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Cancer cells within a tumor are functionally heterogeneous and specific subpopulations, defined as cancer initiating cells (CICs), are endowed with higher tumor forming potential. The CIC state, however, is not hierarchically stable and conversion of non-CICs to CICs under microenvironment signals might represent a determinant of tumor aggressiveness. How plasticity is regulated at the cellular level is however poorly understood. To identify determinants of plasticity in lung cancer we exposed eight different cell lines to TGFβ1 to induce EMT and stimulate modulation of CD133(+) CICs. We show that response to TGFβ1 treatment is heterogeneous with some cells readily switching to stem cell state (1.5-2 fold CICs increase) and others being unresponsive to stimulation. This response is unrelated to original CICs content or extent of EMT engagement but is tightly dependent on balance between epithelial and mesenchymal features as measured by the ratio of expression of CDH1 (E-cadherin) to SNAI2. Epigenetic modulation of this balance can restore sensitivity of unresponsive models to microenvironmental stimuli, including those elicited by cancer-associated fibroblasts both in vitro and in vivo. In particular, tumors with increased prevalence of cells with features of partial EMT (hybrid epithelial/mesenchymal phenotype) are endowed with the highest plasticity and specific patterns of expression of SNAI2 and CDH1 markers identify a subset of tumors with worse prognosis. In conclusion, here we describe a connection between a hybrid epithelial/mesenchymal phenotype and conversion to stem-cell state in response to external stimuli. These findings have implications for current endeavors to identify tumors with increased plasticity., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

TRPM7, a cation channel of the transient receptor potential channel family, has been identified as a ubiquitous magnesium transporter. We here show that TRPM7 is expressed in endothelial cells isolated from the umbilical vein (HUVEC), widely used as a model of macrovascular endothelium. Quiescence and senescence do not modulate TRPM7 amounts, whereas oxidative stress generated by the addition of hydrogen peroxide increases TRPM7 levels. Moreover, high extracellular magnesium decreases the levels of TRPM7 by activating calpains, while low extracellular magnesium, known to promote endothelial dysfunction, stimulates TRPM7 accumulation partly through the action of free radicals. Indeed, the antioxidant trolox prevents TRPM7 increase by low magnesium. We also demonstrate the unique behaviour of HUVEC in responding to pharmacological and genetic inhibition of TRPM7 with an increase of cell growth and migration. Our results indicate that TRPM7 modulates endothelial behavior and that any condition leading to TRPM7 upregulation might impair endothelial function.

Divalent cations, especially calcium and magnesium, have been shown to play an important regulatory role in endothelial and immune cells. To learn more about the interaction of these two metals in the regulation of cell growth, we altered the calcium/magnesium ratio by culturing human endothelial cells, macrophages, and T lymphocytes in media containing different concentrations of magnesium. We observed that the growth of the three cell types was retarded in low extracellular magnesium, and this retardation is particularly evident in highly proliferating cells. High concentrations of magnesium does not exert any effect on cell growth. When (i) calcium influx was blocked by adding the calcium antagonist verapamil, and (ii) calcium release from intracellular stores was inhibited by exposure to TMB-8, the growth of endothelial cells, macrophages, and T lymphocytes was inhibited. In particular, the release of calcium from intracellular stores seems to be more important than its influx in sustaining cell proliferation. Our results indicate that calcium plays a crucial role in mediating cell proliferation independently from the extracellular concentrations of magnesium.

Evidence has accumulated to suggest that magnesium might play a role in controlling angiogenesis. Since microvascular endothelial cells are protagonists in this process, we investigated the behavior of these cells cultured in low extracellular magnesium or silenced for its transporter Transient Receptor Potential Melastatin (TRPM)7, essential for cellular magnesium homeostasis. In particular, we focused on some crucial steps of the angiogenic process, i.e. proliferation, migration, protease production and organization in tridimensional structures. Silencing TRPM7 mimics the effects of low extracellular magnesium on human microvascular endothelial cells (HMEC). Indeed, while no effects were observed on the production of metalloproteases and on tridimensional organization on matrigel, both magnesium deficiency and silencing of TRPM7 impair cell migration and inhibit growth by arresting the cells in the G0/G1 and G2/M phases of the cell cycle. Since low extracellular magnesium markedly decreases TRPM7 in HMEC, we suggest that TRPM7 downregulation might mediate low magnesium-induced inhibition of cell growth and migration. Human endothelial cells from the umbilical vein are growth inhibited by low magnesium and growth stimulated after TRPM7 silencing. An impairment of ERK phosphorylation in HMEC silencing TRPM7 is responsible, in part, for the different proliferative behavior of these two cell types. We broadened our studies also to endothelial colony-forming cells and found that they are sensitive to fluctuations of the concentrations of extracellular magnesium, while their proliferation rate is not modulated by TRPM7 silencing. Our results point to magnesium and TRPM7 as a modulators of the angiogenic phenotype of microvascular endothelial cells.

