Mitochondrial dysfunction after aerobic exposure to the hypoxic cytotoxin tirapazamine.

Tirapazamine (TPZ) is a bioreductive drug that exhibits a high degree of selective toxicity toward hypoxic cells, and at doses that are used clinically, little or no cell killing is observed in aerobic cells. Nonetheless, the effects of TPZ on aerobic tissues are still responsible for the dose limitations on the clinical administration of this drug. Clinical side effects include fatigue, muscle cramping, and reversible ototoxicity. We have investigated TPZ-induced changes in the mitochondria in aerobically exposed cells as a potential mediator of these side effects. Our data show that aerobic administration of TPZ at clinically relevant doses results in a profound loss in the mitochondrial membrane potential (MMP). We show that loss in the MMP occurs in a variety of cell lines in vitro and also occurs in muscle tissues in vivo. The loss in MMP is temporary because recovery occurs within 2 h. TPZ is directly metabolized within mitochondria to a DNA-damaging form, and this metabolism leads to both the cell-killing effects of TPZ on aerobic cells at high doses and to the loss in MMP at clinically relevant doses. Using cell lines derived from genetically modified mice with a targeted deletion in manganese superoxide dismutase, we have further distinguished the phenotypic effects of TPZ in mitochondria at high toxic doses versus those at clinically relevant doses. We have investigated several potential mechanisms for this TPZ-induced loss in MMP. Our results indicate no change in the rate of cellular respiration in TPZ-treated cells. This implies that the loss in MMP results from an inability of the inner mitochondrial membrane to sustain a potential across the membrane after TPZ treatment. Incubation of cells with an inhibitor of the mitochondrial permeability transition suggests that the loss of MMP may result from the regulated opening of a large mitochondria channel.

Oxygen tension measurements of tumors growing in mice.

PURPOSE: Clinical studies using the Eppendorf histograph have shown that patients whose tumors have a low pO2 have worse local control after radiotherapy, and have higher metastatic rates. Because preclinical studies of methods of overcoming, or exploiting, hypoxia generally use transplanted tumors in mice, we have compared the oxygenation of mouse tumors with human tumors to determine the appropriateness of the transplanted mouse model for such preclinical studies. METHODS AND MATERIALS: We evaluated the oxygenation status of subcutaneous (s.c.) tissue and of 12 intradermally (i.d.)- and 7 s.c.-growing mouse or human transplanted tumors in mice using the Eppendorf histograph, and compared the values obtained with measurements of human head and neck nodes. RESULTS: The normal tissue pO2 profile of air-breathing mice showed a nearly Gaussian distribution (38.2+/-14.9 mmHg). Breathing 10% O2 or carbogen resulted in dramatic changes in normal tissue oxygenation. Tumors growing intradermally in the back of air-breathing mice were extremely hypoxic and resistant to expected changes in oxygenation (carbogen breathing, size, and use of anesthetics). Tumors growing s.c. in the foot showed higher oxygen profiles with marked changes in oxygenation when exposing the animals to different levels of oxygen. However, the oxygenation of the mouse tumors transplanted in either site was only a fraction of that of the majority of human tumors. CONCLUSION: Experimental mouse tumors are markedly hypoxic, with median values of 10-20% of those of human tumors. Hence, mouse tumors are probably good models for the most hypoxic human tumors that respond poorly to radiotherapy; however, caution has to be exercised in extrapolating data from mouse to man.

Comparison of the effectiveness of tirapazamine and carbogen with nicotinamide in enhancing the response of a human tumor xenograft to fractionated irradiation.

