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Antiproliferative and antimetastatic effects of the ethanolic extract of Phellinus igniarius (Linnearus: Fries) Quelet
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Journal of Ethnopharmacology 115 (2008) 50–56
Antiproliferative and antimetastatic effects of the ethanolic extract of Phellinus igniarius (Linnearus: Fries) Quelet Tuzz-Ying Song a , Hung-Chi Lin b , Nae-Cherng Yang a , Miao-Lin Hu b,∗ a
b
Department of Nutrition and Health Science, Chungchou Institute of Technology, Changhua, Taiwan, ROC Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan, ROC Received 1 June 2007; received in revised form 20 August 2007; accepted 4 September 2007 Available online 8 September 2007
Abstract Aim of this study: Phellinus igniarius (Linnearus: Fries) Quelet (Phellinus igniarius) has been used in oriental countries for treatment of various diseases including cancer. However, it is unclear how Phellinus igniarius exerts anticancer effects. Materials and methods: In this study the ethanolic extract from the fruiting body of Phellinus igniarius (EEPI) was used to evaluate the antiproliferative and antimetastatic effects in human hepatocarcinoma SK-Hep-1 cells and rat heart vascular endothelial cells (RHE cells). Results: We found that EEPI inhibited the proliferation of both cell lines in a dose-dependent manner, and the IC50 values at 48 h were 72 and 103 g/ml for SK-Hep-1 cells and RHE cells, respectively. EEPI at non- or sub-cytotoxic concentrations (25–100 g/ml) markedly inhibited the migration and invasion of SK-Hep-1 cells. EEPI added at 25 g/ml significantly decreased the secretion of matrix metalloproteinase-2 (MMP2) (49%, p < 0.01) and vascular endothelial growth factor (VEGF) (13%, p < 0.05) in SK-Hep-1 cells. EEPI at 25 g/ml completely inhibited matrigel-induced tube formation in RHE cells. Importantly, EEPI (25 or 50 g/ml) in combination with oxaliplatin (Oxa) or 5-flurouracil (5-FU) synergistically inhibited the proliferation of SK-Hep-1 cells. Conclusion: These results demonstrate the antiproliferative and antimetastatic effects of EEPI in vitro and the potential of EEPI as an adjuvant for chemotherapy. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Antiproliferation; Antimetastasis; Phellinus igniarius; Synergy; Oxaliplatin; 5-Flurouracil
1. Introduction Cancer metastasis is a highly coordinated multistep process in which cancer cells undergo extensive interactions with various host cells before they establish a secondary metastatic colony. In malignant neoplasm, angiogenesis is required for tumor growth, progression, and metastasis (Liotta et al., 1991) and must precede even local invasion (Nguyen, 2004). Well-vascularized tumors expand both locally and by metastasis, whereas avascular tumors do not grow beyond a diameter of 2–3 mm (Folkman, 1990). Phellinus igniarius (Linnearus: Fries) Quelet (Phellinus igniarius), an orange color mushroom grown on mulberry tree, is a well-known fungus of the genus Phellinus in the family
∗
Corresponding author. Tel.: +886 4 2281 2363; fax: +886 4 2287 6211. E-mail address: [email protected] (M.-L. Hu).
0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.09.001
of Hymenochaetaceae and has been used as a traditional herb medicine for years in oriental countries. In folk medicine, several species of Phellinus are known to improve health and to prevent and remedy various diseases, such as gastroenteric disorders, lymphatic diseases, and cancer (Cho et al., 2002; Ajith and Janardhanan, 2003). In the last decade, a few pharmacological actions of Phellinus igniarius have been elucidated. For instance, the butanol extract of Phellinus igniarius was shown to induce relaxation of the phenylephrine-precontracted rat aorta in a dose-dependent manner, and its effect is abolished by the removal of functional endothelium (Kang et al., 2006). Yang et al. (2006) pointed out that Phellinus igniarius extracts have immunoregulatory effects and can prolong life in mice. In addition, the ethanolic extract of Phellinus igniarius was shown to be antimutagenic, partly by inducing quinone oxidoreductase and glutathione S-transferase activities and by increasing GSH levels (Shon and Nam, 2001). Recently, highly oxygenated compounds – phelligridimer A and phelligridin G, H, I, J – have
T.-Y. Song et al. / Journal of Ethnopharmacology 115 (2008) 50–56
been isolated from Phellinus igniarius (Wang et al., 2005a), and these compounds were found to inhibit rat liver microsomal lipid peroxidation and to exhibit cytotoxic activities against human cancer cell lines (Wang et al., 2005b, 2007). The antimetastatic activity for endothelial cells (ECs) and cancer cells has been documented in some mushrooms (Kimura et al., 2004; Lu et al., 2004; Cheng et al., 2005). However, there have been no reports on the antimetastatic effects of Phellinus igniarius. In the present study, we used the ethanolic extract from the fruiting body of Phellinus igniarius (EEPI) to investigate the antimetastatic effects on a human hepatocarcinoma, SK-Hep-1 cells. Hepatocellular carcinoma (HCC), which is one of the most common malignant tumors in the tropics and the far east, especially Taiwan, has been demonstrated to be more invasive and migratory than human hepatoblastoma (Jiang et al., 2001). Because cancer cells do not undergo vascularization in vitro, we used RHE cells to determine the effect of EEPI on matrigel-based tube formation, as a marker of antiangiogenesis. 2. Materials and methods
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of RHE cells) onto a 12-well-plate 24 h prior to EEPI treatment. After incubation, cells were washed with phosphate buffered saline (PBS), and the morphological changes were observed using a phase-contrast inverse microscope (IMT-2, Olympus Co. Ltd., Tokyo, Japan). Viable cell numbers were determined at 24, 48 and 72 h after the addition of EEPI (25–500 g/ml) by the Trypan blue exclusion method using a hemocytometer. 2.4. Matrigel endothelial cell tube formation assay The tube formation in RHE cells was assayed by a modified method described previously (Jones et al., 1999). Matrigel (12.5 mg/ml) was thawed at 4 ◦ C, and 300 l was quickly added to each well of a 12-well-plate and allowed to solidify for 10 min at 37 ◦ C. Once solid, RHE cells (2 × 105 cells/well) were seeded to the wells and incubated for 2 h. After adhesion of the cells, the medium was removed and replaced by fresh medium supplemented with 25 g/ml of EEPI and incubated at 37 ◦ C for 2, 4 and 6 h. The tube formation was visualized with an Olympus IX70 phase-contrast microscope at a magnification of 100×.
