistically, these triterpenes: 1) inhibited activity of farnesyl protein transferase (FPT) (15); 2) demonstrated antioxidative activity (16); 3) inhibited the activity of 5-reductase (17); or 4) suppressed the activities of

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HMG-CoA reductase and acyl-CoA acyltransferase in the mevalonate pathway (18). G. lucidum extract (GLE), containing polysaccharides and triterpenes, was reported to suppress growth and metastatic potential of the highly invasive human breast cancer cells MDA-MB-231 by inhibiting the activity of AKT kinase and transcription factors AP-1 and NF-kB resulting in the downregulation of expression of cyclin D1 and urokinase plasminogen activator (19,20). Moreover, GLE modulated estrogen receptor signaling (21) and inhibited the oxidative stressinduced invasiveness by the suppression of interleukin-8 (IL-8) secretion from breast cancer cells MCF-7 (22). GLE was found to inhibit proliferation of prostate cancer cells PC-3 by downregulating cyclin B and Cdc2 and upregulating p21 expression, which was shown also by cell-cycle arrest at G2/M phase; whereas the induction of apoptosis of PC-3 cells was associated with the upregulation of expression of proapoptotic Bax (23). GLE also inhibited angiogenesis of vascular endothelial cells by suppressing secretion of the proangiogenic factors vascular endothelial growth factor (VEGF) and transforming growth factor- b1 (TGF-b1) from PC-3 cells (24). In this study, we evaluated G. lucidum triterpene extract (GLT) on the in vitro growth and in the xenograft model of human colon cancer cells HT-29 in vivo. Here, we show that GLT suppresses growth of HT-29 cells through cell cycle arrest at the G0/G1 phase and by the induction of the programmed cell death Type II, autophagy. Moreover, GLT also inhibits growth of tumors in a xenograft model of colon cancer. In summary, our data suggest possible molecular mechanism by which triterpene extract from medicinal mushroom Ganoderma lucidum inhibits growth of colon cancer cells.

Cell Proliferation Assay Cell proliferation was determined by the MTT assay, according to the manufacturers instructions (Promega, Madison, WI). Briey, HT-29 cells were cultured in a 96-well plate and treated at indicated times with GLT (01.0 mg/ml). At the end of the incubation period, an absorption was determined with a micro plate reader at 570 nm as described previously (19). Data points represent mean SD in the representative experiment of triplicate determinations. Similar results were obtained in two independent experiments. Flow Cytometry Analysis HT-29 cells (1 106) were seeded in 100 mm dishes and after 24 h treated with GLT (00.25 mg/ml) for 0 to 48 h. The cells were harvested, xed with 3 ml ice-cold ethanol at 4 C for 1 h and stained with 50 g/ml of propidium iodide. Cell cycle analysis was performed on a FACStarPLUS ow cytometer (Becton-Dickinson, San Jose, CA) as previously described (27). Data are the mean SD from 3 independent experiments. Autophagy HT-29 cells were plated in a 6-well plate and treated with GLT (00.5 mg/ml) or SB202190 (20 M) in the absence or presence of 3MA (10 mM). The formation of autophagic vacuoles was observed under the phase contrast microscope at 40 magnication over a period of 0 to 24 h. Immunouorescent Detection of Autophagy HT-29 cells were plated into Lab-Tek II 8 chamber culture slides (Nalge Nunc International, Naperville, IL) and allowed to attach at 37 C for 48 h. Cells were then treated with GLT (0.25 mg/ml) and incubated for 6 h at 37 C. Subsequently, the cells were rinsed briey with PBS and then xed with 4% formaldehyde-PBS for 15 min at room temperature. Later the cells incubated with ice cold methanol for 10 minutes in the freezer, followed by a brief rinse with PBS. Fixed cells were then blocked using 5% normal goat serum in PBS/Triton for 1 h, followed by incubation in LC-3 primary antibody for 24 h at 4 C, washed and incubated with AlexaFluor 488-conjugated goat antirabbit secondary antibody for 2 h at room temperature in the dark, rinsed with PBS, and counterstained with 4 ,6diamidino-2-phenylindole (DAPI). Images were obtained using an Olympus BX40 microscope at 50 magnication with an oil immersion lens. Picture frame software (Optronics, Goleta, CA) was used to obtain all images at similar intensities. Human Colon Tumor Xenograft Experiments HT-29 cells (5 106) in 0.2 ml DMEM were implanted subcutaneously in the right ank on the ventral side of the 6-wk-old male nude mice (Harlan, Indianapolis, IN). After a week of implantation with tumor cells, when tumors reached approximately 20 to 30 mm3, the animals were randomized into control and treatment groups (10 animals/group). The animals received intraperitoneal injections (100 l) of 10% DMSO, 70% mixture

