Possible Mechanisms of Resistance to cis-Diamminedichloroplatinum (II) of Human Ovarian Cancer Cells

The mechanism of cis -diamminedichloroplatinum (II) (CDDP) resistance was examined by using a CDDP-resistant (KFr) cell line established by continuous exposure of KF cells (derived from serous cystadenocarcinoma of the ovary) to escalating doses of CDDP. When KFr cells were incubated with 66.7 μ M CDDP, the uptake of CDDP was significantly inhibited and the cellular content in the KFr cells was about a half of that in KF cells after incubation for 4 h. When the KF or KFr cells were incubated for 4 h with 100 μ M CDDP, the release pattern of CDDP from KFr cells was similar to that from KF cells. In addition, the DNA histogram of both KF and KFr cells revealed that KF cells seemed to contain two clones of cell population and the KFr cells may have been selected by exposure to CDDP. At 3 h after intraperitoneal administration of 0.5 mg of CDDP per mouse to nude mice with KF or KFr tumor, the CDDP content in the KFr tumor was significantly lower than that in the KF tumor. In contrast, at 6 or 9 h after CDDP administration the CDDP content in the KFr tumor was significantly higher than that in the KF tumor. Furthermore, the KFr cells had cross-resistance to various CDDP analogues including carboplatin. It was shown that cellular uptakes of two CDDP analogues into KFr cells were significantly lower than those into KF cells.     

Possible mechanisms of resistance to cis-diamminedichloroplatinum (II) of human ovarian cancer cells

The mechanism of cis-diamminedichloroplatinum (II) (CDDP) resistance was examined by using a CDDP-resistant (KFr) cell line established by continuous exposure of KF cells (derived from serous cystadenocarcinoma of the ovary) to escalating doses of CDDP. When KFr cells were incubated with 66.7 microM CDDP, the uptake of CDDP was significantly inhibited and the cellular content in the KFr cells was about a half of that in KF cells after incubation for 4 h. When the KF or KFr cells were incubated for 4 h with 100 microM CDDP, the release pattern of CDDP from KFr cells was similar to that from KF cells. In addition, the DNA histogram of both KF and KFr cells revealed that KF cells seemed to contain two clones of cell population and the KFr cells may have been selected by exposure to CDDP. At 3 h after intraperitoneal administration of 0.5 mg of CDDP per mouse to nude mice with KF or KFr tumor, the CDDP content in the KFr tumor was significantly lower than that in the KF tumor. In contrast, at 6 or 9 h after CDDP administration the CDDP content in the KFr tumor was significantly higher than that in the KF tumor. Furthermore, the KFr cells had cross-resistance to various CDDP analogues including carboplatin. It was shown that cellular uptakes of two CDDP analogues into KFr cells were significantly lower than those into KF cells.     

Enhanced Topoisomerase I Activity and Increased Topoisomerase IIα Content in Cisplatin-resistant Cancer Cell Lines

Although the combined effects of cisplatin (CDDP) and DNA topoisomerase (Topo) inhibitors have been described in recent literature, little is known about the combined effects and their biological basis in CDDP-resistant cells. The aim of the present study was to elucidate the combined effect of CDDP and Topo inhibitors on CDDP-resistant cells as well as to investigate the biological factors involved in the sensitivity to these anti-cancer agents. We found synergistic actions between CDDP and SN-38 (a Topo I inhibitor) or VP-16 (a Topo II inhibitor) in KFr cells, a CDDP-resistant subline of the KF epithelial ovarian carcinoma cell line, but not in the parent KF cells. We subsequently assayed Topo protein levels and enzymatic activities in two sets of CDDP-sensitive and -resistant cell lines: KF and KFr, and HeLa and HeLa/CDDP. The levels of Topo I protein in the CDDP-resistant cells did not differ from those of their parent cell lines and were unaffected by exposure to CDDP. Topo I enzymatic activity, however, was 2- to 4-fold higher in the CDDP-resistant cell lines than in their respective parent cell lines. In contrast, higher levels of Topo lice protein were observed both before and after CDDP exposure in the CDDP-resistant cells than in their controls. However, no difference in Topo II catalytic activity was observed between the CDDP-resistant and -sensitive cells.     

