Comparative in vitro cytotoxicity of taxol and Taxotere against cisplatin-sensitive and-resistant human ovarian carcinoma cell lines

Summary Using the sulforhodamine B assay, we compared the cytotoxic properties of the novel microtubule agent taxol and the semi-synthetic related compound Taxotere in nine human ovarian-carcinoma cell lines, including three pairs of cell lines rendered resistant to cisplatin or carboplatin. In addition, the cytotoxicity of the commonly used anticancer drugs cisplatin and adriamycin and the topoisomerase II inhibitor etoposide was determined. The results of continuous drug exposure showed that taxol [mean concentration producing 50% growth inhibition (IC₅₀), 1.1×10^–9 m; range, 2.8×10^–9–5×10^–10 m and Taxotere (mean IC₅₀, 5.1×10^–10 m; range, 7.2–3.3×10^–10 m) were >1,000 times more cytotoxic than either cisplatin (mean IC₅₀, 3.1×10^–6 m;P<0.05) or etoposide (mean IC₅₀, 2.3×10^–6 m;P<0.05) and >100 times more cytotoxic than Adriamycin (mean IC₅₀, 6.9×10^–8 m;P<0.05). Taxotere was more cytotoxic than taxol; following continuous exposure, the mean difference across the cell lines was 2 orders of magnitude (range, 1.1–3.9 orders of magnitude for individual lines). Although this difference did not reach statistical significance for any individual cell line (P values ranged from 0.17 for HX/62 to 0.9 for OVCAR-3), when all IC₅₀ values for the 96-h experiments were pooled, Taxotere was found to be significantly more potent than taxol (P=0.05). Following 2 h exposure, the mean cytotoxicity of Taxotere was 3.9-fold > that of taxol across the nine lines (range, 0.75- to 10-fold;P<0.05 for the CH1 cell line; overall pooled IC₅₀ data,P=0.05). Although a 71-fold range of sensitivity to cisplatin was observed across the six parent cell lines (IC₅₀ most resistant line/IC₅₀ most sensitive line), this was largely abolished by treatment with taxol (5.6-fold range) and Taxotere (2.2-fold rante). Following continuous exposure of the three pairs of lines exhibiting acquired resistance to platinum, no cross-resistance with either Taxotere or taxol was found (resistance factors, <1.5). In the 41M and 41McisR pair of lines, in which previous studies have shown resistance to be due to reduced platinum accumulation, taxol and Taxotere exhibited some collateral sensitivity (resistance factors, 0.69 and 0.66, respectively). Taxotere and, particularly, taxol showed a pronounced concentration times exposure duration (CxT) dependence as compared with cisplatin (P<0.05). The mean loss in potency across the nine lines for 2 vs 96 h exposure was 97 for taxol, 35 for Taxotere, 30 for Adriamycin and only 9.9 for cisplatin. However, these differences in potency loss observed between taxol and Taxotere did not reach statistical significance (P=0.18). These data indicate that Taxotere is approximately 2 times more cytotoxic than taxol and shows an encouraging lack of cross-resistance in three cell lines exhibiting acquired resistance to cisplatin and carboplatin.This study was supported by grants to the Institute of Cancer Research, Royal Cancer Hospital, from the Cancer Research Campaign and, through the European Organisation for Research and Treatment of Cancer (EORTC) Clonogenic Assay Screening Group, by grant 90031 from Rhone-Poulenc Rorer.     

Comparison of cellular accumulation and cytotoxicity of cisplatin with that of tetraplatin and amminedibutyratodichloro(cyclohexylamine)platinum(IV) (JM221) in human ovarian carcinoma cell lines.

