Elena Monti

Role of iron in anthracycline cardiotoxicity: new tunes for an old song?

The clinical use of anticancer anthra‐cyclines is limited by the development of a distinctive and life‐threatening form of cardiomyopathy upon chronic treatment. Commonly accepted mechanistic hypotheses have assigned a pivotal role to iron, which would act as a catalyst for free radical reactions and oxidative stress. Although perhaps involved in acute aspects of anthracycline cardiotoxicity, the role of free radical‐based mechanisms in long‐term effects has been challenged on both experimental and clinical grounds, and alternative hypotheses independent of iron and free radicals have flourished. More recently, studies of the role of C‐13 hydroxy metabolites of anthracyclines have provided new perspectives on the role of iron in the cardio‐toxicity of these drugs, showing that such metabolites can impair intracellular iron handling and homeostasis. The present review applies a multisided approach to the critical evaluation of various hypotheses proposed over the last decade for the role of iron in anthracycline‐induced cardiotoxicity. The main goal of the authors is to build a unifying pattern that would both account for hitherto unexplained experimental observations and help design novel and more rational strategies toward a much‐needed improvement in the therapeutic index of anthracyclines.—Minotti, G., Cairo, G., Monti, E. Role of iron in anthracycline cardiotoxicity: new tunes for an old song? FASEB J. 13, 199–212 (1999)

Ruthenium-arene complexes of curcumin: X-ray and density functional theory structure, synthesis, and spectroscopic characterization, in vitro antitumor activity, and DNA docking studies of (p-cymene)Ru(curcuminato)chloro.

The in vitro antiproliferative activity of the title compound on five tumor cell lines shows preference for the colon-rectal tumor HCT116, IC(50) = 13.98 μM, followed by breast MCF7 (19.58 μM) and ovarian A2780 (23.38 μM) cell lines; human glioblastoma U-87 and lung carcinoma A549 are less sensitive. A commercial curcumin reagent, also containing demethoxy and bis-demethoxy curcumin, was used to synthesize the title compound, and so (p-cymene)Ru(demethoxy-curcuminato)chloro was also isolated and chemically characterized. The crystal structure of the title compound shows (1) the chlorine atom linking two neighboring complexes through H-bonds with two O(hydroxyl), forming an infinite two-step network; (2) significant twist in the curcuminato, 20° between the planes of the two phenyl rings. This was also seen in the docking of the Ru-complex onto a rich guanine B-DNA decamer, where a Ru-N7(guanine) interaction is detected. This Ru-N7(guanine) interaction is also seen with ESI-MS on a Ru-complex-guanosine derivative.

Nitroxide TEMPOL impairs mitochondrial function and induces apoptosis in HL60 cells

The piperidine nitroxide TEMPOL induces apoptosis in a number of tumor cell lines through free radical‐dependent mechanisms. As mitochondria play a major role in apoptosis as both source and target for free radicals, the present study focuses on mitochondrial effects of TEMPOL in a human promyelocytic leukemic cell line (HL‐60). On 24‐h exposure to TEMPOL, the following alterations were observed: 1) decrease in both the intracellular and mitochondrial glutathione pools; 2) impairment of oxidative phosphorylation; and 3) decrease in mitochondrial membrane potential. In addition, TEMPOL was found to specifically target complex I of the respiratory chain, with minor effects on complexes II and IV, suggesting that mitochondrial effects might play a role in TEMPOL‐induced oxidative stress and apoptosis, and that TEMPOL might sensitize tumor cells to the pro‐apoptotic effects of cytotoxic agents. J. Cell. Biochem. 82: 271–276, 2001.

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.

Role of the lung resistance-related protein (LRP) in the drug sensitivity of cultured tumor cells.

Drug resistance, one of the major obstacle in the successful anticancer therapy, can be observed at the outset of therapy (intrinsic resistance) or after exposure to the antitumor agent (acquired resistance). To gain a better insight into the mechanisms of intrinsic resistance we have analyzed two human cell types derived from untreated tumors: MCF-7 breast cancer and A549 non small cell lung cancer (NSCLC). We have examined: the cytotoxic effect induced by doxorubicin (DOX); the time course of drug accumulation by flow cytometry and intracellular drug distribution by confocal microscopy; the expression and distribution of proteins related to anthracycline resistance, such as P-gp (P-glycoprotein), MRP1 (multidrug resistance-associated protein) and LRP (lung resistance-related protein). The cytotoxicity assays showed that A549 cells were less sensitive than MCF-7 cells to the DOX treatment in agreement with the different DOX uptake. Moreover, while in A549 cells DOX was mostly located in well defined intracytoplasmic vesicles, in MCF-7 cells it was mainly revealed inside the nuclei. The analysis of P-gp and MRP expression did not show significant differences between the two cell lines while a high expression of LRP was detected at the nuclear envelope and cytoplasmic levels in A549 cells. These findings suggest that the lower sensitivity to DOX treatment showed by lung carcinoma cells could be ascribed to drug sequestration by LRP inside the cytoplasmic compartments.

