Ruthenium(II)-Arene Curcuminoid Complexes as Photosensitizer Agents for Antineoplastic and Antimicrobial Photodynamic Therapy: In Vitro and In Vivo Insights.

Photodynamic therapy (PDT) is an anticancer/antibacterial strategy in which photosensitizers (PSs), light, and molecular oxygen generate reactive oxygen species and induce cell death. PDT presents greater selectivity towards tumor cells than conventional chemotherapy; however, PSs have limitations that have prompted the search for new molecules featuring more favorable chemical-physical characteristics. Curcumin and its derivatives have been used in PDT. However, low water solubility, rapid metabolism, interference with other drugs, and low stability limit curcumin use. Chemical modifications have been proposed to improve curcumin activity, and metal-based PSs, especially ruthenium(II) complexes, have attracted considerable attention. This study aimed to characterize six Ru(II)-arene curcuminoids for anticancer and/or antibacterial PDT. The hydrophilicity, photodegradation rates, and singlet oxygen generation of the compounds were evaluated. The photodynamic effects on human colorectal cancer cell lines were also assessed, along with the ability of the compounds to induce ROS production, apoptotic, necrotic, and/or autophagic cell death. Overall, our encouraging results indicate that the Ru(II)-arene curcuminoid derivatives are worthy of further investigation and could represent an interesting option for cancer PDT. Additionally, the lack of significant in vivo toxicity on the larvae of Galleria mellonella is an important finding. Finally, the photoantimicrobial activity of HCurc I against Gram-positive bacteria is indeed promising.

Phototoxicity of two positive-charged diaryl porphyrins in multicellular tumor spheroids.

Photodynamic therapy (PDT) is a clinically approved cancer treatment in which reactive oxygen species are formed only when three harmless components, a photosensitizer (PS), light and molecular oxygen, are present at the same time, leading to cell death. Most of the PSs were tested on monolayer cells, but differences between 2D cells and solid tumors significantly limit the value of in vitro PDT studies, whereas the use of 3D spheroid might be more suitable for drug development and preclinical drug testing for PDT. In a previous work we have shown that two positive-charged diaryl porphyrins (2 and 4) were more potent than the corresponding neutral molecules (1 and 3) on a panel of 2D-cultured cancer cell lines. In the present study the photodynamic effects of these molecules have been evaluated on HCT116 and MCF7 spheroids. Induction of apoptotic and necrotic cell death, and generation of reactive oxygen species (ROS) have been also evaluated, along with accumulation and localization of PSs into spheroids. Our findings indicate that 2 and 4 retained their phototoxic effects also in 3D spheroids; furthermore, they were more potent than 1 and 3 and as potent as Foscan (m-THPC), the most successful PS approved for clinical PDT of cancer, used as reference. Although further aspects of their mechanisms of action need to be addressed, our results strongly suggest a potential in vivo photodynamic application of 2 and 4, considering that spheroids represent a more realistic indicator of in vivo therapeutic efficacy than 2D cell lines.

New BODIPYs for photodynamic therapy (PDT): Synthesis and activity on human cancer cell lines.

A new class of compounds based on the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene core, known as BODIPYs, has attracted significant attention as photosensitizers suitable for application in photodynamic therapy (PDT), which is a minimally invasive procedure to treat cancer. In PDT the combination of a photosensitizer (PS), light, and oxygen leads to a series of photochemical reactions generating reactive oxygen species (ROS) exerting cytotoxic action on tumor cells. Here we present the synthesis and the study of the in vitro photodynamic effects of two BODIPYs which differ in the structure of the substituent placed on the meso (or 8) position of the dipyrrolylmethenic nucleus. The two compounds were tested on three human cancer cell lines of different origin and degree of malignancy. Our results indicate that the BODIPYs are very effective in reducing the growth/viability of HCT116, SKOV3 and MCF7 cells when irradiated with a green LED source, whereas they are practically devoid of activity in the dark. Phototoxicity occurs mainly through apoptotic cell death, however necrotic cell death also seems to play a role. Furthermore, singlet oxygen generation and induction of the increase of reactive oxygen species also appear to be involved in the photodynamic effect of the BODIPYs. Finally, it is worth noting that the two BODIPYs are also able to exert anti-migratory activity.

In Vivo Effects of A Pro-PO System Inhibitor on the Phagocytosis of Xenorhabdus Nematophila in Galleria Mellonella Larvae.