2.1. Materials 2.5. Cell migration assay The hepatocarcinoma SK-Hep-1 cells were purchased from Food Industry Research & Development Institute (Hsin Chu, Taiwan). Rat heart vascular endoethelial cells (RHE cells) were kindly provided by Dr. Shih-Lan Hsu (Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan). Fruiting body of Phellinus igniarius (Linnearus: Fries) Quelet (Phellinus igniarius) was obtained from Biotechnology Center, KO DA Pharmaceutical Co. Ltd. (Taoyuan, Taiwan), and was further confirmed by Dr. Chin-Chuan Tsai of the Chinese Medical University, Taichung, Taiwan. All chemicals used were of reagent or higher grade. Dulbecco’s modified essential medium (DMEM), fetal bovine serum (FBS), trypsin, penicillin, streptomycin, sodium pyruvate, nonessential amino acids, and Giemsa stain were from Gibco/BRL. Transwells were from FALCON (Becton Dickinson, NJ, USA).
Tumor cell migration was assayed in transwell chambers (Costar) according to the method reported by Repesh (1989) with some modifications. Briefly, transwell chambers with 6.5 mm polycarbonate filters of 8 m pore size were used. SKHep-1 cells (5 × 105 ml−1 ) and 25–100 g/ml of EEPI were suspended in DMEM (100 l, serum free), placed in the upper transwell chamber, and incubated for 24 h at 37 ◦ C. Then, the cells on the upper surface of the filter were completely wiped away with a cotton swab, and the lower surface of the filter was fixed in methanol, stained with Giemsa, and counted under a microscope at a magnification of 200×. For each replicate, the tumor cells in 10 randomly selected fields were determined, and the counts were averaged. 2.6. Cell invasion assay
2.2. Preparation of ethanolic extract of Phellinus igniarius (EEPI) Fruiting bodies of Phellinus igniarius were ground under liquid nitrogen and extracted with five volumes of 95% ethanol for three times at room temperature. The extracts were filtered through Whatman No. 2 filter paper, and then concentrated in vacuum to dryness. The yield of EEPI was 2.3%. 2.3. Cell culture and cell antiproliferation assay Human hepatocarcinoma, SK-Hep-1 cells and RHE cells were maintained in DMEM, supplemented with 10% FBS, 2 mM glutamine and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin), at 37 ◦ C in a humidified atmosphere of 5% CO2 . The medium was changed every 2 days. Cells were seeded at a density of 1 × 105 cells/well (or 2 × 104 cells/well in the case
The invasion of tumor cells was assessed in transwell chambers with a 6.5 mm polyvinyl/pyrrolidone-free polycarbonate filter of 8 m pore size, as described in the cell migration assay (Repesh, 1989) except that each filter was coated with 100 l of a 1:20 diluted matrigel in cold DMEM to form a thin continuous film on the top of the filter. The number of cells was adjusted to 5 × 105 ml−1 and 100 l (containing 5 × 104 cells) was transferred to each of triplicate wells in DMEM containing 10% FBS. After incubation for 24 h, the cells were stained and counted as described above, and the number of cells invading the lower side of the filter was measured. 2.7. Gelatin zymography assay MMP in the medium released from SK-Hep-1 cells was assayed using gelatin zymography (7.5% zymogram gelatin
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gels) according to the methods reported by Hwang et al. (2006) with some modification. Briefly, the culture medium was electrophoresed (125 V for 90 min) in a 10% SDS-PAGE gel containing 0.1% gelatin. The gel was then washed at room temperature in a solution containing 2.5% (v/v) Triton X-100 with two changes and subsequently transferred to a reaction buffer for enzymatic reaction containing 1% NaN3 , 10 mM CaCl2 and 40 mM Tris–HCl, pH 8.0, at 37 ◦ C with shaking overnight (for 12 and 15 h). Finally, the MMP gel was stained for 30 min with 0.25% (w/v) Coomassie blue in 10% acetic acid (v/v) and 50% methanol (v/v) and destained in 10% acetic acid (v/v) and 50% methanol (v/v). The relative MMP activities were quantified by Matrix Inspector 2.1 software. 2.8. VEGF release assay To determine the effects of EEPI on VEGF levels, the SKHep-1 cells grown to 85% confluence were treated with 25 g/ml of EEPI for 24 h. Then, the medium was aspirated from the flasks and centrifuged at 500 × g (10 min) to remove cells from the medium. The level of VEGF released into the incubation medium was estimated using an ELISA kit (Chemicon International Inc., Temecula, CA). 2.9. Statistical analyses and calculation of synergy Data are expressed as means ± S.D. and analyzed statistically using one-way ANOVA followed by Duncan’s Multiple Range test for comparison of group means. A p value of <0.05 is considered statistically significant. Potential synergistic or antagonistic effects of EEPI plus Oxa or 5-FU were evaluated by comparing the total inhibition obtained by the sum of the individual treatment’s effects with the extent of inhibition obtained by a combination of treatments (Meyer et al., 1998), i.e., using observed percentage inhibition and the formula: [control − (EEPI + chemotherapeutic drug)]/[(control − EEPI) + (control − chemotherapeutic drug)]. According to this formula, a value greater than 1.0 is synergistic, a value of 0.5–1.0 is additive, while a value less than 0.5 is antagonistic. However, interactions between EEPI and Oxa or 5-FU were considered valid, only when the differences between observed inhibition and expected inhibition were statistically significant, as evaluated by Student’s t-test at the 5% significance level (Montgomery, 1991; Chuang and Hu, 2006). 3. Results 3.1. Effect of EEPI on proliferation of SK-Hep-1 cells and RHE cells As shown in Fig. 1, EEPI inhibited the proliferation of SK-Hep-1 and RHE cells in a dose-dependent manner during incubation for 72 h. After incubation for 48 h, the IC50 of SK-Hep-1 cells and RHE cells were approximately 72 and 103 g/ml, respectively. At the concentration of 200 g/ml,
Fig. 1. The antiproliferative effect of ethanol extract of Phellinus igniarius (EEPI) in SK-Hep-1 cells (A) and RHE cells (B). Cells were treated with EEPI (0–200 g/ml) for 24, 48 and 72 h. After treatment, cell viability was estimated by Trypan blue dye exclusion method.
