Intracellular Acidification Abrogates the Heat Shock Response and Compromises Survival of Human Melanoma Cells¹

Human melanoma cells were cultured at pHe³ 6.7 to model the acidic tumor microenvironment. To increase cell killing at low pHe, the inhibition of transporters that impact on pHi regulation was tested as a strategy to inhibit the induction of HSPs in cells grown at pHe 6.7 and to sensitize these cells to 42°C cell killing. The stress from growth at low pHe induces HSPs in Chinese hamster ovary cells and human melanoma cells (1–3). Human melanoma cells that are grown at pHe 6.7 and heated (42°C) are more resistant to 42°C-induced cell killing compared with cells grown and heated at pHe 7.3 (Ref. 3; Coss, unpublished observations⁴). Acute extracellular acidification abrogates 42°C-induction of HSPs in DB-1 melanoma cells and enhances cell killing (3). Hyperglycemia induced by oral administration of glucose has been used to transiently reduce the pHe of human tumors by ∼0.2 pH units (4); however, this degree of extracellular acidification alone does not sensitize DB-1 melanoma cells to hyperthermia (3). The degree of reduction in pHi that accompanies acute extracellular acidification is thought to be the critical factor, because it correlates with hyperthermic sensitization more than pHe (5–10). In vitro studies using melanoma cells indicate that pHi must be reduced less than 6.6 to sensitize cells to hyperthermia (Ref. 3 and Coss and Wahl, unpublished observations⁵). Using ³¹P-magnetic resonance spectroscopy, two independent groups have demonstrated the feasibility of lowering tumor pHi to 6.5 or lower in rodents. Tirapazamine was used to lower the pHi of RIF-1 and SCCVII tumors in C3H/HeN mice to 6.5 (11). A combination of hyperglycemia, meta-iodobenzylguanidine, and CNCn was used to lower the pHi of DB-1 xenografts in severe combined immunodeficient mice to less than 6.4 for 30 min before returning to pretreatment levels (12). Therefore, strategies were tested that would enhance the effect of an acute acidification of a 0.2-pHe unit on the reduction of pHi and the sensitization to hyperthermia in vitro.

The MCTs carry lactate or pyruvate, together with protons, out of cells (13). The MCT-1 isoform, which has an affinity for lactate as its primary substrate (14), also has a higher activity in DB-1 melanoma cells when they are grown at pHe 6.7 (15). MCTs are quiescent at pHe above 7.0 in melanoma (15), and the higher activity at pHe 6.7 occurs because the cells are in an acidic environment. Melanoma cells are defective in terms of sodium/hydrogen antiporter function (15). They are also deficient in bicarbonate-chloride exchange (15). Because the inhibition of lactic acid transport can lower pHi and because melanoma cells do not have the usual mechanisms designed for pHi regulation, they may be particularly vulnerable to MCT inhibitors such as CNCn as a therapeutic approach (16–17). We combined CNCn with a mild extracellular acidification to test for selective 42°C sensitization of DB-1 melanoma cells grown at pHe 6.7 compared with cells grown at pHe 7.3. The results of this experimental treatment strategy on pHi, abrogation of 42°C-induction of HSPs³ (HSP70 and HSP27), and cytotoxicity are reported.

The fluorescent pH indicator, BCECF-AM, and the detergent Pluronic F127 were obtained from Molecular Probes (Eugene, OR). Type I rat tail collagen was obtained from Collaborative Biomedical Products (Bedford, MA). Fibronectin-like engineered protein polymer (Arginine-Glycine-Aspartic acid repeating peptides) and all other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

The human melanoma cell line, DB-1, was derived from a patient biopsy at Thomas Jefferson University. The melanoma (metastatic to lymph nodes) was excised before treatment. The cells were maintained in logarithmic growth at 37°C as monolayers in αMEM supplemented with glucose (1 g/liter), glutamine (0.292 g/liter), nonessential amino acids (10 ml/liter; CellGro 100X), and 10% fetal bovine serum (Atlanta Biological, Norcross, GA) in a humidified atmosphere of 5% CO₂. Cells used in this study were cultured at pHe 7.3 for 52–76 days or at pHe 6.7 for 42–121 days before heating. The pH of the αMEM was adjusted by varying the amount of sodium bicarbonate in the medium. Sodium bicarbonate was added at a concentration of 2.2 g/liter for pH 7.3, 1.29 g/liter for pH 7.1, 0.46 g/liter for pH 6.7, and 0.30 g/liter for pH 6.5. Iso-osmotic sodium levels were maintained in the reduced bicarbonate/pH medium by supplementation with NaCl (e.g., 0.60 g/liter for pH 7.1, 1.21 g/liter for pH 6.7, and 1.32 g/liter for pH 6.5. Penicillin G potassium (0.07 g/liter) and streptomycin sulfate (0.1 g/liter) were used regularly as antibiotics. DB-1 cells have a doubling time of ∼30 h at pH 7.3 and 44 h at pH 6.7.

