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Cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis in human colorectal cancer cell line COLO 205: role of FLIP and metabolic inhibitors

100%

Pajak B., Gajkowska B., Orzechowski A.

Journal of Physiology and Pharmacology. Supplement

2005

tom 56

nr 3

101-118

Current efforts are focused on revealing the cellular factors that determine the “immune escape” of cancer cells. As an example in our study human colorectal cancer cell line COLO 205 was resistant to TNF-alpha - a death inducing ligand. Nonetheless, co-incubation TNF-alpha (10 ng/ml) with cycloheximide (5 µg/ml, CHX) caused time-dependent (6, 12, 24 hours) cell death even though, at that concentration cycloheximide did not exert cytotoxic effect unless 24 hour of treatment. After additional pretreatment it is concluded that CHX sensitizes cells to TNF-alpha-induced apoptosis. The aim of this study was to investigate the role and properties of cellular proteins that granted COLO 205 cells survival as well as to assess the effect of several metabolic inhibitors on cell viability at the above-mentioned time-points. PKCs inhibitor staurosporine (5 µM) did not influence, whereas cPKC activator PMA (100 µM) prevented TNF-alpha-induced apoptosis in the presence of CHX. Although EDTA (2 mM) and to a lesser degree EGTA (5 mM) were individually cytotoxic, they exerted protective effect at 6 and partially at 12 hour of combined TNF-alpha and CHX treatment. Ionophore A23187 (1 µM) protected cells against cell death at 12 and 24 but only partially at 6 hour of treatment. On the other hand, AD (10 ng/ml) acted synergistically with TNF-alpha and CHX at 6 and 12 hour. It appears that resistance of COLO 205 cells is determined on genomic level by a few reaction steps in which both Ca2+-dependent and antiapoptotic proteins are involved. Further studies revealed that CHX treatment reduces the level of FLIP but not other members of TNF-alpha-dependent death signalosome.

Position of STAT-1alpha in cycloheximide-dependent apoptosis triggered by TNF-alpha in human colorectal COLO 205 cancer cell line: role of polyphenolic compounds

80%

Pajak B., Gajkowska B., Orzechowski A.

Journal of Physiology and Pharmacology. Supplement

2005

tom 56

nr 3

119-141

“Immune escape” is a crucial instrument used by carcinoma cells to overcome numerous strategies of immune system to delete transformed cells. Cellular factors that make cancer cells immune to defence mechanisms are incompletely understood while some remain ambiguous. Up to date evidence points to some proteins and/or signaling molecules that might be a basis for unusual behavior of cancer cells. In particular STAT kinases are currently in the main focuse of attention since they were both shown to accelerate and/or to inhibit apoptosis. In our studies we observed that human colorectal COLO 205 cancer cells were resistant to TNF-alpha- or cycloheximide-induced cytotoxicity. However, when TNF-alpha (10 ng/ml) has been given along with cycloheximide (5 µg/ml, CHX) COLO 205 cells died extensively from apoptosis. Apparently, cycloheximide sensitized cells to TNF-alpha-induced programmed cell death. To investigate the role of STAT-1alpha in CHX-mediated TNF-alpha-induced COLO 205 cell death certain polyphenolic compounds were studied if they modulate STAT-1alpha phosphorylation status and STAT-1alpha-protein interaction at the level of TNF-alpha signalosome in the 6th, 12th, and 24th hour of experiment. Neither of phenolic compound, namely PI-3K inhibitor (LY294002, 20 µM) nor MEK inhibitor (PD98059, 50 µM), nor flavonol quercetin or kaempferol (10, 100 µM) in contrast to apigenin (20 µM) influenced COLO 205 cell viability during individual or combined treatment with TNF-alpha and CHX. We conclude, that some antiapoptotic proteins were involved but not STAT-1alpha kinase to resist TNF-alpha-dependent cell death promoting activity. Summing up, except apigenin, the above-mentioned polyphenolic componds were unable to modulate survival signal in COLO 205 cells initially believed to be suppressed by STAT-1alpha.

Ograniczanie wyników

2 Journal of Physiology and Pharmacology. Supplement

2 Gajkowska B.

2 Orzechowski A.

2 Pajak B.

2 2005

Wyniki wyszukiwania

Cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis in human colorectal cancer cell line COLO 205: role of FLIP and metabolic inhibitors

Position of STAT-1alpha in cycloheximide-dependent apoptosis triggered by TNF-alpha in human colorectal COLO 205 cancer cell line: role of polyphenolic compounds