Antitumor Effects and Immunomodulating Activities of Phellinus linteus Extract in a CT-26 Cell-Injected Colon Cancer Mouse Model

Abstract

The antitumor effects of Phellinus linteus extract (Keumsa Linteusan) were investigated in a CT-26 cell-injected colon cancer mouse model. When administered orally (250~1,000 mg/kg body weight), Keumsa Linteusan significantly inhibited the growth of solid colon cancer. The highest dose was highly effective, reducing tumor formation by 26% compared with the control group. The anticomplementary activity of Keumsa Linteusan increased in a dose-dependent manner. Lysosomal enzyme activity of macrophages was increased by 2-fold (100 µg/ml) compared with the control group. Keumsa Linteusan can be regarded as a potent enhancer of the innate immune response, and can be considered as a very promising candidate for antitumor action.

The increasing global prevalence and mortality rate of cancer has been linked to excessive consumption of certain foods, lack of exercise, environmental pollution, and excessive stress (Feron et al., 1997). In particular, colon cancer prevalence has trended upward due to intake of animal fat and a diet rich in meat (Franks and Teich, 1997). Treatments range from folk remedies to the use of sophisticated drugs. Despite these options, colon cancer remains difficult to treat; the great majority of chemical compounds that are cytotoxic to cancer cells are also toxic to normal cells (Maroun et al., 2007). The discovery and identification of drugs that have potent antitumor activity and minimal side effects have become important goals of research in the biomedical sciences (Takeda et al., 1969).

Studies concerning the antitumor activity of natural compounds have included mushroom-derived compounds (Kodama et al., 2002; Wasser, 2002). Mushroom polymers exert their antitumor action mainly by activating the immune response of the host (Chihara et al., 1970a). Since the 1968 description that hot water extracts from Polyporaceae mushrooms inhibit the growth of sarcoma 180 (Ikekawa et al., 1968), a large number of antitumor polymers have been isolated from various mushrooms. The polymers include Lentinan from Lentinus edodes (Chihara et al., 1970b), Schizoplyllan from Schizophyllum commune (Tabata et al., 1981), and Krestin from Coriolus versicolor (Tsuru et al., 1991).

Phellinus linteus, which belongs to the family Hymenochaetaceae, has been used for millennia in Eastern countries including Korea, China, and Japan in the treatment of various human diseases, such as an alimentary disease and lymphatic disorders. Since the antitumor activity of P. linteus was first reported (Ikegawa et al., 1968), other reports on antitumor effects (Chung et al., 1993; Mizuno, 2000; Rhee et al., 2000) and immunostimulatory activities (Oh et al., 1992; Kim et al., 1996; Lee et al., 1996; Song et al., 1998) have been published. However, antitumor activities of extracts from fruiting body of P. linteus in mouse models of colon cancer have not yet been reported.

The present study reports the dose-dependent antitumor effects of P. linteus extracts (Keumsa Linteusan) following the oral administration of artificially cultivated P. linteus to mice in which colon cancer had been generated by injection of CT-26 cells. As well, the anticomplementary and macrophage lysosomal enzyme activities of Keumsa Linteusan are described.

Materials and Methods

Strain and extract preparation

Fruiting bodies of P. linteus obtained from the Keumsa Sanghwang Mushroom & Farm (Yeoju, Korea) were cut into small pieces, smashed, and extracted using hot water. The obtained extract (Keumsa Linteusan) was filtered and lyophilized.

Cell culture

The CT-26 cell line was supplied by the Korean Cell Line Bank located at the Seoul National University College of Medicine. The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; GIBCO BRL, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; INVITROGEN, USA), 1% penicillin-streptomycin, and 0.2% fungizone in a humidified atmosphere of 5% CO₂ at 37℃. The medium was replaced with DMEM, and cells were maintained at the same incubation conditions for 5~7 days, with replacement of the medium every 48 h, prior to use in experiments.

Experimental animals and breeding condition

Male 5-week-old BALB/c mice weighing approximately 22 g were purchased from Samtako (Osan, Korea). The mice were individually housed in plastic cages in a room with constant temperature (22 ± 2℃) and humidity (55 ± 5%), and a 12 h light-dark cycle. The mice were fed a commercial pellet diet (SCF, Dangjin, Korea) throughout the experimental period.

