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.     

Betulinic acid sensitization of low pH adapted human melanoma cells to hyperthermia.

Betulinic acid is a known inducer of apoptosis in human melanoma that is most effective under conditions of low pH. It was hypothesized that betulinic acid, in combination with acute acidification and/or hyperthermia, would induce higher levels of apoptosis and cytotoxicity in low pH-adapted human melanoma cells than in cells grown at pH 7.3. DB-1 human melanoma cells, adapted to a tumour-like growth pH of 6.7, were exposed to hyperthermia (2h at 42 degrees C) and/or betulinic acid (4-10 microg/ml) and compared with cells grown at a physiological pH of 7.3 or after acute acidification from pH 7.3-6.3 or pH 6.7-6.3. Betulinic acid induced higher levels of apoptosis and cytotoxicity in low pH-adapted cells than in cells grown at pH 7.3, as measured by the terminal deoxynucleotidyl transferase (TdT) DNA fragmentation assay (TUNEL), the MTS cell viability assay, and single cell survival. Acute acidification of low pH adapted cells rendered them more susceptible to betulinic acid-induced apoptosis and cytotoxicity. In the presence of hyperthermia at 42 degrees C for 2 h, cells grown at pH 7.3 were not sensitized to heat killing by betulinic acid, whereas cells grown at pH 7.3 and acutely acidified to pH 6.3, cells adapted to growth at pH 6.7 and cells adapted to growth at pH 6.7 and acutely acidified to pH 6.3 were all similarly sensitized to heat killing by betulinic acid, with survival values of 5, 9 and 2%, respectively. It is concluded that betulinic acid may be useful in potentiating the therapeutic efficacy of hyperthermia as a cytotoxic agent in acidotic areas of tumours with minimal effect in normal tissues growing at pH 7.3.     

Acute extracellular acidification increases nuclear associated protein levels in human melanoma cells during 42°C hyperthermia and enhances cell killing

Acute acidification is being investigated as a strategy to sensitize human melanoma to 42°C hyperthermia. The present study was conducted to determine the effect of hyperthermia and acute extracellular acidification on the nuclear associated protein (NAP) levels, heat shock protein (hsp) 70 and hsp27 content, and cell survival of human melanoma cells cultured at pH 7.3 or pH 6.7. It was observed that NAP levels increased slightly in both populations after 2h of heating and then decreased to control levels with increasing time of heating at the growth pH. However, the NAP levels continued to increase in cells acutely acidified to pH 6.3 prior to and during heating. Hsp70 was induced to comparable levels in cells heated at their growth pH; however, the hsp27 levels were greater in cells cultured and heated at pH 6.7 than in cells cultured and heated at pH 7.3. Acute acidification to pH 6.3 prior to and during heating suppressed the 42°C induction of hsp70 and hsp27 in both cell populations. The melanoma cells cultured and heated at pH 6.7 were more resistant to cell killing than cells cultured and heated at pH 7.3. Both populations were sensitized to cell killing by acute acidification to pH 6.3. The results suggest that hsps induced during 42°C treatment associate with aggregating NAPs, enhancing their detergent solubility, and that abrogation of induced expression of hsps during heating at pH 6.3 contributes to increased levels of insoluble NAPS. In conclusion, acute extracellular acidification inhibits 42°C induction of hsps, increases NAP levels, and decreases cell survival in DB-1 human melanoma cells.     

Hsp27 protects the cytoskeleton and nucleus from the effects of 42°C at pH 6.7 in CHO cells adapted to growth at pH 6.7

