Biomedical subjects

C Courage

Publications and source records attributed to C Courage.

6 recordsLinked to original sources

PubMed1999

Decrease in susceptibility toward induction of apoptosis and alteration in G1 checkpoint function as determinants of resistance of human lung cancer cells against the antisignaling drug UCN-01 (7-Hydroxystaurosporine).

7-Hydroxystaurosporine (UCN-01) is a protein kinase inhibitor that is under development as an anticancer agent in the United States and Japan. Long-term exposure of human A549 non-small cell lung cancer cells to UCN-01 furnished cells (A549/UCN) with acquired resistance against UCN-01. In this study, the sensitivity of these cells toward the growth-arresting properties of certain conventional cytotoxic agents was explored. Cells were not cross-resistant against adriamycin, Taxol, staurosporine, and UCN-02, but they displayed 14- and 4.4-fold resistance against cisplatin and mitomycin C, respectively. Previous studies on the mechanism(s) of action of UCN-01 suggest that induction of apoptosis and G1 phase accumulation are important for its anticancer activity; therefore, we compared induction of apoptosis and cell cycle distribution caused by UCN-01 in wild-type A549 and A549/UCN cells using flow cytometry. UCN-01 (0.4 microM) induced apoptosis (62% terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells) in A549 cells, but not in A549/UCN cells. The percentages of cells that accumulated in G1 when exposed to UCN-01 (0.4 microM) were 22% in A549 cells and 67% in A549/UCN cells. These results suggest that acquired resistance of cancer cells against UCN-01 is characterized by attenuation of apoptosis induction associated with reinforcement of the G1 checkpoint and that apoptosis regulation is drastically altered in A549/UCN cells as compared with A549 cells. Cyclin-dependent kinase (CDK) inhibitor proteins p21 and p27 in A549/UCN cells were up-regulated, which was accompanied by overexpression of G1 cyclins D1 and E, but UCN-01 hardly affected levels of these proteins. In contrast, cyclin A, cyclin B1, retinoblastoma, and CDK2 proteins were apparently down-regulated, without changes in CDK4/6. UCN-01 hardly affected the expression level of cyclin B1 and induced dephosphorylation of retinoblastoma in both cell types. UCN-01 induced down-regulation of cyclin A level and CDK2 activity accompanied with its dephosphorylation in A549/UCN cells, but not in A549 cells. The antiapoptotic protein bcl-2 was apparently up-regulated in A549/UCN cells, however, bcl-xL, another antiapoptotic protein, was down-regulated, without changes in bak and bax. Taken together, these results are consistent with the notion that induction of apoptosis and block of cell cycle in G1 are important determinants of the sensitivity of cancer cells to UCN-01 and suggest that inhibition of CDK2 activity accompanied by its dephosphorylation and decrease of expression level of cyclin A might play an important role in the G1 phase accumulation induced by UCN-01.

Alkaloids↗

PubMed1997

Characterisation of novel human lung carcinoma cell lines selected for resistance to anti-neoplastic analogues of staurosporine.

The staurosporine analogues CGP 41251, UCN-01 and Ro 31-8220 are specific inhibitors of protein kinase C (PKC). CGP 41251 and UCN-01 exert anti-neoplastic activity against human tumours grown in rodents, and CGP 41251 reverses multidrug resistance. The hypothesis was tested that these agents can induce drug resistance and alter cellular levels of target kinases. Human-derived A549 lung carcinoma cells were exposed for 6 months to CGP 41251, UCN-01 or Ro 31-8220 at gradually increasing concentrations. Cells acquired resistance against these agents, 4.3-fold against CGP 41251 (A549/CGP cells), 4.0-fold against UCN-01 (A549/UCN cells) and 14-fold against Ro 31-8220 (A549/Ro cells). Cells were neither collaterally cross-resistant towards the PKC inhibitors nor resistant against the growth-inhibitory properties of 12-O-tetradecanoylphorbol-13-acetate. However, cross-resistance was observed in A549/CGP cells against staurosporine (13-fold) and in A549/Ro cells against doxorubicin (26-fold). All 3 cell types expressed multidrug resistance-associated protein, and A549/Ro cells expressed P-glycoprotein, as adjudged by Western blot analysis. Phorbol ester-stimulated PKC activity in these cells was decreased by between 57% and 96% compared to wild-type A549 cells. Levels of the PKC isoenzymes alpha and theta in all 3 resistant cell types and of PKC-epsilon in A549/UCN cells were concomitantly reduced. Cells regained drug sensitivity after culture in the absence of drug for 6 (A549/Ro cells), 5 (A549/CGP cells) and 1 (A549/UCN cells) months. Our results suggest the following features of this type of anti-signalling drug: (i) they can induce drug resistance, (ii) they may be potentially useful in combination because of the lack of cross-resistance between them and (iii) they can down-regulate PKC, which may have pharmacological or toxicological consequences.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Cellular relocalisation of protein kinase C-theta caused by staurosporine and some of its analogues.

