Effects of Motexafin gadolinium on tumor metabolism and radiation sensitivity

Purpose: Experiments were undertaken to determine if metabolic changes induced by Motexafin gadolinium (Gd-Tex⁺², XCYTRIN) predict time intervals between drug and radiation wherein there is enhancement of radiation efficacy.

Methods and Materials: We evaluated the effect of Gd-Tex⁺² on tumor metabolism and on tumor growth using a mouse mammary carcinoma model and ³¹P nuclear magnetic resonance (NMR) experiments. Response to therapy was evaluated based on time for the tumor to regrow to pretreatment size and also tumor doubling time.

Results: ³¹P NMR experiments indicated that Gd-Tex⁺² effected tumor energy metabolism during the first 24 hours postadministration. A decrease in phosphocreatine was noted at 2 (p < 0.04), 6 (p < 0.006), and 24 (p < 0.001) hours post Gd-Tex⁺². A decrease in nucleoside triphosphates was noted only at 2 hours (p < 0.02), with subsequent recovery at 6 hours. Phosphocreatine in control (saline treated) tumors showed a significant decrease only at 24 hours (p < 0.01). Irradiation at 2 and 6 hours post Gd-Tex⁺² induced an enhanced effect compared to radiation alone as measured by analyzing the growth curves, maximum tumor volumes, and the time for the tumors to regrow to their initial volumes. Irradiation at 24 hours post Gd-Tex⁺² induced a modest enhancement in tumor growth delay compared to radiation alone.

Discussion: NMR spectroscopy may be useful for monitoring tumor metabolism after treatment with Gd-Tex⁺² and administering radiation during the time of maximal efficacy of Gd-Tex⁺².     

Effects of mitomycin C alone and in combination with X-rays on EMT6 mouse mammary tumors in vivo

The effects of mitomycin C alone and in combination with radiation on the cells of EMT6 mouse mammary tumors in BALB/cKaRw mice were examined. At doses near the toxic level, approximately 98% of the tumor cells were killed by a single injection of mitomycin C. Both proliferating and quiescent cells and both hypoxic and aerobic cells were killed by the drug. Cytotoxicity with mitomycin C occurred rapidly and was apparently complete within 30 minutes after injection of the drug . No evidence was found for repair of potentially lethal mitomycin C damage or sublethal mitomycin C damage by the tumor cells. Mitomycin C and radiation in combination produced an additive cytotoxicity; neither agent was found to alter significantly the shape of the dose-response curve for the other agent. The cytotoxicity of mitomycin C and radiation in combination depended on the sequence and timing of the treatments; additive toxicities were obtained when mitomycin C was given just after, just before, or up to 24 hours before irradiation, but the combination was less effective when mitomycin C was given 2-12 hours after irradiation.     

Pentoxifylline improves the oxygenation and radiation response of BA1112 rat rhabdomyosarcomas and EMT6 mouse mammary carcinomas

Tumor hypoxia can significantly impact the efficacy of cancer therapy. Pentoxifylline, a methylxanthine derivative, can improve oxygen delivery to tissues and is widely used in the treatment of peripheral vascular disease and various cerebrovascular disorders. In this article, we show that pentoxifylline, combined with oxygen breathing, significantly improves the radiation response of two experimental tumors in vivo through improved tumor oxygenation. We also demonstrate that pentoxifylline does not directly radiosensitize EMT6 cells in vitro and does not modify the tumor radiation response when administered postirradiation to solid EMT6 tumors. Our findings confirm that preirradiation administration of pentoxifylline can improve radiation efficacy, but suggest that its role as a postirradiation modifier of treatment response, reported by others, may be tumor-specific.     

Bioactivation of mitomycin C by xanthine dehydrogenase from EMT6 mouse mammary carcinoma tumors.