CHO cells are normally sensitized to hyperthermia by acidic pH. However, CHO cells adapted to growth in pH 6.7 medium become less sensitive to heat killing at the reduced pH. The adapted cells maintain their ability to develop thermotolerance at pH 6.7 and their steady state intracellular pH is elevated. Furthermore, the small molecular weight stress chaperone, hsp27, is elevated in unheated cells maintained at pH 6.7. This report documents that the cytoskeletal and nuclear components of the low pH adapted CHO cells are resistant to 42°C-induced collapse and protein accretion, respectively. Hyperthermia induced a perinuclear collapse of the microtubular cytoskeleton and an increase in the amount of insoluble protein associated with the nuclei and nuclear matrix fractions in the control cells heated at pH 7.3 or heated after acute acidification to pH 6.7. Protection from these effects was observed in the low pH adapted cells heated at pH 6.7. Hsp70 does not appear to play a dominant role in the response of the adapted cells to 42°C. The induction of hsp70 during heating is abrogated by pH 6.7 in cells cultured at either pH 7.3 or pH 6.7. The resistance of the microtubular cytoskeleton to perinuclear collapse and the absence of protein aggregation in the nucleus during 42°C may be due to the elevated levels of hsp27 both before heating and during the heat treatment. In summary, the phenotype of CHO cells adapted to growth at low pH includes resistance of the cytoskeleton to 42°C-induced perinuclear collapse and resistance to 42°C-induced aggregation of nuclear proteins, in addition to the reduction in heat cytotoxicity, upregulation of intracellular pH and upregulation of hsp27.     

Acute extracellular acidification increases nuclear associated protein levels in human melanoma cells during 42 degrees C hyperthermia and enhances cell killing.

Acute acidification is being investigated as a strategy to sensitize human melanoma to 42 degrees C hyperthermia. The present study was conducted to determine the effect of hyperthermia and acute extracellular acidification on the nuclear associated protein (NAP) levels, heat shock protein (hsp) 70 and hsp27 content, and cell survival of human melanoma cells cultured at pH 7.3 or pH 6.7. It was observed that NAP levels increased slightly in both populations after 2 h of heating and then decreased to control levels with increasing time of heating at the growth pH. However, the NAP levels continued to increase in cells acutely acidified to pH 6.3 prior to and during heating. Hsp70 was induced to comparable levels in cells heated at their growth pH; however, the hsp27 levels were greater in cells cultured and heated at pH 6.7 than in cells cultured and heated at pH 7.3. Acute acidification to pH 6.3 prior to and during heating suppressed the 42 degrees C induction of hsp70 and hsp27 in both cell populations. The melanoma cells cultured and heated at pH 6.7 were more resistant to cell killing than cells cultured and heated at pH 7.3. Both populations were sensitized to cell killing by acute acidification to pH 6.3. The results suggest that hsps induced during 42 degrees C treatment associate with aggregating NAPs, enhancing their detergent solubility, and that abrogation of induced expression of hsps during heating at pH 6.3 contributes to increased levels of insoluble NAPS. In conclusion, acute extracellular acidification inhibits 42 degrees C induction of hsps, increases NAP levels, and decreases cell survival in DB-1 human melanoma cells.     

Hsp27 protects the cytoskeleton and nucleus from the effects of 42 degrees C at pH 6.7 in CHO cells adapted to growth at pH 6.7.

CHO cells are normally sensitized to hyperthermia by acidic pH. However, CHO cells adapted to growth in pH 6.7 medium become less sensitive to heat killing at the reduced pH. The adapted cells maintain their ability to develop thermotolerance at pH 6.7 and their steady state intracellular pH is elevated. Furthermore, the small molecular weight stress chaperone, hsp27, is elevated in unheated cells maintained at pH 6.7. This report documents that the cytoskeletal and nuclear components of the low pH adapted CHO cells are resistant to 42 degrees C-induced collapse and protein accretion, respectively. Hyperthermia induced a perinuclear collapse of the microtubular cytoskeleton and an increase in the amount of insoluble protein associated with the nuclei and nuclear matrix fractions in the control cells heated at pH 7.3 or heated after acute acidification to pH 6.7. Protection from these effects was observed in the low pH adapted cells heated at pH 6.7. Hsp70 does not appear to play a dominant role in the response of the adapted cells to 42 degrees C. The induction of hsp70 during heating is abrogated by pH 6.7 in cells cultured at either pH 7.3 or pH 6.7. The resistance of the microtubular cytoskeleton to perinuclear collapse and the absence of protein aggregation in the nucleus during 42 degrees C may be due to the elevated levels of hsp27 both before heating and during the heat treatment. In summary, the phenotype of CHO cells adapted to growth at low pH includes resistance of the cytoskeleton to 42 degrees C-induced perinuclear collapse and resistance to 42 degrees C-induced aggregation of nuclear proteins, in addition to the reduction in heat cytotoxicity, upregulation of intracellular pH and upregulation of hsp27.     