MATERIALS AND METHODS Cell Culture and Reagents Human colon cancer cells HT-29 were obtained from ATCC (Manassas, VA) and were maintained in Dulbeccos modied eagle medium (DMEM) containing penicillin (50 U/ml), streptomycin (50 U/ml), and 10% fetal bovine serum (FBS). Media and supplements came from GIBCO BRL (Grand Island, NY). FBS was obtained from Hyclone (Logan, UT). GLT was obtained from Pharmanex (Provo, UT; batch number 050607; Shanghai R&D, Pharmanex). GLT contains a mixture of lanostanoid triterpenes, which were previously isolated and identied by nuclear magnetic resonance and mass spectrometry (18,25,26). GLT was dissolved in DMSO (Sigma, St. Louis, MO) at the concentration 40 mg/ml and stored at 4 C (the nal concentration of DMSO in controls and GLT was 0.3%). 3-methyl adenine (3MA) was purchased from Sigma (St. Louis, MO) and dissolved in Dulbeccos phosphate-buffered saline (PBS; Cambrex Bio Science Walkersville, Inc., Walkersville, MD). Propidium iodide, absolute ethanol, and cremophor were purchased from Sigma. SB202190 was from Calbiochem (San Diego, CA).

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of ethanol and cremophor (3:1), and 20% PBS (control) or 6 mg GLT/kg of body weight dissolved in DMSO and adjusted to the nal concentration of 10% DMSO, 70% mixture of ethanol and cremophor (3:1), and 20% PBS (treatment group) daily for 23 days. The tumor size was measured using calipers, and the tumor volume was estimated by the formula tumor volume (mm3) = (W L) 2 1/2, where L is the length and W is the width of the tumor. At the end of the experiment (Day 23), the tumors were dissected, xed in 10% neutral formalin or snap frozen, and stored separately in liquid nitrogen. The tumor sections were analyzed by immunohistochemistry. The protocol for animal experiments was approved by the Animal Research Committee at the Methodist Hospital according to the NIH guidelines for the care and use of laboratory animals. Tumor Extracts Tumors from control and GLT-treated mice were washed with ice-cold PBS, minced, and homogenized in the lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM PMSF, 0.1 mM sodium vanadate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, and 50X Complete solution (Roche, Indianapolis, IN). The homogenate was incubated at 4 C for 30 min and the lysate cleared by centrifugation at 14,000 rpm for 30 min. Western Blot Analysis HT-29 cells treated with GLT (01.0 mg/ml) as indicated in the text and whole cell extracts prepared as previously described (19). Equal amounts of cellular (20 g/lane) and tissue (30 g/ml) proteins were separated on NuPAGE 4 to 12% BisTris gel or 12% Tris-Glycine gel (Invitrogen, Carlsbad, CA) and transferred to a PVDF membrane (Millipore, Bedford, MA). The protein expression was detected with the corresponding primary antibodies: anti-Beclin-1, anti-LC-3, anti-p38, anti-phosphop38 (Cell Signaling, Beverly, MA), and anti-GAPDH antibody (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. Protein expression was visualized using the ECL Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, UK). Immunohistochemistry Immunohistochemistry was performed on 5 m sections of formalin-xed, parafn-embedded samples. For antigen retrieval, the slides were boiled for 10 min in citrate buffer followed by an overnight incubation at 4 C with a primary monoclonal antibody against Ki-67 (NeoMarker, Fremont, CA), Beclin-1, and LC-3 (Abgent, San Diego, CA). Densitometric Analysis Autoradiograms of the Western blots were scanned with HP Scanjet 5470c scanner (Hewlett-Packard, Palo Alto, CA). The optical densities of Beclin-1, LC-3, phospho-p38, p38, and GAPDH proteins on the lms were quantied and analyzed with the UN-SCAN-IT software (Silk Scientic, Orem, UT).

