The anti-hepatocellular carcinoma cell activity by a novel mTOR kinase inhibitor CZ415.

Dysregulation of mammalian target of rapamycin (mTOR) in hepatocellular carcinoma (HCC) represents a valuable treatment target. Recent studies have developed a highly-selective and potent mTOR kinase inhibitor, CZ415. Here, we showed that nM concentrations of CZ415 efficiently inhibited survival and induced apoptosis in HCC cell lines (HepG2 and Huh-7) and primary-cultured human HCC cells. Meanwhile, CZ415 inhibited proliferation of HCC cells, more potently than mTORC1 inhibitors (rapamycin and RAD001). CZ415 was yet non-cytotoxic to the L02 human hepatocytes. Mechanistic studies showed that CZ415 disrupted assembly of mTOR complex 1 (mTORC1) and mTORC2 in HepG2 cells. Meanwhile, activation of mTORC1 (p-S6K1) and mTORC2 (p-AKT, Ser-473) was almost blocked by CZ415. In vivo studies revealed that oral administration of CZ415 significantly suppressed HepG2 xenograft tumor growth in severe combined immuno-deficient (SCID) mice. Activation of mTORC1/2 was also largely inhibited in CZ415-treated HepG2 tumor tissue. Together, these results show that CZ415 blocks mTORC1/2 activation and efficiently inhibits HCC cell growth in vitro and in vivo.

A comparative QSAR on 1,2,5-thiadiazolidin-3-one 1,1-dioxide compounds as selective inhibitors of human serine proteinases.

Selective inhibitors of target serine proteinases have a potential therapeutic role for the treatment of various inflammatory and related diseases. We develop a comparative quantitative structure-activity relationships based analysis on compounds embodying the 1,2,5-thiadiazolidin-3-one 1,1-dioxide scaffold. By means of classical Molecular Dynamics we obtain the conformation of each lowest-energy molecular structure from which we derive more than a thousand of structural descriptors necessary for building predictive QSAR models. We resort to two different modeling approaches with the purpose of testing the consistency of our results: (a) multivariable linear regressions based on the replacement method and forward stepwise regression, and (b) the calculation of flexible descriptors with the CORAL program. All the models are properly validated by means of standard procedures. The resulting QSAR models are supposed to be of great utility for the rational search and design (including synthesis and/or in vitro biochemical studies) of new effective non-peptidyl inhibitors of serine proteinases.

Reduced cAMP, Akt activation and p65-c-Rel dimerization: mechanisms involved in the protective effects of mGluR3 agonists in cultured astrocytes.

In recent decades, astrocytes have emerged as key pieces in the maintenance of normal functioning of the central nervous system. Any impairment in astroglial function can ultimately lead to generalized disturbance in the brain, thus pharmacological targets associated with prevention of astrocyte death are actually promising. Subtype 3 of metabotropic glutamate receptors (mGluR3) is present in astrocytes, its activation exerting neuroprotective roles. In fact, we have previously demonstrated that mGluR3 selective agonists prevent nitric oxide (NO)-induced astrocyte death. However, mechanisms responsible for that cytoprotective property are still subject to study. Although inhibition of adenylyl cyclase by mGluR3 activation was extensively reported, the involvement of reduced cAMP levels in the effects of mGluR3 agonists and the association between cAMP decrease and the downstream pathways activated by mGluR3 remain neglected. Thus, we studied intracellular signaling mediating anti-apoptotic actions of mGluR3 in cultured rat astrocytes exposed to NO. In the present work, we showed that the cytoprotective effect of mGluR3 agonists (LY379268 and LY404039) requires both the reduction of intracellular cAMP levels and activation of Akt, as assessed by MTT and TUNEL techniques. Moreover, dibutyryl-cAMP impairs Akt phosphorylation induced by LY404039, indicating a relationship between mGluR3-reduced cAMP levels and PI3K/Akt pathway activation. We also demonstrated, by co-immunoprecipitation followed by western-blot, that the mGluR3 agonists not only induce per se survival-linked interaction between members of the NF-κB family p65 and c-Rel, but also impede reduction of levels of p65-c-Rel dimers caused by NO, suggesting a possible anti-apoptotic role for p65-c-Rel. All together, these data suggest that mGluR3 agonists may regulate cAMP/Akt/p65-c-Rel pathway, which would contribute to the protective effect of mGluR3 against NO challenge in astrocytes. Our results widen the knowledge about mechanisms of action of mGluR3, potential targets for the treatment of neurodegenerative disorders where a pathophysiological role for NO has been established.

Ethopharmacological analysis of 5-HT ligands on the rat elevated plus-maze.

The present study investigated the behavioral effects of five 5-HT agonists and antagonists in the rat elevated-plus-maze using conventional and ethologically derived measures. An anxiolytic effect of the 5-HT1A agonist ipsapirone (0.25, 0.75, and 2.25 mg/kg) was detected by risk-assessment and scanning but not by percentage of open-arm entries and time spent on open arms. Anxiogenic effects of the 5-HT2C agonist TFMPP (0.1, 0.2, and 0.4 mg/kg) and 5-HT2A antagonist SR 46349B (1, 3, and 10 mg/kg) were detected by percentage of open-arm entries, time spent on open arms, scanning, end exploring, but not by risk assessment. Finally, the effects of the 5-HT3 antagonist BRL 46470 A (0.001, 0.01, and 0.1 mg/kg) and 5-HT(2A/C) antagonist RP 62203 (0.25, 1, and 4 mg/kg) were scarce in both conventional and ethologically derived measures. These results are indicative that ethological measures may sometimes be more sensitive than the standard ones, and should be used together with them when assessing serotonergic or any other novel drugs in the elevated plus-maze.

Effect of central acute administration of cadmium on drinking behavior.

The effect of acute third ventricle cadmium administration on the drinking behavior of adult male rats under different situations was studied. Injections of cadmium chloride (0.07, 0.7,and 7.0 ng/rat) significantly attenuated water intake in dehydrated rats. Drinking behavior induced by acute intracerebroventricular injections of carbachol (2 micrograms/rat) or angiotensin II (5 ng/rat) was also inhibited by central cadmium injections. Cadmium-induced blockade in water intake in dehydrated animals was reverted by the previous administration of a 5-HT2 antagonist (RP62203) in different doses (5 and 10 micrograms/rat). The data clearly reveal that cadmium elicits very fast actions on the central nervous system. It is suggested that cadmium-induced attenuation of water intake may rely on at least three different mechanisms: impairment of cholinergic and angiotensinergic systems in the brain and stimulation of a central serotonergic drive acting on 5-HT2 receptors. The study of cadmium neurotoxicity by observation of drinking behavior, a behavioral parameter easy to be recorded and measured, is proposed.