The effect of the nitroxide pirolin on oxidative stress induced by doxorubicin and taxanes in the rat brain.

The anticancer drugs doxorubicin (DOX), paclitaxel (PTX) and docetaxel (DTX) have been proven to induce oxidative stress (OS)-dependent side-effects in non-targeted tissues. In normal conditions, the blood-brain barrier (BBB) prevents these drugs from penetrating into the brain. However, some studies have demonstrated that small amounts of DOX can penetrate the brain via an oxidatively impaired BBB and cause damage, which suggests that including antioxidants in chemotherapy could possibly protect the brain against the toxicity of anticancer drugs. We investigated whether DOX, DTX and PTX can induce oxidative damage in rat brains in vivo and whether inclusion of the nitroxyl antioxidant Pirolin (PL) to DOX/taxane chemotherapy can protect the brain from the OS toxicity of these drugs. Wistar rats received i.p. a single dose (10 mg/kg b.w.) of DOX, DTX, PTX or PL alone or a combination of a drug + PL. After four days, the rats were anesthetized, the brains were excised, homogenized and used for the measurements of lipid peroxidation (LPO), thiol groups, activities of antioxidant enzymes, DNA damage and tumor necrosis factor-α (TNF-α), neuronal nitric oxide synthase (nNOS) and poly (ADP-ribose) polymerase-1 (PARP-1) expression. The results were analyzed using the Kruskal-Wallis and Conover-Inman tests or ANOVA and the Tukey-Kramer test. Doxorubicin, PTX and DTX induced OS, DNA damage and changes in expression of TNF-α, nNOS and PARP-1 in the rat brain. Pirolin alone increased LPO, manganese superoxide dismutase (MnSOD) and catalase (CAT) activities and the expression of PARP-1 but decreased TNF-α expression. PL, in combination with anticancer drugs, partially protected the rat brain against the toxic effects of DOX and taxanes. The best protective effects of PL were obtained with PTX. Pirolin partially attenuated brain damage caused by DOX/taxanes, highlighting its potential application in protecting the brain against DOX-, DTX- and PTX-evoked OS.

Oxidative stress induced in rat liver by anticancer drugs doxorubicin, paclitaxel and docetaxel.

PURPOSE: Oxidative stress generated by anticancer drugs in non-targeted tissues, is considered as a significant factor responsible for their severe side effects, e.g. cardiotoxicity, neurotoxicity and hepatotoxicity. Lack of data on the effect of concurrent administration of commonly used anticancer drugs: doxorubicin (DOX), paclitaxel (PTX) and docetaxel (DTX) on normal tissue, prompted us to examine the markers of oxidative stress in the liver of rats treated with these drugs. MATERIAL/METHODS: Male Wistar rats of average weight 200 g were injected intraperitoneally (i.p.) with 10 mg/kg of body weight (b.w.) of DOX, PTX and DTX. The drugs were given alone or in combinations DOX+taxane. The activities of superoxide dismutase (SOD), catalase (CAT), low molecular weight and total thiols and thiobarbituric acid-reactive substances (TBARS) were estimated. RESULTS: Combination of two drugs generated greater changes than single agents. Concurrent administration of DOX and PTX increased SOD activity and TBARS, decreased the amount of low molecular weight and total thiols, but did not cause any changes in the activity of catalase. Combination of DOX and DTX induced similar changes except for the activity of catalase, which decreased after the treatment. Of the three drugs only DTX significantly decreased the activity of SOD. However, both taxanes increased the activity of catalase. Although a decrease in concentration of -SH groups, depletion of glutathione and an increase of TBARS were observed after treatment with single drugs, the changes were not statistically significant. CONCLUSION: Concurrent administration of DOX and taxane induced enhanced oxidative stress in comparison to single drugs, which suggests their synergistic prooxidant mode of action in liver.

Nitroxide pirolin reduces oxidative stress generated by doxorubicin and docetaxel in blood plasma of rats bearing mammary tumor.

