Chronic exendin-4 treatment prevents the development of cancer cachexia symptoms in male rats bearing the Yoshida sarcoma.

Cancer cachexia is the syndrome of weight loss, loss of appetite, and wasting of skeletal muscle and adipose tissue experienced by many individuals with cancer. Currently, few effective treatment and prevention strategies are available for these patients, due in part to a poor understanding of the mechanisms contributing to cachexia. Insulin resistance has been associated with cancer cachexia in epidemiological, human, and animal research. The present experiment was designed to examine the ability of Exendin-4, a GLP-1 agonist and insulin sensitizing agent, to prevent the development of cachexia symptoms in male Sprague Dawley rats bearing the Yoshida sarcoma. Following tumor implantation or sham surgery, rats were treated daily with saline or Exendin-4 (3 µg/kg body weight/day) and were monitored for tumor growth and cachexia symptoms for 21-23 days. As a result of large variability in treatment effects, data were analyzed separately for animals with large and small tumors. Exendin-4 treatment reduced tumor growth and prevented the development of cancer cachexia symptoms in animals with small, but not large, tumors. In addition, insulin levels were preserved in Exendin-4-treated tumor-bearing animals. The results of this experiment demonstrate a novel preventative therapy for cancer cachexia and a novel use of Exendin-4. Further research is necessary to determine the mechanisms through which Exendin-4 exerts these potent effects.

Vital microscopic analysis of polymeric micelle extravasation from tumor vessels: macromolecular delivery according to tumor vascular growth stage.

Particles larger than a specific size have been thought to extravasate from tumor vessels but not from normal vessels. Therefore, various nanoparticles incorporating anticancer drugs have been developed to realize selective drug delivery to solid tumors. However, it is not yet clear whether nanoparticles extravasate readily from all tumor vessels including vessels of microtumors. To answer this question, we synthesized new polymeric micelles labeled with fluorescein isothiocyanate (FITC) and injected them into the tail vein of rats with implanted skinfold transparent chambers. We also analyzed, by means of time-lapse vital microscopy with image analysis, extravasation of FITC micelles from tumor vessels at different stages of growth of Yoshida ascites sarcoma LY80. Polymeric micelles readily leaked from vessels at the interface between normal and tumor tissues and those at the interface between tumor tissues and necrotic areas. The micelles showed negligible extravasation, however, from the vascular network of microtumors less than 1 mm in diameter and did not accumulate in the microtumor. Our results suggest that we must develop a novel therapeutic strategy that can deliver sufficient nanomedicine to microtumors.

[Antitumor activity and function of S-1, a new oral tegafur-based formulation].

TS-1 (S-1), developed by the scientific theory of both potentiating antitumor activity of 5-fluorouracil (5-FU) and reducing gastrointestinal toxicity induced by 5-FU, is a new oral formulation consisting of 1 M tegafur, 0.4 M gimeracil and 1 M oteracil potassium. We investigated the antitumor efficacy of S-1 alone and in combination with other cytotoxic anticancer drugs using subcutaneously or orthotopically implanted murine and human tumors in rodents. As a single agent, S-1 showed higher antitumor activity with its low intestinal toxicity compared to continuous venous infusion 5-FU, the most effective dosing method of 5-FU, and/or to clinically available oral fluoropyrimidines such as UFT, doxyfluridine and capecitabine on various murine tumors and human tumor xenografts. Especially, it was noteworthy that S-1 as a DPD-inhibitory fluoropyrimidine markedly affected human tumor xenografts with high expression levels of DPD on which other fluoropyrimidines showed a low antitumor activity. In combination with other anticancer drugs such as CPT-11 and taxanes, S-1 exercised synergistic antitumor efficacy not only on 5-FU-sensitive tumors with low expression levels of thymidylate synthase (TS) but also on 5-FU-resistant tumors with originally higher and/or elevated levels of TS expression. As one of the reasonable mechanism of antitumor synergism by the combination, CPT-11 and taxanes were found to reduce the expression of TS in human tumor resistant to 5-FU with high expression TS levels. Throughout our preclinical antitumor studies of S-1, alone and/or in combination with other anticancer drugs, it would be expected to contribute greatly to the treatment of cancer patients.