We have compared the cellular accumulation and cytotoxicity of three platinum compounds in a panel of five human ovarian carcinoma cell lines. The cell lines, which were established from both untreated and pretreated patients, showed a wide range in sensitivity to cisplatin and other platinum drugs. The panel consisted of two sensitive (41M, CH1), one in vivo acquired resistant (PXN/94) with moderate sensitivity, and two intrinsically resistant (SKOV-3, HX/62) cell lines. The cisplatin 2-h concentration of drug required to inhibit cell growth by 50% compared with vehicle treated control cells (IC50 values) for these cell lines were in the following order: CH1 < 41M < PXN/94 < SKOV-3 < HX/62. None of the cell lines showed saturation of platinum accumulation (per mg protein) at 2 h after exposure to cisplatin concentrations of up to 500 microM. The highest cellular platinum accumulation was observed in the sensitive 41M cell line which was established from an untreated patient. The lowest accumulation was found in the intrinsically resistant HX/62 cell line. The rate of platinum accumulation at an equimolar concentration of cisplatin was 41M > SKOV-3 > CH1 > PXN/94 > HX/62. The relationship between drug accumulation and cytotoxicity was evaluated by comparing 2-h IC50 values with platinum accumulation following exposure to both equimolar and equitoxic doses of the agent. The results suggest that reduced drug accumulation may play a partial role in the mechanism of intrinsic resistance to cisplatin in one cell line (SKOV-3) and a major role in another (HX/62), where reduced accumulation is attributable to reduced uptake rather than enhanced efflux. Decreased drug accumulation may also contribute significantly to the lower sensitivity of the PXN/94 cell line to cisplatin. Interestingly, both the PXN/94 and the sensitive CH1 cell lines, which were established from patients pretreated with platinum drugs, showed reduced drug accumulation relative to the 41M cell line. Cellular accumulation of tetraplatin and JM221 [(ammine)dibutyratodichloro(cyclohexylamine)platinum(IV)], a novel platinum(IV) dicarboxylate complex exhibiting enhanced cytotoxicity compared to cisplatin, was also examined. Comparison with platinum accumulation from cisplatin suggests that the increased cytotoxicity of tetraplatin and JM221 may be related to their increased accumulation. Significantly both agents are more lipophilic than cisplatin, which may account partially for their improved uptake in cisplatin resistant cells.     

Potentiation of cisplatin and carboplatin cytotoxicity by amphotericin B in different human ovarian carcinoma and malignant peritoneal mesothelioma cells

An in vitro study of the combined cytotoxicity of either cisplatin (CDDP) or carboplatin and amphotericin B (AmB) was undertaken on a set of different ovarian carcinoma (IGROVI, IGROVI-C10, OAW42) and peritoneal malignant mesothelioma (CFB-CARP1) cell lines and ascitic cells freshly obtained from ovarian cancer patients so as to investigate the possibility of overcoming their resistance to platinum compounds. Growth-inhibition curves obtained 6 days after a 2-h period of exposure to the drugs showed that AmB at 5–10 mg/l allowed a 5- to 10-fold decrease in the 50% growth-inhibitory concentrations (IC₅₀) of CDDP and carboplatin on either sensitive or resistant cells. Intracellular platinum assays with IGROVI cells showed that AmB acted by increasing dramatically the platinum uptake at a proportion that accounted for the increase in cytotoxicity. In the subline IGROVI-C10, a 10-fold resistant subline of IGROVI, AmB at 10 mg/l allowed recovery to the level of sensitivity seen in the parental cell line in the absence of AmB but not to the level observed in the presence of AmB. Acquisition of resistance mechanisms that are independent of the regulation of platinum uptake might be involved in this cell line. Thus, AmB might act by increasing the intracellular concentration of platinum without modifying the resistance mechanism involved downstream. However, in our models an increase in the intracellular level of platinum was always sufficient for the recovery of chemosensivity in vitro. We also show that the phosphodiesterase inhibiting methylxanthines act synergistically with AmB. The latter drugs are weakly toxic and could also attenuate the nephrotoxicity of AmB. Received: 11 August 1996 / Accepted: 7 March 1997     

Comparative evaluation of cisplatin and carboplatin sensitivity in endometrial adenocarcinoma cell lines.