Combination of doxorubicin (DOX) and docetaxel (DTX) is clinically effective against many drug-refractory cancers, nevertheless, enhanced side effects, e.g. cardiotoxicity related to oxidative damage of tissue macromolecules is observed. Nitroxides represent an attractive class of synthetic compounds to ameliorate DOX-DTX toxicity in non-targeted tissues due to their antioxidant and iron-oxidizing properties. The aim of the study was to define the ability of 3-carbamoylpyrroline nitroxyl derivative pirolin (PL) to mitigate oxidative damage to blood plasma proteins and lipids induced by DOX-DTX chemotherapy in Sprague-Dawley rats bearing DMBA-induced mammary tumor. Additionally we also evaluated: i) pro-oxidant and antioxidant activity of pirolin administered as a single agent according to different regimens and ii) differences in biomarkers of the oxidative stress between healthy rats and rats with DMBA-induced mammary tumors. The extent of oxidative stress was evaluated on the basis of its foremost biomarkers: thiol and carbonyl groups, lipid peroxidation products (hydroperoxides, TBARS), activity of antioxidant defense enzyme superoxide dismutase (SOD) and non-enzymatic antioxidant capacity (NEAC). We have found that pirolin alone displayed dual, antioxidant and pro-oxidant activity depending on the regimen of treatment. Daily treatment for 2 weeks increased the amount of thiols, and decreased the protein carbonyl groups. Three administrations of pirolin at 3-week intervals did not influence thiol content but increased hydroperoxides, TBARS and carbonyl groups. Chemotherapy employing DOX-DTX combination caused considerable oxidative stress in the plasma. Significant and dose-dependent oxidative damage to lipids and proteins with concomitant thiol depletion were evident in treated animals. Drugs also increased SOD activity and NEAC. Association of pirolin with DOX-DTX chemotherapy resulted in a partial amelioration of oxidative stress generated by anticancer drugs. This study indicates that a nitroxyl compound pirolin applied as a single agent in vivo can display both antioxidant and pro-oxidant properties but in conjunction with DOX-DTX it is able to protect partially blood plasma against oxidative stress generated by chemotherapy. The outcome, however, seems to be highly dependent on the ratio between the doses of employed anticancer drugs and the nitroxide.

Pharmacological doses of melatonin reduce the glycosaminoglycan level within the infarcted heart scar.

The aim of the study was to define the effect of pharmacological doses of melatonin, an agent known to be a scavenger of reactive oxygen species, on the extracellular matrix composition (glycosaminoglycans and collagen) in the infarcted heart scar. Rats were administered with melatonin at doses of 300 µg/100 g b.w. or 3 mg/100 g b.w. once daily (between 5:00 and 6:00 in the afternoon) or with 1.5 mg/100 g b.w. twice daily (between 8:00 and 9:00 in the morning and additionally between 5:00 and 6:00 in the afternoon). The levels of collagen, glycosaminoglycans (GAG) and some oxidative stress markers (lipid oxidation, the content of sulphydryl groups in proteins and glutathione) were evaluated. In the second part of the experiment, cells were isolated from the scar, identified as myofibroblasts, cultured and treated with melatonin at concentrations ranging from 10⁻7 M to 10⁻¹° M. The pineal indoleamine was seen to reduce the GAG content of the scar, while the collagen content of the scar remained unchanged. A 10⁻7 M concentration of melatonin caused an increase in the GAG level in the myofibroblast cultures, while lower concentrations (10⁻8 M-10⁻¹° M) of pineal indoleamine were not effective. Melatonin decreased lipid oxidation and increased the sulphydryl groups of total proteins and glutathione, which suggests its antioxidative activity in the applied doses. The present study shows that pharmacological doses of melatonin reduce the GAG level in an infarcted heart scar. Since the mechanism of GAG content reduction cannot be explained by direct action of the pineal indoleamine on myofibroblasts in the myocardial infarction scar, we hypothesise that changes in GAG content could be indirectly induced by melatonin, that is caused by changes in regulatory systems or reduction of the inflammatory reaction in the area of the infarction. In addition, this paper shows that long-term treatment with melatonin of rats affected by myocardial infarction may reduce oxidative stress in the infarction area.

Different effectiveness of piperidine nitroxides against oxidative stress induced by doxorubicin and hydrogen peroxide.