EEPI almost completely suppressed the increase in cell numbers of SK-Hep-1 and RHE cells. 3.2. Synergy of antiproliferation between EEPI and Oxa or 5-FU The possible synergy in antiproliferation of SK-Hep-1 cells co-incubated with EEPI (25 g/ml) and Oxa (0.1 M) or 5-FU (1 M) for 72 h. None of the treatment alone at the indicated concentration caused significant inhibition of proliferation (Table 1). However, the combination of EEPI with Oxa or 5-FU increased the inhibition of proliferation (from 3.0%) to 13.7% and to 20.9%. These values were significantly higher than the values of expected inhibition (4.6 and 9.6% for EEPI + Oxa and EEPI + 5-FU, respectively). As calculated by the synergy formula, EEPI + Oxa and EEPI + 5-FU caused 2.9-fold (p < 0.05) and 2.2-fold (p < 0.05) inhibition, respectively, which represented synergistic effects. To confirm these synergistic effects, we incubated SK-Hep-1 cells with a higher concentration of EEPI (50 g/ml) with or without Oxa (at 0.1 M) or 5-FU (1 M). After incubation for 72 h, we found similar synergy, i.e., EEPI + Oxa and EEPI + 5-FU caused 2.4-fold (p < 0.05) and 2.1-fold (p < 0.05) inhibition of proliferation, respectively. 3.3. Effect of EEPI on tube formation of RHE cells We tested the effect of EEPI on matrigel-induced endothelial cell tube formation, an assay that is useful for detecting angiogenesis. As shown in Fig. 2, EEPI at the concentration of 25 g/ml significantly inhibited tube formation after incubation for 2, 4, and 6 h, as compared to the untreated controls, indicating its potent anti-angiogenic action.
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Table 1 Synergistic effects on the antiproliferation of SK-Hep-1 cells between the ethanol extract of Phellinus igniarius (EEPI) and oxaliplatin (Oxa) or 5-flurouracil (5-FU)a Treatments
Cell proliferation (×104 )b
Control Oxa (0.1 M) 5-FU (1 M) EEPI (25 g/ml) EEPI + Oxa EEPI + 5-FU
44.5 43.8 41.6 43.1 38.4 35.2
EEPI (50 g/ml) EEPI + Oxa EEPI + 5-FU
39.4 ± 3.2 30.4 ± 2.8 27.9 ± 3.2
a b c
± ± ± ± ± ±
2.8 2.3 2.2 2.7 2.0 1.5
Observed inhibition (%) – 1.6 ± 6.6 ± 3.0 ± 13.7 ± 20.9 ±
Expected inhibition (%)
Interaction (p < 0.05)
Fold of inhibition (synergistic effects)c
0.2 2.3 0.6 2.6 1.2
– – – – 4.6 ± 0.8 9.6 ± 2.1
– – – – Synergistic Synergistic
– – – – 2.9 2.2
11.5 ± 3.2 31.7 ± 6.3 37.3 ± 7.2
– 13.1 ± 3.4 18.1 ± 3.9
– Synergistic Synergistic
– 2.4 2.1
Data are given as mean values of triplicate analyses ± S.D. Concentrations of Oxa and 5-flurouracil were 0.1 and 1 M, respectively. SK-Hep-1 cells were incubated with 25 or 50 g/ml of EEPI in combination with Oxa or 5-FU for 72 h. The synergistic effect is calculated as [control − (EEPI + chemotherapy drug)]/[(control − EEPI) + (control − chemotherapy drug)].
Fig. 2. The effect of ethanol extract of Phellinus igniarius (EEPI) on the angiogenesis of RHE cells. After RHE cells were seeded into matrigel matrix-coated plates for 2 h, then treated without or with 25 g/ml of EEPI and incubated continuously for indicated time periods. Photomicrographs were obtained using an Olympus IX70 phase-contrast microscope (100×).
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Fig. 4. Effect of ethanol extract of Phellinus igniarius (EEPI) on the activities of matrix metalloproteinase-2 (MMP-2) in SK-Hep-1 cells. Cells were treated with EEPI (0–100 g/ml) for 24 h. Conditioned medium was collected and electrophoresed using SDS-PAGE. MMP activity in the gels was quantified using an Image System 2000 densiometer. Asterisks indicate significant difference from the control, **p < 0.01.
Fig. 3. Effects of ethanol extract of Phellinus igniarius (EEPI) on transwell migration and invasion assay of SK-Hep-1 cells. SK-Hep-1 cells were incubated with EEPI (0, 25, 50 and 100 g/ml) for 24 h, and calculated the migration cells (A) and invasion cells (B) of the transwell. Curcumin (10 M) served as positive control. Asterisks indicate significant difference from the control, **p < 0.01.
3.4. Effect of EEPI on in vitro migration and invasion of SK-Hep-1 cells The transwell assay was used to investigate the migration and invasion of SK-Hep-1 cells at 24 h after EEPI treatment. We found that EEPI added at 25–100 g/ml significantly decreased both the migration (Fig. 3A) and invasion (Fig. 3B) of SK-Hep-1 cells and that these effects of EEPI were concentrationdependent. As expected, curcumin (added at 10 M) as a positive control strongly inhibited the migration and invasion of SK-Hep1 cells. The IC50 values for EEPI on migration and invasion of SK-Hep-1 by EEPI was approximately 41 and 54 g/ml, respectively. These IC50 values are much lower than that for EEPI on viability (133 g/ml, Fig. 1), indicating that the inhibition of EEPI on migration and invasion of SK-Hep-1 cells is not due to decreased cell proliferation. 3.5. Effect of EEPI on MMP-2 activity of SK-Hep-1 cells To examine the effect of EEPI on MMPs, SK-Hep-1 cells were treated with EEPI (25–100 g/ml) for 24 h in serumfree medium. The resulting medium (conditioned medium) was
collected and examined for MMP activity using gelatin zymography. Gelatin zymograms of serum-free conditioned medium revealed the band of lysis at 72 kDa (MMP-2) (Fig. 4A). As shown in Fig. 4B, treatment with EEPI (25–100 g/ml) decreased the MMP-2 activity of SK-Hep-1 cells in a dosedependent manner. We also investigated the release of MMP-9 from SK-Hep-1 cells incubated with EEPI, but we found that EEPI treatment did not significantly affect the activity of MMP-9 in the conditioned medium (data not shown). 3.6. Effect of EEPI on VEGF levels of SK-Hep-1 cells As shown in Fig. 5, SK-Hep-1 cells not treated with EEPI released detectable levels of VEGF into the serum-free media at approximately 120 pg/106 SK-Hep-1 cells. Treatment of cells with EEPI (25–100 g/ml) for 24 h resulted in significant and dose-dependent decrease in the release of VEGF (13, 24 and 54%) decrease for 25, 50 (p < 0.05) and 100 g/ml of EEPI (p < 0.01). 4. Discussion Phellinus ignairis has been shown to have anti-tumor effects in vitro, but the mechanism underlying such an effect is unclear. In this study, we demonstrated that EEPI inhibited the proliferation of SK-Hep-1 cells and RHE cells in a concentration-dependent manner, with IC50 values (obtained at 48 h of incubation) of 72 and 103 g/ml, respectively. EEPI added at non- or sub-cytotoxic concentrations (25–100 g/ml) significantly and concentration-dependently inhibited and invasion of SK-Hep-1 cells. In addition, we showed that EEPI at 25 g/ml completely inhibited matrigel-induced tube formation
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Fig. 5. Effect of ethanol extract of Phellinus igniarius (EEPI) on the VEGF release in SK-Hep-1 cells. Concentration of VEGF released into the medium was determined by ELISA. The data are means ± S.D., n = 3. Asterisks indicate significant difference from the control, *p < 0.05; **p < 0.01.