A comparative pharmacology study between the intracolonic and oral routes of 5-FU administration in a colon cancer-bearing Yoshida sarcoma rat model.

We prepared a colon cancer-bearing Yoshida sarcoma rat model to examine the dose-response relationship of antitumor activity of intracolonically or orally administered 5-fluorouracil (5-FU; 45, 30, 20, 13, and 8 mg/kg). At doses of > or =20 mg/kg and > or =30 mg/kg, the 5-FU intracolonic and oral administration groups each showed a statistically significant difference in antitumor activity against the control group (P<0.05, Williams' test). A statistically significant dose-response relationship was noted in the two routes of administration, with an ED(50) value of 29 mg/kg. White blood cell count tended to decrease at high doses when 5-FU was administered intracolonically and showed a statistically significant decrease at doses of > or =30 mg/kg when 5-FU was administered orally. Regarding the time-course of body weight, even the 5-FU highest dose (45 mg/kg) intracolonic administration group showed no inhibited body weight increase compared to the control group. However, the 5-FU (> or =20 mg/kg) oral administration groups showed a statistically significant difference in body weight increase against the control group. These facts suggested that the intracolonic administration of 5-FU, while exhibiting more potent antitumor activity than that observed in oral administration, allows an extensive reduction in its toxicities compared to oral administration.

Preliminary study of the optimal dosing schedule for oral UFT/leucovorin chemotherapy.

BACKGROUND: We evaluated the optimal dosage schedule for combined oral chemotherapy using uracil/tegafur (UFT) and leucovorin (LV) in Yoshida sarcoma-bearing rats. MATERIALS AND METHODS: The antitumor activity and survival effect were compared between two schedules, thrice daily administration on 5 days of the week followed by 2 drug-free days (schedule A) and twice daily on 7 days of the week (schedule B). RESULTS: Significant tumor growth inhibition and improved survival rate were seen with both schedules of the UFT/LV group as compared to their respective control groups. Tumor growth inhibition was significantly greater in schedule A than schedule B. The number of survivors on day 60 after the tumor inoculation was higher in schedule A although the survival rate did not differ significantly. CONCLUSION: Administration of UFT/LV for 5 days of the week seemed to exhibit superior antitumor activity, with no increase in the incidence of adverse effects, as compared with the consecutive daily dosing schedule.

Inhibition of induced chemoresistance by cotreatment with (E)-5-(2-bromovinyl)-2'-deoxyuridine (RP101).

Induced chemoresistance leads to the reduction of apoptotic responses. Although several drugs are in development that circumvent or decrease existing chemoresistance, none has the potential to prevent or reduce its induction. Here, we present data from a drug that could perhaps fill this gap. Cotreatment of chemotherapy with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU, RP101) prevented the decrease of apoptotic effects during the course of chemotherapy and reduced nonspecific toxicity. Amplification of chemoresistance genes (Mdr1 and Dhfr) and overexpression of gene products involved in proliferation (DDX1) or DNA repair (UBE2N and APEX) were inhibited, whereas activity of NAD(P)H: quinone oxidoreductase 1 (NQO1) was enhanced. During recovery, when treatment was with BVDU only, microfilamental proteins were up-regulated, and proteins involved in ATP generation or cell survival (STAT3 and JUN-D) were down-regulated. That way, in three different rat tumor models, the antitumor efficiency of chemotherapy was optimized, and toxic side effects were reduced. Because of these beneficial properties of BVDU, a clinical pilot Phase I/II study with five human tumor entities has been started at the University of Dresden (Dresden, Germany). So far, no unwanted side effects have been observed.

Effects of the phosphodiesterase-IV inhibitor EMD 95832/3 on tumour growth and cachexia in rats bearing the Yoshida AH-130 ascites hepatoma.