Sensitization of human Ewing's tumor cells to chemotherapy and heat treatment by the bioflavonoid quercetin

BACKGROUND: The bioflavonoid quercetin, a polyphenolic compound widely distributed in the plant kingdom, has been demonstrated to exert cytostatic activity against a variety of tumor cells in vitro and in vivo. It may be useful in cancer therapy as a thermosensitizer by increasing the cell killing effect of hyperthermia and chemotherapy because of its ability to suppress heat-shock protein expression. MATERIALS AND METHODS: We investigated the effect of quercetin combined with two cytotoxic agents, cDDP (cis-diamminedichloroplatinum II) and VP-16 (etoposide), under various heat-shock conditions in two Ewing's tumor cell lines SK-ES-1 and RD-ES, using XTT-assay and Western blot analysis. RESULTS: Induction of thermotolerance by a sublethal heat-shock (42 degrees C, 1 hour) led to a transient resistance against subsequent heat treatment alone or combined thermochemotherapy with the crosslinking agent cDDP or the topoisomerase II inhibitor VP-16. Quercetin (> or = 50 microM) applied for 24 hours inhibited cell proliferation, increased the cytotoxic activity of cDDP or VP-16 alone or combined with simultaneous hyperthermia and suppressed the development of thermotolerance. Hyperthermia (43 degrees C, 45 degrees C for 1 hour) induced high expression of the inducible form of HSP70, whereas HSP27, which is constitutively expressed at normothermic conditions, is only slightly induced by 43 degrees C and nearly completely suppressed at 45 degrees C. Induction of thermotolerance is accompanied by an elevated expression of both HSP70 and HSP27. Quercetin (> or = 50 microM), alone as well as in combination with thermochemotherapy, inhibited the expression of both HSP70 and HSP27. CONCLUSION: These data suggest that the bioflavonoid quercetin potentially may be useful in clinical trials for optimizing the efficacy of hyperthermia in combination with chemotherapy.     

Quercetin, an inhibitor of heat shock protein synthesis, inhibits the acquisition of thermotolerance in a human colon carcinoma cell line.

Here, we describe the effects of quercetin on the induction of thermotolerance as examined by colony forming assay in a cell line derived from human colon carcinoma (COLO320 DM). Cells became resistant to heat treatment at 45 degrees C when they were preheated at 42 degrees C for 1.5 h or at 45 degrees C for 10 min. This induction of thermotolerance was almost completely inhibited by continuous treatment with 100 microM quercetin during the first and second heating sessions, and the interval between. This effect of quercetin was demonstrated to be dose-dependent over a concentration range of 50-200 microM. Quercetin did not increase the thermosensitivity of non-tolerant cells. The presence of quercetin during the first conditioning heating was more effective in inhibiting thermotolerance than its presence during the second heating. Quercetin was also found to inhibit the acquisition of thermotolerance induced by sodium arsenite. Cycloheximide, a nonspecific inhibitor of protein synthesis, did not affect the acquisition of thermotolerance by the same cell line. Quercetin specifically inhibits the synthesis of all heat shock proteins so far reported previously, and this leads to inhibition of the induction of thermotolerance. Such inhibition of thermotolerance by quercetin may improve the efficacy of clinical fractionated hyperthermia.     

Quercetin, an Inhibitor of Heat Shock Protein Synthesis, Inhibits the Acquisition of Thermotolerance in a Human Colon Carcinoma Cell Line

Here, we describe the effects of quercetin on the induction of thermotolerance as examined by colony forming assay in a cell line derived from human colon carcinoma (COLO320 DM). Cells became resistant to heat treatment at 45°C when they were preheated at 42°C for 1.5 h or at 45°C for 10 min. This induction of thermotolerance was almost completely inhibited by continuous treatment with 100 μM quercetin during the first and second heating sessions, and the interval between. This effect of quercetin was demonstrated to be dose-dependent over a concentration range of 50 200 μM, Quercetin did not increase the thermosensitivity of non-tolerant cells. The presence of quercetin during the first conditioning heating was more effective in inhibiting thermotolerance than its presence during the second heating. Quercetin was also found to inhibit the acquisition of thermotolerance induced by sodium arsenite. Cycloheximide, a nonspecific inhibitor of protein synthesis, did not affect the acquisition of thermotolerance by the same cell line, Quercetin specifically inhibits the synthesis of all heat shock proteins so far reported previously, and this leads to inhibition of the induction of thermotolerance. Such inhibition of thermotolerance by quercetin may improve the efficacy of clinical fractionated hyperthermia.     

