Cajanus Cajan (l.) Millsp Aqueous Extracts against Melanoma Cell Line and their Proteases

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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Insight into Mechanistic Action of Thymoquinone Induced Melanogenesis in Cultured Melanocytes

Protein and Peptide Letters ◽

10.2174/0929866526666190506114604 ◽

2019 ◽

Vol 26 (12) ◽

pp. 910-918

Author(s):

Kamal U. Zaidi ◽

Firoz N. Khan ◽

Sharique A. Ali ◽

Kausar P. Khan

Keyword(s):

Western Blot ◽

Signaling Pathways ◽

Cell Line ◽

Melanoma Cell ◽

Melanoma Cell Line ◽

Protein Kinase Inhibitors ◽

Tyrosinase Activity ◽

Dependent Manner ◽

B16f10 Melanoma ◽

B16f10 Melanoma Cell

Background: Melanin plays a crucial role in camouflage, social communication and protection against harmful ultraviolet radiations. Melanin is synthesized by melanocytes through melanogenesis and several intrinsic and extrinsic factors are involved during the process. Any change occuring in the normal melanogenesis process can cause severe pigmentation problems of hypopigmentation or hyperpigmentation. Objective: The present study is based on the evaluation of the effect of thymoquinone on melanogenesis and their possible mechanism of action using the B16F10 melanoma cell line for the production via blocking signaling pathways. Methods: Phase contrast microscopy, cell viability, tyrosinase activity, melanin content and western blot analysis were used in the present study. Results: In the present investigation, cultured melanocytes exhibit that the stimulation of melanin synthesis when treated with thymoquinone. Tyrosinase activity and melanin production in B16F10 melanoma cell line was increased in doze-dependent manner. In western blot, we investigated the involvement of the cAMP/PKA pathway in thymoquinone induced melanogenesis. It was observed protein kinase inhibitors PKA, PKC, PKB and MEK1 decreased the stimulatory effects of thymoquinone from 11.45- fold value to 8.312, 6.631, 4.51, and 7.211-fold value, respectively. However, the results also prove that thymoquinone may partially induce tyrosinase expression via PKA, PKB, PKC and MEK1 signaling pathways. Conclusion: The present finding proposed that thymoquinone is a protective challenger for melanogenesis and it might be useful for the treatment of hypopigmentary disorders.

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In Vitro Anticancer Potential of Jasione montana and Its Main Components Against Human Amelanotic Melanoma Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22073345 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3345

Author(s):

Aleksandra Maria Juszczak ◽

Robert Czarnomysy ◽

Jakub Władysław Strawa ◽

Marijana Zovko Končić ◽

Krzysztof Bielawski ◽

...

Keyword(s):

Cell Line ◽

Melanoma Cell ◽

Melanoma Cell Line ◽

Binding Assay ◽

Annexin V ◽

Melanoma Cells ◽

Amelanotic Melanoma ◽

Cytotoxic Activities ◽

Ionization Mass ◽

Caspase 9

Jasione montana L. (Campanulaceae) is used in traditional Belarusian herbal medicine for sleep disorders in children, but the chemical composition and biological activity have not been investigated. In this study, the activities of J. montana extracts, their fractions and main compounds were evaluated in amelanotic melanoma C32 (CRL-1585) cells and normal fibroblasts (PCS-201-012). The extracts and fractions were analyzed using liquid chromatography–photodiode array detection–electrospray ionization–mass spectrometry (LC–PDA–ESI–MS/TOF) to characterize 25 compounds. Further, three major and known constituents, luteolin (22) and its derivatives such as 7-O-glucoside (12) and 7-O-sambubioside (9) were isolated and identified. The cytotoxic activities against fibroblasts and the amelanotic melanoma cell line were determined using the fixable viability stain (FVS) assay. The influence of diethyl ether (Et2O) fraction (JM4) and 22 on apoptosis induction was investigated using an annexin V binding assay. The obtained results showed significant cytotoxicity of JM4 and 22 with IC50 values of 119.7 ± 3.2 and 95.1 ± 7.2 μg/mL, respectively. The proapoptotic potential after 22 treatment in the C32 human amelanotic melanoma cell line was comparable to that of vinblastine sulfate (VLB), detecting 29.2 ± 3.0% apoptotic cells. Moreover, 22 displayed less necrotic potential against melanoma cells than VLB. In addition, the influences of JM4 and 22 on the dysfunction of the mitochondrial membrane potential (MMP), cell cycle and activity of caspases 3, 8, 9, and 10 were established. The effects of JM4 on MMP change (74.5 ± 3.0% of the cells showed a reduced MMP) corresponded to the results obtained from the annexin V binding assay and activation of caspase-9. JM4 and 22 displayed a significant impact on caspase-9 (40.9 ± 2.4% of the cells contained active caspase-9 after JM4 treatment and 16.6 ± 0.8% after incubation with 22) and the intrinsic (mitochondrial) apoptotic pathway. Moreover, studies have shown that JM4 and 22 affect the activation of external apoptosis pathways by inducing the caspase-8 and caspase-10 cascades. Thus, activation of caspase-3 and DNA damage via external and internal apoptotic pathways were observed after treatment with JM4 and 22. The obtained results suggest that J. montana extracts could be developed as new topical preparations with potential anticancer properties due to their promising cytotoxic and proapoptotic potential.

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