Extracellular Citrate is a Trojan Horse for Cancer Cells

The first intermediate in the mitochondrial tricarboxylic acid (TCA) cycle is citrate, which is essential and acts as a metabolic regulator for glycolysis, TCA cycle, gluconeogenesis, and fatty acid synthesis. Within the cytosol, citrate is cleaved by ATP citrate lyase (ACLY) into oxaloacetate (OAA) and acetyl-CoA; OAA can be used for neoglucogenesis or in the TCA cycle, while acetyl-CoA is the precursor of some biosynthetic processes, including the synthesis of fatty acids. Accumulating evidence suggests that citrate is involved in numerous physiological and pathophysiological processes such as inflammation, insulin secretion, neurological disorders, and cancer. Considering the crucial role of citrate to supply the acetyl-CoA pool for fatty acid synthesis and histone acetylation in tumors, in this study we evaluated the effect of citrate added to the growth medium on lipid deposition and histone H4 acetylation in hepatoma cells (HepG2). At low concentration, citrate increased both histone H4 acetylation and lipid deposition; at high concentration, citrate inhibited both, thus suggesting a crucial role of acetyl-CoA availability, which prompted us to investigate the effect of citrate on ACLY. In HepG2 cells, the expression of ACLY is correlated with histone acetylation, which, in turn, depends on citrate concentration. A decrease in H4 acetylation was also observed when citrate was added at a high concentration to immortalized human hepatic cells, whereas ACLY expression was unaffected, indicating a lack of control by histone acetylation. Considering the strong demand for acetyl-CoA but not for OAA in tumor cells, the exogenous citrate would behave like a trojan horse that carries OAA inside the cells and reduces ACLY expression and cellular metabolism. In addition, this study confirmed the already reported dual role of citrate both as a promoter of cell proliferation (at lower concentrations) and as an anticancer agent (at higher concentrations), providing useful tips on the use of citrate for the treatment of tumors

Structural and Functional Characterization of the ABCC6 Transporter in Hepatic Cells: Role on PXE, Cancer Therapy and Drug Resistance

Pseudoxanthoma elasticum (PXE) is a complex autosomal recessive disease caused by mutations of ABCC6 transporter and characterized by ectopic mineralization of soft connective tissues. Compared to the other ABC transporters, very few studies are available to explain the structural components and working of a full ABCC6 transporter, which may provide some idea about its physiological role in humans. Some studies suggest that mutations of ABCC6 in the liver lead to a decrease in some circulating factor and indicate that PXE is a metabolic disease. It has been reported that ABCC6 mediates the efflux of ATP, which is hydrolyzed in PPi and AMP; in the extracellular milieu, PPi gives potent anti-mineralization effect, whereas AMP is hydrolyzed to Pi and adenosine which affects some cellular properties by modulating the purinergic pathway. Structural and functional studies have demonstrated that silencing or inhibition of ABCC6 with probenecid changed the expression of several genes and proteins such as NT5E and TNAP, as well as Lamin, and CDK1, which are involved in cell motility and cell cycle. Furthermore, a change in cytoskeleton rearrangement and decreased motility of HepG2 cells makes ABCC6 a potential target for anti-cancer therapy. Collectively, these findings suggested that ABCC6 transporter performs functions that modify both the external and internal compartments of the cells

The Expression Level of ABCC6 Transporter in Colon Cancer Cells Correlates with the Activation of Different Intracellular Signaling Pathways

The ATP-binding cassette sub-family C member 6 transporter (ABCC6) is mainly found in the basolateral plasma membrane of hepatic and kidney cells. In hepatocarcinoma HepG2 cells, ABCC6 was involved in cell migration. In the present study, we investigated the role of ABCC6 in colon cancer evaluating the effect of Quercetin and Probenecid, inhibitors of the ectonucleotidase NT5E and ABCC6, respectively, on migration rate of Caco2 and HT29 cell lines. Both drugs reduced cell migration analyzed by scratch test. Gene and protein expression were evaluated by quantitative reverse-transcription PCR (RT-qPCR) and Western blot, respectively. In Caco2 cells, in which ABCC6 is significantly expressed, the addition of ATP restored motility, suggesting the involvement of P2 receptors. Contrary to HT29 cells, where the expression of ABCC6 is negligible but remarkable to the level of NT5E, no effect of ATP addition was detected, suggesting a main role on their migration by the phosphatidylinositol 3′-kinase (PI3K)/Akt system. Therefore, in some colon cancers in which ABCC6 is overexpressed, it may have a primary role in controlling the extracellular purinergic system by feeding it with ATP, thus representing a potential target for a therapy aimed at mitigating invasiveness of those type of cancers

Identification of a New Splice Variant of the Human ABCC6 Transporter

ABCC6 is a member of the adenosine triphosphate-binding cassette (ABC) gene subfamily C that encodes a protein (MRP6) involved in active transport of intracellular compounds to the extracellular environment. Mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), an autosomal recessive disorder of the connective tissue characterized by progressive calcification of elastic structures in the skin, the eyes, and the cardiovascular system. MRP6 is codified by 31 exons and contains 1503 amino acids. In addition to a full-length transcript of ABCC6, we have identified an alternatively spliced variant of ABCC6 from a cDNA of human liver that lacks exons 19 and 24. The novel isoform was named ABCC6 Δ19Δ24. PCR analysis from cDNA of cell cultures of primary human hepatocites and embryonic kidney confirms the presence of the ABCC6Δ19Δ24 isoform. Western blot analysis of the embryonic kidney cells shows a band corresponding to the molecular weight of the truncated protein

Effect of Quercetin on ABCC6 Transporter: Implication in HepG2 Migration.