of RHE cells. Importantly, we demonstrated that EEPI (25 and 50 g/ml) in combination with Oxa or 5-FU exhibited synergistic antiproliferative effects. The MMPs form a family of highly homologous, zinc- and calcium-dependent endopeptidase participating in extracellular matrix degradation, which eventually leads to tumor metastasis including migration, invasion, and angiogenesis (Belotti et al., 2003). It has been shown that MMP-2 activity in the tissue homogenate of HCC is the highest in HCC with metastasis and has been suggested to play a key role in the degradation of the basement membrane, thereby promoting migration of endothelial cells (Nakatsukasa, 1986). Our results showed that EEPI added at 25–100 g/ml significantly inhibited MMP-2 activity of SK-Hep-1 cells. Because EEPI also significantly inhibited the invasion of SK-Hep-1 cells at these concentrations, it is likely that EEPI’s inhibition of MMP-2 activity is responsible for its inhibition of invasion of the SK-Hep-1 cells. In addition to MMPs, VEGF contributes to the angiogenic process by stimulation of the migration, proliferation, and the formation of new blood vessels in endothelial cells (Ferrara and Davis-Smyth, 1997). Anti-angiogenic activity in endothelial cells has been documented in numerous mushrooms (Lu et al., 2004; Cheng et al., 2005). In HCC, the expression of VEGF has been reported as an essential tumor angiogenesis factor, and its down-regulation results in decreased stimulation of tumor angiogenesis (Suzuki et al., 1996). We found that EEPI added at 25–100 g/ml significantly and dose-dependently inhibited the secretion of VEGF from the SK-Hep-1 cells. The results suggest that the inhibition of EEPI on angiogenesis is attributable to decreased secretion of VEGF, as VEGF has been shown to increase MMP secretion of cancer cells and to enhance tumor metastasis (Ferrara and Davis-Smyth, 1997). Thus, the molecular mechanism of the in vitro antimetastatic properties of EEPI may be due to its ability to inhibit release of VEGF and activation of MMP-2 of SK-Hep-1 cells, leading to anti-angiogenesis, as evidenced by the inhibition of matrigel-induced tube formation in RHE cells.
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It has been known that chemotherapy in cancer patients is often associated with serious short-term and long-term adverse effects, such as leukoencephalopathy and cognitive impairment (Dietrich et al., 2006). Thus, one strategy of cancer therapy is to use non-toxic natural herbs as adjuvant for chemotherapeutic drugs to reduce the adverse effects. The results presented here revealed that EEPI at non- or sub-cytotoxic concentrations in combination with Oxa or 5-FU exhibited synergistic antiproliferative effects. The results demonstrate the potential of EEPI as an adjuvant for chemotherapy. Many polyphenols and flavonoids have been shown to inhibit carcinogenesis and tumorigenesis in animal experiments (Hertog et al., 1993; Elangovan et al., 1994) and to inhibit proliferation and angiogenesis of tumor cells in vitro (Fotsis et al., 1997). Although the active ingredient(s) in EEPI exerting the anti-tumor and anti-angiogenic effects are not yet identified, the EEPI is abundant in flavonoids and polyphenols (7.9 and 16.7%) (data not shown). Many of the flavonoids and polyphenols have been shown to exert antiproliferation and anti-angiogenic effects on tumor cells and endothelial cells (Ren et al., 2003; Oak et al., 2005). For instance, hispolon isolated from Phellinus linteus (Berkeley & Curtis) Teng has been shown to induce the death of human epidermoid KB cells through mitochondria-mediated apoptotic pathway (Chen et al., 2006). In addition, synthetic hispidin, another polyphenol found in Phellinus linteus, has been shown to be more cytotoxic toward cancer cells than normal cells in vitro by inhibition of protein kinase C (Gonindard et al., 1997). Activation of PKC induces phenotypic changes in the morphology of microvascular endothelial cells that affect major functions of the microvasculature, which include the first stages of sprouting in angiogenesis, cell migration following wounding, and vascular permeability (Bokhari et al., 2006). Thus, it is possible that polyphenols/flavonoids in EEPI may play an important role in its antimetastatic actions. In conclusion, we have demonstrated that the ethanolic extract of the mushroom Phellinus igniarius exerts potent antiproliferation and antimetastatic effects on SK-Hep-1 cells and RHE cells and that these effects of EEPI are likely mediated by suppression of MMP-2 and VEGF secretion. In addition, the combination of EEPI with Oxa or 5-FU exhibits synergistic antiproliferative effects on SK-Hep-1 cells. In vivo studies are needed to confirm the pharmacological efficacy and safety of Phellinus igniarius. Acknowledgement This work was supported by the grant NSC 93-2313-B-041008 from the National Science Council, Taiwan, ROC. References Ajith, T.A., Janardhanan, K.K., 2003. Cytotoxic and anti-tumor activities of a polypore macrofungus, Phellinus rimosus (Berk) Pilat. Journal of Ethnopharmacology 84, 157–162. Belotti, D., Paganoni, P., Manenti, L., Garofalo, A., Marchini, S., Taraboletti, G., Giavazzi, R., 2003. Matrix metalloproteinases (MMP-9 and MMP-2) induce the release of vascular endothelial growth factor (VEGF) by ovarian
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Antiproliferative and antimetastatic effects of the ethanolic extract of Phellinus igniarius (Linnearus: Fries) Quelet Tuzz-Ying Song a , Hung-Chi Lin b , Nae-Cherng Yang a , Miao-Lin Hu b,∗ a
b
Department of Nutrition and Health Science, Chungchou Institute of Technology, Changhua, Taiwan, ROC Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan, ROC Received 1 June 2007; received in revised form 20 August 2007; accepted 4 September 2007 Available online 8 September 2007
