Combined effect of temozolomide and hyperthermia on human melanoma cell growth and O6-methylguanine-DNA methyltransferase activity

Hyperthermic isolated limb perfusion (HILP) with L-phenylalanine mustard (L-PAM) represents an effective treatment for locally advanced melanoma of the limbs. However, regional chemotherapy of melanoma still needs to be improved. Temozolomide (TMZ) is a methylating agent that spontaneously decomposes into the active metabolite of dacarbazine, the most effective agent for the systemic treatment of melanoma. Tumor cells with high levels of O6-methylguanine-DNA methyltransferase (MGMT) and/or with a defective DNA mismatch repair (MMR) are resistant to TMZ. Inhibition of MGMT activity increases TMZ sensitivity of MMR-proficient, but not of MMR-deficient cells, while inhibition of base excision repair (BER) potentiates TMZ cytotoxicity in both cell types. Recent studies, performed in an animal model, have shown that TMZ is more effective than L-PAM when applied regionally and that hyperthermia can increase the antitumor activity of TMZ. In this study, three thermoresistant human melanoma cell lines, endowed with different MGMT activity and functional status of the MMR system, were treated with TMZ at 37°C or 41.5°C for 90 min, and then analyzed for cell growth and MGMT activity. Hyperthermia significantly enhanced TMZ cytotoxicity in MMR-proficient cells, either endowed or not with MGMT activity, and in MMR-deficient cells. Endogenous MGMT activity was not affected by hyperthermia that, however, enhanced the enzyme depletion induced by TMZ treatment. Moreover, MGMT recovery after drug removal was delayed in cells that had been treated at 41.5°C. Taken together, these findings confirm the therapeutic potential of a combined treatment of hyperthermia and TMZ. They also suggest that inhibition of BER and/or increased DNA methylation may be involved in the thermal enhancement of TMZ cytotoxicity. Additional studies are necessary to better clarify the mechanisms underlying hyperthermia-induced potentiation of TMZ activity. However, the present investigation provides further support to the development of clinical trials of HILP with TMZ.