The ratios of specic proteins to GAPDH were calculated by standardizing the ratios of each control to the unit value. Statistical Analysis Cell cycle distribution data and proliferation data were summarized using descriptive statistics (mean, SD). For each cell cycle phase, the effect of GLT over time on cell cycle distribution was analyzed using a 2-way ANOVA model, and the interaction between time and treatment was assessed. If a signicant interaction was found, Tukeys post hoc test was then used to compare mean values at different levels of the 2-way interaction. Proliferation data was compared across treatment groups at both time points (24 h and 48 h) using 1-way ANOVA models. Dunnetts post hoc test was used to compare mean values for each treatment group to the control. Tumor volume measurements were summarized by treatment group using descriptive statistics (mean, SD). The comparison of the change in tumor volume, immunohistochemical staining, and vacuole formation between groups was performed using a 2-sample Student t-test. Results were considered signicant at P 0.05.

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RESULTS GLT Inhibits Proliferation of Colon Cancer Cells and Growth of Xenograft Tumors In Vivo As mentioned previously, G. lucidum demonstrated cytostatic/cytotoxic effects against a variety of cancer cells. Therefore, we evaluated whether GLT also affects growth of human colon cancer cells HT-29. As seen in Fig. 1A, increased concentration of GLT (01.0 mg/ml) markedly suppressed proliferation of HT-29 cells. At the low/moderate dosage, GLT exhibits cytostatic activity; whereas at higher concentrations, cytotoxic activity is observed. Both the cytostatic and cytotoxic effects of GLT are statistically signicant (P < 0.001). To investigate whether GLT suppresses growth of colon tumors in vivo, we determined the effect of GLT on the growth of established colon cancer HT-29 xenograft tumors. Therefore, HT-29 cells were subcutaneously injected in the right ank of the nude mice as described in Materials and Methods. After the tumors reached size to 20 to 30 mm3, mice were randomly assigned to control (vehicle) and GLT treatment groups. Mice bearing established HT-29 tumors were given intraperitoneal injections of vehicle only or 6 mg/kg of GLT daily for 23 days. As seen in Fig. 1B and Table 1, GLT treatment signicantly (P = 0.012) suppressed the growth of HT-29 tumors (nal volume = 650.2 283.8 mm3; change in volume = 631.7 276.5 mm3) when compared with the vehicle control group (nal volume = 1,227.1 584.3 mm3; change in volume = 1,203.6 589.9 mm3). In addition, GLT markedly suppressed proliferation of colon cancer cells in vivo as demonstrated by the decrease in the staining with Ki-67 in the tumor tissue from the animals treated with GLT (Fig. 1C; P < 0.005).

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FIG. 1. Ganoderma lucidum triterpene extract (GLT) inhibits growth of colon cancer cells in vitro and in vivo. HT-29 cells were treated with GLT (0, 0.06, 0.125, 0.250, 0.5, and 1.0 mg/ml) for 24 and 48 h. Proliferation was assessed as described in Materials and Methods. Data are the mean SD of triplicate determinations and compared across treatment groups at each time point using ANOVA. Dunnetts post hoc test was used to compare each treatment group to the control. indicates a signicant difference between treatment and control. Statistical signicance, , P < 0.001. Similar results were received in at least two additional experiments. B: HT-29 cells were injected subcutaneously into nude mice and treated with vehicle (empty circles) or 6 mg GLT/kg of body weight daily for 23 days as described in Materials and Methods. Data are the mean SD (n = 10 mice/group). Statistical analysis for the changes in the nal tumor volumes is described in the Table 1. C: Dissected tumors from animals treated with vehicle (control) or 6 mg GLT/kg of body weight (GLT) were sectioned and stained with Ki-67 as described in Materials and Methods. Data are the mean SD (n = 4). , P < 0.005. D: Body weight of nude mice with HT-29 xenografts, which were treated with vehicle (empty circles) or 6 mg GLT/kg of body weight daily for 23 days as described in Materials and Methods. Data are the mean SD (n = 10 mice/group).