Platinum analogues are frequently used in the treatment of advanced or recurrent endometrial cancer. To study the sensitivity of endometrial cancer to cisplatin and carboplatin, we tested two long-established (RL95-2, KLE) and six new cell lines (UM-EC-1, UM-EC-2, UM-EC-3, UT-EC-2A, UT-EC-2B, UT-EC-3) using the 96-well-plate clonogenic assay. This assay has proven to be suitable for testing chemosensitivity of both adenocarcinoma and squamous cell carcinoma. The chemosensitivity was expressed as an IC50 value, the drug concentration causing 50% inhibition of clonogenic survival. IC50 values were obtained from dose-response curves after fitting the data by the linear quadratic equation, F = exp[-(alpha D + beta D2)]. The IC50 values of the two platinum derivatives varied considerably. The values for cisplatin varied between 0.022 microgram ml-1 and 0.56 microgram ml-1 and the corresponding values for carboplatin were 0.096-1.20 microgram ml-1. The range of the ratios between carboplatin IC50 and cisplatin IC50, from 1.5:1 to 4.4:1, was rather narrow. However, no constant ratio between carboplatin IC50 and cisplatin IC50 could be detected. The equivalent doses with regard to efficacy of these two platinum analogues remain to be determined.     

DHMEQ enhances the cytotoxic effect of cisplatin and carboplatin in ovarian cancer cell lines

Ovarian cancer (OvCa) is one of the most lethal gynaecological malignancies. It is diagnosed mostly in advanced stages. Due to a lack of appropriate early detection markers and non-ambiguous symptoms, the five-year survival rate is significantly reduced. Despite a primary good response to platinum-based therapy, approximately 70% of patients will develop a chemoresistance phenotype. The activation of the NF-κB signalling pathway plays a crucial role in this process. It is responsible for increasing cell viability, cell cycle progression and induces growth and migration of neoplastic cells. A few independent studies have yet suggested a high correlation between activation of NF-κB and poor outcome in OvCa patients. Thus, developing inhibitors of the NF-κB pathway has become a new target of cancer therapies. One of the promising compounds is DHMEQ (dehydroxymethylepoxyquinomicin). Our preliminary studies indicated that DHMEQ combined with cisplatin (CDDP) or carboplatin (CBP) enhanced apoptosis in the A2780 cell line and caused cell cycle arrest in the G2/M phase in the SKOV3 cell line, but not in the normal cell line MRC-5 pd19. Moreover, the combination of those agents caused decreased motility of cells, especially with the CBP. However, the invasion of cells was not changed significantly. The analysis of drug interactions using CompuSyn software has revealed that observed effect of the doses used in the study was antagonistic, but the DRI guidelines and in vitro observation of biological response indicate that a combination of DHMEQ with CDDP or CBP could be a novel proposal in ovarian cancer treatment.     

Hepatocyte growth factor sensitizes human ovarian carcinoma cell lines to paclitaxel and cisplatin

The hepatocyte growth factor (HGF) receptor, encoded by the MET oncogene, is expressed in approximately 70% of human ovarian carcinomas and overexpressed in 30% of cases. Because HGF is known to protect cells from apoptosis, we investigated whether receptor expression modifies ovarian cancer cell response to chemotherapy. The apoptotic effect of the front-line chemotherapeutic drugs paclitaxel and cisplatin on cells treated with HGF was studied. In ovarian cancer cell lines, pretreatment with HGF surprisingly enhances the apoptotic response to low doses of paclitaxel and cisplatin. HGF empowers specifically the intrinsic apoptotic pathway, whereas it protects cells from extrinsic Fas-induced apoptosis. Chemotherapy sensitization is specific for HGF because another growth factor (e.g., epidermal growth factor) increases ovarian cancer cell survival. In nonovarian cancer cell models, as expected, HGF provides protection from drug-induced apoptosis. These data show that HGF sensitizes ovarian carcinoma cells to low-dose chemotherapeutic agents. This suggests that HGF may be used to improve response to chemotherapy in a set of human ovarian carcinomas molecularly classified based on the MET oncogene expression.     