Administration of the phosphodiesterase-IV inhibitor EMD 95832/3 (Merck KGaA, Darmstadt, Germany) to rats bearing the ascites hepatoma Yoshida AH-130, a highly cachectic tumour, could not prevent either the anorexia nor the massive weight loss (affecting both adipose and skeletal muscle tissues) present in the tumour-bearing animals. This compound did not have any effects on the fractional rates of protein turnover in skeletal muscle, and did not affect circulating triacylglycerols or lipoprotein lipase activity in adipose tissue. Although the administration of EMD 95832/3 did not influence tumour growth either, it did increase the number of tumour cells undergoing apoptosis. It is concluded that the drug is unable to reverse the cachectic state in this particular experimental tumour model.

A novel combretastatin A-4 derivative, AC7700, strongly stanches tumour blood flow and inhibits growth of tumours developing in various tissues and organs.

In a previous study, we used subcutaneous LY80 tumours (a subline of Yoshida sarcoma), Sato lung carcinoma, and methylcholanthrene-induced primary tumours, to demonstrate that a novel water-soluble combretastatin A-4 derivative, AC7700, abruptly and irreversibly stopped tumour blood flow. As a result of this interrupted supply of nutrients, extensive necrosis was induced within the tumour. In the present study, we investigated whether AC7700 acts in the same way against solid tumours growing in the liver, stomach, kidney, muscle, and lymph nodes. Tumour blood flow and the change in tumour blood flow induced by AC7700 were measured by the hydrogen clearance method. In a model of cancer chemotherapy against metastases, LY80 cells (2x10(6)) were injected into the lateral tail vein, and AC7700 at 10 mg x kg(-1) was injected i.v. five times at intervals of 2 days, starting on day 7 after tumour cell injection. The number and size of tumours were compared with those in the control group. The change in tumour blood flow and the therapeutic effect of AC7700 on microtumours were observed directly by using Sato lung carcinoma implanted in a rat transparent chamber. AC7700 caused a marked decrease in the tumour blood flow of all LY80 tumours developing in various tissues and organs and growth of all tumours including lymph node metastases and microtumours was inhibited. In every tumour, tumour blood flow began to decrease immediately after AC7700 administration and reached a minimum at approximately 30 min after injection. In many tumour capillaries, blood flow completely stopped within 3 min after AC7700 administration. These results demonstrate that AC7700 is effective for tumours growing in various tissues and organs and for metastases. We conclude that tumour blood flow stanching induced by AC7700 may become an effective therapeutic strategy for all cancers, including refractory cancers because the therapeutic effect is independent of tumour site and specific type of cancer.

[Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use]. / Citotoxicidad de la beta-lapachona: una o-naftoquinona con posibles usos terapéuticos.

beta-lapachone (beta-lap) is a lipophilic o-naphthoquinone isolated from the bark of the lapacho tree. Initial observations proved its capability for inhibiting growth of Yoshida tumor and Walker 256 carcinosarcoma. beta-Lap redox-cycling in the presence of reductants and oxygen yields "reactive oxygen species" (ROS: O2-, OH and H2O2) which cytotoxicity led to assume its role in beta-lap activity in cells. beta-Lap inhibited DNA synthesis in Trypanosoma cruzi as well as topoisomerases I and II, poly(ADP-ribose) polymerase (PARP) in different cells. These enzymes are essential for maintaining DNA structure. beta-Lap inhibited growth of a large variety of tumor cells including epidermoid laringeal cancer, prostate, colon, ovary and breast cancer and also different types of leukemia cells. Advances in knowledge of apoptosis ("programmed cell death") and necrosis provided useful information for understanding the mechanism of beta-lap cytotoxicity. Thiol-dependent proteases (Calpaine), kinases (e.g. c-JUN NH2-terminal kinase), caspases and nucleases are involved in beta-lap cytotoxicity. These enzymes activity, as well as ROS production by beta-lap redox-cycling, would be essential for beta-lap cytotoxicity. Diaphorase and NAD(P)H-quinone reductase, which catalyse beta-lap redox-cycling and ROS production, seem to play an essential role in beta-lap activity. On these grounds, clinical applications of beta-lap have been suggested.