The microbial product staurosporine is a protein kinase C (PKC) inhibitor with some phorbol ester-agonistic properties. It is known to cause the translocation of the PKC isoenzymes epsilon and delta from the cellular cytosol to the membrane and nucleus. We tested the hypothesis that it also affects the cellular localisation of the novel PKC isoenzyme theta, and that staurosporine analogues, some of which are currently under clinical evaluation as potential anticancer drugs, have a similar effect. Their ability to alter PKC-theta distribution was studied in human-derived A549 lung carcinoma cells. Western blot analysis and confocal microscopy after indirect immunofluorescence staining showed that staurosporine (100 nM), like the phorbol ester 12-O-tetradecanoylphorhol-13-acetate (25 nM) caused the translocation of PKC-theta from the cytosol to the membrane and the nucleus. The bisindolylmaleimide GF 109203X mimicked staurosporine, but had a weaker effect. Ro 31-8220 and UCN-01 decreased cytosolic PKC-theta only at 1 microM. CGP 41251 had no effect on PKC-theta in either experimental design. The results show that some, but not all, staurosporine analogues share the partial phorbol ester-agonistic PKC-translocatory activity of the parent molecule.

Alkaloids↗

PubMed1996

Differential effects of staurosporine analogues on cell cycle, growth and viability in A549 cells.

Staurosporine is a potent but non-specific kinase inhibitor. It has served as synthetic template for a variety of analogues, the indolocarbazoles, UCN-01 and CGP 41251, and the bisindolylmaleimides, Ro 31-8220 and GF 109203X, were investigated as growth inhibitors of human-derived A549 human lung adenocarcinoma cells. They were compared with respect to (1) effect on the cell cycle, (2) time dependency of growth arrest and (3) cytotoxic potency. Cells were exposed for 1, 2 and 4 days, or for 6, 12 and 24 h in the case of cycle-synchronised cells, to staurosporine analogues at concentrations at which they inhibited growth by 80% after 4 day exposure. Staurosporine and UCN-01 retarded cells in G0/1, and CGP 41251 appeared to inhibit cell growth without cell cycle specificity. Ro 31-8220 slowed progression of synchronised cells through the cycle; over a longer time period it induced a weak block in G2/M. GF 109203X induced potent G2/M arrest in synchronised cells. This was not so apparent in asynchronous cells, which by day 4 were slowed in G0/1 instead. Growth arrest induced by these inhibitors was more potent after incubation for 4 rather than 2 days. Incubation for 1 day followed by maintenance in drug-free medium for 3 days was sufficient to exert some cytostasis. The differences between cytotoxic and cytostatic concentrations, the former measured by release from cells of lactate dehydrogenase, were 15 000-fold for staurosporine, 300-fold for UCN-01, approximately 400-fold for CGP 41251, 25-fold for Ro 31-8220 and approximately 4-fold for GF 109203X. The results show that PKC-selective staurosporine analogues differ with respect to the mechanisms by which they interfere with the cell cycle. The necessity of long-term exposure for effective growth inhibition and the considerable margin between cytostatic and acute cytotoxic indolocarbazole concentrations are findings which might influence the planning and interpretation of clinical trials of these kinase inhibitors.

