Killing lung cancer cells at cell-cycle phase by a new indium-111-bleomycin complex

The efficacy of killing small cell lung cancer (SCLC) cells at the G1, S, and G2-M phase of the cell-cycle by a new 111In-bleomycin complex (111In-BLMC) was investigated. SCLC cells (N417, H526, H209) were synchronized by double thymidine block and assessed by DNA content with flow cytometry, and the period for the maximal accumulation of cells in S, G1, or G2-M phase was determined. Cells in different cell cycle phases were exposed to 0.9% NaCl, BLM, or 111In-BLMC for 1 hour and observed for colony formation. The survival of H526 cells treated with 111In-BLMC was 71% (for enriched S phase), 46% (G1), and 31% (G2-M). For N417 cells, it was 25% (S), 20% (G1), and 8% (G2-M) for 111In-BLMC and 18% (S), 33% (G1), and 10% (G2-M) for BLM. These results indicated that SCLC cells in G2-M were most sensitive and those in S phase were least sensitive to 111In-BLMC; cells in G1 phase were the least sensitive to BLM.     

Chromosome aberrations of human small cell lung cancer induced by a new 111In-bleomycin complex.

A new 111Indium labeled bleomycin complex (111In-BLMC) was prepared and found to be effective for tumor imaging and therapy both in mouse glioma and human small cell lung cancer (SCLC) cells. Chromosome aberrations were studied in human SCLC cells to explore its mechanisms of killing cancer cells. SCLC cells (N417) were exposed to 111In-BLMC, BLM, or 111InCl3 (for control) for 1 hour, treated with colcemid, and chromosomal changes were analyzed. A dramatic increase in chromatic gaps, breaks, chromosome breaks, double minutes, rings, triradii, quadriradii, and chromosome stickiness were observed in the cells treated by 111In-BLMC compared to BLM or 111InCl3. These results indicated that 111In-BLMC has therapeutic potential for combination chemo-radiotherapy of cancer (e.g., by Auger electrons and local energy deposition).     

Distribution of 111In-bleomycin complex in small cell lung cancer cells by autoradiography.

The distribution of 111In-bleomycin Complex (111In-BLMC) in small cell lung cancer (SCLC) cells was studied by autoradiography. SCLC cells were exposed to 111In-BLMC and 111Indium chloride (111InCl3) for 1 hour, 3 hours, and 4 hours; washed with fresh medium; and spread on slides. The slides were smeared with NTB2 (NTB3) emulsion by wet or dry-mount technique and exposed 3 to 15 days. 111In-BLMC was found to localize in the cell nucleus and nuclear membrane (78.3%); 111InCl3 located mainly in the cytoplasm (52.3%). This distribution of labeled BLM may explain the mechanism of killing SCLC cells by 111In-BLMC.     

Killing of Human Lung Cancer Cells Using a New [111In]Bleomycin Complex [111In]BLMC

The ability of a [¹¹¹In]bleomycin complex ([¹¹¹In]BLMC) to kill five cell lines of human lung cancer (small cell lung cancer) was investigated. Cells were exposed to either 0.9% NaC1, [¹¹¹In]C1₃, BLM, [¹¹¹In]BLMC, nonradioactive InC1₃, or In-BLMC for 60 minutes, plated in soft agarose, and assessed for colony formation. [¹¹¹In]BLMC (40-200 μ;C) carried by 15-25 μ;g BLM/ml) was more cytotoxic than BLM (15-25 μ;g BLM/ml) by a factor of 1.6-5.3 for five cell lines. The percent survival of N417 cells was 28.4 for [¹¹¹In]BLMC (40 μ;Ci/15 μ;g BLM/ml) and 54.3 for BLM (15 μ;g/ml); 1.9 for [¹¹¹In]BLMC (200 μ;Ci/25 μ;g BLM/ml), and 10.0 for BLM (25 μ;g/ml). ¹¹¹InC1₃ (200 μ;Ci/ml) and nonradioactive InC1₃ failed to inhibit colony formation. The new [¹¹¹In]BLMC may be useful for therapy of some lung cancer patients.     

