Reduction of cellular cisplatin resistance by hyperthermia--a review.

Resistance to cisplatin (cDDP) is a major limitation to its clinical effectiveness. Review of literature data indicates that cDDP resistance is a multifactorial phenomenon. This provides an explanation why attempts to reverse or circumvent resistance using cDDP-analogues or combination therapy with modulators of specific resistance mechanisms have had limited success so far. It therefore provides a rationale to use hyperthermia, an agent with pleiotropic effects on cells, in trying to modulate cDDP resistance. In this review the effects of hyperthermia on cDDP cytotoxicity and resistance as well as underlying mechanisms are discussed. Hyperthermia is found to be a powerful modulator of cDDP cytotoxicity, both in sensitive and resistant cells. Relatively high heat doses (60 min 43 degrees C) seem to specifically interfere with cDDP resistance. The mechanism of interaction has not been fully elucidated so far, but seems to consist of multiple (simultaneous) effects on drug accumulation, adduct-formation and -repair. This may explain why hyperthermia seems to be so effective in increasing cDDP cytotoxicity, irrespective of the presence of resistance mechanisms. Therefore, the combination of hyperthermia and cDDP deserves further attention.

Sensitizing for cis-diamminedichloroplatinum(II) action by hyperthermia in resistant cells.

cDDP-resistant Ehrlich ascites tumour (EAT) cells (ER cells) were tested for cellular content of total glutathione, heat sensitivity, cDDP sensitivity and synergistic effects of a combined treatment of heat and chemotherapy. In comparison with the non-resistant EAT cells (EN) the ER cells had an elevated level of glutathione. Treatment with D,L-buthionine-(S,R)-sulphoximine (BSO), resulting in almost complete depletion of cellular glutathione, did not cause drug sensitization. The ER cells were somewhat less heat sensitive compared with the EN cells. Heat chemosensitization was observed for the EN cells as well as for the ER cells. At 43 degrees C (but not at 42 degrees C) the thermal enhancement ratio (TER) for cDDP toxicity was significantly higher in the ER cells. The total number of cells killed by the combined treatment was less in the ER cells than in the EN cells. After analysing existing literature, combined with the current results, it is concluded that although cDDP-resistant cells can often considerably be chemosensitized by hyperthermia, in most cases the difference in cDDP sensitivity cannot be overcome totally. In those situations where cDDP-resistant cells are more sensitive to heat and also show a high TER, especially at clinically relevant temperatures, hyperthermia as added modality is indicated for clinical treatment.

Cisplatin sensitivity and thermochemosensitisation in thermotolerant cDDP-sensitive and -resistant cell lines.

Development of thermotolerance is an important phenomenon that must be considered when thermochemotherapy with multiple heat treatments is used clinically. To study the effect of thermotolerance on cellular cisplatin (cDDP) sensitivity at 37 degrees C and 43 degrees C in cell lines with different cDDP sensitivities, two Ehrlich ascites tumour cell lines (one with high cDDP sensitivity and one with in vitro acquired cDDP resistance) were used. The results indicate that in both cell lines the state of thermotolerance per se did not affect the cDDP sensitivity at 37 degrees C. Thus, general elevations in 'all' heat shock protein levels as found in thermotolerant cells apparently do not influence cDDP sensitivity to a considerable extent. The sensitising effect of a (second) heat treatment given simultaneously with a cDDP treatment was less in thermotolerant cells. Thermal enhancement ratios (TERs) at the 10% survival level for heat doses of 43 degrees C for 30 min or 43 degrees C for 60 min were reduced by a factor of 1.6 and 2.1 in cDDP-resistant and -sensitive thermotolerant cells respectively, as compared with control cells. Thus, protection against heat damage in thermotolerant cells seems to be paralleled by diminished thermal chemosensitisation. Although the effect of thermotolerance on the cDDP-sensitising effect was less pronounced in the resistant cells, a modifying effect on the resistance factor was not achieved.

Sensitization to cisplatin action by step-down heating in cDDP-sensitive and -resistant cells.

Hyperthermia treatment (> or = 43 degrees C) has been shown to be able to (partially) reverse acquired cDDP resistance. However, such heat treatment is difficult to achieve in the clinic. Short pre-treatment at a high temperature (> 42 degrees C), immediately before a treatment at a lower temperature (< 42 degrees C) can enhance the heat toxicity of the lower temperatures. This "step-down heating schedule" was explored for its possible drug-sensitizing potential in in vitro-cultured cDDP-sensitive and -resistant murine and human tumour cells. A 10-min pre-treatment at 44 degrees C enhanced the cytotoxicity of 41 degrees C hyperthermia alone. It also enhanced sensitivity to cDDP when given at 37 degrees C. However, it did not increase the 41 degrees C-induced cDDP sensitization. Thus, no correlation was found between heat kill and cDDP sensitization for step-down heating schedules. The observed effects of step-down heating were comparable in sensitive and in resistant cells, so the step-down heating schedule, unlike the 43 degrees C treatment, did not lead to a decrease of the cDDP-resistance factor. Yet the total cytotoxicity caused by this treatment protocol was 10-fold more than for cDDP with 41 degrees C alone, due to the extra hyperthermic cell killing and the cDDP-sensitizing effect of the pre-treatment. This treatment could have a substantial impact on cDDP efficacy in the clinic even when cDDP resistance has developed.

