Simultaneous action of UV light and hyperthermia on survival and recombination of yeast: effect of intensity of agents on their synergistic interaction

Synergistic effects of simultaneous application of ultraviolet (UV) light and hyperthermia on survival and recombination of diploid yeast cells were studied. For both test-systems the dependence of the synergistic interaction on UV light fluence rate and exposure temperature was revealed: the temperature range synergistically increasing the action of UV light is shifted towards low temperature values with decreasing of UV light fluence rate. For cell survival, the dependence of the synergistic enhancement ratio on the exposure temperature passes through a maximum. A possible qualitative interpretation of these results is discussed.     

Effect of hyperthermia on the antitumor actions of interferons.

Hyperthermia treatment has been shown to enhance the in vitro antiproliferative effects of IFN-alpha, IFN-beta, and IFN-gamma, with IFN-gamma being more strongly enhanced than IFN-alpha. The comparative effects of hyperthermia on the in vivo antitumor activities of IFN-alpha and IFN-gamma were evaluated in the murine system using both subcutaneous and intraperitoneal B16 melanoma tumor model systems. Heat-induced whole body hyperthermia, resulting in a 2 degree C rise in body temperature, was administered by incubating the mice for 8 hours in a dry incubator at 37.1 degrees C. Whole body hyperthermia was found to enhance the antitumor activity of IFN-alpha by approximately 1.0 fold and 1.2 fold for the subcutaneous and intraperitoneal tumor models, respectively. This represented an additive effect of hyperthermia and IFN-alpha. Hyperthermia was found to enhance the antitumor activity of IFN-gamma by approximately 2.9 fold and 2.2 fold for the subcutaneous and intraperitoneal tumor models, respectively. This represented a synergistic effect of hyperthermia and IFN-gamma. The results of this in vivo study confirm and extend the in vitro observation that hyperthermia more strongly enhances the antitumor action of IFN-gamma than IFN-alpha. These results may have clinical importance because they suggest that hyperthermia may be used in combination with IFN-gamma to provide a synergistically enhanced antitumor action.     

Effect of elevated temperatures on the radiation sensitivity of yeast cells of different species

Summary The influence of hyperthermia on the survival of irradiated yeast cells of different species has been studied. The experiments reported in the paper have shown: (1) simultaneous action of ionizing radiation and high temperatures appeared to increase the radiation response by a factor of approximately 2.7 for diploid and only by a factor of 1.5 for haploid cells of wild-type; (2) the combined action of high temperature and ionizing radiation had no synergistic effect for rad51 mutant diploid yeast cells; (3) heating before or after irradiation did not alter the radiation response of yeast cells; (4) enhancement of yeast cell sensitivity by simultaneous action of hyperthermia and²³⁹Pu--particles was negligible; (5) the magnitude and the rate of liquid holding recovery is lowered with increasing of irradiation temperature. On this basis, it was concluded that possible mechanism for thermal sensitization of yeast cells may involve the reduced capacity of cells to recover damages resulted from the combined action of both modalities.     

Prognosis of yeast cells recovery after simultaneous exposure to UV-radiation and hyperthermia

The results of experimental investigations of survival of diploid yeast cells Saccharomyces cerevisiae (strain XS800) after simultaneous exposure to UV-radiation (254 nm) and hyperthermia (53-57 degrees C) have been described. It was shown that the portion of cells capable of recovery in innutrient medium after the action of these agents decreased with the increasing of temperature under which the irradiation was occurred. Mathematical model taking into account the synergistic interaction was suggested for quantitative prediction of irreversible component after combined actions of these agents. A good correspondence between experimental data and model predictions has been demonstrated. The importance of the results obtained for the interpretation of the synergistic interaction mechanisms are discussed.     

The prognosis of the portion of irreversible radiation damages after simultaneous action of hyperthermia and ionizing radiation on yeast cells

The results of experimental research of diploid yeasts cells survival after simultaneous action of hyperthermia and ionizing radiation (60Co) have been described. It was shown that the cell ability to liquid holding recovery decreased with an increase in the temperature, at which the exposure was carried out. due to the increase in the irreversible component determining the relative part of radiation damage which cells are incapable to recover. To predict theoretically the relative part of irreversible radiation damage after combined action, the mathematical model was suggested taking into account the synergistic interaction of agents. Good correlation between experimental results and model prediction was demonstrated. The importance of the results obtained for the interpretation of the mechanism of synergistic interaction of various factors is discussed.     