加温和抗癌药物消瘤芥对恶性HEp—2细胞的毒性作用

Cytotoxic effect of hyperthermia combined with Nitrocaphane (NC) on HEp-2 cells was detected by colony formation assay. The results showed: 1. There was significant synergetic effect on survival fraction of HEp-2 cells when the heat was at 43 degrees C and 44 degrees C and NC at 0.5-4.0 micrograms/ml; 2. There was almost no effect on the cell survival when the heat was at 39 degrees C and 41 degrees C for 1 hr without NC; however, the cytotoxicity of NC (1.0 micrograms/ml) was enhanced when temperature was at 39 degrees C and 41 degrees C; 3. The sequence between heat and NC could influence the synergism. When the heat at 43 degrees C, 60 min and NC at 1.0 micrograms/ml were used simultaneously, the cytotoxicity was the strongest (SF = 0.025). When the heat was given 1-4 hr before or after the drug, the cytotoxicity was decreased. These results suggest that, over the range of 39-44 degrees C, the synergetic effects of hyperthermia and NC on malignant HEp-2 cells provide certain cell biologic basis for clinical application of local or systemic hyperthermia combined with NC.     

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.     

Flavone acetic acid increases the antitumor effect of hyperthermia in mice.

The combined effects of flavone acetic acid (FAA), a synthetic flavonoid, and hyperthermia on B16 melanoma cells were investigated. In vitro, FAA alone at concentrations below 100 micrograms/ml was not cytotoxic with a 60-min exposure at 37 degrees C. Hyperthermia at 43 degrees C for 60 min enhanced the cytotoxicity of FAA only at concentrations over 100 micrograms/ml. Inhibition of the growth of B16 melanoma solid tumor by FAA and/or hyperthermia was examined in vivo. FAA (100-200 mg/kg) inhibited tumor growth in a dose-dependent manner. The combined treatment of FAA (200 mg/kg) and hyperthermia (43 degrees C, 15 min) significantly inhibited tumor growth compared to a treatment of FAA or hyperthermia alone. The maximum antitumor effect of FAA combined with hperthermia was obtained when FAA was administered 2 or 4 h before heat. The significantly increased cytotoxicity of FAA combined with hyperthermia seems to relate to specific decreases in tumor blood flow, a reduction in tumor pH, and an increased tumor temperature, without altering pH in the normal tissues. This combined treatment of FAA and hyperthermia warrants further study for treating subjects with solid tumors.     

A new 111In-bleomycin complex for combined radiotherapy and chemotherapy

Six days after tumor transplantation three daily intraperitoneal doses of 0.9% NaCl, bleomycin (BLM), or a new 111In-bleomycin complex (BLMC, 15 microCi/g body weight) were administered to glioma-bearing mice. After therapy, tumors in mice treated with 111In-BLMC were smaller than those treated with BLM. Sixteen days after the first injection tumor size for 111In-BLMC-treated mice was 560 (240-1,030) mm3, 1,980 (1,400-3,290) mm3 for BLM (P less than 0.025), and 4,830 (2,580-9,180) mm3 for NaCl (0.1 less than P less than 0.2). Thirteen days after tumor transplantation glioma-bearing mice received single intratumor injection of 0.9% NaCl, BLM, or 111In-BLMC (1.5 mCi, carried by 0.5 mg BLM/g tumor weight). The average tumor size for 111In-BLMC was smaller than that for BLM by a factor of 2.5-3.7. Host weights for these two groups were similar, and morphologic abnormalities were not found in kidney or liver.     

Interaction between the kinetics of thermotolerance and effect of cis-diamminedichloroplatinum(II) or bleomycin given at 37 or 43 degrees C.