BACKGROUND: Mitomycin C is an antineoplastic antibiotic requiring bioactivation to an alkylating species or to an intermediate capable of generating oxygen radicals for its toxic effect. The enzymes responsible for the in vivo activation of mitomycin C have been proposed to include NADPH-cytochrome-c reductase, DT-diaphorase, and xanthine oxidase. PURPOSE: In this study, xanthine dehydrogenase, an enzyme structurally similar to xanthine oxidase, was assessed for its ability to activate mitomycin C. Partially purified xanthine dehydrogenase, from EMT6 mouse mammary tumors, was investigated for its ability to bioactivate mitomycin C under both aerobic and hypoxic conditions. METHODS: We conducted this analysis by measuring mitomycin C-induced oxygen consumption, alkylating potential, and mitomycin C consumption and metabolite formation as determined by high-pressure liquid chromatography analysis. RESULTS: Bioactivation of mitomycin C by xanthine dehydrogenase under both aerobic and hypoxic conditions gave rise to the formation of a metabolite, 2,7-diaminomitosene. Formation of this metabolite and alkylating ability were greater under hypoxic than under aerobic conditions and were increased when the pH was decreased from 7.4 to 6.0. Mitomycin C consumption was the same under both aerobic and hypoxic conditions and was independent of pH. Oxygen consumption studies showed that xanthine dehydrogenase-activated mitomycin C consumed oxygen at a much lower rate than xanthine oxidase-activated mitomycin C. CONCLUSIONS: Xanthine dehydrogenase-activated mitomycin C appears to be a good alkylating species but a relatively poor generator of reactive oxygen when compared with xanthine oxidase activation under aerobic conditions. IMPLICATION: Xanthine dehydrogenase may play an important role in the bioactivation of mitomycin C to an alkylating species under both aerobic and hypoxic conditions.     

Effect of DL-buthionine-S,R-sulfoximine on the growth of EMT6 and RIF mouse tumors

The response of murine EMT6 and RIF tumors to DL-buthionine-S, R-sulfoximine (BSO), a glutathione (GSH) depletor, given either as a single dose, continuous oral administration, or in multiple doses, was determined with the use of a tumor-growth-delay assay. BSO consistently caused significant tumor growth delay in EMT6 tumors, which reached 5-7 days (two doubling times) after a single BSO dose (40 mumol/kg or 4 mmol/kg), 3 days (one doubling time) with continuous oral administration (20 mM), and 9 days (three doubling times) with daily BSO administrations (4 mmol/kg) starting at the time of tumor inoculation. Growth inhibition persisted after discontinuation of BSO treatment. Some complete tumor regressions were observed. Only slight tumor growth delay (one doubling time) was observed in RIF tumors at all treatment modes. No direct correlation was observed between tumor GSH content and the effects on tumor growth. In vitro BSO pretreatment (2 mM, 24 hr) of EMT6 tumor cells prior to tumor inoculation, which reduced cellular GSH levels to 34% of controls, did not influence subsequent tumor growth. Pretreatment of mice with BSO (4 mmol/kg, 1 daily sc injection for 7 days) prior to tumor inoculation led to a reduction of tumor takes by 25% when compared to 100% tumor takes in untreated mice. These data imply a BSO-induced change in the host response to tumor development.     

Lymphocytic infiltration and cytotoxicity under hypoxic conditions in the EMT6 mouse mammary tumor

Infiltration of lymphocytes, neutrophils and macrophages was evaluated in hypoxic and well-oxygenated areas of the EMT6 mouse mammary adenocarcinoma, by in vivo staining with the fluorescent dye Hoechst 33342 followed by cell sorting on the basis of fluorescence intensity. Tumors were grouped by days post-injection (days 11-14, 15-17 and 20-27). As lymphocytes are the only host cell population in this tumor model to possess lytic activity against EMT6 tumor cells, the ability of sensitized T lymphocytes to lyse syngeneic EMT6 cells was examined under conditions of varying oxygen concentrations. Infiltrating lymphocytes were detected to the same extent in cell fractions from both areas in all tumors. In contrast, neutrophils were found in significantly higher percentages in the hypoxic population than in the well-oxygenated cell fraction of all but the largest tumors. Macrophages were present in significantly higher percentages in the well-oxygenated fraction than in the hypoxic fraction of day-11 to -14 tumors. Extreme radiobiological hypoxia (0% O2) resulted in a significant decrease in T-cell-mediated lysis of EMT6 tumor cells, compared to lysis in room air (20% O2), but lysis was not impaired under conditions of mild radiobiological hypoxia (1% O2). Our study indicates that host-cell infiltration into areas of differing oxygenation may be quantitated via in situ Hoechst staining followed by cell sorting; in the EMT6 tumor, lymphocytes appear to infiltrate hypoxic areas to the same extent as well-oxygenated areas, and T-lymphocyte killing of syngeneic tumor cells is significantly reduced, although still present, under these hypoxic conditions.     

