Interactions of BCNU, low pH, glucose and hyperthermia in cultured RIF cells

The interactions of BCNU (1,3-bis[2-chloroethyl]-1-nitrosourea) with low pH, glucose and hyperthermia were studied in cultured RIF tumor cells. The effect of a mild heat treatment of 43 degrees C, 1 h at pH 7.4 on cell killing [surviving fraction (S) = 0.27 +/- 0.05, standard error of the mean (S.E.)] was significantly enhanced by pH 6.5 (S = 0.11 +/- 0.02, S.E.) and 50 mM D-glucose (S = 0.14 +/- 0.01, S.E.). When heat (43 degrees C, 1 h) was added to BCNU, cytotoxicity was increased approximately 14-fold over BCNU alone. Moreover, pH 6.5 increased killing with BCNU and heat by an additional factor of 28. The presence of glucose at 37 degrees C at either pH 6.5 or 7.4 reduced BCNU toxicity in a dose dependent fashion. However, the presence of glucose did not reduce cell killing by BCNU at 43 degrees C. As a result BCNU cytotoxicity was enhanced by approximately 2 orders of magnitude when tumor cell acidification (glucose and low pH) was combined with BCNU and heat.     

Development of thermotolerance in CHO cells: modification by procaine

We have tested the reported ability of procaine to inhibit the induction and the development of thermotolerance in Chinese hamster ovary cells. Thermotolerance was induced either by hyperthermia alone (10 min, 45 degrees C) or by combining hyperthermia and procaine (5 min, 45 degrees C + 10 mM procaine) with heating times adjusted to yield similar cell survival after the conditioning treatments. Both the kinetics of thermotolerance development in fresh medium without procaine and the magnitude of thermotolerance 6 h after heat conditioning were similar for the two treatment groups. Development of thermotolerance in the presence of procaine was tested by adding the drug at 5 or 10 mM to culture medium between, but not during two fractionated heat treatments. Thermotolerance development was observed even in the presence of 10 mM procaine, but only if cell survival was corrected for the 37 degrees C-procaine toxicity. Complete survival curves of cells incubated for 6 h at 37 degrees C in 7.5 mM procaine between heat conditioning and test heating showed a D0 that was only 35 per cent lower than that of thermotolerant controls. The data are consistent with the reported sensitization to heat killing by procaine, but show that thermotolerance induction and development were only minimally perturbed by procaine.     

Cytotoxicity of mitomycin C and carboquone combined with hyperthermia against hypoxic tumor cells in vitro.

Enhancement of the cytotoxicity of mitomycin C (MMC) and carboquone (CQ) by hypoxia at elevated temperature was examined using the SDI test of mouse Sarcoma 180 and Ehrlich cells and clonogenic assay of HeLa cells. When Sarcoma 180 and Ehrlich cells were incubated at 43 degrees C for 2-10 h, the hyperthermic effect was enhanced by hypoxia. The succinate dehydrogenase activity of the cells was reduced by hyperthermia to a greater extent in the presence of hypoxia (O2:5%) than under conditions of aeration (O2:20%). When the cells were exposed to various concentrations of MMC and CQ, under hypoxia, activity of the drugs was enhanced compared to the findings under conditions of aeration. The enhancement was prominent in case of drugs and hyperthermia combined. Clonogenicity of hypoxic HeLa cells was also reduced to a greater extent with this combination than in case of aerated cells. We tentatively speculate that hyperthermo-chemotherapy using MMC and CQ has a potential to attack selectively hypoxic cells present in a solid tumor.     

Interaction of hyperthermia with bleomycin and liblomycin: effects on CHO cells in vitro.

