Assessment of the anti-cancer potential of mushroom extracts in A549 lung cancer cells and the efficacy of doxorubicin

Abstract

Introduction: The five-year survival rate for metastatic lung cancer is 18.6%. In Eastern medicine, mushrooms have been used as a treatment for various cancer types. Among these are Ganoderma lucidum and Trametes versicolour, which contain bioactive molecules with proposed anticancer and immunomodulatory properties. In this study, commercially available mushroom products were evaluated for their potential cytotoxic effects against the A549 lung cancer cell line and the results were compared to a normal cell line (HEK293). Methods: The mushroom products underwent dual extraction using methanol and dichloromethane which was evaporated. The final dried extracts were redissolved in sterile distilled water (I. obliquus) or a DMSO:ethanol mixture (T. versicolour and G. lucidum).A549 cells were maintained aseptically in culture. The MTT cell viability assay was used to determine the cytotoxic effect and the 50% inhibitory concentrations (IC50) of the mushroom extracts. Doxorubicin was used as a positive control. Cell morphology was evaluated using fluorescent probes, Hoechst 33342, propidium iodide, and acridine orange, to assess the mode of cell death when cells were exposed to concentrations equivalent to IC80 values of the mushroom extracts and doxorubicin. The expression of phosphatidylserine on cell membranes was detected with FITC-labelled annexin-V, caspase-3/7 expression was assessed using the CellEvent™ Caspase-3/7 Green Detection Reagent, and reactive oxygen species formation was measured using the CellROX™ Deep Red Reagent. P-glycoprotein expression levels were determined using immunofluorescence techniques. Results: G. lucidum was the most active mushroom extract in A549 and HEK293 cells at 24 h (IC50 value = 0.255 mg/mL and 0.591 mg/mL, respectively). When combined with doxorubicin in A549 cells, both G. lucidum and T. versicolour decreased the efficacy of doxorubicin. In HEK293 cells, the mushroom extracts increased the efficacy of doxorubicin. According to FTIR analysis, extracts were most stable when stored at 4℃. Morphological evaluation showed apoptosis in A549 cells treated with T. versicolour at 0.5 mg/ml, whereas G. lucidum caused concentration dependent necrosis. Annexin-V staining confirmed apoptosis in G. lucidum and T. versicolour treated cells, along with an increase in caspase-3/7 activity in A549 cells. HEK293 cells underwent necrosis when treated with the extracts for 24 h. Reactive oxygen species production was also elevated in both A549 and HEK293 treated cells. Immunofluorescence analysis showed no significant changes in P-glycoprotein expression. iv Conclusion: The cytotoxic effects of G. lucidum and T. versicolour on both cancerous and non- cancerous cells highlight their lack of selectivity, raising significant safety concerns regarding their use. Given their potential to harm normal tissues, further research is necessary to establish safe exposure limits and regulatory guidelines are needed to protect consumers from unverified or misleading health claims.