Cellular efflux pump and interaction between cisplatin and paclitaxel in ovarian cancer cells

OBJECTIVE: The aim of this study was to evaluate the combination effect of paclitaxel (PTX) and cisplatin (CDDP) and to determine the mechanisms of interaction between these agents. METHODS AND RESULTS: We used human ovarian adenocarcinoma cell lines, namely a parent cell line (KF), a CDDP-resistant cell line (KFr) and a PTX-resistant cell line (KFTx).The combination effect of PTX and CDDP was synergistic on KF and KFTx and additive on KFr. The incidence of anaphase or telophase, evaluated by immunofluorescence microscopy, decreased with PTX and significantly decreased with PTX and CDDP in KF and KFTx. The concentration of PTX, which was measured by high-performance liquid chromatography, was higher in KF and KFTx cells treated with a combination of PTX and CDDP than those treated with PTX alone. Multidrug resistance gene mRNA appeared in KFTx and its expression decreased after exposure to PTX and CDDP. After exposure to CDDP, the expression of multidrug resistance-associated protein (MRP) and the concentration of glutathione increased in KF, but not in KFr or KFTx. MRP expression slightly increased in KF and KFTx after exposure to PTX. In contrast, its expression decreased in KFr. CONCLUSION: The present study suggests that CDDP enhances PTX accumulation and that the interaction of these agents is synergistic in CDDP-sensitive cells.     

Reduced cisplatin-uptake by cisplatin-resistant human ovarian cancer cells

We have developed a human ovarian carcinoma cell line (KFr) which is relatively stable and resistant to cis-diamminedichloroplatinum (cisplatin) after repeated exposure to escalating doses of the drug. This resistant cell line (KFr) was 9.7-fold resistant to carboplatin (JM-8), 8.8-fold resistant to (glycolato-O, O') diammineplatinum (II) (254S), and 7.1-fold resistant to Cis-1, 1-cyclobutanedicarboxylato (2R)-2-methyl-1, 4-butanediammineplatinum (II) (NK121), respectively. The KFr cell line proved to be 3.1-fold resistant to L-phenylalanine mustard (L-PAM). On the other hand, no cross-resistance to vincristine (VCR), 5-fluorouracil (5-FU) and daunomycin (DM) was observed. In addition, we examined the level of platinum in whole cells after 1-16 h exposure to 20 micrograms cisplatin. The parent cell line (KF) took up cisplatin in a time-dependent manner until 4 h. The uptake reached the plateau level after 4 h. On the other hand, the uptake by the KFr cell line reached the plateau level at 1 h-exposure time; after 4 h the uptake was about 1/3 of that by the KF cell line. The release of cisplatin from both cell lines showed a similar pattern, reaching the plateau within the first 30 min. Based upon these results, we assumed that the impairment of transport of influx systems of cisplatin into cells was the mechanism of resistance.     

Hammerhead Ribozyme against γ-Glutamylcysteine Synthetase Attenuates Resistance to Ionizing Radiation and Cisplatin in Human T98G Glioblastoma Cells

Glioblastoma cells are highly malignant and show resistance to ionizing radiation, as well as anti-cancer drugs. This resistance to cancer therapy is often associated with a high concentration of glutathione (GSH). In this study, the effect of continuous down-regulation of γ-glutamylcysteine synthetase (γ-GCS) expression, a rate-limiting enzyme for GSH synthesis, on resistance to ionizing radiation and cisplatin (CDDP) was studied in T98G human glioblastoma cells. We constructed a hammerhead ribozyme against a γ-GCS heavy subunit (γ-GCSh) mRNA and transfected it into T98G cells. (1) The transfection of the ribozyme decreased the concentration of GSH and resulted in G1 cell cycle arrest of T98G cells. (2) The transfection of the ribozyme increased the cytotoxicity of ionizing radiation and CDDP in T98G cells. Thus, hammerhead ribozyme against γ-GCS is suggested to have potential as a cancer gene therapy to reduce the resistance of malignant cells to ionizing radiation and anti-cancer drugs.     