Cytotoxicity of tetraplatin and cisplatin for human and rodent cell lines cultured as monolayers and multicellular spheroids

The cytotoxicity of tetraplatin (dl-trans), its d- and l-isomers, and cisplatin for four human tumor cell lines (myeloma 8226, ovarian 2008, A2780, and OVCAR-3), their cisplatin-resistant variants, and three rodent cell lines (V79, EMT6/Ro, and L1210) were compared. Tetraplatin was more, or equally as, potent as cisplatin for the human cell lines and for L1210 but was clearly less potent for V79 and EMT6/Ro. The d-trans tetraplatin was more potent than the l-trans. Cisplatin resistant human tumor cells were less resistant to tetraplatin. On comparing sensitivity of V79 and EMT6/Ro cells in two growth models, we observed that all of the platinum compounds were more cytotoxic to cells in multicellular spheroids than in exponentially growing monolayers. Uptake studies, however, showed that tetraplatin was more cytotoxic to spheroids because spheroids accumulated more drug than monolayers.     

Augmentation of cisplatin cytotoxicity using cytokines on cervical carcinoma cell lines

We investigated whether the biological response modifiers like IFN-alpha, IFN-gamma and TNF-alpha could enhance the cytotoxic action of cisplatin on cervical carcinoma cell lines in vitro. The sensitivity of three cell lines SiHa, ME 180 and C33A to these agents was tested using colorimetric MTT assay as well as tritiated thymidine uptake. All the three cell lines demonstrated range of sensitivity to cisplatin and cytokines. Interferons and TNF when used in combination with lower dose of cisplatin showed a significant enhancement of cytotoxic action of the drug in all the cell lines. Thus these data indicate that cytokines in concert with the drug may have a potential to improve the 'in vivo' therapy in these patients.     

Effect of topoisomerase modulators on cisplatin cytotoxicity in human ovarian carcinoma cells

The in vitro interaction of modulators of topoisomerase I and II with cisplatin in human ovarian carcinoma cells might be synergistic. The interactions were evaluated by median effect analysis of survival data derived from continuous exposure to drug combinations for 10 days in colony-forming assays. The interaction between cisplatin and the topoisomerase I inhibitor camptothecin and the topoisomerase I activator β-lapachone was additive, as was that between cisplatin and the topoisomerase II inhibitor novobiocin. Despite the clinical efficacy of the combination of etoposide (a topoisomerase II inhibitor) and cisplatin, the combination index at 50% cell kill indicated antagonism between these two drugs. Thus, biochemical synergism at the cellular level is not a prerequisite of improved therapeutic efficacy.     

Additive and supra-additive cytotoxicity of cisplatin-taxane combinations in ovarian carcinoma cell lines

The purpose of this study was to compare the growth-inhibitory effect of cisplatin–paclitaxel with that obtained with a cisplatin–docetaxel combination and to assess the type of interaction. Concomitant use of taxanes and cisplatin was studied in seven human ovarian carcinoma cell lines, using the 96-well plate clonogenic assay. Chemosensitivity was expressed in terms of IC₅₀ values, the drug concentration causing 50% inhibition of clonogenic survival. The type of interaction was studied using the area under the survival curve ratios (AUC ratios) obtained by numerical integration. Comparison of the AUC ratio and the surviving fraction (SF) value after taxane alone was made using Student''s t-test. The influence of the drug concentration was tested by one-way analysis of variance (Anova). A supra-additive or additive effect was seen when seven ovarian carcinoma cell lines were exposed to paclitaxel or docetaxel concomitantly with cisplatin. A supra-additive effect was found in four cell lines (UT-OC-3, UT-OC-4, UT-OC-5 and SK-OV-3) after simultaneous use of cisplatin with all docetaxel concentrations tested, and in two cell lines (UT-OC-4 and SK-OV-3) when cisplatin was used concomitantly with paclitaxel. A more pronounced supra-additive effect was seen with the combination of cisplatin and docetaxel. The degree of supra-additivity was dose dependent, with increasing synergy after a higher taxane dose. The data obtained in this study suggest that a supra-additive or additive effect can be achieved in ovarian carcinoma with the concomitant use of cisplatin and a taxane. © 1999 Cancer Research Campaign     