Induction of colon cancer and experimental design

Tumors were induced by an intradermal injection of CT-26 cells (100 µl, 1.5 × 10⁶ cells/ml in phosphate buffered saline) into the right back of BALB/c mice. After 7 days, mice received saline (control) and Keumsa Linteusan at doses of 250, 500, and 1,000 mg/kg body weight (BW), using an oral zonde needle daily for 4 weeks. The solid tumors were allowed to grow in the mice for 4 weeks before they were removed and weighed. Spleens and liver were dissected from each mouse and weights determined. The antitumor activity of the tested samples was expressed as an inhibition ratio (%) calculated as [(A - B) ÷ A] × 100, where A and B are the average tumor weight of the control and treated groups, respectively (Yang et al., 1992).

Assay of anticomplementary activity

Anticomplementary activity was measured by the complement fixation test based on complement consumption and the degree of red blood cell lysis by the residual complement (Kabat and Mayer, 1964). Fifty microliters of a Keumsa Linteusan solution in water was mixed with equal volumes of normal human serum (NHS) and gelatin veronal buffered saline (GVB, pH 7.4) containing 500 µg Mg⁺⁺ and 150 µg Ca⁺⁺. The mixtures were incubated at 37℃ for 30 min and the residual total complement hemolysis (TCH₅₀) was determined using IgM hemolysin sensitized sheep erythrocytes (1 × 10⁸ cells/ml). At the same time, the NHS was incubated with deionized water and GVB⁺⁺ (GVB containing 500 µg Mg⁺⁺ and 150 µg Ca⁺⁺) to provide a control. The anticomplementary activity of Keumsa Linteusan was expressed as the percentage inhibition of the TCH₅₀ of control according to the calculation:

Inhibition of TCH₅₀ (%) = [(TCH₅₀ of control - TCH₅₀ of treated sample) ÷ TCH₅₀ of control] × 100

Preparation of mouse macrophage

Macrophages were harvested from mice 3 days after an intraperitoneal injection with 3 ml of 10% thioglycolate medium. Cell density was adjusted to 1 × 10⁶ cells/ml with DMEM supplemented with 10% FBS. Thereafter, each well of a 96-well microplate was inoculated with 200 µl of the cell suspension (2 × 10⁵ cells/well). Adherent macrophages were isolated by incubating the cells for 2 h at 37℃ in an atmosphere of 5% CO₂, followed by vigorous shaking and washing of wells to remove non-adherent cells. Cultures were maintained untreated or with the addition of Keumsa Linteusan (10, 50, and 100 µg/ml) and lipopolysaccharide (LPS; SIGMA, USA), applied at the same concentration as Keumsa Linteusan at 37℃ and 5% CO₂ in a humidified incubator. Incubation was carried out for 48 h to determine the production of lysosomal enzyme activity.

Determination of macrophage cellular lysosomal enzyme activity

Lysosomal enzyme activity was assayed using 96-well flat-bottomed tissue culture plates (Suzuki et al., 1990). Macrophage monolayers in the microplate (2 × 10⁵ cells/well) were solubilized by the addition of 25 µl of 0.1% Triton X-100. One hundred fifty microliters of 10 mM p-nitrophenyl phosphate solution was added as a substrate for acid phosphatase. Subsequently, 50 µl of citrate buffer was added to each well. After incubation for 1 h at 37℃, 25 µl of 0.2M borate buffer (pH 9.8) was added to the reaction mixture, and the optical density was measured at 405 nm.

Statistical analyses

Each data value was expressed as the mean ± SE for 10 mice. The group means were compared by one-way analysis of variance and by Duncan's multiple-range test (Duncan, 1957). The statistical differences were considered significant at p < 0.05.

Results and Discussion

Effects of body weight and organ weight

The effect of Keumsa Linteusan on the weight of immune-related organs and BW in the CT-26 cell-injected colon cancer mouse model is presented in Table 1. No significant differences in BW were observed under the influence of Keumsa Linteusan. Generally, splenic macrophages have immune responses towards a foreign substance in the human body and Kupffer cells in the liver involving the production of a variety of cytokines stimulated by foreign antigens (Arthur et al., 1989). The relative spleen weight is an important index for nonspecific immunity (Zheng et al., 2005). However, presently, the liver and spleen weights did not differ significantly within the experimental groups.

Table 1.

