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Background: The immunotherapy has revolutionized the world of oncology in the last decades with considerable advantages in terms of overall survival in cancer patients. The association of Pembrolizumab and Trastuzumab was recently proposed in clinical trials for the treatment of Trastuzumab-resistant advanced HER2-positive breast cancer. Although immunotherapies are frequently associated with a wide spectrum of immune-related adverse events, the cardiac toxicity has not been properly studied., Purpose: We studied, for the first time, the putative cardiotoxic and pro-inflammatory effects of Pembrolizumab associated to Trastuzumab., Methods: Cell viability, intracellular calcium quantification and pro-inflammatory studies (analyses of the production of Interleukin 1β, 6 and 8, the expression of NF-kB and Leukotriene B4) were performed in human fetal cardiomyocytes. Preclinical studies were also performed in C57BL6 mice by analyzing fibrosis and inflammation in heart tissues., Results: The combination of Pembrolizumab and Trastuzumab leads to an increase of the intracellular calcium overload (of 3 times compared to untreated cells) and to a reduction of the cardiomyocytes viability (of 65 and 20-25%, compared to untreated and Pembrolizumab or Trastuzumab treated cells, respectively) indicating cardiotoxic effects. Notably, combination therapy increases the inflammation of cardiomyocytes by enhancing the expression of NF-kB and Interleukins. Moreover, in preclinical models, the association of Pembrolizumab and Trastuzumab increases the Interleukins expression of 40-50% compared to the single treatments; the expression of NF-kB and Leukotriene B4 was also increased., Conclusion: Pembrolizumab associated to Trastuzumab leads to strong cardiac pro-inflammatory effects mediated by overexpression of NF-kB and Leukotriene B4 related pathways., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The growth inhibition that occurs in cisplatin-sensitive 2008 human ovarian cancer cells in response to the spermine analogue, N1,N12-bis(ethyl)spermine (BESpm), is associated with a potent induction of spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in polyamine catabolism. Conversely, in cisplatin-resistant C13* cells, which are less responsive to BESpm, enzyme induction does not occur at comparable levels after exposure to the bis(ethyl)-derivative. In this study, we investigated the molecular mechanisms underlying the differential induction of SSAT activity in cisplatin-sensitive and -resistant cells. Northern blot analysis revealed a difference in the level of SSAT mRNA expression in the two cell lines; in particular, 2008 cells treated with 10 microM BESpm for progressively increasing periods of time accumulated more heteronuclear (3.5 kb) and mature (1.3/1.5 kb) SSAT mRNAs than its resistant variant. SSAT mRNA accumulation paralleled enzyme activity and both were almost completely prevented in the two lines by co-treatment with 5 microg/ml actinomycin-D (Act-D), suggesting that transcription plays a major role in the analogue-mediated induction of SSAT. Moreover, when Act-D was added 48 h after BESpm exposure, SSAT mRNA and enzyme activity were stabilised in both cell lines. Therefore, the marked difference in the induction of SSAT activity seems to be related to increased enzyme synthesis, particularly in sensitive cells, whose SSAT protein turnover was also greatly reduced (half-life >12 h in 2008 cells versus 5 h in C13* cells) in the presence of BESpm. These findings suggest that cisplatin-resistance modulates the SSAT response to BESpm at transcriptional and post-transcriptional levels.

We investigated the involvement of natural polyamines in HL-60 cell death triggered by exposure to 2-deoxy-D-ribose (dRib). In contrast to previous studies, exogenous polyamines failed to protect HL-60 cells against apoptosis caused by dRib. Moreover, in our experimental conditions, depletion of intracellular levels of putrescine and spermidine by alpha-difluoromethylornithine (DFMO) delayed the onset of apoptosis by at least a day or so. Exogenous polyamines reversed the beneficial effect of DFMO and restored the apoptotic levels observed in dRib-treated cells. We suggested that polyamines, especially putrescine and spermidine, act as facilitating factors in the induction of apoptosis triggered by dRib in HL-60 cells.

