Melphalan resistance and photoaffinity labelling of P-glycoprotein in multidrug-resistant Chinese hamster ovary cells: Reversal of resistance by cyclosporin A and hyperthermia

doi:10.1016/S0006-2952(99)00094-5

Biochemical Pharmacology

Volume 58, Issue 2, 15 July 1999, Pages 291-302

https://doi.org/10.1016/S0006-2952(99)00094-5 Get rights and content

Abstract

The multidrug resistance phenotype is often associated with overexpression of P-glycoprotein, an energy-dependent efflux pump responsible for decreased intracellular accumulation of chemotherapeutic agents. The role of P-glycoprotein in the mechanism of cross-resistance to melphalan in multidrug-resistant Chinese hamster ovary cells (CH^RC5) was investigated by photoaffinity labelling of P-glycoprotein using [³H]azidopine. We investigated whether the chemosensitiser cyclosporin A and hyperthermia, either used alone or combined, could reverse melphalan resistance and alter transport processes for [¹⁴C]melphalan in CH^RC5 cells. Melphalan inhibited azidopine photolabelling of P-glycoprotein, implicating drug efflux mediated by P-glycoprotein in the mechanism of melphalan resistance in CH^RC5 cells. Azidopine photolabelling also was inhibited by the chemosensitiser cyclosporin A, which binds to P-glycoprotein. Cyclosporin A alone reversed melphalan resistance in CH^RC5 cells, but had no effect in drug-sensitive AuxB1 cells. Hyperthermia (40–45°) alone increased melphalan cytotoxicity in both cell lines. When hyperthermia was combined with cyclosporin A, a large increase in melphalan cytotoxicity occurred, but only in CH^RC5 cells. This effect increased with temperature and exposure time. Sensitisation to melphalan cytotoxicity by heat and cyclosporin A in CH^RC5 cells appeared to be explained by altered drug transport processes. Lower accumulation of melphalan occurred in CH^RC5 cells than in drug-sensitive cells. At 37°, cyclosporin A increased drug accumulation in CH^RC5 cells, but not in AuxB1 cells, by slowing drug efflux from cells. Heat alone increased both melphalan uptake and drug efflux for both cell lines. Our findings suggest that the combination of cyclosporin A and hyperthermia could be very useful in overcoming melphalan resistance by increasing intracellular drug accumulation in multidrug-resistant cells.

Section snippets

Tissue culture

The MDR cell line CH^RC5 was selected for resistance to colchicine from the drug-sensitive AuxB1 parent cell line [3]. The resistance factor to colchicine is about 300-fold, but CH^RC5 cells are also cross-resistant to other anticancer agents including Vinca alkaloids, Adriamycin, and melphalan [39]. We have obtained a resistance factor to melphalan of about 2- to 3-fold. The CHO cell lines AuxB1 and CH^RC5 were grown in monolayers in 75-cm² tissue culture flasks at 37° under 5% CO₂ in MEM Alpha

Results

We determined whether P-glycoprotein is involved in resistance to melphalan in CH^RC5 cells. To achieve this, MDR cells were labelled with the photoreactive agent [³H]azidopine, which has a high binding affinity for P-glycoprotein 43, 44, 45. Several studies showed that agents with a binding affinity for P-glycoprotein can inhibit photoaffinity labelling of this glycoprotein by azidopine. In drug-sensitive AuxB1 cells, no photolabelling of protein at 170 kDa was seen (Fig. 1 ), indicating that

Discussion

Drug resistance in the well-characterised CH^RC5 cell line has been associated with enhanced efflux of structurally unrelated drugs by an energy-dependent process 3, 39. The mechanism of resistance to melphalan in CH^RC5 cells has been attributed mainly to enhanced drug efflux, although the precise mechanism involved is unclear 24, 46, 47. Melphalan is not usually considered to be a substrate for P-glycoprotein, although this is also unclear. We previously showed that verapamil, a calcium channel

Acknowledgements

Financial support was obtained from the National Cancer Institute of Canada. The authors thank Julie Poirier for technical assistance and André Lévesque for review of the manuscript.