The goal of this study was to compare, with a human tumor xenograft, two different strategies for increasing tumor response to fractionated irradiation, namely, oxygenating the hypoxic tumor cells with carbogen and nicotinamide, or killing these cells with the hypoxic cytotoxin, tirapazamine (TPZ). We used the human hypopharyngeal squamous cell carcinoma cell line FaDu implanted in immune-deficient SCID mice and assessed its response to radiation by cell survival and by growth delay. The tumors were irradiated either once or twice daily with 2 or 2.5 Gy/fraction with either TPZ (0.08 mmol/kg) or nicotinamide (1,000 mg/kg) with carbogen breathing. We also tested the effect of giving TPZ on alternate days, or daily during the first half of the course, the second half, or for the whole course of radiation. We found that adding TPZ or nicotinamide with carbogen to the fractionated radiation regimen enhanced the response of the human xenograft. The enhancement was somewhat greater (though not significantly so) for TPZ, especially when given with each radiation dose. In conclusion, adding TPZ, or nicotinamide plus carbogen, to fractionated irradiation enhanced the response of this human tumor xenograft to fractionated irradiation. Consistent with theoretical modeling, there was a greater enhancement of the radiation response of the tumor when TPZ was given with each radiation dose than when given with only half of the radiation doses.

DNA damage measured by the comet assay in head and neck cancer patients treated with tirapazamine.

Tirapazamine (TPZ) [3-amino-1,2,4-benzotriazine 1,4-dioxide, SR4233, WIN 59075, and Tirazone] is a novel anticancer drug that is selectively activated by the low oxygen environment in solid tumors. By killing the radioresistant hypoxic cells, TPZ potentiates the antitumor efficacy of fractionated irradiation of transplanted tumors in mice. As this cell kill is closely correlated with TPZ-induced DNA damage, we investigated whether human head and neck cancers would show DNA damage similar to that seen in mouse tumors following TPZ administration. TPZ-induced DNA damage in both transplanted tumors in mice and in neck nodes of 13 patients with head and neck cancer was assessed using the alkaline comet assay on cells obtained from fine-needle aspirates. The oxygen levels of the patients' tumors were also measured using a polarographic oxygen electrode. Cells from the patients' tumors showed DNA damage immediately following TPZ administration that was comparable to, or greater than, that seen with transplanted mouse tumors. The heterogeneity of DNA damage in the patients' tumors was greater than that of individual mouse tumors and correlated with tumor hypoxia. The similarity of TPZ-induced DNA damage in human and rodent tumors suggests that tirapazamine should be effective when added to radiotherapy or to cisplatin-based chemotherapy in head and neck cancers.

1,2,4-Benzotriazine 1,4-dioxides. An important class of hypoxic cytotoxins with antitumor activity.

Tirapazamine (1,2,4-benzotriazin-3-amine 1,4-dioxide, SR 4233, WIN 59075) is the lead compound representing this class of anticancer drugs. It is also the first compound to be introduced in the clinic as a pure bioreductive cytotoxic agent. Tirapazamine represents a completely novel approach to the treatment of solid tumors and has generated considerable interest, with research being carried out on all aspects of the its anticancer activity. Phase III trials of tirapazamine in combination with cisplatin (cDDP) have recently been concluded, and phase II trials of triapazamine in combination with irradiation are presently being performed. We developed a drug discovery program into this class of compounds designed to produce derivatives with improved in vivo activity against solid tumors. Based on the hypothesis that these compounds require bioreductive activation for antitumor activity, the research was primarily directed at producing analogues with greater electron affinity and improved aqueous solubility. The in vitro and in vivo data for a variety of structural analogues clearly show that 1,2,4-benzotriazine 1,4-dioxides have considerable potential as anticancer agents. When their activity is compared directly with the activity observed for triapazamine, the most promising series of analogues appears to be the 3-alkyl-substituted derivatives, especially the 3-ethyl- and 3-(2'-methoxyethyl)-derivatives, SR 4895 and SR 4941 respectively.

Tirapazamine-induced cytotoxicity and DNA damage in transplanted tumors: relationship to tumor hypoxia.