Intracellular P‐glycoprotein expression is associated with the intrinsic multidrug resistance phenotype in human colon adenocarcinoma cells

The 2 clones, LoVo 5 and LoVo 7, derived from untreated LoVo WT human colon adenocarcinoma cells and exhibiting different sensitivity to doxorubicin (DOX), were compared in order to identify possible determinants of intrinsic drug resistance. A multidrug resistant variant cell line, selected from LoVo WT cells by continuous exposure to DOX (LoVo DX), was also included in the study. Analysis of the expression and organization of cytoskeletal elements by flow cytometry and fluorescence microscopy evidenced a positive correlation between vimentin expression and DOX resistance in LoVo 7 and LoVo DX cells, whereas differences in actin, tubulin or cytokeratin did not seem to relate to drug response. The expression and localization of different drug transporters commonly implicated in drug resistance, i.e., the MDR1 gene product P‐glycoprotein (P‐gp), the multidrug resistance‐related protein MRP and the lung resistance‐related protein LRP were also investigated by means of flow cytometry and fluorescence microscopy, following labeling with specific monoclonal antibodies. Surface expression of P‐gp was only detected in LoVo DX cells, which also exhibited increased MRP and LRP protein levels. However, significant amounts of P‐gp were found at intracellular sites in the intrinsically resistant LoVo 7 clone. Modulation of P‐gp function by cyclosporin A was found to alter DOX accumulation and efflux in LoVo 7 cells, indicating that intracellular P‐gp plays a functional role in drug trafficking and suggesting possible implications in determining the intrinsic resistance displayed by this clone. Int. J. Cancer 87:615–628, 2000.

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.

Role of the p53/p21 system in the response of human colon carcinoma cells to Doxorubicin

BackgroundColon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX) and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels.MethodsThe relationship between p53 and p21 protein levels and the cytotoxic effect of DOX was investigated, by MTT assay and western blot analysis, in HCT116 (p53-positive) and HT29 (p53-negative) colon cancer cells. We then assessed the effects of DOX in two isogenic cell lines derived from HCT116 by abrogating the expression and/or function of p53 and p21 (HCT116-E6 and HCT116 p21-/-, respectively). Finally, we evaluated the effect of pre-treatment with the piperidine nitroxide Tempol (TPL), an agent that was reported to induce p21 expression irrespective of p53 status, on the cytotoxicity of DOX in the four cell lines. Comparisons of IC50 values and apoptotic cell percentages were performed by ANOVA and Bonferronis test for independent samples. C.I. calculations were performed by the combination Index method.ResultsOur results indicate that, in the colon carcinoma cell lines tested, sensitivity to DOX is associated with p21 upregulation upon drug exposure, and DOX cytotoxicity is potentiated by pre-treatment with TPL, but only in those cell lines in which p21 can be upregulated.Conclusionsp21 induction may significantly contribute to the response of colon adenocarcinomas cells to DOX treatment; and small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms.

The nitroxide tempol induces oxidative stress, p21WAF1/CIP1, and cell death in HL60 cells

The antiproliferative effect of Tempol, a stable nitroxide free radical, was investigated on the p53-negative human leukemia cell line HL60. A concentration- and time-dependent inhibition of cell growth was observed that appears to be due to induction of apoptosis. Involvement of oxidative stress is indicated by a concentration-dependent increase in intracellular peroxides and a parallel decrease in total cellular glutathione; in addition, increased survival rates were observed in cells simultaneously treated with Tempol and the antioxidant N-acetylcysteine. Tempol did not affect the relative levels of Bax and Bcl2, whereas p21(WAF1/CIP1) was enhanced in a concentration- and time-dependent fashion; this effect was partially inhibited by N-acetylcysteine, was maintained for up to 8 h after Tempol removal, and seemed to depend on continuing protein synthesis. The increase in p21(WAF1/CIP1) was accompanied by a parallel accumulation of cells in the G(1) phase of the cycle and by a decrease in the 110 kDa form of pRb. Our results suggest that p53-independent induction of p21(WAF1/CIP1) mediates the antiproliferative effect of Tempol; on the basis of this observation, the nitroxide could be proposed as an useful adjunct to the treatment of p53-deficient tumors, which are often refractory to standard chemotherapy.

Study of in vitro and in vivo effects of the piperidine nitroxide Tempol--a potential new therapeutic agent for gliomas.

The identification of novel therapeutic agents for the management of malignant gliomas represents an area of active research. Here, we show that Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; TPL), a stable nitroxide free radical, inhibits the growth of C6 glioma cells both in vitro and in vivo. Morphological features of apoptosis were apparent in C6 cells following in vitro treatment with TPL. Cell death was preceded by dose-dependent increase in p21(WAF1/CIP1) expression, without apparent stabilisation of the TP53 gene product. When C6 cells were grown as xenografts in nude mice, treatment with TPL induced a significant dose-dependent decrease in tumour growth, without signs of general or organ toxicity. Tumours from treated mice showed an increase in the number of apoptotic cells and a decrease in the rate of neo-vascularisation compared with tumours from control mice. Our findings suggest a potential use for TPL as a novel antiproliferative agent for the treatment of malignant gliomas.