The subcommissural organ (SCO) is a circumventricular organ of glial origin typical of all vertebrates. The SCO releases its secretion into the third ventricle to constitute Reissner's fibre (RF). Reportedly, in reptiles, SCO has cyclic secretory activity related to the reproductive cycle. In this immunocytochemical study we show that, in females of oviparous reptiles (Lacertidae: Podarcis sicula) and in a viviparous species (Scincidae: Chalcides chalcides), SCO secretion consists of hormones, in part of the oxytocin-like (OXY-like) type. The amount of OXY-like material in the cells and in the third ventricle varies according to the different stages of the reproductive cycle. In the oviparous species, OXY-like hormone secretion can be induced by FSH administration at 28 degrees C, in the period of winter reproductive stasis as well. In the viviparous skink, showing an annual single ovulatory cycle, OXY-like secretion is present in the basal region of the cells, and is released into the third ventricle only at delivery. The role of an OXY-like hormone in the SCO is here discussed in relation to the different stages of the reproductive cycle. Its influence on the hypothalamus-hypophysis-gonad axis and its role in the transport of eggs into the ducts in the oviparous species, and at delivery in the viviparous one, are also suggested.

Exposure of HL-60 cells to millimolar levels of spermine resulted in the inhibition of cell growth. Flow cytometry revealed that the addition of exogenous spermine prevented the accumulation of cells in the S and G2/M phases of the cell cycle as observed in the control cells. High intracellular levels of spermine completely suppressed the early onset of ornithine decarboxylase activity and, consequently, the intracellular increase in spermidine and putrescine. On the other hand, the addition of exogenous spermidine or putrescine also abolished ornithine decarboxylase activity, but in this case neither the growth of spermidine- or putrescine-treated cells nor the cell cycle phase distribution was affected. In the latter cells, intracellular levels of spermidine were not significantly different from control ones. These results suggest that the addition of exogenous spermine inhibits cell proliferation by hindering the increase in cellular spermidine needed to accelerate the G1 to S phase transition.

We have previously reported that polyamines interfere with protein kinase C-membrane interactions. With the aim of clarifying the influence of the relationship between calcium and polyamines on this process we have investigated the effect of spermine on the formation of active protein kinase C-membrane complexes as a function of Ca++ concentrations. Protein kinase C, purified from rat brain, was allowed to interact with phospholipid vesicles of defined composition. The active complex protein kinase C-liposomes was determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. The results show that, at Ca++ levels below 0.1 microM, spermine inhibits the formation of complexes between protein kinase C and membranes. At higher Ca++ concentrations, spermine does not prevent the association process but does influence the ratio between the enzyme molecules irreversibly inserted into the membrane and those reversibly associated with it. We have also demonstrated that spermine, by reducing the density of acidic component of liposomes, influences the calcium requirement for protein kinase C-membrane binding. This study indicates that spermine may regulate the activation of protein kinase C and affects the calcium requirement for the association of this enzyme with the phospholipid bilayer. The results suggest a possible role for polyamines in signal transduction when protein kinase C is involved.

Protein kinase C is reported to exist in two membrane-bound states: a reversible one which can be dissociated by calcium chelators (membrane-associated form) and an irreversible one which is chelator stable (membrane-inserted form). In the present work the effects of a naturally occurring polyamine (spermine) on the membrane-associated and membrane-inserted forms of protein kinase C were investigated using a reconstituted system consisting of partially purified protein kinase C from rat brain and phospholipid vesicles of defined composition. The active membrane-bound complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. Our experimental data show that, in the absence of calcium ions, the amount of enzyme bound to phospholipids vesicles was dramatically reduced by the presence of spermine whereas the PDBu binding affinity was not significantly affected. The addition of the divalent cation increased the affinity of phorbol ester for the active complex but had no effect on Nmax; spermine added in this experimental conditions was no longer able to decrease the total number of enzyme molecules bound to liposomes. Moreover gel filtration experiments of the protein kinase C-phospholipids complex formed in the presence of calcium, indicated that polyamine added during the association process was able to reduce the extent of enzyme insertion into liposomes. Since the increase in phospholipid concentration resulted in a higher level of non-dissociable protein kinase C-liposomes complex we propose that spermine, complexing to membrane binding sites both in the absence and in the presence of Ca++, could promote binding conditions that oppose to the formation of the inserted form of the enzyme. As a consequence the distribution between the reversible and the irreversible membrane-bound forms of protein kinase C is affected.