Xenorhabdus nematophila is a Gram-negative bacterium symbiont of the entomopathogen nematode Steinernema carpocapsae whose immunosuppressive properties over host's immune response have been thoroughly investigated. In particular, live X. nematophila actively impairs phagocytosis in host's hemocytes through the secretion of inhibitors of eicosanoids synthesis. In this article we have investigated the cell surface structural features of X. nematophila responsible for the elusion from phagocytosis. To this end we have studied the uptake of heat-killed (hk), fluorescein isothiocyanate (FITC)-labeled X. nematophila by phagocytes from both a host insect and a mammalian species. In vitro dead X. nematophila passively resists engulfment by insect hemocytes without impairing the phagocytosis machinery whereas, unexpectedly, in vivo a significant phagocytosis of dead X. nematophila was observed. X. nematophila in vivo phagocytosis was increased by the co-injection of the specific inhibitor of pro-phenoloxidase (PO) system phenylthiourea (PTU), even if these effects were not observed in in vitro tests. Furthermore, biochemical modifications of X. nematophila cell wall implement in vivo phagocytosis, suggesting that this bacterium avoid phagocytosis because the ligand of phagocytic receptors is somehow buried or disguised in the cell wall. Finally, dead X. nematophila escapes engulfment even by human phagocytes suggesting that X. nematophila could be a useful model to investigate escape from phagocytosis by mammalian macrophages.

Vitamin D Deficiency has a Negative Impact on Cetuximab-Mediated Cellular Cytotoxicity against Human Colon Carcinoma Cells.

BACKGROUND: Hypovitaminosis D is associated with an adverse prognosis in colon cancer patients, possibly due to the effects of the vitamin on the immune system. Antibody-dependent cell-mediated cytotoxicity (ADCC) significantly contributes to the anti-tumor effects of monoclonal antibodies, including cetuximab, an epidermal growth factor receptor (EGFR)-targeted monoclonal antibody that is frequently added to chemotherapy in the treatment of colon cancer. OBJECTIVE: The present study evaluates the association between vitamin D serum levels and the ability of ex vivo NK cells to support cetuximab-mediated ADCC in colon cancer cell lines. METHODS: Blood samples were obtained from 124 healthy volunteers and serum vitamin D was determined by RIA. NK cells were isolated from each sample and added to human colorectal carcinoma cells with or without cetuximab, and ADCC was assessed using a colorimetric lactate dehydrogenase assay. RESULTS: Correlation analysis indicates a significant, gender- and age-independent association between vitamin D levels and cetuximab-induced ADCC on HT29 cells, where NK cells from samples with vitamin D < 20 ng/mL are significantly less efficient in inducing ADCC. A confirmatory study on two additional colon cancer cell lines yielded similar results. CONCLUSIONS: These data suggest that vitamin D supplementation in vitamin-deficient/insufficient colorectal cancer patients could improve cetuximab-induced ADCC.

The cisplatin-based Pt(iv)-diclorofibrato multi-action anticancer prodrug exhibits excellent performances also under hypoxic conditions.

Multi-action cisplatin-based mono- (1) and di-clofibric acid (2) Pt(iv) "combo" derivatives were synthesized via both traditional and microwave assisted procedures. The two complexes offered very good performances (IC50 values in a nanomolar range) on a panel of human tumor cell lines, including the highly chemoresistant malignant pleural mesothelioma ones. Moreover, both 1 and 2 bypass the cisplatin resistance. Indeed, cisplatin and clofibric acid, the metabolites of the Pt(iv) → Pt(ii) intracellular reduction, proved to act synergistically. The adjuvant action of clofibric acid relies on the activation of peroxisome proliferator-activated receptor α (PPARα) that, in turn, decreases the level of Hypoxia-Inducible Factor-1α. Both compounds induced extensive apoptosis in tumor cells, also via oxidative stress. Finally, 2 exhibited excellent performances also under the hypoxic conditions typical of solid tumors, where cisplatin is less effective.

The BH3-mimetic obatoclax reduces HIF-1α levels and HIF-1 transcriptional activity and sensitizes hypoxic colon adenocarcinoma cells to 5-fluorouracil.

