calcimycin and Colorectal-Neoplasms

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 micro g/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 microM) did not influence, whereas cPKC activator PMA (100 microM) 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 microM) 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 Ca(2+)-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.

Topics: Apoptosis; Calcimycin; Calcium; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Cell Survival; Chelating Agents; Colorectal Neoplasms; Cycloheximide; Dactinomycin; Drug Synergism; Edetic Acid; Egtazic Acid; Enzyme Activation; Enzyme Activators; Humans; Ionophores; Protein Kinase C; Protein Kinase Inhibitors; Protein Synthesis Inhibitors; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate; Time Factors; Tumor Escape; Tumor Necrosis Factor-alpha

Peripheral blood monocytes obtained from 8 colorectal cancer patients and 6 normal controls were incubated in vitro with interferon-r (IFN-r) in the presence of bacterial lipopolysaccharide (LPS). The cytotoxic properties of the monocyte were determined subsequent to the interaction with radiolabeled autologous, allogeneic, as well as cultured colorectal cancer cells. Monocytes from normal controls and all colorectal cancer patients were activated in vitro to become tumoricidal; monocytes lysed tumorigenic cells but not nontumorigenic cells. Activators of protein kinase C (e.g. phorbol esters, PMA) and Ca2+ ionophores (A23187) when added alone did not effect the activation state of the monocyte. Whereas, PMA and A23187 cooperatively reproduced the ability of IFN-r to prime monocytes for tumoricidal activity. In the presence of PMA, A23187, and EGTA, the addition of excessive Ca2+ was sufficient for priming, whereas the addition of excessive Mg2+ was much less efficient. Priming by IFN-r, however, was not blocked by EGTA. An efflux of Ca2+ from preloaded monocytes was significantly increased by A23187 and by IFN-r. Quin-2/AM, an intracellular chelator of Ca2+, blocked priming by IFN-r. The results suggest that priming of monocytes for tumoricidal function by IFN-r may be involved in the activation of protein kinase C and mobilization of intracellular Ca2+.

Topics: Aged; Aminoquinolines; Calcimycin; Calcium; Colorectal Neoplasms; Cytotoxicity, Immunologic; Enzyme Activation; Female; Humans; In Vitro Techniques; Interferons; Lipopolysaccharides; Macrophage Activation; Male; Middle Aged; Monocytes, Activated Killer; Protein Kinase C; Recombinant Proteins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Peripheral blood mononuclear cells (PBMC) were cultured with phytohemagglutinin (PHA) in 46 colorectal cancer patients and 27 normal controls. By collectively comparing the cancer patients with the normal controls, the cancer patients had: (1) decreased interleukin-2 (IL-2) secretion, (2) fewer interleukin-2 receptor (IL-2R) expression on cell surface, and (3) less 3H thymidine incorporation. The ability of the phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the calcium ionophore, A23187, in co-stimulation with PHA, to enhance the IL-2 secretion, IL-2R expression and 3H thymidine incorporation were studied in the PBMC of colorectal cancer patients. By adding A23187 into the PHA-stimulated PBMC from colorectal cancer patients, IL-2 production and IL-2R expression were increased up to levels equal to that obtained in PHA-stimulated PBMC from normal controls. Whereas there was no significant increase in proliferative response. However, PMA has no effect on all three parameters. A23187 is known to be effective in elevating cytosolic free Ca2+ and PMA is regarded as an activator of protein kinase C. Therefore, we may conclude that the impairment of IL-2 production and IL-2R expression in PHA-stimulated cultures is mainly due to failure of the free Ca2+ release which can be repaired by A23187. While protein kinase C activation may not be impaired since its deficit in IL-2 production and IL-2R expression can not be corrected by PMA.

Topics: Aged; Calcimycin; Colorectal Neoplasms; Female; Humans; Interleukin-2; Killer Cells, Natural; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Middle Aged; Receptors, Interleukin-2; Tetradecanoylphorbol Acetate