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Aims: Extract proteins with protease inhibitor (PI) activity from fresh organs of Cajanus cajan, using aqueous systems; study the activity of melanoma secreted proteases; investigate inhibitory effect of C. cajan extracts on melanoma proteases; and evaluated the effect of the extract, with the most protease inhibitor activity, on melanoma cell line (SK-MEL-28) viability.
Material and Methods: Extracts of C. cajan leaves, stems, and roots were prepared using different aqueous systems. Protein content was evaluated by Bradford method, protein profile by gel electrophoresis by Laemmli method and, extracts PI activity against trypsin, papain, and pepsin. Melanoma cell line was cultured in Dulbecco's medium, and secreted proteases was obtained from culture supernatant. Characterization of melanoma proteases included substrates activity, optimum pH, and effect of specific PIs and cations on protease activity. Anticancer activity was investigated using C. cajan extracts (containing PIs) and SK-MEL-28 extracellular fraction (containing proteases). Cytotoxic effect of extract was assayed on SK-MEL-28 cells line using methylthiazol tetrazolium method. CC-P was submitted to thin layer chromatography to identify alkaloids, coumarins, flavonoids, saponins and terpenes.
Results: C. cajan extracts showed different protein contents and protein profiles in electrophoresis analysis. C. cajan organs presented PIs activities against serine, cysteine, and aspartic proteases. Leaf extract prepared using phosphate buffer (CC-P) and stems extract prepared with water (CC-CA) had the best inhibitory activities against trypsin (~58%). Pepsin was the lowest inhibited (11-29%) and papain was the most inhibited (14-100%). Protease activity of melanoma fraction was the highest using casein as substrate, and two proteins with 150 and 100 kDa with gelatinase activity. These proteases has maximal activity at pH 7.0 and 9.0, and was importantly inhibited by benzamidine, 1,10-phenanthroline and EDTA, suggesting that serine and metalloproteases are secreted by SK-MEL-28 cells. CC-P was the most important inhibitor of melanoma proteases, and induced cytotoxicity on SK-MEL-28 cells in culture. Although there is correlation between melanoma protease inhibition and cell death, CC-P has secondary metabolites, as coumarins, flavonoids and terpenes that can have synergy of antitumor activity.
Conclusion: C. cajan extracts have serine, cysteine, and aspartic protease inhibitor activities. CC-P had the best inhibition on melanoma proteases and it was also cytotoxic to melanoma cell in culture. Therefore, these PIs can be important strategy for cancer treatment because tumor cells secrete proteases that are crucial to cancer progression.
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