Activation of hypoxia-inducible factor (HIF)-1 is a feature of hypoxic solid tumors that has been associated with drug resistance, mainly due to disruption of Bcl-2 family dynamics. Resetting the balance in favor of proapoptotic family members is an attractive therapeutic goal that has been pursued by developing BH3-mimetic compounds. In the present study we evaluated the response of human colon adenocarcinoma cells to the BH3-mimetic obatoclax (OBX), in terms of growth arrest, apoptosis and autophagy, in the presence or absence of HIF-1α-stabilizing conditions; its possible effect on HIF-1α expression and HIF-1 activity; and the possibility to improve the response of colon cancer cells to cytotoxic chemotherapeutics by combining them with OBX. Colon cancer cell response to the BH3-mimetic was unmodified by HIF-1 activation and OBX induced a decrease in HIF-1α protein levels and HIF-1 transcriptional activity, probably by decreasing HIF-1α synthesis and facilitating a VHL-independent proteasomal degradation pathway. Finally, a chemosensitizing effect of OBX with respect to 5-fluorouracil or oxaliplatin treatment was observed, highlighting the possibility that patients with hypoxic colon tumors might benefit from combined regimens including OBX.

Study of the synthesis, antiproliferative properties, and interaction with DNA and polynucleotides of cisplatin-like Pt(II) complexes containing carcinogenic polyaromatic amines.

The chemical and biological features of two newly synthesized [PtCl2(L)(2-aminonaphthalene)] complexes (L is NH3 or 2-aminonaphthalene) were compared with those of two already reported enantiomeric complexes of formula [PtCl2(DABN)] [DABN is (R)-1,1'-binaphthyl-2,2'-diamine or (S)-1,1'-binaphthyl-2,2'-diamine]. Solution behavior, lipophilicity, cytotoxicity with regard to one colorectal (HCT116) and two ovarian (A2780 and A2780Cp8) human carcinoma cell lines, and in vitro DNA- and G-quadruplex-binding properties were evaluated. In particular, the cytotoxicity of [PtCl2(NH3)(2-aminonaphthalene)] was better than that of cisplatin for all cell lines, and rather resembled that of oxaliplatin. The solution behavior of the whole series of complexes and the absence of an evident relationship between lipophilicity and cytotoxicity seem to suggest that all these experimental parameters are probably smoothed out during the 3-day cytotoxicity experiments and do not strongly affect the half-maximal inhibitory concentrations. The results of electrophoretic studies indicate that different kinds of interaction with DNA can be involved in the mode of action of these complexes, with intercalation in double-stranded DNA and stacking on G-quadruplex DNA being strongly implicated in particular for [PtCl2(NH3)(2-aminonaphthalene)].

Synthesis and photodynamic activity of a panel of BODIPY dyes.

Eight BODIPY dyes were synthesized and used as photosensitizers (PSs) on the human colon carcinoma cell line HCT116. In this panel of molecules, the structure varies in the substituents on pyrrole 2, 6 positions and on the phenyl ring at the indacene 8 position. For these compounds relevant physico-chemical parameters, such as singlet oxygen production, fluorescent quantum yield, absorbance profile and a relative rank of lipophilicity were determined. Our results indicate that some of these novel PSs are very effective in reducing the growth/viability of HCT116 cells when irradiated with a green LED source, whereas they are practically devoid of activity in the dark, up to 5 µM. To evaluate whether cell death is induced under these conditions, flow cytometric analysis of the percentage of apoptotic and autophagic cells was performed on four molecules, chosen for their efficacy/structural characteristics. Our data indicate that phototoxicity likely occurs mainly through apoptotic cell death, whereas autophagy seems to play a minor role in determining cell fate. Furthermore, the relationship between singlet oxygen generation and the PS efficacy is confirmed, thus underscoring the importance of the heavy-atom effect and of the presence of an aryl substituent at dipyrromethene 8 (meso) position. Among the PSs here described, the most efficient BODIPY was successfully tested on three other human cancer cell lines of different tissue origin, MCF7 (breast), A2780 and A2780/CP8 (ovary, sensitive and resistant to cisplatin, respectively), yielding IC(50) values comparable to those obtained on HCT116.

Synthesis, characterization, structure, molecular modeling studies and biological activity of sterically crowded Pt(II) complexes containing bis(imidazole) ligands.

The syntheses, structures and biological evaluation of a series of cisplatin-like complexes containing bis(imidazole) derivatives - the so-called Joseph ligands - are described. Their cytotoxicity is discussed in terms of their polar surface area, rate of aquation, and lipophilicity. The X-ray crystal structure of the platinum diiodido derivative of dimethyl 2-(di(1H-imidazol-2-yl)methyl)malonate) is reported and compared to those of related systems. Molecular modeling studies are focused on the hydrogen bonding properties of such systems, and their relevance to antitumor activity.