Determinants of Drug Response in a Cisplatin-resistant Human Lung Cancer Cell Line

To elucidate the mechanism(s) of cisplatin resistance, we have characterized a human non-small cell lung cancer cell line (PC-9/CDDP) selected from the wild type (PC-9) for acquired resistance to cisplatin. PC-9/CDDP demonstrated 28-fold resistance to cisplatin, with cross resistance to other chemotherapeutic drugs including chlorambucil (× 6.3), melphalan (× 3.7) and 3-[(4-amino-2-methyl-5-pyrimidinyl)]methyl-1-(2-chloroethyl)-1-nitrosourea (ACNU) (× 3.9). There was no expression of mdr-1 mRNA in either wild-type or resistant cells. The mRNA and protein levels of glutathione S -transferase (GST) × were similar in the two lines. A GST-μ isozyme was present in equal amounts and the activities of selenium-dependent and independent glutathione peroxidase and glutathione reductase were unchanged. The mRNA level of human metallothionein IIA and the total intracellular metallothionein levels were reduced in the resistant cells. Significantly increased intracellular glutathione (GSH) levels were found in the resistant cells (20.0 vs. 63.5 nmol/mg protein) and manipulation of these levels with buthionine sulfoximine produced a partial sensitization to either cisplatin or chlorambucil. Increased GSH probably also played a role in determining cadmium chloride resistance of the PC-9/CDDP, even though this cell line had a reduced metallothionein level. Also contributing to the cisplatin resistance phenotype was a reduced intracellular level of platinum in the PC-9/CDDP. Thus, at least two distinct mechanisms have been selected in the resistant cells which confer the phenotype and allow degrees of cross resistance to other electrophilic drugs.     

The Mechanism of Acquired Resistance to Cisplatin by a Human Ovarian Cancer Cell Line

The present study was designed to elucidate the mechanism of resistance to cisplatin. A cisplatin-resistant cell line (KFr) was established from KF cells derived from human serous cystadenocarcinoma of the ovary. The DNA histogram revealed an increase of S-phase cells and a decrease of G₁-phase cells in cultured KFr cells, compared to that in cultured KF cells. Although the cisplatin content in the KF cells incubated with cisplatin at 10 μg/ml increased in a time-dependent manner, that in the KFr cells remained unchanged during the experimental period. When 0.5 mg of cisplatin was administered ip to nude mice with KF or KFr tumor, the cisplatin content in the KFr tumor was significantly lower than that in the KF tumor. The KFr cells showed a cross-resistance to L-phenylalanine mustard, while no cross-resistance to vincristine or 5-fluorouracil was observed. These findings suggest that the mechanism of cisplatin resistance in the KFr cells involves a decrease of cisplatin accumulation in the tumor cells.     

Metallothionein content correlates with the sensitivity of human small cell lung cancer cell lines to cisplatin.

We have established cis-diamminedichloroplatinum(II) (cisplatin) resistant human small cell lung cancer cell lines, H69/CDDP0.2 and H69/CDDP, to investigate the mechanism of acquired resistance to cisplatin. H69/CDDP0.2 and H69/CDDP were 6- and 11-fold resistant to cisplatin compared with the H69 parental cell line. H69/CDDP was also resistant to cadmium chloride (2-fold), cis-diammine(glycolato)platinum (4-fold), 4-hydroperoxycyclophosphamide (3-fold) and 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosour ea (4-fold) if the drug concentrations that inhibit cell growth by 50% from growth inhibition assay were compared. There was no significant difference in the cisplatin accumulation among these cell lines. Although DNA interstrand cross-link formations, determined by filter elution assay in H69/CDDP0.2 and H69/CDDP, was decreased to 20 to 30% of that in H69 parental cells, the repair capacity of DNA interstrand cross-links was equivalent in all three cell lines. Intracellular glutathione content was equal in all cell lines. H69/CDDP had the highest glutathione S-transferase activity (H69, 11 nmol/min/mg protein, H69/CDDP0.2, 12 nmol/min/mg protein; H69/CDDP, 74 nmol/min/mg protein, respectively) and an overexpression of glutathione S-transferase pi mRNA. The drug concentrations that inhibit cell growth by 50% for cisplatin in all cell lines were decreased by treatment with ethacrynic acid, an inhibitor of glutathione S-transferase pi, but this did not alter the relative degree of resistance. Intracellular metallothionein content (H69, 14 pmol/mg protein, H69/CDDP0.2, 22 pmol/mg protein; H69/CDDP, 33 pmol/mg protein, respectively) and expression of metallothionein mRNA were correlated with the drug concentrations that inhibit cell growth by 50% of the three cell lines for cisplatin and cadmium chloride. The present study suggested the importance of metallothionein in the mechanisms of cisplatin resistance.     

