Retention of activity by the new generation platinum agent AMD0473 in four human tumour cell lines possessing acquired resistance to oxaliplatin.

Four models of acquired resistance to the clinically-used platinum drug, oxaliplatin, have been established using human tumour cell lines in vitro; two colon (HCT116 and HT29) and two ovarian (A2780 and CH1). Levels of acquired resistance ranged from 3.0- to 15.8-fold with levels of resistance higher in the colon relative to the ovarian carcinoma cell lines. Notably, the platinum analogue, AMD0473, currently undergoing clinical evaluation, exhibited superior circumvention of acquired oxaliplatin resistance in comparison to either cisplatin or the trinuclear platinum BBR3464. Resistance in the two colon cell lines was unique to oxaliplatin itself among the platinum drugs studied. Acquired oxaliplatin resistance was not due to either reduced drug membrane transport or increased levels of glutathione in any of the four resistant lines. Following exposure to oxaliplatin, a lower level of platinum-DNA adducts was present in acquired oxaliplatin-resistant HT29 cells. In the remaining resistant lines, there was no change in the levels of platinum-DNA adducts relative to the parent lines. There was no change in hMLH1 DNA mismatch repair gene status in any of the four cell line pairs. However, in an A2780 subline where loss of hMLH1 and a p53phe172 mutation occurred, 5-fold resistance to cisplatin was observed, but only 1.7-fold resistance to oxaliplatin and no resistance to AMD0473 were observed. Re-introduction of hMLH1 into these cells caused no significant change in the sensitivity to cisplatin, oxaliplatin or AMD0473. These data show that acquired resistance to oxaliplatin may occur in cell lines (and therefore probably in the clinic) and in the four independent cell lines studied this was circumvented by AMD0473. Alongside previously described models of acquired resistance to cisplatin, these oxaliplatin-resistant cell line models may be useful in the evaluation of further novel platinum agents.

Establishment of an isogenic human colon tumor model for NQO1 gene expression: application to investigate the role of DT-diaphorase in bioreductive drug activation in vitro and in vivo.

Many tumors overexpress the NQO1 gene, which encodes DT-diaphorase (NADPH:quinone oxidoreductase; EC 1.6.99.2). This obligate two-electron reductase deactivates toxins and activates bioreductive anticancer drugs. We describe the establishment of an isogenic human tumor cell model for DT-diaphorase expression. An expression vector was used in which the human elongation factor 1alpha promoter produces a bicistronic message containing the genes for human NQO1 and puromycin resistance. This was transfected into the human colon BE tumor line, which has a disabling point mutation in NQO1. Two clones, BE2 and BE5, were selected that were shown by immunoblotting and enzyme activity to stably express high levels of DT-diaphorase. Drug response was determined using 96-h exposures compared with the BE vector control. Functional validation of the isogenic model was provided by the much greater sensitivity of the NQO1-transfected cells to the known DT-diaphorase substrates and bioreductive agents streptonigrin (113- to 132-fold) and indoloquinone EO9 (17- to 25-fold) and the inhibition of this potentiation by the DT-diaphorase inhibitor dicoumarol. A lower degree of potentiation was seen with the clinically used agent mitomycin C (6- to 7-fold) and the EO9 analogs, EO7 and EO2, that are poorer substrates for DT-diaphorase (5- to 8-fold and 2- to 3-fold potentiation, respectively), and there was no potentiation or protection with menadione and tirapazamine. Exposure time-dependent potentiation was seen with the diaziquone analogs methyl-diaziquone and RH1 [2, 5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], the latter being an agent in preclinical development. In contrast to the in vitro potentiation, there was no difference in the response to mitomycin C when BE2 and BE vector control were treated as tumor xenografts in vivo. This isogenic model should be valuable for mechanistic studies and bioreductive drug development.

Mechanisms of drug resistance to the platinum complex ZD0473 in ovarian cancer cell lines.