The piperidine nitroxides Tempamine and Tempace have been studied for their effect on doxorubicin (DOX) and hydrogen peroxide (H(2)O(2)) cytotoxicity in immortalized B14 cells, a model for neoplastic phenotype. The significance for nitroxide performance of the substituent in position 4 of the piperidine ring was evaluated. The cells were exposed to DOX/H(2)O(2) alone or in combination with the nitroxides Tempamine or Tempace. Two other piperidine nitroxides, Tempo and Tempol, were used for comparison. All the nitroxides except Tempamine modestly reduced DOX cytotoxicity. Tempamine evoked a biphasic response: at concentrations lower than 200 micromol/L the nitroxide decreased DOX cytotoxicity, while at concentrations higher than 200 micromol/L, it enhanced DOX cytotoxicity. In contrast to Tempo and Tempol, Tempamine and Tempace ameliorated hydrogen peroxide cytotoxicity, but none of the nitroxides influenced TBARS stimulated by hydrogen peroxide. The cytoprotective effect of Tempace, Tempo and Tempol in DOX-treated cells correlated with the inhibition of DOX-induced lipid peroxidation. The bioreduction rates of the investigated nitroxides differed significantly and were variously affected by DOX depending on the nitroxide substituent. In combination with DOX, Tempo and Tempol were reduced significantly more slowly, while no influence of DOX on Tempamine and Tempace bioreduction was observed. Our results suggest that the structure of the 4-position substituent is an important factor for biological activity of piperidine nitroxides. Among the investigated nitroxides, Tempace displayed the best protective properties in vitro but Tempamine was the only nitroxide that potentiated cytotoxicity of DOX and did not influence DOX-induced lipid peroxidation. However, this nitroxide showed different performance depending on its concentration and conditions of oxidative stress.

Mechanisms of induction of apoptosis by anthraquinone anticancer drugs aclarubicin and mitoxantrone in comparison with doxorubicin: relation to drug cytotoxicity and caspase-3 activation.

We examined molecular events and morphological features associated with apoptosis induced by anthraquinone anticancer drugs aclarubicin, mitoxantrone and doxorubicin in two spontaneously immortalized cell lines (NIH 3T3 and B14) in relation to cytotoxicity of these drugs. The investigated cells showed similar sensitivity to aclarubicin but different sensitivity to doxorubicin and mitoxantrone: mitoxantrone was the most cytotoxic drug in both cell lines. All three drugs triggered both apoptosis and necrosis but none of these processes was positively correlated with their cytotoxicity. Apoptosis was the prevalent form of cell kill by aclarubicin, while doxorubicin and mitoxantrone induced mainly the necrotic mode of cell death. The extent and the timing of apoptosis were strongly dependent on the cell line, the type of the drug and its dose, and were mediated by caspase-3 activation. A significant increase in caspase-3 activity and the percentage of apoptotic cells, oligonucleosomal DNA fragmentation, chromatin condensation and formation of apoptotic bodies was observed predominantly in B14 cells. NIH 3T3 cells showed lesser changes and a lack of DNA fragmentation. Aclarubicin was the fastest acting drug, inducing DNA fragmentation 12 h earlier than doxorubicin, and 24 h earlier than mitoxantrone. Caspase-3 inhibitor Ac-DEVD-CHO did not show any significant effect on drug cytotoxicity and DNA nucleosomal fragmentation.

Cytotoxicity of daunorubicin in trisomic (+21) human fibroblasts: relation to drug uptake and cell membrane fluidity.

The influence of daunorubicin (DNR) on survival of human normal (S-126) and trisomic, with respect to chromosome 21 (T-164; S-240), skin fibroblasts and some parameters related to it, such as intracellular drug accumulation, distribution and interaction with cell membrane, were studied. The in vitro growth-inhibition assay indicated that DNR was less cytotoxic for trisomic than for normal cells. Comparison of kinetic parameters and intracellular distribution of this compound showed that the uptake and the amount of intracellular free DNR were greater in normal than in trisomic cells. Contrary to this, there were no significant differences between the amount of DNA-bound drug in both types of cells. TMA-DPH and 12-AS fluorescence anisotropy measurements demonstrated that DNR decreased lipid fluidity in the inner hydrophobic region of plasma membrane in both cell types, but did not influence the fluidity of the outer surface of membrane. We conclude that fibroblasts derived from individuals affected with Down's syndrome are better protected from the damage induced by DNR than normal cells.

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.

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.

