Francesco Piccinini

Protein kinase Cε is oncogenic in colon epithelial cells by interaction with the ras signal transduction pathway

We have shown previously that overexpression of the ε isoform of protein kinase C (PKCε) in rat colonic epithelial cells causes malignant transformation, possibly by interacting with the ras signal transduction pathway (Oncogene 12: 847, 1996). We have now performed experiments to examine certain early steps in the ras signaling pathway. A marked increase of Raf-1 phosphorylation was detected in tumorigenic ras-transformed D/ras as well as in D/ε cells (overexpressing PKCε), compared to the nontumorigenic D/WT parental line. Moreover, in the PKCε-transformed D/ε cell line, stable transfection with a dominant-negative raf-1 (DNraf) sequence caused complete regression of the neoplastic phenotype. These results suggested that PKCε-induced transformation was associated with increased Raf-1 activation, and that DNraf could block the oncogenic effect of PKCε. Furthermore, transfection of D/WT cells with dominant-negative ras induced arrest of cell growth, and subsequent transfection with PKCε cDNA enhanced cell proliferation and induced neoplastic transformation. These results suggest that ras acts upstream of PKCε, and that overexpression of PKCε circumvents the block in cell proliferation caused by dominant-negative ras. We conclude that PKCε exerts its oncogenic activity in rat colonic cells by affecting the ras signaling cascade at the level of Raf-1 activation.

PKCδ acts as a growth and tumor suppressor in rat colonic epithelial cells

We have analysed the expression of three calcium-independent isoforms of protein kinase C (PKC), PKCδ, PKCε and PKCζ, in an in vitro model of colon carcinogenesis consisting of the nontumorigenic rat colonic epithelial cell line D/WT, and a derivative src-transformed line D/src. While PKCζ and PKCε showed similar protein levels, PKCδ was markedly decreased in D/src cells when compared to the D/WT line. To assess whether down-regulation of PKCδ was causally involved in the neoplastic phenotype in D/src cells, we prepared a kinase-defective mutant of PKCδ. Stable transfection of this sequence caused morphological and growth changes characteristic of partial transformation in D/WT cells. Moreover, to test whether PKCδ was involved in growth control and transformation in this model, we overexpressed PKCδ in D/src cells. Transfected cells underwent marked growth and morphological modifications toward the D/WT phenotype. In a late stage in culture, transfected cells ceased to proliferate, rounded up and degenerated into multinucleated, giant-like cells. We conclude that PKCδ can reverse the transformed phenotype and act as a suppressor of cell growth in D/src cells. Moreover, our data show that downregulation of this isoenzyme of PKC may cooperate in the neoplastic transformation induced by the src oncogene in D/WT cells.

Protective activity of the spin trap tert-bytyl-α-phenyl nitrone (PBN) in reperfused rat heart

Abstract The aim of this work was to ascettain whether free radicals play a causal role in the injury occurring in myocardial ischemia and reperfusion. To this purpose we observed whether spin-trapping compounds protect the heart when used at a concentration capable of reacting with free radicals. The lipophilic spin trap α-phenyl-t-butyl nitrone (PBN) was used because it is taken up by the myocites. Isolated Langendorff rat hearts were subjected to ischemia according to two schemes: “Model A” = 30 min zero-flow ischemia followed by 30 min reperfusion; “Model B” = 60 min of low-flow ischemia (10% of the individual value; N 2 saturated) followed by 30 min reperfusion. Treated groups received in addition 5.0 m m PBN which was supplied continuously. The following parameters were measured throughout the experiment: contractile performance (RPP); coronary flow (CF); CPK; phosphocreatine (PCr), ATP, inorganic phosphate ( P i ), intracellular pH (pH i ). The pathology obtained by “Model A” is more severe than that of Model B, and partly irreversible. During the ischemic phase in “Model A”, contractility, PCr and ATP dropped to near zero; during initial reflow CPK rose about 13-fold and P i rose 2.5-fold, while pH i decreased to 6.1. During reperfusion, a partial recovery of PCr, P i and pH i was observed, while RPP and ATP did not increase; PBN treatment improved significantly PCr and CPK, while the other parameters were unaffected. During ischemia, “Model B” hearts showed a drop of contractility to near zero, of PCr to 35%, of ATP to 50%; CPK rose 7-fold and P i 1.5-fold; pH i was not modified. During reperfusion, all parameters recovered in part, with exception of P i . PBN developed a marked protective activity on all tested parameters, which gained a nearly normal value. The results of the present investigations show that the lipophilic spin trap PBN partly protects the heart from the ischemia/reperfusion injury, thus confirming that free radicals play a causal role in this pathology; the continuous loading of the tissue with the drug can be an important factor for obtaining the protective effect.