The interaction between the cytotoxic effect of bleomycin (BLM) or cis-diamminedichloroplatinum(II) (cis-DDP) and the kinetics of thermotolerance was studied in cultured Chinese hamster ovary (CHO) cells. Pre-heated cells were treated with cis-DDP or BLM at 37 or 43 degrees C for various times after heating. Pre-heating enhanced cis-DDP cytotoxicity given immediately after heating, but this enhancement decreased within 24 h to an additive level. Cell survival following the initial heating and the second treatment of 'cis-DDP at 43 degrees C was minimal when cis-DDP at 43 degrees C was given immediately after the initial heating, but became higher with increasing treatment interval and reached 'less than additive' level when the treatment interval was extended to more than 24 h. This alteration in cell survival appeared to follow the kinetics of thermotolerance. The interaction between BLM treatment and the kinetics of thermotolerance was similar to that of cis-DDP. However, pre-heating enhanced BLM cytotoxicity much less extensively than cis-DDP cytotoxicity. These results indicate that: (a) pre-heating of cells enhanced drug-toxicity when the drug was given shortly after heating, but the magnitude of this enhancement depended on the drug; (b) pre-heating did not influence the cytotoxicity of drugs given at 37 degrees C; and (c) pre-heating decreased the magnitude of thermal sensitization of drug cytotoxicity. The magnitude of the decrease in thermal sensitization appeared to be parallel to the kinetics of thermotolerance. In this study it was also demonstrated that pre-treatment of CHO cells by cis-DDP or BLM did not alter sensitivity to subsequent drug treatment, hyperthermia or thermochemotherapy.     

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.     

Evaluation of hypoxia and acidosis on the cytotoxicity of the stereoisomers of tetraplatin at normal and hyperthermic temperatures

Tetraplatin is a second-generation platinum analogue that will be introduced into clinical trials during 1990. We have evaluated the cytotoxicity of tetraplatin and its stereoisomers under a variety of conditions in a human solid tumour cell line (A549). The d-trans stereoisomer of tetraplatin is the most cyutotoxic form of the drug under all conditions examined. Simultaneous treatment with hyperthermia (43 degrees C) resulted in a greater than 1 log increase in cytotoxicity over tetraplatin (10 micrograms/ml) alone. Lowering the pH of the media from 7.4 to 6.4 did not significantly modify the cytotoxicity of tetraplatin at 37 degrees C or at 43 degrees C. Under acidic pH (6.4) hypoxia did not alter the cytotoxicity of tetraplatin at 37 degrees C or 43 degrees C. These results indicate a potential therapeutic advantage of combining tetraplatin chemotherapy with hyperthermia, which appears to be uneffected by acidic and/or hypoxic conditions.     

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.     

Effect of pH, oxygenation, and temperature on the cytotoxicity and radiosensitization by etanidazole.

The effect of etanidazole was examined in vitro and in vivo in the FSaIIC tumor system. At pH 7.40 and 37 degrees C, etanidazole at 5-500 microM for 1 hr was minimally cytotoxic. At 42 degrees C and 43 degrees C, however, the cytotoxicity of etanidazole increased. Etanidazole was more cytotoxic at pH 6.45 and 37 degrees than at pH 7.40 by about 1 log. Increasing the temperature to 42 degrees C or 43 degrees C at pH 6.45 during drug exposure, however, caused little increase in drug killing above the lethality of hyperthermia. When the radiosensitizing abilities of etanidazole were tested in vitro, there was a radiation dose modifying factor of 2.40 at pH 7.40, but only 1.70 at pH 6.45. In vivo, etanidazole (1 g/kg) produced a radiation dose modifying factor of 1.47, whereas 43 degrees C for 30 min produced a radiation dose modifying factor of 1.38. The combination resulted in a radiation dose modifying factor of 2.29. When the cytotoxicities of hyperthermia (43 degrees C x 30 min), etanidazole (500 mg/kg or 1 mg/kg), and radiation (10 Gy) combinations were assayed by Hoechst 33342 dye selected tumor subpopulations, 43 degrees C x 30 min increased the killing of irradiated dim cells by approximately 9.2-fold but by only 2.9-fold in bright cells. Etanidazole (1 g/kg) increased radiation killing of bright cells by about 3-fold and dim cells by about 4.3-fold. The combination of hyperthermia and etanidazole increased the killing of both dim and bright cells exposed to radiation by approximately 10-fold versus 10 Gy alone.     