Abstract Aim of this study: Phellinus igniarius (Linnearus: Fries) Quelet (Phellinus igniarius) has been used in oriental countries for treatment of various diseases including cancer. However, it is unclear how Phellinus igniarius exerts anticancer effects. Materials and methods: In this study the ethanolic extract from the fruiting body of Phellinus igniarius (EEPI) was used to evaluate the antiproliferative and antimetastatic effects in human hepatocarcinoma SK-Hep-1 cells and rat heart vascular endothelial cells (RHE cells). Results: We found that EEPI inhibited the proliferation of both cell lines in a dose-dependent manner, and the IC50 values at 48 h were 72 and 103 g/ml for SK-Hep-1 cells and RHE cells, respectively. EEPI at non- or sub-cytotoxic concentrations (25–100 g/ml) markedly inhibited the migration and invasion of SK-Hep-1 cells. EEPI added at 25 g/ml significantly decreased the secretion of matrix metalloproteinase-2 (MMP2) (49%, p < 0.01) and vascular endothelial growth factor (VEGF) (13%, p < 0.05) in SK-Hep-1 cells. EEPI at 25 g/ml completely inhibited matrigel-induced tube formation in RHE cells. Importantly, EEPI (25 or 50 g/ml) in combination with oxaliplatin (Oxa) or 5-flurouracil (5-FU) synergistically inhibited the proliferation of SK-Hep-1 cells. Conclusion: These results demonstrate the antiproliferative and antimetastatic effects of EEPI in vitro and the potential of EEPI as an adjuvant for chemotherapy. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Antiproliferation; Antimetastasis; Phellinus igniarius; Synergy; Oxaliplatin; 5-Flurouracil
1. Introduction Cancer metastasis is a highly coordinated multistep process in which cancer cells undergo extensive interactions with various host cells before they establish a secondary metastatic colony. In malignant neoplasm, angiogenesis is required for tumor growth, progression, and metastasis (Liotta et al., 1991) and must precede even local invasion (Nguyen, 2004). Well-vascularized tumors expand both locally and by metastasis, whereas avascular tumors do not grow beyond a diameter of 2–3 mm (Folkman, 1990). Phellinus igniarius (Linnearus: Fries) Quelet (Phellinus igniarius), an orange color mushroom grown on mulberry tree, is a well-known fungus of the genus Phellinus in the family
∗
Corresponding author. Tel.: +886 4 2281 2363; fax: +886 4 2287 6211. E-mail address: [email protected] (M.-L. Hu).
0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.09.001
of Hymenochaetaceae and has been used as a traditional herb medicine for years in oriental countries. In folk medicine, several species of Phellinus are known to improve health and to prevent and remedy various diseases, such as gastroenteric disorders, lymphatic diseases, and cancer (Cho et al., 2002; Ajith and Janardhanan, 2003). In the last decade, a few pharmacological actions of Phellinus igniarius have been elucidated. For instance, the butanol extract of Phellinus igniarius was shown to induce relaxation of the phenylephrine-precontracted rat aorta in a dose-dependent manner, and its effect is abolished by the removal of functional endothelium (Kang et al., 2006). Yang et al. (2006) pointed out that Phellinus igniarius extracts have immunoregulatory effects and can prolong life in mice. In addition, the ethanolic extract of Phellinus igniarius was shown to be antimutagenic, partly by inducing quinone oxidoreductase and glutathione S-transferase activities and by increasing GSH levels (Shon and Nam, 2001). Recently, highly oxygenated compounds – phelligridimer A and phelligridin G, H, I, J – have
T.-Y. Song et al. / Journal of Ethnopharmacology 115 (2008) 50–56
been isolated from Phellinus igniarius (Wang et al., 2005a), and these compounds were found to inhibit rat liver microsomal lipid peroxidation and to exhibit cytotoxic activities against human cancer cell lines (Wang et al., 2005b, 2007). The antimetastatic activity for endothelial cells (ECs) and cancer cells has been documented in some mushrooms (Kimura et al., 2004; Lu et al., 2004; Cheng et al., 2005). However, there have been no reports on the antimetastatic effects of Phellinus igniarius. In the present study, we used the ethanolic extract from the fruiting body of Phellinus igniarius (EEPI) to investigate the antimetastatic effects on a human hepatocarcinoma, SK-Hep-1 cells. Hepatocellular carcinoma (HCC), which is one of the most common malignant tumors in the tropics and the far east, especially Taiwan, has been demonstrated to be more invasive and migratory than human hepatoblastoma (Jiang et al., 2001). Because cancer cells do not undergo vascularization in vitro, we used RHE cells to determine the effect of EEPI on matrigel-based tube formation, as a marker of antiangiogenesis. 2. Materials and methods
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of RHE cells) onto a 12-well-plate 24 h prior to EEPI treatment. After incubation, cells were washed with phosphate buffered saline (PBS), and the morphological changes were observed using a phase-contrast inverse microscope (IMT-2, Olympus Co. Ltd., Tokyo, Japan). Viable cell numbers were determined at 24, 48 and 72 h after the addition of EEPI (25–500 g/ml) by the Trypan blue exclusion method using a hemocytometer. 2.4. Matrigel endothelial cell tube formation assay The tube formation in RHE cells was assayed by a modified method described previously (Jones et al., 1999). Matrigel (12.5 mg/ml) was thawed at 4 ◦ C, and 300 l was quickly added to each well of a 12-well-plate and allowed to solidify for 10 min at 37 ◦ C. Once solid, RHE cells (2 × 105 cells/well) were seeded to the wells and incubated for 2 h. After adhesion of the cells, the medium was removed and replaced by fresh medium supplemented with 25 g/ml of EEPI and incubated at 37 ◦ C for 2, 4 and 6 h. The tube formation was visualized with an Olympus IX70 phase-contrast microscope at a magnification of 100×.
