Search

EDIZIONE ITALIANA DEL VOLUME "BIOCHEMISTRY" DI CAMPBELL/FARREL ISBN 88 7959 292 0

The differentiation program of a cell is the result of events affecting the expression levels both of genes involved in the modification of chromatin structure and of genes specific of the cell type. We have utilized human carcinoma colon cells (CaCO-2) because they spontaneously reach the final phenotype of enterocyte-type polarized cells upon confluence. Among genes involved in modification of chromatin structure we have investigated on those controlling the methylation state of DNA. To this aim, we analyzed the levels of gene expression both of methylating enzymes (the maintenance Dnmt1 and the “de novo” methyltransferases Dnmt3a and Dnmt3b) and demethylating enzymes (demethylases MBD2, MBD4 and the T:G mismatch-specific thymine-DNA glycosylase) because DNA methylation state is defined by the equilibrium between methylating and demethylating activities. We have analyzed also the time-courses of apolipoprotein A1, villin (a brush border molecular marker), H-ferritin and angiogenin (typical of late differentiation steps of carcinoma colon cells) as typical of genes specific of the cell type and GAPDH and beta-actin as typical of constitutive genes. During differentiation of CaCO-2 cells to enterocyte-like phenotype, we examined the general methylation pattern of DNA by Southern blot analysis of genomic DNA extracted from cells at different days in cultures, digested with methylation sensitive enzymes and hybridized with Sat II probe. These experiments show that, during the investigated period, no variation of global genome methylation is detectable indicating that no general change of chromatin structure was occurring. Analysis by RT-PCR of gene expression showed a complex pattern consistent with the presence of four different classes of genes with different expression time courses. These are: 1. genes, such as apolipoprotein A-1, villin and H-ferritin, that are expressed already in the seeded cells at 1 day in culture, but their expression increases constantly during differentiation; 2. genes, such as Dnmt1 and MBD4, that switch from a lower to a higher expression level that remains thereafter constant during following days in culture. 3. genes, such as Dnmt3b, MBD2 and T:G mismatch glycosylase, that change in expression levels during differentiation with peculiar patterns in correlation with cell confluence and the appearance of a brush border phenotype typical of enterocyte-type polarized cells, indicating that these genes might be involved in activities controlling the phenotype changes. 4. genes, such as angiogenin, that are specifically expressed only late during differentiation. The invariance of the global methylation pattern indicates that the general chromatin reorganization to produce the final phenotype, has already occurred in the fresh cultured CaCO-2 cells. The variations in the activity of Dnmt3b and MBD2 and T:G mismatch glycosylase suggest that local change might still occur, to modify the expression of specific genes necessary to implement the differentation program. Work is in progress to assess if this is the case.

Hypoxia-inducible factor (HIF-1α) protein is over-expressed in many human cancers and is a major cause of resistance to drugs. HIF-1α up-regulation decreases the effectiveness of several anticancer agents, including 5-fluorouracil (5-FU), because it induces the expression of drug efflux transporters, alters DNA repair mechanisms and modifies the balance between pro- and antiapoptotic factors. These findings suggest that inhibition of HIF-1α activity may sensitize cancer cells to cytotoxic drugs. We previously reported that l-carnosine reduces HIF-1α expression by inhibiting the proliferation of colon cancer cells. In the present study we investigated the effect of l-carnosine on HT29 colon cancer cells with acquired resistance to 5-FU. We found that l-carnosine reduces colon cancer cell viability, decreases HIF-1α and multi-drug resistant protein MDR1-pg expression, and induces apoptosis. Moreover, the l-carnosine/5-FU combination lowers the expression of some chemoresistance markers. The combination index evaluated in vitro on the HT29-5FU cell line by median drug effect analysis reveals a significant synergistic effect.

Oncolytic viruses are able to specifically replicate, infect, and kill only cancer cells. Their combination with chemotherapeutic drugs has shown promising results due to the synergistic action of virus and drugs; the combinatorial therapy is considered a potential clinically relevant approach for cancer. In this study, we optimized a strategy to absorb peptides on the viral capsid, based on electrostatic interaction, and used this strategy to deliver an active antitumor drug. We used L-carnosine, a naturally occurring histidine dipeptide with a significant antiproliferative activity. An ad hoc modified, positively charged L-carnosine was combined with the capsid of an oncolytic adenovirus to generate an electrostatic virus–carnosine complex. This complex showed enhanced antitumor efficacy in vitro and in vivo in different tumor models. In HCT-116 colorectal and A549 lung cancer cell lines, the complex showed higher transduction ratio and infectious titer compared with an uncoated oncolytic adenovirus. The in vivo efficacy of the complex was tested in lung and colon cancer xenograft models, showing a significant reduction in tumor growth. Importantly, we investigated the molecular mechanisms underlying the effects of complex on tumor growth reduction. We found that complex induces apoptosis in both cell lines, by using two different mechanisms, enhancing viral replication and affecting the expression of Hsp27. Our system could be used in future studies also for delivery of other bioactive drugs. Mol Cancer Ther; 15(4); 651–60. ©2016 AACR.