Response of EMT6 multicellular tumour spheroids to hyperthermia and cytotoxic drugs

The response of multicellular tumour spheroids of the EMT6 cell line to combinations of hyperthermia and Bleomycin (BLM) or Adriamycin (ADM) has been investigated. Using this model system, we have demonstrated enhanced BLM cytotoxicity at 43 degrees C and also heat-induced drug tolerance to BLM at 43 degrees C. ADM cytotoxicity was not significantly increased after 43 degrees C x 1 h but after 6 h at 42 degrees C greatly enhanced cell-killing was evident. These results are discussed in relation to our previous data for EMT6 cells growing either as monolayer cultures in vitro or as solid tumours in mice.     

Combined and complex treatment of patients with II-stage malignant skin melanoma using hyperthermia

Combined and complex treatment for stage II (T3-4N1M0) malignant melanoma of the skin was carried out 111 cases. In 73 of them, local and/or general hyperthermia was employed as cytostatic and radiotherapy modifier. Local super-high frequency hyperthermia was shown to potentiate the antitumor action of ionizing radiation on melanoma cells. Its effect increased with temperature rising from 42 to 43 degrees C. Application of hyperthermia in the said treatment was followed by a considerable increase in 5-year survival, viz. 49.4%. It was significantly higher than with standard treatment procedures (P less than 0.05).     

Potentiation of adriamycin cytotoxicity in P388 murine leukemia sensitive and resistant to adriamycin by use of lonidamine and hyperthermia

The in vitro effect of adriamycin (ADR) and lonidamine alone and in combination, at 37 degrees C and 43 degrees C, was investigated on murine leukemia P388 sensitive (P388/S) and resistant (P388/ADR) to adriamycin. The sensitive and the resistant cells were exposed in vitro with and without the drugs for 1 h at 37 degrees C and 43 degrees C. These cells were inoculated ip (10(6) cells/mouse) into groups of BDF1 mice. Cytotoxic effect of the treatment was assessed on the basis of percentage increase in life span (% ILS) of these animals, compared to the animals receiving cells which did not receive any treatment but exposed only to 37 degrees C for 1 h. It was observed that exposure of P388/ADR cells to lonidamine or adriamycin alone at 43 degrees C for 1 h resulted in greater cell kill, thus enhancing the % ILS of the experimental animals receiving those cells, compared to that of mice receiving the cells exposed to the same drugs for 1 h at 37 degrees C. However, the combination of lonidamine (0.02 mM) and adriamycin (10 micrograms/ml) at 43 degrees C for 1 h showed more than a synergistic effect, resulting in a % ILS of 120. Similar results were seen in the case of P388/S; however, the observations pertaining to P388/ADR are encouraging, since the mode of treatment has reversed the acquired resistance of P388 leukemia cells to adriamycin.     

The cytotoxic effect of cis-diamminedichloroplatinum(II) on cultured Chinese hamster ovary cells at elevated temperatures: Arrhenius plot analysis

