In vitro platinum drug chemosensitivity of human cervical squamous cell carcinoma cell lines with intrinsic and acquired resistance to cisplatin.

The platinum drug chemosensitivity of five human cervical squamous cell carcinoma cell lines (HX/151, HX/155, HX/156, HX/160 and HX/171) derived from previously untreated patients has been determined. Compared to our data obtained previously using human ovarian carcinoma cell lines, all five lines were relatively resistant to cisplatin, carboplatin, iproplatin and tetraplatin. One of the lines (HX/156) was exceptionally sensitive to the novel platinum (IV) ammine/amine dicarboxylates JM216 [bis-acetatoammine dichloro (cyclohexylamine) platinum (IV)] and JM221 [ammine dibutyrato dichloro (cyclohexylamine) platinum (IV)]. The range in IC50 values across the five lines was approximately 2.5-fold for cisplatin, carboplatin and iproplatin, 13-fold for tetraplatin and JM216, and 25-fold for JM221. No significant correlation (P > 0.05) was observed between platinum drug chemosensitivity and either glutathione levels or cadmium chloride sensitivity, an indicator of metallothionein levels. In addition, there was no significant correlation (P > 0.05) between cisplatin cytotoxicity and intracellular cisplatin accumulation or JM216 cytotoxicity and intracellular JM216 accumulation over the dose range 5-100 microM (2 h exposure). The exceptional sensitivity of HX/156 to JM216 appears, at least partially, to be a result of enhanced accumulation of JM216. An 8.6-fold acquired cisplatin resistant stable variant of HX/155 has been generated in vitro. Intracellular cisplatin accumulation was reduced by 2.4 +/- 0.3-fold (mean +/- s.d.) in HX/155cisR across the dose range 1-100 microM (2 h exposure). Glutathione levels in HX/155cisR were elevated by 1.3-fold in terms of protein content and by 1.6-fold in terms of cell number. HX/155cisR was 1.9-fold resistant to cadmium chloride. Total platinum bound to DNA after cisplatin exposure (10, 25, 50 or 100 microM for 2 h) was 3.6 +/- 0.6-fold (mean +/- s.d.) lower in HX/155cisR. Hence the mechanism of acquired cisplatin resistance in HX/155cisR appears to be multifocal, with reduced intracellular drug accumulation and elevated glutathione and metallothionein levels combining to reduce DNA platination levels. While HX/155cisR was cross-resistant to tetraplatin and carboplatin, novel platinum (II) and (IV) ammine/amine complexes, including JM216 and JM221, partially circumvented resistance (resistance factors of 1.5-2). Non cross-resistance was observed to iproplatin and nine non-platinum anticancer agents. Intracellular tetraplatin accumulation was reduced by 1.8 +/- 0.1-fold (mean +/- s.d.) in HX/155cisR across the dose range 1-100 microM (2 h exposure). In contrast, after JM216 exposure (1-100 microM for 2 h), no significant difference in intracellular platinum levels between HX/155 and HX/155cisR was observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro circumvention of cisplatin resistance by the novel sterically hindered platinum complex AMD473.

