In vitro evaluation of cytotoxic, anti-proliferative, anti-oxidant, apoptotic, and anti-microbial activities of Cladonia pocillum.

The aim of this study was to investigate the anti-proliferative, apoptotic, cytotoxic, and anti-oxidant effects of extracts from the lichen Cladonia pocillumon human breast cancer cells (MCF-7), and to characterize the anti-microbial features.  MCF-7 cells were treated with methanolic C. pocillum extract for 24h. The cytotoxicity of the extract was tested with MTT. Moreover, its anti-proliferative effects were examined with immunocytochemical method. Apoptosis and biochemical parameters were detected in MCF-7. The methanol and chloroform extracts of the lichen were tested for anti-microbial activity against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans using the disc diffusion method and calculation of minimal inhibitory concentrations. Although BrdU incorporation was not observed in MCF-7 cells treated with methanol extract at a concentration above 0.2 mg/mL, a significant decrease was observed int he percentage of PCNA immunoreactive cells in groups treated with 0.2, 0.4, 06, and 0.8 mg/mL methanol extracts of C.pocillum (49±6.3, 44±5.2, 23±2.5, 0, respectively) compared to that of control (85±4.5). The percentage of apoptotic cells significantly increased in groups treated with 0.2, 0.4, 0.6, and 0.8 mg/mL extracts of the C.pocillum (54±3.5, 76±2.6, 77±1.8, 82±4.2, respectively) compared with that of control group (3.9±1.5).The half-maximal inhibitory concentration of the methanol extract against MCF-7 cells was 0.802 mg/mL .Although the chloroform extract showed more effective anti-microbial activity overall, the methanol extract showed higher anti-fungal activity. Collectively, the results of our study indicate that C.pocillum extracts have strong anti-microbial and apoptotic effects. This lichen therefore shows potential for development as a natural anti-microbial, anti-oxidant, and apoptotic agent.

Anti-Proliferative and Apoptotic Effects of Methanolic Extracts from Different Cladonia Species on Human Breast Cancer Cells.

This study tries to elucidate the anti-proliferative and apoptotic effects of methanolic lichen extracts from Cladonia rangiformis and Cladonia convolute in MCF-7 human breast cancer cells. Lichen extracts (0-2 mg/ml) were added to MCF-7 cells for 24 h. Cell viability was tested using 3-(4,5-dimethylthiazol- 2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Cell proliferation was observed using bromodeoxyuridine (BrdU) labelling and proliferating cell nuclear antigen (PCNA) by immunocytochemistry. The TUNEL method was used for cell death detection. The effective dose (ED50) values of methanolic extracts from C. rangiformis and C. convolute were found to be 0.905 and 0.977 mg/ml, respectively. Treatment with C. rangiformis methanolic extract (0.2-0.8 mg/ml) dose-dependently inhibited proliferation of MCF-7 cells as detected by BrdU incorporation. The inhibition was started in 0.2 mg/ml concentration of C. convoluta methanolic extract. The percent of PCNA immunopositive cells showed a decrease in MCF-7 cells treated with two lichen extracts compared to control MCF-7. Both methanolic extracts showed a significant increase in percentage of apoptosis-positive cells. These results indicate that methanolic lichen extracts from C. rangiformis and C. convolute inhibited proliferation of MCF-7 cells and caused apoptosis in MCF-7 cells. The lichens may be novel natural agents for treating breast cancer disease.