Quercetin sensitizes cells in a tumour-like low pH environment to hyperthermia.

Quercetin has been shown to act as a hyperthermia sensitizer by inhibiting the synthesis of heat shock protein 70 (HSP70) in a variety of tumour cell lines. It is most effective under conditions of low pH. This study was designed to test the hypothesis that quercetin suppresses thermotolerance development in cells adapted to growth at low pH and renders them as responsive as acutely acidified cells to hyperthermia-induced cytotoxicity. Chinese hamster ovarian carcinoma cells (OvCa) were exposed to 42 degrees C hyperthermia and/or quercetin (50-200 mm) at their growth pH of either 7.3 or 6.7 or after acute acidification from 7.3 to 6.7. Thermotolerance development was measured by colony survival. HSP70 synthesis and total protein synthesis were measured by radioactive precursor pulse labelling techniques. Quercetin, in a concentration-dependent manner, reduced the rate of total protein synthesis and increased cytotoxicity equally after acute acidification to pH 6.7 or growth at pH 6.7 at 37 degrees C, and to a greater extent than it did in cells at pH 7.3. At 42 degrees C, 100 mm quercetin inhibited total protein synthesis, HSP70 synthesis and thermotolerance development to a similar extent in cells grown at pH 6.7 or acutely acidified to pH 6.7. In contrast, quercetin reduced but did not completely inhibit HSP70 synthesis and thermotolerance development in cells grown and heated at pH 7.3. These results support the hypothesis that quercetin can specifically reduce thermotolerance development in tumour cells adapted to growth at pHe 6.7 so that they respond similarly to acutely acidified cells. Since many tumours are adapted to growth at low pH and may resist a wide variety of therapeutic modalities, inhibition of thermotolerance expression by quercetin may not only enhance the response to hyperthermia but the response to commonly used therapies such as chemotherapy and radiation.

Variability in glucose transporter-1 levels and hexokinase activity in human melanoma.

Melanoma exhibits heterogeneous growth patterns and widely varying sensitivities to multiple treatment modalities. This variability may reflect intrinsic genetic differences in factors giving rise to altered metabolism. Glucose is the primary energy source of tumours, including melanoma, and glucose transporter isoform 1 (Glut-1) and hexokinase are key rate-limiting factors in glucose metabolism. The levels of Glut-1 and total hexokinase activity were measured in 31 melanoma biopsies to determine the extent of tumour-to-tumour variability in these parameters. Relative Glut-1 levels were determined by Western immunoblot analysis using human anti-Glut-1 rabbit polyclonal antibody, and hexokinase activity was measured in the same samples by an enzymatic assay monitoring the reduction in the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) (in nmol NADP+ reduced/min per mg protein). All melanomas were from patients who had received no therapy prior to surgery. Immediately after excision, tumour biopsies were disaggregated to single cells by collagenase and DNase and frozen in liquid nitrogen. Thirty human melanomas exhibited a 22-fold variation in levels of Glut-1 and 29 exhibited a nine-fold variation in total cellular hexokinase activity. Glut-1 levels and hexokinase activity were not correlated with one another. The broad range in Glut-1 levels and hexokinase activity observed between melanomas suggests that these glycolytic rate-limiting parameters that influence the rate of glucose metabolism may contribute to the variability in melanoma response to treatment modalities.

Effects of 42 degrees C hyperthermia on intracellular pH in ovarian carcinoma cells during acute or chronic exposure to low extracellular pH.