A comparison of hyperthermia cisplatin sensitization in human ovarian carcinoma and glioma cell lines sensitive and resistant to cisplatin treatment

 Two pairs of human tumor cell lines (glioma and ovarian carcinoma (OvCa)) each having a parental cell line and cisplatin-resistant variant, were evaluated for (a) cisplatin response, (b) hyperthermia response, and (c) combined hyperthermia and cisplatin response. The two resistant lines had comparable resistant responses while for the parental lines, the OvCa was more sensitive than the glioma to cisplatin doses up to 14 μg/ml. For the hyperthermia response, the OvCa parental line was more resistant than the variant line at low-temperature hyperthermia (41° C or 42°C) but became more sensitive at high temperature (45°C). For the glioma, the parental line was more sensitive to hyperthermia at all temperatures tested. Hyperthermia caused sensitization to cisplatin in all cell lines but was generally greater in the glioma cell lines. In the OvCa system, hyperthermia had a slightly greater sensitizing effect on the resistant cell lines, while in the glioma the opposite was true. The degree of sensitization increased with hyperthermia temperature. In summary, the results showed that there is no cross-resistance for hyperthermia and cisplatin, that the degree of thermal sensitization is not reduced in cisplatin-resistant cell lines, and that cisplatin thermal sensitization is cell-line and temperature dependent. Thus, hyperthermia can effectively improve tumor cell response to cisplatin and may be useful in overcoming resistance to cisplatin. Received: 10 March 1995/Accepted: 21 July 1995     

Thermal enhancement of tetraplatin and carboplatin in human leukaemic cells

The thermal enhancement of tetraplatin (racemic, d-trans and l-trans isomers) and carboplatin was studied as a function of temperature in vitro in JM, a human acute lymphoblastic leukaemia cell line. Exponentially growing JM cells were exposed to tetraplatin (0-10 micrograms/ml) or carboplatin (0-45 micrograms/ml) for 1 h at 37-43 degrees C in 1 degree C increments. Graphic analysis demonstrated a breakpoint for the onset of thermal enhancement for tetraplatin at 40 degrees C. Thermal enhancement was maximal at 42 degrees C with no significant increase at 43 degrees C. The tetraplatin thermal enhancement ratio (TER) was 2.7 at 42 degrees C. The TERs for d and l isomers at 41.8 degrees C were not significantly different. The relationship of TER to temperature for carboplatin closely paralleled that of tetraplatin. The implications of these results are discussed in the context of tetraplatin's unique properties.     

Thermal enhancement of tetraplatin and carboplatin in human leukaemic cells

The thermal enhancement of tetraplatin (racemic, d-trans and l-trans isomers) and carboplatin was studied as a function of temperature in vitro in JM, a human acute lymphoblastic leukaemia cell line. Exponentially growing JM cells were exposed to tetraplatin (0–10 μg/ml) or carboplatin (0–45 μg/ml) for 1 h at 37–43°C in 1°C increments. Graphic analysis demonstrated a breakpoint for the onset of thermal enhancement for tetraplatin at 40°C. Thermal enhancement was maximal at 42°C with no significant increase at 43°C. The tetraplatin thermal enhancement ratio (TER) was 2·7 at 42°C. The TERs for d and / isomers at 41·8°C were not significantly different. The relationship of TER to temperature for carboplatin closely paralleled that of tetraplatin. The implications of these results are discussed in the context of tetraplatin's unique properties.     

Arsenic trioxide-mediated cytotoxicity and apoptosis in prostate and ovarian carcinoma cell lines.