A novel sterically hindered platinum complex, AMD473 [cis-aminedichloro(2-methylpyridine) platinum (II)], has been selected for phase I clinical trials due to commence in 1997. AMD473 was rationally designed to react preferentially with nucleic acids over sulphur ligands such as glutathione. This report documents the in vitro circumvention of acquired cisplatin resistance mechanisms in human ovarian carcinoma (HOC) cell lines by AMD473. In a panel of 11 HOC cell lines, AMD473 showed intermediate growth inhibition potency (mean IC50 of 8.1 microM) in comparison to cisplatin (mean IC50 of 2.6 microM) and carboplatin (mean IC50 of 20.3 microM). AMD473 showed only a 30.7-fold increase in IC50 value from the most sensitive to the most resistant HOC cell line, whereas for cisplatin it was 117.9-fold and for carboplatin 119.7-fold. AMD473 also showed significantly (P < 0.05) reduced cross-resistance to cisplatin in a panel of three cell lines with known acquired platinum drug resistance mechanisms (mean RF for AMD473 was 1.9, for cisplatin 9.1). Cellular accumulation of AMD473 was not reduced in two HOC cell lines (A2780cisR and 41McisR), in which reduced cisplatin accumulation is a major mechanism of acquired cisplatin resistance. AMD473 naked-DNA binding was significantly less affected (P < 0.05) than that of cisplatin by the presence of 5 mM glutathione. Also, AMD473 almost completely circumvented acquired cisplatin resistance in a cell line (A2780cisR) with fivefold elevated intracellular glutathione levels compared with the parent A2780 cell line when measured by clonogenic assay (RF 4.5 for AMD473 vs RF 18 for cisplatin). AMD473 also showed a lower increase in IC50 than cisplatin in an A2780 cell line model with artificially elevated glutathione levels. AMD473 DNA binding was slower than that of cisplatin on both naked and cellular DNA. AMD473 also formed DNA interstrand cross-links (ICLs) at a slower rate than cisplatin (peak ICL formation was at 5 h for cisplatin vs > or = 14 h for AMD473) after equitoxic doses were exposed to HOC cells for 2 h. AMD473 ICLs in the CH1 HOC cell line were slowly formed and showed no visible signs of being repaired 24 h after removal of drug. This was paralleled by a slower, longer lasting induction of p53 protein by equitoxic doses of AMD473 in HOC cell lines with wild-type p53. This new class of sterically hindered platinum compound, selected for clinical trial in 1997, may therefore elicit improved clinical response in intrinsically and acquired cisplatin-resistant tumours in the clinic.     

Preclinical activity of a new platinum analogue,lobaplatin, in cisplatin-sensitive and -resistant human testicular,ovarian, and gastric carcinoma cell lines

Lobaplatin [1,2-diamminomethylcyclobutane-platinum(II) lactate] is a new platinum compound with interesting preclinical activity and apparently no nephro- or neurotoxicity that is currently undergoing clinical phase II studies. Little is known about the cross-resistance between cisplatin and lobaplatin. The activity of this new compound in comparison with cisplatin and carboplatin was evaluated in cisplatin-sensitive and cisplatin-resistant human testicular, gastric, and ovarian carcinoma cell lines using 96 h continuous drug exposure in a sulforhodamine-B assay. In three cisplatin-sensitive testicular carcinoma cell lines, lobaplatin and cisplatin showed comparable antitumor activity. The 50% growth-inhibitory concentrations (IC₅₀ values) determined for cisplatin ranged from 0.1 to 0.4 μM, and those found for lobaplatin ranged from 0.25 to 0.5 μM. Carboplatin showed markedly lower cytotoxicity in all cell lines tested. Lobaplatin was not cross-resistant to cisplatin in a 10-fold cisplatin-resistant testicular carcinoma cell line and showed only weak cross-resistance in a 20-fold cisplatin-resistant ovarian carcinoma cell line. In contrast, complete cross-resistance between cisplatin and lobaplatin occurred in two cisplatin-resistant human gastric carcinoma cell lines, which were 3.3- and 9-fold resistant to cisplatin and 3.1- and 6.5-fold resistant to lobaplatin, respectively. Furthermore, lobaplatin showed significant activity against cisplatin-resistant human ovarian and testicular carcinoma xenografts in vivo. These data indicate a high level of activity for lobaplatin at clinically achievable concentrations in drug-sensitive testicular, ovarian, and gastric carcinoma cell lines and a lack of complete cross-resistance to cisplatin. Further clinical development of lobaplatin is clearly warranted.     

Mechanism-related circumvention of acquired cis-diamminedichloroplatinum(II) resistance using two pairs of human ovarian carcinoma cell lines by ammine/amine platinum(IV) dicarboxylates.