Citotoxicidad de la ß-Lapachona: una O-Naftoquinona con posibles usos terapéuticos / Citotoxicity of ß-Lapachone: an O-naphthoquinone with possible therapeutic uses

La ß-lapachona (ß-lap) es una o-naftoquinona extraída de la madera del lapacho. Las observaciones iniciales mostraron su acción inhibidora del crecimiento del sarcoma de Yoshida, del carcinosarcoma de Walker 256 y del Trypanosoma cruzi. La ß-lap genera productos reactivos del oxígeno (EROS: anión superóxido, radical hidroxilo y peróxido de hidrógeno) a los que inicialmente se atribuyó su citotoxicidad. ß-lap resultó un potente inhibidor de la síntesis de ADN en T. cruzi, de las topoisomerasas I y II de la poli(ADP-ribosa) polimerasa (PARP) de diferentes orígenes, enzimas responsables de la reparación y mantenimiento de la estructura del ADN. Se investigó la citotoxicidad de ß-lap en células de cáncer epidermoide de laringe, melanoma, cáncer de ovario, de mama, de próstata, de pulmón, adenocarcinoma de colon y diferentes formas de leucemia aportando un mejor conocimiento de los mecanismos moleculares involucrados en la acción de ß-lap y su relación con los procesos de apoptosis y necrosis. Entre esos mecanismos se comprobó la activación de la calpaina, proteasa cuya actividad depende de tioles, seguida por la activación de quinasas (c-JUN), caspasas y nucleasas, que finalmente degradan al ADN y a las proteínas celulares. Una reacción importante para la actividad de la ß-lap es su reducción enzimática, especialmente por la diaforasa y la NAD(P)H-quinona reductasa, que inician la producción de EROS. La acción de ß-lap sobre células tumorales resultaría de la inhibición directa de enzimas como las topoisomerasas, PARP y el factor TNF, sumada a la acción de radicales libres generados por la ß-lap. Los efectos citostáticos de ß-lap han abierto interesantes perspectivas para la quimioterapia del cáncer. (AU)

D-24851, a novel synthetic microtubule inhibitor, exerts curative antitumoral activity in vivo, shows efficacy toward multidrug-resistant tumor cells, and lacks neurotoxicity.

N-(pyridin-4-yl)-[1-(4-chlorbenzyl)-indol-3-yl]-glyoxyl-amid (D-24851) is a novel synthetic compound that was identified in a cell-based screening assay to discover cytotoxic drugs. D-24851 destabilizes microtubules and blocks cell cycle transition specifically at G2-M phase. The binding site of D-24851 does not overlap with the tubulin binding sites of known microtubule-destabilizing agents like vincristine or colchicine. In vitro, D-24851 has potent cytotoxic activity toward a panel of established human tumor cell lines including SKOV3 ovarian cancer, U87 glioblastoma, and ASPC-1 pancreatic cancer cells. In vivo, oral D-24851 treatment induced complete tumor regressions (cures) in rats bearing Yoshida AH13 sarcomas. Of importance is that the administration of curative doses of D-24851 to the animals revealed no systemic toxicity in terms of body weight loss and neurotoxicity in contrast to the administration of paclitaxel or vincristine. Interestingly, multidrug-resistant cell lines generated by vincristine-driven selection or transfection with the Mr 170,000 P-glycoprotein encoding cDNA were rendered resistant toward paclitaxel, vincristine, or doxorubicin but not towards D-24851 when compared with the parental cells. Because of its synthetic nature, its oral applicability, its potent in vitro and in vivo antitumoral activity, its efficacy against multidrug-resistant tumors, and the lack of neurotoxicity, D-24851 may have significant potential for the treatment of various malignancies.