For determination of pHi, cells were plated on 35-mm microwell plastic Petri dishes with 18-mm glass coverslips glued to 1-cm diameter holes in the center of each dish (Mattek Corp., Ashland, MA). The coverslips were coated with a 1:1 mixture of collagen I and fibronectin-like Arg-Gly-Asp polymeric repeating peptides at final concentrations of 50 μg/ml each.

Cells were incubated for 4 min with 5 μm BCECF-AM in medium at room temperature and ambient air as described previously (18–19). After a change of medium, the cells were further incubated for 20 min at 37°C, 5% CO₂ to complete hydrolysis of the dye ester and to allow recovery time from the dye loading. The Petri dish was then mounted on the temperature-controlled microscope stage for study at 37°C and/or 42°C under humidified air containing 5% CO₂.

pHi values were calculated based on data from whole excitation spectra on cells attached to substrate on an inverted microscope interfaced with a spectrofluorimeter, as has been described previously (15, 20).

An aqueous stock of CNCn was diluted to a final concentration of 5 mm in αMEM. The pH of the 5 mm solution was readjusted with HCl before use in experiments, and the pH of the solution was stable for the duration of the experiments.

Dye-loaded cells in complete medium were mounted on the microscope stage, and steady-state values for pHi were determined. The initial steady-state pHi at pHe 6.7 or 7.3 was measured several times on a field of 8–15 cells, and then the medium was replaced with medium containing inhibitors at normal or low pHe. The cells were incubated at 37°C with 5 mm CNCn at pHe 7.3, 7.1, 6.7, or 6.5, for 1 h to measure the effects on pHi. The cells were then heated at 42°C for two h on the microscope stage. Results from these experiments were always compared with experiments in which pHe was lowered in the absence of CNCn. The pHi was measured on a single field of cells three to five times every 15 min for 3 h. Results from two to five different experiments are presented.

Samples were prepared for SDS-PAGE and Western immunoblot analysis as described previously (3). Cells in 25 square cm tissue culture flasks were washed once with PBS immediately after heating, and 500 μl of SDS sample lysate buffer [62.5 mm Tris HCl, 10% glycerol, 0.5% SDS, 1 mm EDTA, 40 mm DTT, 14 mg/liter aprotinin, 0.7 mg/liter pepstatin, and 5 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride] was layered over the cells in each flask. Cell lysates were collected in 1.5-ml tubes on ice, sonicated for 5 s, boiled for 7 min, and then kept at −20°C until analysis by SDS-PAGE. Samples (10 μg/lane) were resolved on 12% SDS-PAGE gels. The proteins were transferred onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ) using a semidry transfer apparatus (Pharmacia-LKB multi-phor II) containing transfer buffer (48 mm Tris, 39 mm glycine, 20% of methanol, and 0.0376% of SDS). Immunoblotting was performed with monoclonal antibodies: antihuman HSP27 antibody (1:500 dilution), antihuman HSP70 antibody (1:1,000 dilution; StressGen Biotech Corp., Victoria, British Columbia, Canada), and mouse anti-GAPDH (1:100,000 dilution; Chemicon, Temecula, CA). Immunodetection was performed by enhanced chemiluminescence using a Tropix Western-Star protein detection kit (Applied Biosystems, Foster City, CA). The relative protein content of individual bands on X-ray film was determined by scanning laser densitometry using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The absorbance (A) obtained for each HSP band was normalized to the A of the GAPDH band in that lane and was expressed as a percentage of the respective normalized HSP band from unheated cells grown at pH 7.3.

Tissue culture flasks (25 square cm) used for survival assays contained a total of 10⁵ cells, composed of irradiated (30 Gy) Chinese hamster ovarian carcinoma feeder cells and unirradiated DB-1 cells. All of the medium in tissue culture flasks was replaced with fresh, prewarmed (37°C), CO₂-equilibrated medium at the growth pHe, or at pHe 7.1 or 6.5 with and without 5 mm CNCn, 1 h before the hyperthermic treatment. After the medium change, the flasks were gassed with 5% CO₂, plugged with sterile rubber stoppers, and returned to the CO₂ incubator. After the 1-h incubation, the flasks were placed into weighted test tube racks, and submerged in a 37°C waterbath for 5 min. The racks were then resubmerged in a second waterbath at 42°C for 2 h. The pH of the medium did not change during heating. For clonal survival assays, medium in the heated flasks was replaced with fresh, equilibrated medium at the growth pH within 15 min after heating, the caps placed onto the flasks, and the flasks placed into a CO₂ incubator for colony formation.