Acquired drug resistance to the sterically hindered platinum drug ZD0473 (formerly known as JM473 and AMD473) and currently being tested in phase I clinical trials, has been studied in two human ovarian carcinoma cell lines (CH1 and A2780) where previously, acquired cisplatin resistance has been described. Common mechanisms of resistance were observed in A2780 acquired cisplatin and ZD0473R (resistant) lines (including reduced drug transport and DNA platination, increased glutathione (GSH) and loss of the MLH1 DNA mismatch repair gene). However, contrasting mechanisms were observed in the CH1 sublines. While ZD0473 retained activity against the acquired cisplatin resistant sublines, cisplatin did not circumvent acquired ZD0473 resistance. The trans platinum complex JM335 circumvented resistance in CH1cisR and A2780ZD0473R lines, but not in A2780cisR or CH1ZD0473R cells. Overexpression of metallothionein (MT) in A2780 cells by stable gene transfection resulted in protection from the growth-inhibitory effects of cadmium chloride (3. 8-fold) and a range in protection with platinum drugs (from 7-fold with cisplatin, but only 1.3-fold with ZD0473). Overall, the results show that some mechanisms of resistance to ZD0473 are shared with those previously described in the same parental lines for cisplatin (e.g. in A2780), but in the CH1 lines, differing mechanisms were apparent. Moreover, ZD0473 possesses distinct cellular pharmacological properties in comparison with cisplatin with respect to reduced interactions with MTs, a thiol-containing species associated with tumour resistance to cisplatin.

BCL-2 family protein expression and platinum drug resistance in ovarian carcinoma.

The expression of the BCL-2 family proteins, BCL-2, BAX, BCL(XL) and BAK have been determined in a panel of 12 human ovarian carcinoma cell lines encompassing a wide range in sensitivity to cisplatin. Whereas BAX, BCL(XL) and BAK levels did not correlate with sensitivity, there was a statistically significant inverse correlation (r = -0.81; P = 0.002) between growth inhibition by cisplatin and BCL-2 levels. In sublines possessing acquired resistance to various platinum-based drugs or across a panel of human ovarian carcinoma xenografts, there was no consistent pattern of BCL-2 expression. Two relatively sensitive lines (A2780 and CH1) have been stably transfected with bcl-2 and bcl(XL) respectively and two relatively resistant lines (A2780cisR and SKOV-3) stably transfected with bax. Overexpression of BCL-2 in A2780 cells led to resistance to cisplatin compared to the vector control when assayed at 48 h post-drug incubation but a significant increase in sensitivity at 96 h. Relative rates of apoptosis at 48- and 96-h post-cisplatin exposure mirrored the growth inhibition. There was no significant difference in sensitivity of the pair of lines by clonogenic assay. No significant changes in chemosensitivity to a variety of DNA-damaging or tubulin-interactive agents were observed in the remaining transfected lines. Taken together, these results suggest that, in human ovarian carcinoma cells, high BCL-2 levels (either naturally occurring or through gene transfection) confers a trend towards sensitivity not resistance to platinum drugs.

DT-Diaphorase expression and tumor cell sensitivity to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat shock protein 90.

BACKGROUND: To our knowledge, 17-allylamino,17-demethoxygeldanamycin (17AAG) is the first inhibitor of heat shock protein 90 (Hsp90) to enter a phase I clinical trial in cancer. Inhibition of Hsp90, a chaperone protein (a protein that helps other proteins avoid misfolding pathways that produce inactive or aggregated states), leads to depletion of important oncogenic proteins, including Raf-1 and mutant p53 (also known as TP53). Given its ansamycin benzoquinone structure, we questioned whether the antitumor activity of 17AAG was affected by expression of the NQO1 gene, which encodes the quinone-metabolizing enzyme DT-diaphorase. METHODS: The antitumor activity of 17AAG and other Hsp90 inhibitors was determined by use of a sulforhodamine B-based cell growth inhibition assay in culture and by the arrest of xenograft tumor growth in nude mice. DT-diaphorase activity was determined by use of a spectrophotometric assay, and protein expression was determined by means of western immunoblotting. RESULTS: In two independent in vitro human tumor cell panels, we observed a positive relationship between DT-diaphorase expression level and growth inhibition by 17AAG. Stable, high-level expression of the active NQO1 gene transfected into the DT-diaphorase-deficient (by NQO1 mutation) BE human colon carcinoma cell line resulted in a 32-fold increase in 17AAG growth-inhibition activity. Increased sensitivity to 17AAG in the transfected cell line was also confirmed in xenografts. The extent of depletion of Raf-1 and mutant p53 protein confirmed that the Hsp90 inhibition mechanism was maintained in cells with high and low levels of DT-diaphorase. 17AAG was shown to be a substrate for purified human DT-diaphorase. CONCLUSION: These results suggest that the antitumor activity and possibly the toxicologic properties of 17AAG in humans may be influenced by the expression of DT-diaphorase. Careful monitoring for NQO1 polymorphism and the level of tumor DT-diaphorase activity is therefore recommended in clinical trials with 17AAG.

A molecular cytogenetic approach to studying platinum resistance.

