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Complex assessment of in vitro anti-melanoma action of medicinal plant Cotinus coggygria Scop
Abstract
Purpose: To elucidate the anticancer mechanism and molecular targets of the aqueous fraction from Cotinus coggygria leaf extract in A375 malignant melanoma cells.
Methods: Aqueous ethanol extract from the leaves of C. coggygria was fractionated into the aqueous and chloroform fractions by solvent–solvent partition. A375 cells were treated with the aqueous fraction at various concentrations and incubated for 72 h. The effect of the aqueous fraction on programmed cell death by staining with Annexin V/Propidium iodide ias well as on cell viability and adhesion through labeling with fluorescein diacetate were determined under fluorescence microscopy. In addition, the clonogenicity assessment, comet assay evaluation of genotoxic properties, characterization of thermodynamic behavior of cells by differential scanning calorimetry, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on the expression of genes involved in processes of apoptosis and cell cycle control were determined.
Results: The number of viable adherent cells was reduced by 38.00 and 85.06 % at 20 and 80 µg/mL, respectively compared to untreated cells. In addition, a significant, dose-dependent reduction in the clonogenicity of melanoma cells was observed, culminating in an almost complete inhibition of cancer cells colony-forming ability at 40 and 60 µg/mL of the fraction. Treatment with the fraction increased comets of Classes 3 and 4 to 35 %, while comets of Class 5, which corresponds to the highest level of DNA fragmentation, increased to 2.25 %. Also, the gene damage index (GDI) was highest (1.2) at the concentration of 40 μg/mL. Furthermore, the expression levels of BAX, CASP8 and CDKN1A were upregulated and MYC expression decreased after treatment with the aqueous fraction.
Conclusion: Cotinus coggygria possesses significant in vitro anti-melanoma potential. Future studies will be focused on the in vivo assessment of its therapeutic qualities in experimental animals.