We examined the effects of some cytokinins and cytokinin ribosides including a series of adenosine analogs differently substituted in the N(6) position, along with some hypoxanthine derivatives on the viability of normal and neoplastic human cells. Cytokinins such as trans-zeatin, isopentenyladenine and benzyladenine do not show any effect, while cytokinin ribosides such as trans-zeatin riboside, isopentenyladenosine, and benzylaminopurine riboside impair the viability of normal and neoplastic cells, apart from colon carcinoma LoVo cells.

A series of adenosine analogues differently substituted in N⁶-position were synthesized to continue our studies on the relationships between structure and biological activity of iPA. The structures of the compounds were confirmed by standard studies of ¹H NMR, MS and elemental analysis. These molecules were then evaluated for their anti-proliferative activity on bladder cancer cells. We found that some of these compounds possess anti-proliferative activity but have no effect on cell invasion and metalloprotease activity., (Copyright © 2010. Published by Elsevier Ltd.)

Phenotypic modulation of endothelium to a dysfunctional state contributes to the pathogenesis of atherosclerosis, partly through the activation of the transcription factor NFkB. Several data indicate that magnesium deficiency caused by prolonged insufficient intake and/or defects in its homeostasis may be a missing link between diverse cardiovascular risk factors and atherosclerosis. Here we report that endothelial cells cultured in low magnesium rapidly activate NFkB, an event which is prevented by exposure to the anti-oxidant trolox. It is well known that NFkB activation correlates with marked alterations of the cytokine network. In the present study, we show that exposure of endothelial cells to low magnesium increases the secretion of RANTES, interleukin 8 and platelet derived growth factor BB, all important players in atherogenesis. Moreover, we describe the increased secretion of matrix metalloprotease-2 and -9 and of their inhibitor TIMP-2. Interestingly, by zymography we show that metalloprotease activity predominated over the inhibitory effect of TIMP-2. These results indicate that low magnesium promotes endothelial dysfunction by inducing pro-inflammatory and pro-atherogenic events., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Magnesium promotes endothelial migration, an event which is orchestrated by a complex interplay between protein tyrosine kinases and phosphatases. We found that high extracellular concentrations of magnesium do not modulate the levels and the activation of FAK and Src, two tyrosine kinases involved in driving cell migration. Interestingly, we show that magnesium induced-endothelial motility correlates with the downregulation of HD-PTP, a potential tyrosine phopshatase previously shown to be involved in modulating cell migration. The decreased amounts of HD-PTP are not dependent upon transcriptional mechanisms. In contrast to Fibroblast Growth Factor-induced HD-PTP downregulation, the proteasome seems not to be involved in regulating HD-PTP levels in endothelial cells cultured in high magnesium containing medium. Our results indicate that, in the presence of high magnesium concentrations, endothelial cells are stimulated to migrate through complex mechanisms involving also HD-PTP.

The present study describes the synthesis, the characterization and the evaluation of some derivatives of N(6)-isopentenyladenosine on T24 human bladder carcinoma cells. In particular we have modified the hydroxyl groups in the ribose moiety, the position of the isopentenyl chain in the purine ring and the base moiety. The structures of the compounds were confirmed by standard studies of NMR, MS and elemental analysis. We here show that only two derivatives, 1-(3-methyl-2-butenylamino)-9-(3'-deoxy-beta-d-ribofuranosyl)-purine hydrobromide and 2-amino-6-(3-methyl-2- butenylamino)-9-(beta-d-ribofuranosyl)-purine, inhibit the growth of T24 cells, although to a lower extent than N(6)-isopentenyladenosine. We conclude that the integrity of ribosidic and purine moiety and the N(6) position of the chain are essential for maintaining the antiproliferative activity., (Copyright 2010 Elsevier Ltd. All rights reserved.)

N-(2,5-Dimethyl-1H-pyrrol-1-yl)-6-(4-morpholinyl)-3-pyridazinamine hydrochloride (MDL-899) is a new long-acting antihypertensive vasodilator which reduces the blood pressure of conscious hypertensive rats and dogs to normal levels. The oral doses that reduce blood pressure by 50 mmHg are: 4.4 mg/kg in conscious spontaneously hypertensive rats (SHR), 18 mg/kg in conscious Milan hypertensive strain (MHS) and 1.7 mg/kg in conscious renal hypertensive dog (RHD). The i.v. doses are 1.26, 3.2 and 0.9 mg/kg. The reduction in blood pressure is slow (peak effect at 3 h) and long-lasting (more than 7 h) after p.o. or i.v. administration. Tolerance to MDL-899 is seen to develop in hypertensive dogs, whereas in hypertensive rats this phenomenon never occurs. The compound antagonizes the development of hypertension when given to SHR between days 25 and 88. The haemodynamic study in conscious normotensive rats (labelled microspheres) demonstrated that the fall in blood pressure is accompanied by increases in heart rate and cardiac output and a decrease in total peripheral resistance. The lack of alpha-blocking activity, in the rat caudal artery "in vitro"; beta 2-stimulating activity, in SHR pretreated with propranolol, and prostaglandin (PG) release activity, in SHR pretreated with indomethacin, excludes the possibility that the hypotension is due to one of these mechanisms. MDL-899 given orally to rats has no important depressant effects on the CNS at hypotensive or higher doses and induces no adrenergic system stimulation symptoms (midriasis, exophthalmus). In comparison with hydralazine, it is slower in onset and longer lasting, devoid of adrenergic system stimulation, less toxic and nonmutagenic. They are equipotent after p.o. treatment.

