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.     

Effects of pre-heating on cis-diamminedichloroplatinum(II)-hyperthermia-induced tumour growth depression of transplantable human oesophageal cancer to nude mice

The effects of the combination of cis-diamminedichloroplatinum(II) (CCDP) and hyperthermia on tumour growth were examined using transplantable human oesophageal cancer (ESO-2), having a histological type of moderately differentiated squamous cell carcinoma, in nude mice. Eighteen days after the inoculation of the tumour fragment into the subcutaneous tissue of the right hind foot, the treatment of CDDP and/or hyperthermia was performed and the antitumour effect was evaluated 21 days after the treatment. The combination of 4 mg/kg of CDDP and 43?C heating for 30 min effectively depressed tumour growth in comparison with the individual treatment. The mean relative tumour weight of the combination group at 3 weeks after the treatment was 15% of that of the control group without treatment. On the other hand, pre-heating at 42?C for 30 min did not influence the inhibition of tumour growth by CDDP alone or the concentration of CDDP in tumour. When pre-heating at 42?C for 30 min was performed at 6 or 12 h prior to the combined treatments of 2 mg/kg of CDDP and 43?C hyperthermia for 30 min, however, tumour growth depression by CDDP-hyperthermia was diminished. When pre-heating was performed 4 days prior to CDDP-hyperthermia, however, tumour growth depression occurred. These results showed that thermotolerance of tumour cells induced by pre-heating diminished hyperthermic potentiation for cytotoxicity of CDDP, though the thermotolerance did not affect the cytotoxicity of CDDP.     

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.     

Cytotoxicity of hyperthermia combined with bleomycin or cis-platinum in cultured RIF cells: modification by thermotolerance and by polyhydroxy compounds

The effect of thermotolerance and of polyhydroxy compounds on the cytotoxicity of bleomycin and cis-platinum was studied in cultured RIF tumor cells. Cell survival in response to drug-heat treatments was compared in cells not previously exposed to hyperthermia and in preheated cells that had developed thermotolerance. Since cellular accumulation of polyhydroxy compounds is a potential mechanistic basis of thermotolerance, we also compared cell survival of thermotolerant cells and chemically heat-protected cells. The cytotoxicity of bleomycin and cis-platinum in control cells treated with drug plus heat (43 degrees C, 1 h) was increased synergistically over the cytotoxicity of drug and heat alone. In thermotolerant cells, the synergistic interaction was largely reversed with the bleomycin-heat combination but retained with cis-platinum at 43 degrees C. In the absence of heat, bleomycin and cis-platinum showed similar cytotoxicity in control and thermotolerant cells. The addition of heat protectors (erythritol or galactose) modified the drug-heat cytotoxicity similar to thermotolerance. The synergistic interaction of bleomycin-43 degrees C, but not cis-platinum-43 degrees C, was reversed by the polyhydroxy compounds.     

Effect of hyperthermia on cis-diamminedichloroplatinum(II) (rhodamine 123)2[tetrachloroplatinum(II)] in a human squamous cell carcinoma line and a cis-diamminedichloroplatinum(II)-resistant subline

The effect of concomitant hyperthermia on the cytotoxicities of cis-diamminedichloroplatinum(II) (CDDP), a newly synthesized drug, Pt(Rh-123)2, and its chemical components, K2PtCl4 and rhodamine 123, was examined in vitro in a squamous cell tumor line of human origin (SCC-25) and in a CDDP-resistant subline (SCC-25/CP). No difference in the cytotoxicity of hyperthermia alone was observed between these cell lines. The dose-dependent cytotoxicities of 1-h exposures to CDDP and Pt(Rh-123)2 were markedly increased at 42 degrees C and 43 degrees C in comparison to 37 degrees C, and this effect was of the same magnitude in both cell lines (enhancements of approximately 1.5 logs at 42 degrees C and 2.5 logs at 43 degrees C for CDDP and 1.5 logs at 42 degrees C and greater than 3 logs at 43 degrees C for Pt(Rh-123)2). The use of hyperthermia with CDDP, however, did not lower survivals in the SCC-25/CP cells even to the levels seen in the parent line at 37 degrees C. The cytotoxicities of K2PtCl4 and rhodamine 123 were essentially the same in the CDDP-sensitive and -resistant cells at all temperatures tested. The magnitude of the temperature effect was significantly greater for Pt(Rh-123)2 than for its chemical components. No significant effect on CDDP or Pt(Rh-123)2 accumulation was observed at 42, 43, 44 or 45 degrees C in either cell line. DNA lesions, measured by alkaline elution, were significantly enhanced for CDDP in the SCC-25 cells at 42 degrees C. These results suggest that treatment with hyperthermia and either CDDP or Pt(Rh-123)2 should result in supraadditive anti-tumor effects, although the efficacy of CDDP plus hyperthermia will be significantly less once resistance to CDDP has developed. Since resistance to CDDP does not imply cross-resistance to Pt(Rh-123)2, and since the effect of hyperthermia is somewhat greater for Pt(Rh-123)2 than for CDDP at 43 degrees C, Pt(Rh-123)2 may be more selectively toxic to tumor cells when used with local hyperthermia versus normal cells outside the treated area, especially if resistance to CDDP has already developed.     