The effect of cis-diamminedichroloplatinum (II) (cis-DDP) and hyperthermia on cultured Chinese hamster ovary (CHO) cells were investigated. Cells were treated with 6 microM cis-DDP for various durations of time at temperatures ranging from 37 to 43 degrees C, and cell survival curves were determined as a function of treatment time. The cytotoxic effect of cis-DDP increased with increasing temperatures, indicating that hyperthermia enhanced cytotoxicity of cis-DDP. Arrhenius analysis of surviving fraction data (6 microM cis-DDP) yielded activation energies of 61 kcal/M between 37 and 41 degrees C and 213 kcal/M between 41 and 43 degrees C. Further experiments using two different drug concentrations (3 and 12 microM) confirmed these activation energies in these two different temperature ranges. However, the activation energy for 12 microM cis-DDP given in the temperature range 33-37 degrees C was 22 kcal/M, which is smaller than those found above 37 degrees C. This activation energy appeared to be identical to that reported for the degradation or depurination of DNA. The activation energy between 37 and 41 degrees C, i.e. approximately 61 kcal/M, was twice as great as that found for alkylation of thio-TEPA, an alkylating agent. This may indicate that the mechanism of action of cis-DDP differs from that of thio-TEPA. A greater activation energy observed in the range of 41-43 degrees C is most likely attributable to the additive effect of hyperthermia and thermal enhancement for cis-DDP. Although only one data point is available at the temperature above 43 degrees C, it suggests that the activation energy is identical to that for hyperthermia alone.     

Hyperthermia inhibits the repair of DNA double-strand breaks induced by ionizing radiation as determined by pulsed-field gel electrophoresis

Hyperthermia is known to synergistically interact with X-rays to kill cells. We have used pulsed-field gel electrophoresis to investigate the effects of hyperthermia on cell survival and on repair of radiation-induced DNA double-strand breaks (dsbs). Combining hyperthermia (43°C, 45 min) with radiation (7.5 Gy) resulted in a complete inhibition of dsb repair and a surviving fraction of 0·9%. Cells treated with hyperthermia alone resulted in a 55% cell survival with no increase in dsb levels over background. Cells treated with 7·5 Gy alone demonstrated 11% survival and exponential dsb repair. Dsb repair was equally inhibited by hyperthermia whether administered immediately before or after the radiation. We compared the rejoining of dsbs resulting from 7·5 Gy at 37 and 43°C to determine whether dsbs were being repaired during hyperthermia. While repair occurred at 37°C, no dsbs were repaired at 43°C. Our results indicate that hyperthermia completely inhibits dsb repair.     

Influence of oxidative stress induced by cysteamine upon the induction and development of thermotolerance in Chinese hamster ovary cells

Chinese hamster ovary cells exposed to the sulfhydryl compound cysteamine combined with heat treatment at 44 degrees C developed thermotolerance within 8 h. After initial treatment either with 15 min cysteamine (0.4 mM) at 37 degrees C immediately followed by 15 min heat at 44 degrees C or with 15 min cysteamine (0.4 mM) at 44 degrees C, the magnitude of thermotolerance developed was identical. The D0 of the subsequent 44 degrees C heat survival curves increased by factors of 8.9 and 7.9, respectively. The kinetics of thermotolerance induction and the time to reach the maximum of thermotolerance expression after combined cysteamine treatment at 44 degrees C for 15 min was found to be comparable to the effects of 44 degrees C treatment alone for 30 min. The synergistic effect of cysteamine with the conditioning heat treatment at 44 degrees C was blocked by catalase (50 micrograms/ml). Following initial treatment with cysteamine at 37 degrees C, cells became thermotolerant within 2 h. The D0 of the survival curves for 44 degrees C heat treatments increased with duration (t1 = min, 37 degrees C) of the cysteamine (0.4 mM) exposure; e.g., the D0 increased by factors of 1.5, 1.6, 2.2, and 2.6 for t1 = 30, 60, 90, and 120 min. The induction of thermotolerance by cysteamine at 37 degrees C was completely blocked by the addition of catalase (50 micrograms/ml), present during the initial period of drug treatment. Combined cysteamine and heat treatment at 44 degrees C, but also cysteamine exposure at 37 degrees C, enhanced synthesis of heat shock proteins. The data suggest that oxidative stress by cysteamine can be synergistic with the conditioning heat treatment at 44 degrees C which induces thermotolerance. At 37 degrees C, cysteamine itself induces thermotolerance and the enhanced synthesis of heat shock proteins under these conditions.     

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.