HIF-1 as a target for cancer chemotherapy, chemosensitization and chemoprevention.

Cells in rapidly growing solid tumors are commonly exposed to chronic or intermittent hypoxia. Hypoxia can induce cell death by multiple mechanisms; however, some cells may adapt by orchestrating dramatic changes in gene expression patterns. In addition, hypoxia exerts a powerful selective pressure on tumor cells, resulting in the emergence of clonal populations whose defects in DNA repair mechanisms favor genomic instability and tumor progression, whereas disabling of apoptotic pathways makes them more resistant to both environmental stresses and therapeutic interventions. The transcriptional factor HIF-1 (Hypoxia-Inducible Factor 1) is generally considered as the major regulator of the hypoxic adaptive response, and as such it is viewed as a viable prospective target for novel pharmacologic approaches to the clinical management of solid tumors. Several agents have been identified that inhibit HIF1 transcriptional activity, and some of them are currently undergoing clinical trials, mostly based on their antiangiogenic properties. This article reviews the role played by HIF-1 in tumorigenesis and chemoresistance and provides an overview of current and prospective pharmacologic strategies designed to inhibit HIF-1 activity, emphasizing their direct and indirect effects on tumor growth, as well as their potential for chemoprevention and chemosensitization.

Changes in the two-dimensional electrophoresis pattern of the Parkinson's disease related protein DJ-1 in human SH-SY5Y neuroblastoma cells after dopamine treatment.

DJ-1 is a mitochondrial protein linked to Parkinson's disease. DJ-1 has been suggested to have several possible functions, although it has been mainly associated to oxidative stress defence. Changes in the two-dimensional electrophoresis pattern have been thoroughly described as a consequence of oxidative modification of the Cys106 residue. There is accumulating evidence supporting a specific role of DJ-1 in protecting dopaminergic neurons from dopamine itself. By exposing SH-SY5Y human neuroblastoma catecholaminergic cells to dopamine, we observed a specific increase in the most acidic forms in the DJ-1 two-dimensional electrophoresis pattern together with a significant decrease of the most basic spot. Unlike cells exposed to generic oxidative conditions, no additional shift was observed. The results are corroborated by a meta-analysis of the literature showing that in the absence of dopamine treatment the specific acidic form is underrepresented.

Antiproliferative Pt(IV) complexes: synthesis, biological activity, and quantitative structure-activity relationship modeling.

Several Pt(IV) complexes of the general formula [Pt(L)2(L')2(L'')2] [axial ligands L are Cl-, RCOO-, or OH-; equatorial ligands L' are two am(m)ine or one diamine; and equatorial ligands L'' are Cl- or glycolato] were rationally designed and synthesized in the attempt to develop a predictive quantitative structure-activity relationship (QSAR) model. Numerous theoretical molecular descriptors were used alongside physicochemical data (i.e., reduction peak potential, Ep, and partition coefficient, log Po/w) to obtain a validated QSAR between in vitro cytotoxicity (half maximal inhibitory concentrations, IC50, on A2780 ovarian and HCT116 colon carcinoma cell lines) and some features of Pt(IV) complexes. In the resulting best models, a lipophilic descriptor (log Po/w or the number of secondary sp3 carbon atoms) plus an electronic descriptor (Ep, the number of oxygen atoms, or the topological polar surface area expressed as the N,O polar contribution) is necessary for modeling, supporting the general finding that the biological behavior of Pt(IV) complexes can be rationalized on the basis of their cellular uptake, the Pt(IV)-->Pt(II) reduction, and the structure of the corresponding Pt(II) metabolites. Novel compounds were synthesized on the basis of their predicted cytotoxicity in the preliminary QSAR model, and were experimentally tested. A final QSAR model, based solely on theoretical molecular descriptors to ensure its general applicability, is proposed.

The IGFR1 inhibitor NVP-AEW541 disrupts a pro-survival and pro-angiogenic IGF-STAT3-HIF1 pathway in human glioblastoma cells.

