l-carnosine dipeptide overcomes acquired resistance to 5-fluorouracil in HT29 human colon cancer cells via downregulation of HIF1-alpha and induction of apoptosis.

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 Adenovirus Loaded with L-carnosine as Novel Strategy to Enhance the Antitumor Activity.

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.

The anti-proliferative effect of L-carnosine correlates with a decreased expression of hypoxia inducible factor 1 alpha in human colon cancer cells.

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.

A comparative analysis of the photo-protective effects of soy isoflavones in their aglycone and glucoside forms.

Isoflavones exist in nature predominantly as glucosides such as daidzin or genistin and are rarely found in their corresponding aglycone forms daidzein and genistein. The metabolism and absorption of isoflavones ingested with food is well documented, but little is known about their use as topical photo-protective agents. The aim of this study was to investigate in a comparative analysis the photo-protective effects of isoflavones in both their aglycone and glucoside forms. In human skin fibroblasts irradiated with 60 mJ/cm2 ultraviolet B (UVB), we measured the expression levels of COX-2 and Gadd45, which are involved in inflammation and DNA repair, respectively. We also determined the cellular response to UVB-induced DNA damage using the comet assay. Our findings suggest that both the isoflavone glucosides at a specific concentration and combination with an aglycone mixture exerted an anti-inflammatory and photo-protective effect that prevented 41% and 71% of UVB-induced DNA damage, respectively. The advantages of using either isoflavone glucosides or an aglycone mixture in applications in the field of dermatology will depend on their properties and their different potential uses.

Carnosine inhibits KRAS-mediated HCT116 proliferation by affecting ATP and ROS production.

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.

Damage-specific DNA binding protein 1 (DDB1): a protein with a wide range of functions.

Damage-specific DNA binding protein 1 (DDB1) is a multifunctional protein that was first isolated as a subunit of a heterodimeric complex that recognises the UV-induced DNA lesions in the nucleotide excision repair pathway. DDB1 and DDB2 form a complex that promotes the global genome repair (GG-NER), whereas DDB1 and Cockayne syndrome group A protein (CSA) form a complex that contributes to the transcription-coupled repair (TC-NER) pathway. DDB1 is also a component of an ubiquitin-E3 ligase complex and functions as substrate or adapter protein between Cullin 4A (Cul4A) and CUL4-associated factors (DCAFs) to target substrates for ubiquitination. CUL4-DDB1 E3-ligase complex regulates the selective proteolysis of key proteins in DNA repair, replication and transcription. In addition, DDB1 plays a role in transcriptional regulation of UV-induced genes. It is conceivable that DDB1 acts as a sensor of damage to maintain the balance between genome integrity and cell cycle progression. However, the temporal order between these two events remains to be established.

Synergic Effect of Genistein and Daidzein on UVB-Induced DNA Damage: An Effective Photoprotective Combination.

The anti-inflammatory effects and antioxidant activities of individual isoflavones are well established although little is known about the photoprotective effect of their combination. The aim of this study was to investigate the photoprotective effects of different concentrations of genistein and daidzein individually or combined. We measured the expression levels of the cyclo-oxygenase-2 (COX-2) and growth arrest and DNA-damage inducible (Gadd45) genes, which are involved in inflammation and DNA repair, respectively, in BJ-5ta human skin fibroblasts irradiated with 60 mJ/cm(2) UVB. We also determined the cellular response to UVB-induced DNA damage by Comet assay. We report that genistein and daidzein when administered combined, and at a specific concentration and ratio, exerted a synergistic photoprotective effect that was greater than the effect obtained with each isoflavone alone. The results reported herein suggest that low concentrations of genistein and daidzein combined may be good candidate ingredients for protective agents against UV-induced photodamage.

Damage-specific DNA binding protein 1 (DDB1) is involved in ubiquitin-mediated proteolysis of p27Kip1 in response to UV irradiation.