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

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°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°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 (pH_i) of cells grown at pH 6.7 was less than the pH_i of cells grown at pH 7.3 both before and after acute acidification. A pH_i threshold existed for melanoma cells growing at pH 7.3 below which they became sensitized to 42°C. This pH_i threshold differed between the SK-Mel-28 and DB-1 cells. In contrast, a pH_i threshold for heat sensitization did not exist for cells growing at pH 6.7: any reduction in pH_i before heating resulted in increased cell killing. Since cells grown at low pH lack a pH_i threshold for heat sensitization, they are sensitized more to 42°C per unit decrease in pH_i than cells grown at pH 7.3. Acute acidification abrogated the 42°C-induction of Hsp70 and Hsp27 in the melanoma cells. The pH_i 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 pH_i 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 pH_i in melanoma cells growing at low pH, such as in acidotic regions of tumours, could selectively sensitize them to hyperthermia because they lack a pH_i threshold for heat sensitization.     

Defect in the development of thermotolerance and enhanced heat shock protein synthesis in the mouse temperature-sensitive mutant ts85 cells upon moderate hyperthermia

The effect of exposure to moderate hyperthermia on the induction of thermotolerance and heat shock protein (HSP) synthesis was investigated using mouse FM3A cells and the temperature-sensitive mutant ts85 cells. The thermal sensitivity of the two cell lines was markedly different; the mutant ts85 cells were more sensitive than the parental wild-type FM3A cells to heating at 41 and 44°C. The shift-up treatment of FM3A cells for 3 h at 39 5°C from 33°C induced thermotolerance development to subsequent heating at 44°C, with little if any enhancement of major HSP synthesis. On the other hand, the similar treatment of ts85 cells at the non-permissive temperature of 39 5°C induced significantly enhanced HSP synthesis, but could not induce thermotolerance. The exposure to 41°C also induced thermotolerance in the wild-type cells, but failed to induce tolerance in the mutant ts85 cells. These results suggest that enhanced major-HSP synthesis is neither a sufficient or necessary condition for thermotolerance development upon moderate heat shock. The mechanism of thermotolerance is discussed by relating the observed defect in thermotolerance development to the known defect in ubiquitindependent protein degradation system of the mutant ts85 cells at non-permissive temperature.     

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

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.     

Thermotolerance in normal and tumor tissues

Thermotolerance, or increased resistance of tissues to hyperthermia following a prior heat treatment, has been demonstrated not only in normal but also in tumor tissues. The kinetics of thermotolerance depends on the prior heating conditions, i.e. the greater the initial heat damage, the greater is the magnitude of thermotolerance and the longer is the time to reach maximum and the time to decay. Although thermotolerance develops less extensively in cultured cells in low pH medium and average tumor tissue pH is lower than the normal tissue pH, the magnitude of thermotolerance in tumor thus far examined is at least equal to that in the normal tissues. It is known that development of thermotolerance is well correlated with enhanced synthesis of heat shock proteins (HSPs). Recently, we purified HSP 70 (70 kilo dalton HSP) and prepared a rabbit antiserum against the HSP 70. In our preliminary experiments, HSP 70 could be detected and analyzed quantitatively not only in cultured cells but also in normal and tumor tissues by immunoblotting using the antiserum.     

Defect in the development of thermotolerance and enhanced heat shock protein synthesis in the mouse temperature-sensitive mutant ts85 cells upon moderate hyperthermia

The effect of exposure to moderate hyperthermia on the induction of thermotolerance and heat shock protein (HSP) synthesis was investigated using mouse FM3A cells and the temperature-sensitive mutant ts85 cells. The thermal sensitivity of the two cell lines was markedly different; the mutant ts85 cells were more sensitive than the parental wild-type FM3A cells to heating at 41 and 44 degrees C. The shift-up treatment of FM3A cells for 3 h at 39.5 degrees C from 33 degrees C induced thermotolerance development to subsequent heating at 44 degrees C, with little if any enhancement of major HSP synthesis. On the other hand, the similar treatment of ts85 cells at the non-permissive temperature of 39.5 degrees C induced significantly enhanced HSP synthesis, but could not induce thermotolerance. The exposure to 41 degrees C also induced thermotolerance in the wild-type cells, but failed to induce tolerance in the mutant ts85 cells. These results suggest that enhanced major-HSP synthesis is neither a sufficient or necessary condition for thermotolerance development upon moderate heat shock. The mechanism of thermotolerance is discussed by relating the observed defect in thermotolerance development to the known defect in ubiquitin-dependent protein degradation system of the mutant ts85 cells at non-permissive temperature.     