Tirapazamine (TPZ) is a hypoxia-selective bioreductive drug currently in Phases II and III clinical trials with both radiotherapy and chemotherapy. The response of tumors to TPZ is expected to depend both on the levels of reductive enzymes that activate the drug to a DNA-damaging and toxic species and on tumor oxygenation. Both of these parameters are likely to vary between individual tumors. In this study, we examined whether the enhancement of radiation damage to tumors by TPZ can be predicted from TPZ-induced DNA damage measured using the comet assay. DNA damage provides a functional end point that is directly related to cell killing and should be dependent on both reductive enzyme activity and hypoxia. We demonstrate that TPZ potentiates tumor cell kill by fractionated radiation in three murine tumors (SCCVII, RIF-1, and EMT6) and two human tumor xenografts (A549 and HT29), with no potentiation observed in a third xenograft (HT1080). Overall, there was no correlation of radiation potentiation and TPZ-induced DNA damage in the tumors, except that the nonresponsive tumor xenograft had significantly lower levels of DNA damage than the other five tumor types. However, there was a large tumor-to-tumor variability in DNA damage within each tumor type. This variability appeared not to result from differences in activity of the reductive enzymes but largely from differences in oxygenation between individual tumors, measured using fluorescent detection of the hypoxia marker EF5. The results, therefore, suggest that the sensitivity of individual tumors to TPZ, although not necessarily the response to TPZ plus radiation, might be assessed from measurements of DNA damage using the comet assay.

Modification of the antitumor activity of chemotherapeutic drugs by the hypoxic cytotoxic agent tirapazamine.

PURPOSE: Preclinical studies were performed to examine the interaction of the hypoxic cell toxin tirapazamine (TPZ), a benzotriazine di-N-oxide, with several chemotherapeutic agents, including carboplatin, cyclophosphamide, doxorubicin, etoposide, 5-fluorouracil (5-FU), taxol, and navelbine. METHODS: The modification by TPZ of the antitumor drug activity and the effect of schedule were determined with an in vivo/in vitro clonogenic assay using well-established RIF-1 murine tumors transplanted into C3H mice. RESULTS: Additive, or greater than additive, tumor cell killing was observed when TPZ was combined with carboplatin, cyclophosphamide, doxorubicin, etoposide, 5-FU and taxol. With the exception of 5-FU there were only small, or no, enhancements of the systemic toxicities of the drugs by TPZ. The greatest enhancement of antitumor activity was with carboplatin, with the maximum effectiveness when TPZ was given 2-3 h before the carboplatin. The activity of cyclophosphamide, doxorubicin, etoposide and taxol were most enhanced when TPZ was given 24 h before the drug. Additional investigations with three-drug combination treatments using cisplatin and TPZ with either etoposide or navelbine indicated a substantial therapeutic gain from the addition of TPZ. CONCLUSIONS: The data for each of the drugs tested in combination with TPZ, with the exception of 5-FU, indicate that potential clinical benefit may be obtained from therapies combining TPZ with conventional chemotherapy.

Interleukin 1 increases thymidine labeling index of normal tissues of mice but not the tumor.

PURPOSE: This study was conducted to investigate the action of human recombinant interleukin 1 as a radioprotector for different mouse normal cells other than bone marrow cells. METHODS AND MATERIALS: Semi-continuous injections of tritiated thymidine were administered every 6 h, over 24 h to determine thymidine labeling index. Mice were injected with recombinant human interleukin 1 24 h prior to tritiated thymidine and were compared to control animals that did not receive interleukin 1. Mice were killed 1 h after the last thymidine injection. The 24 h thymidine labeling index for normal tissues and RIF-1 tumor was determined. Labeling indices were also determined 1-14 days after a series of fractionated irradiations with or without pretreatment with a single dose of interleukin 1 administered 24 h prior to the first radiation. RESULTS: The thymidine labeling index of normal tissues was higher following the injection of recombinant human interleukin 1 24 h before radiolabeling. This was found in all normal tissues tested, including the lip and tongue mucosal basal cell layers, crypt cells of the duodenum, alveolar cells of the lung, hepatocytes, and basal skin cells. The thymidine labeling index of RIF-1 fibrosarcoma was not affected by interleukin 1 injection. A single interleukin 1 injection 24 h before the first radiation fraction also increased the thymidine labeling indices of normal tissues after localized fractionated irradiation. The thymidine labeling index of RIF-1 tumor was not increased by interleukin 1 administration except after relatively high radiation doses (20 Gy in five fractions). The ability of interleukin 1 to enhance the thymidine labeling index declined after the first day following the completion of fractionated irradiation. CONCLUSION: Recombinant human interleukin 1 increased the 24 h thymidine labeling index in normal tissues in mice, but not in RIF-1 tumor. Fractionated irradiation could maintain the effect of a single dose of interleukin 1, administered 24 h prior to the first fraction, up to 24 h after the end of radiation.