Damage-specific DNA binding protein 1 (DDB1) is a conserved protein component of the damaged DNA binding protein complex (DDB) that recognizes UV-induced DNA lesions and initiates the nucleotide excision repair process. DDB1 is also part of an E3 ubiquitin-ligase complex that targets a variety of substrates for proteolysis including the cyclin-dependent kinase inhibitor p27(Kip1). The mechanism regulating the trafficking of DDB1 and its relationship with UV irradiation is not known, although cell cycle progression is implicated in the molecular machinery driving DDB1 into the nucleus. We evaluated the involvement of DDB1 in ubiquitination of the cdk inhibitor p27(Kip1) in response to UV irradiation. First, we observed that low and high doses of UV irradiation exert different effects on p27(Kip1) protein levels. Indeed, low but not high UV doses induced p27(Kip1) protein proteolysis in several human cell lines and UV-dependent degradation is dominant over other genotoxic agents such as cisplatin. We also demonstrate that p27(Kip1) reduction is not due to transcriptional regulation and that the proteasome inhibitor MG132 affects p27(Kip1) degradation. We observed that at low UV doses the decrease in p27(Kip1) nuclear protein related with DDB1 translocation into the nucleus; conversely, high doses of UV-induced p27(Kip1) accumulation and unchanged level of DDB1. The knockdown of DDB1 or Skp2 prevents UV-induced degradation of p27(Kip1) suggesting that DDB1 is essential to regulation of p27(kip1) turnover after a mild DNA damage. Our findings support the concept that DDB1 contributes to the activation of DNA repair mechanisms and could be a key factor in regulating the cell cycle in response to UV-induced DNA damage. Although the temporal order with which DDB1 contributes to ubiquitination of p27(Kip1) or initiates the nucleotide excision repair process remains to be established, our results represent a major step towards clarifying these issues.

Ultraviolet B and A irradiation induces fibromodulin expression in human fibroblasts in vitro.

Ultraviolet (UV) radiation affects the extracellular matrix (ECM) of the human skin. The small leucine-rich repeat protein fibromodulin interacts with type I and II collagen fibrils, thereby affecting ECM assembly. The aim of this study was to evaluate whether short wave UV (UVB) or long wave UV (UVA) irradiation influences fibromodulin expression. Exponentially growing human fibroblasts (IMR-90 cells) were exposed to increasing doses of UVB (2.5-60 mJ/cm(2)) or UVA (0.5-10 J/cm(2)). After UV irradiation fibromodulin, p21 and GADD45 levels were evaluated as well as cell viability, reactive oxygen species formation (ROS) and DNA damage. We found that fibromodulin expression: (i) increased after UVB and UVA irradiation; (ii) was 10-fold higher after UVA (10 J/cm(2)) versus 5-fold with UVB (10 mJ/cm(2)); (iii) correlated with reactive oxygen species formation, particularly after UVA; and (iv) was linked to the DNA damage binding protein (DDB1) translocation in the nucleus, particularly after UVB. These results further suggest that the UV-induced fibromodulin increase could counteract the UV-induced connective tissue damage, promoting the assembly of new collagen fibrils.

Induction of H-ferritin synthesis by oxalomalate is regulated at both the transcriptional and post-transcriptional levels.

Ferritin gene expression is complex and is controlled at transcriptional level in response to a variety of stimuli such as hormones, cytokines and cAMP. Iron, hemin and several compounds, chemically different, also activate the transcription of the ferritin gene. Ferritin biosynthesis is mainly regulated at post-transcriptional level by iron regulatory proteins (IRP1 and IRP2). We previously reported that oxalomalate, a competitive inhibitor of aconitase, remarkably decreases the IRP1 RNA-binding activity and induces a significant increase of ferritin expression. Here, we examined in cells cultured in presence of OMA the IRP1 intracellular content, ferritin biosynthesis and the transcriptional efficiency of H-ferritin gene promoter. Our results demonstrate a peculiar role of OMA that rapidly inactivates IRP1 without affecting IRP1 protein content and subsequently activates H-ferritin gene transcription leading to an overall increase of ferritin biosynthesis. We conclude that OMA regulates H-ferritin biosynthesis acting early at the post-transcriptional level and later on at transcriptional level.

The role of NF-kappaB, IRF-1, and STAT-1alpha transcription factors in the iNOS gene induction by gliadin and IFN-gamma in RAW 264.7 macrophages.