The relative ability of hyperthermia to potentiate the cytotoxicity of bleomycin A2 (BLM) and the lipophilic bleomycin analog liblomycin (LBM) was examined in chinese hamster ovary (CHO) cells. A 60-min exposure to either BLM or LBM inhibited CHO clonogenic survival in a concentration-dependent manner (IC50 values: 11 microM and 2 microM, respectively). Hyperthermia alone also inhibited CHO cell survival in a temperature- and time-dependent manner. In combination with hyperthermia, the cytotoxicity of both drugs was enhanced, however, combination of LBM with hyperthermia exhibited a greater synergistic effect than that observed with the BLM + heat regimen. The greatest inhibitory effects of combined drug + heat treatments occurred when hyperthermia was applied simultaneously with drug exposure. Hyperthermic potentiation of drug cytotoxicity could not be explained by increased uptake of radiolabeled BLM or LBM by CHO cells, or heat-mediated potentiation of the ability of these drugs to cleave plasmid pBR322 DNA in vitro. Our data demonstrate the greater efficacy of LBM, as well as the greater synergy between LBM and hyperthermia against the BLM-sensitive CHO cell line.     

Induction of apoptosis by carboplatin and hyperthermia alone or combined in WERI human retinoblastoma cells

This paper investigated the induction of apoptosis and perturbation of cell cycle progression caused by carboplatin (CPt) and hyperthermia alone or combined in WERI human retinoblastoma cells in vitro . An incubation of the cells with 25 or 50 µ m of CPt at 37°C caused apoptosis, which progressively increased during the 24-72 h treatment. Hyperthermia at 42.5°C for 1 h induced apoptosis, which became significant from 24 h after the heating. Heating the cells in the presence of CPt and subsequent incubation with CPt was far more effective than treating the cells with hyperthermia or CPt treatment alone in inducing apoptosis in the WERI cells, indicating that the combination of these two modalities is potentially useful for the treatment of retinoblastoma. It appeared that the apoptosis in WERI cells caused by hyperthermia and CPt occurs during G1 phase. An interesting observation was that caspase 9 activation preceded the release of cytochrome C from mitochondria during apoptosis in WERI cells, contrary to the general notion that caspase 9 is activated by cytochrome C.     

Cytotoxicity of perillyl alcohol against cancer cells is potentiated by hyperthermia

PURPOSE: Perillyl alcohol (POH) (4-isopropenyl-cyclohexenecarbinol) is a member of the monoterpenes, which are present in various fruits and vegetables. POH has been demonstrated to be cytotoxic against a variety of experimental cancer cells in vitro and in vivo. Phase I clinical trials have indicated that POH may be useful for human tumor treatment. The purpose of our study was to reveal whether the anticancer effect of POH could be enhanced by hyperthermia. METHODS AND MATERIALS: SCK mammary carcinoma cells of A/J mice were used. The effects of POH or hyperthermia alone were studied by incubating the cells during exponential growth phase in culture with 0.25-1.0 mM of POH at 37 degrees C for varying lengths of time or heating cells at 41-43 degrees C for varying lengths of time. The combined effect of POH and hyperthermia was investigated by heating the cells with 1 mM of POH at 41-43 degrees C for varying lengths of time. The effects of the treatments were evaluated using the clonogenic cell survival assay and three types of apoptosis assays. RESULTS: An incubation of SCK cells with 1 mM of POH at 37 degrees C for 60 min or hyperthermia at 43 degrees C for 1 h decreased clonogenic cell survival to 40% and 60%, respectively. When the cells were heated at 43 degrees C for 1 h in the presence of 1 mM of POH, clonogenic cell survival decreased to 0.2%, indicating that hyperthermia potentiated the effect of POH to cause clonogenic cell death. Hyperthermia also markedly increased the degree of POH-induced apoptosis. CONCLUSION: Hyperthermia synergistically potentiates the cytotoxicity of naturally occurring POH against cancer cells.     

Induction of apoptosis by carboplatin and hyperthermia alone or combined in WERI human retinoblastoma cells.

This paper investigated the induction of apoptosis and perturbation of cell cycle progression caused by carboplatin (CPt) and hyperthermia alone or combined in WERI human retinoblastoma cells in vitro. An incubation of the cells with 25 or 50 microm of CPt at 37 degrees C caused apoptosis, which progressively increased during the 24-72 h treatment. Hyperthermia at 42.5 degrees C for 1 h induced apoptosis, which became significant from 24 h after the heating. Heating the cells in the presence of CPt and subsequent incubation with CPt was far more effective than treating the cells with hyperthermia or CPt treatment alone in inducing apoptosis in the WERI cells, indicating that the combination of these two modalities is potentially useful for the treatment of retinoblastoma. It appeared that the apoptosis in WERI cells caused by hyperthermia and CPt occurs during G1 phase. An interesting observation was that caspase 9 activation preceded the release of cytochrome C from mitochondria during apoptosis in WERI cells, contrary to the general notion that caspase 9 is activated by cytochrome C.     

