Accumulation Of DDP Research Articles

Modulation of cis‐diamminedichloroplatinum(II) accumulation and sensitivity by forskolin and 3‐isobutyl‐1‐methylxanthine in sensitive and resistant human ovarian carcinoma cells

We have determined the effect of forskolin, an adenyl cyclase agonist, and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, on the accumulation and cytotoxicity of cisplatin (DDP) in 2008 human ovarian carcinoma cells. In DDP-sensitive 2008 cells, forskolin and IBMX caused 2.1-fold and 2.3-fold increases, respectively, in the short-term accumulation of DDP relative to untreated cells. The inactive analogue, 1,9-dideoxyforskolin, decreased DDP accumulation. Forskolin and IBMX also increased accumulation in A2780 cells. Neither forskolin nor IBMX had any effect on DDP accumulation in DDP-resistant 2008 cells. The effects were detectable as early as 1 min and persisted at 60 min. The concentrations for half-maximal stimulation of DDP accumulation were approximately 0.2 microM for forskolin and 0.2 mM for IBMX. Forskolin caused marked increases in cAMP levels in both sensitive and resistant 2008 cells within 1 min, although there were differences in the subsequent time-courses of the response. Both 2008 cell types had identical cAMP-dependent protein kinase (PKA) activity. These results suggest that there is a target downstream of PKA that is an important participant in DDP accumulation, and that this target is defective or missing in DDP-resistant cells. Following a 1-hr exposure to drugs, forskolin and IBMX at concentrations that were by themselves completely non-toxic increased the slopes of the clonogenic survival vs. DDP concentration curves in 2008 cells 1.9-fold and 3.3-fold, respectively. In DDP-resistant 2008 cells, however, forskolin and IBMX increased the slopes only 1.2 and 2.6-fold, respectively. These effects of forskolin and IBMX on DDP cytotoxicity did not directly correlate with the effects on the 1-hr DDP accumulation which suggested that, in addition to modulating DDP accumulation, these agents increase the cytotoxicity of the intracellular platinum. The results indicate that modulation of cAMP levels can have important effects on DDP accumulation and cytotoxicity in 2008 cells and that these effects are significantly diminished in DDP-resistant cells.

Increased sensitivity to cis-diamminedichloroplatinum(II) in human ovarian carcinoma cells in response to treatment with 12-O-tetradecanoylphorbol 13-acetate.

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) enhanced sensitivity to cis-diamminedichloroplatinum(II) (DPP) in human ovarina carcinoma 2008 cells by a factor of 2.53 +/- 0.74 fold (S.D.). Sensitization was maximum 3 h after a 1-h exposure to TPA and had disappeared completely by 7 h after treatment. An equivalent degree of sensitization was produced in a 2008 variant selected for 10-fold resistance to DDP. TPA neither increased nor decreased cellular accumulation of DDP. Phorbol, a TPA analog which does not activate protein kinase C, did not cause sensitization. This synergistic interaction between TPA and DDP was completely inhibited by pretreatment with staurosporine, a protein kinase C inhibitor. Cellular cAMP was not altered by TPA stimulation. Furthermore, cycloheximide, a potent protein synthesis inhibitor, did not block the TPA-induced enhancement of drug sensitivity. These results strongly suggest that DDP sensitivity can be modulated by protein kinase C and regulated by phosphorylation of a protein kinase C substrate in both intrinsically sensitive and DDP-resistant cells.

Rapid Emergence of Acquired cis-Diamminedichloroplatinum(II) Resistance in an In Vivo Model of Human Ovarian Carcinoma

We have characterized the acquisition of resistance to cisplatin (DDP) in an in vivo solid tumor model. Human ovarian carcinoma cells (2008 cell line) were grown s.c. as xenografts in athymic mice. Tumors were selected with 3.0 mg/kg of DDP i.p. given once per week for four weeks. One week after the last dose of DDP, cell lines were generated from the tumors. Cells from these lines were then re-inoculated into athymic mice, and the DDP selection process was repeated. This procedure was continued for a total of four passages (16 doses). Although this chemotherapy did not affect the tumors' growth, cell lines derived from these tumors after the first passage displayed low-level resistance to DDP as determined by the concentration causing 50% inhibition of colony formation in a clonogenic assay. The mean resistance (+/- SD) of cell lines derived from tumors treated with four doses of DDP was 1.6 +/- 0.06 (n = 5). A minimum of only two doses of DDP was required to generate significant resistance (1.5 fold). The DDP resistance slowly increased with further selections so that after four passages with chemotherapy, the cells were 3.0-fold resistant. The DDP resistance was not stable; after three passages, resistance slowly declined over 104 days from 2.2- to 1.4-fold. Resistant cells obtained after both three and four passages did not have elevated glutathione as determined by flow cytometry of monochlorobimane-stained cells. After three passages, DDP-resistant cells were not resistant to CdCl2, suggesting that metallothioneins were also not elevated. A key biochemical change found in these cells was a decrease in DDP accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cisplatin-resistant COLO 316 human ovarian carcinoma cells

The biochemical changes responsible for acquired resistance to cisplatin (DDP) are not fully understood. We have developed DDP-resistant sublines of COLO 316 human ovarian carcinoma cells in vitro and characterized a number of biochemical features of these cells. Following selection with either continuous 50 nM DDP (COLO/DDP 50 cells) or intermittent 1 μ M DDP (COLO/B, COLO/C, or COLO/D cells) the onset of resistance was rapid. The resistance of the COLO/B cells gradually fell from 14-fold to 5-fold over 6 months in drug-free media. Both selection procedures produced cells exhibiting broad cross-resistance to other platinum analogs, natural products and alkylating agents. There was no significant change in the growth rate (doubling time = 36 h, cloning efficiency ( 28%), protein content ( 0.55 mg/10 6 cells), or morphology of these cells. Cell cycle distributions of log-phase cells were similar ( 60% G 0 G 1 , 35% S, 5% G 2 M ) as determined by flow cytometry. Glutathione (GSH) levels, while not elevated in COLO-B cells at low levels of resistance ( 2–3-fold), were 30% elevated at higher levels of resistance ( 9-fold). However, GSH levels in COLO/DDP 50 cells with 13-fold resistance were 2.3-fold elevated. The resistance of both cell types could be partially reversed by extended depletion of GSH with d,l -buthionine-S,R-sulfoximine. COLO/D cells had a 48% decrease in DDP accumulation at 1 h while COLO/DDP 50 cells had no change in DDP accumulation. The cross-resistance profiles, GSH biochemistry and DDP accumulation data indicate that acquired DDP-resistance is a complex, multifactorial response in these cells. The specific combination of mechanisms expressed in these cells appears to depend upon the selection procedure.