Antiproliferative and cytotoxic effects of N,N-diethylaminoethyl ethers of flavanone oximes: a comparison with anthracycline(s), anthraquinone and nitroxides action.

Since flavanone oximes derivatives (ethers) have been shown to modulate the growth of Yoshida Sarcoma cells in vivo and to induce apoptosis, the effects of these substances on immortalized cell lines growth were examined. Cell viability and sensitivity to investigated substances was measured by the modified tetrazolium salt (MTT) assay. The antiproliferative effects were expressed as IC50 and IC90, respectively. There were very substantial differences in the dose-dependency of the observed antiproliferative and cytotoxic effects. The structure-activity relationship was evident and revealed that the substitution at B-ring of molecule seems to be an important factor in flavanone oxime (ether) potency. Compared to anticancer drugs (doxorubicin, aclarubicin and mitoxantrone) flavanone oximes displayed cytotoxicity at considerably higher concentrations. The antiproliferative action of the investigated model nitroxides depended on the free radical part of the molecule. N-hydroxy derivative (reduced cation form) did not influence cells proliferation and nor display any cytotoxicity at the applied range about 60 times higher than those of flavanone derivatives. Taken together it seems reasonable to suggest that flavanone oxime(s) (ethers) as compared with antracycline(s), anthraquinone and nitroxides might be especially good candidates for in the future development of new therapeutic techniques.

Anticancer potential of N,N-diethylaminoethyl ethers of flavanone oximes: a comparison with mitoxantrone action on rat Yoshida sarcoma cells in vivo.

This study was performed to evaluate the anticancer abilities of four biologically active N,N-diethylaminoethyl ethers of flavanone oximes against rat Yoshida Sarcoma cells in vivo, and to investigate the mechanism(s) involved. The effects were compared with those of anthraquinone drug (mitoxantrone) action. The presented results provide the first evidence that all the investigated substances induce programmed cell death (apoptosis) of Yoshida Sarcoma cells in vivo. On interpretative grounds, the administration of investigated flavanone derivatives in the promotion phase of the disease led to both growth inhibition (cell cycle perturbation) and apoptosis. A correlation was found between structure of the substituent(s) at B-ring of substances and the revealed anticancer activity. The data suggest that flavanone derivatives (oxime ethers) besides their antiradical, antioxidant and radioprotector properties observed before, may act as promising anticancer agents acting in the promotion phase of disease. This finding prompted us to consider the development of a new strategy: modulation of effects using combination therapy involving mitoxantrone and flavanone oximes.

Changes in plasma membrane fluidity of immortal rodent cells induced by anticancer drugs doxorubicin, aclarubicin and mitoxantrone.

The aim of this study was to examine the effect of three structurally different anticancer drugs-the pro-oxidative anthracyclines doxorubicin (DOX) and aclarubicin (ACL), and antioxidative anthraquinone mitoxantrone (MTX) on the fluidity of plasma membrane of immortalized rodent fibroblasts using fluorescence spectroscopy and electron spin resonance (ESR) techniques. Two kinds of fluorescent probes (TMA-DPH and 12-AS) and spin labels (5-DS and methyl-12-DS) were used to monitor fluidity in the hydrophobic core and in the polar headgroup region of the lipid bilayer. Immortalized hamster B14 and NIH 3T3 mouse fibroblasts were exposed to DOX, ACL and MTX. We demonstrate that these drugs influence predominantly the hydrophobic core of the lipid bilayer, inducing significant decrease in its fluidity at low concentrations (2-5 microM). A decreased membrane fluidity at the surface of the lipid bilayer was observed only at a higher concentration (20 microM) of the drugs, which indicates that DOX, ACL and MTX intercalate mainly into the hydrophobic core of the membrane, thereby perturbing its structure.

TTT, a novel free radical spin trap, induces apoptosis in sarcoma Yoshida cells in vivo: comparative investigations of its cytotoxicity in vitro.