Differential effects of hyperthermia on human leukocyte production of interferon-alpha and interferon-gamma

Hyperthermia is being used clinically in the treatment of neoplasms. However, there are insufficient data regarding effects of hyperthermia on leukocyte functions potentially important in antitumor immunity. In order to provide such data, human mononuclear leukocytes were exposed to moderate (40.7 degrees C) and marked (42.7 degrees C) hyperthermia for 2 hr. Leukocyte viability, measured by dye exclusion, was not altered by such exposures. Exposure of the cells to moderate hyperthermia did not alter leukocyte production of interferon-alpha in response to influenza virus or interferon-gamma in response to the mitogen phytohemagglutinin. Exposure of the cells to marked hyperthermia significantly depressed production of interferon-alpha. In contrast, production of interferon-gamma was not altered by exposure of the leukocytes to marked hyperthermia. Many studies support a role for interferons (alpha as well as gamma) in antitumor immunity. The current and other data suggest that marked hyperthermia in cancer therapy should be applied locally whenever possible, rather than to the whole body, in order to limit adverse effects on immunity. The data suggest further that interferon-gamma may be a heat shock (stress) protein for human leukocytes.     

Dependence of synergism of the combined effect of ultrasound and hyperthermia upon intensity of ultrasound

The inactivation of wild-type yeast Saccharomyces cerevisiae was studied after simultaneous treatment with ultrasound and hyperthermia. A temperature range was established within which ultrasound and hyperthermia exert a synergistic action. The effect was shown to depend on ultrasound intensity and the temperature at which the treatment takes place. The temperature range enhancing the ultrasound effect shifted forward higher temperature with increasing ultrasound intensity. For every intensity value, an optimal temperature exists at which the synergetic effect is maximum. The biophysical interpretation of the results obtained is based on the assumption that synergism is due to an additional lethal damage, which arises from the interaction of some sub-lesions induced by both agents. These sublesions are considered non-lethal if the agents are applied separately.     

Protective effect of a commercial vaccine, poludan and their combinations in an experimental herpes infection

The antiviral action of herpes vaccine and poludan, an interferon-inducing agent, has been studied in experimental herpes infection, and the synergic effect of these preparations has been obtained. The best result (survival rate equal to 100% after the intraperitoneal inoculation of herpes simplex virus and 85.7% after its intracerebral inoculation) has been registered after the multiple vaccination of experimental animals and the injection of poludan 1 day before the challenge of the animals with the virus. The protective effect of the synergic action of the preparations under study coincides with the period of the maximum content of antibodies in the blood sera of the animals.     

Antitumor effects of combined therapy of recombinant heat shock protein 70 and hyperthermia using magnetic nanoparticles in an experimental subcutaneous murine melanoma

Heat shock proteins (HSPs) are recognized as significant participants in cancer immunity. We previously reported that HSP70 expression following hyperthermia using magnetic nanoparticles induces antitumor immunity. In the present study, we examine whether the antitumor immunity induced by hyperthermia is enhanced by administration of recombinant HSP70 protein into the tumor in situ. Hyperthermia was conducted using our original magnetite cationic liposomes (MCLs), which have a positive surface charge and generate heat in an alternating magnetic field (AMF) due to hysteresis loss. MCLs and recombinant mouse HSP70 (rmHSP70) were injected into melanoma nodules in C57BL/6 mice, which were subjected to AMF for 30 min. Temperature within the tumor reached 43°C and was maintained by controlling the magnetic field intensity. The combined treatment strongly inhibited tumor growth over a 30-day period and complete regression of tumors was observed in 20% (2/10) of mice. It was also found that systemic antitumor immunity was induced in the cured mice. This study suggests that novel combined therapy using exogenous HSP70 and hyperthermia has great potential in cancer treatment.     