Comparison of the enhancement of tumor responses to fractionated irradiation by SR 4233 (tirapazamine) and by nicotinamide with carbogen.

PURPOSE: This study was undertaken to compare in a fractionated regimen, with clinically relevant radiation doses, two radiation response modifiers that function by different mechanisms: SR 4233, a bioreductive agent toxic to hypoxic cells, and nicotinamide with carbogen, a combination that has been shown to improve tumor oxygenation. METHODS AND MATERIALS: Cell survival assays were used to examine the response of three different tumors: KHT, RIF-1 and SCCVII/St in C3H/Km mice. Regrowth delay studies were also performed with the RIF-1 tumor. A fractionated irradiation schedule, consisting of twice daily 2.5 Gy treatments was investigated with and without drug pretreatment. SR 4233 was given IP at 0.12 mmol/kg one half hour before each irradiation. Nicotinamide (250, 500, 1000 mg/kg) was given IP 1 h before each irradiation with carbogen exposure 5 min prior to and during the irradiation. RESULTS: Both treatment strategies enhanced the response of all three tumors to the fractionated radiation regimen. However, for two of the tumors (KHT and SCCVII), SR 4233 produced a significantly greater enhancement than did the combination of nicotinamide + carbogen. For the RIF-1 tumor (which has the lowest hypoxic fraction of the three), the response was comparable for the two modalities. For nicotinamide + carbogen, there was no significant change in the radiation enhancement at nicotinamide doses between 250 and 1000 mg/kg. CONCLUSION: Adding the bioreductive cytotoxin SR 4233 or nicotinamide + carbogen to fractionated irradiation enhances the response of the three transplanted tumors used in this study to fractionated irradiation. The radiation enhancement was significantly greater, however, for SR 4233 for two of the tumors with comparable results in the third. The data are consistent with the prediction that killing tumor hypoxic cells can produce a similar or greater enhancement of the efficacy of fractionated radiation in enhancing tumor response than either oxygenating or radiosensitizing these cells.

Tumor-specific, schedule-dependent interaction between tirapazamine (SR 4233) and cisplatin.

Tirapazamine (SR 4233), a benzotriazine di-N-oxide, a potent and selective killer of hypoxic cells, is currently in Phase I clinical trials with the expectation that it will be combined with radiation therapy. However, because of the likelihood that hypoxic tumor cells may also be resistant to some commonly used chemotherapeutic agents, we have tested tirapazamine in combination with cisplatin (c-DDP) in the mouse RIF-1 tumor. A large, schedule-dependent enhancement of tumor cell killing was observed both in vivo and in vitro, with a maximal response observed when the SR 4233 was given 2-3 h before c-DDP. Assay of serum blood urea nitrogen levels following treatment with these two drugs indicates that SR 4233 does not enhance the kidney damage which can result from high doses of c-DDP. Leukopenia induced by the two drugs in combination was equal to that predicted from an additive effect of the responses to the individual drugs. Also, there was no change in the systemic toxicity of c-DDP (as judged by 50% lethal dose) when SR 4233 was combined with c-DDP at a dose and timing that produced the maximum tumor interaction. These observations point to a promising new combination therapy with considerable therapeutic advantage.

Interleukin-1 modulatory effect on the action of chemotherapeutic drugs and localized irradiation of the lip, duodenum, and tumor.