Acquired resistance to cisplatin has been generated in vitro in two human ovarian carcinoma cell lines: 41M, established from a previously untreated patient; and CH1, from a patient previously treated with cisplatin and cis-diammine-1,1-cyclobutane dicarboxylatoplatinum(II) (carboplatin). In neither cell line with acquired resistance did intracellular detoxification (via increased glutathione or metallothioneins) appear to be a major determinant of resistance. Resistance in 41McisR (resistance factor of 4.7) appeared to be due predominantly to a reduced platinum accumulation (levels were only 23.8% in 41McisR versus 41M). This was also reflected at the DNA level by a similar level of reduced DNA interstrand cross-links and total platinum-DNA adducts measured immediately after a 2-h exposure to cisplatin in 41McisR versus 41M. Conversely, for CH1cisR (resistance factor of 6.5), platinum accumulation, and initial numbers of DNA-interstrand cross-links and total DNA-platinum adducts were not significantly different from the parent CH1 line. This is suggestive of a resistance mechanism involving increased DNA repair or tolerance to platinum-DNA adducts operating in the CH1cisR/CH1 pair of lines. Cross-resistance to carboplatin and partial cross-resistance to the 1,2-diaminocyclohexane-containing agent, (trans-d,l)-1,2-diaminocyclohexane tetrachloroplatinum(IV) (tetraplatin), was observed in both pairs. However, two novel platinum(IV) ammine/amine dicarboxylates, ammine dibutyratodichloro(cyclohexylamine)platinum(IV) (JM221) and ammine dibenzoatodichloro(propylamine)platinum(IV) (JM244), completely circumvented resistance in 41McisR to produce some collateral sensitivity (resistance factors of 0.67 and 0.54, respectively) but showed cross-resistance in CH1cisR (resistance factors of 3.7 and 4.6). In contrast to the data for cisplatin, intracellular platinum levels were not significantly different between the 41M and 41McisR pair of cell lines after exposure to JM244. These results suggest that the ammine/amine platinum(IV) dicarboxylates, which show considerably greater in vitro cytotoxicity than cisplatin, are capable of circumventing acquired cisplatin resistance which is due to decreased intracellular accumulation but are not able to overcome resistance at the level of DNA platination and removal.     

The relative activity of cisplatin, oxaliplatin and satraplatin in testicular germ cell tumour sensitive and resistant cell lines

Background  Germ cell tumours (GCT) can become resistant to cisplatin, which is associated with a relatively poor prognosis. Oxaliplatin and satraplatin have been developed to overcome cisplatin resistance in other cancers, but their effect in cisplatin resistant (cisR) GCTs is unclear. In this work we address this issue by comparing their efficacy in three paired sensitive and cisR GCT cell lines. Methods  Three established cisplatin sensitive (cisS) and resistant cell line pairs were used (GCT27, GCT27r: SUSA, SUSAr: 833k, 833kr). Viability was assessed using a luciferase based ATP assay and EC₅₀ and EC₈₀ concentrations were calculated. Western blot analysis and flow cytometry was used for further assessment. Results  Sensitivity to the three platinum compounds was broadly similar in the three cisS lines GCT cell lines (EC₅₀ = 0.27–0.51 μM for cisplatin, 0.52–0.79 μM for oxaliplatin, 0.31–1.26 μM for satraplatin). EC₅₀ values for cisplatin in the three cisR sub lines were 1.8- to 3.8-fold higher than in the sensitive parental lines. Cross resistance to satraplatin and oxaliplatin occurred in all three cisR cell lines (resistance factor 1.9–4.4), with the exception of oxaliplatin in the 833Kr (resistance factor 0.9). Differences in the effect of specific drugs on cell cycle distribution, p53, p21 and MDM2 were observed. Conclusions  These data suggest that satraplatin and oxaliplatin could theoretically be used in chemo-naive GCTs and support the further clinical evaluation of these agents in this setting. The mechanism of cross resistance to these drugs appears multifactorial.     

Preclinical activity of a new platinum analogue, lobaplatin, in cisplatin-sensitive and -resistant human testicular, ovarian, and gastric carcinoma cell lines

Lobaplatin [1,2-diamminomethylcyclobutane-platinum(II) lactate] is a new platinum compound with interesting preclinical activity and apparently no nephro- or neurotoxicity that is currently undergoing clinical phase II studies. Little is known about the cross-resistance between cisplatin and lobaplatin. The activity of this new compound in comparison with cisplatin and carboplatin was evaluated in cisplatin-sensitive and cisplatin-resistant human testicular, gastric, and ovarian carcinoma cell lines using 96 h continuous drug exposure in a sulforhodamine-B assay. In three cisplatin-sensitive testicular carcinoma cell lines, lobaplatin and cisplatin showed comparable antitumor activity. The 50% growth-inhibitory concentrations (IC₅₀ values) determined for cisplatin ranged from 0.1 to 0.4 M, and those found for lobaplatin ranged from 0.25 to 0.5 M. Carboplatin showed markedly lower cytotoxicity in all cell lines tested. Lobaplatin was not cross-resistant to cisplatin in a 10-fold cisplatin-resistant testicular carcinoma cell line and showed only weak cross-resistance in a 20-fold cisplatin-resistant ovarian carcinoma cell line. In contrast, complete cross-resistance between cisplatin and lobaplatin occurred in two cisplatin-resistant human gastric carcinoma cell lines, which were 3.3- and 9-fold resistant to cisplatin and 3.1- and 6.5-fold resistant to lobaplatin, respectively. Furthermore, lobaplatin showed significant activity against cisplatin-resistant human ovarian and testicular carcinoma xenografts in vivo. These data indicate a high level of activity for lobaplatin at clinically achievable concentrations in drug-sensitive testicular, ovarian, and gastric carcinoma cell lines and a lack of complete cross-resistance to cisplatin. Further clinical development of lobaplatin is clearly warranted.This work was supported in part by a grant from Asta Medica, D-6000 Frankfurt 1, Germany     

