Relevance of small GTPase Rac1 pathway in drug and radio-resistance mechanisms: Opportunities in cancer therapeutics.

Rac1 GTPase signaling pathway has a critical role in the regulation of a plethora of cellular functions governing cancer cell behavior. Recently, it has been shown a critical role of Rac1 in the emergence of resistance mechanisms to cancer therapy. This review describes the current knowledge regarding Rac1 pathway deregulation and its association with chemoresistance, radioresistance, resistance to targeted therapies and immune evasion. This supports the idea that interfering Rac1 signaling pathway could be an interesting approach to tackle cancer resistance.

Rho GTPases as therapeutic targets in cancer (Review).

Rho GTPases are key molecular switches controlling the transduction of external signals to cytoplasmic and nuclear effectors. In the last few years, the development of genetic and pharmacological tools has allowed a more precise definition of the specific roles of Rho GTPases in cancer. The aim of the present review is to describe the cellular functions regulated by these proteins with focus in deregulated signals present in malignant tumors. Finally, we describe the state of the art in search of different experimental therapeutic strategies with Rho GTPases as molecular targets.

Pharmacological inhibition of Rac1-PAK1 axis restores tamoxifen sensitivity in human resistant breast cancer cells.

Tamoxifen is a standard endocrine therapy for estrogen receptor positive breast cancer patients. Despite its success, development of resistance mechanisms is still a serious clinical problem. Deregulation of survival signaling pathways play a key role in tamoxifen resistance, being upregulation of Rac1-PAK1 signaling pathway one of the most important. Here, we report the development of the breast cancer cell model MCF7::C1199 having Rac1 enhanced activity with the aim of evaluating the role of Rac1 in acquired endocrine resistance. These cells not only showed distinctive features of Rac1-regulated process as increased migration and proliferation rates, but also showed that upregulation of Rac1 activity triggered a hormonal-independent and tamoxifen resistant phenotype. We also demonstrated that PAK1 activity increases in response to Tamoxifen, increasing phosphorylation levels of estrogen receptor at Ser305, a key phosphorylation site involved in tamoxifen resistance. Finally, we evaluated the effect of 1A-116, a specific Rac1 inhibitor developed by our group, in tamoxifen-resistant cells. 1A-116 effectively restored tamoxifen anti-proliferative effects, switched off PAK1 activity and decreased estrogen receptor phospho-Ser305 levels. Since combination schemes of novel targeted agents with endocrine therapy could be potential new strategies to restore tamoxifen sensibility, these results show that inhibition of Rac1-PAK1 signaling pathway may provides benefits to revert resistance mechanisms in endocrine therapies.

In vitro modeling of the structure-activity determinants of anthracycline cardiotoxicity.

Doxorubicin and other anthracyclines rank among the most effective anticancer drugs ever developed. Unfortunately, the clinical use of anthracyclines is limited by a dose-related life-threatening cardiotoxicity. Understanding how anthracyclines induce cardiotoxicity is essential to improve their therapeutic index or to identify analogues that retain activity while also inducing less severe cardiac damage. Here, we briefly review the prevailing hypotheses on anthracycline-induced cardiotoxicity. We also attempt to establish cause-and-effect relations between the structure of a given anthracycline and its cardiotoxicity when administered as a single agent or during the course of multiagent chemotherapies. Finally, we discuss how the hypotheses generated by preclinical models eventually translate into phase I-II clinical trials.

Doxorubicin irreversibly inactivates iron regulatory proteins 1 and 2 in cardiomyocytes: evidence for distinct metabolic pathways and implications for iron-mediated cardiotoxicity of antitumor therapy.

Changes in iron homeostasis have been implicated in cardiotoxicity induced by the anticancer anthracycline doxorubicin (DOX). Certain products of DOX metabolism, like the secondary alcohol doxorubicinol (DOXol) or reactive oxygen species (ROS), may contribute to cardiotoxicity by inactivating iron regulatory proteins (IRP) that modulate the fate of mRNAs for transferrin receptor and ferritin. It is important to know whether DOXol and ROS act by independent or combined mechanisms. Therefore, we monitored IRP activities in H9c2 rat embryo cardiomyocytes exposed to DOX or to analogues which were selected to achieve a higher formation of secondary alcohol metabolite (daunorubicin), a concomitant increase of alcohol metabolite and decrease of ROS (5-iminodaunorubicin), or a defective conversion to alcohol metabolite (mitoxantrone). On the basis of such multiple comparisons, we characterized that DOXol was able to remove iron from the catalytic Fe-S cluster of cytoplasmic aconitase, making this enzyme switch to the cluster-free IRP-1. ROS were not involved in this step, but they converted the IRP-1 produced by DOXol into a null protein which did not bind to mRNA, nor was it able to switch back to aconitase. DOX was also shown to inactivate IRP-2, which does not assemble or disassemble a Fe-S cluster. Comparisons between DOX and the analogues revealed that IRP-2 was inactivated only by ROS. Thus, DOX can inactivate both IRP through a sequential action of DOXol and ROS on IRP-1 or an independent action of ROS on IRP-2. This information serves guidelines for designing anthracyclines that spare iron homeostasis and induce less severe cardiotoxicity.