槲皮素提高肝癌HepG2细胞热化疗疗效的实验研究

  目的 研究热休克对人肝癌HepG2细胞耐药性的影响，合用槲皮素（Qu）能否提高肝癌细胞热化疗的疗效。方法 42 ℃恒温水浴法热休克传代人肝癌细胞系HepG2细胞90 min。MTT检测半数抑制浓度（IC50），求得耐药倍数、增敏倍数。荧光染色检测凋亡率。流式细胞术检测HSP70和P-gp表达率。结果 Qu能诱导HepG2细胞凋亡。HepG2细胞热休克诱导后4 h对ADM耐药性升高2.78倍（P＜ 0.05），HSP70阳性细胞数升高，4 h达到11.47 ％，升高近1倍（P ＜0.01），P-gp阳性细胞数12 h达到96.31 ％，升高近2倍（P ＜0.01）。热休克前应用Qu能有效抑制热休克诱导HSP70和P-gp的过量表达，抑制细胞对ADM耐药性的产生，起增敏作用（P ＜0.05），呈浓度依赖性。结论 槲皮素可阻断热休克诱导HepG2细胞HSP70和P-gp的过表达，抑制耐药性的产生，起到热化疗的增敏作用，可成为肿瘤耐药逆转剂。     

Hsp27 anti-sense oligonucleotides sensitize the microtubular cytoskeleton of Chinese hamster ovary cells grown at low pH to 42°C-induced reorganization

Chinese hamster ovary (CHO) cells maintained in vitro at pH 6.7 were used to model cells in the acidic environment of tumours. CHO cells grown at pH 6.7 develop thermotolerance during 42°C heating at pH 6.7 and their cytoskeletal systems are resistant to 42°C-induced perinuclear collapse. Hsp27 levels are elevated in cells grown at pH 6.7 and are further induced during 42°C heating, while Hsp70 levels remain low or undetectable, suggesting that Hsp27 is responsible for some of the novel characteristics of these cells. An anti-sense oligonucleotide strategy was used to test the importance of Hsp27 by lowering heat-induced levels of the protein. The response of the microtubular cytoskeleton to heat was used as an endpoint to assess the effectiveness of the anti-sense strategy. Treatment with anti-sense oligonucleotides prevented the heat-induced increase of Hsp27 levels measured immediately following heat. Treatment with anti-sense oligonucleotides also sensitized the cytoskeleton of cells grown at low pH to heat-induced perinuclear collapse. However, cytoskeletal collapse was not evident in cells grown at pH 6.7 and treated with 4-nt mismatch oligonucleotides or in control cells maintained and heated at pH 6.7. The cytoskeleton collapsed around the nucleus in cells cultured and heated at pH 7.3. These results confirm that over-expression of Hsp27 confers heat protection to the microtubular cytoskeleton in CHO cells grown at low pH.     

Influence of induction of HSP70 on the cytotoxicity of oxazaphosphorine compounds and cisplatin

Induction of HSP70 and thermotolerance may also decrease the cytotoxicity of cytostatic agents or their combination with hyperthermia in clinically used thermochemotherapy. HSP70 and thermotolerance were induced by hyperthermia (42 degrees C, 1 h) in two human tumor cell lines in vitro and in vivo. The influence of thermotolerance on the cytotoxicity of CDDP and the oxazaphosphorine compounds Mafo and Ifo and their combination with hyperthermia (42 degrees C or 43 degrees C, 1 h) were studied. The results show that neither thermotolerance nor HSP70 affects the tumor cell sensitivity to CDDP or oxazaphosphorine compounds. However, the additive effect of hyperthermia and CDDP was found to be attenuated in thermotolerant cells. The cytotoxicity of oxazaphosphorine compounds combined with hyperthermia was not altered after preheating, suggesting a different mechanism may be responsible for the drug-hyperthermia interaction of CDDP and oxazaphosphorine compounds. There were no differences between in vitro and in vivo results suggesting mechanisms at the cellular level being responsible for the influence of thermotolerance on drug- and drug-hyperthermia action.     