Citotoxidad de la beta lapachona: una o-naftoquinona con posibles usos terapeuticos / Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use

La Beta-lapachona (Beta-lap) es una o-naftoquinona extraída de la madera del lapacho. Las observaciones iniciales mostraron su acción inhibidora del crecimiento del sarcoma de Yoshida y del carcinosarcoma de Walker 256. La Beta-lap genera productos reactivos del oxígeno (ROS: anión superóxido, radical hidroxilo y peróxido de hidrógeno) a los que inicialmente se atribuyó su citotoxicidad. Beta-Lap resultó un potente inhibidor de la síntesis de ADN en T. cruzi, de la topoisomerasas I y II y de la poli(ADP-ribosa) polimerasa (PARP) de diferentes orígenes, enzimas responsables de la conservación del ADN. Se investigó la citotoxicidad de Beta-lap en células de cáncer epidermoide de laringe, melanoma, cáncer de ovario, de mama, de próstata, de pulmón, adenocarcinoma de colon y leucemia, aportando un mejor conocimiento de los mecanismos moleculares involucrados en la acción de Beta-lap y su relación con los procesos de apoptosis y de necrosis. Se comprobó la activación de la calpaina, proteasa cuya actividad depende de tioles, seguida por la activación de quinasas (c-JUN NH2 -quinasa terminal), caspasas y nucleasas, enzimas que degradan al ADN y a las proteínas celulares. Una reacción importante para la actividad de la Beta-lap es su reducción, especialmente por la diaforasa y la NAD(P)H-quinona reductasa, que inician la producción de ROS. La acción de Beta-lap sobre células tumorales resultaría de la inhibición directa de enzimas como las topoisomerasas, PARP y el factor TNF, sumada a la acción de radicales libres. Los efectos citostáticos de ß-lap han abierto interesantes perspectivas para la quimioterapia del cáncer. (Au)

Citotoxidad de la beta lapachona: una o-naftoquinona con posibles usos terapeuticos / Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use

La Beta-lapachona (Beta-lap) es una o-naftoquinona extraída de la madera del lapacho. Las observaciones iniciales mostraron su acción inhibidora del crecimiento del sarcoma de Yoshida y del carcinosarcoma de Walker 256. La Beta-lap genera productos reactivos del oxígeno (ROS: anión superóxido, radical hidroxilo y peróxido de hidrógeno) a los que inicialmente se atribuyó su citotoxicidad. Beta-Lap resultó un potente inhibidor de la síntesis de ADN en T. cruzi, de la topoisomerasas I y II y de la poli(ADP-ribosa) polimerasa (PARP) de diferentes orígenes, enzimas responsables de la conservación del ADN. Se investigó la citotoxicidad de Beta-lap en células de cáncer epidermoide de laringe, melanoma, cáncer de ovario, de mama, de próstata, de pulmón, adenocarcinoma de colon y leucemia, aportando un mejor conocimiento de los mecanismos moleculares involucrados en la acción de Beta-lap y su relación con los procesos de apoptosis y de necrosis. Se comprobó la activación de la calpaina, proteasa cuya actividad depende de tioles, seguida por la activación de quinasas (c-JUN NH2 -quinasa terminal), caspasas y nucleasas, enzimas que degradan al ADN y a las proteínas celulares. Una reacción importante para la actividad de la Beta-lap es su reducción, especialmente por la diaforasa y la NAD(P)H-quinona reductasa, que inician la producción de ROS. La acción de Beta-lap sobre células tumorales resultaría de la inhibición directa de enzimas como las topoisomerasas, PARP y el factor TNF, sumada a la acción de radicales libres. Los efectos citostáticos de ß-lap han abierto interesantes perspectivas para la quimioterapia del cáncer.

Effect of consecutive lower-dose cisplatin in enhancement of 5-fluorouracil cytotoxicity in experimental tumor cells in vivo.