Inappropriate activation of the IGF (insulin-like growth factor) system has been implicated in the growth and progression of a number of tumor types. Recent evidence indicates a possible role for the IGF system in modulating/mediating tumor cell response to hypoxia, a common occurrence in solid tumors, and particularly in malignant gliomas, causing tumor cells either to die, or to mount a pleiotropic adaptive response that is mainly orchestrated through activation of the hypoxia-inducible transcription factor HIF1. Experimental evidence suggests possible links between IGF- and HIF1-dependent signaling pathways, as well as a role for activated STAT3 in mediating their activities. Interestingly, igf2 is among the target genes transactivated by HIF1, thereby providing the missing link in a hypothetical autocrine self-amplifying circuit. The present study investigates the presence of the IGF-HIF1-VEGF axis in the human glioma cell line U-87 MG, and characterizes its molecular effectors. Our results show that exogenous IGF-I causes IGF1R and STAT3 activation, and increases HIF1alpha protein levels and HIF1 trascriptional activity, inducing VEGF release; a similar response, mediated by IGF-II release, is observed following HIF1alpha stabilization. The existence of an autocrine loop is confirmed by its down-regulation following inactivation of IGF1R (using the IGF1R-specific tyrosine kinase inhibitor NVP-AEW541), STAT3 (transfecting the cells with an expression vector encoding a dominant negative form of STAT3), or HIF1 (using the small molecule inhibitor YC-1). The ability of NVP-AEW541 to block this circuit could be beneficial in suppressing the growth and angiogenic potential of hypoxic glial tumors.

The relevance of polar surface area (PSA) in rationalizing biological properties of several cis-diamminemalonatoplatinum(II) derivatives.

A panel of six cis-diamminemalonatoplatinum(II) derivatives were designed and synthesized, and their physicochemical properties and in vitro biological activity were experimentally evaluated and studied in silico. All the complexes showed higher IC(50) values (> or =20 microM) than those observed for cisplatin and its malonato analogue on three different human tumor cell lines, namely A2780 ovarian carcinoma, A549 lung carcinoma, and MCF-7 breast carcinoma. In silico studies revealed that polar surface area (PSA) is the best descriptor to explain the poor biological activity observed for this series of new compounds, which in turn is likely due to poor cellular uptake. This finding is in line with general rules that assign a major role to PSA in characterizing the transport properties of drugs, in the actual case of antiproliferative metallopharmaceuticals.

Photodynamic effects of novel 5,15-diaryl-tetrapyrrole derivatives on human colon carcinoma cells.

Preliminary in vitro cytotoxicity studies on a panel of meso diaryl-substituted tetrapyrrole derivatives newly synthesized in our laboratory have shown that these compounds are photodynamically active on the human colon carcinoma cell line HCT116. In the present study, we investigate some mechanistic aspects of the photodynamic action of the most active compounds in the series, namely the 5-phenyl-15-(3-methoxyphenyl)porphyrin (1), the 5-phenyl-15-(3-hydroxyphenyl)porphyrin (2) and the 5,15-diphenylporphyrin (3). The results of the cytotoxicity studies indicate that the novel photosensitisers (PSs) are more potent in vitro than m-THPC (Foscan), a powerful PS already approved for clinical use in photodynamic therapy (PDT). A series of experiments were performed to elucidate a number of aspects in the mechanism of PS-induced phototoxicity, including, intracellular accumulation and subcellular localization of the PSs, induction of apoptosis, and generation of reactive oxygen species (ROS) and NO*. All the compounds tested exhibit similar singlet oxygen quantum yields; differential intracellular accumulation can contribute to the observed differences in phototoxicity. Flow cytometric studies indicate that all the tested compounds induce apoptosis; however, their cytotoxic effect does not seem to rely solely on this process. Generation of significant amounts of reactive oxygen species (ROS) and NO* were also observed; however, the contribution of this latter effect to the overall phototoxicity is unclear. Taken together, our observations suggest that the diaryl derivatives included in the present study could represent promising leads for the development of novel photosensitizing agents.

Effect of HIF-1 modulation on the response of two- and three-dimensional cultures of human colon cancer cells to 5-fluorouracil.

Tumour hypoxia represents a major obstacle to the success of radiotherapy and chemotherapy. The discovery that the hypoxia-inducible factor 1 (HIF-1) is a master regulator of cellular response to low oxygen led to the concept that inhibiting HIF-1 activity may sensitise hypoxic cancer cells to radiation and cytotoxic drugs. In the present study we investigate the effects of HIF-1 modulation on the response of the human colon adenocarcinoma cell line HCT116 to 5-fluorouracil (5FU). Increasing HIF-1 activity, either by exposing cells to hypoxia or by forced expression of a degradation-resistant form of HIF-1alpha, results in poor cell response to 5FU; conversely, knockdown of HIF-1alpha by RNA interference prevents hypoxia-induced resistance to 5FU. PMX290, a thioredoxin-1 inhibitor, significantly inhibits HIF-1 activity and concomitantly sensitises hypoxic cells to 5FU. These results were confirmed in HCT116 cells grown as three-dimensional spheroids, a model that more closely reproduces the hypoxic environment of solid tumours.