Protective effects of spin-trapping agents on adriamycin-induced cardiotoxicity in isolated rat atria

Adriamycin (ADR) is known to exert a severe negative inotropic effect on isolated myocardial preparations; a role for free radical generation has been hypothesized. Spin-trapping of free radicals has been extensively exploited in ESR studies, both in cell-free systems and in intact tissues. The interaction between spin-traps and free radicals should in principle stop the reaction cascade leading to cellular damage. Based on this hypothesis, the possible cardioprotective action of three spin-trapping agents, 5,5-dimethyl-l-pyrroline-N-oxide (DMPO), N-tert-butyl-alpha-phenylnitrone (PBN) and alpha-(4-pyridyl 1-oxide) N-tert-butylnitrone (POBN), was tested on isolated rat atria incubated in the presence of ADR; maximal non-cardiotoxic concentrations were used (50, 10 and 50 mM respectively) in order to achieve a maximal spin-trapping effect. A varying degree of protection was observed with the three compounds, directly correlated to their hydrophobicity, as assessed by chloroform/water partition coefficients. It is proposed that ADR-induced free radical generation is responsible for the acute cardiotoxic effects of the drug; this seems to be a site-specific mechanism restricted to one or more hydrophobic cellular compartment/s, since only lipophilic spin-trapping agents are able to prevent the development of the negative inotropic effect of ADR.

EFFECT OF GLUTATHIONE AND N- ACETYLCYSTEINE ON IN VITRO AND IN VIVO CARDIAC TOXICITY OF DOXORUBICIN

The effects of two sulfhydryl compounds, glutathione (GSH) and N-acetylcysteine (NAC), on the cardiotoxicity of doxorubicin (DXR) were tested on in vitro and in vivo models. DXR was administered to rats as 4 weekly i.v. doses of 3 mg/kg. GSH (1.5 mmoles/kg), given i.v. 10 min before and 1 hr after DXR, was found to prevent the development of the delayed cardiotoxic effects of DXR, as assessed by electrocardiographic and mechanical parameters, as well as by histological examination of left ventricular preparations. In contrast, equimolar oral doses of NAC (1 hr before and 2 hrs after DXR) were found to be ineffective. Both GSH and NAC prevented the negative inotropic effect produced by DXR on isolated rat atria. A good correlation exists between the cardioprotective effects of the two agents and their ability to enhance the non-protein sulfhydryl group content of the myocardium. Differences observed in vivo between GSH and NAC might be accounted for by pharmacokinetic factors.

Relationship between doxorubicin-induced ECG changes and myocardial alterations in rats.

The aim of the present study was to evaluate the dose- and time-dependence of the effect displayed by doxorubicin (DXR) on the electrocardiogram (ECG) and to establish the relationship between structural alterations of the myocardium and ECG changes in rats administered DXR, at a dose of 1.5 or 3.0 mg/kg, every 3 days for a total of three administrations. The most interesting findings consisted of a dose-dependent, but reversible prolongation of the QRS complex, and in a dose-dependent and progressive irreversible increase in QaT and, in particular, in SaT duration. Furthermore, animals treated with the higher DXR dose showed a slight increase in serum K+ concentration and a significant decrease in serum Ca2+ levels. A good correlation was found between the morphologic score indicating the degree of observed tissue damage and SaT prolongation. These results therefore support the usefulness of measuring this ECG parameter for monitoring the development of DXR-induced cardiotoxicity in rats.

Myocardial contractility and heart pharmacokinetics of adriamycin following a single administration in rat

SummaryA single administration of adriamycin (DXR) 6.0 mg/kg i. v. to rats brings about a biphasic impairment of the maximal myocardial contractile performance, measured as dF/dt of ex vivo isolated atria incubated in the presence of calcium concentrations varying up to 12 mM. The initial impairment of the contractile performance peaks 1 week after DXR administration and recovers within 3 weeks (acute phase of cardiotoxicity). After this time and up to the end of the observation period (8 weeks after treatment), delayed cardiotoxicity occurs, showing a progressive and irreversible impairment of the contractile performance of the atria. This behaviour parallels the previously shown ECG and morphological abnormalities. Tissue determinations of DXR showed that the drug is present in myocardium during the acute phase of cardiotoxicity, while the metabolite adriamycinol is not detectable 1 week after DXR administration. These data show that the presence of DXR and/or metabolites in heart muscle is not necessary for the delayed form of cardiotoxicity to become apparent and suggest that this form of cardiotoxicity is related to a mechanism different from that involved in acute cardiotoxicity.

