Boron-based inhibitors of acyl protein thioesterases 1 and 2.

Ras proteins are of importance in cell proliferation, and hence their mutated forms play causative roles in many kinds of cancer in different tissues. Inhibition of the Ras-depalmitoylating enzyme acyl protein thioesterases APT1 and -2 is a new approach to modulating the Ras cycle. Here we present boronic and borinic acid derivatives as a new class of potent and nontoxic APT inhibitors. These compounds were detected by extensive library screening using chemical arrays and turned out to inhibit human APT1 and -2 in a competitive mode. Furthermore, one of the molecules was demonstrated to inhibit Erk1/2 phosphorylation significantly.

Low concentration of ethanol induce apoptosis in HepG2 cells: role of various signal transduction pathways.

As we previously demonstrated in human hepatocellular carcinoma (HepG2) cells, ethanol at low concentration triggers the Fas apoptotic pathway. However, its role in other intracellular signaling pathways remains unknown. Therefore, the aim of the present study was to evaluate the role of low concentration of ethanol on different intracellular signaling pathways. For this purpose, HepG2 cells were treated with 1 mM ethanol for 10 min and the phosphorylation state of protein kinases was determined. In addition, the mRNA levels of transcription factors and genes associated with the Fas apoptotic pathway were determined. Our data demonstrated that ethanol-induced phosphorylation of protein kinases modulates both anti-apoptotic and pro-apoptotic mechanisms in HepG2 cells. Pro-apoptosis resulted mainly from the strong inhibition of the G-protein couple receptor signaling pathway. Moreover, the signal transduction initiated by ethanol-induced protein kinases phosphorylation lead to increased expression of the transcription factors with subsequent expression of genes associated with the Fas apoptotic pathway (Fas receptor, Fas ligand, FADD and caspase 8). These results indicate that low concentration of ethanol exert their effect by predominant activation of pro-apoptotic events that can be divided in two phases. An early phase characterized by a rapid transient effect on protein kinases phosphorylation, after 10 min exposure, with subsequent increased expression of transcription factors for up to 6 hr. This early phase is followed by a second phase associated with increased gene expression that began after 6 hr and persisted for more than 24 hr. This information provided a novel insight into the mechanisms of action of ethanol (1mM) in human hepatocellular carcinoma cells.

Functional genomics analysis of low concentration of ethanol in human hepatocellular carcinoma (HepG2) cells. Role of genes involved in transcriptional and translational processes.

We previously found that ethanol at millimolar level (1 mM) activates the expression of transcription factors with subsequent regulation of apoptotic genes in human hepatocellular carcinoma (HCC) HepG2 cells. However, the role of ethanol on the expression of genes implicated in transcriptional and translational processes remains unknown. Therefore, the aim of this study was to characterize the effect of low concentration of ethanol on gene expression profiling in HepG2 cells using cDNA microarrays with especial interest in genes with transcriptional and translational function. The gene expression pattern observed in the ethanol-treated HepG2 cells revealed a relatively similar pattern to that found in the untreated control cells. The pairwise comparison analysis demonstrated four significantly up-regulated (COBRA1, ITGB4, STAU2, and HMGN3) genes and one down-regulated (ANK3) gene. All these genes exert their function on transcriptional and translational processes and until now none of these genes have been associated with ethanol. This functional genomic analysis demonstrates the reported interaction between ethanol and ethanol-regulated genes. Moreover, it confirms the relationship between ethanol-regulated genes and various signaling pathways associated with ethanol-induced apoptosis. The data presented in this study represents an important contribution toward the understanding of the molecular mechanisms of ethanol at low concentration in HepG2 cells, a HCC-derived cell line.

Sodium-phosphate cotransporter in human salivary glands: molecular evidence for the involvement of NPT2b in acinar phosphate secretion and ductal phosphate reabsorption.

OBJECTIVE: In order to elucidate the cellular and molecular mechanisms of phosphate secretion by human salivary glands, the expression and intracellular distribution of sodium-phosphate cotransporters was investigated. DESIGN: Total RNA was extracted from 33 parotid gland (PG) and 35 submandibular gland (SMG) samples and RT-PCR was performed using gene specific primers for all known sodium-phosphate cotransporters. An antibody was raised against an NPT2b epitope and the cellular and intracellular distribution was investigated by immunohistochemistry. RESULTS: No mRNA for the type I cotransporter NPT1 was found. Out of the type II phosphate cotransporters only message for NPT2b but not for NPT2a or NPT2c could be detected in about the same number of samples (76% in PG versus 69% in SMG). Type III cotransporter mRNA was also found in both glands, PIT1 gave positive results for 93% of PG samples compared to 69% of SMG samples. For PIT2 also, a higher expression was found in PG than in SMG, although the difference was smaller (79% versus 51%). Immunostaining for NPT2b was found both in the acini and in the ducts, with a stronger reaction in the latter. In acinar cells, NPT2b was restricted to the basal-lateral plasma membrane, in duct cells, a broad band of reactivity was located in the apical part of the cell. CONCLUSIONS: These findings suggest a secondary active secretion of phosphate into the primary saliva. Ductal cells appear to be able to reabsorb phosphate, thereby modifying the phosphate concentration in the final saliva.