Use of a perfluorochemical emulsion to improve oxygenation in a solid tumor

The ability of the perfluorochemical emulsion, Fluosol-DA, to oxygenate the naturally-occurring hypoxic cells of solid tumors was examined. BALB/c mice bearing EMT6 mouse mammary tumors were given in a single i.v. injection of 0.015 ml/g of Fluosol, and were then treated with 95% O2/5% CO2 for 30 min before irradiation with graded doses of X rays. The pretreatment increased the cytotoxic effect of the radiation, as assayed by colony formation in vitro. The dose-response curve for the combination regimen was parallel to that for radiation alone, but was offset towards lower survivals. This change is consistent with the change which would be expected if the pretreatment decreased the proportion of hypoxic cells in the tumors. Studies with unoxygenated perfluorochemical emulsions in vivo and in vitro and studies with O2/CO2 alone in vivo suggested that the survival curve changes did not reflect either direct cytotoxic effects of the perfluorochemical emulsion or the O2/CO2-breathing, or radiosensitizing or physiologic effects of either agent alone. The data reported here suggest that oxygenated perfluorochemical emulsions may decrease the proportion of hypoxic cells in solid animal tumors and encourage further preclinical evaluation of these agents as potential adjuvants to radiotherapy.     

Effects of glutathione and ethylxanthate on mitomycin C activation by isolated rat hepatic or EMT6 mouse mammary tumor nuclei

Mitomycin C (MC) activation to a reactive species was studied in nuclei isolated from rat liver and EMT6 tumor cells. Both preparations were similar in the rate of 4-(p-nitrobenzyl)pyridine (NBP) alkylation by MC and the levels of NADPH-cytochrome P-450 reductase. MC activation by both hepatic and EMT6 cell nuclei was inhibited by the presence of O2 and by heat inactivation. NADPH was preferred over NADH as the source of reducing equivalents by both types of isolated nuclei. MC activation to alkylating metabolites was not affected when EDTA or diethylenetriaminepentaacetic acid, two Fe2+ chelating agents, was present in the incubation system with either preparation of isolated nuclei. Glutathione (1 and 5 mM) and N-acetylcysteine (1 and 10 mM) both inhibited MC alkylation of NBP in nuclear preparations from rat liver and EMT6 tumor cells by 50-60%. Ethylxanthate (1 mM) effectively inhibited the MC alkylation of NBP by hepatic nuclei but was unable to inhibit MC alkylation of NBP by tumor cell nuclei. At 100 mM, ethylxanthate produced a slight stimulation in the rate of MC alkylation of NBP. These data are consistent with the hypothesis that MC activation in EMT6 tumor cells proceeds via a one electron reduction pathway which is inhibitable by glutathione but not inhibitable by ethylxanthate. Hepatic nuclei are apparently able to activate MC by either a one- or two-electron pathway.     

Hypoxia in cancer: significance and impact on clinical outcome

Hypoxia, a characteristic feature of locally advanced solid tumors, has emerged as a pivotal factor of the tumor (patho-)physiome since it can promote tumor progression and resistance to therapy. Hypoxia represents a “Janus face” in tumor biology because (a) it is associated with restrained proliferation, differentiation, necrosis or apoptosis, and (b) it can also lead to the development of an aggressive phenotype. Independent of standard prognostic factors, such as tumor stage and nodal status, hypoxia has been suggested as an adverse prognostic factor for patient outcome. Studies of tumor hypoxia involving the direct assessment of the oxygenation status have suggested worse disease-free survival for patients with hypoxic cervical cancers or soft tissue sarcomas. In head & neck cancers the studies suggest that hypoxia is prognostic for survival and local control. Technical limitations of the direct O₂ sensing technique have prompted the use of surrogate markers for tumor hypoxia, such as hypoxia-related endogenous proteins (e.g., HIF-1α, GLUT-1, CA IX) or exogenous bioreductive drugs. In many—albeit not in all—studies endogenous markers showed prognostic significance for patient outcome. The prognostic relevance of exogenous markers, however, appears to be limited. Noninvasive assessment of hypoxia using imaging techniques can be achieved with PET or SPECT detection of radiolabeled tracers or with MRI techniques (e.g., BOLD). Clinical experience with these methods regarding patient prognosis is so far only limited. In the clinical studies performed up until now, the lack of standardized treatment protocols, inconsistencies of the endpoints characterizing the oxygenation status and methodological differences (e.g., different immunohistochemical staining procedures) may compromise the power of the prognostic parameter used.     