Cellular pharmacology of novel C8-linked anthramycin-based sequence-selective DNA minor groove cross-linking agents.

The cellular pharmacology of a series of C8-linked pyrrolobenzodiazepine dimers with polymethylene linkers of n = 3-6 (compounds 1-4) has been studied in a range of human tumour cell lines. The four compounds showed the same pattern of relative activity in five ovarian carcinoma cell lines and one cervical carcinoma cell line with the order of IC50 values of 1 < or = 3 < 4 < 2, which correlated with the previously demonstrated DNA interstrand cross-linking ability of the compounds in plasmid DNA. In human leukaemic K562 cells the agents produced a block in the G2/M phase of the cell cycle characteristic of cross-linking drugs, and extensive interstrand cross-linking was observed in cells by alkaline elution with no evidence of single-strand breaks. Cross-links continued to increase up to 24 h following a 1 h exposure to drug, and no repair was evident by 48 h. In a series of ovarian and cervical carcinoma cell lines with acquired resistance to cisplatin no cross-resistance to the most potent compound 1 was observed in two lines whose major mechanism of resistance to cisplatin was reduced platinum transport. Cross-resistance to 1 was observed in a cell line (A2780cisR) possessing elevated glutathione, and depletion of intracellular glutathione using D,L-buthionine-S,R-sulphoximine (BSO) from 10.25 nmol to 2.8 nmol 10(-6) cells reduced the level of resistance from 11-fold to 2-fold compared with sensitive cells. Cross-linking in the resistant cells was restored to 80% of the level in the parent line by BSO pretreatment. There was also a correlation between glutathione levels and sensitivity to 1 measured in several other ovarian cell lines. Compound 1 also showed cross-resistance in the doxorubicin-resistant cell line 41MdoxR and partial cross-resistance in CH1doxR cells. Both these lines possess elevated levels of p170 glycoprotein. Following treatment with 6 microM verapamil, the resistance in these lines decreased almost 2-fold and 8-fold respectively.     

Relationship of cellular glutathione to the cytotoxicity and resistance of seven platinum compounds.

The role of glutathione (GSH) in the effectiveness of and resistance to 7 platinum compounds [5 Pt(II) and 2 Pt(IV) drugs] was evaluated in a 8.6-fold cisplatin (CDDP)-resistant human small cell lung cancer cell line (GLC4/CDDP), the parent GLC4 line, a 3.7-fold CDDP-resistant human embryonal carcinoma cell line (Tera-CP), and the parent Tera line (NTera2/D1). Resistance factors for both CDDP-resistant cell lines were determined after continuous incubation (4 days) with CDDP. Continuous incubation with the other studied platinum drugs revealed complete cross-resistance for carboplatin (CBDCA) and zeniplatin but less for enloplatin (ENLO) and iproplatin in both models. Tetraplatin and lobaplatin showed, respectively, partial and complete cross-resistance in GLC4/CDDP but no cross-resistance in Tera-CP. GSH level, but not glutathione S-transferase activity, of the 4 cell lines correlated with platinum drug concentrations inhibiting cell survival by 50% after continuous incubation (r = 0.86, P < 0.05). GSH depletion by DL-buthionine-S,R-sulfoximine (BSO) increased sensitivity, as measured after a 4-h exposure to the drugs, of GLC4/CDDP for CDDP 2.0-fold, for CBDCA 1.7-fold, for zeniplatin 1.7-fold, and almost to the level of the sensitive GLC4 for ENLO, whereas no effect was observed for lobaplatin and the Pt(IV) compounds iproplatin and tetraplatin. BSO-modulating effect was higher in the sensitive GLC4 line for most compounds; therefore reduction of resistance could be achieved only for CDDP and ENLO. In contrast to GLC4, no modulation occurred in Tera. In Tera-CP BSO increased sensitivity for CDDP 1.5-fold, for CBDCA 1.9-fold, and for zeniplatin 1.2-fold; no effect was observed for ENLO, lobaplatin, and the Pt(IV) compounds. Reduction of CDDP resistance by BSO was known to occur with identical cellular platinum levels and higher Pt-DNA binding in GLC4/CDDP. However, pretreatment with BSO followed by 4 h ENLO incubation increased cellular platinum levels in both GLC4 and GLC4/CDDP while Pt-DNA binding remained unchanged. In conclusion, GSH reflected sensitivity to platinum-containing drugs. However, since the involvement of GSH differed between the models and the various platinum drugs, the effect of modulation with BSO was unpredictable.     