Heat shock protein 70 expression induces antitumor immunity during intracellular hyperthermia using magnetite nanoparticles

In this study we demonstrated that heat shock protein (HSP) 70 expression by hyperthermia induced antitumor immunity in the T-9 rat glioma. Our hyperthermic system using magnetic nanoparticles induced necrotic cell death that correlated with HSP70 expression. We purified the HSP70-peptide complexes from the tumor after hyperthermia to investigate whether HSP70 was involved in the antitumor immunity, and we found that in the F344 rats immunized with T-9-derived HSP70 the tumor growth of T-9 was significantly suppressed. Tumor rejection assay after hyperthermic treatment of implanted T-9 cells with incorporated magnetite cationic liposomes (MCL) was performed to investigate whether antitumor immunity was induced by release of HSP70 from the necrotic cells in the F344 rat. Tumor growth was strongly suppressed in the rats subjected to hyperthermia of implanted T-9 cells, and 50% of rats were protected from challenge with T-9 cells. Immunogenicity was enhanced when the HSP70-overexpressing T-9 cells were killed via necrosis in rats by hyperthermia, after which all rats were completely protected from challenge with T-9 cells. Our hyperthermic system produces vaccination with HSP70-peptide via necrotic tumor cell death in vivo, resulting in antitumor immunity. This phenomenon, which may be termed in situ vaccination, has important implications for the development of novel antitumor therapies.     

Combined effect of propofol and GSNO on oxidative phosphorylation of isolated rat liver mitochondria.

Isolated rat liver mitochondria have been treated with the general anaesthetic propofol (2,6-diisopropylphenol, 200 microM) and the physiological NO donor nitrosoglutathione (GSNO, 200 or 250 microM). The efficiency of the oxidative phosphorylation has been evaluated by measuring the respiration and ATP synthesis rates and the behavior of transmembrane electrical potential. In mitochondria energized by succinate, the simultaneous presence of both propofol and GSNO gives rise to a synergic action in affecting the resting and the ADP-stimulated respiration, the respiratory control ratio, the ATP synthesis, and the formation and utilization of the electrochemical transmembrane potential.     

Thermosensitization by methylglyoxal bis(guanylhydrazone) in Chinese hamster cells

The modification of methylglyoxal bis(guanylhydrazone) (MGBG) by 42 degrees C hyperthermia-and/or radiation-induced cell killing was examined in Chinese hamster V-79 cells. At concentrations of more than 10 microM, cell survival decreased exponentially with increased MGBG exposure times. Cell lethality of MGBG (10 microM) was not specific for cell-cycle phases tested from G1/S through G2. When cells were treated with MGBG (10 microM) for 6 hr and then exposed to 42 degrees C hyperthermia with or without a 24-hr interval, cell survival decreased markedly compared with that for 42 degrees C alone. Cells became thermosensitive after MGBG treatment. Cells exposed to MGBG (10 microM) for 6 hr before or after X irradiation were slightly radiosensitive. When X irradiation was combined with MGBG and 42 degrees C hyperthermia, cells became more radiosensitive. From these results, it is suggested that MGBG may change the intracellular state to sensitize cells to the cytotoxic action(s) of hyperthermia.     

Role of extracellular calcium in the hyperthermic killing of CHL V79 cells

Two major questions are addressed by this study: Can an influx of calcium ion sensitize CHL V79 cells to hyperthermia, and, if so, does this occur during heating and does it play a crucial role in cell death? V79 cells are sensitized to hyperthermia by the calcium ionophore A23187 which also induces an influx of calcium at both 37 and 43 degrees C. Sensitization is at least partially dependent on the presence of extracellular calcium. In the absence of A23187, survival is independent of calcium concentration (from 0 to 25 mM) during heating, which differs from the behavior of hepatocytes which are sensitized to hyperthermia by 15 mM CaCl2. Calcium influx, as assayed by uptake of 45Ca measured after washing in LaCl3, is detectable in 3 mM CaCl2 only after 30 min at 45 degrees C, an exposure which reduces reproductive survival to less than 0.1%. Calcium uptake reaches 6 nmol/10(6) cells after 180 min at 45 degrees C. This is not due to a general loss of membrane permeability since there is no trypan blue staining during this time. In 15 mM CaCl2, influx occurs earlier (15 min) but still succeeds the loss of reproductive survival which is less than 1% at this time. Uptake is much higher in 15 mM CaCl2, reaching 10 nmol/10(6) cells by 30 min and 25 nmol/10(6) cells at 180 min, but the temporal pattern of uptake does not correlate with loss of reproductive survival. Thus, although A23187 sensitizes V79 cells to hyperthermia, probably by increased influx of calcium ion, and increased influx occurs during exposure to 45 degrees C, influx is not a crucial early event in the killing of V79 cells. This does not eliminate the possibility of intracellular calcium redistribution during hyperthermia.     

