Uptake Rate of Cationic Mitochondrial Inhibitor MKT-077 Determines Cellular Oxygen Consumption Change in Carcinoma Cells

<div><h3>Objective</h3><p>Since tumor radiation response is oxygen-dependent, radiosensitivity can be enhanced by increasing tumor oxygenation. Theoretically, inhibiting cellular oxygen consumption is the most efficient way to increase oxygen levels. The cationic, rhodacyanine dye-analog MKT-077 inhibits mitochondrial respiration and could be an effective metabolic inhibitor. However, the relationship between cellular MKT-077 uptake and metabolic inhibition is unknown. We hypothesized that rat and human mammary carcinoma cells would take up MKT-077, causing a decrease in oxygen metabolism related to drug uptake.</p> <h3>Methods</h3><p>R3230Ac rat breast adenocarcinoma cells were exposed to MKT-077. Cellular MKT-077 concentration was quantified using spectroscopy, and oxygen consumption was measured using polarographic electrodes. MKT-077 uptake kinetics were modeled by accounting for uptake due to both the concentration and potential gradients across the plasma and mitochondrial membranes. These kinetic parameters were used to model the relationship between MKT-077 uptake and metabolic inhibition. MKT-077-induced changes in oxygen consumption were also characterized in MDA-MB231 human breast carcinoma cells.</p> <h3>Results</h3><p>Cells took up MKT-077 with a time constant of ∼1 hr, and modeling showed that over 90% of intracellular MKT-077 was bound or sequestered, likely by the mitochondria. The uptake resulted in a rapid decrease in oxygen consumption, with a time constant of ∼30 minutes. Surprisingly the change in oxygen consumption was proportional to uptake rate, not cellular concentration. MKT-077 proved a potent metabolic inhibitor, with dose-dependent decreases of 45–73% (p = 0.003).</p> <h3>Conclusions</h3><p>MKT-077 caused an uptake rate-dependent decrease in cellular metabolism, suggesting potential efficacy for increasing tumor oxygen levels and radiosensitivity <em>in vivo</em>.</p> </div

Nitric Oxide Synthase Inhibition Enhances the Antitumor Effect of Radiation in the Treatment of Squamous Carcinoma Xenografts

This study tests whether the nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NNA), combines favorably with ionizing radiation (IR) in controlling squamous carcinoma tumor growth. Animals bearing FaDu and A431 xenografts were treated with L-NNA in the drinking water. IR exposure was 10 Gy for tumor growth and survival studies and 4 Gy for ex vivo clonogenic assays. Cryosections were examined immunohistochemically for markers of apoptosis and hypoxia. Blood flow was assayed by fluorescent microscopy of tissue cryosections after i.v. injection of fluorospheres. Orally administered L-NNA for 24 hrs reduces tumor blood flow by 80% (p<0.01). Within 24 hrs L-NNA treatment stopped tumor growth for at least 10 days before tumor growth again ensued. The growth arrest was in part due to increased cell killing since a combination of L-NNA and a single 4 Gy IR caused 82% tumor cell killing measured by an ex vivo clonogenic assay compared to 49% by L-NNA or 29% by IR alone. A Kaplan-Meyer analysis of animal survival revealed a distinct survival advantage for the combined treatment. Combining L-NNA and IR was also found to be at least as effective as a single i.p. dose of cisplatin plus IR. In contrast to the in vivo studies, exposure of cells to L-NNA in vitro was without effect on clonogenicity with or without IR. Western and immunochemical analysis of expression of a number of proteins involved in NO signaling indicated that L-NNA treatment enhanced arginase-2 expression and that this may represent vasculature remodeling and escape from NOS inhibition. For tumors such as head and neck squamous carcinomas that show only modest responses to inhibitors of specific angiogenic pathways, targeting NO-dependent pro-survival and angiogenic mechanisms in both tumor and supporting stromal cells may present a potential new strategy for tumor control

Multi-parametric assessment of the anti-angiogenic effects of liposomal glucocorticoids

Inflammation plays a prominent role in tumor growth. Anti-inflammatory drugs have therefore been proposed as anti-cancer therapeutics. In this study, we determined the anti-angiogenic activity of a single dose of liposomal prednisolone phosphate (PLP-L), by monitoring tumor vascular function and viability over a period of one week. C57BL/6 mice were inoculated subcutaneously with B16F10 melanoma cells. Six animals were PLP-L-treated and six served as control. Tumor tissue and vascular function were probed using MRI before and at three timepoints after treatment. DCE-MRI was used to determine Ktrans, ve, time-to-peak, initial slope and the fraction of non-enhancing pixels, complemented with immunohistochemistry. The apparent diffusion coefficient (ADC), T2 and tumor size were assessed with MRI as well. PLP-L treatment resulted in smaller tumors and caused a significant drop in Ktrans 48 h post-treatment, which was maintained until one week after drug administration. However, this effect was not sufficient to significantly distinguish treated from non-treated animals. The therapy did not affect tumor tissue viability but did prevent the ADC decrease observed in the control group. No evidence for PLP-L-induced tumor vessel normalization was found on histology. Treatment with PLP-L altered tumor vascular function. This effect did not fully explain the tumor growth inhibition, suggesting a broader spectrum of PLP-L activities