2.1. Materials 2.5. Cell migration assay The hepatocarcinoma SK-Hep-1 cells were purchased from Food Industry Research & Development Institute (Hsin Chu, Taiwan). Rat heart vascular endoethelial cells (RHE cells) were kindly provided by Dr. Shih-Lan Hsu (Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan). Fruiting body of Phellinus igniarius (Linnearus: Fries) Quelet (Phellinus igniarius) was obtained from Biotechnology Center, KO DA Pharmaceutical Co. Ltd. (Taoyuan, Taiwan), and was further confirmed by Dr. Chin-Chuan Tsai of the Chinese Medical University, Taichung, Taiwan. All chemicals used were of reagent or higher grade. Dulbecco’s modified essential medium (DMEM), fetal bovine serum (FBS), trypsin, penicillin, streptomycin, sodium pyruvate, nonessential amino acids, and Giemsa stain were from Gibco/BRL. Transwells were from FALCON (Becton Dickinson, NJ, USA).
Tumor cell migration was assayed in transwell chambers (Costar) according to the method reported by Repesh (1989) with some modifications. Briefly, transwell chambers with 6.5 mm polycarbonate filters of 8 m pore size were used. SKHep-1 cells (5 × 105 ml−1 ) and 25–100 g/ml of EEPI were suspended in DMEM (100 l, serum free), placed in the upper transwell chamber, and incubated for 24 h at 37 ◦ C. Then, the cells on the upper surface of the filter were completely wiped away with a cotton swab, and the lower surface of the filter was fixed in methanol, stained with Giemsa, and counted under a microscope at a magnification of 200×. For each replicate, the tumor cells in 10 randomly selected fields were determined, and the counts were averaged. 2.6. Cell invasion assay
2.2. Preparation of ethanolic extract of Phellinus igniarius (EEPI) Fruiting bodies of Phellinus igniarius were ground under liquid nitrogen and extracted with five volumes of 95% ethanol for three times at room temperature. The extracts were filtered through Whatman No. 2 filter paper, and then concentrated in vacuum to dryness. The yield of EEPI was 2.3%. 2.3. Cell culture and cell antiproliferation assay Human hepatocarcinoma, SK-Hep-1 cells and RHE cells were maintained in DMEM, supplemented with 10% FBS, 2 mM glutamine and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin), at 37 ◦ C in a humidified atmosphere of 5% CO2 . The medium was changed every 2 days. Cells were seeded at a density of 1 × 105 cells/well (or 2 × 104 cells/well in the case
The invasion of tumor cells was assessed in transwell chambers with a 6.5 mm polyvinyl/pyrrolidone-free polycarbonate filter of 8 m pore size, as described in the cell migration assay (Repesh, 1989) except that each filter was coated with 100 l of a 1:20 diluted matrigel in cold DMEM to form a thin continuous film on the top of the filter. The number of cells was adjusted to 5 × 105 ml−1 and 100 l (containing 5 × 104 cells) was transferred to each of triplicate wells in DMEM containing 10% FBS. After incubation for 24 h, the cells were stained and counted as described above, and the number of cells invading the lower side of the filter was measured. 2.7. Gelatin zymography assay MMP in the medium released from SK-Hep-1 cells was assayed using gelatin zymography (7.5% zymogram gelatin
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gels) according to the methods reported by Hwang et al. (2006) with some modification. Briefly, the culture medium was electrophoresed (125 V for 90 min) in a 10% SDS-PAGE gel containing 0.1% gelatin. The gel was then washed at room temperature in a solution containing 2.5% (v/v) Triton X-100 with two changes and subsequently transferred to a reaction buffer for enzymatic reaction containing 1% NaN3 , 10 mM CaCl2 and 40 mM Tris–HCl, pH 8.0, at 37 ◦ C with shaking overnight (for 12 and 15 h). Finally, the MMP gel was stained for 30 min with 0.25% (w/v) Coomassie blue in 10% acetic acid (v/v) and 50% methanol (v/v) and destained in 10% acetic acid (v/v) and 50% methanol (v/v). The relative MMP activities were quantified by Matrix Inspector 2.1 software. 2.8. VEGF release assay To determine the effects of EEPI on VEGF levels, the SKHep-1 cells grown to 85% confluence were treated with 25 g/ml of EEPI for 24 h. Then, the medium was aspirated from the flasks and centrifuged at 500 × g (10 min) to remove cells from the medium. The level of VEGF released into the incubation medium was estimated using an ELISA kit (Chemicon International Inc., Temecula, CA). 2.9. Statistical analyses and calculation of synergy Data are expressed as means ± S.D. and analyzed statistically using one-way ANOVA followed by Duncan’s Multiple Range test for comparison of group means. A p value of <0.05 is considered statistically significant. Potential synergistic or antagonistic effects of EEPI plus Oxa or 5-FU were evaluated by comparing the total inhibition obtained by the sum of the individual treatment’s effects with the extent of inhibition obtained by a combination of treatments (Meyer et al., 1998), i.e., using observed percentage inhibition and the formula: [control − (EEPI + chemotherapeutic drug)]/[(control − EEPI) + (control − chemotherapeutic drug)]. According to this formula, a value greater than 1.0 is synergistic, a value of 0.5–1.0 is additive, while a value less than 0.5 is antagonistic. However, interactions between EEPI and Oxa or 5-FU were considered valid, only when the differences between observed inhibition and expected inhibition were statistically significant, as evaluated by Student’s t-test at the 5% significance level (Montgomery, 1991; Chuang and Hu, 2006). 3. Results 3.1. Effect of EEPI on proliferation of SK-Hep-1 cells and RHE cells As shown in Fig. 1, EEPI inhibited the proliferation of SK-Hep-1 and RHE cells in a dose-dependent manner during incubation for 72 h. After incubation for 48 h, the IC50 of SK-Hep-1 cells and RHE cells were approximately 72 and 103 g/ml, respectively. At the concentration of 200 g/ml,
Fig. 1. The antiproliferative effect of ethanol extract of Phellinus igniarius (EEPI) in SK-Hep-1 cells (A) and RHE cells (B). Cells were treated with EEPI (0–200 g/ml) for 24, 48 and 72 h. After treatment, cell viability was estimated by Trypan blue dye exclusion method.