We studied the effect of arsenic trioxide (As2O3) on prostate and ovarian carcinoma cell lines. As2O3 has been shown to be effective in leukemia, and acute promyelocytic leukemia in particular, both in vitro and in vivo. As model cell lines, we used DU145 and PC-3 for prostate cancer and MDAH 2774 for ovarian cancer. New modalities of treatment are essential in these kinds of cancers, which produce a high death toll. The 3-(4,5-dimethyl-thiazoyl-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to evaluate cytotoxicity. Flow cytometric analysis and mono-oligo nucleosome detection-based ELISA were used to determine the apoptosis. Isobologram analysis was used to evaluate synergism and/or the additive effects of As2O3 and conventional chemotherapeutic agents. We clearly demonstrated that As2O3 has significant cytotoxic effect on both prostate and ovarian carcinoma cell lines. The dose range of As2O3 in all three cell lines was approximately 10(-6) M. The mechanism underlying cytotoxicity of As2O3 was shown to be apoptosis. The experiments by butylated hydroxyanisole showed that the cytotoxic effect of As2O3 was not through superoxide generation. There was no synergism, but the additive effects of As2O3 were demonstrated with cisplatin, adriamycin, and etoposide. We strongly suggest that As2O3 alone or in combination with conventional chemotherapeutic agents be evaluated further as a new agent for the treatment of prostate and ovarian cancers.     

The relationships between glutathione, glutathione-S-transferase and cytotoxicity of platinum drugs and melphalan in eight human ovarian carcinoma cell lines

The role of glutathione (GSH) and GSH-S-transferase (GST) activity in modulating the cytotoxicity of four platinum drugs and melphalan was evaluated in eight human ovarian carcinoma cell lines. The cell lines were established from solid and ascitic tumours from pretreated and untreated patients, and showed a wide spectrum of sensitivity to several platinum II and platinum IV drugs; cisplatin, carboplatin, CHIP and tetraplatin. Intracellular glutathione concentration measured in the cell lines showed a significant (P = 0.05) correlation with IC50 values for cisplatin (r = 0.91), carboplatin (r = 0.87) and CHIP (r = 0.88). The correlation between GSH levels and IC50 values for melphalan (r = 0.76) or tetraplatin (r = 0.60) was not as significant. GST activity showed no correlation with IC50 values, for the four platinum drugs. To determine the significance of the elevated GSH concentration in the refractory cell lines, the effect of D,L-buthionine-S, R-sulfoximine (BSO) mediated GSH depletion on platinum drug cytotoxicity was examined in one of the most sensitive (CH1) and two of the least sensitive (relatively resistant; SKOV-3, HX/62) cell lines. Comparison was made with the effect of GSH depletion on melphalan cytotoxicity in these three lines. These lines were differentially sensitive to BSO, with the two most platinum drug resistant lines being more tolerant to BSO than the sensitive CH1 line. Depletion of cellular GSH, ranging between 61 and 88%, had a differential effect on the sensitivity to PtII vs PtIV drugs in the three cell lines: cytotoxicity of the PtIV drugs, tetraplatin and CHIP, was substantially enhanced in both the resistant and sensitive cell lines; in contrast, the cytotoxicity of the PtII drugs, cisplatin and carboplatin, was only significantly increased in one of the two relatively resistant lines (SKOV-3) and in the sensitive (CH1) line after GSH depletion. Moreover the dose modification factor (DMF) for the PtII agents were lower than those for PtIV agents in the three cell lines. The dose modification factor for tetraplatin after BSO treatment was similar to that observed for melphalan in all three cell lines. In the SKOV-3 cell line extending the BSO pretreatment period to 48 h from 24 h marginally reduced the cytotoxicity of cisplatin, whereas the cytotoxicity of the other three drugs remained similar to that observed after 24 h BSO pretreatment. In contrast, extending the BSO treatment to 24 h after drug exposure potentiated the cytotoxicity of cisplatin, CHIP and tetraplatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of carboplatin, cisplatin, and ionizing radiation on a human cell line of ovarian cancer]