Cytostatic effects of bleomycin and cis-platinum in A549 human lung tumour cells using autofeeder conditions

Residual colony forming ability (CFA) of cells from a human alveolar carcinoma (A549) was determined after 48 hours of contact with bleomycin (BLM) or cisdichlorodiammineplatinum (II) (DDP) using feeder cells arising from parallel cultures left in contact with sublethal BLM or DDP concentrations. The CFA of untreated cells was 60 to 80% (45% with X-irradiated feeder cells). Growing cultures were more resistant to DDP and more sensitive to BLM than plateau phase cultures. Subclones arising from a first treatment were resistant to a second BLM or X-ray treatment, but more sensitive to a second DDP treatment.     

Rhabdomyosarcoma of the urinary bladder: complete remission induced by vinblastine, cis-platinum, and bleomycin

Combination therapy consisting of vincristine, actinomycin-D, and cyclophosphamide with or without adriamycin is the most common chemotherapy for rhabdomyosarcoma in childhood. But the effective chemotherapy for rhabdomyosarcoma resistant to these four drugs has not been established. We report a case with rhabdomyosarcoma, which was resistant to these four drugs but responded completely to three drug combination chemotherapy consisting of vinblastine, cis-platinum, and bleomycin (VPB therapy). A 11-months-old boy was referred to us because of giant abdominal tumor. Postoperative diagnosis was Group III embryonal rhabdomyosarcoma of the urinary bladder. Partial resection was followed by vincristine, actinomycin-D, cyclophosphamide, and adriamycin, but his residual tumor was growing. Then VPB therapy was administered and the first course of the chemotherapy reduced the size of tumor. After three courses of VPB therapy the second-look operation was performed. At operation no residual tumor was found and a complete remission was confirmed. During the course of VPB therapy no severe adverse effect was detectable.     

Effects of combined treatment with 40 degrees C hyperthermia and bleomycin on the accumulation of heat shock protein in murine L cells.

Our research group has reported the enhanced cytotoxicity of combined treatment with bleomycin (BLM) and low hyperthermia at 40 degrees C, using murine L cells, and suggested that post-heating could inhibit BLM-induced sublethal damage repair. For further understanding of the involved mechanisms, we subsequently investigated the kinetics of the cellular accumulations of inducible 72-kDa heat shock protein (hsp72) after 40 degrees C hyperthermia and/or BLM treatment using the same cell line. Western blot analysis showed significantly enhanced accumulation of hsp72 after a low hyperthermia at 40 degrees C for 40, 105 or 180 min, and no significant enhancement of it after exposure to 10 microg/ml BLM at 37 degrees C for either 40 or 105 min. When the cells were heated in the presence of BLM, the accumulations of hsp72 were markedly suppressed, with the maxima of hsp72 accumulation decreasing to 38% and 63% of those induced by hyperthermia alone for 40 or 105 min, respectively. On the other hand, sequential treatment with hyperthermia either before or after BLM treatment did not show significant alteration of the heat-induced accumulations of hsp72. It was demonstrated that BLM was necessary during heating to effectively suppress the heat-induced accumulation of hsp72. This study indicates that the suppression of heat-induced accumulation of hsp72 by BLM may partially contribute to enhance cytotoxicity of the simultaneous treatment of 40 degrees C hyperthermia and BLM.     