Impairment of myocardial contractility by anticancer anthracyclines: role of secondary alcohol metabolites and evidence of reduced toxicity by a novel disaccharide analogue.

1. The anticancer anthracycline doxorubicin (DOX) causes cardiotoxicity. Enzymatic reduction of a side chain carbonyl group converts DOX to a secondary alcohol metabolite that has been implicated in cardiotoxicity. We therefore monitored negative inotropism, assessed as inhibition of post-rest contractions, in rat right ventricle strips exposed to DOX or to analogues forming fewer amounts of their alcohol metabolites (epirubicin, EPI, and the novel disaccharide anthracycline MEN 10755). 2. Thirty microM EPI exhibited higher uptake than equimolar DOX, but formed comparable amounts of alcohol metabolite due to its resistance to carbonyl reduction. MEN 10755 exhibited also an impaired uptake, and consequently formed the lowest levels of alcohol metabolite. Accordingly, DOX and EPI inhibited post-rest contractions by approximately 40-50%, whereas MEN 10755 inhibited by approximately 6%. 3. One hundred microM EPI exhibited the same uptake as equimolar DOX, but formed approximately 50% less alcohol metabolite. One hundred microM MEN 10755 still exhibited the lowest uptake, forming approximately 60% less alcohol metabolite than EPI. Under these conditions DOX inhibited post-rest contractions by 88%. EPI and MEN 10755 were approximately 18% (P<0.05) or approximately 80% (P<0.001) less inhibitory than DOX, respectively. 4. The negative inotropism of 30-100 microM DOX, EPI, or MEN 10755 correlated with cellular levels of both alcohol metabolites (r=0.88, P<0.0001) and carbonyl anthracyclines (r=0.79, P<0.0001). Nonetheless, multiple comparisons showed that alcohol metabolites were approximately 20-40 times more effective than carbonyl anthracyclines in inhibiting contractility. The negative inotropism of MEN 10755 was therefore increased by chemical procedures, like side chain valeryl esterification, that facilitated its uptake and conversion to alcohol metabolite but not its retention in a carbonyl form. 5. These results demonstrate that secondary alcohol metabolites are important mediators of cardiotoxicity. A combination of reduced uptake and limited conversion to alcohol metabolite formation might therefore render MEN 10755 more cardiac tolerable than DOX and EPI.

Paclitaxel and docetaxel enhance the metabolism of doxorubicin to toxic species in human myocardium.

Doxorubicin cardiotoxicity is a multifactorial process in which the alcohol metabolite doxorubicinol mediates the transition from reversible to irreversible damage. We investigated whether the tubulin-active taxane paclitaxel increases conversion of doxorubicin to doxorubicinol, thus explaining the high incidence of congestive heart failure when doxorubicin is used with paclitaxel. Specimens of human myocardium from patients undergoing bypass surgery were processed to obtain cytosolic fractions in which doxorubicin was converted to doxorubicinol by NADPH-dependent aldo/keto or carbonyl reductases. In this model, clinically relevant concentrations of paclitaxel (1-2.5 microM) increased doxorubicinol formation by mechanisms consistent with allosteric modulation of the reductases. Stimulation was observed over a broad range of basal enzymatic activity, and was accompanied by a similar pattern of enhanced formation of doxorubicinol aglycone, a metabolite potentially involved in the reversible phase of cardiotoxicity. The closely related analogue docetaxel had effects similar to paclitaxel, but increased doxorubicinol formation over a narrower range of enzymatic activity. The unrelated tubulin-active alkaloid vinorelbine had no effect. These results demonstrate that taxanes have a unique potential for enhancing doxorubicin metabolism to toxic species in human myocardium. The effects on doxorubicinol formation provide clues to explain the clinical pattern of doxorubicin-paclitaxel cardiotoxicity and also caution against the potential toxicity of combining docetaxel with high cumulative doses of doxorubicin.

In vitro activity of a Solanum tuberosum extract against mammary carcinoma cells.