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

A Regulator Role for the ATP-Binding Cassette Subfamily C Member 6 Transporter in HepG2 Cells: Effect on the Dynamics of Cell-Cell and Cell-Matrix Interactions

There is growing evidence that various ATP-binding cassette (ABC) transporters contribute to the growth and development of tumors, but relatively little is known about how the ABC transporter family behaves in hepatocellular carcinoma (HCC), one of the most common cancers worldwide. Cellular model studies have shown that ABCC6, which belongs to the ABC subfamily C (ABCC), plays a role in the cytoskeleton rearrangement and migration of HepG2 hepatocarcinoma cells, thus highlighting its role in cancer biology. Deep knowledge on the molecular mechanisms underlying the observed results could provide therapeutic insights into the tumors in which ABCC6 is modulated. In this study, differential expression levels of mRNA transcripts between ABCC6-silenced HepG2 and control groups were measured, and subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Real-Time PCR and Western blot analyses confirmed bioinformatics; functional studies support the molecular mechanisms underlying the observed effects. The results provide valuable information on the dysregulation of fundamental cellular processes, such as the focal adhesion pathway, which allowed us to obtain detailed information on the active role that the down-regulation of ABCC6 could play in the biology of liver tumors, as it is involved not only in cell migration but also in cell adhesion and invasion

New heteroaryl carbamates: synthesis and biological screening in vitro and in mammalian cells of wild-type and mutant HIV-protease inhibitors

New heteroaryl HIV-protease inhibitors bearing a carbamoyl spacer were synthesized in few steps and high yield, from commercially available homochiral epoxides. Different substitution patterns were introduced onto a given isopropanoyl-sulfonamide core that can have either H or benzyl group. The in vitro inhibition activity against recombinant protease showed a general beneficial effect of both carbamoyl moiety and the benzyl group, ranging the IC50 values between 11 and 0.6 nM. In particular, benzofuryl and indolyl derivatives showed IC50 values among the best for such structurally simple inhibitors. Docking analysis allowed to identify the favorable situation of such derivatives in terms of number of interactions in the active site, supporting the experimental results. The inhibition activity was also confirmed in HEK293 mammalian cells and was maintained against protease mutants. Furthermore, the metabolic stability was comparable with that of the commercially available inhibitors

Antioxidant and Proapoptotic Activities of Sclerocarya birrea [(A. Rich.) Hochst.] Methanolic Root Extract on the Hepatocellular Carcinoma Cell Line HepG2

The main goal of this study was to characterize the in vitro antioxidant activity and the apoptotic potential of S. birrea methanolic root extract (MRE). Among four tested extracts, obtained with different solvents, MRE showed the highest content of polyphenols, flavonoids, and tannins together with antioxidant activities tested with superoxide, nitric oxide, ABTS, and beta-carotene bleaching assays. Moreover, the cytotoxic effect of MRE was evaluated on the hepatocarcinoma cell line HepG2. In these cells, MRE treatment induced apoptosis and generated reactive oxygen species (ROS) in dose-dependent manner. The cytotoxic effect promoted by MRE was prevented by pretreatment of HepG2 cells with N-acetyl-L-cysteine (NAC), suggesting that oxidative stress was pivotal in MRE-mediated cell death. Moreover, we showed that the MRE treatment induced the mitochondrial membrane depolarization and the cytochrome c release from mitochondria into the cytosol. It suggests that the apoptosis occurred in a mitochondrial-dependent pathway. Interestingly, MRE showed a sensibly lower cytotoxicity, associated with a low increase of ROS, in normal human dermal fibroblasts compared to HepG2 cells. It is suggested that the methanolic root extract of S. Birrea is able to selectively increase intracellular ROS levels in cancer cells, promoting cell death

The hepatitis B x antigen anti-apoptotic effector URG7 is localized to the endoplasmic reticulum membrane

Hepatitis B x antigen up-regulates the liver expression of URG7 that contributes to sustain chronic virus infection and to increase the risk for hepatocellular carcinoma by its anti-apoptotic activity. We have investigated the subcellular localization of URG7 expressed in HepG2 cells and determined its membrane topology by glycosylation mapping in vitro. The results demonstrate that URG7 is N-glycosylated and located to the endoplasmic reticulum membrane with an Nlumen–Ccytosol orientation. The results imply that the anti-apoptotic effect of URG7 could arise from the C-terminal cytosolic tail binding a pro-apoptotic signaling factor and retaining it to the endoplasmic reticulum membrane