Statistical analyses were performed using StatXact-4 (Cytel Software Corp., Cambridge, MA). All of the Ps are based on the exact version of the nonparametric Wilcoxon/Mann-Whitney test (to account for the nonnormal measurements and the small number of observations per condition). The test essentially compares group medians rather than group means.

The steady-state pHi of DB-1 cells cultured at pH 7.3 and pH 6.7 were 7.25 and 6.94, respectively. The 0.2-pHe-unit acute acidification had negligible effects on the pHi of cells grown at pH 7.3. However, the pHi decreased to 6.70 after acute acidification of cells grown at pH 6.7. The pHi of cells that were grown at pHe 7.3, acidified to pHe 7.1, and exposed to CNCn remained above 7.0 (7.11) at 37.0°C. However, the steady-state pHi of cells grown and monitored at pHe 6.7 was reduced from 6.94 to 6.65 after 1 h in the presence of CNCn. This decrease in steady-state indicates that MCT-1 transport is constitutively active at pHe 6.7. The steady-state pHi is further reduced to 6.45 when cells grown at pHe 6.7 are exposed to CNCn at pHe 6.5.

The 42°C, 2-h heat treatment did not reduce pHi below the preheat levels in cells heated at pHe 7.3 and 7.1. The pHi was reduced from 6.94 to 6.75 in cells heated at pHe 6.7 and from pHi 6.70 to 6.52 in cells acidified to pHe 6.5 before heating. The heat treatment lowered pHi, at most, an additional 0.04-pH unit in cells pretreated with CNCn. Table 1 summarizes the results of all of the pHi studies. An example of a time course experiment is shown in Fig. 1. This figure illustrates the effects of various treatments on pHi in cells grown and heated at pHe 6.7.

Figs. 2 and 3 summarize the effects of 5 mm CNCn on the induction of HSP70 and HSP27 during the experimental treatments. The acute extracellular acidification of 0.2 pH unit with or without CNCn had little effect on HSP levels at 37°C. HSP70 and HSP27 are significantly induced during the 42°C (2-h) heat shock without CNCn at both pHe 7.3 and 6.7 (Ps < 0.03), with some indication of higher induction levels in cells grown and heated at pHe 6.7. The acute extracellular acidification of a 0.2-pH unit has little effect on HSP levels in heated cells (Ps > 0.15).

Five mm CNCn suppressed the 42°C-induction of HSP70 and HSP27 in cells grown at pHe 7.3 and heated at pHe 7.3 and 7.1. However, the reduced levels of HSPs are not statistically significant (all Ps > 0.15). Five mm CNCn further reduced the induction of HSP27 in cells grown at pHe 6.7 and heated at pHe 6.7 and at pHe 6.5, although only the latter is statistically significant (P = 0.029). However, 5 mm CNCn significantly reduced the levels of HSP70 in cells grown at pHe 6.7 and heated at pHe 6.7 (P = 0.022) and at pHe 6.5 (P = 0.008). The combination of CNCn with acute acidification to pHe 6.5 completely suppressed the heat-induction of HSP70 in cells grown at low pHe.

Fig. 4 summarizes the effects of the experimental treatments on cell survival. The acute extracellular acidification of a 0.2-pH unit with or without CNCn had little effect on survival at 37°C. None of the differences in survival in the presence or absence of CNCn are significant (Ps were always >0.3). Heating alone shows a strong effect for all conditions: the SFs are all statistically lower after 42°C compared with 37°C (Ps less than 0.05). However, the acute extracellular acidification of 0.2 pH unit had little sensitizing effect on the SF of the heated cells (Ps >0.3). Whereas the effects of CNCn on survival are much stronger in the presence of heating (Ps <0.05), CNCn did not sensitize cells grown at pHe 7.3 and heated at pHe 7.1 compared with cells heated at pHe 7.3 (P = 0.238). However, this inhibitor did have a dramatic, statistically significant effect on survival when combined with acute acidification and heating of cells grown at pHe 6.7 (SF of 0.17 versus 0.00001; P = 0.004). Fig. 4 also confirms the observation noted earlier (3) that DB-1 cells grown at pHe 6.7 are more resistant (P, 0.036) to 42°C (SF, 0.56) than cells grown at pHe 7.3 (SF, 0.17).