Synergistic effects of topoisomerase I inhibitor, 7-ethyl-10-hydroxycamptothecin, and irradiation in a cisplatin-resistant human small cell lung cancer cell line.

7-ethyl-10-[4-(1-piperidyl)-1-piperidyl] carbonyloxy-camptothecin, a topoisomerase I (topo I) inhibitor, is one of the most active agent against lung cancer, and its radiosensitizing effect has been reported recently. We evaluated a combination in vitro effect of irradiation and 7-ethyl-10-hydroxy-CPT (SN-38), an active metabolite of 7-ethyl-10-[4- (1-piperidyl)-1-piperidyl] carbonyloxy-camptothecin, on a human small cell lung cancer cell line (SBC-3) and its cisplatin-resistant subline (SBC-3/CDDP). Growth-inhibitory effects of irradiation with or without SN-38 were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A modified isobologram method was used to evaluate the treatment interaction. The combination of irradiation and SN-38 showed a synergistic inhibitory effect on the growth of SBC-3/CDDP despite its cross-resistance to irradiation and SN-38. In contrast, the same combination showed only an additive effect on the growth of parental SBC-3 cells. There was no significant difference in topo I protein expression between these two cell lines. In SBC-3 cells, topo I catalytic activity was suppressed by 4 Gy of irradiation, without a decrease of nuclear topo I protein, whereas the exposure of SBC-3 cells to 1 microM SN-38 subsequent to irradiation showed no remarkable additional effects on both topo I activity and protein content. On the other hand, in SBC-3/CDDP cells, topo I activity was unchanged by irradiation, but the subsequent exposure to SN-38 gave rise to a decrease in topo I activity, which was accompanied by a significant decrease in the topo I protein content (P = 0.02). These observations may indicate that SN-38 induces sequestration of topo I onto DNA in radiation-treated SBC-3/CDDP cells and suggest that the synergistic effect of irradiation and SN-38 in SBC-3/CDDP cells was considered attributable to DNA repair-related enhanced recruitment of topo I onto the damaged DNA.     

Modulation of cisplatin sensitivity and accumulation by interferon α-2A in human squamous carcinoma cell lines

This study was undertaken to elucidate the mechanism(s) of potentiation of cisplatin (CDDP) cytotoxicity by interferon α-2a (IFN α-2a) in human squamous carcinoma cell lines SCC-25 and SCC-4. IFN α-2a treatment significantly increased the cytotoxicity of CDDP in both cell lines in a dose-dependent manner. In SCC-25 cells, the cytotoxicity of CDDP was increased by about 2- and 4-fold, respectively, by treating the cells with 400 and 800 1U/ml IFN α-2a. Sensitivity of SCC-4 cells to CDDP was increased by about 3- and 7-fold, respectively, by 400 and 800 1U/ml IFN α-2a treatment. Drug uptake experiments revealed approximately 1.4- to 5-fold higher platinum accumulation in IFN α-2a-treated cells as compared to respective controls. Cellular levels of glutathione (GSH) and GSH transferase, which have been suggested to be important determinants of tumor cell sensitivity to CDDP, were not altered by IFN α-2a treatment in either of the cell lines. Northern blot analysis showed a moderate increase (about 30–40%) in the level of MT-II_A mRNA by IFN α-2a treatment in these cells. Our results suggest that IFN α-2a-mediated sensitization of SCC-25 and SCC-4 cell lines to CDDP in vitro may be due to an increase in intracellular platinum accumulation.     