Sensitivity to novel platinum compounds of panels of human lung cancer cell lines with acquired and inherent resistance to cisplatin.

We have developed panels of human lung cancer cell lines with acquired and inherent resistance to cisplatin. Three parental cell lines, NCI-H69/P (small cell), COR-L23/P (large cell), and MOR/P (adenocarcinoma), were grown in increasing concentrations of cisplatin over a period of 6-9 months. This resulted in the development of sublines, H69/CPR, L23/CPR, and MOR/CPR which were 3- to 8-fold resistant to cisplatin as determined by a 6-day 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. None of the resistant sublines showed a significant change in cellular glutathione content or sensitivity to cadmium chloride (an indicator of metallothionein content), although changes in glutathione-S-transferase activity were seen. The sublines each showed cross-resistance to melphalan. Cisplatin accumulation was unchanged in H69/CPR, 1.3-fold reduced in L23/CPR, and 2.0-fold reduced in MOR/CPR compared with their respective parent lines. In a panel of 10 small cell lung cancer cell lines, there was a 16-fold range of sensitivities to cisplatin. The panels have been used to examine cross-resistance between cisplatin, carboplatin, iproplatin, tetraplatin, and a series of 10 novel ammine/amine dicarboxylate platinum(IV) compounds. Whereas H69/CPR and MOR/CPR showed little or no cross-resistance to any of the other compounds, L23/CPR was generally cross-resistant to all of them. In the panel of small cell lines, whereas the ranking of sensitivity to carboplatin and cisplatin were similar, each of the other compounds provided individual patterns of sensitivity. There was always a wide range of sensitivities among the panel, ranging from 8- to 28-fold. Among the dicarboxylate compounds, there was a great range of potencies, with two compounds (JM273 and JM274) being approximately 100-fold more potent than cisplatin.     

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     

Gene-specific repair of Pt/DNA lesions and induction of apoptosis by the oral platinum drug JM216 in three human ovarian carcinoma cell lines sensitive and resistant to cisplatin

JM216, an oral platinum drug entering into phase III clinical trial, exhibited comparable cytotoxicity to cisplatin in three human ovarian carcinoma cell lines: the sensitive (CH1), acquired resistant (CH1cisR) and intrinsically resistant (SKOV-3). Platinum accumulation and binding to DNA were similar in each of the three cell lines at equimolar doses, indicating that the resistant cell lines could tolerate higher intracellular platinum levels and platinum bound to DNA at IC50 concentrations of drug. Comparison with cisplatin demonstrated that intracellular platinum levels were marginally higher with JM216, but that platinum binding to DNA was similar for the two drugs in each of the cell lines. Each of the cell lines exhibited an ability to repair JM216 induced platinum/DNA lesions in the N-ras gene (gene-specific repair) at equitoxic concentrations of drug. However, this occurred to a greater extent in the two resistant cell lines such that by 24 h the CH1cisR and SKOV-3 had removed 72% and 67% respectively compared with approximately 32% for the CH1. Reduced gene-specific repair capacity in CH1 cells was also seen following incubation with 25 microM (or 5 microM - 2 x IC50) cisplatin, whereas the CH1cisR and SKOV-3 cell lines were repair proficient. JM216 induced apoptosis in the three cell lines following a 2h incubation with 2 x the IC50 of drug. Fluorescent microscopy of cells stained with propidium iodide showed that the detached cell population displayed typical apoptotic nuclei. Furthermore, field inversion gel electrophoresis demonstrated the presence of DNA fragments approximately 23-50 kb in size, indicative of apoptosis, in the detached cells. JM216 induced an S phase slow down in each of the three cell lines accompanied by a G2 block in the CH1 pair. Incubation with this concentration of JM216 also resulted in the induction of p53 in the CH1 and CH1cisR. These studies suggest that the relative sensitivity of the CH1 cell line to cisplatin and JM216 is at least partly attributable to a deficiency in gene-specific repair. The oral platinum drug, JM216, exerts its cytotoxic effects through the induction of apoptosis following a slow-down in S phase in both the sensitive and resistant lines.     