The in vitro mechanism by which polyamines affect protein kinase C (PK C) activation process was investigated in a reconstituted system consisting of purified enzyme and phospholipid vesicles of various phosphatidylserine content. It was found that the addition of spermine greatly interferes with the association of PK C to liposomes. This tetramine, at micromolar concentrations, was most potently effective while other polyamines such as spermidine and putrescine were almost ineffective; therefore the modulatory action appeared to be structure specific. The spermine effect is dramatically influenced by the density of the phosphatidylserine present on the liposome, suggesting the complex formation with the acidic component on phospholipid vesicles to be the mechanism by which this polyamine exerts its modulatory action.

The spermine-binding activity of a cytosolic protein from chick intestine increases during embryogenesis and in the first week of life. Ornithine and S-adenosylmethionine decarboxylase activities assayed under the same experimental conditions increase showing a maximum at day 18 and 20 respectively. The behaviour of either enzyme activity is reflected in the pattern of duodenal polyamine concentration measured during the same period. The possibility that duodenal spermine-binding protein may be correlated with spermine accumulation in the tissue is discussed.

A protein fraction extracted from the liver of rats accustomed to eating during the first 8 h of a daily 12 h dark period is able to increase homologous form-B RNA polymerase activity. When the protein factor is added to RNA polymerase extracted at the end of the daily fasted period (0.9:OOh) the sensitivity of enzyme to ionic strength is modified, and resembles that of the enzyme extracted during the eating period (15:00 h) either using liver deproteinized DNA or chromatin as template. The chromatin-bound RNA polymerase activity is also modified in the presence of the factor, showing a modification of enzyme sensitivity to ionic strength similar to the one induced by food intake.

Chick duodenal mucosa contains an endogenous factor which is capable to inhibit selectively a homologous polyamine-sensitive protein kinase. The inhibitor was partially purified and characterized, and it was found to contain typical mucopolysaccharidic components. Glycosidases digestion studies, selective degradation analysis and spectrophotometric titrations with metachromatic dyes indicated that the inhibitor preparation contained two major moieties identified as heparin-like and heparan sulfate-like structures. In chick intestine the inhibitor was specific for polyamine-sensitive protein kinase since selectively interacted with it and was inert towards other cAMP-independent and cAMP-dependent protein kinases. The inhibitory effect of the endogenous factor was counteracted by naturally occurring polyamines such as spermine. The order of potency of various polyamines was: spermine greater than thermine much greater than spermidine much greater than diamines. The release of inhibition by addition of physiological concentrations of spermine was also apparent when using cytosolic proteins as endogenous phosphate acceptors. These results suggest that a possible role of polyamine in the regulation of polyamine-sensitive protein kinase in the intestine is to protect the enzyme from the inhibitory action of endogenous heparinoids.

Human histiocytic lymphoma cells (U-937) contain high affinity binding protein for 1 alpha, 25-dihydroxycholecalciferol. The Kd (0.2 nM) and sedimentation coefficient on sucrose gradients (3.7 S) are similar to those reported for 1 alpha, 25-dihydroxycholecalciferol receptor in other tissues. This secosteroid added to the culture was able to inhibit cell proliferation in a dose dependent manner. Differentiation-associated properties such as phagocytic ability, C3 rosette formation, positive reaction for alpha- naphtyl acetate esterase as well as electron microscopy examination indicate that 1 alpha, 25-dihydroxycholecalciferol induces in vitro monocyte-macrophage differentiation in this monoblast-like cell line.

The effect of polyamines on the catalytic domain of protein kinase C from rat brain was investigated. It was found that the addition of spermine strongly inhibited phosphorylation activity toward histone H1 as substrate. This tetramine, at millimolar concentrations, was most potently effective while triamines and diamines were almost uneffective, therefore the inhibitory action appeared to be structural specific. Data shown here suggest that polyamine by interacting with the catalytic domain of the enzyme may contribute to its regulation.

Nuclei from liver of rats accustomed to eating during the first 8h of a daily 12h dark period demonstrate an increased capacity to synthesize RNA 6H after the beginning of the feeding period. 2. This increase is accompanied by a higher yield of extractable form-B DNA-dependent RNA polymerase activity. 3. The endogenous RNA polymerase activity associated with nuclear chromatin is also stimulated by food intake. Both purified and chromatin-associated form-B enzyme activities exhibit different ionic strength requirements after food intake. 4. The sensitivity of exogenous (added) form-B-enzyme to changes in ionic strength changes after feeding when chromatin is used as template. 5. Chromatin extracted from the liver of fed rats is a better template for form-B-enzyme than chromatin extracted from starved rats.