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°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°C, and to a greater extent than it did in cells at pH 7.3. At 42°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 pH e 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.     

Interaction of platinum complexes of thiazin and xanthene dyes with hyperthermia

Summary In an attempt to develop platinum-containing drugs for use with hyperthermia that would be relatively nontoxic at 37° C but would become very cytotoxic at 42° or 43° C, several nuclear dyes were complexed to the tetrachloroplatinum(II) dianion (PtCl₄) at a ratio of 2:1. The cytotoxicity of PtCl₄ complexes of three thiazin dyes (thionin, azure B, and methylene blue), the xanthene dye pyronin Y, and the thiazole dye thioflavin was examined in exponentially growing euoxic and hypoxic EMT6 cells in vitro at 37°, 42°, and 43° C and at pH 7.40 and 6.45. Of the thiazin dye complexes, the cytotoxicity of Pt(methylene blue)₂ was most enhanced at hyperthermic temperatures. Both Pt(pyronin Y)₂ and Pt(thioflavin)₂ also became markedly more cytotoxic at 42° and 43° C at pH 6.45 vs pH 7.40. In vivo tumor excision assays in the FSaIIC fibrosarcoma showed that with each of the thiazin dye-platinum complexes, hyperthermia enhanced cell kill [most effectively on Pt(methylene blue)₂] but was not dose-modifying. For both Pt(pyronin Y)₂ and Pt(thioflavin)₂, however, administration of 43° C, 30-min hyperthermia to the tumor immediately after i.p. drug injection was dose-modifying. Tumor growth delay studies in the FSaIIC tumor system demonstrated that, as with the in vitro studies, Pt(pyronin Y)₂ and Pt(methylene blue)₂ were most enhanced by hyperthermia [tumor growth delay increased by 4.8- and 3.0-fold, respectively, vs only 1.3-fold for cisplatin (CDDP)]. Examination of intracellular platinum levels after exposure of EMT6 cells to 25 μM of drug for 1 h at 37° and 42° C and at pH 7.40 and 6.45 showed that each platinum-dye complex achieved platinum levels that were 100–600 times higher at 37° C and pH 7.40 than those obtained using CDDP. The platinum levels for each drug dropped markedly when exposure took place at pH 6.45. Exposure at 42°C only moderately increased platinum levels in cells exposed to these drugs. Thus, for several of these drugs the level of cytotoxicity observed was in great part independent of the intracellular platinum levels achieved. Pt(pyronin Y)₂ is an effective drug for use with hyperthermia, and further studies using this combination with and without radiation are under way. This work was supported by NCI grants ROI-CA47379 and ROI-CA36508     

Addition of mitomycin C to cis-diamminedichloroplatinum(II)/hyperthermia/radiation therapy in the FSaIIC fibrosarcoma.