It is known that cisplatin (CDDP) potentiates the cytotoxicity of 5-fluorouracil (5-FU), and that the biochemical mechanism is an increase in the intracellular reduced folate levels in the tumor cells. We investigated the effect of consecutive administration with lower-dose CDDP on intracellular accumulation of reduced folate and the activity of methionine synthase, a key enzyme in intracellular methionine synthesis. When CDDP (1 mg/kg) was administered i.p. to ascitic Yoshida sarcoma-bearing rats for 4 consecutive days, both the reduced folate levels and methionine synthase activity in the cells significantly increased, as the same as a single 5 mg/kg dose of CDDP. Furthermore, when Yoshida sarcoma-bearing rats were pre-treated with 1 mg/kg CDDP for 5 consecutive days, [14C]L-methionine incorporation into the isolated ascitic cells was significantly inhibited as compared to that in non-treated cells, suggesting that consecutive administration of lower-dose CDDP is capable of inducing the intracellular modulation of reduced folate levels and methionine synthase activity via inhibition of cellular uptake of methionine. In addition, 5-day administration of lower-dose (1 mg/kg) CDDP potentiated the antitumor effect of 5 mg/kg S-1, a new oral preparation of tegafur, given for 7 consecutive days, and this combined effect was almost similar to the antitumor effect of a combination of S-1 and a single conventional dose (5 mg/kg) of CDDP. Consecutive lower-dose CDDP also may be concluded to act as an important modulator of the enhancement of 5-FU cytotoxicity in experimental tumors.

Tissue distribution and biotransformation of potassium oxonate after oral administration of a novel antitumor agent (drug combination of tegafur, 5-chloro-2,4-dihydroxypyridine, and potassium oxonate) to rats.

S-1, a new oral 5-fluorouracil (5-FU)-derivative antitumor agent, is composed of tegafur, 5-chloro-2,4-dihydropyridine, and potassium oxonate (Oxo). Oxo, which inhibits the phosphorylation of 5-FU, is added to reduce the gastrointestinal (GI) toxicity of the agent. In this study, we investigated the tissue distribution and the metabolic fate of Oxo in rats after oral administration of S-1. Oxo was mainly distributed to the intracellular sites of the small intestines in a much higher concentration than 5-FU, but little distributed to other tissues, including tumorous ones in which 5-FU was observed after oral administration of S-1. Plasma concentration-time profiles of Oxo and its metabolites after i.v. and oral administration of S-1 revealed that Oxo was mainly converted to cyanuric acid in the GI tract. Furthermore, the analysis of drug-related radioactivity in GI contents and in vitro studies suggested that Oxo was converted to cyanuric acid by two routes, the first being direct conversion by the gut flora in the cecum, and the second, conversion by xanthine oxidase or perhaps by aldehyde oxidase after degradation to 5-azauracil (5-AZU) by the gastric acid. These results indicate that, although a part of the administered Oxo was degraded in the GI tract, Oxo was mainly distributed to the intracellular sites of the small intestines in a much higher concentration than 5-FU and that little was distributed to other tissues, including tumors. We conclude that this is the reason why Oxo suppresses the GI toxicity of 5-FU without affecting its antitumor activity.

[Tumor microcirculation and selective enhancement of drug delivery--clinical applications based on pathophysiological experiments].