Finally, the therapeutic dose (6 mg GLT/kg of body weight) was not toxic to tested animals as assessed by body weight determinations of the treated mice when compared with the control group (Fig. 1D). In addition, GLT treatment did not change the weight of spleen and liver as compared to the control group (data not shown). Therefore, GLT inhibits growth of colon cancer cells in vitro and suppresses growth of colon tumors in animal experiments.

GLT Treatment Induces Cell Cycle Arrest at G0/G1 and Autophagy in HT-29 Cells Dietary/natural, agent-induced, cell cycle arrest is usually prerequisite to the demise of cancer cells, which can be mediated by the apoptotic or nonapoptotic, autophagy or necrosis, pathways (28). To determine whether GLT induces cell cycle arrest, HT-29 cells were treated with GLT at indicated times and analyzed by ow cytometry as described in Materials and

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TABLE 1 Effect of GLT on the tumor growth in vivoa Volume Baseline (mm3) Final (mm3) Change (mm3) Control 23.5 7.8 1, 227.1 584.3 1, 203.6 589.9 GLT (6 mg/kg) 18.5 10.0 650.2 238.8 631.7 276.5b

a Abbreviation is as follows: GLT, Ganoderma lucidum triterpene extract. Tumor volume was summarized by treatment group using descriptive statistics (mean, SD). The comparison of the change in tumor volume between groups was performed using a 2-sample Student t-test. b Statistical signicance P = 0.012 for the change in tumor volume in animals treated with GLT (6 mg/kg of body weight) vs. control (0 mg/kg of body weight; n = 10 mice per group).

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Methods. As seen in Table 2, the treatment of HT-29 cells with 0.25 mg/ml of GLT signicantly increased the cell population at G0/G1 after 24 and 48 h, respectively (68% compared to 48% in control at 24 h and 73% compared to 62% in control at 48 h; P < 0.001). Interestingly, GLT treatment did not induce the amount of cells at sub-G0/G1 phase, suggesting that the inhibition of growth of HT-29 cells is independent of apoptosis (Table 2, Fig. 2). In addition, we were not able to detect any apoptotic DNAladdering in HT-29 cells treated with GLT (data not shown), suggesting that nonapoptotic cell death can be induced by GLT treatment. To examine this hypothesis, HT-29 cells were treated with GLT (01.0 mg/ml) for 3, 6, and 24 h and the formation of autophagic vacuoles evaluated by the phase microscopy. Therefore, GLT induced the formation of vacuoles in HT-29 cells in a time- and dose-response manner (Figs. 3a, 3b, 3c; only 6 h treatment is shown; P < 0.001). As recently demonstrated, inhibition of p38 pathway can induce autophagic death in colon cancer cells HT-29 (29). As expected, the treatment of HT-29