Hyperthermia (temperatures greater than or equal to 42 degrees C) is used clinically to improve the effectiveness of radiation therapy and, although therapeutic gains have been reported, efficacy is limited when tumours are large and/or radiation tolerance is reduced. In order to improve the utility of the hyperthermia/radiation combination we have tested the addition of cisplatin (CDDP) in the laboratory and in the clinic. Our clinical studies have shown that the CDDP/hyperthermia/radiation combination is tolerable and effective, but laboratory investigations demonstrated a relative lack of cytotoxicity in the hypoxic tumour subpopulation. In order to improve the effectiveness of the CDDP/hyperthermia/radiation combination against hypoxic cells we have evaluated the addition of mitomycin C, a hypoxic cell cytotoxic agent to this combination. Mitomycin C (5 mg/kg) i.p. produced a tumour growth delay (TGD) of about 5.3 days in the FSaIIC murine fibrosarcoma; hyperthermia (43 degrees C x 30 min) caused only about 1.4 day TGD and the combination of mitomycin C followed immediately by hyperthermia caused a TGD of about 8.6 days. CDDP (5 mg/kg) i.p. followed by hyperthermia and then 3 Gy on day 1 only of a 5 day x 3 Gy radiation protocol produced a TGD of about 25 days. With the addition of mitomycin C just before CDDP a TGD of about 44 days resulted. Whole tumour excision experiments demonstrated that mitomycin C was highly interactive with CDDP at 37 degrees C and was dose-modifying. When used with CDDP and hyperthermia, however, mitomycin C added little additional cytotoxicity. Hoechst 33342 dye diffusion-determined tumour subpopulation studies indicated a marked effect of the addition of mitomycin C in the dim (enriched in hypoxic cells) subpopulation and nearby equal cytotoxicity in both bright (enriched in euoxic cells) and dim cells resulted. These investigations suggest considerable potential therapeutic efficacy to the addition of mitomycin C to the CDDP/hyperthermia/radiation combination.     

Kinetics and possible mechanism of thermotolerance: a review

Thermotolerance examined in vitro studies was reviewed in related to their kinetics, modifying factors and also the possible mechanisms. Heat treatments which reduce the survivals at same level at different temperature 42, 43, 44, 45 degrees C induces a same degree of thermotolerance and the thermotolerance decayed in same manner. These findings showed that thermotolerance induced by acute hyperthermia and that by chronic hyperthermia are not different in the nature. Examinations of heat sensitivities and kinetics of thermotolerance among different cell lines indicated that there is a possible relationship between the cellular sensitivity to heat and the magnitude of thermotolerance. The HSP has been found to be the most likely related factor in thermotolerance. But, their functions have not yet been clearly observed. Further research on HSP and thermotolerance is necessary to elucidate the possible mechanism of thermotolerance development.     

Combined modality therapy with bleomycin, hyperthermia, and radiation

In an attempt to develop better combination therapies for use with local radiation, the interaction between bleomycin and hyperthermia +/- radiation was studied in the FSaIIC tumor system. In cells exposed in vitro to bleomycin at 37 degrees C and at pH 7.40, the drug was substantially more toxic toward normally oxygenated than hypoxic cells. At hyperthermic temperatures (42 degrees or 43 degrees C), however, the differential killing between the normally oxygenated and hypoxic cells disappeared and bleomycin became significantly more toxic. Exposure to bleomycin at pH 6.45 did not substantially alter the cytotoxicity of the drug at 42 degrees or 43 degrees C. In tumor growth delay experiments, combining bleomycin, hyperthermia, and radiation induced long delays, and the more successful sequences were bleomycin----radiation----hyperthermia or bleomycin----hyperthermia----radiation. If radiation was given prior to drug and hyperthermia, however, the sequence was significantly less effective. In tumor excision experiments performed 24 h after treatment, increasing doses of bleomycin produced a shallow, log-linear increase in tumor cell kill at 37 degrees C, but bleomycin followed by hyperthermia (43 degrees C, 30 min) led to about 1 log more cell killing. Administration of bleomycin just prior to treatment with a single dose of radiation was cytotoxically additive. In this assay the most effective trimodality treatment sequence was bleomycin----hyperthermia----radiation. In tumor subpopulations defined by Hoechst 33342 dye staining, bleomycin at 37 degrees C was about two-fold more toxic toward the bright (presumably well-oxygenated) cells than toward the dim (presumably hypoxic) cell subpopulation. The addition of hyperthermia following bleomycin produced nearly a log more tumor cell killing in both the bright and dim tumor cells. The combination of bleomycin followed by hyperthermia and then radiation was at least additive in the bright cells and caused a large cell kill, but in comparison, there was marked sparing of the dim cells. These results indicate that treatment with bleomycin and hyperthermia in conjunction with radiation can add substantially to tumor cell killing. This combination is significantly less effective in the hypoxic than oxic tumor regions, however, in spite of in vitro data which demonstrate that the cytotoxicity of bleomycin at hyperthermic temperatures is not oxygen-dependent.     