In vitro and in vivo activity and cross resistance profiles of novel ruthenium (II) organometallic arene complexes in human ovarian cancer

Ruthenium complexes offer the potential of reduced toxicity, a novel mechanism of action, non-cross resistance and a different spectrum of activity compared to platinum containing compounds. Thirteen novel ruthenium(II) organometallic arene complexes have been evaluated for activity (in vitro and in vivo) in models of human ovarian cancer, and cross-resistance profiles established in cisplatin and multi-drug-resistant variants. A broad range of IC50 values was obtained (0.5 to >100 microM) in A2780 parental cells with two compounds (RM175 and HC29) equipotent to carboplatin (6 microM), and the most active compound (HC11) equipotent to cisplatin (0.6 microM). Stable bi-dentate chelating ligands (ethylenediamine), a more hydrophobic arene ligand (tetrahydroanthracene) and a single ligand exchange centre (chloride) were associated with increased activity. None of the six active ruthenium(II) compounds were cross-resistant in the A2780cis cell line, demonstrated to be 10-fold resistant to cisplatin/carboplatin by a mechanism involving, at least in part, silencing of MLH1 protein expression via methylation. Varying degrees of cross-resistance were observed in the P-170 glycoprotein overexpressing multi-drug-resistant cell line 2780AD that could be reversed by co-treatment with verapamil. In vivo activity was established with RM175 in the A2780 xenograft together with non-cross-resistance in the A2780cis xenograft and a lack of activity in the 2780AD xenograft. High activity coupled to non cross-resistance in cisplatin resistant models merit further development of this novel group of anticancer compounds.     

Acquisition of platinum drug resistance and platinum cross resistance patterns in a panel of human ovarian carcinoma xenografts.

In vivo models of acquired resistance to the platinum-based agents cisplatin (CDDP), carboplatin (CBDCA), iproplatin (CHIP) and tetraplatin have been established using a panel of six parent human ovarian carcinoma lines, two (HX/110 and PXN/87) being derived from previously untreated patients. Resistance has been generated to CDDP (three lines), CBDCA (one line), CHIP (three lines) and tetraplatin (one line) either by treatment in vivo or (for one line to CDDP) through exposure in vitro and subsequent transfer to mice. With the four tumours where resistance was generated using CDDP or CBDCA, a complete cross-resistance to the remaining platinum agents studied was observed. In contrast, in one of three lines with derived resistance to the platinum (IV) agent, CHIP, (PXN/951) a retention in sensitivity was observed with CDDP and CBDCA. Only one of the six parent tumour lines (PXN/100) was markedly sensitive to tetraplatin. Where resistance was generated to tetraplatin (PXN/100T) there was some retention of activity by CDDP. For the CDDP-resistant line established in vitro, there was a close agreement between the cross-resistance profile obtained in vitro vs that obtained in vivo. This tumour panel may be useful in the elucidation of cellular and molecular resistance mechanisms to platinum drugs operative in vivo. Moreover, as they appear to mimic the clinical observations of shared cross-resistance between CDDP, CBDCA and CHIP, they may represent valuable preclinical evaluation models for the discovery of drugs capable of conferring responses in CDDP-refractory ovarian cancer.     

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.     

The role of murine tumour models and their acquired platinum-resistant counterparts in the evaluation of novel platinum antitumour agents: a cautionary note.