AIM OF THE STUDY: Cisplatin (CDDP) and radiotherapy are frequently used concomitantly in the treatment of various malignant conditions. Because of its toxicity, cisplatin tends to be replaced by carboplatin (CBDCA) in several indications. Available data regarding the combined effects of cisplatin and carboplatin with ionising radiation are contradictory. MATERIALS AND METHODS: Various concentrations of cisplatin and carboplatin and various timing of association with radiation have been tested in vitro in a human ovarian cancer cell line. The parental cell line (AOvC-0) and a cisplatin-resistant stable subline (AOvC-CDDP/0) (De Pooter et al., Canc Res, 1991) were exposed to carboplatin (2.5, 5 and 10 M) and to CDDP (1, 2.5 and 5 M), 16 h and 4 h before and 4 h and 16 h after irradiation, respectively. Cell survival was evaluated by a classical clonogenic assay. RESULTS: Exposure of AOvC-0 to 5 M CBDCA and of AOvC-CDDP/0 to 10 M CBDCA, before or shortly after radiation exposure, increased cell lethality in a clear supra-additive way, with the highest DEF in the shoulder region of the survival curve and at radiation doses relevant to clinical radiotherapy. In the sensitive cell line, 5 M carboplatin resulted in an additional lethality equivalent to 4.5 Gy; in the resistant cells, 10 M carboplatin was equivalent to 3.6 Gy. Replacing carboplatin by cisplatin in an identical set-up demonstrated exclusively simple additivity (DEF = 1). CONCLUSION: These data suggest that carboplatin and cisplatin delivered at equitoxic doses interact with radiation in a different way and that, in the present set-up, only carboplatin enhanced the effects of radiation. Carboplatin might consequently be a better candidate than cisplatin in some concomitant combinations with radiotherapy.     

Enhanced cisplatin cytotoxicity by disturbing the nucleotide excision repair pathway in ovarian cancer cell lines

Ovarian cancer is the leading cause of death among women from gynecological malignancies inthe United States. Resistance to the chemotherapeutic agent cisplatin isa major limitation for the successful treatment of ovarian cancer. In an effort to overcome the cisplatin resistance problem in ovarian cancer treatment, we have sought to enhance cisplatin cytotoxicity by perturbing the nucleotide excision repair (NER) pathway. The NER pathway is responsible for repairing cisplatin bound to DNA. Expression of one of the NER components, ERCC1, is correlated with cisplatin drug resistance. Hence, we targeted ERCC1 by antisense RNA methodologies, and we show that we could sensitize a relatively sensitive A2780 cell line and also the highly resistant OVCAR10 cell line to cisplatin by expressing antisense ERCC1 RNA in them as measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The A2780 cell lines expressing antisense ERCC1 had 1.9-8.1-fold enhancements in cisplatin sensitivity. The OVCAR10 antisense ERCC1 cell lines had IC(50) values ranging from 2.28 microM to 2.7 microM cisplatin as compared with 9.52 micro M for control OVCAR10 cells. The OVCAR10 antisense ERCC1 cells also show reduced DNA-damage repair capacity as assessed by host cell reactivation. Furthermore, immunocompromised mice transplanted with the antisense cell lines survived longer than the mice bearing control cells after response to cisplatin treatment. These data suggest that it is possible to substantially enhance the cisplatin cytotoxicity by disturbing the NER pathway in cisplatin-resistant cell lines and to enhance the survival capacity of mice in an ovarian cancer xenograft model.     

P-glycoprotein expression in multidrug-resistant human ovarian carcinoma cell lines

Multiple selections with either vinblastine or vincristine in the human ovarian carcinoma cell line SKOV3 resulted in variants with increasing degrees of multidrug resistance. SKOV3 derivatives that span a wide range in resistance (4- to 2000-fold) were obtained and analyzed for P-glycoprotein expression. In general, we observed a progressive increase in P-glycoprotein level (detected by Western blot) that paralleled the increase in multidrug resistance. However, a more detailed analysis of the P-glycoprotein mRNA and gene level indicated that the amount of P-glycoprotein expressed may be under complex control. At low levels of resistance, only an increase in P-glycoprotein mRNA and protein was observed. At intermediate to high levels of resistance P-glycoprotein gene amplification became evident. At the high level of resistance, an example was observed where only the amount of P-glycoprotein was increased without a concomitant increase in mRNA or gene copy. The mechanisms through which the content of P-glycoprotein in the plasma membrane is mediated are not understood; it is possible that the resistant variants identified here represent perturbations at different levels of regulation.     

