Effect of Na+, K+ ion concentration on the production of plasminogen activator from a melanoma cell line

1985 ◽  
Vol 38 (5) ◽  
pp. 577-584
Author(s):  
O. Matsuo ◽  
H. Kikuchi ◽  
S. Ueshima ◽  
K. Okada ◽  
C. Matsuo ◽  
...  
Keyword(s):  
Cell Line ◽  
Plasminogen Activator ◽  
Melanoma Cell ◽  
Melanoma Cell Line ◽  
Ion Concentration

scholarly journals Determination of tissue-type plasminogen-activator mRNA in human and non-human cell lines by dot-blot hybridization

1985 ◽  
Vol 231 (2) ◽  
pp. 309-313 ◽  
Author(s):  
G Opdenakker ◽  
A Billiau ◽  
G Volckaert ◽  
P de Somer
Keyword(s):  
Cell Line ◽  
Plasminogen Activator ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Human Cell ◽  
Melanoma Cell Line ◽  
Tissue Type ◽  
Human Cell Lines ◽  
Dot Blot ◽  
Blot Technique

A labelled cDNA clone was used in DNA-RNA hybridization on nitrocellulose filter paper (dot-blot technique) to detect and quantify mRNA for endogenous tissue plasminogen activator (PA) in cell extracts and samples of RNA purification runs. Although, for detection purposes, the assay was less sensitive than translation in Xenopus oocytes, it was at least as reliable and much more convenient for the purpose of quantitative determination. In particular, the technique was used to study the kinetics of PA mRNA formation in a human melanoma cell line (Bowes) after exposure to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Incubation of the cells with TPA resulted in a 15-20-fold increase in cellular PA mRNA content. The effect was time- and dose-dependent: the increase in PA-specific mRNA was clearly visible as early as 4 h after initiation of TPA treatment in Bowes cells. It was blocked completely by pretreatment of the cells with actinomycin D, indicating that TPA caused enhancement of synthesis of PA mRNA rather than inhibition of PA mRNA degradation. The use of the nitrocellulose dot-blot technique also revealed that two non-human cell lines produce mRNAs which cross-react with the human PA mRNA, namely the mouse melanoma cell line B16 and the rat brain-tumour cell line, RT4-71-1. TPA was found to exert similar stimulatory effects on the synthesis of mRNAs in these cell lines as in Bowes cells.

ANALYSIS OF THE SECRETION MECHANISM OF TISSUE-TYPE PLASMINOGEN ACTIVATOR IN A HUMAN MELANOMA CELL LINE (BOWES)

1987 ◽  
Author(s):  
T Sakai ◽  
K Okada ◽  
H Bando ◽  
S Ueshima ◽  
N Tanaka ◽  
...  
Keyword(s):  
Cell Line ◽  
Plasminogen Activator ◽  
Melanoma Cell ◽  
Melanoma Cell Line ◽  
Human Melanoma ◽  
Human Melanoma Cell ◽  
Tissue Type ◽  
Cell Organelles ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

The human melanoma cell line (Bowes) secretes tissue-type plasminogen activator (t-PA) into the culture medium. As reported previously, the secretion of t-PA was depressed and t-PA was accumulated in the intracellular compartment at alternative sodium and potassium ion concentrations and also in the presence of monensin, an ionophore for monovalent cations. In the present study, the secretion and intracellular distribution of t-PA were investigated by radioisotope labeling and fractionation of the cell organelles under normal and monensin-treated conditions. Cell homogenate was fractioneted by discontinuous sucrose density gradient ultracentrifugation. The plasminogen activator (PA) activity or t-PA antigenicity in these fractions was not uniformly distributed, but rather localized in those fractions where cell organelles were rich. This implied that t-PA was enclosed by the intracellular membranous system. Cells were incubated with 35s-methionine and/or 3H-mannose to produce labeled intracellular glycoproteins including t-PA and its premature intermediates, which were separated by immunological adsorption to rabbit anti t-PA IgG-protein A Sepharose. Pulse labeling with 35s-methionine (3 min) demonstrated that intracellular t-PA was transported from the heavier fractions (rER), via intermediate ones (Golgi complex), to lighter ones. The radioactivity of the intracellular t-PA reached a maximum in 30 min, while that of secreted t-PA was observed in 30 min and increased linearly at least for the following 20 min. Enzymography revealed the major intracellular PA activity at 72 kDa and minor activity at 50 kDa. Monensin treatment (10 μM, 6 hr) caused accumulation of the 72 kDa component which was immunologically homologous to secreted t-PA. After long term (3 hr) simultaneous 35s-methionine and 3H-mannose labeling, the radioactivity ratio (3H/35S) the intracellular t-PA was increased in the presence of monensin. These results suggest that some sugar chain(s) in the t-PA molecule are of high mannose type, which is interfered with by monensin in the course of the intracellular processing and transport of t-PA.

Insight into Mechanistic Action of Thymoquinone Induced Melanogenesis in Cultured Melanocytes

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.

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

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.

scholarly journals Combined p14ARF and Interferon-β Gene Transfer to the Human Melanoma Cell Line SK-MEL-147 Promotes Oncolysis and Immune Activation

2020 ◽  
Vol 11 ◽  
Author(s):  
Otto Luiz Dutra Cerqueira ◽  
Maria Alejandra Clavijo-Salomon ◽  
Elaine Cristina Cardoso ◽  
Tharcisio Citrangulo Tortelli Junior ◽  
Samir Andrade Mendonça ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cell Line ◽  
Melanoma Cell ◽  
Immune Activation ◽  
Melanoma Cell Line ◽  
Human Melanoma ◽  
Human Melanoma Cell Line ◽  
Human Melanoma Cell ◽  
Interferon Β
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