NMR evaluation of changes in myocardial high energy metabolism produced by repeated short periods of ischemia.

Following our previous results which demonstrated that repeated short periods (2 min) of ischemia are capable of protecting the isolated rat heart from a subsequent global ischemia (30 min), in the present study we have concentrated on the metabolic changes occurring in rat hearts during six 2 min ischemia/3 min reperfusion cycles. Cardiac high-energy phosphates were monitored using 31P-NMR. Phosphocreatine levels fell (50-60%) during each ischemic period, and recovered to 70-80% of their initial values during reperfusion. P(i) rose by 59% during the first ischemic period, but increased less during subsequent ischemias (30% during the 6th occlusion, P < 0.05 vs. the first ischemic period) returning to baseline levels after each reperfusion. [ATP], pH, and [Mg2+] remained almost unaffected, but there was a decrease in HPLC-determined effluent ATP catabolites. The first occlusion led to a 95% drop in contractile function (P < 0.001 vs. baseline), but this recovered to 73% upon reperfusion (P < 0.02 vs. baseline), and was 65% at the end of the protocol. Phosphorylation potential (PP = [ATP]/([ADP].[P(i)]) correlated exponentially with total purine (r = 0.90) and with adenosine + inosine release (r = 0.81), and by the 6th ischemia/reperfusion cycle, exceeded that observed in controls by 21% (P < 0.05). We conclude that repeated short periods of ischemia do not lead to any significant alteration in the absolute myocardial ATP, but are associated with an enhanced cytosolic energy state in the heart, that enables the myocardium to reach a steady albeit lower functional state. Adenosine (+inosine) release may be involved in the regulation of the energy supply-demand balance.

Protective effect of dietary selenium supplementation on delayed cardiotoxicity of adriamycin in rat: Is PHGPX but not GPX involved?

The involvement of Se enzymes in the protection against the oxidative stress induced by adriamycin (ADR) in rat heart has been studied in animals fed for 10 weeks at three different levels of Se content (low = 0.02 ppm; normal = 0.5 ppm; high = 1.0 ppm) and receiving a weekly injection of 3 mg/kg ADR for 4 weeks. ECG (QaT duration) and contractility of isolated atria were measured. The high-Se diet showed a significant protection on both parameters. To assess the hypothesis that an increase of specific activity of antioxidant Se enzymes may account for the cardioprotective effect of selenium, glutathione peroxidase (GPX), and phospholipid hydroperoxide glutathione peroxidase (PHGPX) were tested. The assays were performed on ventricles isolated from treated rats. At the end of the experimental period, GPX (cytosolic enzyme) did not show any significant difference between controls and ADR-treated at any level of Se content, thus excluding its involvement in the cardioprotection observed in high-Se ADR-treated animals. PHGPX, which is present both in cytosol and in the cell membrane, showed a trend to increase its activity in the presence of ADR treatment only in the membrane fraction; however, the statistical significance was reached only in the low-Se group (+100%). This observation suggests that membrane PHGPX might be involved in the cellular mechanism of adaptation of the heart to the toxic effects of ADR; however, the behavior of these enzymes does not seem to account for the significant protection of selenium supplementation both on ECG and on contractile indices of ADR cardiotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of myofibrillar ATPase activity and isomyosin shift in delayed doxorubicin cardiotoxicity

The aim of this study was to determine whether variations of isomyosin expression occurred during doxorubicin-induced cardiomyopathy. A suitable experimental model in which pure delayed cardiotoxic effects could be easily studied was adopted. Young adult female Sprague Dawley rats received 9 mg/kg of doxorubicin (DXR) i.v. divided into three subdoses of 3 mg/kg every third day. Control animals received equal volumes of saline. The animals were examined 9 weeks after treatment. At this time the animals treated with DXR showed ECG alterations, reduction of body weight and a marked decrease of both atrial and ventricular mass, but were still fully hemodynamically compensated. Loss of myofibrillar material could be documented by the reduced recovery of myofibril and myosin. The contractile response of papillary muscles isolated from the right ventricle of treated animals was markedly impaired. Ca-Mg-activated and Mg-activated myofibrillar ATPase activity and Ca-activated myosin ATPase activity were determined on ventricular myocardium of control and treated animals. Both myofibrillar and myosin ATPase activities were found to be significantly reduced. Pyrophosphate gel electrophoresis of purified myosin was carried out. The isomyosin pattern of DXR-treated animals showed a pronounced shift towards V3, the percent of alpha heavy chains being 54.6% in treated rats (80.5% in control rats). This isomyosin shift can explain the reduced myofibrillar and myosin ATPase activity found in treated animals.