PURPOSE: To determine whether intracellular pH (pHi) is affected during hyperthermia in substrate-attached cells and whether acute extracellular acidification potentiates the cytotoxicity of hyperthermia via an effect on pHi. METHODS AND MATERIALS: The pHi was determined in cells attached to extracellular matrix proteins loaded with the fluorescent indicator dye BCECF at 37 degrees C and during 42 degrees C hyperthermia at an extracellular pH (pHe) of 6.7 or 7.3 in cells. Effects on pHi during hyperthermia are compared to effects on clonogenic survival after hyperthermia at pHe 7.3 and 6.7 of cells grown at pHe 7.3, or of cells grown and monitored at pHe 6.7. RESULTS: The results show that pHi values are affected by substrate attachments. Cells attached to extracellular matrix proteins had better signal stability, low dye leakage and evidence of homeostatic regulation of pHi during heating. The net decrease in pHi in cells grown and assayed at pHe = 7.3 during 42 degrees C hyperthermia was 0.28 units and the decrease in low pH adapted cells heated at pHe = 6.7 was 0.14 units. Acute acidification from pHe = 7.3 to pHe = 6.7 at 37 degrees C caused an initial reduction of 0.5-0.8 unit in pHi, but a partial recovery followed during the next 60-90 min. Concurrent 42 degrees C hyperthermia caused the same initial reduction in pHi in acutely acidified cells, but inhibited the partial recovery that occurred during the next 60-90 min at 37 degrees C. After 4 h at 37 degrees C, the net change in pHi in acutely acidified cells was 0.30 pH unit, but at 42 degrees C is 0.63 pH units. The net change in pHi correlated inversely with clonogenic survival. CONCLUSIONS: Hyperthermia causes a pHi reduction in cells which was smaller in magnitude by 50% in low pH adapted cells. Hyperthermia inhibited the partial recovery from acute acidification that was observed at 37 degrees C in substrate attached cells, in parallel with a lower subsequent clonogenic survival.

Thermotolerance and intracellular pH in two Chinese hamster cell lines adapted to growth at low pH.

As an in vitro model for the low extracellular pH (pHe) which has frequently been observed in tumors, cell lines have been grown in a low-pH medium in order to allow cell adaptation to that milieu. Two Chinese hamster cell lines [Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa)] were compared, both of which acquired thermotolerance during 42 degrees C heating in pHe = 7.3 buffer, but not in pHe = 6.7 medium unless grown at that pH long enough to become adapted. CHO cells, even when acutely acidified, showed higher intracellular pH (pHi) values in a suspension assay than OvCa cells, which confirmed the danger of comparing absolute values of pHi between cell lines. Despite this fundamental difference, relative changes in pHi were similar in that both lines showed a higher pHi in adapted than in unadapted cells, over the range of pHe values tested. The upregulation of pHi was statistically significant, but the two lines differed in the time frame over which adaptation occurred. OvCa cells acquired an enhanced ability to develop tolerance to 42 degrees heat at pHe = 6.7 in 4 days, but the CHO cells acquired this ability more progressively, achieving a maximum ability at approximately 100 days. In contrast, both lines were able to upregulate their pHi within 4 hours of being exposed to pH 6.7 medium. A further indication of different biochemical mechanisms at work was the opposite effects seen on pHi in the two cell lines upon the removal of extracellular CO2/HCO3-. The differential between adapted and unadapted OvCa cells was enhanced by removal of bicarbonate, whereas CHO cells seemed less stable and the data with greater scatter failed to show any difference between adapted and unadapted cells.

Accurate whole-spectrum measurements of intracellular pH and [Na(+)].

Fluorescent measurements of intracellular H(+) and Na(+) are improved by using whole spectra of the fluorescent indicators BCECF and SBFI, respectively. The extra data in whole spectra enable both an accurate calibration and a ready detection of artifacts which are not possible to identify using a more conventional data analysis that relies upon only two wavelength "windows" in the fluorescence spectra. The whole-spectrum technique is applicable to cell suspensions in a conventional fluorimeter (as is reported here with SBFI), as well as to attached cells using a fluorimeter combined with an inverted epifluorescence microscope. The spectral method was highly reproducible in that pairs of successive pH measurements differed, on average, by only 0.01±0.02 U. Random uncertainty from sample to sample was estimated numerically from the standard deviation of measurements on ionophore-treated cells. When full-spectrum analysis was employed, this scatter showed a two-fold improvement over results obtained using the two-wavelength ratio method. Because SBFI has a relatively narrow dynamic range, whole-spectrum analysis has been applied to improve the accuracy of sodium determinations. The calibrated system measured [Na(+)]i with excellent linearity over the range 2-150 mM and with an accuracy of approximately 5 mM.