EEPI almost completely suppressed the increase in cell numbers of SK-Hep-1 and RHE cells. 3.2. Synergy of antiproliferation between EEPI and Oxa or 5-FU The possible synergy in antiproliferation of SK-Hep-1 cells co-incubated with EEPI (25 g/ml) and Oxa (0.1 M) or 5-FU (1 M) for 72 h. None of the treatment alone at the indicated concentration caused significant inhibition of proliferation (Table 1). However, the combination of EEPI with Oxa or 5-FU increased the inhibition of proliferation (from 3.0%) to 13.7% and to 20.9%. These values were significantly higher than the values of expected inhibition (4.6 and 9.6% for EEPI + Oxa and EEPI + 5-FU, respectively). As calculated by the synergy formula, EEPI + Oxa and EEPI + 5-FU caused 2.9-fold (p < 0.05) and 2.2-fold (p < 0.05) inhibition, respectively, which represented synergistic effects. To confirm these synergistic effects, we incubated SK-Hep-1 cells with a higher concentration of EEPI (50 g/ml) with or without Oxa (at 0.1 M) or 5-FU (1 M). After incubation for 72 h, we found similar synergy, i.e., EEPI + Oxa and EEPI + 5-FU caused 2.4-fold (p < 0.05) and 2.1-fold (p < 0.05) inhibition of proliferation, respectively. 3.3. Effect of EEPI on tube formation of RHE cells We tested the effect of EEPI on matrigel-induced endothelial cell tube formation, an assay that is useful for detecting angiogenesis. As shown in Fig. 2, EEPI at the concentration of 25 g/ml significantly inhibited tube formation after incubation for 2, 4, and 6 h, as compared to the untreated controls, indicating its potent anti-angiogenic action.
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Table 1 Synergistic effects on the antiproliferation of SK-Hep-1 cells between the ethanol extract of Phellinus igniarius (EEPI) and oxaliplatin (Oxa) or 5-flurouracil (5-FU)a Treatments
Cell proliferation (×104 )b
Control Oxa (0.1 M) 5-FU (1 M) EEPI (25 g/ml) EEPI + Oxa EEPI + 5-FU
44.5 43.8 41.6 43.1 38.4 35.2
EEPI (50 g/ml) EEPI + Oxa EEPI + 5-FU
39.4 ± 3.2 30.4 ± 2.8 27.9 ± 3.2
a b c
± ± ± ± ± ±
2.8 2.3 2.2 2.7 2.0 1.5
Observed inhibition (%) – 1.6 ± 6.6 ± 3.0 ± 13.7 ± 20.9 ±
Expected inhibition (%)
Interaction (p < 0.05)
Fold of inhibition (synergistic effects)c
0.2 2.3 0.6 2.6 1.2
– – – – 4.6 ± 0.8 9.6 ± 2.1
– – – – Synergistic Synergistic
– – – – 2.9 2.2
11.5 ± 3.2 31.7 ± 6.3 37.3 ± 7.2
– 13.1 ± 3.4 18.1 ± 3.9
– Synergistic Synergistic
– 2.4 2.1
Data are given as mean values of triplicate analyses ± S.D. Concentrations of Oxa and 5-flurouracil were 0.1 and 1 M, respectively. SK-Hep-1 cells were incubated with 25 or 50 g/ml of EEPI in combination with Oxa or 5-FU for 72 h. The synergistic effect is calculated as [control − (EEPI + chemotherapy drug)]/[(control − EEPI) + (control − chemotherapy drug)].
Fig. 2. The effect of ethanol extract of Phellinus igniarius (EEPI) on the angiogenesis of RHE cells. After RHE cells were seeded into matrigel matrix-coated plates for 2 h, then treated without or with 25 g/ml of EEPI and incubated continuously for indicated time periods. Photomicrographs were obtained using an Olympus IX70 phase-contrast microscope (100×).
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Fig. 4. Effect of ethanol extract of Phellinus igniarius (EEPI) on the activities of matrix metalloproteinase-2 (MMP-2) in SK-Hep-1 cells. Cells were treated with EEPI (0–100 g/ml) for 24 h. Conditioned medium was collected and electrophoresed using SDS-PAGE. MMP activity in the gels was quantified using an Image System 2000 densiometer. Asterisks indicate significant difference from the control, **p < 0.01.
Fig. 3. Effects of ethanol extract of Phellinus igniarius (EEPI) on transwell migration and invasion assay of SK-Hep-1 cells. SK-Hep-1 cells were incubated with EEPI (0, 25, 50 and 100 g/ml) for 24 h, and calculated the migration cells (A) and invasion cells (B) of the transwell. Curcumin (10 M) served as positive control. Asterisks indicate significant difference from the control, **p < 0.01.
3.4. Effect of EEPI on in vitro migration and invasion of SK-Hep-1 cells The transwell assay was used to investigate the migration and invasion of SK-Hep-1 cells at 24 h after EEPI treatment. We found that EEPI added at 25–100 g/ml significantly decreased both the migration (Fig. 3A) and invasion (Fig. 3B) of SK-Hep-1 cells and that these effects of EEPI were concentrationdependent. As expected, curcumin (added at 10 M) as a positive control strongly inhibited the migration and invasion of SK-Hep1 cells. The IC50 values for EEPI on migration and invasion of SK-Hep-1 by EEPI was approximately 41 and 54 g/ml, respectively. These IC50 values are much lower than that for EEPI on viability (133 g/ml, Fig. 1), indicating that the inhibition of EEPI on migration and invasion of SK-Hep-1 cells is not due to decreased cell proliferation. 3.5. Effect of EEPI on MMP-2 activity of SK-Hep-1 cells To examine the effect of EEPI on MMPs, SK-Hep-1 cells were treated with EEPI (25–100 g/ml) for 24 h in serumfree medium. The resulting medium (conditioned medium) was
collected and examined for MMP activity using gelatin zymography. Gelatin zymograms of serum-free conditioned medium revealed the band of lysis at 72 kDa (MMP-2) (Fig. 4A). As shown in Fig. 4B, treatment with EEPI (25–100 g/ml) decreased the MMP-2 activity of SK-Hep-1 cells in a dosedependent manner. We also investigated the release of MMP-9 from SK-Hep-1 cells incubated with EEPI, but we found that EEPI treatment did not significantly affect the activity of MMP-9 in the conditioned medium (data not shown). 3.6. Effect of EEPI on VEGF levels of SK-Hep-1 cells As shown in Fig. 5, SK-Hep-1 cells not treated with EEPI released detectable levels of VEGF into the serum-free media at approximately 120 pg/106 SK-Hep-1 cells. Treatment of cells with EEPI (25–100 g/ml) for 24 h resulted in significant and dose-dependent decrease in the release of VEGF (13, 24 and 54%) decrease for 25, 50 (p < 0.05) and 100 g/ml of EEPI (p < 0.01). 4. Discussion Phellinus ignairis has been shown to have anti-tumor effects in vitro, but the mechanism underlying such an effect is unclear. In this study, we demonstrated that EEPI inhibited the proliferation of SK-Hep-1 cells and RHE cells in a concentration-dependent manner, with IC50 values (obtained at 48 h of incubation) of 72 and 103 g/ml, respectively. EEPI added at non- or sub-cytotoxic concentrations (25–100 g/ml) significantly and concentration-dependently inhibited and invasion of SK-Hep-1 cells. In addition, we showed that EEPI at 25 g/ml completely inhibited matrigel-induced tube formation
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Fig. 5. Effect of ethanol extract of Phellinus igniarius (EEPI) on the VEGF release in SK-Hep-1 cells. Concentration of VEGF released into the medium was determined by ELISA. The data are means ± S.D., n = 3. Asterisks indicate significant difference from the control, *p < 0.05; **p < 0.01.