The mechanism of acquired resistance to cisplatin by a human ovarian cancer cell line

The present study was designed to elucidate the mechanism of resistance to cisplatin. A cisplatin-resistant cell line (KFr) was established from KF cells derived from human serous cystadenocarcinoma of the ovary. The DNA histogram revealed an increase of S-phase cells and a decrease of G1-phase cells in cultured KFr cells, compared to that in cultured KF cells. Although the cisplatin content in the KF cells incubated with cisplatin at 10 micrograms/ml increased in a time-dependent manner, that in the KFr cells remained unchanged during the experimental period. When 0.5 mg of cisplatin was administered ip to nude mice with KF or KFr tumor, the cisplatin content in the KFr tumor was significantly lower than that in the KF tumor. The KFr cells showed a cross-resistance to L-phenylalanine mustard, while no cross-resistance to vincristine or 5-fluorouracil was observed. These findings suggest that the mechanism of cisplatin resistance in the KFr cells involves a decrease of cisplatin accumulation in the tumor cells.     

Correlated Expression of Glutathione S-Transferase-π and c-Jun or Other Oncogene Products in Human Squamous Cell Carcinomas of the Head and Neck: Relevance to Relapse after Radiation Therapy

The expression of glutathione S -transferase (GST)-π and four oncogene products, c-Jun, c-Fos, c-H-Ras, and c-Myc, in human squamons cell carcinomas of the head and neck was investigated immunohistochemically before and after radiation therapy, to examine whether these oncogene products might be involved in GST-π expression, and also to examine the relationship between their expression and therapeutic response. Clinical response to radiation was evaluated in terms of both tumor regression and relapse over two-year follow-up periods. The overall positive rates in 83 carcinoma specimens before therapy were 60.2% for GST-π and 28.9–51.8% for the individual oncogene products, the positive rates for the oncogene products being higher in GST-π-positive than in GST-π-negative cancers. c-Jun was most highly correlated with GST-π expression. Following radiation, the expression of GST-π and the oncogene products was altered in about a half of 30 patients. Eleven of the 18 patients who exhibited prior positivity for GST-π showed negative conversion, while 4 of the 12 patients with prior negativity demonstrated positive conversion. In most cases, changes in c-Jun staining coincided with those in GST-π. Regarding clinical response to radiation therapy, the positive rates for GST-π and c-Jun before radiation were higher in the residual cancer or relapse cases than in the group showing complete response without relapse. Examination of 26 patients with laryngeal cancer revealed that relapse occurred more frequently in cases exhibiting positive reactions for GST-π,c-Jun, or c-H-Ras. These results suggest a direct link between c-Jun and GST-π in head and neck cancers before and after radiation. Although GST-π and the oncogene products can be influenced by radiation, GST-π and c-H-Ras expression may be a risk factor for relapse of laryngeal cancer.     

Up-regulation of γ-glutamylcysteine synthetase activity in melphalan-resistant human multiple myeloma cells expressing increased glutathione levels

Levels of intracellular glutathione (GSH) and the GSH-related enzymes -glutamylcysteine synthetase (-GCS) and -glutamyltranspeptidase (-GT) were measured in the melphalan-resistant human multiple myeloma cell line 8226/LR-5 and were compared to those measured in the drug-sensitive 8226/S and doxorubicin-resistant 8226/Dox40 cell lines. Both GSH and -GCS activity, the rate-limiting step in the de novo synthesis of GSH, were elevated by a factor of approximately 2 in the melphalanresistant 8226/LR-5 cells relative to the other two lines. -GT activity was not elevated significantly in the /LR-5 cells. Northern analysis with a probe specific for the large subunit of human liver -GCS identified two bands (3.2 and 4.0 kb), both of which were increased by a factor of 2–3 in the 8226/LR-5 line. Levels of -GCS mRNA expression were comparable in the /S and /Dox40 cell lines. Levels of -GT mRNA were similar in the /S and /LR-5 lines but were reduced in the /Dox40 cells. These data suggest that the increased GSH levels associated with resistance to melphalan in the 8226/LR-5 myeloma cells is attributable to up-regulation of -GCS. This observation is consistent with recent demonstrations of up-regulation of -GCS in melphalan-resistant prostate carcinoma cells and cisplatinum-resistant ovarian carcinoma cells, suggesting that increased expression of -GCS may be an important mediator of GSH-associated resistance mechanisms.     