Adenocarcinoma↗

PubMed1995

Comparison of ability of protein kinase C inhibitors to arrest cell growth and to alter cellular protein kinase C localisation.

Inhibitors of protein kinase C (PKC) such as the staurosporine analogues UCN-01 and CGP 41251 possess antineoplastic properties, but the mechanism of their cytostatic action is not understood. We tested the hypothesis that the ability of these compounds to arrest growth is intrinsically linked with their propensity to inhibit PKC. Compounds with varying degrees of potency and specificity for PKC were investigated in A549 and MCF-7 carcinoma cells. When the log values of drug concentration which arrested cell growth by 50% (IC50) were plotted against the logs of the IC50 values for inhibition of cytosolic PKC activity, two groups of compound could be distinguished. The group which comprised the more potent inhibitors of enzyme activity (calphostin C, staurosporine and its analogues UCN-01, RO 31-8220, CGP 41251) were the stronger growth inhibitors, whereas the weaker enzyme inhibitors (trimethylsphingosine, miltefosine, NPC-15437, H-7, H-7I) affected proliferation less potently. GF 109203X was exceptional in that it inhibited PKC with an IC50 in the 10(-8) M range, yet was only weakly cytostatic. To substantiate the role of PKC in the growth inhibition caused by these agents, cells were depleted of PKC by incubation with bryostatin 1 (1 microM). The susceptibility of these enzyme-depleted cells towards growth arrest induced by staurosporine, RO 31-8220, UCN-01 or H-7 was studied. The drug concentrations which inhibited incorporation of [3H]thymidine into PKC-depleted A549 cells by 50% were slightly, but not significantly, lower than significantly, lower than those observed in control cells. These results suggest that PKC is unlikely to play a direct role in the arrest of the growth of A549 and MCF-7 cells mediated by these agents. Staurosporine is not only a strong inhibitor of PKC but also mimics activators of this enzyme in that it elicits the cellular redistribution of certain PKC isoenzymes. The ability of kinase inhibitors other than staurosporine to exert a similar effect was investigated. Calphostin C, H-7, H-7I, miltefosine, staurosporine, UCN-01, RO 31-8220, CGP 41251 or GF 109203X were incubated for 30 min with A549 cells in the absence or presence of the PKC activator 12-O-tetradecanoyl phorbol-13-acetate. The subcellular distribution of PKC-alpha-, -epsilon and -zeta was measured by Western blot analysis. None of the agents affected PKC-alpha or -zeta.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaloids↗

PubMed1994

Establishment of a murine leukaemia cell line resistant to the growth-inhibitory effect of bryostatin 1.

Bryostatin 1 is a novel macrocyclic lactone activator of protein kinase C (PKC) which has clinical potential as an anti-cancer agent. The mechanism of action of this agent is unknown, but protein kinase C has been implicated. In order to investigate this possibility, we have developed P388 sublines resistant to bryostatin 1, by continuous challenge of the parent cell line with increasing incremental concentrations of the drug over 4 months. Cell lines were established at monthly intervals yielding four sublines: P388/BR/A, which were removed at 1 month; P388/BR/B, obtained after 2 months; P388/BR/C, obtained after 3 months; and P388/BR/D, which were established after 4 months. All four P388/BR sublines show an equal degree of resistance to the growth inhibitory effects of bryostatin 1, with a relative resistance ratio (RR) IC50 of approximately 4,000. The ability of the cytosol of cells to phosphorylate PKC-specific substrate is decreased by 41% for BR/A, 57% for BR/B 80% for BR/C and 94% for BR/D compared with the parental cell line, even when grown in the absence of bryostatin 1 for up to 4 weeks. Similar decreases are seen for cytosolic phorbol ester binding and whole-cell PKC isoenzyme expression. All four P388/BR sublines show high and equal levels of cross-resistance to the PKC activatory phorbol ester, phorbol 12-myristate 13-acetate (PMA). There is no loss of resistance to either bryostatin 1 or PMA up to 3 months after termination of exposure of the sublines to bryostatin 1. There was no significant degree of cross-resistance to daunorubicin in the bryosatin 1-resistant cell lines, P388/BR/A, B, C or D, when compared with the parent cell line, P388.

Animals↗