Two murine tumour models, the L1210 leukaemia and the ADJ/PC6 plasmacytoma, have featured prominently in the preclinical development of platinum drugs. Mindful of the unequivocal need to discover new platinum-based drugs exhibiting activity in cisplatin/carboplatin refractory and relapsed cancers, and to devise clinically-predictive screening models, we have generated resistance in vivo in the ADJ/PC6 plasmacytoma to cisplatin (19- to 21-fold), to carboplatin (25-fold), iproplatin (greater than 14-fold) and tetraplatin (10-fold). The chemo-sensitivity profiles of these tumours have been compared with L1210 leukaemia lines resistant to either cisplatin (10-fold) or tetraplatin (34-fold). In cross-resistance studies, the L1210 and ADJ/PC6 resistant variants provided conflicting predictions of structures likely to circumvent cisplatin-acquired resistance. In particular, the L1210/cisplatin resistant model exhibited cross-resistance to carboplatin and iproplatin, whereas the diaminocyclohexane (DACH)-containing complex, tetraplatin, was even more active in the cisplatin resistant tumour than in the 'wild-type' tumour. The ADJ/PC6/cisplatin resistant tumour, however, was cross-resistant, not only to carboplatin and iproplatin, but also to tetraplatin. These data provide an important caveat on the adoption of single acquired resistant animal tumour models for platinum drug development.     

Synthesis of trans-bis-(2-hydroxypyridine)dichloroplatinum(II) and its activity in human ovarian tumour models

We report the synthesis and in vitro activity of trans-bis-(2-hydroxypyridine)dichloroplatinum(II) (coded as DH3) in humour ovarian tumour models. The compound is less active than cisplatin against the parent cell line A2780 but more so against the cisplatin-resistant A2780(cisR) cell line, thus indicating that it is better able to overcome mechanisms of resistance operating in the A2780(cisR) cell line. DH3 is marginally less active than cisplatin against ZD0473-resistant A2780(ZD0473R) cell line but with a much lower resistance factor than cisplatin. DH3 has higher platinum-DNA binding levels than cisplatin in the A2780 and A2780(ZD0473) cell lines and a lower value in the A2780(cisR) cell line. Even though DH3 has a lower activity than cisplatin, the higher platinum-DNA binding levels observed for DH3 than cisplatin in A2780 and A2780(ZD0473R) cell lines may not be entirely unexpected when we note that the two compounds are likely to differ in their nature of binding with DNA. Whereas cisplatin binds with DNA forming mainly intrastrand 1,2-Pt(GG) and 1,2-Pt(AG) adducts, DH3 is expected to form more 1,2-interstrand Pt(GG) and monofunctional adducts. The higher activity of DH3 than cisplatin in the A2780(cisR) cell line despite its lower level of platinum-DNA binding can also be seen to indicate the complexity of the situation. Although platinum-DNA binding may be an essential requirement for apoptosis, it is not sufficient to cause cell death that is actually brought about by downstream processes in the cycle. The results of interaction with pBR322 plasmid DNA combined with BamH1 digestion show that DH3 is less able to prevent BamH1 digestion than is cisplatin, indicating that cisplatin causes a greater conformational change in the DNA than DH3. CONCLUSION: DH3 is less active than cisplatin against the parent cell line A2780, but more so against the cisplatin-resistant A2780(cisR) cell line, thus indicating that it is better able to overcome mechanisms of resistance operating in the A2780(cisR) cell line.     

Lack of a role for MRP1 in platinum drug resistance in human ovarian cancer cell lines.

The level of expression of the multidrug resistance-associated protein (MRP1) in a panel of human ovarian carcinoma cell lines and their variants with acquired cisplatin resistance was determined using Western blotting. No overexpression of MRP1 was detected in any of the cell lines. In addition, we have transfected the MRP1 gene into an intrinsically cisplatin-resistant cell line SKOV3, previously shown to have elevated levels of glutathione (GSH). The MRP1-transfected line SKOV3-S2 was shown to be cross-resistant to doxorubicin, vincristine and etoposide but not to paclitaxel, vinblastine and platinum agents, such as cisplatin, JM216 [bis-acetato-ammine-dichloro-cyclohexylamine platinum (IV)] and AMD473 [cis-ammine dichloro (2-methyl-pyridine) platinum (II)]. No cross-resistance to any of the platinum agents was observed in a MRP1-overexpressing human lung cancer cell line with acquired doxorubicin resistance. Reduction of GSH levels (80-90%) by buthionine sulphoximine (BSO) produced significant potentiation in cisplatin sensitivity in the parental SKOV3, the vector-alone control SKOV3-puro and the MRP1-transfected line SKOV3-S2. The degree of sensitization was similar in all cell lines (1.6-fold). However, selective sensitization by BSO to vincristine was observed in the MRP1-transfected line (4.1-fold) but not in the vector control. No significant differences were observed in cisplatin accumulation in the SKOV3-puro and the SKOV3-S2 cells, although both these transfected lines accumulated significantly more than the parental line. Our results suggest that MRP1 does not play a significant role in platinum resistance in the human tumour cell lines investigated in this study.     