Hyperthermic potentiation of cisplatin toxicity in a human small cell lung carcinoma cell line and a cisplatin resistant subline.

A human small cell lung carcinoma cell line (GLC4) and its subline with in vitro acquired cisplatin (cDDP) resistance (GLC4-cDDP) were used to study the applicability of hyperthermia to interfere with acquired cDDP resistance. GLC4 and GLC4-cDDP did not differ in heat sensitivity (clonogenic ability). Both cell lines could be sensitized to cisplatin to a considerable extent, both at 42 and 43 degrees C. For 42 degrees C hyperthermia treatments up to 90 min no differences in TER between the cell lines were observed. Only prolonged (> or = 45 min) exposures to 43 degrees C hyperthermia sensitized the resistant cell line to a greater extent than the parent cell line, resulting in a reduction of the resistance factor from 3.6 (at 37 degrees C) to 1.7 (60 min 43 degrees C). The finding in this human system that for treatments up to 90 min, 43 degrees C heat is more suitable than 42 degrees C heat to reduce cDDP resistance, is in accordance with earlier findings with murine cells (Konings et al. 1993). Effects of heat, cisplatin and combined treatments on cell killing were not only measured with the clonogenic assay, but also with the microculture tetrazolium method (MTT assay), an assay of potential use in the clinic for rapid screening of cells obtained from patients. The data with the latter assay were comparable to those obtained with the clonogenic assay. However, its applicability to measure thermo-chemosensitization is limited due to its inability to measure more than one log of cell killing.

Heat-shock protein expression in cisplatin-sensitive and -resistant human tumor cells.

It has been suggested that the expression of certain heat-shock proteins (HSPs) may be prognostic markers in several tumor types. Since HSPs may be involved in determining cellular sensitivity to chemotherapeutic drugs, the possible relation between HSP expression and cisplatin (cDDP) sensitivity was studied. Three human germ-cell tumor cell lines, 1 human small-cell lung carcinoma (SCLC) cell line and 3 human colon carcinoma cell lines were used as a model for differences in intrinsic cDDP sensitivity. The constitutive expression of a panel of HSPs was studied by immunoblotting. No correlation was found between expression of HSP90, HSP73, HSP72, HSP60 and HSP27 and the extent of intrinsic cDDP sensitivity when all cell lines studied were considered. However, for the 3 cell lines derived from germ-cell carcinomas, HSP27 expression was inversely related to cDDP sensitivity; ie. decreased HSP27 levels were associated with decreased sensitivity. Constitutive HSP expression was also studied in 2 sets of human cell lines with in vitro acquired cDDP resistance. In both resistant cell lines, decreased expression of HSP27 (as determined by Western blotting) was found as compared to the sensitive parent cell lines. Thus, acquired resistance to cDDP was also accompanied by decreased HSP27 expression. Interestingly, when basal HSP27 mRNA levels were measured in the SCLC cell line (GLC4) and its subline with acquired resistance (GLC4-cDDP), no significant differences were detected. Continuous cDDP incubation increased HSP27 levels and induced HSP27 phosphorylation in GLC4 cells, but not in the resistant subline. Thus, although no general relationships between HSP expression and cDDP sensitivity are apparent, high HSP27 expression in vitro relates to high sensitivity to cDDP treatment in some tumor types. This is in accordance with reported clinical data on high HSP27 levels in tumors correlating with good prognosis.

Mechanism of hyperthermic potentiation of cisplatin action in cisplatin-sensitive and -resistant tumour cells.

In this study, the mechanism(s) by which heat increases cis-diamminedichloroplatinum (cisplatin, cDDP) sensitivity in cDDP-sensitive and -resistant cell lines of murine as well as human origin were investigated. Heating cells at 43 degrees C during cDDP exposure was found to increase drug accumulation significantly in the cDDP-resistant cell lines but had little effect on drug accumulation in the cDDP-sensitive cell lines. DNA adduct formation, however, was significantly increased in all cell lines studied. Furthermore, ongoing formation of platinum (Pt)-DNA adducts after the end of cDDP treatment was enhanced and/or adduct removal was decreased in heated cells, resulting in relatively more DNA damage remaining at 24 h after the end of cDDP exposure. Correlation plots with survival revealed weak correlations with cellular Pt accumulation (r2 = 0.59) and initial Pt-DNA adduct formation (r2 = 0.64). Strong correlations, however, were found with Pt-DNA adducts at 6 h (r2 = 0.97) and 24 h (r2 = 0.89) after the incubation with the drug. In conclusion, the mechanism by which heat sensitizes cells for cDDP action seems to be the sum of multiple factors, which comprise heat effects on accumulation, adduct formation and adduct processing. This mechanism did not seem to differ between cDDP-sensitive and -resistant cells, emphasizing the potential of hyperthermia to reduce cDDP resistance.