Immunological studies on mouse mammary tumors. II. Characterization of tumor-specific antibodies against mouse mammary tumors

After injection of isografted mammary tumor MM2 sensitized with rabbit antiserum, resistance was induced in C3H/He mice after repeated challenges with MM2. The serum taken from these mice was found to be cytotoxic against MM2 cells. The serum also inhibited the outgrowth of transplant of primary tissue culture cells of spontaneous mammary tumor of C3H/He mice. A series of transplanted mammary tumors recently converted into ascitic form in our laboratory (MM3, MM4-1, MM4-2, MM4-3, MM6, MM8, MM9, MM11, MM12 and MM13), and Ehrlich ascites tumor were found to be susceptible to this serum, as tested by trypan blue uptake in vitro. Outgrowth of these tumors was also inhibited when tumor premixed with this serum was injected. No cytotoxic effect was observed against normal mouse mammary gland cells of a C3H/He mouse. Sera obtained from hyperimmunized syngeneic C3H/He mice were able to fix complement with MM2 tumors. They were partially inactivated by heating at 56° C and by treatment with 2-mercaptoethanol. After gel filtration through Sephadex G-200, complement fixing and cytotoxic activity were found in both 19S and 7S fractions. The 7S fractions, after DEAE cellulose column chromatography, gave a precipititin line at the IgG position in immunoelectrophoresis. From the above evidence, it is concluded that the target cells of the cytotoxic factors of this serum are primary cultured cells or isografted cells of mammary tumors of C3H/He mice. The cytotoxic factors present in the serum are considered to be antibodies against isografts of mammary tumors in C3H/He mice.     

Phase I and pharmacokinetic study of the novel redox-active agent, motexafin gadolinium, with concurrent radiation therapy in patients with locally advanced pancreatic or biliary cancers

Purpose: To determine the maximum tolerated dose and dose-limiting toxicity (DLT) of the novel anticancer agent, motexafin gadolinium (MGd), administered concurrently with radiation therapy (RT) in patients with locally advanced pancreatic or biliary tumors. The pharmacokinetics of MGd were also evaluated. Methods: Cohorts of three to six patients were treated with escalating doses of MGd, administered three times per week for a total of 16 doses concurrent with RT. The dose of RT was fixed at 5,040 cGy, and given in 28 fractions, from Monday to Friday of every week. Plasma MGd concentrations were measured by high performance liquid chromatography. Results: Eight patients were treated at dose level 1 (2.9 mg/kg), with one DLT (grade 3 fever). Three patients were treated at dose level 2 (3.6 mg/kg), and two DLTs were noted. One DLT was grade 3 nausea and vomiting (N/V), and the other was grade 3 skin toxicity. The most common toxicity was N/V. There were no objective responses. The median survival was 6 months. The MGd plasma concentration versus time profile in each patient was best fit by a two-compartment, open, linear model. There was minimal accumulation of MGd in plasma with the three-times/week dosing schedule. Simulation of the time course of MGd in the peripheral compartment indicated that maximal MGd concentrations of 1–2 μmol/kg occurred between 4 and 6 h after MGd infusion. Conclusion: Dose level 1 (2.9 mg/kg of MGd) is the recommended dose for combination with (RT) in phase II studies for locally advanced pancreatic and biliary cancers. Patient tolerance might be improved by modification of the RT schedule and antiemetic prophylaxis.     

Effects of hypoxia on tumor metabolism

Rapidly growing tumors invariably contain hypoxic regions. Adaptive response to hypoxia through angiogenesis, enhanced glucose metabolism and diminished but optimized mitochondrial respiration confers survival and growth advantage to hypoxic tumor cells. In this review, the roles of hypoxia, the hypoxia inducible factors, oncogenes and tumor suppressors in metabolic adaptation of tumors are discussed. These new insights into hypoxic metabolic alterations in tumors will hopefully lead us to target tumor bioenergetics for the treatment of cancers.     

Modulation of the cytotoxicity of mitomycin C to EMT6 mouse mammary tumor cells by dicoumarol in vitro

Aerobic and hypoxic cultures of EMT6 mouse mammary tumor cells were used to study the effects of dicoumarol (DIC) on the cytotoxicity of mitomycin C (MC). DIC protected aerobic cells from MC toxicity, but sensitized hypoxic cells to the cytotoxic actions of this antibiotic. Survival curves for cells treated with 1.5 microM MC +/- 100 microM DIC for different periods of time under aerobic or hypoxic conditions showed that DIC acted as a dose-modifying agent, that is, an agent which changed the slopes, but not the shapes, of the MC survival curves. Experiments that examined the effects of the DIC concentration on the modulation of MC cytotoxicity revealed significant perturbations in MC toxicity with a DIC concentration of 100 microM and increased sensitization/protection with increasing levels of DIC. DIC altered the toxicity of MC only when it was present during exposure of the cells to MC. Treatment with DIC before or after (but not during) MC did not alter the amount of cytotoxicity. Addition of DIC to cell cultures seconds before the addition of MC was as effective as addition of DIC 30 min to 2 h before MC. Taken together, these findings suggest that DIC reversibly inhibits one or more enzymes involved in the activation and inactivation of MC, and that this modulation of the enzymatic processing of MC alters the cytotoxicity of the drug.     