Enhancement of cisplatin sensitivity and platinum uptake by 40 degrees C hyperthermia in resistant cells

We studied the cytotoxic and pharmacological properties of 40 degrees C hyperthermia and CDDP in CDDP-sensitive (IMC-3) and CDDP-resistant (IMC-3-DDP) human maxillary carcinoma cells. Heating at 40 degrees C alone caused almost no cell killing to IMC-3 and IMC-3-DDP cells. In both cell lines, the dose-dependent cytotoxicity of 2-h exposures to CDDP was increased at 40 degrees C in comparison to 37 degrees C. Heating at 40 degrees C also potentiated CDDP cytotoxicity in both IMC-3 and IMC-3-DDP cells with thermal chemoenhancement ratios (CER) of 1.48 and 1.94, respectively. The intracellular CDDP uptake level of IMC-3-DDP at 37 degrees C was significantly reduced compared with IMC-3 cells. At 40 degrees C, however, hyperthermia increased platinum accumulation by factors of 1.4 and 1.8 in IMC-3 and IMC-3-DDP cells, respectively. These findings indicated that CDDP sensitivity was hyperthermically chemopotentiated in CDDP-resistant variants rather than in the control clones. Thus, clinical cancer chemotherapy with CDDP may be improved by an appropriate combination with hyperthermia even at 40 degrees C.     

Absence of acquired thermotolerance in murine tumors unable to increase the expression of heat shock proteins following stress stimuli

The induction of thermotolerance was studied in two groups of murine tumors, one able to produce heat shock proteins (HSP) and the other entirely lacking HSP expression in response to various stress inducers. Heat treatments were performed in vitro and the development of thermotolerance was then evaluated in vivo. The data obtained on the death rate of mice inoculated with tumor cells previously conditioned at 42 degrees C for 1 h and then challenged at 45 degrees C for 30 min following 2 h of reincubation at 37 degrees C, show that the rate of survival is far higher in mice inoculated with HSP negative tumor cells. This indicates that a large number of cells able to increase HSP synthesis following stress escape heat killing, whereas cells unable to express HSP after adequate stimuli are less tolerant against heat challenge.     

Effects of antineoplastic agents and hyperthermia on cytotoxicity toward chronically hypoxic glioma cells.

The effects of hyperthermia and antineoplastic agents on the cytotoxicity to normally oxygenated and chronically hypoxic glioma cells were investigated in vitro. Exposure to temperatures above 43.0 degrees C was less cytotoxic to hypoxic cells which predominantly accumulated in the G0/G1 phase fraction. On the other hand, mitomycin C (MMC) and adriamycin (ADM) were preferentially cytotoxic to hypoxic cells not only at 37 degrees C but also at elevated temperatures (42 degrees C and 43 degrees C). These two agents showed marked synergistic effects with hyperthermia under both oxygenated and hypoxic conditions. In contrast, bleomycin (BLM), cis-diamminedichloroplatinum(II) (CDDP), and vincristine (VCR) were preferentially cytotoxic to oxygenated cells at both 37 degrees C and elevated temperatures. CDDP showed cytotoxic synergism with hyperthermia that appeared to be oxygen-dependent. A nitrosourea derivative, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), showed no major preferential toxicity under either oxygenated or hypoxic conditions. This study suggests that hyperthermia in combination with MMC or ADM would have a greater cytotoxic effect on hypoxic cell subpopulations of malignant gliomas.     

Re-induction of hsp70 synthesis: an assay for thermotolerance

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp 70, and examined the relationship between hsp70 and cells' thermal sensitivity during the development and decay of thermotolerance in model systems. Here, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance. Specifically, we studied the relationship between the retained thermotolerance in a murine tumor cell line SQ-1 and a human tumor cell line, HCT-8, after fractionated heat doses and the cells' ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro. Monolayers of cells were exposed to a first heat treatment (e.g., 41 degrees C, 4 h) and then incubated at 37 degrees C for 0-72 h. At various times after the first heat treatment, cells were either challenged with a 45 degrees C, 45 min heat shock to assess the residual thermotolerance by colony formation, or labelled with [35S]methionine before or after an additional test heat dose (e.g. 43.5 degrees C, 15 min). We found that the cells' ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. Our data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during a course of fractionated hyperthermia.     