Effect of Glutathione Depletion on Cisplatin Resistance in Cancer Cells Transfected with the γ-Glutamylcysteine Synthetase Gene

Overexpression of the human γ-glutamylcysteine synthetase (γ-GCS) gene resulted in cisplatin resistance with an increased glutathione (GSH) content, increased ATP-dependent glutatbione S-conjugate export pump (GS-X pump) activity and decreased platinum accumulation in human lung cancer cells transfected with a γ-GCS cDNA expression vector, as we previously reported. In this study, we examined the effects of buthionine sulfoximine (BSO), a specific inhibitor of γ-GCS, to determine whether GSH depletion alters cisplatin resistance in a γ-GCS-transfected cell line, SBC-3/ GCS. In the presence of 10 μM BSO for 4 days, SBC-3/GCS still showed resistance to cisplatin, although it was partially reversed. Under these conditions, GS-X pump activity remained up-regulated in spite of low GSH content, and the platinum content was decreased. These data suggest that the GS-X pump itself influences cisplatin resistance, as well as cellular GSH content.     

Resistance-associated factors in human small-cell lung-carcinoma GLC4 sub-lines with increasing adriamycin resistance

Previous studies have shown that the in vitro-selected adriamycin-resistant human small-cell lung-carcinoma cell line GLC₄-ADR₁₅₀ displays multidrug resistance as the result of 3-fold decreased DNA-topoisomerase II (topo II) activity and a 6-fold reduction in adriamycin accumulation. Not the MDRI gene, but the MRP gene, was over-expressed in this cell line. The aim of our study was to establish which of these drug-resistance-associated factors are already involved in drug resistance occurring at early steps of selection with adriamycin. To address this question, changes in expression of topo IIα/topo IIβ, MRP and drug accumulation were measured along with adriamycin resistance (from 2- to 10- to 150-fold) and in a partial revertant cell line (10-fold resistant). Topo IIα and IIβ mRNA and protein levels were decreased in the resistant sub-lines, except in the 10-fold-resistant cell line. Cellular daunorubicin accumulation was decreased 1.2- to 5-fold with increasing resistance. MRP mRNA was over-expressed in all resistant sub-lines, with a marked increase in the 10-fold-resistant cells (level of expression as high as in the GLC₄-ADR₁₅₀ cells). Expression of an ATP-binding 190-kDa membrane protein and Western-blot analysis with anti-MRP anti-serum ASPKE, was in accordance with the expression of MRP mRNA in all cell lines. Expression of MRP mRNA and protein, however, was not proportional with the decrease in drug accumulation in all resistant sub-lines. This study also shows that drug accumulation, topo II and MRP expression were all changed at the earliest stage of resistance development of GLC₄ cells upon adriamycin selection. © 1995 Wiley-Liss, Inc.     

Circumvention of glutathione-mediated mitomycin C resistance by a novel mitomycin C analogue,KW-2149

A novel antitumor antibiotic 7-N-[2-[[2-(γ-l-glutamyl-amino)ethyl]dithio]ethyl] mitomycin C (KW-2149), an analogue of mitomycin C (MMC), is activated by thiol molecules, such as glutathione (GSH). To clarify the relationship between cellular GSH levels and the cytotoxicity of KW-2149, a murine fibroblast cell line (NIH/3T3) was transfected with human γ-glutamylcysteine synthetase (γ-GCS) cDNA, which codes a rate-limiting enzyme of GSH synthesis. Transfected cells (3T3/GCS) displayed increased γ-GCS mRNA levels, γ-GCS activity and GSH content, compared with NIH/3T3 cells. 3T3/GCS cells exhibited a 4.4-fold resistance to MMC, but not to KW-2149 (×0.69), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, suggesting that the increased cellular GSH levels did not affect the growth-inhibitory effect of KW-2149. KW-2149 exerted a greater growth-inhibitory effect than MMC on cisplatin- and doxorubicin-resistant cells with cross-resistance to MMC. KW-2149 exhibited a greater growth inhibitory effect than MMC not only on cells with GSH-mediated MMC resistance but also on cells with acquired resistance. We thus conclude that KW-2149 might be a clinically useful drug. Int. J. Cancer 72:865–870, 1997. © 1997 Wiley-Liss, Inc.