Carnosine is a natural dipeptide that has generated particular interest for its antioxidant, anti-aging and especially for its antiproliferative properties. In this study, we demonstrate that carnosine inhibits the proliferation of human HCT116 colon cancer cells. In this cell line, the activating KRAS mutation induces mitochondrial ROS, the signaling molecules for cell proliferation. We observed that 50-100 mM carnosine decreases ATP and ROS concentration and induces cell cycle arrest in G1 phase. In HCT116 cells these effects are related to decreased ERK1/2 phosphorylation and increased p21waf1 protein. Our findings support the concept that carnosine could inhibit HCT116 cell growth via its antioxidant activity and its ability to affect glycolysis.

The CaCo-2 cell line is used to study the molecular mechanisms underlying differentiation of intestinal epithelial cells. These cells undergo a gradual differentiation process that is growth-related and depends on cellular density. CaCo-2 cells acquire a morphological polarity and express such markers of mature enterocytes as sucrase-isomaltase, apolipoproteins, alkaline phosphatase, and H-ferritin. Because the NF-Y transcription factor is required for H-ferritin gene expression, we investigated whether it is involved in the expression of the other CaCo-2 differentiation markers. We observed that subunit NF-YA increases during CaCo-2 differentiation and that the constitutive expression of NF-YA, obtained in stably transfected CaCo-2 cells, results in the expression of differentiation markers. In fact, sucrase-isomaltase, apolipoprotein A1, and H-ferritin were constitutively expressed in NF-YA-transfected cells and their levels did not increase during prolonged culture, while these markers were not expressed in mock-transfected CaCo-2 cells or transfected with an inactive NF-YA expression vector until the onset of differentiation.

c-Jun is a member of the activator protein 1 family, and its interaction with different nuclear factors generates a wide spectrum of complexes that regulate transcription of different promoters. H ferritin promoter transcription is tightly dependent on nuclear factor Y (NFY). Ferritin transcription is activated by c-Jun, although the promoter does not contain a canonical binding site. NFY, on the other hand, does not bind c-Jun in vitro, whereas in vivo c-Jun is found in the complex containing NFY. Moreover, a c-Jun—GCN4 chimaeric construct containing only the transactivation domain of Jun and the basic-region leucine-zipper domain of GCN4 stimulates the H ferritin promoter. A synthetic GAL4 promoter and the cognate activator, the fusion protein NFY—GAL4, are potently activated by c-Jun. Titration of p300 by co-expressing E1A abolishes the stimulatory effect. Moreover, another p300-dependent promoter, the cAMP-response element, can be superactivated by c-Jun using the same mechanism. These data indicate that c-Jun, when activated or overexpressed, is recruited to the H ferritin promoter by p300, which links NFY, bound to DNA, to the complex. These results add a new level of complexity to transcriptional regulation by c-Jun, which can activate p300-dependent promoters without binding directly to the target DNA.

In recent years considerable attention has been given to the use of natural substances as anticancer drugs. The natural antioxidant dipeptide L-carnosine belongs to this class of molecules because it has been proved to have a significant anticancer activity both in vitro and in vivo. Previous studies have shown that L-carnosine inhibits the proliferation of human colorectal carcinoma cells by affecting the ATP and Reactive Oxygen Species (ROS) production. In the present study we identified the Hypoxia-Inducible Factor 1α (HIF-1α) as a possible target of L-carnosine in HCT-116 cell line. HIF-1α protein is over-expressed in multiple types of human cancer and is the major cause of resistance to drugs and radiation in solid tumours. Of particular interest are experimental data supporting the concept that generation of ROS provides a redox signal for HIF-1α induction, and it is known that some antioxidants are able to suppress tumorigenesis by inhibiting HIF-1α. In the current study we found that L-carnosine reduces the HIF-1α protein level affecting its stability and decreases the HIF-1 transcriptional activity. In addition, we demonstrated that L-carnosine is involved in ubiquitin-proteasome system promoting HIF-1α degradation. Finally, we compared the antioxidant activity of L-carnosine with that of two synthetic anti-oxidant bis-diaminotriazoles (namely 1 and 2, respectively). Despite these three compounds have the same ability in reducing intracellular ROS, 1 and 2 are more potent scavengers and have no effect on HIF-1α expression and cancer cell proliferation. These findings suggest that an analysis of L-carnosine antioxidant pathway will clarify the mechanism underlying the anti-proliferative effects of this dipeptide on colon cancer cells. However, although the molecular mechanism by which L-carnosine down regulates or inhibits the HIF-1α activity has not been yet elucidated, this ability may be promising in treating hypoxia-related diseases. ispartof: PLoS One vol:9 issue:5 ispartof: location:United States status: published