Thermal sensitisation by lonidamine of human melanoma cells grown at low extracellular pH.

PURPOSE: This study tested the ability of lonidamine (LND), a clinically applicable inhibitor of monocarboxylate transporters (MCT), to thermally sensitise human melanoma cells cultured at a tumour-like extracellular pH (pHe) 6.7. MATERIALS AND METHODS: Human melanoma DB-1 cells cultured at pHe 6.7 and pHe 7.3 were exposed to 150 µM LND for 3 h, beginning 1 h prior to heating at 42 °C (2 h). Intracellular pH (pHi) was determined using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and whole spectrum analysis. Levels of heat shock proteins (HSPs) were determined by immunoblot analysis. Cell survival was determined by colony formation. RESULTS: Treatment with LND at pHe 6.7 reduced pHi to 6.30 ± 0.21, reduced thermal induction of HSPs, and sensitised cells growing at pHe 6.7 to 42 °C. When LND was combined with an acute acidification from pHe 6.7 to pHe 6.5, pHi was reduced to 6.09 ± 0.26, and additional sensitisation was observed. LND had negligible effects on cells cultured at pH 7.3. CONCLUSIONS: The results show that LND can reduce pHi in human melanoma cells cultured at a tumour-like low pHe so that the 42 °C induction of HSPs are abrogated and the cells are sensitised to thermal therapy. Cells cultured at a normal tissue-like pHe 7.3 were not sensitised to 42 °C by LND. These findings support the strategy that human melanoma cells growing in an acidic environment can be sensitised to thermal therapy in vivo by exposure to an MCT inhibitor such as LND.

Intracellular acidification abrogates the heat shock response and compromises survival of human melanoma cells.

This study tests the hypothesis that lowering intracellular pH (pHi) in melanoma cells grown at low extracellular pH (pHe) selectively abrogates 42 degrees C-induced heat shock protein (HSP) expression and reduces survival. Cells were acidified by a combination of a 0.2-pH-unit decrease in pHe coupled with the lactate/H+ transport inhibitor alpha-cyano-4-hydroxy-cinnamic acid (CNCn). A mild acute extracellular acidification was used to mimic the acute extracellular acidification observed in tumors that can be induced in vivo by oral glucose administration. CNCn blocks the activity of H(+)-linked monocarboxylate transporters (MCTs), particularly MCT isoform 1 (MCT-1). This transporter removes lactic acid from cells and has a high activity in DB-1 melanoma cells grown at low pHe. The effect of extracellular acidification combined with CNCn on pHi was measured in cells grown at pHe 6.7 and pHe 7.3. Cells grown at pHe 6.7 serve as an in vitro model for cells in an acidic tumor microenvironment. When cells were grown at pHe 6.7 and incubated with CNCn at pHe 6.5, the pHi decreased from 6.9 to below 6.5, and the 42 degrees C induction of HSP70 and HSP27 was blocked. The abrogation of HSP induction correlated positively with decreased clonogenic survival. In contrast, when cells growing at pHe 7.3 were acidified by a 0.2-pH unit to pHe 7.1, the inhibitor had less effect on pHi, which remained above 7.0. Under these conditions, the 42 degrees C-induction of HSPs was not inhibited, and cytotoxicity was not enhanced. These results indicate that a significant decrease in the pHi of melanoma cells can selectively sensitize the cells to 42 degrees C hyperthermia, possibly through the inhibition of HSP expression. This strategy could result in a therapeutic gain, because normal tissues, existing at a pHe above 7.0, would not be sensitized.