Human histiocytic lymphoma cells (U-937) undergo similar differentiation when incubated with the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate (TPA) and 1,25-dihydroxycholecalciferol. In this action, TPA somehow implicates calcium-sensitive and phospholipid-dependent protein kinase (protein kinase C), which is rapidly and significantly affected by this inducer. On the contrary, 1,25-dihydroxycholecalciferol in its differentiating action does not involve protein kinase C thus suggesting that the secosteroid induces monocytic differentiation possible through a different mechanism of that of phorbol ester.

Physiological activation of protein kinase C requires the interaction of this enzyme with cellular membranes [Nishizuka (1986) Science 233, 305-312]. In the present work a reconstituted system of protein kinase C and human inside-out erythrocyte vesicles was utilized to study the effect in vitro of naturally occurring polyamines on the activation process of protein kinase C. The active membrane-associated complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. The association reaction of the enzyme to membrane was rapid, being complete within 1 min at 25 degrees C. The addition of polyamines, particularly spermine, greatly decreased in a dose-dependent manner the amount of protein kinase C bound to membranes (i.e. in the activated form). The effect observed was quite specific, since it was dependent on the chemical structure of the polyamine and it was manifest at micromolar concentrations of the polycation; the order of potency was spermine greater than spermidine greater than putrescine. A characterization of this effect is presented and possible physiological implications are discussed.

Partially hepatectomised rats have been exposed immediately after surgery and every 12 hours thereafter to pulsed extremely low frequency electromagnetic fields. Stereological analysis performed on electron micrographs has shown that the treatment limits the depletion of liver glycogen found in control untreated rats within the first post-operative day. The massive accumulation of lipid droplets found in control rats is also limited to about one half by exposing the animals to pulsed magnetic fields. The time taken for glycogen and lipid content to recover to values found at zero time decreases from seven to five days in animals undergoing treatment with pulsed electromagnetic fields. The liver wet weight and the total protein content show a pattern of changes which is consistent with the behaviour of glycogen and lipid content. Five days after operation the treated rats reach the values found at zero time, while control animals need seven days to reach the same values.

A cyclic nucleotide-independent protein kinase which phosphorylates preferentially acidic proteins such as casein or phosvitin was isolated from cytosol of chick duodenal mucosa. The enzyme was purified more than 633 fold to apparent homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and by sucrose density gradient centrifugation. The native enzyme has a molecular weight of 131 000 as measured by gel filtration. The enzyme is a complex protein containing three polypeptides of molecular weight of 39 000, 36 000 and 27 000. It behaves as a complex throughout its purification and gel filtration but its components are readily separated by electrophoresis in denaturing buffer. The 27 000 molecular weight band was selectively autophosphorylated when the enzyme was incubated in the presence of [gamma-32P]ATP. When casein was used as substrate, physiological concentrations of naturally occurring polyamines such as spermine and spermidine markedly stimulated enzyme activity. However with phosvitin as substrate polyamines were strong inhibitors of the enzyme activity. This contrasting effect on intestinal kinase activity was also apparent using cytoplasmic proteins as endogenous phosphate acceptors. A characterization of this differential effect is presented and some possible physiological implications are discussed.

Mammary explants from pregnant mice cultured in the presence of lactogenic hormones express their differentiated function by producing milk components in vitro. In the present paper radioactive calcium uptake was studied in the mammary gland during terminal differentiation, when lactogenesis was initiated. Under these conditions a relevant accumulation of calcium in the tissue was clearly demonstrable. We have characterized the calcium uptake by the tissue as a saturable and energy dependent process. When 1,25-dihydroxycholecalciferol [1,25-(OH)2D3], the major calcium regulator hormone, was added to the incubation medium the secosteroid strongly stimulated this process by increasing the maximal velocity without significantly altering the Michaelis Menten constant (Km). Detectable increases in calcium uptake could be measured after the explants were cultured with 1,25-(OH)2D3 for 30 min, with the response continuing to increase sharply over time and reaching a plateau at 3 h. The increase was not affected by the presence of actinomycin D or cycloheximide suggesting that the 1,25-(OH)2D3 stimulation of calcium uptake may be independent of de novo protein synthesis. These results provide evidence for early and direct action of the hormonal form of vitamin D in affecting calcium transport across membranes of functionally differentiated epithelial cells of the mammary gland.