Selective enhancement of the cytotoxicity of the bleomycin derivative, peplomycin, by local anesthetics alone and combined with hyperthermia

The effect of local anesthetics alone and combined with hyperthermia on the cytotoxic effect of the bleomycin derivative, peplomycin, was studied in FM3A and HeLa cells. Noncytotoxic doses of the local anesthetics procaine, lidocaine, butacaine, tetracaine, and dibucaine enhanced peplomycin cytotoxicity. This enhancement correlated with the reported anesthetic potency of these agents. Combination of lidocaine (3 to 6 mM) and moderate hyperthermia (40 and 41 degrees) greatly enhanced peplomycin cytotoxicity, although these doses of lidocaine alone were ineffective at 37 degrees, and the temperatures alone enhanced the cytotoxicity only slightly. Cell sensitization to peplomycin cytotoxicity induced by lidocaine combined with 41 degrees hyperthermia produced a decrease in cell survival that depended on the dose of lidocaine and the dose and duration of peplomycin treatment. Lidocaine at 37 or 41 degrees did not enhance the cytotoxicity of Adriamycin, mitomycin C, and cis-diamminedichloroplatinum(II), suggesting a unique interaction with peplomycin. The enhancing effect of lidocaine on peplomycin-induced cell killing was found to increase as pH increased within the range of 7.0 to 8.0.     

Poly-D-lysine enhances the genotoxicity of bleomycin in cultured CHO cells

Cultured CHO cells were treated with the radiomimetic antitumoragent bleomycin (BLM) and post-treated with the polycationiccompound poly-D-lysine (PDL), recently reported by us as ableto potentiate chromosome damage induced by X-rays and chemicalmutagens in both plant and mammallan cells. Our results seemto indicate that PDL enhances the genotoxic action of BLM measuredas induced chromosomal aberrations, colony-forming ability andDNA strand breakage. Taking into account the reported low efficiencyof BLM treatment due to problems with cellular uptake, enzymaticdegradation and efficient repair, the possibility of optimizingthe dose-effectiveness for cancer therapy is discussed.     

Carcinoma of the Penis Treatment by surgery or combined bleomycin and radiation therapy

Forty-four patients with squamous cell carcinoma of the penis stage T1-T2, N0 were either treated surgically (n = 19) or with a combination of irradiation and bleomycin (n = 25). the overall actuarial survival rate was 80% at 3 years, 77% at 5 years and 60% at 10 years. the result of irradiation treatment combined with bleomycin was in stage NO equivalent to that of surgical therapy. the non-surgical treatment had the advantage of preserved sexual ability.     

Induction chemotherapy in advanced squamous head and neck carcinoma with high-dose cis-platinum and bleomycin infusion.

Forty patients with advanced head and neck cancer were treated with combined Cis-platinum-Bleomycin chemotherapy. Cis-diammine dichloroplatinum (DDP) 120 mg/m2 iv was given after prehydration, with mannitol diuresis on Day 1. On Day 3, an initial loading dose of Bleomycin 15 mg/m2 was given by rapid iv push followed by continuous 24 hour intravenous infusion of Bleomycin 15 mg/m2 Day 3 through Day 10. DDP 120 mg/m2 iv was administered again on Day 22. The patients were evaluated for tumor response and resectability between Day 29 to Day 35. Of 39 patients who were evaluable, there were 8 complete responses or CR (20%) and 22 partial responses or PR (56%), for a major response rate of 76%. Nineteen patients had surgery (14 patients whose lesions were initially inoperable and 5 patients who were initially operable). Chemotherapy toxicity in 40 patients included alopecia (40), vomiting (39), mucositis (11), skin rash (10), fever (17), weight loss of more than 5 lbs. (25), WBC less than 3,000 (2), platelets less than 100,000 (1), peak serum creatinine of 2 mg% (3), severe-hearing loss (1), hypersensitivity reaction (2). Surgical complication in 19 patients were pharyngocutaneous fistulae (2), wound dehiscence (1), meningitis and brain abscess (1). There was one death secondary to nephrotoxicity. This particular combination chemotherapy when given as initial treatment, appears very effective in reduction of tumor bulk. Long-term follow-up and randomization is necessary to determine effect upon survival.     

Enhanced effectiveness of adriamycin and bleomycin combined with local hyperthermia in neck node metastases from head and neck cancers.