Influence of low pH on the development and decay of 42°C thermotolerance in cho cells

The development, magnitude and decay of thermotolerance in Chinese hamster ovary cells heated to 42°C at pH 6.7 was examined. The cells were exposed to single or fractionated heat treatments with 0 to 168 hours elapsing between the treatments. Administration of a specified heat treatment in two fractions at low pH substantially reduced the lethal effect of heat. The rate of hyperthermic cell killing was reduced by a factor of approximately 2.5 in preheated cells compared to cells not receiving prior heat treatment. Tolerance to second heat treatments was apparent when 3 hours at 37°C separated the treatments, and was near maximum when 6 hours separated the treatments. Beginning about 96 hours after the initial heat treatment, resistance to second heat treatments began to decline, and was not evident when 168 hours at 37°C separated the treatments. In contrast to these results, a sparing effect as a result of dose fraction was not observed at pH 7.4. At pH 7.4, cells developed thermotolerance during the initial heat treatment and additional culturing at 37°C was without additional effect. Nevertheless, the sensitivity of cells to hyperthermia was greater at pH 6.7 than at pH 7.4. This pH sensitizing effect was more pronounced in cells exposed to single heat treatments than in cells exposed to fractionated treatments at 42°C.     

Effects of the flavonoid drug quercetin on the response of human prostate tumours to hyperthermia in vitro and in vivo.

Tumour hyperthermia, although potentially a powerful therapeutic agent and radiation sensitizer, is hindered by a number of considerations including inhomogeneous heating of deep seated tumours due to energy deposition and perfusion issues. One solution is to design hyperthermia sensitizers to amplify the effects of hyperthermia, particularly at cold spots within the tumour undergoing treatment. This study examined the use of Quercetin, a flavonoid drug shown previously to antagonize the expression of HSP72 and induce apoptosis as a sensitizer of prostate cancer growth in vivo. Quercetin dose-dependently suppressed PC-3 tumour growth in vitro and in vivo. When combined in a treatment protocol with hyperthermia, quercetin drastically inhibited tumour growth and potently amplified the effects of hyperthermia on two prostate tumour types, PC-3 and DU-145 in vivo. These experiments, thus, suggest the use of Quercetin as a hyperthermia sensitizer in the treatment of prostate carcinoma.     

Effect of cycloheximide or puromycin on induction of thermotolerance by heat in Chinese hamster ovary cells: dose fractionation at 45.5 degrees C1

While studying the quantitative relationship between hyperthermia-induced heat shock proteins (HSPs) and thermotolerance (TT), we observed that heat induced a family of HSPs, particularly an HSP 70 family, that might be involved in the development of TT. When cells were heated for 10 min at 45.5 degrees C, they became thermotolerant to a second heat exposure at 45.5 degrees C, with a thermotolerance ratio of 5-6 at 10(-3) isosurvival at 12 h after heating. In parallel, during the 12-h interval, heat shock resulted in a 2-fold relative increase in the synthesis of three major HSP families (Mr = 110,000, 87,000, and 70,000). Rate of synthesis was expressed relative to total protein synthesis, as studied with one-dimensional polyacrylamide gels analyzed by counting radioactivity in selected protein bands. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. Furthermore, even though the development of TT was partially suppressed by treatment with cycloheximide (10 micrograms/ml) or puromycin (100 micrograms/ml) at concentrations that inhibited total protein synthesis by 96 or 99%, respectively, a family of HSP 70 was still preferentially synthesized. Nevertheless, when cells were labeled for 3 days, the total level of HSP families did not change either when TT developed after a triggering heat treatment or as the development of TT was partially inhibited by suppressing protein synthesis with cycloheximide or puromycin. Thus, TT could still occur when total levels of HSP families did not change and when synthesis of HSP families was less than in unheated control cells, which may imply that TT is unrelated to HSPs. However, the finding that the amount of TT increased with increased synthesis of both total protein and HSP families, as studied with different concentrations of cycloheximide or puromycin, suggests that heat-inducible proteins, in particular the observed preferential synthesis of the HSP 70 family, may be necessary for the development of TT.