cells with p38 inhibitor SB202190 (20 M, 6 h) also induced autophagy (Fig. 3d; P < 0.001). The pretreatment of HT-29 cells with 3MA, a well known inhibitor of autophagic process (30), prevented the formation of vacuoles in cells treated with GLT and SB202190 (Figs. 3e, 3f, 3g; P < 0.005). Since the induction of autophagy in mammalian cells is associated with the upregulation of expression of autophagic proteins Beclin-1 and LC-3 (31,32), we examined whether GLT and SB2092190 induces their expression in colon cancer cells. HT-29 cells were treated with GLT (01.0 mg/ml, 6 h), and the expression of Beclin-1 and LC-3 was evaluated in the whole cell lysates by Western blot analysis. As seen in Fig. 4A, although the GLT treatment only slightly induced the expression of Beclin-1, the expression of LC-3 was markedly induced in HT-29 cells treated with GLT. To examine whether the inhibition of p38 induces expression of Beclin-1 and LC-3 in colon cancer cells, HT-29 cells were treated with p38 inhibitor SB202190 (20 M) at indicated times and the expression of Beclin-1 and LC-3 evaluated as described previously. As expected, the inhibition of p38 markedly induced expression of LC-3, whereas the same treatment only slightly induced the expression of Beclin1 in HT-29 cells (Fig. 4B). To further evaluate whether GLT itself modulates the activity of p38 in colon cancer cells, HT29 cells were treated with GLT (01.0 mg/ml) for 0, 3, and 6 h, and the expression of phospho-p38 was evaluated in the whole cell lysates by Western blot analysis. As seen in Fig. 4C, GLT markedly decreased phosphorylation of p38 in a dose- and time-dependent manner. The localization of LC-3 protein to the membranes of the autophagic vacuoles is an additional conrmation of autophagy. Therefore, we used an immunohistochemical approach to detect the localization of LC-3 in HT-29 cells treated with GLT and SB202190. As seen in Fig. 5, both GLT and SB202190 induced the expression of LC-3 in HT-29 cells; and LC-3 was mainly localized in the autophagic vacuoles. Therefore, our data suggest

TABLE 2 Effect of GLT on cell cycle distributiona Time (h) 0 24 48 0 24 48 P value GLT (mg/ml) 0 0 0 0.25 0.25 0.25 G0/G1 40 0.5a 48 0.6b 62 1.4c 40 0.5a 68 1.1d 73 1.2e <0.001 S 46 0.8a 33 0.9b 21 1.3c 47 1.0a 19 1.0c 10 0.6d <0.001 G2/M 14 1.0a 19 1.0b 16 0.3c 13 1.2a 13 0.6a 16 0.6c <0.001 sub-G0/G1 0.6 0.28 1.9 0.30 1.6 0.17 1.6 1.20 2.1 0.89 2.4 0.20 0.520

a Abbreviation is as follows: GLT, Ganoderma lucidum triterpene extract. Cell cycle distribution G0/G1, S, G2/M, and sub-G0/G1 in percent. Data summarized using mean (SD) and compared across all time points and treatment groups using a 2-way analysis of variance (ANOVA) for each cell cycle phase. The P value presented is for the interaction between time and treatment from the ANOVA model. Post hoc tests between pairs of means were performed using Tukeys multiple comparison test. Within cell cycle phase, means sharing the same subscript letter are not signicantly different. Those with differing subscript letters were found to be signicantly different.

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FIG. 2. Ganoderma lucidum triterpene extract (GLT) induces cell cycle arrest at G0/G1. HT-29 cells were treated with A: 0 mg/ml of GLT or B: 0.25 mg/ml of GLT for 48 h. Cell cycle analysis was performed as described in Materials and Methods. The results are representative of 3 separate experiments.

FIG. 3. Induction of autophagy in HT-29 cells treated with Ganoderma lucidum triterpene extract (GLT). HT-29 cells were treated with a: vehicle, b: 0.25 mg/ml GLT, c: 0.5 mg/ml GLT, d: 20 M SB202190 in the absence (b, c, d) or presence of 10 mM 3-methyl adenine (3MA) for 6 h. The autophagic vacuoles were detected under the phase microscope as described in Materials and Methods. Data are representative from 3 independent experiments. The amount of vacuoles in b, c, and d were compared with the control; , P < 0.001. The amount of vacuoles in the absence or presence of 3MA (b vs. e; c vs. f; d vs. g) was also compared; , P < 0.005.