Characterization of the effect of hyperthermia on nanoparticle extravasation from tumor vasculature

The efficacy of novel cancer therapeutics can be hampered by inefficient delivery of agents to the tumor at effective concentrations. Liposomes have been used as a method to overcome some delivery issues and, in combination with hyperthermia, have been shown to increase drug delivery to tumors. This study investigates the effects of a range of temperatures (34-42 degrees C) and hyperthermia treatment scheduling (time between hyperthermia and drug administration as well as between consecutive hyperthermia treatments) on the extravasation of nanoparticles (100-nm liposomes) from tumor microvasculature in a human tumor (SKOV-3 ovarian carcinoma) xenograft grown in athymic nude mouse window chambers. Under normothermic conditions (34 degrees C) and at 39 degrees C, nanoparticles were unable to extravasate into the tumor interstitium. From 40 to 42 degrees C, nanoparticle extravasation increased with temperature, reaching maximal extravasation at 42 degrees C. Temperatures higher than 42 degrees C led to hemorrhage and stasis in tumor vessels. Enhanced nanoparticle extravasation was observed several hours after heating, decaying back to baseline at 6 h postheating. Reheating (42 degrees C for 1 h) 8 h after an initial heating (42 degrees C for 1 h) did not result in any increased nanoparticle extravasation, indicating development of vascular thermotolerance. The results of this study have implications for the application and scheduling of hyperthermia combined with other therapeutics (e.g., liposomes, antibodies, and viral vectors) for the treatment of cancer.     

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.     

Thermotolerance and heat shock protein induction by slow rates of heating

The magnitude of thermotolerance and the level of heat shock protein (HSP) expression have been measured in Chinese hamster ovary cells after gradual temperature transients from 37 degrees or 39 degrees to 42 degrees or 43 degrees C. When the level of thermotolerance was measured by clonogenic survival after challenging temperatures between 42 degrees and 43 degrees, substantial thermotolerance was observed. However, when the challenging temperature was raised to 45 degrees C, proportionally less thermotolerance was apparent. Heat shock proteins were quantitated by scanning densitometry of radiographs and, in the case of HSP 70, by immunoassay. Scanning densitometry revealed that low levels of heat shock proteins were synthesized during the heating gradients, but less than after a heat shock at 45 degrees C that delivered an equivalent heat dose. The immunoassay of HSP 70 levels measures both pre-existing and newly synthesized protein, and showed that there was net increase in HSP 70 during two of the heating gradients tested, despite the increase in synthesis noted on the gels. Higher turnover of HSP 70 at the elevated temperatures possibly accounted for the failure to detect a net gain in total protein. In contrast, the total amount of HSP 70 doubled during the 6 hr following a heat shock of 45 degrees for 10 min, an equivalent heat dose to one of the gradients where no net increase in HSP 70 was measured by immunoassay. It appears, then, that tolerance to hyperthemia at 43 degrees C or below may occur under some conditions in the absence of elevated levels of HSP 70, but tolerance to higher temperatures is more closely correlated with increased levels of heat shock proteins. However, even at higher temperatures, our data show disparities between the levels of HSP measured and the thermotolerance expressed.     

Effect of hyperthermia on the action of cis-diamminedichloroplatinum(II), rhodamine 123(2) [tetrachloroplatinum(II)], rhodamine 123, and potassium tetrachloroplatinate in vitro and in vivo

Platinum rhodamine 123 [Pt(Rh-123)2] was synthesized in an effort to produce a new drug which would have the selective uptake into carcinoma cells of Rh-123 and the alkylating and radiosensitizing properties of the chloroplatinum moiety. Because both Rh-123 and cis-diamminedichloroplatinum(II) (CDDP) have been shown to become more cytotoxic at elevated temperatures, we tested the interactions between Pt(Rh-123)2 and hyperthermia both in EMT6 cells in vitro and in the Lewis lung carcinoma in vivo. In the EMT6 cells, CDDP was far more cytotoxic than Pt(Rh-123)2 at 37 degrees C, but its cytotoxicity was less enhanced by exposure of cells to the drug at 42 degrees C than was true for Pt(Rh-123)2 [about 2 logs of increased killing at 42 degrees C after exposure to 10 microM CDDP versus over 3 logs of increased killing at 42 degrees C after exposure to 500 microM Pt(Rh-123)2]. Both Rh-123 and K2PtCl4 also are more cytotoxic to EMT6 cells at 42 degrees C than at 37 degrees C, but the hyperthermic enhancement was far less striking. In the Lewis lung carcinoma, the growth delay produced by CDDP (8 mg/kg) increased by a factor of approximately 2.5 when the drug was given i.p. just prior to local heating of the s.c. thigh tumor to 43 degrees C for 30 min, but the growth delay produced by Pt(Rh-123)2 (100 mg/kg) given i.p. 1 h before local hyperthermia increased by a factor of 5. In contrast, K2PtCl4 and Rh-123 given i.p. produced very short growth delays at normal temperatures and these growth delays were not enhanced by hyperthermia. The effect of these drugs at 37 degrees C and 42 degrees C on the conformation of superhelical pBR322 DNA was also examined. Exposure to CDDP caused progressive alteration from the supercoiled to the linear form of the DNA over time. In contrast, Pt(Rh-123)2 apparently produced progressive degradation of the DNA. Hyperthermia did not alter the qualitative damage produced by the drugs but increased the rate at which the changes occurred. These results suggest both that Pt(Rh-123)2 probably has a different mechanism of action at the DNA than does CDDP and that Pt(Rh-123)2 may be a good drug to use with local hyperthermia and radiation.     