PURPOSE: This study was conducted to examine the radioprotective and radiochemoprotective capabilities of interleukin 1 beta (IL-1) on two acute-reacting normal tissues of the C3H mouse, the mucosa of the lip and the duodenum. Also assessed was the modulating effect of IL-1 on tumor growth in the same strain of mice. METHODS AND MATERIALS: IL-1 was administered to C3H/Km mice in combination with fractionated irradiation, or with cyclophosphamide, cisplatin, or 5-fluorouracil (5FU) followed by irradiation. Normal tissue damage was evaluated in the mouse lip, using a subjective scoring system for tissue reaction, and in the duodenum, using the crypt cell survival assay. RIF-1 fibrosarcoma tumor response was assayed with the regrowth delay method. RESULTS: IL-1 protected against the acute reaction produced by fractionated irradiation in the lip mucosa, shifting the dose-response curve by 3.8 Gy. IL-1 was protective when injected intraperitoneally 24 hr before CY or c-DDP, which were given immediately before the first of five daily radiation dose fractions. The dose-response curves for cyclophosphamide and cisplatin were shifted 4.0 Gy and 1.6 Gy, respectively. IL-1 did not protect against 5FU toxicity when treatments were administered in that same sequence; however, when 5FU was given 4 or 8 hr before IL-1 and the first radiation dose fraction followed 20 or 16 hr later, there was significant protection and the curves were separated by 1.5 Gy or 3.5 Gy. IL-1 also protected duodenal crypt cells against the cytocidal effect of fractionated irradiation, with a dose difference of 1.5 Gy and an improvement of crypt survival of 11.7%. It was even more protective for these cells against the enhanced drug toxicity when cyclophosphamide or 5FU were administered immediately before the first of five daily radiation doses, with the dose differences of 4.4 and 5.3 Gy, respectively, and improvements of crypt survival of 33.8 and 29.9%, respectively. There was no modification by IL-1 of the effect of irradiation alone on the RIF-1 tumor. CONCLUSION: This study demonstrates the potential for use of IL-1 as an auxiliary in combinations with chemotherapeutic agents and radiation. It also indicates that for some drugs, such as 5FU, IL-1 effects may be sequence dependent.

Reoxygenation in the RIF-1 tumor after chemotherapy.

The proportion of hypoxic cells in the RIF-1 tumor was observed for 24 hr after treatments with bleomycin, cisplatin, cyclophosphamide, and mitomycin C. The assays were based upon the paired survival curve method for determining the hypoxic fraction using irradiation of aerobic and artificially hypoxic tumors. It was observed that at 1/2 hr after bleomycin, hypoxic fraction was elevated but returned to baseline levels by 2 hr. Following cisplatin, hypoxic fraction did not rise above baseline. However, after cyclophosphamide, hypoxic fraction was elevated and did not return to baseline over the 24-hr observation period of this study. At 1/2 hr after mitomycin treatment, the hypoxic fraction was raised but within 1 hr returned to baseline. These results indicate that tumor reoxygenation varies after different drug treatments and that the determination of drug specificity for aerobic versus hypoxic cells may be strongly biased by the choice of the time after treatment for making the determination.

Protection by interleukin 1 against lung toxicity caused by cyclophosphamide and irradiation.

Interleukin 1 has been shown to provide protection against the toxic effects of cyclophosphamide given in combination with localized irradiation of the lung. A single dose of 15 micrograms/kg interleukin 1 was given 24 h before cyclophosphamide (25-125 mg/kg) which was followed 1 day later by five daily exposures of 4.5 or 5.0 Gy, localized to the lung. Interleukin 1 significantly reduced early measurements of breathing rate for mice treated with high cyclophosphamide doses and irradiation, and at cyclophosphamide doses above 50 mg/kg, there was a significant reduction in the lethality from the combined treatment. At lower levels of cyclophosphamide, however, there seemed to be no effect.

Interleukin-1 modification of the effects of cyclophosphamide and fractionated irradiation.

Studies were performed to determine whether recombinant human interleukin-1 (IL-1) modifies the tumor cytotoxicity of cyclophosphamide (CY) combined with fractionated X-irradiation. RIF-1 tumors were implanted intradermally in C3H/Km mice and therapeutic effect was evaluated by the regrowth delay method, that is, the time for treated tumors to grow to 3 times their volume at the start of treatment relative to that for untreated tumors. A single intraperitoneal treatment of 15 micrograms/kg IL-1 given 24 hr after 100 or 200 mg/kg CY and immediately before the first of 5 daily fractionated treatments of 1-4 Gy increased tumor growth delay beyond that produced by CY and irradiation without the IL-1. However, the IL-1 given with either CY or fractionated irradiation did not extend the time for tumor regrowth beyond that produced by the agents themselves. Thus, while CY and fractionated irradiation together produce a greater than additive effect, IL-1 seems to extend this phenomenon. From these findings, it appears that IL-1 enhances the cytotoxic effects of CY and X ray against tumors, an effect that would have considerable practical significance in the light of the protective effects shown elsewhere for the same lymphokine on normal tissues.

