M M Oosthuizen1, M J Nel, D Greyling. 1. Department of Surgery, University of the Witwatersrand, Health Science Faculty, Johannesburg, South Africa.
Abstract
BACKGROUND: Cancer of the oesophagus is the most common gastrointestinal malignancy in South African blacks. The aim of this study was to determine whether repetitive heat-shock (HS) treatment of oesophageal cancer cells would induce multi-drug resistance (MDR) as was previously found with human renal carcinoma cells. METHODS: The oesophageal cancer-line WHCO-3 was heat-shocked in sequence, on five occasions, for 90 min each at 42 degrees C, followed by a recovery period of 24 h between the consecutive HSs. After each shock and recovery period the cells were divided; one part was used for the next shock treatment and the other, designated the HS fraction, was used for assessments of drug cytotoxicity, enhanced mdr-1 and mrp gene expression by RT-PCR, and enhanced isoenzyme GsT-P activity. The IC50s of the drugs doxorubicin, amsacrine, melphalan, and cisplatin were determined after each HS using the sulphorhodamine B(SRB) cell-proliferation assay which was able to assess cytotoxicity. A drug accumulation assay was conducted by measuring 3H-Vinblastin uptake in surviving cells using the SRB assay to quantitate the viable cells. RESULTS: Multiple heat-shocks did not introduce MDR via the MDR-1 or MRP mechanisms because these genes were not over-expressed after consecutive HS treatments. Deminished cytotoxicity of the drugs, as measured by increased IC50, did not occur, as it would have been if MDR was introduced. Therefore neither the topoisomerase drug resistant mechanism nor the enhanced GsT-P detoxification mechanism were introduced with repetitive heat-shocks. On the contrary the IC50s of doxorubucin and amsacrine decreased after five HSs, whereas melphalan and cisplatin, had no cytotoxic effects. The GsT-P levels were dramatically reduced by 90% after five HSs and an 8 day recovery period, indicating that the cancer cells became more sensitive towards toxic drugs and not drug resistant as expected. However, with the drug influx assay the uptake of 3H-vinblastin was reduced, with each consecutive HS, and not reversed by verapamil thereby confirming the finding that the efflux mechanisms of MDR-1 and MRP were not introduced. CONCLUSIONS: Repetitive heat shocks did not introduce multi-drug resistance, but on the contrary, sensitized the oesophageal cancer cells against toxic anticancer drugs and they therefore became thermosensitized.
BACKGROUND:Cancer of the oesophagus is the most common gastrointestinal malignancy in South African blacks. The aim of this study was to determine whether repetitive heat-shock (HS) treatment of oesophageal cancer cells would induce multi-drug resistance (MDR) as was previously found with humanrenal carcinoma cells. METHODS: The oesophageal cancer-line WHCO-3 was heat-shocked in sequence, on five occasions, for 90 min each at 42 degrees C, followed by a recovery period of 24 h between the consecutive HSs. After each shock and recovery period the cells were divided; one part was used for the next shock treatment and the other, designated the HS fraction, was used for assessments of drug cytotoxicity, enhanced mdr-1 and mrp gene expression by RT-PCR, and enhanced isoenzyme GsT-P activity. The IC50s of the drugs doxorubicin, amsacrine, melphalan, and cisplatin were determined after each HS using the sulphorhodamine B(SRB) cell-proliferation assay which was able to assess cytotoxicity. A drug accumulation assay was conducted by measuring 3H-Vinblastin uptake in surviving cells using the SRB assay to quantitate the viable cells. RESULTS: Multiple heat-shocks did not introduce MDR via the MDR-1 or MRP mechanisms because these genes were not over-expressed after consecutive HS treatments. Deminished cytotoxicity of the drugs, as measured by increased IC50, did not occur, as it would have been if MDR was introduced. Therefore neither the topoisomerase drug resistant mechanism nor the enhanced GsT-P detoxification mechanism were introduced with repetitive heat-shocks. On the contrary the IC50s of doxorubucin and amsacrine decreased after five HSs, whereas melphalan and cisplatin, had no cytotoxic effects. The GsT-P levels were dramatically reduced by 90% after five HSs and an 8 day recovery period, indicating that the cancer cells became more sensitive towards toxic drugs and not drug resistant as expected. However, with the drug influx assay the uptake of 3H-vinblastin was reduced, with each consecutive HS, and not reversed by verapamil thereby confirming the finding that the efflux mechanisms of MDR-1 and MRP were not introduced. CONCLUSIONS: Repetitive heat shocks did not introduce multi-drug resistance, but on the contrary, sensitized the oesophageal cancer cells against toxic anticancer drugs and they therefore became thermosensitized.