Nitric oxide (NO) plays an important role in the pathogenesis of celiac disease. We have examined the involvement of nuclear factor-kappaB (NF-kappaB), interferon regulatory factor-1 (IRF-1), and signal transducer and activator of transcription-1alpha (STAT-1alpha) on the synergistic induction of inducible nitric oxide synthase (iNOS) gene expression by gliadin (G) in association with interferon-gamma (IFN-gamma) in RAW 264.7 macrophages. We found that IFN-gamma was efficient in enhancing the basal transcription of the iNOS promoter at 1, 6, and 24 h, whereas G had no effect. The G plus IFN-gamma association caused an increase in iNOS promoter activity which was inhibited by pyrrolidine dithiocarbammate (PDTC) at 6 and 24 h as well as by genistein (Gen) and tyrphostine B42 (TB42) at 1 h, inhibitors of NF-kappaB, IRF-1, and STAT-1alpha activation, respectively. Similarly, the IFN-gamma and G combination treatment led to a higher increase in iNOS mRNA levels at 1, 6, and 24 h compared with IFN-gamma alone. Gen and TB42 inhibited iNOS mRNA levels at 1 h, whereas PDTC inhibited iNOS mRNA levels at 6 and 24 h. In addition, the synergistic induction of iNOS gene expression by G plus IFN-gamma correlated with the induction of NF-kappaB, IRF-1, and STAT-1alpha/DNA binding activity and mRNA expression. In conclusion, our study, which provides evidence that the effect of G on iNOS gene transcription in IFN-gamma-stimulated RAW 264.7 cells can be ascribed to all three transcription factors, may contribute to lead to new insights into the molecular mechanisms governing the inflammatory process in celiac disease.

Fibromodulin gene transcription is induced by ultraviolet irradiation, and its regulation is impaired in senescent human fibroblasts.

Cells undergoing replicative senescence display an altered pattern of gene expression. Senescent fibroblasts show significant changes in the expression of mRNAs encoding extracellular matrix-remodeling proteins; among these mRNAs, the mRNA encoding fibromodulin is highly decreased in these cells. To understand the molecular basis of this phenomenon, we explored the regulatory mechanisms of the human fibromodulin gene. We found that fibromodulin gene promoter contains a cis-element, crucial for its basal expression, that forms a DNA-protein complex when exposed to nuclear extracts from exponentially growing human fibroblasts and not to extracts from cells undergoing senescence by repeated in vitro passages or by mild oxidative stress. The purification of this complex showed that it contains the damage-specific DNA-binding protein DDB-1. The latter is known to be induced by UV irradiation; therefore we checked whether fibromodulin gene promoter is regulated upon the exposure of the cells to UV rays. The results showed that, in exponentially growing fibroblasts, the promoter efficiency is increased by UV irradiation and the DDB-1-containing complex is robustly enriched in cells exposed to UV light. Accordingly, in these experimental conditions the endogenous fibromodulin mRNA accumulates to very high levels. On the contrary, senescent cells did not show any activation of the fibromodulin gene promoter, any induction of the DDB-1-containing complex, or any accumulation of fibromodulin mRNA. These phenomena are accompanied in senescent cells by a decrease of the UV-damaged DNA binding activity.

Gliadin increases iNOS gene expression in interferon-gamma-stimulated RAW 264.7 cells through a mechanism involving NF-kappa B.

Nitric oxide (NO) plays an important role in the pathogenesis of the histological changes seen in coeliac disease. We have investigated the effect of peptic-tryptic digest of gliadin (Pt-G) and gliadin (G) on inducible nitric oxide synthase (iNOS) protein expression in RAW 264.7 macrophages stimulated with interferon-gamma (IFN-gamma). Pt-G and G enhanced in a concentration and time-dependent manner NO production by IFN-gamma-stimulated RAW 264.7 cells. The increase of iNOS protein expression was correlated with NF-kappa B/DNA binding activity and occurred at transcriptional level. Pyrrolidine dithiocarbamate and N-alpha-para-tosyl-L-lysine chloromethyl ketone, two known inhibitors of NF-kappa B activation, decreased significantly NO production and iNOS protein expression as well as NF-kappa B/DNA binding activity. Our results show that the effect of Pt-G and G on enhancement of iNOS protein expression in IFN-gamma-treated RAW 264.7 cells is mainly mediated through NF-kappa B and suggest that blockage of NF-kappa B activation reduces enhancing effect of gluten on NO production in inflamed mucosa of coeliac patients.