In light of our previous SAR studies on nitroxides acting as less toxic anticancer agents, antioxidants and radioprotectors, we designed and tested, in vivo an in vitro, a new triradical spin trap -N,N',N"-tris-(l-oxyl-2,2,6,6-tetramethylpiperidine-4-yl)-1,3, 5-tnazine-2,4,6-triamine (TTT). The ability of TTT to act as an antitumor agent in vivo was investigated in pharmacological tests. The administration of TTT to rats bearing 3 day-old Sarcoma Yoshida (promotion phase of the disease) led to induction of apoptotic cell(s) death. Our results clearly indicated the suggested involvement of the free radical moiety of piperidine ring(s), thus indicating that the anticancer activity of nitroxide(s) may involve its intracellular redox reactions. To assess the relationship between the apoptotic effects of TTT in vivo and its possible cytotoxicity, we determined the relative antiproliferative and cytostatic potential of TTT in vitro as compared with this of the anticancer drugs: doxorubicin, aclarubicin and mitoxantrone, currently used in clinical practice in Poland. We found that TTT inhibits the growth and proliferation of two immortalized cell types-hamster B14 cell line and mouse NIH 3T3 fibroblasts which we used as a model for neoplastic phenotype, in a dose-dependent manner.

Changes in DNA supercoiling in fibroblasts cultured in the presence of hydralazine.

We have analyzed changes in the supercoiling of nucleoid DNA of murine fibroblasts cultured in the presence of hydralazine. The entire DNA attached to the nuclear matrix was extracted from the cells and sedimented in neutral sucrose density gradients containing ethidium bromide. Nucleoids from cells treated with hydralazine responded to increasing ethidium bromide concentrations in a different way than those from control cultures. That is, supercoiled loops of DNA unwound with lower concentrations of ethidium bromide sedimented less rapidly than those of control cells, indicating that hydralazine reduced the degree of DNA supercoiling. Also, nucleoids from the drug-treated cells resisted the transition from relaxed to positive supercoiling at higher concentrations of ethidium bromide. Changes in nucleoid DNA supercoiling correlated directly with the dose of hydralazine in the fibroblast culture.

Single-strand breaks and repair in nuclear DNA in the presence of hydralazine assayed by the nucleoid technique.

The nucleoid sedimentation assay was used to study hydralazine-induced DNA structural changes and repair in the fibroblasts cultured in vitro. The drug induced a dose dependent loss in negative DNA supercoiling due to the physical breakage of the DNA. Relaxation of supercoiled DNA resulted in the nucleoids sedimenting with lower velocities than those of undamaged control cultures. Repair incubation of the cells did not cause the restoration of DNA supercoiling to control level. Unsuccessful repair of DNA damaged by hydralazine may result in maintaining the damaged DNA in the cell which could have immunologic consequences.

Development of murine epidermal cell lines which contain an activated rasHa oncogene and form papillomas in skin grafts on athymic nude mouse hosts.

We have developed four murine epidermal cell lines which form squamous papillomas when grafted to athymic nude mice in a reconstituted skin. Two of the lines, SP-1 and BP-4, were derived from pools of papillomas produced on SENCAR and BALB/c mouse skin, respectively, by initiation with 7,12-dimethylbenz(a)anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate. Line 308 was derived from BALB/c mouse skin initiated in vivo with 7,12-dimethylbenz(a)anthracene, culture of the epidermal cells, and selection of cells resistant to Ca2+-induced terminal differentiation. Line LC14 was derived from untreated, cultured newborn BALB/c mouse primary epidermal cells which spontaneously developed resistance to Ca2+-induced terminal differentiation. Each line has an activated rasHa gene with a mutation within codon 61. Cells from all four lines, in contrast to normal primary epidermal cells, survive in medium with Ca2+ levels greater than 0.1 mM. Clonal growth studies in culture showed a unique growth pattern for each of the four lines in medium with 1.4 mM and 0.05 mM Ca2+, with or without 12-O-tetradecanoylphorbol-13-acetate. Early passage cells of these lines should provide a valuable resource for detecting genes or genetic alterations which complement an activated ras gene to cause malignant conversion and for studying the biology of tumor promotion.

Effect of heparin on the porcine lymphocyte chromatin--II. Comparative study of sedimentation of chromatin DNA and isolated DNA.

1. Sedimentation of chromatin DNA and isolated deproteinized DNA was compared in neutral and alkaline sucrose density gradients after incubation of chromatin or DNA with various concentrations of heparin. 2. Irrespective of the molecular weight of DNA, an increase in the sedimentation constant of DNA was found with increasing concentration of the polyanion employed.