We investigated the antitumor properties of a Solanum tuberosum extract (STE) on F3II mouse mammary carcinoma cells. STE significantly inhibited adhesion on fibronectin-coated surfaces and blocked migration of tumor cells in vitro. A major gelatinolytic activity (gelatinase) of 82 kD was identified in STE by zymographic analysis and characterized by exposure to different experimental conditions. Proteolytic activity of STE may be responsible, at least in part, for the in vitro effects on mammary carcinoma cells.

Anthracycline metabolism and toxicity in human myocardium: comparisons between doxorubicin, epirubicin, and a novel disaccharide analogue with a reduced level of formation and [4Fe-4S] reactivity of its secondary alcohol metabolite.

Secondary alcohol metabolites have been proposed to mediate chronic cardiotoxicity induced by doxorubicin (DOX) and other anticancer anthracyclines. In this study, NADPH-supplemented human cardiac cytosol was found to reduce the carbonyl group in the side chain of the tetracyclic ring of DOX, producing the secondary alcohol metabolite doxorubicinol (DOXol). A decrease in the level of alcohol metabolite formation was observed by replacing DOX with epirubicin (EPI), a less cardiotoxic analogue characterized by an axial-to-equatorial epimerization of the hydroxyl group at C-4 in the amino sugar bound to the tetracyclic ring (daunosamine). A similar decrease was observed by replacing DOX with MEN 10755, a novel anthracycline with preclinical evidence of reduced cardiotoxicity. MEN 10755 is characterized by the lack of a methoxy group at C-4 in the tetracyclic ring and by intercalation of 2, 6-dideoxy-L-fucose between daunosamine and the aglycone. Multiple comparisons with methoxy- or 4-demethoxyaglycones, and a number of mono- or disaccharide 4-demethoxyanthracyclines, showed that both the lack of the methoxy group and the presence of a disaccharide moiety limited alcohol metabolite formation by MEN 10755. Studies with enzymatically generated or purified anthracycline secondary alcohols also showed that the presence of a disaccharide moiety, but not the lack of a methoxy group, made the metabolite of MEN 10755 less reactive with the [4Fe-4S] cluster of cytoplasmic aconitase, as evidenced by its limited reoxidation to the parent carbonyl anthracycline and by a reduced level of delocalization of Fe(II) from the cluster. Collectively, these studies (i) characterize the different influence of methoxy and sugar substituents on the formation and [4Fe-4S] reactivity of anthracycline secondary alcohols, (ii) lend support to the role of alcohol metabolites in anthracycline-induced cardiotoxicity, as they demonstrate that the less cardiotoxic EPI and MEN 10755 share a reduction in the level of formation of such metabolites, and (iii) suggest that the cardiotoxicity of MEN 10755 might be further decreased by the reduced [4Fe-4S] reactivity of its alcohol metabolite.

Role of tumor-derived granulocyte-macrophage colony-stimulating factor in mice bearing a highly invasive and metastatic mammary carcinoma.

We have examined the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in tumor-bearing BALB/c mice using the syngeneic F3II mammary carcinoma. In the present model, progression of subcutaneous tumors induced massive myelopoiesis in bone marrow and spleen due to GM-CSF secretion by tumor cells. In vitro, the addition of recombinant mouse GM-CSF (5- 25 ng/ml) caused a significant increase in F3II cell growth, either in the presence or absence of serum. Zymographic analysis of conditioned media from F3II monolayers showed that GM-CSF exerted a dose-dependent enhancement in the metalloproteinases MMP-9 (105 kD) and MMP-2 (70 kD), key enzymes in mammary tumor cell invasion. Our data suggest that ectopic GM-CSF production stimulates myelopoiesis and may also play an important role in tumor progression and metastasis formation.

[Differential aspects of the dynamics of the renin-angiotensin-aldosterone system in obesity with and without hypertension and in essential arterial hypertension]. / Aspetti differenziali della dinamica del sistema renina-angiotensina-aldosterone nell'obesità con e senza ipertensione e nella ipertensione arteriosa essenziale.

This report describes the impulsive function of the RAAS investigated in obese and non-obese hypertensives as compared to obese and non-obese normotensives. The aim of the investigation was to clarify whether or not the hypertensive vascular disease accompanying the ponderal excess can be regarded as a well-defined pathophysiologic entity. Data obtained showed that the behavior of the RAAS in hypertensive obese patients is quite different from that of non-obese hypertensives and obese normotensives. Such a difference implies that hypertension of obese people is a biochemically distinguishable entity. This observation corroborates the concept that the clinical association of obesity-hypertension might be regarded as a syndrome with a proper nosographic dignity.