This study documents in vitro that an experimental treatment combining a MCT inhibitor with mild extracellular acidification can reduce pHi in melanoma cells growing in an acidic pH environment, such that they are sensitized to hyperthermia. Furthermore, the sensitization is selective for melanoma cells growing at low pH. The pHi of melanoma cells cultured at pHe 7.3 and acidified to pHe 7.1 is not lowered below 7.0 by the MCT inhibitor, and the cells are not sensitized to hyperthermia.

A comparison of the SF and pHi data indicates that lowering the pHi to 6.65 does not significantly sensitize cells growing at pHe 6.7 to 42°C hyperthermia. Significant sensitization was observed when the pHi was lowered to 6.45 before hyperthermia treatment. The cells may be sensitized to heat because induction of HSPs is prevented at this pHi. The levels of both HSP70 and HSP27 were comparable with those in unheated cells after the heat treatment when pHi was reduced to 6.45 before heating. The pHi of 6.45 was achieved by combining the MCT-1 inhibitor with acute extracellular acidification of cells growing at pH 6.7.

CNCn may have another effect on the induction of HSP70 in addition to this transport inhibitor’s ability to decrease the steady-state pHi of cells grown at low pHe. The steady-state pHi was lowered to 6.70 by acute acidification alone (pHe 6.7→pHe 6.5) and to 6.65 by exposure of cells to CNCn at the growth pHe of 6.7. However, the heat-induced levels of HSP70 were abrogated less in cells heated at pHe 6.5 in the absence of CNCn than in cells heated at pHe 6.7 in the presence of CNCn (Fig. 2). We are unaware of other modes of action of CNCn that may affect the induction of HSPs. Quercetin, another MCT inhibitor (21–22), is known to independently inhibit HSP70 expression by interfering with the binding of the heat shock factor to the heat shock element in the hsp70 promoter (23–24). Furthermore, quercetin, like CNCn, is more effective under conditions of acute acidification (25–26). It is unknown whether CNCn also interferes with the interaction of the heat shock factor and heat shock element. Regardless, statistically significant and selective heat sensitization was observed when the induction of both HSPs was inhibited. The 42°C-induction of HSP70 and HSP27 was abrogated when pHi was reduced to 6.45 before heating. In vivo studies are in progress that examine the ability of CNCn combined with hyperglycemia-induced tumor acidification and hyperthermia to enhance tumor growth delay of DB-1 melanoma xenografts.

Studies using isolated mitochondria have shown that CNCn is two orders of magnitude more potent at inhibiting pyruvate transport into mitochondria than in preventing lactate transport across the plasma membrane (13, 27). The possibility exists that CNCn may reduce pHi by inducing a large increase in lactic acid production rather than by any effect on plasma-membrane MCT activity (13, 28). However, results with 4,4′-dibenzyl-amidostilbene-2,2′-disulfonic acid, a MCT inhibitor that does not enter cells, support the proposition that the inhibition of MCT activity induces an acute intracellular acidification when combined with acute extracellular acidification (15). CNCn may reduce pHi both by increasing lactic acid production and by inhibiting plasma membrane MCT activity in melanoma cells at a low pHe.

It takes at least a month to generate clonal survival data in human melanoma cells. Therefore, we have examined various surrogates of clonal survival for use in screening potential heat-sensitizers of human melanoma cells adapted to low pHe. One of the potential determinants of heat response is the inhibition of heat-induced HSP expression. The 5-mm concentration of CNCn used in this study was selected on the basis of a preliminary screen of concentrations ranging from 1 mm to 10 mm that reduced HSP70 expression when combined with mild acute acidification before heat shock. The abrogation of induction of HSP70 correlated positively with decreased clonal survival. The results of these experiments support the use of reduction of HSP70 expression as a rapid screen for the effectiveness of various combinations of drugs, acute acidification, and heat treatments on human melanoma cells grown at low pH.

In summary, we show that a combination of 5 mm CNCn with acute acidification of melanoma cells grown at pHe 6.7 to pHe 6.5 reduced the pHi from 6.9 to less than 6.5. The induction of HSP70 and HSP27 was inhibited during the 42°C treatment, and cytotoxicity was dramatically enhanced. 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 of an effect on pHi, which remained above 7.0. Neither the 42°C-induction of HSP70 was inhibited nor was the cytotoxicity significantly enhanced. Finally, these studies confirm that MCT-1 is constitutively active in DB-1 cells grown at low pHe.

We thank Angela R. Page, Michael L. Zahaczewsky, Shari Lee, and Jenel Nixon for excellent technical assistance.