of RHE cells. Importantly, we demonstrated that EEPI (25 and 50 g/ml) in combination with Oxa or 5-FU exhibited synergistic antiproliferative effects. The MMPs form a family of highly homologous, zinc- and calcium-dependent endopeptidase participating in extracellular matrix degradation, which eventually leads to tumor metastasis including migration, invasion, and angiogenesis (Belotti et al., 2003). It has been shown that MMP-2 activity in the tissue homogenate of HCC is the highest in HCC with metastasis and has been suggested to play a key role in the degradation of the basement membrane, thereby promoting migration of endothelial cells (Nakatsukasa, 1986). Our results showed that EEPI added at 25–100 g/ml significantly inhibited MMP-2 activity of SK-Hep-1 cells. Because EEPI also significantly inhibited the invasion of SK-Hep-1 cells at these concentrations, it is likely that EEPI’s inhibition of MMP-2 activity is responsible for its inhibition of invasion of the SK-Hep-1 cells. In addition to MMPs, VEGF contributes to the angiogenic process by stimulation of the migration, proliferation, and the formation of new blood vessels in endothelial cells (Ferrara and Davis-Smyth, 1997). Anti-angiogenic activity in endothelial cells has been documented in numerous mushrooms (Lu et al., 2004; Cheng et al., 2005). In HCC, the expression of VEGF has been reported as an essential tumor angiogenesis factor, and its down-regulation results in decreased stimulation of tumor angiogenesis (Suzuki et al., 1996). We found that EEPI added at 25–100 g/ml significantly and dose-dependently inhibited the secretion of VEGF from the SK-Hep-1 cells. The results suggest that the inhibition of EEPI on angiogenesis is attributable to decreased secretion of VEGF, as VEGF has been shown to increase MMP secretion of cancer cells and to enhance tumor metastasis (Ferrara and Davis-Smyth, 1997). Thus, the molecular mechanism of the in vitro antimetastatic properties of EEPI may be due to its ability to inhibit release of VEGF and activation of MMP-2 of SK-Hep-1 cells, leading to anti-angiogenesis, as evidenced by the inhibition of matrigel-induced tube formation in RHE cells.
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It has been known that chemotherapy in cancer patients is often associated with serious short-term and long-term adverse effects, such as leukoencephalopathy and cognitive impairment (Dietrich et al., 2006). Thus, one strategy of cancer therapy is to use non-toxic natural herbs as adjuvant for chemotherapeutic drugs to reduce the adverse effects. The results presented here revealed that EEPI at non- or sub-cytotoxic concentrations in combination with Oxa or 5-FU exhibited synergistic antiproliferative effects. The results demonstrate the potential of EEPI as an adjuvant for chemotherapy. Many polyphenols and flavonoids have been shown to inhibit carcinogenesis and tumorigenesis in animal experiments (Hertog et al., 1993; Elangovan et al., 1994) and to inhibit proliferation and angiogenesis of tumor cells in vitro (Fotsis et al., 1997). Although the active ingredient(s) in EEPI exerting the anti-tumor and anti-angiogenic effects are not yet identified, the EEPI is abundant in flavonoids and polyphenols (7.9 and 16.7%) (data not shown). Many of the flavonoids and polyphenols have been shown to exert antiproliferation and anti-angiogenic effects on tumor cells and endothelial cells (Ren et al., 2003; Oak et al., 2005). For instance, hispolon isolated from Phellinus linteus (Berkeley & Curtis) Teng has been shown to induce the death of human epidermoid KB cells through mitochondria-mediated apoptotic pathway (Chen et al., 2006). In addition, synthetic hispidin, another polyphenol found in Phellinus linteus, has been shown to be more cytotoxic toward cancer cells than normal cells in vitro by inhibition of protein kinase C (Gonindard et al., 1997). Activation of PKC induces phenotypic changes in the morphology of microvascular endothelial cells that affect major functions of the microvasculature, which include the first stages of sprouting in angiogenesis, cell migration following wounding, and vascular permeability (Bokhari et al., 2006). Thus, it is possible that polyphenols/flavonoids in EEPI may play an important role in its antimetastatic actions. In conclusion, we have demonstrated that the ethanolic extract of the mushroom Phellinus igniarius exerts potent antiproliferation and antimetastatic effects on SK-Hep-1 cells and RHE cells and that these effects of EEPI are likely mediated by suppression of MMP-2 and VEGF secretion. In addition, the combination of EEPI with Oxa or 5-FU exhibits synergistic antiproliferative effects on SK-Hep-1 cells. In vivo studies are needed to confirm the pharmacological efficacy and safety of Phellinus igniarius. Acknowledgement This work was supported by the grant NSC 93-2313-B-041008 from the National Science Council, Taiwan, ROC. References Ajith, T.A., Janardhanan, K.K., 2003. Cytotoxic and anti-tumor activities of a polypore macrofungus, Phellinus rimosus (Berk) Pilat. Journal of Ethnopharmacology 84, 157–162. Belotti, D., Paganoni, P., Manenti, L., Garofalo, A., Marchini, S., Taraboletti, G., Giavazzi, R., 2003. Matrix metalloproteinases (MMP-9 and MMP-2) induce the release of vascular endothelial growth factor (VEGF) by ovarian
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