The technique of comparative genomic hybridisation (CGH) has until recently been used to screen for common genomic abnormalities in fresh tumour material; it has identified previously unrecognised regions of amplification associated with poor prognosis subtypes of breast cancer and lymphoma. Our group has applied this technique to resistant cell lines and their sensitive counterparts in order to define chromosomal abnormalities associated with acquired drug resistance. We have demonstrated the applicability of this technique to the study of drug resistance using cell lines with known mechanisms of resistance. The ability to detect novel genomic alterations in cell lines with novel mechanisms of resistance was also demonstrated. We subsequently examined the CGH profiles of seven different cell lines made resistant to three platinum analogues and showed the most consistent abnormalities to involve over-representation of regions 4q and 6q. More recently, we have applied the CGH technique to a series of testicular germ cell tumours (TGCTs) collected as formalin-fixed paraffin-embedded biopsy specimens from patients, both pre- and post-therapy using a platinum-based regimen (POMB/ACE). Previous reports have shown over-representation of X, 7q, 8q and 12p and loss of 13q to occur in 25% of primary TGCTs. Over-representation of 12p was confirmed in the majority of these biopsy samples; deletion of 13q was noted in the initial biopsies of several patients. We also demonstrated alterations of 4p, 4q, 5q and 6q in this series of patients. Newly acquired deletions of 2q and 18q and amplifications of 8q were frequently observed in post-chemotherapy samples from resistant tumours. The CGH studies on these patients with TGCT will not only enable us to correlate our observations on clinical material with those from long-term cell lines, but should also identify sites of key genes involved in clinical platinum resistance.

Mini-review: discovery and development of platinum complexes designed to circumvent cisplatin resistance.

The discovery and development of new platinum-containing anticancer drugs have represented an integral part of anticancer drug development at the Institute of Cancer Research, Sutton, over almost 20 years. As part of a collaboration with chemists at Johnson Matthey, later AnorMED, four major new classes of platinum drug have been discovered, three of which have entered clinical trial. Earlier studies led to the clinical development of the less toxic analogue carboplatin and JM216, the first orally administerable platinum drug. In recent years, the focus has been on two lead complexes designed to overcome the major mechanisms of tumour resistance to cisplatin: JM335 (trans-ammine (cyclohexylaminedichlorodihydroxo) platinum(IV)), an active trans platinum complex; and ZD0473 (cis-amminedichloro(2-methylpyridine) platinum(II)), a sterically hindered complex shown to be less reactive towards thiol-containing molecules than cisplatin. JM335 shows some circumvention of acquired cisplatin resistance in vitro and exhibits unique cellular pharmacological properties in comparison to cisplatin or its cis-isomer in terms gene-specific repair of adducts on DNA and the rate of induction of apoptosis. ZD0473 is now in phase I clinical trial. Myelosuppression is the dose-limiting toxicity at a dose of 130 mg/m2 given i.v. every 3 weeks and there has been evidence of antitumour activity. ZD0473-resistant human ovarian carcinoma cell lines have been established in vitro. Some mechanisms of resistance common to those described for cisplatin (decreased drug uptake, increased glutathione) have been observed plus, in one cell line, increased BCL2 levels and loss of the DNA mismatch repair protein MLH1.

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.

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.

Transport of cisplatin and bis-acetato-ammine-dichlorocyclohexylamine Platinum(IV) (JM216) in human ovarian carcinoma cell lines: identification of a plasma membrane protein associated with cisplatin resistance.

