Anticancer activity of Amauroderma rude

Abstract

More and more medicinal mushrooms have been widely used as a miraculous herb for health promotion, especially by cancer patients. Here we report screening thirteen mushrooms for anti-cancer cell activities in eleven different cell lines. Of the herbal products tested, we found that the extract of Amauroderma rude exerted the highest activity in killing most of these cancer cell lines. Amauroderma rude is a fungus belonging to the Ganodermataceae family. The Amauroderma genus contains approximately 30 species widespread throughout the tropical areas. Since the biological function of Amauroderma rude is unknown, we examined its anti-cancer effect on breast carcinoma cell lines. We compared the anti-cancer activity of Amauroderma rude and Ganoderma lucidum, the most well-known medicinal mushrooms with anti-cancer activity and found that Amauroderma rude had significantly higher activity in killing cancer cells than Ganoderma lucidum. We then examined the effect of Amauroderma rude on breast cancer cells and found that at low concentrations, Amauroderma rude could inhibit cancer cell survival and induce apoptosis. Treated cancer cells also formed fewer and smaller colonies than the untreated cells. When nude mice bearing tumors were injected with Amauroderma rude extract, the tumors grew at a slower rate than the control. Examination of these tumors revealed extensive cell death, decreased proliferation rate as stained by Ki67, and increased apoptosis as stained by TUNEL. Suppression of c-myc expression appeared to be associated with these effects. Taken together, Amauroderma rude represented a powerful medicinal mushroom with anti-cancer activities.

Figure 1. Preparation of thirteen types of mushrooms.

(a) Photograph of thirteen types of mushrooms used in the study. (b) The thirteen types of mushrooms were dried and subject to hot water extract. The concentrations of polysaccharides were also measured.

Figure 2. Effects of the thirteen mushroom extracts on breast cancer cell activity.

(a) Cells of MDA-MB231, MDA-MB468, MT-1, MCF-7, 4T1, and BEAS-2B were inoculated in 24-well tissue culture plates at a density of the 8×10⁴ cells/well. Four hours after cell inoculation, extracts from the thirteen different mushrooms were added to each well at a concentration of 400 µg/ml and incubated at 37°C in a tissue culture incubator for 48 hours. Cell survival was determined by trypan blue staining and cell counting. n = 3, *p<0.05; **p<0.01. (b) The number of cells was normalized to the same concentration of polysaccharides listed in Figure 1b using MB231 cells to examine the pattern of effects of total extract and polysaccharides.

Figure 3. Effects of the thirteen mushroom extracts on other cancer cell activity.

Cells of A549, U87, Jurkat, HepG2, DU145, and Hela were inoculated in 24-well tissue culture plates at a density of the 8×10⁴ cells/well. Four hours after cell inoculation, extracts from the thirteen different mushrooms were added (400 µg/ml) and incubated for 48 hours. Cell survival was determined. n = 3, *p<0.05; **p<0.01.

Figure 4. Effects of polysaccharide extracts from Ganoderma lucidum and Amauroderma rude on breast cancer cell death.

The life cycle of Amauroderma rude contains spore, mycelia, and fruit body (a). Cells of MT-1 (b), MDA-MB231 (c), 4T1 (d), MDA-MB468 (e), and MCF-7 (f) were inoculated in 24-well tissue culture plates at a density of the 8×10⁴ cells/well. Four hours after cell inoculation, extracts from Ganoderma lucidum and Amauroderma rude were added to each well at different concentrations (0, 50, 100, 200, 400, 600, 800 and 1000 µg/ml) and incubated for 48 hours. Cell survival was determined by trypan blue staining and cell counting. n = 3, *p<0.05; **p<0.01.

Figure 5. The effects of Amauroderma rude extracts on breast cancer cell survival and apoptosis.

(a) The breast cancer cell line MDA-MB231 were cultured in normal medium treated with different concentrations of Amauroderma rude extract as indicated for 24 hours. Even at low concentrations, Amauroderma rude extract could effectively induce tumor cell death. Cell counting indicated a significant effect of Amauroderma rude at the concentration of 50 µg/ml (left). n = 3, **p<0.01. Typical photos of cell death are shown (right). (b) MDA-MB231 cells were cultured in normal medium treated with Amauroderma rude extract (400 µg/ml) for different periods of time. Extensive cell death was detected after eight hours of treatment. (c) MDA-MB231 cells were cultured in normal medium treated with different concentrations of Amauroderma rude extract as indicated for or 48 hours, followed by apoptosis assay. Treatment with Amauroderma rude induced apoptosis.

Figure 6. The effects of Amauroderma rude extract on colony formation.

(a) MDA-MB231 cells were mixed with low-melting agarose at the density of 1000 cells/ml and cultured in normal medium. The cultures were treated with different concentrations of Amauroderma rude extract as indicated for 20 days. Colonies formed in the agarose gel were counted. Treatment with Amauroderma rude inhibited colony formation significantly (a). n = 3, **p<0.001. (b) Typical sizes of colonies. (c) Typical photos of culture plates containing colonies were stained blue.

Figure 7. Tumorigenic activity of polysaccharide extracts from Amauroderma rude.

(a) Balb/c regular mice were injected with 4T1 cells (2×10⁵ cells/mouse). Three days after the injection, mice were injected with Amauroderma rude (20 mg/site) locally or the buffer vehicle. This was repeated every other day. On days 15, mice were sacrificed and tumors were removed for further analysis. Treatment with Amauroderma rude reduced tumor sizes. (b) Tumor sections were subject to H&E staining and examined under a light microscope. Injection with Amauroderma rude induced extensive cell death and bleeding.

Figure 8. The effects of Amauroderma rude extract on tumor cell proliferation and apoptosis in vivo.

(a) Tumor sections were subject to TUNEL staining and examined under a light microscope. Injection with Amauroderma rude induced cell death. (b) The TUNEL stained sections were scanned for quantification of apoptotic cells. Treatment with Amauroderma rude induced significantly more cell death than the control. **p<0.01. (c) Tumor sections were subject to Ki67 staining followed by examination and photograph under a light microscope to count the Ki67-positive cells. Injection with Amauroderma rude decreased Ki67 staining. (d) Typical photos of Ki67 staining are shown.

Figure 9. Suppression of c-myc expression by Amauroderma rude.

(a) Cell lysate prepared from cells treated with or without Amauroderma rude was subject to Western blot analysis probed with anti-c-myc antibody. Expression of c-myc was suppressed by Amauroderma rude treatment. (b) Lysate prepared from tumors treated with or without Amauroderma rude was subject to Western blotting probing with anti-c-myc antibody. Expression of c-myc was suppressed by Amauroderma rude treatment.