Linalool, a plant-derived monoterpene alcohol, reverses doxorubicin resistance in human breast adenocarcinoma cells.

Essential oils from various aromatic plants have been reported to exert chemopreventive and/or antitumor effects. In addition, a number of studies have shown the ability of chemopreventive phytochemicals to increase the sensitivity of cancer cells to conventional anticancer drugs. The success of chemotherapeutic agents is often hindered by the development of drug resistance, with multidrug resistant (MDR) phenotypes reported in a number of tumors, generally involving reduced intracellular drug accumulation due to increased drug efflux by membrane transporters. In the present study, the effects of linalool (LIN), a monoterpene alcohol found in the essential oils from many aromatic plants, on the growth of two human breast adenocarcinoma cell lines, MCF7 WT and multidrug resistant MCF7 AdrR, were investigated, both as a single agent and in combination with doxorubicin (DOX). The results reported here show that LIN only moderately inhibits cell proliferation; interestingly, however, subtoxic concentrations of LIN potentiate DOX-induced cytotoxicity and pro-apoptotic effects in both cell lines. A significant synergism can be observed in MCF7 AdrR cells, which may be due, at least in part, to the ability of LIN to increase DOX accumulation and to induce a decrease in Bcl-xL levels. In summary, the results of the present study suggest that LIN may improve the therapeutic index of anthracyclines in the management of breast cancer, especially in MDR tumors.

Inhibition of Stat3 increases doxorubicin sensitivity in a human metastatic breast cancer cell line.

Metastatic breast cancer is an incurable disease, often characterized by poor response to standard chemotherapy, which is mainly based on anthracyclines and taxanes. Thus, increasing tumor cell sensitivity to these agents is an attractive goal towards improving the clinical management of this disease. The present study investigates the effects of signal transducer and activator of transcription 3 (Stat3) inhibition on the response of the highly metastatic MDA-MB-231 human breast adenocarcinoma cell line to doxorubicin (DOX). Stat3 is a transcription factor often constitutively activated in breast tumors and cancer cell lines, and is thought to contribute to malignant transformation and progression by transactivation of a host of target genes involved in cell proliferation and survival, angiogenesis and invasiveness. Our results indicate that (a) untreated MDA-MB-231 cells express higher baseline levels of (activated) pTyr(705)Stat3, that are further upregulated following exposure to DOX, than the non-metastatic MCF-7 cell line; (b) inhibiting the Stat3 signaling pathway, by exposure to the tyrphostin AG490 (an inhibitor of the upstream activating Janus kinases), by transfection with a dominant-negative form of Stat3 or by treatment with satraplatin (a tetravalent platinum derivative that inhibits Stat3 activation), increases breast cancer cell response to the proapoptotic effect of DOX (to different extents). In addition, the latter two approaches have been shown to interfere with expression of one or more antiapoptotic proteins. Overall, these observations suggest that suppression of Stat3 signaling may provide a potential therapeutic approach to overcoming DOX resistance in metastatic breast cancer cells.

The nitroxide Tempol modulates anthracycline resistance in breast cancer cells.

The occurrence of multidrug resistance (MDR) is the major obstacle to successful anthracycline-based cancer chemotherapy. In the present study, we assessed the effects of Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, TPL), a piperidine nitroxide with growth-inhibitory properties in tumor cell lines, on a number of molecular mechanisms involved in the resistance of human breast adenocarcinoma cell lines to doxorubicin (DOX). Cytotoxicity studies in MCF-7 wildtype and their MDR variant MCF-7 Adr(R) cells showed a synergistic effect between TPL and DOX when exposure to TPL preceded or was simultaneous with DOX treatment in MCF-7 Adr(R) cells. This effect of TPL seems to be due in part to its ability to increase peroxide levels and to deplete cellular glutathione pools. In addition, TPL increased DOX accumulation in MCF-7 Adr(R) cells by interfering with P-glycoprotein-mediated DOX efflux, as evidenced using a specific antibody that recognizes the active form of the protein. TPL was also found to affect the expression levels of proteins involved in response to drug treatment (e.g., p53, bcl2, bax, p21). Taken together, our results indicate that TPL is a potential new agent that may improve the clinical effect of DOX in tumors exhibiting a MDR phenotype.