Maria Ausiliatrice Puglisi

Therapeutic Implications of Mesenchymal Stem Cells in Liver Injury

Mesenchymal stem cells (MSCs), represent an attractive tool for the establishment of a successful stem-cell-based therapy of liver diseases. A number of different mechanisms contribute to the therapeutic effects exerted by MSCs, since these cells can differentiate into functional hepatic cells and can also produce a series of growth factors and cytokines able to suppress inflammatory responses, reduce hepatocyte apoptosis, regress liver fibrosis, and enhance hepatocyte functionality. To date, the infusion of MSCs or MSC-conditioned medium has shown encouraging results in the treatment of fulminant hepatic failure and in end-stage liver disease in experimental settings. However, some issues under debate hamper the use of MSCs in clinical trials. This paper summarizes the biological relevance of MSCs and the potential benefits and risks that can result from translating the MSC research to the treatment of liver diseases.

Colon cancer stem cells: controversies and perspectives

Tumors have long been viewed as a population in which all cells have the equal propensity to form new tumors, the so called conventional stochastic model. The cutting-edge theory on tumor origin and progression, tends to consider cancer as a stem cell disease. Stem cells are actively involved in the onset and maintenance of colon cancer. This review is intended to examine the state of the art on colon cancer stem cells (CSCs), with regard to the recent achievements of basic research and to the corresponding translational consequences. Specific prominence is given to the hypothesized origin of CSCs and to the methods for their identification. The growing understanding of CSC biology is driving the optimization of novel anti-cancer targeted drugs.

Post-translational modulation of CD133 expression during sodium butyrate-induced differentiation of HT29 human colon cancer cells: implications for its detection.

The CD133 molecule has been proposed as a surface marker of cancer stem cells in several human malignancies, including colon cancers. The function and the mechanisms regulating CD133 expression remain unknown. The HT29 human colon cancer cells undergo differentiation following treatment with various agents and represent a useful in vitro model of colon differentiation. This study evaluated the behavior of CD133 during sodium butyrate‐induced differentiation of HT29 cells. Treatment with sodium butyrate induced a progressive decrease of CD133 expression, as assessed by flow cytometry using the AC133 monoclonal antibody. Indeed, expression of CD133, which was about 47% in untreated control cells, gradually decreased down to about 3% after 72 h in a time‐ and dose‐dependent manner. No relationship was observed between CD133 protein evaluated by flow cytometry and mRNA expression level, and no changes were detected in the methylation status of the CD133 gene promoter during HT29 differentiation. Moreover, the expression of the CD133 protein, evaluated by Western blot analysis using a specific anti‐CD133 antibody directed against the C‐terminal intracytoplasmic region of human CD133 protein, did not correlate with flow cytometry results. Different results were also obtained using the two antibodies to analyze the expression of the CD133 molecule in human colon cancers. These findings demonstrate that membrane expression of the CD133 stem cell marker might undergo a complex regulation during differentiation of colon cells and suggest that HT29 cells are a useful in vitro model to study the mechanisms involved in this regulation which likely occurs at a post‐transcriptional level. J. Cell. Physiol. 224:234–241, 2010

Neurotrophic features of human adipose tissue-derived stromal cells: in vitro and in vivo studies.

Due to its abundance, easy retrieval, and plasticity characteristics, adipose-tissue-derived stromal cells (ATSCs) present unquestionable advantages over other adult-tissue-derived stem cells. Based on the in silico analysis of our previous data reporting the ATSC-specific expression profiles, the present study attempted to clarify and validate at the functional level the expression of the neurospecific genes expressed by ATSC both in vitro and in vivo. This allowed evidencing that ATSCs express neuro-specific trophins, metabolic genes, and neuroprotective molecules. They were in fact able to induce neurite outgrowth in vitro, along with tissue-specific commitment along the neural lineage and the expression of the TRKA neurotrophin receptor in vivo. Our observation adds useful information to recent evidence proposing these cells as a suitable tool for cell-based applications in neuroregenerative medicine.

