Apoptotic activity of ethanolic extract of Thai indigenous mushroom Russula alboareolata against L929, HeLa and HepG2 cells by MMP assay

Objectives: This study was carried out to investigate apoptotic activity of Russula alboareolata ethanolic extract against L929, HepG2 and HeLa cells Methods: Fresh samples of R alboareolata from Kalasin, Mukdahan and Yasothon in the Northeastern part of Thailand. Dried samples were individually macerated with 95% ethanol with sonication for 1 hour to obtain crude extracts. Three cell lines including L929, HepG2 and HeLa purchased from ATCC were grown as adherent in DMEM at 37oC in 5% CO2 incubator. Cells (1x105 cells/ml, 200 µl) were seeded onto 24 well plate and further grown for 24 hr. Apoptotic activity of R alboareolata extract was determined by mitochondrial membrane potential (MMP) assay using JC-1 fluorescent method. The L929, HeLa and HepG2 cells were treated for 24 hr with the extract at 600, 500 and 1,000 µg/ml,respectively. The chemotherapeutic drug, valinomycin was used as an apoptotic inducer and kept as a positive control. Apoptotic cells of L929, HeLa and HepG2 were analysed by fluorescent imaging system using the InCell 2200 Analyser (GE Healthcare, UK) following being stained with JC-1 and Hoechst 3342 reagents. Results: Valinomycin (1µg/ml) clearly induced apoptosis in L929, HeLa and HepG2 at 43.86, 36.85 and 40.00%, respectively. The percentages of apoptosis induced by R alboareolata extract was found as follows: L929 (77.20%, tested at 600 ug/ml), HeLa (73.69%, tested at 500 ug/ml),   and HepG2 (30.00%, tested at 1,000 ug/ml), Conclusion: The ethanolic extract R. alboareolata possesses apoptotic activity against both normal (L929) and cancerous cells (HeLa and HepG2). However, we were unable to compare the degree of its apoptotic induction among these three cell lines due to the different concentrations used. Further studies including active constituents and in vivo experiments are required if this mushroom will be used as a novel dietary supplement and/or botanical-drug for chemoprevention.