Interleukin 1 protects against the lethal effects of irradiation of mice but has no effect on tumors in the same animals.

Interleukin 1 (IL-1) is a radioprotector of bone marrow and is cytotoxic to some tumor cells. This investigation examines these two properties in the same host animals and gives evidence of radioprotection against localized x-irradiation of the head and neck region. By LD50 analyses, recombinant human IL-1 (100 ng/mouse, approximately 3 micrograms/kg) was found to be radioprotective against whole-body irradiation for both C3H/Km and C57BL/Ka mice. The combined potency ratio for the two strains was 1.07 (95% confidence limit: 1.02-1.12). It was also radioprotective against the injury leading to acute lethality resulting from localized head and neck irradiation of C3H/Km mice; 100 ng of IL-1/mouse produced a potency ratio of 1.05 (95 confidence limit: 1.03-1.07). However, two tumors that originated in C3H/Km mice, RIF-1 and SCCVII, showed neither in vitro nor in vivo response to IL-1. Also, there was no IL-1-induced reduction in in vivo growth of the RL 12NP lymphoma in C57BL/Ka mice.

Reoxygenation of the RIF-1 tumor after fractionated radiotherapy.

The regrowth delay assay was used to assess hypoxic fraction in the RIF-1 tumor. Results approximating those of earlier paired survival curve data were obtained for previously untreated tumors and tumors treated with a single dose of 15 Gy. Further studies showed that after 5 daily fractions of 5 Gy, the hypoxic fraction returned to approximate pretreatment values within 24 hr.

Effect of cytochalasin B, nocodazole and irradiation on migration and internalization of cells and microspheres in tumor cell spheroids.

The effect of cytochalasin B (CB), nocodazole, and irradiation on the adherence and internalization of 3H-labeled EMT6 spheroid-derived single cells and inert microspheres in unlabeled, intact EMT6 multicellular spheroids has been examined. CB inhibited adhesion and internal migration, whereas nocodazole did not stop adhesion but did prevent later internalization. Treatment of labeled cells with 5, 15 and 25 Gy 250 kV X-rays before adherence did not effect their adherence or later internalization. The same radiation treatments administered to the spheroids either immediately before or after the introduction of unirradiated single cells did not affect adherence, but the depths reached by labeled cells and microspheres were reduced largely because of the consequent reduction in spheroid growth. Microsphere size (9, 15, or 25 microns) and surface charge (negative, or non-ionic) had minimal, if any, effect on the adherence and internalization of these particles.

Tumor oxygenation and reoxygenation during radiation therapy: their importance in predicting tumor response.

The degree of oxygenation of a tumor is a major determinant of the effectiveness of radiotherapy. From animal experiments, it is known that hypoxia is common in tumors, that there can be marked heterogeneity in cellular oxygenation within a given tumor, and that the hypoxic fraction is influenced by tumor size and site. The three methods used in obtaining such information are discussed. They are not applicable to tumors in man, but other kinds of evidence suggest qualitatively that the patterns of oxygenation in human tumors are not dissimilar to those in animals. Reoxygenation of animal tumors appears to proceed rapidly after single conditioning radiation doses, but its rate and extent depend upon the size and scheduling of the conditioning dose(s). The importance of reoxygenation in determining the outcome of fractionated radiotherapy may be inferred from the results of recent tumor growth delay experiments wherein we have found that reoxygenation in the RIF-1 sarcoma is nearly complete by 24 hr. after the completion of 5 daily 5 Gy fractional doses.

Reoxygenation in the RIF-1 tumor.

The proportion of hypoxic cells in the RIF-1 tumor was examined for 13 days following a 15 Gy conditioning dose. The paired survival curve technique indicated that 100% of the surviving cells were hypoxic immediately following this treatment. However, within 1 hour, only about 50% remained hypoxic; this proportion continued to drop to about 10% but did not reach the pretreatment level of 1.1% for the duration of the study.