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FIG. 4. Ganoderma lucidum triterpene extract (GLT) induces expression of Beclin-1 and LC-3 and inhibits p38 mitogen-activated protein kinase in HT-29 cells. A: HT-29 cells were treated with GLT (01.0 mg/ml) for 6 h, and the expression of Beclin-1 and LC-3 was evaluated by Western blot analysis as described in Materials and Methods. The same blots were stripped and probed for glyderaldehyde-3-phosphate dehydrogenase (GAPDH) for the loading of equal proteins. The results are representative of 3 separate experiments. B: HT-29 cells were treated with p38 inhibitor SB202190 (20 M) for 0 to 6 h, and the expression of Beclin-1 and LC-3 was evaluated by Western blot analysis as described in Materials and Methods. The same blots were stripped and probed for GAPDH for the loading of equal proteins. The results are representative of 3 separate experiments. C: HT-29 cells were treated with GLT (01.0 mg/ml) for 0, 3, and 6 h; and the expression of phospho-p38 and p38 was evaluated by Western blot analysis as described in Materials and Methods. The results are representative of 3 separate experiments.

that GLT induces autophagy in HT-29 cells, and this process is associated with increased expression of LC-3 protein and its localization in vacuoles.

GLT Induces Autophagy in Tumors As we demonstrated previously (Fig. 1C, Table 1), GLT suppresses the growth of colon cancer HT-29 xenograft tumors. To examine whether GLT also induces autophagy in vivo, we examined the expression of LC-3 and Beclin-1 in colon tumors. To evaluate whether Beclin-1 and LC-3 expression is induced in the tumor tissue from the animals treated with GLT, the tumor tissue lysates were prepared and analyzed by Western blotting. GLT treatment induced expression of Beclin-1 and LC-3 in colon tumors in a xenograft model of HT-29 cells in mice (Fig. 6A; only representative samples are shown). Moreover, immunohistochemical analysis of the tissue sections stained with Beclin-1 and LC-3 antibodies conrmed increased levels of Beclin-1 (P < 0.001) and LC-3 (P < 0.005) in tumors in animals treated with GLT (Fig. 6B). Therefore, GLT treatment induces autophagy of colon cancer cells in vivo, and this effect is associated with increased expression of Beclin-1 and LC-3, respectively.

DISCUSSION In spite of the early detection by colonoscopy, surgical intervention, chemotherapy, and radiation therapy, colorectal cancer remains the third leading cause of cancer mortality in the United States (1). Colorectal cancer is mainly a disease of high income countries (North America, parts of Europe, Australia, New Zealand, and Japan), where overall rates are nearly 4 times higher than in middle- to low-income countries (Africa, Central America, and parts of Asia) (2). In addition to the nonWesternized dietary patterns some of the natural products are commonly used in the forms of extracts or teas in Asian countries. One of these natural products is a medicinal mushroom Ganoderma lucidum, a well known natural agent recognized in traditional Chinese medicine (4). Although recent studies have demonstrated chemopreventative effects of G. lucidum in chemically induced colon carcinogenesis in animals (33,34), and the inhibition of growth and induction of apoptosis in human colon cancer cells (35), the molecular mechanism(s) responsible for the demise of colon cancer cells by G. lucidum have not been fully addressed. In this study, we showed that GLT suppresses proliferation of HT-29 colon cancer cells in vitro and inhibits growth of colon cancer tumors in an animal xenograft model through the

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FIG. 5. Ganoderma lucidum triterpene extract (GLT) induces expression of LC-3 to autophagic vacuoles. HT-29 cells were treated with a: vehicle, b: 0.25 mg/ml GLT, and c: 20 M SB202190 for 6 h; and the expression of LC-3 was evaluated by the immunouorescent staining as described in Materials and Methods. Data are representative from 3 independent experiments. DAPI, 4 ,6-diamidino-2-phenylindole.