Effects of Keumsa Linteusan on the body, liver, and spleen weight in a CT-26 cell-injected colon cancer mouse model after 4 weeks

CT-26 cells (1.5 × 10⁶ cell/ml) were intradermally injected to BALB/c mice. Mice were orally administrated vehicle (control) or Keumsa Linteusan (250, 500, and 1,000 mg/kg) daily for 4 weeks.

Each value is the mean ± SE of 10 mice.

^aNot significant.

Antitumor effects

The dose-dependent effects of Keumsa Linteusan were investigated in the mouse model and relative antitumor activities were evaluated with respect to that evident in the saline control group. Tumor growth was significantly decreased in all the Keumsa Linteusan groups compared with the control group (Table 2). The inhibition rate increased significantly even at a Keumsa Linteusan dose as low as 250 mg/kg BW. The maximum increase in tumor inhibition rate (26%) was evident at the highest dose of 1,000 mg/kg BW (Table 2). The effects of Keumsa Linteusan due to mushroom protein-polysaccharide could be a major factor in the colon cancer antitumor action, by promoting cell mediated immune response (Maeda and Chihara, 1971) and reestablishing the blunted antibody production (Nomoto et al., 1975). Supporting data comes from the observations that cancer cell inhibition rate increases in accordance with increasing doses of L. edodes and Pleurotus eryngii extracts in colon cancer cells (Hwang et al., 2003).

Table 2.

Effects of Keumsa Linteusan on tumor weight and inhibition rate in a CT-26 cell-injected colon cancer mouse model after 4 weeks

^a,bValues with different superscript letters in the same column are significantly different among the groups at p < 0.05.

^b[(Control tumor weight - Treated tumor weight) ÷ Control tumor weight] × 100.

CT-26 cells (1.5 × 10⁶ cell/ml) were intradermally injected into BALB/c mice. Mice were orally administrated vehicle (control) or Keumsa Linteusan (250, 500, and 1,000 mg/kg) daily for 4 weeks.

Each value is the mean ± SE of 10 mice.

Anticomplementary activity

The anticomplementary activities of Keumsa Linteusan were compared with positive control (LPS) at various concentrations (100, 500, and 1,000 µg/ml). Activity increased with increasing concentration (Fig. 1), reaching 64.7% at 1,000 µg/ml. These anticomplementary activities were lower than LPS at all concentrations. It has been reported that a mushroom polymer is closely related with antitumor actions via activation of the complementary system (Okuda et al., 1972; Jeong et al., 2008). Furthermore, activation of the complementary system is closely related with the antitumor effect exerted by host immune defenses (Lee et al., 1994). These results suggest that Keumsa Linteusan could play an important role as an antitumor substance.

Fig. 1.

Anticomplementary activities of Keumsa Linteusan. LPS was used for the positive control. Each value is the mean ± SD of triplicates.

Macrophage lysosomal enzyme activity

Lysosomal enzyme and phagocytic activities are crucial aspects of macrophage functional assessments (Jung et al., 2008). The selective release of lysosomal enzyme by mononuclear phagocytes occurs in response to numerous exogenous stimuli (Page et al., 1978). Effects of 10, 50, and 100 µg/ml Keumsa Linteusan on lysosomal enzyme activity of peritoneal macrophages at different concentration levels are shown in Fig. 2. A dose-dependent response was evident, with relative enzyme activity of Keumsa Linteusan increasing by 59%, 69%, and 103%, respectively, as compared to the negative control (physiological saline). The Keumsa Linteusan-mediated macrophage activation, and subsequent lysis and phagocytosis of foreign substances, is similar to that found with the biopolymer from P. pini on the production of macrophage cellular lysosomal enzyme in mice (Jeong et al., 2004). Also, sarcoma 180 antitumor activity is related to macrophage lysosomal enzyme activation (Kiho et al., 1992). Therefore, Keumsa Linteusan can be regarded as a potent enhancer of the innate immune response, and can be considered as a very promising candidate for antitumor action.

Fig. 2.

Macrophage cellular lysosomal enzyme activities of Keumsa Linteusan. NC denotes normal saline, which was used for the negative control. LPS was used for the positive control. Concentration of macrophages was 2.5 × 10⁶ cells/ml. Each value is the mean ± SD of triplicates. The asterisk (^*) indicates values that are significantly different when compared with the values for NC (p < 0.05).