Gene profiling of bone marrow- and adipose tissue-derived stromal cells: a key role of Kruppel-like factor 4 in cell fate regulation

BACKGROUND AIMS Bone marrow- and adipose tissue-derived mesenchymal stromal cells (MSC) represent promising sources for regenerative medicine. However, the precise molecular mechanisms underlying MSC stemness maintenance versus differentiation are not fully understood. The aim of this study was to compare the genome-wide expression profiles of bone marrow-and adipose tissue-derived MSC, in order to identify a common molecular stemness core. METHODS Molecular profiling was carried out using Affymetrix microarray and relevant genes were further validated by Q-PCR. RESULTS We identified an overlapping dataset of 190 transcripts commonly regulated in both cell populations, which included several genes involved in stemness regulation (i.e. self-renewal potential and the ability to generate differentiated cells), various signaling pathways and transcription factors. In particular, we identified a central role of the Kruppel-like factor 4 (KLF4) DNA-binding protein in regulating MSC transcriptional activity. CONCLUSIONS Our results provide new insights toward understanding the molecular basis of MSC stemness maintenance and underline the ability of KLF4 to maintain cells in an undifferentiated state.

The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing

Although the only effective drug against primary hepatocarcinoma, the multikinase inhibitor Sorafenib (SFB) usually fails to eradicate liver cancer. Since SFB targets mitochondria, cell metabolic reprogramming may underlie intrinsic tumor resistance. To characterize cancer cell metabolic response to SFB, we measured oxygen consumption, generation of reactive oxygen species (ROS) and ATP content in rat LCSC (Liver Cancer Stem Cells) -2 cells exposed to the drug. Genome wide analysis of gene expression was performed by Affymetrix technology. SFB cytotoxicity was evaluated by multiple assays in the presence or absence of metabolic inhibitors, or in cells genetically depleted of mitochondria. We found that low concentrations (2.5–5 μM) of SFB had a relatively modest effect on LCSC-2 or 293 T cell growth, but damaged mitochondria and increased intracellular ROS. Gene expression profiling of SFB-treated cells was consistent with a shift toward aerobic glycolysis and, accordingly, SFB cytotoxicity was dramatically increased by glucose withdrawal or the glycolytic inhibitor 2-DG. Under metabolic stress, activation of the AMP dependent Protein Kinase (AMPK), but not ROS blockade, protected cells from death. We conclude that mitochondrial damage and ROS drive cell killing by SFB, while glycolytic cell reprogramming may represent a resistance strategy potentially targetable by combination therapies.

Tumor Initiating Cells and Chemoresistance: Which Is the Best Strategy to Target Colon Cancer Stem Cells?

There is an emerging body of evidence that chemoresistance and minimal residual disease result from selective resistance of a cell subpopulation from the original tumor that is molecularly and phenotypically distinct. These cells are called “cancer stem cells” (CSCs). In this review, we analyze the potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies for metastatic colon cancer. These include induction of terminal epithelial differentiation of CSCs or targeting some genes expressed only in CSCs and involved in self-renewal and chemoresistance. Ideal targets could be cell regulators that simultaneously control the stemness and the resistance of CSCs. Another important aspect of cancer biology, which can also be harnessed to create novel broad-spectrum anticancer agents, is the Warburg effect, also known as aerobic glycolysis. Actually, little is yet known with regard to the metabolism of CSCs population, leaving an exciting unstudied avenue in the dawn of the emerging field of metabolomics.