induction of programmed cell death (PCD): autophagy. Apoptosis is a well-known form of PCD and is widely accepted as the main mechanism of cancer cell death. Apoptosis is classied as a Type I PCD, whereas autophagy is classied as Type II PCD (28). Apoptosis as a demise of cancer cells induced by G. lucidum extracts or isolated triterpenes has been previously described (23,3544). However, we were unable to detect apoptosis in human colon cancer cells treated with GLT. Instead, we have found that GLT induces autophagy in HT-29 cells and in colon tumors as we conrmed by the presence of autophagic vacuoles and by the increased expression of proautophagic proteins Beclin-1 and LC-3. Comes et al. (29) demonstrated induction of autophagy of colon cancer cells by the inhibition of p38 MAPK. As we mentioned previously, our data clearly demonstrate that GLT inhibits activity of p38 in HT-29 cells. Therefore, inhibition of p38 by GLT may result in the induction of expression of Beclin-1 and LC-3 proteins and formation of autophagic vacuoles, nally leading to the autophagy of colon cancer cells. The induction of autophagy of colon cancer cells by the soybean B-group triterpenoid saponins though the induction of LC-3 expression has been previously reported (45). However,

the autophagy has been linked to the inhibition of AKT signaling and enhanced extracellular-regulated kinase 1/2 activity (46). In addition, a plant triterpenoid, avicin D, induced autophagy of osteosarcoma cells by activation of adenosine monophosphateactivated protein kinase (47). Alternatively, activation of p38 induced autophagy in astrocytes (48) and melanoma cells (49). Therefore, autophagy can be induced by a variety of stimuli through different cellular targets and signaling pathways, and the activation or inhibition of specic signaling pathway depends on the molecular characteristic of the particular cancer cell (50). Here, we demonstrated the anticancer effect of G. lucidum triterpenes after the intraperitoneal injection of GLT. Although this route of administration is not optimal for dietary intervention, we are able to demonstrate that GLT can suppress growth of human colon cancer cells in vivo and that the effect of GLT is systemic. Moreover, a study evaluating an oral administration of GLT in an animal model of the food-borne, carcinogenand inammation-induced colon carcinogenesis is in progress. G. lucidum is generally used in the forms of tea, powder, and dietary supplements to promote health (51). Although there is

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FIG. 6. Ganoderma lucidum triterpene extract (GLT) induces autophagy in colon tumors. A: Protein extracts were prepared from tumors from animals treated with vehicle (-) or 6 mg GLT/kg of body weight (GLT). The expression of Beclin-1 and LC-3 was evaluated by Western blot analysis as described in Materials and Methods. Representative sample is shown. B: Dissected tumors from animals treated with vehicle (control) or 6 mg GLT/kg of body weight (GLT) were sectioned and stained with Beclin-1 and LC-3 as described in Materials and Methods. Each section from 3 different tumors for each group was analyzed at 200 magnication, and the stain positive cells were scored from 4 independent places on the slide depending upon the intensity of the brown coloration (0 = no change, 1 = slight brown, 2 = light brown, 3 = brown, and 4 = dark brown). Statistical signicance was Beclin-1: , P < 0.001; LC-3: , P < 0.005. GAPDH, glyderaldehyde-3-phosphate dehydrogenase.

anecdotal evidence of the use of G. lucidum in humans, and the recommended dosage in the Pharmacopeia of the Republic of China is 6 to 12 g/day, only a recent controlled human supplementation study demonstrated that an oral application of 1.44 g of G. lucidum extract/day for 4 wk did not show any sign of toxicity (52). Moreover, daily oral use of an herbal extract containing 4 g of G. lucidum for 24 wk by patients with rheumatoid arthritis and an oral application of 5.4 g of G. lucidum polysaccharide extract for 12 wk by patients with advanced lung cancer was well tolerated, respectively (53,54). Based on our current nding, further studies that have evaluated the bioavailability of GLT are justied and needed prior to initiating clinical studies with GLT. In summary, our report suggests GLT as a natural agent that possesses the activity to inhibit growth of colon cancer cells in

vitro and in vivo by the induction of autophagy in colon cancer cells. ACKNOWLEDGMENTS This work was supported by the Methodist Research Institute, Clarian Health, Inc. We thank Shailesh Dudhgaonkar for his help with animal experiments. REFERENCES
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