Tumor tissue is composed of cancer cells (parenchyma) and tumor vessels (interstitium). Many investigators have pointed out that blood flow in tumors has a very inhomogenous distribution, and that this inhomogeneity in blood flow increases as tumors grew. This would be a certain cause of insufficient drug delivery to tumor tissues. Among the experimental evidence using Yoshida Sarcoma and Ascites Hepatomas, functional differences in microcirculation between tumor and normal tissues were found by Suzuki et al. (1977). Under hypertensive state induced by the continuous infusion of angiotensin II, tumor blood flow increased remarkably, while there was no change or decrease in blood flow in normal tissues such as the brain, bone marrow, liver and kidney. Moreover, the increase in blood flow in tumors was selective, as the mean blood pressure remained at the level of 150 mmHg. Increases were confirmed not only in many growing sites such as in the liver, muscle, subcutis, and even microfoci, but also in various kinds of xenografted human tumors and autochthonous tumors. Augmentation of the anti-tumor effects of angiotensin II-induced hypertension chemotherapy (IHC) for advanced gastric carcinoma was revealed in two randomized controlled trials (RCT-1 & 2) of collaborative study groups in Japan. The response rates were 42.9% vs 10.5% in RCT-1 and 31.3% vs 6.7% in RCT-2. The frequencies of toxicities were not statistically different. In the results of phase II studies from 1978 to 1994 (OPN-1) and 1995 to 1999 (OPN-2) for advanced gastric carcinoma (GC), the response rates were 37.9% and 35.7%. Down staging in which the conclusive stage score was lower than the score of the clinical stage, was observed in 8 out of 94 cases (19%) with primary lesions in total and in 30 patients (63%) receiving reduction surgery after IHC, since 1978. It is very important for exact evaluation after chemotherapy to understand or estimate the pathohistological changes in the tumor and its degenerated or repaired tissues, which present various clinical images. In the present study, the actual administered dose intensity of adriamycin (aDIadm) was 5.9 +/- 2.4 mg/sqm/w, and the ratio of aDIadm to the proposed DIadm of reported FAM/FAP schedules was 0.78 +/- 0.32. IHC with smaller DI could lead to a reduction in the accumulation of toxicities of anti-cancer drugs in the host. In conclusion, IHC might be applied to all kinds of tumors to enhance the chemotherapeutic effects through selective increase of drug delivery to tumors.

Evidence for peroxidative oxidation of substituted piperidine nitroxides, acting as apoptosis inducers in Yoshida Sarcoma cells in vivo.

The results presented herein clearly indicate that nitroxide derivatives--free radicals are effective as substrates for one-electron oxidation in the peroxidase cycle involving hydrogen peroxide, which have been the subject of considerable controversy. This oxidation is catalyzed enzymatically and it might occur in tumor cells (in vivo) where the level of ROS (H2O2 and O2.-) is increased. The result of this reaction involving hydrogen peroxide is the obligative formation of the oxo-ammonium cation involved in the superoxide dismutase-mimic reaction of nitroxides with superoxide and/or in reaction with H2O2 leading to superoxide formation and regeneration of the parent nitroxide molecule. The efficiency of this enzymatically catalyzed oxidation of nitroxide(s) depends on the structure of the substituent in position 4 of nitroxide ring as follows: -OCH3 > -NHCOCH3 > -NHCOCH2CH3. Notably, the reduced nitroxide salt was not substrate for peroxidatic oxidation clearly indicating the importance of the free radical moiety of the nitroxide molecule. These findings may have some relevance in the recent investigations of antioxidant properties/mechanisms of nitroxides. Based on these considerations we hypothesize that the administration of oxidizable free radical nitroxide compounds--antioxidants may be a useful strategy in the treatment and investigations of cancer diseases. An in vivo study ("Screening test of chemicals employing Yoshida Sarcoma animals") was carried out to verify whether the structure and/or the chain length of substituent of oxidizable nitroxide derivatives--antioxidants could influence their apoptotic activity. The results reported in this study are encouraging as we found a limited correlation between the molecular oxidative properties of nitroxides under study, their structure and antitumor (apoptotic) action. In conclusion, this work demonstrates that investigation of the structure-dependent oxidation of antioxidatively acting nitroxides can become a very important step in their future screening and selection for applications in vivo and in vitro.

The paradoxical apoptotic effects of novel nitroxide antioxidants on Yoshida sarcoma cells in vivo: a commentary.

