Athanasios Karapetsas

Metal-induced carcinogenesis, oxidative stress and hypoxia signalling

Heavy metal-induced carcinogenesis is well documented by epidemiological studies. Several diverse mechanisms of cancer induction may be involved, depending on the form of every metal and the tissue that is exposed. Over the recent years, induction of signalling pathways that regulate key cellular responses related to cancer growth and progression by metals has been the focus of many studies. The unravelling of these pathways and the deciphering of their interplay with metals should allow a better understanding of metal toxicity and hopefully will enable development of prophylactic strategies and therapeutic approaches. In this work, we review the mechanisms of carcinogenesis caused by heavy metals emphasizing on the involvement of the hypoxia signalling pathway by metal-induced generation of reactive oxygen species and oxidative stress generation in cancer progression.

Effect of probiotic-fermented milk administration on gastrointestinal survival of Lactobacillus casei ATCC 393 and modulation of intestinal microbial flora.

The aim of the present study was to assess the survival of free and immobilized Lactobacillus casei ATCC 393 on apple pieces, contained in probiotic-fermented milk, after gastrointestinal (GI) transit and to investigate the potential regulation of intestinal microbial flora in a rat model. In in vitro GI stress tolerance tests, immobilized L. casei ATCC 393 exhibited significantly higher survival rates compared to free cells. At a second stage, probiotic-fermented milk produced by either free or immobilized cells was administered orally at a single dose or daily for 9 days in Wistar rats. By 12 h after single-dose administration, both free and immobilized cells were detected by microbiological and molecular analysis at levels ≧6 logCFU/g of feces. Moreover, daily administration led to significant reduction of staphylococci, enterobacteria, coliforms and streptococci counts. In conclusion, L. casei ATCC 393 contained in fermented milk survived GI transit and modulated intestinal microbiota.

Effective survival of immobilized Lactobacillus casei during ripening and heat treatment of probiotic dry-fermented sausages and investigation of the microbial dynamics.

The aim was the assessment of immobilized Lactobacillus casei ATCC 393 on wheat in the production of probiotic dry-fermented sausages and the investigation of the microbial dynamics. For comparison, sausages containing either free L. casei ATCC 393 or no starter culture were also prepared. During ripening, the numbers of lactobacilli exceeded 7 log cfu/g, while a drastic decrease was observed in enterobacteria, staphylococci and pseudomonas counts. Microbial diversity was further studied applying a PCR-DGGE protocol. Members of Lactobacillus, Leuconostoc, Lactococcus, Carnobacterium, Brochothrix, Bacillus and Debaryomyces were the main microbial populations detected. Microbiological and strain-specific multiplex PCR analysis confirmed that the levels of L. casei ATCC 393 in the samples after 66 days of ripening were above the minimum concentration for conferring a probiotic effect (≥ 6 log cfu/g). However, after heat treatment, this strain was detected at the above levels, only in sausages containing immobilized cells.

Rapid Detection and Identification of Probiotic Lactobacillus casei ATCC 393 by Multiplex PCR

Many functional foods containing probiotic strains have been developed recently. Lactobacillus casei ATCC 393 is one of the most frequently used cultures in probiotic products. The present study aimed to develop a method for the detection and identification of L. casei ATCC 393 based on genetic polymorphisms of the hsp60 gene. A multiplex polymerase chain reaction (PCR) assay was designed, utilizing two novel strain-specific primer sets that enable identification of L. casei ATCC 393.The accuracy of our method was further confirmed by successful identification of our strain in probiotic cheese. The method described is an easy to use, rapid, inexpensive and accurate tool that may be readily applied to food, fecal and intestinal samples.

Isolation, characterization and evaluation of the probiotic potential of a novel Lactobacillus strain isolated from Feta-type cheese

In the present study 45 lactic acid bacteria (LAB) strains were isolated from Feta-type cheese and were screened for probiotic potential in a series of established in vitro tests, including resistance to low pH, resistance to pepsin and pancreatin and tolerance to bile salts. The strain K5, which displayed properties similar to or even better than the reference strain Lactobacillus plantarum ATCC 14917, was chosen for further analysis. Firstly, multiplex PCR analysis indicated that the novel strain belongs to the paracasei species. Secondly, the susceptibility against common antibiotics was determined to ensure a safe exploitation of the potentially probiotic strain. Additionally, the performance of L. paracasei K5 as starter in the fermentation of pomegranate juice was studied to evaluate its technological properties. Finally, a novel multiplex PCR assay, based on random amplified polymorphic DNA (RAPD) analysis was developed for its efficient and accurate detection in food products.