Molecular mechanisms underlying human adipose tissue-derived stromal cells differentiation into a hepatocyte-like phenotype

BACKGROUND Adipose tissue-derived stromal cells (ATSCs) hold great promises in regenerative medicine. In the last decade, several studies have reported the plasticity of ATSCs toward a hepatocyte-like phenotype. Nonetheless, the molecular mechanisms underlying the conversion from a mesenchymal to an epithelial phenotype remain poorly understood. AIM In this study, we compared the full genome expression profiles of ATSCs cultured for 4 weeks under pro-hepatogenic conditions to undifferentiated ATSCs, in order to depict the molecular events involved in ATSC hepatic transdifferentiation. METHODS Analysis was performed using the Affymetrix human focus arrays. Sets of differentially expressed genes were functionally categorized in order to understand which pathways drive the hepatic conversion and interesting targets were validated by Q-PCR. RESULTS ATSC-derived hepatocyte-like cells activate several genes associated with specific liver functions, including protein metabolism, innate immune response regulation, and biodegradation of toxic compounds. Furthermore, microarray analysis highlighted downregulation of transcripts associated with the mesenchymal lineage, while epithelial-related genes were overexpressed. CONCLUSION Our data suggest that the in vitro system used in this study drove ATSCs toward a hepatic conversion through a subtle regulation of molecular pathways controlling lineage commitment that promote mesenchymal-epithelial transition.

High nitric oxide production, secondary to inducible nitric oxide synthase expression, is essential for regulation of the tumour-initiating properties of colon cancer stem cells

Chronic inflammation is a leading cause of neoplastic transformation in many human cancers and especially in colon cancer (CC), in part due to tumour promotion by nitric oxide (NO) generated at inflammatory sites. It has also been suggested that high NO synthesis, secondary to inducible NO synthase (iNOS) expression, is a distinctive feature of cancer stem cells (CSCs), a small subset of tumour cells with self‐renewal capacity. In this study we explored the contribution of NO to the development of colon CSC features and evaluated potential strategies to treat CC by modulating NO production. Our data show an integral role for endogenous NO and iNOS activity in the biology of colon CSCs. Indeed, colon CSCs with high endogenous NO production (NOhigh) displayed higher tumourigenic abilities than NOlow fractions. The blockade of endogenous NO availability, using either a specific iNOS inhibitor or a genetic knock‐down of iNOS, resulted in a significant reduction of colon CSC tumourigenic capacities in vitro and in vivo. Interestingly, analysis of genes altered by iNOS‐directed shRNA showed that the knockdown of iNOS expression was associated with a significant down‐regulation of signalling pathways involved in stemness and tumour progression in colon CSCs. These findings confirm that endogenous NO plays an important role in defining the stemness properties of colon CSCs through cross‐regulation of several cellular signalling pathways. This discovery could shed light on the mechanisms by which NO induces the growth and invasiveness of CC, providing new insights into the link between inflammation and colon tumourigenesis. Copyright

Identification of Endothelin-1 and NR4A2 as CD133-regulated genes in colon cancer cells

Several in vitro assays have been proposed to identify cancer stem cells (CSCs), including immunophenotyping, sphere assay and side population (SP) assay. CD133 antigen has been proposed as a CSC marker in colon cancer (CC). However, no functional data are available to date and conflicting results have been reported regarding its role as true CSC marker. Here we set out to identify a molecular signature associated with potential CSC. CD133+ cells isolated from the CaCo‐2 CC cell line were analysed by microarray molecular profiling compared to CD133− counterparts. Various differentially expressed genes were identified and the most relevant transcripts found to be over‐expressed in CD133+ cells were evaluated by quantitative RT‐PCR in the CD133+ fractions isolated from several CC cell lines. In the attempt to find a correlation between putative CSCs, isolated by means of CD133 immunophenotyping and the SP approach, we demonstrated a significant enrichment of CD133+ cells within the SP fraction of CC cells, and comparison of the gene expression profiles revealed that Endothelin‐1 (END‐1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts are highly expressed in both CD133+ and SP fractions of CC cells. Moreover, depletion of CD133 by siRNA induced a significant attenuation of END‐1 and NR4A2 expression levels in CaCo‐2 cells, while expression of all three molecules decreased during sodium butyrate‐induced differentiation. In conclusion, we have identified a molecular signature associated with potential CSCs and showed for the first time the existence of a functional relationship between CD133, END‐1 and NR4A2 expression in colon cancer cells. Copyright