Here we show for the first time that the model nitroxide derivatives, free radical or its reduced piperidinium salt, suppressed cytotoxicity of ROS (O2 and H2O2) generated outside the cells (B14 line, model for neoplastic phenotype) in ***. The nitroxides prevented the decrease in the number of *** caused by exogenous O2- and H2O2 at concentrations which were not themselves cytotoxic. In the present study, we have also shown that a very substantial difference in the cell response occurred when the model rat tumor cells (Yoshida Sarcoma ascites) were treated in vivo with six novel synthesized nitroxide antioxidants. A number of tumor cells displayed morphological characteristics of apoptosis. This effect was comparable to those observed for other nitroxyls under similar experimental conditions. Since the increase in the ROS generation followed by apoptotic changes of nuclei is the consistent recent finding in various experimental models of apoptosis, one fundamental question was raised: why nitroxide antioxidants paradoxically act as apoptosis inducers in vivo? Taking together the results presented here and in our previous works, it seems reasonable to suggest that nitroxide-antioxidants improve the endogenous "antioxidants reserve" and action can induce a reductive stress as opposed to an oxidative stress, triggering a cascade of dose-dependent processes involving indirectly an antioxidant mechanism(s) and resulting in the apoptotic death of cancer cells in vivo. The SAR (structure activity relationship) revealed that either the substituent structure at 4-position of the nitroxide ring or its oxidation state are determinant for the degree of the observed differences in the apoptotic potency of nitroxide derivates in vivo.

Lack of effect of the cytokine suppressive agent FR167653 on tumour growth and cachexia in rats bearing the Yoshida AH-130 ascites hepatoma.

Daily s.c. administration of 6 mg/kg of FR167653 (an inhibitor of the synthesis of interleukin-1 and tumour necrosis factor-alpha) to rats bearing the ascites hepatoma Yoshida AH-130 (a highly cachectic tumour) did not prevent either the anorexia or the massive weight loss - affecting both adipose tissue and skeletal muscle - present in the cachectic animals. The compound did not affect the circulating levels of triacylglycerols or other metabolites such as glucose or lactate. Nor did the administration of FR167653 influence tumour growth. It is concluded that the drug is unable to reverse the cachectic state in this particular experimental tumour model.

Tempicol-3, a novel piperidine-N-oxide stable radical and antioxidant, with low toxicity acts as apoptosis inducer and cell proliferation modifier of Yoshida Sarcoma cells in vivo.

The novel nitroxyl, Tempicol-3 (nitroxide-N-oxide) was synthesized and its capacity to act as a scavenger of hydroxyl radicals was tested. The concentration-dependent reducibility of this novel compound was also examined and compared with those of previously characterized nitroxides, Tempo and Tempace. The cytotoxicity of Tempicol-3 in vitro was measured by the modified tetrazolium assay (MTT), using, model cells for neoplastic phenotype (mouse NIH 3T3 fibroblast line). The ability of Tempicol-3 to act as an antitumor agent in vivo was also investigated in a pharmacological test, using rats bearing 3-day old Yoshida Sarcoma (promotion phase of the disease). Our results clearly indicated that Tempicol-3 acts as an effective and promising hydroxyl radical scavenger-antioxidant. Structure- and concentration-dependent bioreduction of Tempicol-3 by ascorbic acid may account for some of its biological effects, causing modulation of the antioxidant status of cells. The presence of one nitrone moiety per molecule of Tempicol-3 caused a significant decrease in nitroxide cytotoxicity as compared with Tempo, in vitro. The results clearly confirmed that the toxic effect could result either from the presence or structure of substituent(s) at position 4 of the free radical piperidine moiety. It can be stated that Tempicol-3 is a lowtoxicity nitroxide, which could be effective in providing antioxidative activity. We have also observed that lowtoxic Tempicol-3, at m.e.d. (minimal effective dose) suppressed tumorigenesis, acting as a cell proliferation modifier and apoptosis inducer in vivo. This work provides the base for further investigations on nitroxide-N-oxide derivatives since the serious question remains to be solved as to what is the molecular mechanism of action of the nitroxide-N-oxides.