The effects of N-(2,5-dimethyl-1H-pyrrol-1-yl)6-(4-morpholinyl)-3-pyridazinamine hydrochloride (MDL-899) on systemic, pulmonary, renal and coronary circulation and on myocardial contractility have been investigated in conscious dogs treated orally. The compound induced a decrease in systemic arterial blood pressure, slow in onset (peak effect at 4 h) and long-lasting (more than 7 h), by reducing total peripheral resistance, while the heart rate, cardiac output, cardiac work and myocardial contractility increased because of the reflex increase in sympathetic drive evoked by the blood pressure fall. Coronary and renal blood flows were increased for a long time. Pulmonary resistance was markedly decreased, but the pulmonary pressure was increased, probably due to the hyperkinetic activity triggered by the increases of cardiac output and heart rate. The hemodynamic changes induced by MDL-899 are qualitatively similar to those induced by hydralazine. The results suggest that the compound belongs to the class of vasodilators that act primarily on systemic arterioles to produce arteriolar dilation.

3-(1-Methylethyl)-2-(4-methoxyphenyl)-3H-naphth[1,2-d] imidazole (MDL-035) has antiinflammatory activity in various antiinflammatory models such as carrageenin and nystatin oedemas, cotton pellet granuloma and adjuvant arthritis. The antiinflammatory potency of MDL-035 is greater than that of acetylsalicylic acid and phenylbutazone, but lower than that of indomethacin. MDL-035 has practically no gastroulcerogenic activity in rats, does not affect water or salt excretion, has no hormonal or antihormonal effects and has no other unwanted pharmacological effects. Its acute toxicity is very low.

The synthesis of a few derivatives of the angiotensin converting enzyme inhibitor enalapril is described. In these molecules the chelating carbethoxy group is substituted with an imidazolyl one. The compounds were tested both in vitro and in vivo. None of them showed in vivo activity but only a marginal in vitro inhibitory activity.

The synthesis of a new class of potential angiotensin converting enzyme (ACE) inhibitors, analogs of enalapril, is reported. In these molecules the C-terminal amino acidic sequence Ala-Pro of enalapril was replaced by the sequence Pro-Pro. The compounds were tested both in vitro and in vivo. They showed no in vivo activity but only a week in vitro inhibitory activity.

The hypothesis that the side effects of hydralazine, such as mutagenicity and lupus erythematosus like syndrome, might be due to the NHNH2 group prompted us to incorporate part of this moiety into a pyrrole ring. Therefore, we prepared a series of N-1H-pyrrol-1-yl-3-pyridazinamines and a limited number of N-1H-pyrrol-1-yl-1-phthalazinamines by reaction of 3-hydrazinopyridazines and 1-hydrazinophthalazines with gamma-diketones. Most of these compounds, especially in the pyridazine series, showed moderate to strong antihypertensive activity in spontaneously hypertensive rats. The decrease in blood pressure generally had a slow onset after either oral or intravenous administration. N-(2,5-Dimethyl-1H-pyrrol-1-yl)-6-(4-morpholinyl)-3-pyridazinamine hydrochloride (30) (MDL 899) showed no mutagenic activity in several tests and is now in clinical trials in patients.

The hemodynamic modifications induced by vincamine and by one of its derivatives, apovincaminic acid ethylester (RGH-4405), have been studied in anesthetized and conscious dogs. The two alkaloids induced peripheral vasodilatation in all the experimental models, but their action on systemic blood pressure and heart rate was clearly influenced by anesthesia and was different according to the anesthetic used. In the conscious animals an increase of both heart rate and systemic blood pressure was observed, concomitantly with an increase in femoral and vertebral blood flow and a decrease in renal blood flow. In the renal vascular bed a subsequent decrease in resistance was shown, when all the other measured hemodynamic parameters had returned to control values. The greater increase of vertebral blood flow than of femoral blood flow for the same increment of cardiac output was taken as an indirect demonstration of the selectivity of the two drugs for the cerebral circulation.

A series of new 3-aryl-4,5,6,7-tetrahydropyrazolo[4,3-c]pyridines was synthesized and screened for in vitro [3H]prazosin displacement activity. The results correlated well with their antihypertensive activity in spontaneous hypertensive rats. 1-Benzyl-3-(4-fluorophenyl)-4,5,6,7-tetrahydropyrazolo[4,3-c]pyrid ine (50, L 16052) was selected for further pharmacological evaluations of its potency when administered orally to conscious renal hypertensive dogs.