Acute extracellular acidification reduces intracellular pH, 42°C-induction of heat shock proteins and clonal survival of human melanoma cells grown at pH 6.7

Abstract

Two human melanoma cell lines, SK-Mel-28 and DB-1, were used for in vitro studies of the mechanisms underlying heat resistance of human tumour cells adapted to growth in acidic environments. Adaptation to growth at low pH was characterized by resistance to 42 degrees C cytotoxicity and accompanied by an increase in endogenous levels of Hsp70 and/or Hsp27. Acute extracellular acidification to levels below pH 6.5 was required to sensitize the melanoma cells to 42 degrees C. Furthermore, cells grown at low pH were more resistant to sensitization by acute acidification than cells grown at pH 7.3. The intracellular pH (pHi) of cells grown at pH 6.7 was less than the pHi of cells grown at pH 7.3 both before and after acute acidification. A pHi threshold existed for melanoma cells growing at pH 7.3 below which they became sensitized to 42 degrees C. This pHi threshold differed between the SK-Mel-28 and DB-1 cells. In contrast, a pHi threshold for heat sensitization did not exist for cells growing at pH 6.7: any reduction in pHi before heating resulted in increased cell killing. Since cells grown at low pH lack a pHi threshold for heat sensitization, they are sensitized more to 42 degrees C per unit decrease in pHi than cells grown at pH 7.3. Acute acidification abrogated the 42 degrees C-induction of Hsp70 and Hsp27 in the melanoma cells. The pHi thresholds for abrogation of these HSPs are slightly higher than or comparable with the thresholds for cytoxicity for each cell line grown at pH 7.3, but abrogation occurred over a narrower range of pHi compared with cytotoxicity. Abrogation of heat-induced expression of these HSPs correlates with cytotoxicity in both cell lines with the exception of Hsp27 expression in SK-Mel-28 cells. In conclusion, strategies that reduce pHi in melanoma cells growing at low pH, such as in acidotic regions of tumours, could selectively sensitize them to hyperthermia because they lack a pHi threshold for heat sensitization.

Acute acidification or amiloride treatment suppresses the ability of Hsp70 to inhibit heat-induced apoptosis

“…Exposure to hyperthermia under conditions of acidic extracellular pH also reduced the amount of Hsp70 synthesis that occurred during the 6 h incubation at 37°C. The suppressive effect of intracellular acidification on heat-shock protein synthesis has been observed previously, however the mechanism responsible for this effect is not known [15,33]. Although Hsp70 synthesis occurs in the non-induced cells following exposure to a lethal heat treatment, this is not cytoprotective since it occurs after apoptosis has been initiated [30].…”

“…Exposure of cultured cells to acidic media (pH 6.0-6.8) has been shown to either induce apoptosis [14] or increase sensitivity to stress-induced apoptosis [15]. The extracellular pH of tumors is more acidic (6.2-6.9) than that of normal tissues (7.3-7.4), however, the intracellular pH (pH i ) is maintained slightly alkaline through activation of the Na + /H + exchanger NHE1 resulting in increased acid extrusion [16].…”

Hsp27anti-sense oligonucleotides sensitize the microtubular cytoskeleton of Chinese hamster ovary cells grown at low pH to 42°C-induced reorganization

“…As mentioned previously, numerous studies from different laboratories have shown that Hsp27 14,51-55 and Hsp70 13,15,56-59 act as inhibitor of apoptosis proteins. Cells derived from a variety of tumours show elevated levels of one or more heat shock proteins 60-62 including Hsp27 [61][62][63] . Therefore, heat shock proteins may protect cells from thermal stress by acting both as molecular chaperones of critical cellular structures such as the cytoskeleton and as inhibitors of apoptotic signalling.…”

“…One means of accomplishing this is to decrease the intracellular pH prior to thermal therapy by either reducing the extracellular pH 62,64 or inhibiting hydrogen ion transporters responsible for maintaining the intracellular pH of tumour cells in an acidic environment 65 .…”

Inhibiting induction of heat shock proteins as a strategy to enhance cancer therapy

“…Human melanoma cells cultured at acidic pH, a characteristic of regions of solid tumours that may affect treatment outcome [10][11][12][13][14], are resistant to thermal therapy compared to cells cultured at pH 7.3 [15][16][17]. This is due in part to elevated endogenous levels of HSPs [15,17].…”