Tumor growth retardation and chemosensitizing action of fatty acid synthase inhibitor orlistat on T cell lymphoma: Implication of reconstituted tumor microenvironment and multidrug resistance phenotype

Background

Orlistat, a fatty acid synthase (FASN) inhibitor, has been demonstrated to inhibit tumor cell survival. However, the mechanism(s) of its tumor growth retarding action against malignancies of hematological origin remains unclear. It is also not understood if the antitumor action of orlistat implicates modulated susceptibility of tumor cell to anticancer drugs. Therefore, the present investigation focuses to study the antitumor and chemosensitizing action of orlistat in a murine host bearing a progressively growing T cell lymphoma.

Methods

Tumor-bearing mice were administered with vehicle alone or containing orlistat followed by administration of PBS with or without cisplatin. Tumor progression and survival of tumor-bearing host were monitored along with analysis of tumor cell survival and apoptosis. Tumor ascitic fluid was examined for pH, NO and cytokines. Expression of genes and proteins was investigated by RT-PCR and western blot respectively. ROS was analyzed by DCFDA staining and FASN activity by spectrophotometry.

Results

Orlistat administration to tumor-bearing mice resulted in tumor growth retardation, prolonged life span, declined tumor cell survival and chemosensitization to cisplatin. It was accompanied by increased osmotic fragility, modulated acidosis, expression of ROS, NO, cytokines, MCT-1 and VH⁺ ATPase, Bcl2, Caspase-3, P53, inhibited FASN activity and declined expression of MDR and MRP-1 proteins.

Conclusion

Orlistat manifests antitumor and chemosensitizing action implicating modulated regulation of cell survival, reconstituted-tumor microenvironment and altered MDR phenotype.

General significance

These observations indicate that orlistat could be utilized as an adjunct regimen for improving antitumor efficacy of cisplatin.     

Hyperthermia and tumour necrosis factor-alpha induced apoptosis via mitochondrial damage

Hyperthermia is a potential anti-cancer regimen but the mode of action is far from clear. Based on the flow cytometric analysis with FITC-annexin V and propidium iodide, apoptosis was found to be the major form of cell death after the treatment with hyperthermia (43 degrees C, 3 h) and/or recombinant murine tumour necrosis factor-alpha (TNF-alpha, 50 ng/ml) in L929 cells. Since mitochondria are thought to play a key role in apoptosis, experiments were done to assess their role in the hyperthermia-mediated apoptosis. Our results indicate that hyperthermia was able to depolarize the mitochondrial membrane potential (delta psi m) and release cytochrome c to the cytoplasm, in a way very similar to the action of TNF-alpha. With the use of cyclosporin A to inhibit the delta psi m dissipation, the cytotoxicity mediated by hyperthermia or TNF-alpha was suppressed. Taken together, our results indicate that hyperthermia and TNF-alpha can induce apoptosis in L929 cells and the mitochondrial dysfunction plays a key role in the cell death process.     

Recovery of yeast cells after exposure to ionizing radiation and hyperthermia

Quantitative regularities of recovery of wild-type diploid yeast cells irradiated with gamma-rays (60Co) simultaneously with exposure to high temperatures were studied. It was shown that in conditions of such a combined action the constant of recovery did not depend on the temperature at which the irradiation was carried out. However, with an increase of acting temperature an augmentation in the portion of irreversible component was registered. The analysis of cell inactivation revealed that the augmentation of the irreversible component was accompanied by a continuous increase of cell killing without any postirradiation division after which cells are incapable of recovery. The reproductive death was mainly exerted after ionizing radiation applied alone while in conditions of simultaneous thermoradiation action the interphase killing (cell death without division) predominated. It is concluded on this base that the mechanism of synergistic interaction of ionizing radiation and hyperthermia may be related with cardinal change in mechanisms of cell killing.     

Antitumor activity of recombinant human tumor necrosis factor in combination with hyperthermia against heterotransplanted human prostatic carcinoma and its lymph node metastasis in nude mice.