Evaluation of novel ammine/amine platinum (IV) dicarboxylates in L1210 murine leukaemia cells sensitive and resistant to cisplatin, tetraplatin or carboplatin.

Seventeen alkylamine ammine dicarboxylatodichloroplatinum(IV) complexes of general structure c,t,c-[PtCl2(OCOR1)2NH3(RNH2)], where R = aliphatic or alicyclic and R1 = aliphatic or aromatic, have been evaluated against L1210 cell lines with acquired resistance to cisplatin (10-fold), tetraplatin (34-fold) or carboplatin (14-fold) using an in vitro growth-delay assay. All of these compounds overcame cisplatin, tetraplatin and carboplatin resistance. Potency increased as the number of carbon atoms in the axial aliphatic ligands (R1) increased, for example comparing JM216 (R = cyclohexyl, R1 = CH3, IC50 = 1.2 microM) with JM274 (R = cyclohexyl, R1 = n-C4H9, IC50 = 0.05 microM) against the parent sensitive line (L1210/S). The most active compounds were those possessing aromatic ligands at R1, regardless of whether R = aliphatic or alicyclic, for example JM244 (R = n-C3H7, R1 = C6H5, IC50 = 0.028 microM) and JM2644 (R = c-C6H11, R1 = C6H5, IC50 = 0.031 microM) against L1210/S. For an alicyclic alkylamine series in which R is varied from c-C3H7 to C-C7H13, with R1 = n-C3H7 for each compound, cytotoxic potency was maximised at c-C6H11 (JM221, IC50 = 0.06 microM against L1210/S). Preliminary biochemical studies, at equitoxic doses, comparing JM221 (0.1 microM) with cisplatin (0.6 microM) identified five times more platinum associated with JM221 treated cells and 1.5 times more platinum bound to the DNA of JM221-treated cells. The lipophilic properties of some of these platinum(IV) dicarboxylates may contribute to both the potency and circumvention of resistance by these compounds.     

Carboplatin- and cisplatin-induced potentiation of moderate-dose radiation cytotoxicity in human lung cancer cell lines.

The interaction between moderate-dose radiation and cisplatin or carboplatin was studied in a cisplatin-sensitive (GLC4) and -resistant (GLC4-CDDP) human small-cell lung cancer cell line. Cellular toxicity was analysed under oxic conditions with the microculture tetrazolium assay. For the platinum and radiation toxicity with the clinically relevant dose ranges applied, this assay was used to obtain information on cell survival after the treatments. Apart from effects on cell survival effects on DNA were also investigated. Configurational DNA changes could be induced by platinum drugs and thereby these drugs might change the frequency of DNA double-strand breaks (dsbs). DNA fragmentation assayed with the clamped homogeneous electric field (CHEF) technique was used as a measure for dsbs in DNA. The radiosensitising effect of the platinum drugs was expressed as enhancement ratio (ER) calculated directly from survival levels of the initial slope of the curve. The highest ER for cisplatin in GLC4 was 1.39 and in GLC4-CDDP 1.38. These were all at 75% cell survival. Carboplatin showed increased enhancement with prolonged incubation up to 1.21 in GLC4 and was equally effective as cisplatin in GLC4-CDDP. According to isobologram analysis, prolonged incubation with both platinum drugs showed at least additivity with radiation for both cell lines at clinically achievable doses. GLC4-CDDP showed cross-resistance to radiation. The radiosensitising capacity of both lung cancer cell lines was not dependent on their platinum sensitivity. The formation of dsbs in DNA directly after radiation was not influenced by pretreatment of either drug in the sensitive or in the resistant cell line. Drug treatment resulted in decreased DNA extractability in control as well as in irradiated cells. Modest enhancement ratio for radiosensitisation by platinum drugs cannot be explained on the level of dsb formation in DNA in both cell lines. Interaction of radiation with the clinically less toxic carboplatin can be improved by prolonged low-dose carboplatin exposure before irradiation and is as potent as cisplatin in the resistant lung cancer cell line. This suggests an advantage in combining radiation and carboplatin in lung cancer patients.