Effects of RSR13 and oxygen on the cytotoxicity of cisplatin and carboplatin to EMT6 mouse mammary tumor cells in vitro and in vivo

Purpose RSR13, 2-[4-[2-[(3,5-dimethylphenyl)amino]-2-oxoethyl]phenoxy]-2-methylpropanoic acid monosodium salt, allosterically modifies hemoglobin to increase tumor pO₂, increases the effect of radiation in animal tumor models, and is in phase III clinical trials as an adjuvant to radiotherapy. Cisplatin and carboplatin, two commonly used anticancer drugs have been used in combination with radiotherapy. Some studies have suggested that the cytotoxic effects of these drugs are altered in hypoxia. This study tested whether RSR13 plus oxygen breathing increased the cytotoxicity of cisplatin and carboplatin in vivo.Methods Solid EMT6 tumors in BALB/c Rw mice were treated with cisplatin (5–30 g/g) or carboplatin (5–200 g/g) along with 150 g/g RSR13 in combination with oxygen breathing. Tumor cell survival was assayed using clonogenic assays. The effects of pre- and posttreatments with RSR13 and oxygen breathing on the cytotoxicity of cisplatin or carboplatin were also examined. To assess whether RSR13 had direct effects on the cytotoxicity of the drugs, exponentially growing monolayers of EMT6 mouse mammary carcinoma cells were treated with graded concentrations of cisplatin or carboplatin for 2 h along with simultaneous (2 h) RSR13 treatments or with prolonged (22 h) pre- or posttreatment incubations with 100 M RSR13.Results Single or multiple treatments with 150 g/g RSR13 plus oxygen breathing had no effect on the viability of cells in EMT6 tumors in mice. After treatment with cisplatin or carboplatin, the tumor cell survival tended to be lower in oxygen-breathing mice especially at higher doses of cisplatin. Treatment with RSR13 plus oxygen breathing beginning 15 min before administration of the alkylating agents did not alter the cytotoxicity of cisplatin or carboplatin from that seen with oxygen breathing alone. Pretreatment with RSR13 plus oxygen at 22 and 14 h prior to administration of either cisplatin or carboplatin did not alter the effect of either alkylating agent. Treatment with RSR13 plus oxygen breathing beginning 15 min before administration of the alkylating agents and lasting for 2 or 5 h did not alter the cytotoxicity of either drug from that seen with oxygen breathing alone. The cytotoxicity of cisplatin was not altered by treatment with oxygen alone or with RSR13 plus oxygen for 5 h after cisplatin injection. For carboplatin, treatment with oxygen alone and with RSR13 plus oxygen for 5 h after injection increased to similar extents the response of the tumor cells compared to that seen with assays at 2 h. Neither short simultaneous treatments, prolonged pretreatment incubations, nor prolonged posttreatment incubations with RSR13 altered the survival of EMT6 cells in cultures treated with cisplatin or carboplatin.Conclusions These findings indicate that RSR13 in combination with oxygen breathing does not alter the cytotoxicity of cisplatin or carboplatin when used simultaneously, as a pretreatment or as a posttreatment in vitro or in vivo. Our in vivo findings indicate trends that support previous findings that cisplatin is more cytotoxic to well-oxygenated tumor cells than to hypoxic tumor cells, and that this effect can be improved by improving tumor oxygenation, but the differences seen in our studies did not achieve statistical significance.     

The disaggregation,separation and identification of cells from irradiated and unirradiated EMT6 mouse tumors

A mixture of enzymes has been used to disaggregate experimental mouse tumor #6 (EMT6) tumors grown in BALB/c mice. Cell yields of 5 × 10⁷-10⁸ dye-excluding cells per gram of tumor and in vitro clonogenicities of about 10% have been achieved. The STAPUT procedure, which differentiates cells primarily according to size by sedimentation at unit gravity, has been used to separate two main populations of cells, those which are host derived and those which are clonogenic in vitro and in vivo. The separated cells were 45% EMT6, 35% macrophages and 20% lymphocytes. The host cells may have some antitumor (EMT6) properties which can be assayed in vitro. The effects of in vivo radiation have been assayed in vitro. The size distributions of hypoxic tumor cells (those which survive 2000 tad of in vivo irradiation) was similar to well-oxygenated ones and this suggests that EMT6 tumor cells may not be severely hypoxic for long times.