The antitumor activity of recombinant human tumor necrosis factor (rhTNF) against heterotransplanted human prostatic carcinoma (PC-3) and spontaneous lymphatic tumor metastasis was studied in vivo. The spontaneous lymphatic metastasis of PC-3 tumor was found in approximately 50% of cases. Significant antitumor activity was observed with repeated intratumoral administration of a large dose of rhTNF, not only on the subcutaneous tumor xenografts but also on the lymph node metastases. Strong antitumor activity could be achieved even with the intratumoral administration of a small dose of rhTNF in combination with mild hyperthermia on either the transplanted tumors or on the metastatic tumors.     

Novel 3-aroylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 8-substituted, ligands at GABAA/benzodiazepine receptor complex: synthesis, pharmacological and molecular modeling studies

The synthesis and binding studies of a series of 3-acylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 8-substituted are reported. High-affinity ligands at benzodiazepine site on GABA(A) receptor complex (GABA(A)/BzR complex) were obtained when the 3-aroyl substituent is represented by a five-member heteroaroyl ring (furoyl-, thenoyl-, and pyrroyl-). Moreover the type of heteroaroyl ring at position 3 influences the feature of the substituent at position 8 to obtain high-affinity ligands: a 'hydrogen-bond acceptor ring' at position 3 is synergic with an electron donor substituent at position 8, while a 'hydrogen-bond donor ring' is synergic with a withdrawing substituent. Compounds 8a, 9b, and 11 were deeply studied in vivo for their pharmacological effects considering six potential benzodiazepine actions: motor coordination, anticonvulsant action, spontaneous motor activity and explorative activity, anxiolytic-like effects, mouse learning and memory modulation, and ethanol-potentiating action. To rationalize and qualitatively interpret the GABA(A)/Bz binding affinities of compounds 8a and 11, a dynamic molecular modeling study has been performed, with the aim of assessing the preferred geometry of protein-ligand complex.     

The capacity of lysosomes of cultured mammalian cells to accumulate acridine orange is destroyed by hyperthermia

Summary Lysosomes of cultured mammalian cells, derived from a transplantable murine mammary adenocarcinoma, irreversibly lose their capacity to accumulate the fluorescent dye acridine orange after hyperthermia. As acridine orange may be regarded as a fluorescent probe of the internal pH of the lysosomes, we may conclude that the ability of lysosomes to maintain a low internal pH is destroyed by hyperthermia.The effects of hyperthermia on lysosome fluorescence and on cell survival show several similarities: in both cases hyperthermia is more effective at low pH, below pH 7.0, and CCP (carbonylcyanide-m-chlorophenylhydrazone) enhances effects at low pH, but has no clear effect at pH 8.0. This leads to the conclusion that effects on lysosomes are an important and early event in cellular injury caused by hyperthermia. The activation energy, however, obtained for the effects of hyperthermia on lysosome fluorescence is about a factor of two lower than the activation energy reported for cell survival after hyperthermia. This suggests that the effect on lysosomes is not directly caused by hyperthermia but is triggered by some other hyperthermia-induced cellular damage.Abbreviations CCP carbonylcyanide-m-chlorophenylhydrazone - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 2-(N-morpholino)propanesulfonic acid - Tricine N-tris(hydroxymethyl)methylglycine Supported in part by grants from the KWF (Koningin Wilhelmina Fonds) and the IRS (Interuniversitair Instituut voor Radiopathologie en Stralenbescherming)     

Effect of sequential application of hyperthermia and chemotherapy on the survival of a thermoresistant human melanoma cell line

The human melanoma cell line M14 has been proven in previous experiments to be much less sensitive to the action of heat (42 degrees C, 60 min) than other melanoma lines. In the present study, we have investigated the possibility of increasing the effect of heat by means of drug treatment. Thermochemotherapy was applied to exponentially growing cells according to different schedulings, and was analyzed in its efficacy by measuring the impairment of the cellular colony-forming ability. Findings of the present study point out that: (a) appropriate sequencing between hyperthermia and cis-diamminedichloroplatinum II(DDP), melphalan (L-Pam), 1,2,4-dichlorobenzyl-1H-indazol-3-carboxylic acid (LNA) or 5-3,3-dimethyl-1-triazeno-imidazole-4-carboxamide (DTIC) strongly influences the cytotoxic effect of the two agents; and (b) the optimal combination appears to be the simultaneous application of heat and drugs. However, as far as thermochemotherapy with DDP is concerned, a synergistic effect may also be achieved when hyperthermia precedes DDP.