Suppressing effects of daily oral supplementation of beta-glucan extracted from Agaricus blazei Murill on spontaneous and peritoneal disseminated metastasis in mouse model

Abstract

Purpose: The Basidiomycete fungus Agaricus blazei Murill has traditionally been used as a health food for the prevention of cancer.

Methods: We examined whether beta-(1-6)-D: -glucan extracted from A. blazei is a potential anticancer agent in an in vitro and in vivo animal model.

Results: Here we show that (1) beta-glucan had cytotoxic effect against human ovarian cancer HRA cells, but not against murine Lewis lung cancer 3LL cells, in vitro; (2) beta-glucan promotes p38 MAPK activity for suppressing HRA cell proliferation and amplifying the apoptosis cascade; (3) beta-glucan stimulates translocation of the proapoptotic protein, Bax, from the cytosol to mitochondria, cytochrome c release, and subsequent caspase-9 activation; (4) treatment with SB203580, a p38 MAPK-specific inhibitor, suppresses beta-glucan-induced effects, indicating that activation of p38 MAPK is involved in the suppression of cell proliferation and mitochondrial activation-mediated cell death pathway; (5) in mice, oral supplementation with beta-glucan reduces pulmonary metastasis of 3LL cells and peritoneal disseminated metastasis of HRA cells and inhibits the growth of these metastatic tumors in lung or peritoneal cavity, in part, by suppressing uPA expression; and (6) in an in vivo experimental metastasis assay, however, the oral supplementation with beta-glucan after i.v. tumor cell inoculation did not reduce the number of lung tumor colonies.

Conclusion: Treatment with beta-glucan may be beneficial for cancer patients with or at risk for metastasis. The beta-glucan-dependent signaling pathways are critical for our understanding of anticancer events and development of cancer therapeutic agents.

Fig. 1

Cancer cell proliferation in response to beta-glucan extracted from A. blazei. HRA and 3LL cells were deprived of serum for 24 h and incubated with the control, serum-free medium or beta-glucan (0.4, 2, or 10 μg/ml) for 20 h. a [³H]thymidine incorporation was measured during the last 5 h. b Cell viability was measured by the enzymatic reduction of MTT (OD 550–670 nm) during the last 3 h. Values represent the mean ± SD of quadruplicate samples of a representative experiment. *P<0.05 vs. control

Fig. 2

Effects of beta-glucan on peritoneal disseminated metastasis. HRA cells (5×10⁶) were injected intraperitoneally. a Beta-glucan (0, 20, 100, or 500 μg/mouse) was injected intraperitoneally on days 0, 1, 2, 3, 5, and 7. b Mice received oral beta-glucan by drinking fluid (0, 20, 100, or 500 μg/ml) every day during the experimental period. Nine days after injection of the cells, the animals were sacrificed, intraperitoneal tumor masses were dissected and weighted. Results are the mean ± SD of three different determinations. *P<0.05 vs. control

Fig. 3

Beta-glucan did not inhibit the growth of subcutaneous 3LL tumor. 3LL cells were inoculated subcutaneously into the flank of mice. The tumor volume from animals treated (black squares) or not treated (white squares) with oral beta-glucan was assessed by bilateral Vernier caliper measurement. Values are the mean ± SD of six animals per group and are expressed in mm³

Fig. 4

Effect of beta-glucan on the formation of lung metastases caused by 3LL cells. a The number of lung tumor colonies from mice bearing 3LL cells 28 days after s.c. tumor cell inoculation. b The number of lung tumor colonies from mice bearing 3LL cells 21 days after i.v. tumor cell inoculation. Mice were treated with daily oral administration of beta-glucan (0, 20, 100, or 500 μg/ml) during these experiments. Values are mean ± SD of five to seven animals. *P<0.05 vs. control

Fig. 5

Beta-glucan specifically promotes p38 MAPK activation in HRA cells. Cells were deprived of serum for 24 h and incubated in the serum-free medium containing 10 μg/ml beta-glucan for indicated periods. Phosphorylation of the specific proteins was detected by Western blot analysis using antibodies to total- or phosphorylated-ERK1/2 (a), JNK (b), or p38 MAPK (c). d HRA ovarian cancer cells were deprived of serum for 24 h and pretreated with or without SB203580, a p38 inhibitor, for 30 min. Then cells were treated with 0, 0.4, 2, and 10 μg/ml beta-glucan for 15 min. Phosphorylation of p38 was assessed by Western blot analysis with anti-phospho-p38 antibodies. To normalize p38 phosphorylation levels to total amounts of p38 protein, membranes were then stripped and re-probed with the antibody that recognizes both phosphorylated and non-phosphorylated form of p38. The data represent a typical experiment conducted three times with similar results

Fig. 6

Promotion of p38 activation is required for beta-glucan-mediated suppression of ovarian cancer cell proliferation. After a 24-h serum deprivation, cells were pretreated with beta-glucan or beta-glucan plus SB203580 at indicated concentrations for 20 h. Cell proliferation was measured by [³H]thymidine incorporation during the last 5 h. Values represent the mean ± SD of quadruplicate samples of a representative experiment. Unlike letters (a, b, and c) stand for statistical differences (P<0.05)

Fig. 7

Beta-glucan induces apoptosis through p38 MAPK cascade. HRA cells pretreated with or without pharmacological inhibitors, PD98059 (10 μM), SP600125 (50 μM), and SB203580 (15 μM), were treated with beta-glucan (10 μg/ml) for the time periods indicated (3 and 12 h). Cells were stained with Hoechst 33258, and apoptotic cells were analyzed by fluorescence microscopy. Apoptotic cells containing condensed chromatin fragments were scored and expressed as a percentage of the total cell number measured. Results from three independent experiments are shown as means (bars, ±SD). Unlike letters (a, b, and c; a′, b′, c′, and d′) stand for statistical differences (P<0.05)

Fig. 8

Beta-glucan induces Bax translocation, cytochrome c release, and caspase-9 activation in HRA cells. HRA cells were treated with 10 μg/ml beta-glucan for 3 h. Cell lysates were prepared, and Western blot analysis was performed for caspase-9 and β-actin. Cytosolic fraction was prepared, and cytochrome c was detected by Western blot analysis using anti-cytochrome c antibody. Mitochondrial fraction was analyzed for Bax protein expression using anti-Bax antibody. The data represent a typical experiment conducted three times with similar results

Fig. 9

Effects of beta-glucan on uPA expression and cell invasion in vitro. a HRA cells pretreated with or without beta-glucan were stimulated with 10 ng/ml TGF-β1. Total proteins were obtained and subjected to electrophoresis. Gels were transferred to PVDF membrane and probed with antibodies to uPA and β-actin, respectively. b The monolayer of HRA cells seeded in 96-well plate was pretreated with beta-glucan (2 or 10 μg/ml) in the presence of 10 ng/ml TGF-β1 for 12 h. The monolayers were washed twice with PBS and analyzed by colorimetric assay in the presence of the Spectrozyme UK. Results represent mean ± SD of three experiments and triplicate determination. *P<0.05 compared with lane 3. c A cell suspension, 200 μl (500,000 cells/ml) in medium, was placed at the upper compartment. After a 24-h incubation, cells that invaded through the Matrigel-coated membrane were stained and counted under the microscope. All experiments were performed three times, and typical data are shown. Results represent mean ± SD of three experiments and triplicate determination. *P<0.05 compared with lane 3