Mechanisms underlying early tumor reoxygenation after irradiation and radiosensitizing properties of anti-inflammatory drugs : consequences of their oxygen effect on radiotherapy time schedule

Tumor oxygenation is a key factor of the radiotherapy treatment efficacy because the efficacy for killing cells in oxygenated conditions is three times more effective than in hypoxic conditions. This tumor oxygenation is a balance between oxygen supply and oxygen consumption rate by cells. We made the hypothesis that a reoxygenation of the tumor could occur in the early time after irradiation, or early after administration of anti-inflammatory or glucocorticoid drugs. In the first part of the thesis, we investigated the early reoxygenation effect in tumors, the mechanisms responsible for this effect and its consequences for tumor radiosensitivity. We demonstrated that tumor oxygenation increases rapidly after irradiation and remains high for several days. The maximal reoxygenation time was achieved 3-4 hours after irradiation. At this time, other hemodynamic parameters were studied in order to determine the underlying mechanisms. It was demonstrated that oxygen consumption and oxygen delivery played a role in this early reoxygenation, since we observed an important decrease in oxygen consumption and an increase in blood flow after irradiation. Contrary to the late reoxygenation effect, the early reoxygenation effect was not NO-mediated. We also found that inflammatory vasodilatation played a role in the early oxygen effect. Finally, we demonstrated that two irradiations of 9 Gy, when the second was applied at the maximal reoxygenation time, was more effective than one irradiation of 18 Gy. In the second part of the thesis, we investigated the impact of Non steroidal anti-inflammatory drugs (NSAIDs). In particular, we studied the effects of a selective COX-2 inhibitor (NS-398) on tumor oxygenation. The underlying mechanisms and the impact on radiosensitivity were also studied. We demonstrated that all NSAIDS increased tumor oxygenation rapidly after the administration. At the time of maximal reoxygenation (30 minutes after administration), the oxygen effect was demonstrated to be due to a decrease in tumor oxygen consumption. This decrease in consumption was probably due to the uncoupling of mitochondrial respiration, since this effect was described for NSAIDs in the literature. On the other hand, tumor perfusion was decreased at the same time. Finally, the regrowth delay was much larger (i.e., treatment was more effective) when irradiation was applied at the maximal reoxygenation time. This increase in treatment effectiveness was comparable to that observed for carbogen. The last part of the thesis focused on the effect of glucocorticoid administration on tumor oxygenation. The tumor oxygenation was increased after the administration of these drugs, with maximal reoxygenation time occurring after 30 minutes. At this time the perfusion and the tumor oxygen consumption rate were significantly decreased. The effect on oxygen consumption could be correlated to an effect on the mitochondrial respiratory chain, both via a direct effect on specific mitochondrial complexes or via prostaglandin inhibition (as prostaglandins play important role in mitochondrial respiration). Moreover, this latter effect could explain the surprisingly analogy of timing in oxygenation between glucocorticoids and NSAIDs. Finally, when irradiation was applied at the maximal reoxygenation time, the regrowth delay was much larger (i.e., treatment was more effective) than for irradiation aloneThèse de doctorat en sciences pharmaceutiques (FARM 3) --UCL, 200

Argutissime subtiles et fecunde questiones phisicales magistri Petri de Bruxellis alias Crokart, ... in octo libros Phisicorum et in tres de Anima ... Aristotelis

Marca de Jean Petit en port.Port. a 2 tinta

Argutissime subtiles et fecunde questiones phisicales magistri Petri de Bruxellis alias Crokart ordinis predicatorum In octo libros phisicorum et in tres De animaipsius omnium philosophorum facile principijs Aristotelis

Marca de ed. en port.Texto a 2 col.Inic. grab

Argutissime subtiles et fecunde questiones phisicales magistri Petri de Bruxellis alias Crokart ordinis predicatorum In octo libros phisicorum et in tres De anima ipsius omnium philosophorum facile principijs Aristotelis

A Res. 44/3/1

Preclinical safety and antitumor efficacy of insulin combined with irradiation

BACKGROUND AND PURPOSE: We have previously reported that insulin significantly enhances tumor oxygenation (pO(2)) and increases radiation-induced tumor regrowth delay in experimental models. Considering the large radiosensitizing effect, clinical trials might be envisioned. The aim of the present pre-clinical study was to obtain a more complete set of safety and efficacy data which would further justify the commencement of such clinical trials. MATERIAL AND METHODS: Toxicity on normal (early and late-responding) tissues was measured by the intestinal crypt regeneration assay and the late leg contracture assay. Efficacy in terms of enhancement of pO(2) (measured by in vivo EPR oximetry) and increase in radiation-induced tumor regrowth delay was evaluated with a dose-response study on mice bearing FSaII fibrosarcoma. RESULTS: The effect on regrowth delay was directly correlated with the effect on the tumor pO(2), with a maximal effect using 400 mU kg(-1) insulin. Importantly, there was no increase in the radiation toxicity for normal tissues. Finally, we found that the hypoglycaemia induced by insulin can be corrected by simultaneous glucose infusion without modification of efficacy. CONCLUSION: Insulin here demonstrated a therapeutic gain and a lack of toxicity to normal tissues. The results of this study fully justify further larger preclinical assays such as the use of fractionated irradiation and a tumor control dose assay, before determining the utility of insulin as a radiosensitizer for human patients in the clinic