Selective potentiation of platinum drug cytotoxicity in cisplatin-sensitive and-resistant human ovarian carcinoma cell lines by amphotericin B

Resistance to the clinically used platinum-based drugs cisplatin and carboplatin represents a major limitation to their clinical effectiveness. Using cisplatin-sensitive and-resistant human ovarian carcinoma cell lines previously characterized in terms of their major underlying mechanisms of resistance, we attempted to potentiate the cytotoxic effects of cisplatin and carboplatin using the clinically used antifungal agent amphotericin B (AmB). Using nontoxic concentrations of AmB (up to 15 g/ml) and continuous exposure to cisplatin, a concentration-dependent selective potentiation (maximum of 3.2-fold) of cisplatin cytotoxicity was observed in two cisplatin-resistant cell lines (41 McisR6, acquired resistant, and HX/62, intrinsically resistant). In both these cisplatin-resistant cell lines, previous studies have shown resistance to be due primarily to reduced platinum uptake. Notably, no significant potentiation was observed in the parent 41M cell line, in the intrinsically resistant SKOV-3 cell line (where reduced drug accumulation plays only a partial role in determining resistance) or in a pair of cell lines (CH1 and its acquired-resistant variant CH1cisR6) where reduced drug uptake does not play any role in determining resistance. The potentiating effect of AmB was lower with carboplatin and not significant in all cell lines. Platinum uptake following a 2-h exposure of cells to cisplatin was enhanced 3.5-fold in 41McisR6 cells (producing platinum levels similar to those obtained in the parental line) and 1.7-fold in 41M cells by the concomitant exposure to AmB. These data indicate that the potentiation of cisplatin (and carboplatin) cytotoxicity by AmB is not due to a generalized membrane disruption, as effects were observed only in resistant lines where reduced drug transport was apparent. Moreover, AmB did not increase the cytotoxicity of JM216 [bis-acetatoammine(cyclohexylamine)dichloroplatinum (II)], a recently developed, more lipophilic orally active platinum drug, in the 41M/41McisR6 lines. JM216 has previously been shown to circumvent acquired cisplatin resistance due to decreased drug uptake. In vivo, however, using the HX/62 xenograft, AmB (at its maximum tolerated dose of 20 mg/kg; q7d×4 schedule) did not enhance the antitumour effect of carboplatin (at its maximum tolerated dose of 80 mg/kg; q7d×4 schedule).This study was supported by grants to the Institute of Cancer Research from the Cancer Research Campaign and the Medical Research Council, the Johnson Matthey Technology Centre and Bristol Myers Squibb Oncology     

Comparative properties of five human ovarian adenocarcinoma cell lines

We describe the derivation of three human ovarian carcinoma cell lines and the comparison of their properties with two previously described cell lines of like histology (SKOV-3 and CAOV-3). Two of the new lines (HOC-1 and HOC-7) were derived from separate ascites tumors (at 9-month intervals) of a patient with well-differentiated serous adenocarcinoma of the ovary. The third new line, HEY, was derived from a human ovarian cancer xenograft (HX-62) originally grown from a peritoneal deposit of a patient with moderately differentiated papillary cystadenocarcinoma of the ovary. The cell lines demonstrated differential ability to grow in semisolid culture and as xenografts in immunologically deprived CBA/CJ mice. Dose-response curves were generated for clonogenic cell survival of cells exposed to common chemotherapeutic agents; one of the lines (HEY) shows a degree of resistance to the alkylating agent cis-diamminedichloroplatinum(II) (cis-platinum). Common karyological features included structural abnormalities of chromosomes 3 and 11. Heterogeneity of expression of ovarian tumor-associated antigens was documented.