The results of this study concern the comparison of the clinical effects of adriamycin (ADM) or bleomycin (BLM) alone and combined with local hyperthermia on 15 patients with multiple (29) neck node metastases from head and neck cancers. With repeated low fractional daily doses of drug a significant though transient tumor regression was obtained in 2/8 and in 3/6 of the lesions treated with ADM or BLM alone, respectively. When the drugs were combined with 42-43 degrees C hyperthermia, an overall response, either complete or partial, was seen in all the lesions. Complete regression was observed in 38% (3/8) and 43% (3/7) of the lesions treated with ADM or BLM, respectively, combined with heat. At a 4-month follow-up, 33% (2/6) and 40% (2/5) of the same groups of lesions remained still undetectable. These results suggest that the combined treatment of drugs and local hyperthermia can be advantageously employed in clinical practice for treating local tumors, especially recurrences in previously irradiated areas.     

The induction of thermotolerance in the ear of the mouse by fractionated hyperthermia

The development of thermotolerance in ears of mice was investigated after fractionated hyperthermia. Ears were heated at 43.5 degrees C by immersion in a water bath and the response was measured in terms of the heating time required to cause thermal necrosis in 50% of the ears (NT50). Three types of treatment were given: (1) single treatments, for which the NT50 was 42 minutes; (2) priming treatments, which caused little visible effect but induced thermotolerance. These treatments were given as 1-10 daily fractions, the total heating time ranging from 20-630 minutes; (3) test treatments which were given at various times after priming and were varied to estimate the NT50. Thermotolerance was defined as an increase in the test NT50 for preheated ears relative to the single treatment NT50. It has been suggested that thermotolerance induced by a single priming treatment may be increased by giving additional heat treatments which would not be tolerated by normal cells. In the mouse ear, the maximum thermotolerance induced by a single priming treatment of 20 min at 43.5 degrees C was seen after 24 hr when the test NT50 was about 2.5 times the single NT50. The effect of giving up to nine additional daily treatments of 70 min, each of which would cause necrosis in ears that had not received prior hyperthermia, was measured. The maximum thermotolerance observed was equal to that after a single 20 minute priming treatment but thermotolerance decreased as the number of 70 min treatments was increased from four to nine. The effects of repeating a treatment (20 min or 5 min) which was tolerated by normal ears and induced maximal or less than maximal resistance were compared. The interval between each fraction (24 hr or 12 hr respectively) was equal to the time at which maximal thermotolerance was observed after one treatment. For each regimen, the degree of resistance seen after 2 to 10 exposures was similar to that after the appropriate single treatment. This resistance was maintained throughout the course of priming treatment and decayed after the last fraction. Thus for this regimen, thermotolerance depended on the duration of each treatment rather than on the number of treatments given.     

Effects of hyperthermia and differentiation on cultured Dunn osteosarcoma cells

We investigated the characteristics of morphology, DNA synthesis and argyrophilic nucleolar organizer regions (AgNORs) on murine Dunn osteosarcoma cells in response to heat (42 degrees C, 1 h) or dibutyryl cyclic adenosine 3',5'-monophosphate (Bt(2)cAMP). The cell morphology changes to a fibroblast-like appearance with long and thin protoplastic processes with the reduction of DNA synthesis by heat. It is closely similar to a response by Bt(2)cAMP. In the presence of 3 mM Bt(2)cAMP, the mean number of AgNORs was significantly decreased in 48 h compared with the untreated group. It was increased conversely by heat. Among these responsive cells, we can also find many cells stained without discrimination by the use of AgNORs staining. The present study provides a new clue to support differentiation of osteosarcoma cells from the viewpoint of hyperthermia in vitro.     

A hyperthermia study of differential sensitivity and thermotolerance in AKR murine leukemia and normal bone marrow cells

It has been previously demonstrated that AKR leukemia (lymphoma) cells are more sensitive than normal bone marrow cells to hyperthermic killing at 41.8 degrees C and 42.5 degrees C in vitro. This differential heat sensitivity might be explained by a greater ability to induce thermotolerance (TT) in normal versus neoplastic hematopoetic cells. We tested this hypothesis using the spleen colony methodology in the AKR murine model. A greater heat sensitivity of leukemia in comparison to normal bone marrow cells was observed at 42.5 degrees C; this observation agrees with previous reports. However, using a preincubation temperature of 40.0 degrees C for 120 min did not result in the induction of TT in either normal bone marrow (AKR) cells or AKR leukemia cells. The rationale for the choice of preincubation temperatures and times, as well as the clinical implications of these results are discussed.