Apoptotic properties of polysaccharide isolated from fruiting bodies of medicinal mushroom Fomes fomentarius in human lung carcinoma cell line

Abstract

Mushrooms are known to complement chemotherapy and radiation therapy by countering the side effects of cancer. Recently, there has been great interest in isolation of novel bioactive compounds from mushrooms due to their numerous health beneficial effects. Chemically water-extractable polysaccharide (MFKF-AP1β), with a molecular weight of 12 kDa, was isolated from fruiting bodies of mushroom Fomes fomentarius. In this research, we investigated the anti-tumor effects of MFKF-AP1β on human lung carcinoma A549 cells. Results showed that MFKF-AP1β markedly inhibited A549 cell growth in a dose-dependent manner based on the amount of lactate dehydrogenase (LDH) released and morphological alterations. In addition, MFKF-AP1β induced cellular apoptosis by causing single-stranded DNA breakage, as evidenced by apoptosis assay. Furthermore, MFKF-AP1β (25-100 μg/ml) significantly induced single-stranded DNA breakage in A549 cells, as shown by comet assay. Taken together, our results demonstrate that MFKF-AP1β has strong anti-tumor effects mediated through induction of apoptosis. Therefore, MFKF-AP1β could be useful in lung chemotherapy.

Keywords: Anti-apoptosis; Anti-oxidant; Comet assay; DNA binding; DNA damage.

Figure 1

Effect of MFKF-AP1β on viability of A549 lung cancer cells. The viability of cells pretreated with 1 μM staurosporine (positive control) and MFKF-AP1β at different concentrations (25, 50, and 100 μg/ml) for 24 h was estimated using MTT assay after being cultured for 24 h. Data are represented as the mean ± SD of three independent experiments. An asterisk indicates ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 2

Lactate dehydrogenase (LDH) release assay. There was a significant increase in LDH release from lung cancer cells as measured by colorimetric assay. Pretreatment with 1 μM staurosporine (positive control) and MFKF-AP1β at different concentrations (25, 50, and 100 μg/ml) for 24 h significantly increased LDH release from A549 cells as a measure of cytotoxicity. Data are represented as the mean ± SD of three independent experiments. An asterisk indicates ^∗∗p < 0.01.

Figure 3

Morphological changes induced by (A) control, (B) 1 μM staurosporine, (C) 25 μg/ml of MFKF-AP1β, (D) 50 μg/ml of MFKF-AP1β, and (E) 100 μg/ml of MFKF-AP1β in A549 lung cancer cells after 24 h of treatment.

Figure 4

Representative nuclear stained images of A549 cells following treatment with different concentrations of MFKF-AP1β (25, 50, and 100 μg/ml) and 1 μM staurosporine observed under a fluorescence microscope at a magnification of 40×. (A) Control; (B) treated with staurosporine; (C–E) MFKF-AP1β 25, 50, and 100 μg/ml, respectively.

Figure 5

Representative Comet images of A549 cells following treatment with different concentrations of MFKF-AP1β (25, 50, and 100 μg/ml) and staurosporine (1 μM). (A) Control; (B) treated with staurosporine; (C–E) MFKF-AP1β (25, 50, and 100 μg/ml, respectively).

Figure 6

Induction of cell death by MFKF-AP1β. A549 cells were treated with 1 μM staurosporine and MFKF-AP1β (25, 50, and 100 μg/ml), after which amount of single-stranded DNA was detected by an ApoStrand™ ELISA apoptosis detection kit. An asterisk indicates ^∗p < 0.05, ^∗∗p < 0.01.