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Multidrug resistance (MDR) remains one of the primary obstacles in cancer chemotherapy and often involves overexpression of drug efflux transporters such as P-glycoprotein and multidrug resistance protein 1 (MRP1). Regional hyperthermia is undergoing clinical investigation in combination with chemotherapy or radiotherapy. This study evaluates whether hyperthermia can reverse MDR mediated by MRP1 in human cervical adenocarcinoma (HeLa) cells.
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The major finding is that HeLa and HeLaMRP cells are both sensitive to cytotoxicity of hyperthermia (41–45°C). Hyperthermia induced activation of caspase 3 and chromatin condensation. Although total levels of cell killing were similar, there was a switch from apoptotic to necrotic cell death in MDR cells. This could be explained by decreased glutathione and GPx in MDR cells. MDR cells also contained very low levels of GST and were resistant to etoposide-induced apoptosis. Hyperthermia caused a modest increase in etoposide-induced apoptosis in HeLa and HeLaMRP cells, which required appropriate heat-drug scheduling.
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A possible explanation may be that differences exist in actual cellular melphalan uptake, and thus melphalan–DNA adduct formation, among different cell lines. Differences in active transport systems between cell lines can play a role in cellular melphalan uptake [32–34]. For instance, Harada et al. [33] reported that down-regulation of CD98 results in decreased melphalan uptake, and Larrivee and Averill [34] showed that P-glycoprotein can mediate active melphalan efflux.
Melphalan is a chemotherapeutic drug that exerts its cytotoxic effect mainly through the formation of DNA adducts. We report the specific immunohistochemical detection and visualisation of melphalan–DNA adducts using the monoclonal antibody MP5/73 in cultured tumour cells and solid tumour tissue from colorectal liver metastases from patients treated with melphalan. The human colon cancer cell lines HT29, SW480 and SW1116, and the rat colon cancer cell line CC531 were exposed to different concentrations of melphalan. In addition, tumour samples from 17 patients with colorectal liver metastases treated by isolated hepatic perfusion with high dose melphalan (200 mg) were collected. Cell lines and tumour samples were stained with the MP5/73 antibody against melphalan–DNA adducts and cell viability was determined by an MTT assay. Melphalan–DNA adducts could be visualised by immunohistochemistry in both cultured cells and solid tumour tissue. A correlation between melphalan exposure concentration, the subsequent melphalan–DNA adduct staining intensity, and melphalan cytotoxicity existed for each individual cell line, but the level of both parameters independently differed between cell lines. Specific staining for melphalan–DNA adducts also was feasible in the human solid tumour tissue. There was considerable variation in melphalan–DNA adduct staining, staining intensity, and distribution in the tumour stroma and the tumour epithelium among the different patients. Melphalan–DNA adducts appeared to be more intense in tumour cells at the border of the tumour nodules than in tumour cells in the centre. Thus, visualisation of melphalan–DNA adducts by immunohistochemistry allows the study of distribution of melphalan–DNA adducts in solid tumours.
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^†: Formerly Dr. Diana Averill-Bates.

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Chemotherapy and Metabolic InhibitorsMelphalan resistance and photoaffinity labelling of P-glycoprotein in multidrug-resistant Chinese hamster ovary cells: Reversal of resistance by cyclosporin A and hyperthermia

Abstract

Section snippets

Tissue culture

Results

Discussion

Acknowledgements

Cancer Lett

Biochem Pharmacol

Biochem Biophys Res Commun

Biochem Pharmacol

Cancer Lett

Biochem Pharmacol

Biochim Biophys Acta

J Biol Chem

Biochem Pharmacol

Biochem Pharmacol

Eur J Cancer

Pharmacol Ther

The biochemistry of P-glycoprotein mediated multidrug resistance

Annu Rev Biochem

P-glycoprotein and resistance to anticancer drugs

Cancer

Reduced permeability in CHO cells as a mechanism of resistance to colchicine

J Cell Physiol

Reduced drug accumulation in multiple drug resistant human KB carcinoma cell lines

Cancer Res

Mechanisms of resistance to daunorubicin in Ehrlich ascites tumor cells

Cancer Res

Daunorubicin-resistant Chinese hamster ovary cells expressing multidrug resistance and a cell-surface P-glycoprotein

Cancer Res

Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil

Cancer Res

Cyclosporin A reverses vincristine and daunorubicin resistance in acute lymphatic leukemia in vitro

J Clin Invest

Cyclosporin A and its analogues as modifiers of adriamycin and vincristine resistance in a multidrug resistant human lung cancer cell line

Br J Cancer

Enhancement of anthracycline growth inhibition in parent and multidrug-resistant Chinese hamster ovary cells by cyclosporin A and its analogues

Cancer Res

Planting ASTh, Seynaeve C, Stoter G and Nooter K, A phase II study of epidoxorubicin in colorectal cancer and the use of cyclosporin-A in an attempt to reverse multidrug resistance

Br J Cancer

Phase I–II study of vinblastine and oral cyclosporin A in metastatic renal carcinoma

Am J Clin Oncol

Cyclosporin A as a multidrug-resistant modulator in patients with renal cell carcinoma treated with teniposide

Br J Cancer

Hyperthermia and Cancer

Mechanisms of thermal enhancement of chemotherapeutic cytotoxicity

Thermal biology and physiology in clinical hyperthermiaCurrent status and future needs

Cancer Res

Effect of melphalan and hyperthermia on cell cycle progression and cyclin B expression in human melanoma cells

Cell Prolif

The effect of hyperthermia in combination with melphalan on drug-sensitive and drug-resistant CHO cells in vitro

Br J Cancer

Thermal enhancement of drug cytotoxicity in vivo and in vitro

Recent Results Cancer Res

Potential for therapy of drugs and hyperthermia

Cancer Res

Basic mechanisms in tumor thermochemotherapy

J Mol Med

Effect of thermal adaptation at 40°C on membrane viscosity and the sodium-potassium pump in Chinese hamster ovary cells

Cancer Res

Hyperthermic adjuvant perfusion chemotherapy for Stage I malignant melanoma of the extremity with literature review

Cancer

Hyperthermic perfusion for melanoma of the extremitiesExperience with 165 patients, 1967 to 1979

Ann NY Acad Sci

Chemotherapy and Metabolic Inhibitors
Melphalan resistance and photoaffinity labelling of P-glycoprotein in multidrug-resistant Chinese hamster ovary cells: Reversal of resistance by cyclosporin A and hyperthermia