Biochemical and molecular analysis of the interaction between ERK2 MAP kinase and hypoxia inducible factor-1α

The mitogen activated protein kinase (MAPK) signaling pathways play significant roles in fundamental cellular processes, such as cell growth and differentiation. It has been shown that the specificity and efficacy of phosphorylation by MAP kinases rely upon distinct MAPK-docking domains (D-domains) found in a wide range of MAPK substrates including the ETS-transcription factor Elk-1. Importantly, the MAPK signaling cascade converges with the hypoxia-induced signaling pathway. The key regulator of hypoxia signaling is the heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1). The α-subunit of HIF-1 (HIF-1α) is a substrate for the ERK2 MAP kinase. Unraveling the interplay of these main signaling systems is a prerequisite for understanding their role in tumor growth, a situation sustained by simultaneous mitogenic and hypoxic signals. In this work, we investigated the molecular cues that direct HIF-1α recognition and phosphorylation by ERK2. We showed that HIF-1α possesses a MAPK docking domain. Utilizing surface plasmon resonance (SPR) methodologies we demonstrated efficient binding between HIF-1α and ERK2, with a K(D) value in the low micromolar range. Although, the D-domain did not contribute to the above interaction significantly, it could act in trans by recruiting ERK2 and conferring responsiveness to poor ERK substrates. These results indicate that, via its conserved D-domain, HIF-1α could serve as a platform for ERK2 in the nucleus of the cell, thus potentially facilitating phosphorylation of other ERK2 substrates. The identification of an ERK2 recognition domain on HIF-1α opens new avenues for the analysis of HIF-1α-related ERK2 signaling and may allow designing of interfering compounds.

Overexpression of SMARCE1 is associated with CD8+ T-cell infiltration in early stage ovarian cancer.

T-lymphocyte infiltration in ovarian tumors has been linked to a favorable prognosis, hence, exploring the mechanism of T-cell recruitment in the tumor is warranted. We employed a differential expression analysis to identify genes over-expressed in early stage ovarian cancer samples that contained CD8 infiltrating T-lymphocytes. Among other genes, we discovered that TTF1, a regulator of ribosomal RNA gene expression, and SMARCE1, a factor associated with chromatin remodeling were overexpressed in first stage CD8+ ovarian tumors. TTF1 and SMARCE1 mRNA levels showed a strong correlation with the number of intra-tumoral CD8+ cells in ovarian tumors. Interestingly, forced overexpression of SMARCE1 in SKOV3 ovarian cancer cells resulted in secretion of IL8, MIP1b and RANTES chemokines in the supernatant and triggered chemotaxis of CD8+ lymphocytes in a cell culture assay. The potency of SMARCE1-mediated chemotaxis appeared comparable to that caused by the transfection of the CXCL9 gene, coding for a chemokine known to attract T-cells. Our analysis pinpoints TTF1 and SMARCE1 as genes potentially involved in cancer immunology. Since both TTF1 and SMARCE1 are involved in chromatin remodeling, our results imply an epigenetic regulatory mechanism for T-cell recruitment that invites deciphering.

Overexpression of GPC6 and TMEM132D in Early Stage Ovarian Cancer Correlates with CD8+ T-Lymphocyte Infiltration and Increased Patient Survival

Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (p = 0.0067) and 0.861 (p < 0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (p = 0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling.

The homeodomain transcription factor MEIS1 triggers chemokine expression and is involved in CD8+ T-lymphocyte infiltration in early stage ovarian cancer

CD8+ T‐lymphocytes infiltration is a favorable prognostic marker in ovarian cancer. Recently we identified MEIS1 as a gene overexpressed in early stage ovarian tumors enriched for CD8+ T‐cells. Here, we report the molecular mechanism of the homeodomain transcription factor MEIS1 in lymphocyte recruitment. We validated that MEIS1 expression is a positive predictor of CD8+ T cells in early stage ovarian cancer. We showed that MEIS1 induces the expression of CCL18, CCL4, CXCL7, CCL5, CXCL1, and IL8 chemokines in cancer cells followed by their secretion in the culture medium ultimately triggering CD8+ T‐lymphocyte recruitment in vitro. Knock down of MEIS1 expression by siRNA resulted in downregulation of these chemokines. We verified that MEIS1 binds to the promoters of chemokine genes, both in vitro and in vivo. We also showed that the expression levels of MEIS1 correlated tightly with the mRNA levels of chemokines CCL4 and CCL18 in early stage ovarian cancer patient samples and served as a positive prognostic marker, as shown by Kaplan‐Meyer survival analysis. In conclusion, we propose that MEIS1 plays a pivotal role in the regulatory circuitry governing T‐cell chemo‐attraction during the early stages of ovarian cancer.

Lactobacillus paracasei K5 displays adhesion, anti-proliferative activity and apoptotic effects in human colon cancer cells

Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain, isolated recently from feta-type cheese. Its probiotic potential has been demonstrated in a series of established in vitro tests. Moreover, incorporation of L. paracasei K5 as starter culture offered organoleptic and technological advantages to novel fermented food products. In the present study, further investigation of the potential probiotic activity of L. paracasei K5 was performed and its mechanisms of action were investigated. Employing quantitative analysis and confocal, fluorescent microscopy the adhesion properties of the above strain were studied. L. paracasei K5 displayed efficient adherence capacity to Caco-2 colon cancer cells, similarly to the reference strains Lactobacillus casei ATCC 393 and Lactobacillus rhamnosus GG. Moreover, treatment of Caco-2 cells with L. paracasei K5 inhibited cell proliferation in a time-and dose-dependent manner. The anti-proliferative effects appear to be mediated through induction of apoptosis via modulation of expression of specific Bcl-2 family proteins. These results elucidate the mechanisms of action of L. paracasei K5 and enhance its potential probiotic activity.