The mechanisms by which cis-diamminedichloroplatinum(II) (cisplatin) is transported across the plasma membrane (i.e., passive diffusion versus active transport) were investigated in the 41M and CH1 human ovarian carcinoma cell lines and their acquired cisplatin-resistant variants 41McisR6 and CH1cisR6, respectively. Intracellular cisplatin accumulation was significantly reduced (4.0 +/- 1.7-fold) in the parental 41M line at 4 degrees C when compared to incubations at 37 degrees C. However, no significant differences in platinum uptake were observed in the 41McisR6 and in the CH1 pair of lines at 4 degrees C versus 37 degrees C. Similarly, in the presence of ouabain (an inhibitor of Na+,K+-ATPase), there was a marked reduction (2.0 +/- 0.4-fold) in drug accumulation in the sensitive 41M cells only, and no changes in drug uptake were observed in the other cell lines in the absence or presence of ouabain. Platinum accumulation was significantly enhanced in all cell lines in the presence of metabolic inhibitors (NaF and NaN3). These results suggest that in the parental 41M cell line, cisplatin transport may occur via passive diffusion and active/facilitated transport, whereas in the resistant 41McisR6 variant, cisplatin enters cells by passive diffusion only. The orally active drug bis-acetato-ammine-dichloro-cyclohexylamine platinum(IV) (JM216) is a lipophilic platinum(IV) complex that has been shown to circumvent cisplatin resistance in the 41McisR6 by increasing drug uptake. Across the entire range of concentrations used (5-50 microm), intracellular accumulation of JM216 was significantly reduced in 41M and 41McisR6 cells (3.5 +/- 0.7-fold; P < 0.01), and in CH1 and CH1cisR6 cells (14.2 +/- 6.0-fold; p < 0.01) at 4 degrees C when compared to incubations at 37 degrees C. No significant difference in JM216 uptake was observed in the 41M pair of lines in the absence or presence of ouabain. Additional studies have revealed that the fold reduction observed in cis-ammine(cyclohexylamine)dichloroplatinum(II) (JM118) accumulation in the 41M and 41McisR6 cells at 4 degrees C (3.7 +/- 1.9) reflects similar fold reductions to those observed with JM216 uptake at 4 degrees C. These results suggest that the mechanism of JM216 transport across cell membranes is through passive diffusion, predominantly as a result of its enhanced lipophilicity. Notably, an overexpression of a Mr 36,000 plasma membrane protein was observed in the 41McisR variants when compared to the sensitive 41M line. Increased levels of this Mr 36,000 protein may relate to the observed reduction in active transport of cisplatin in the 41McisR6 variant. Tyrosine phosphorylation of the Mr 36,000 protein appeared to be greater in the resistant 41McisR6 variant than in the parental 41M line. In addition, the constitutive levels of the Mr 36,000 protein in the CH1 pair of lines and in two acquired JM216-resistant variants (41M/JM216R and CH1/JM216R), where resistance in these cell lines is not mediated through reduced drug uptake, were similar to those observed in their respective parental lines. These results suggest that the overexpression of this Mr 36,000 protein in the acquired cisplatin-resistant subline 41McisR6 may play a significant role in cisplatin uptake in resistant cells exhibiting reduced drug accumulation as a major mechanism of cisplatin resistance.

Effects of a new antioestrogen, idoxifene, on cisplatin- and doxorubicin-sensitive and -resistant human ovarian carcinoma cell lines.

Pyrrolidino-4-iodotamoxifen (idoxifene) is a new non-steroidal antioestrogen currently undergoing phase I clinical evaluation. Using idoxifene and tamoxifen and two additional analogues of tamoxifen (3-hydroxytamoxifen and 4-iodotamoxifen) and the imidazole-based calmodulin inhibitor, calmidazolium, a strong positive correlation (r2 > 0.95) was observed between cytotoxicity and inhibition of calmodulin-dependent cyclic AMP phosphodiesterase (e.g. mean IC50 across four human ovarian carcinoma cell lines of 4.5 microM for idoxifene and 6.3 microM for tamoxifen). Using two parent human ovarian carcinoma cell lines (41M and CH1; both oestrogen receptor negative) in which acquired resistance to doxorubicin or cisplatin has been generated, we have determined the ability of idoxifene to overcome resistance in these lines. At a non-toxic concentration of 2 microM, idoxifene appeared at least as effective as the clinically used multidrug resistance modifiers verapamil and tamoxifen in overcoming doxorubicin resistance in two acquired resistant cell lines shown to overexpress the P-170 efflux glycoprotein. Non-cross-resistance between cisplatin and idoxifene was observed in two acquired resistant cell lines possessing contrasting mechanisms of resistance to cisplatin (41McisR6 reduced drug transport and CH1cisR6 resistance mediated at the level of DNA). In one of four cell lines (CH1), synergism between idoxifene and cisplatin was observed by median effect analysis. However, with the 41M and its 6-fold cisplatin-resistant variant, antagonism was observed. These observations made by median effect analysis appeared to be unrelated to platinum uptake or removal of platinum-induced DNA interstrand cross-links. These in vitro data suggest that idoxifene may be usefully combined with doxorubicin in the clinical setting, but caution should be exercised in combining it with cisplatin in the treatment of certain tumours.

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 non-toxic concentrations of AmB (up to 15 micrograms/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 (41McisR6, 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) were 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 x 4 schedule) did not enhance the antitumour effect of carboplatin (at its maximum tolerated dose of 80 mg/kg; q7d x 4 schedule.