Addition of mitomycin C to cis-diamminedichloroplatinum(ii)/ hyperthermia/radiation therapy in the FSaIIC fibrosarcoma

Hyperthermia (temperatures<42°) is used clinically to improve the effectiveness of radiation therapy and, although therapeutic gains have been reported, efficacy is limited when tumours are large and/or radiation tolerance is reduced. In order to improve the utility of the hyperthermia/radiation combination we have tested the addition of cisplatin (CDDP) in the laboratory and in the clinic. Our clinical studies have shown that the CDDP/hyperthermia/radiation combination is tolerable and effective, but laboratory investigations demonstrated a relative lack of cytotoxicity in the hypoxic tumour subpopulation. In order to improve the effectiveness of the CDDP/hyperthermia/radiation combination against hypoxic cells we have evaluated the addition of mitomycin C, a hypoxic cell cytotoxic agent to this combination. Mitomycin C (5 mg/kg) i.p. produced a tumour growth delay (TGD) of about 5.3 days in the FSaIIC murine fibrosarcoma; hyperthermia (43°±30 min) caused only about 1.4 day TGD and the combination of mitomycin C followed immediately by hyperthermia caused a TGD of about 8.6 days. CDDP (5 mg/kg) i.p. followed by hyperthermia and then 3 Gy on day 1 only of a 5 day ± 3 Gy radiation protocol produced a TGD of about 25 days. With the addition of mitomycin C just before CDDP a TGD of about 44 days resulted. Whole tumour excision experiments demonstrated that mitomycin C was highly interactive with CDDP at 37° and was dose-modifying. When used with CDDP and hyperthermia, however, mitomycin C added little additional cytotoxicity. Hoechst 33342 dye diffusion-determined tumour subpopulation studies indicated a marked effect of the addition of mitomycin C in the dim (enriched in hypoxic cells) subpopulation and nearly equal cytotoxicity in both bright (enriched in euoxic cells) and dim cells resulted. These investigations suggest considerable potential therapeutic efficacy to the addition of mitomycin C to the CDDP/hyperthermia/radiation combination.     

The effects of KNK437, a novel inhibitor of heat shock protein synthesis, on the acquisition of thermotolerance in a murine transplantable tumor in vivo.

A newly synthesized reagent, KNK437, has been found specifically to inhibit the synthesis of heat shock proteins in vitro. In this study, we investigated the effects of KNK437 on the synthesis of heat shock proteins and the induction of thermotolerance in transplantable tumors in vivo. SCC VII cells were grown in vivo and transplanted into C3H/He mice. The concentrations of KNK437 in the tumors and the sera of the mice were examined by high-performance liquid chromatography. Hsp72 synthesis was examined by Western immunoblot analysis. The response to hyperthermia was evaluated in terms of the delay in tumor growth. KNK437 had low toxicity in vivo. The concentration of KNK437 in the tumors gradually increased and reached a peak 6 h after i.p. injection. Hsp72 were synthesized 8 h after hyperthermia at 44 degrees C for 10 min, and their synthesis was inhibited by administration of KNK437 6 h before hyperthermia. At a concentration of 200 mg/kg, KNK437 alone showed no antitumor effects and did not increase the thermosensitivity of nontolerant tumors. The same dose of KNK437 enhanced the antitumor effects of fractionated heat treatment at 44 degrees C in a synergistic manner. This study strongly suggests the inhibition of thermotolerance via the inhibition of HSP72 in vivo. The inhibition of thermotolerance by KNK437 may help to improve the efficacy of clinical fractionated hyperthermia.