The ATP-binding cassette sub-family C member 6 transporter (ABCC6) is an ATP dependent transporter mainly found in the basolateral plasma membrane of hepatic and kidney cells. Mutations in ABCC6 gene were associated to the Pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by a progressive ectopic calcification of elastic fibers in dermal, ocular, and vascular tissues. It is reported that the over-expression of ABCC6 in HEK293 cells results in the cellular efflux of ATP and other nucleoside triphosphates, which in turn are rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since PPi is an inhibitor of mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the ectopic mineralization, a typical feature of PXE. In the extracellular environment, ATP is converted, not only into pyrophosphate, but also into AMP by an ectonucleosidase, which in turn is transformed into adenosine and phosphate. ABCC6 protein is thus involved in the production of extracellular adenosine and therefore it could have a role in the activation of the purinergic system. In the liver, purinergic signaling has been shown to regulate key basic cellular functions. Our previous studies showed that in ABCC6 knockdown HepG2 cells the expression of some genes, related with the calcification processes, is dysregulated. In this study, experiments have been carried out in order to verify if ABCC6, besides supplying the pyrophosphate required to prevent the mineralization of soft tissues, also plays a role in the activation of the purinergic system. For this purpose, the transport activity of ABCC6 was blocked with Probenecid and the expression of ABCC6 and NT5E was analyzed with real time PCR and western blotting. The results of this study showed that both proteins are downregulated in the presence of Probenecid and upregulated in the presence of adenosine or ATP.

ABCC6, belonging to sub-family C of ATP-binding cassette transporter, is an ATP-dependent transporter mainly present in the basolateral plasma membrane of hepatic and kidney cells. Although the substrates transported are still uncertain, ABCC6 has been shown to promote ATP release. The extracellular ATP and its derivatives di- and mono-nucleotides and adenosine by acting on specific receptors activate the so-called purinergic pathway, which in turn controls relevant cellular functions such as cell immunity, inflammation, and cancer. Here, we analyzed the effect of Abcc6 knockdown and probenecid-induced ABCC6 inhibition on cell cycle, cytoskeleton, and motility of HepG2 cells. Gene and protein expression were evaluated by quantitative Reverse Transcription PCR (RT-qPCR) and western blot, respectively. Cellular cycle analysis was evaluated by flow cytometry. Actin cytoskeleton dynamics was evaluated by laser confocal microscopy using fluorophore-conjugated phalloidin. Cell motility was analyzed by in vitro wound-healing migration assay. Cell migration is reduced both in Abcc6 knockdown HepG2 cells and in probenecid treated HepG2 cells by interfering with the extracellular reserve of ATP. Therefore, ABCC6 could contribute to cytoskeleton rearrangements and cell motility through purinergic signaling. Altogether, our findings shed light on a new role of the ABCC6 transporter in HepG2 cells and suggest that its inhibitor/s could be considered potential anti-metastatic drugs.

Breast cancer represents the most frequently diagnosed malignancy in women worldwide. Various therapeutics are currently used in order to halt the progression of breast tumor, even though certain side effects may limit the beneficial effects. In recent years, many efforts have been addressed to the usefulness of natural compounds as anticancer agents due to their low toxicity. Resveratrol, a stilbene found in grapes, berries, peanuts and soybeans, has raised a notable interest for its antioxidant, anti-inflammatory, and antitumor properties. Here, we report the design, the synthesis and the characterization of the anticancer activity of a small series of imino N-aryl-substituted compounds that are analogues of resveratrol. In particular, the most active compound, named 3, exhibited anti-tumor activity in diverse types of breast cancer cells through the inhibition of the human topoisomerase II and the induction of apoptotic cell death. Therefore, the abovementioned compound maybe considered as a promising agent in more comprehensive treatments of breast cancer.

The vitamin D and microRNA (miR) systems may play a role in the pathogenesis of cardiometabolic disorders, including hypertension. The HYPODD study was a double-blind placebo-controlled trial aiming to assess the effects of cholecalciferol treatment in patients with well-controlled hypertension and hypovitaminosis D (25OHD levels < 50 nmol/L). In addition to this clinical trial, we also evaluated the effects of cholecalciferol and calcitriol treatment on miR-21 expression in vivo and in vitro, respectively. Changes in the cardiovascular risk profiles were evaluated in HYPODD patients treated with cholecalciferol (C-cohort) or with placebo (P-cohort). The miR-21circulating levels were measured in four C-cohort patients and five P-cohort patients. In vitro, the miR-21 levels were measured in HEK-293 cells treated with calcitriol or with ethanol vehicle control. Cholecalciferol treatment increased 25OHD levels and reduced parathormone, total cholesterol, and low-density lipoprotein cholesterol levels in C-cohort patients, whereas no significant changes in these parameters were observed in P-cohort patients. The miR-21 circulating levels did not change in the C- or the P-cohort patients upon treatment. Calcitriol treatment did not affect miR-21 levels in HEK-293 cells. In conclusion, hypovitaminosis D correction ameliorated the cardiovascular risk profiles in hypertensive patients treated with cholecalciferol but did not influence the miR-21 expression.

Quercetin is a member of the flavonoid group of compounds, which is abundantly present in various dietary sources. It has excellent antioxidant properties and anti-inflammatory activity and is very effective as an anti-cancer agent against various types of tumors, both in vivo and in vitro. Quercetin has been also reported to modulate the activity of some members of the multidrug-resistance transporters family, such as P-gp, ABCC1, ABCC2, and ABCG2, and the activity of ecto-5′-nucleotidase (NT5E/CD73), a key regulator in some tumor processes such as invasion, migration, and metastasis. In this study, we investigated the effect of Quercetin on ABCC6 expression in HepG2 cells. ABCC6 is a member of the superfamily of ATP-binding cassette (ABC) transporters, poorly involved in drug resistance, whose mutations cause pseudoxanthoma elasticum, an inherited disease characterized by ectopic calcification of soft connective tissues. Recently, it has been reported that ABCC6 contributes to cytoskeleton rearrangements and HepG2 cell motility through purinergic signaling. Gene and protein expression were evaluated by quantitative Reverse-Transcription PCR (RT-qPCR) and western blot, respectively. Actin cytoskeleton dynamics was evaluated by laser confocal microscopy using fluorophore-conjugated phalloidin. Cell motility was analyzed by an in vitro wound-healing migration assay. We propose that ABCC6 expression may be controlled by the AKT pathway as part of an adaptative response to oxidative stress, which can be mitigated by the use of Quercetin-like flavonoids.