Stem cells under the influence of alcohol: effects of ethanol consumption on stem/progenitor cells.

Stem cells drive embryonic and fetal development. In several adult tissues, they retain the ability to self-renew and differentiate into a variety of specialized cells, thus contributing to tissue homeostasis and repair throughout life span. Alcohol consumption is associated with an increased risk for several diseases and conditions. Growing and developing tissues are particularly vulnerable to alcohol's influence, suggesting that stem- and progenitor-cell function could be affected. Accordingly, recent studies have revealed the possible relevance of alcohol exposure in impairing stem-cell properties, consequently affecting organ development and injury response in different tissues. Here, we review the main studies describing the effects of alcohol on different types of progenitor/stem cells including neuronal, hepatic, intestinal and adventitial progenitor cells, bone-marrow-derived stromal cell, dental pulp, embryonic and hematopoietic stem cells, and tumor-initiating cells. A better understanding of the nature of the cellular damage induced by chronic and episodic heavy (binge) drinking is critical for the improvement of current therapeutic strategies designed to treat patients suffering from alcohol-related disorders.

Current Biomedical Use of Copper Chelation Therapy.

Copper is an essential microelement that plays an important role in a wide variety of biological processes. Copper concentration has to be finely regulated, as any imbalance in its homeostasis can induce abnormalities. In particular, excess copper plays an important role in the etiopathogenesis of the genetic disease Wilson's syndrome, in neurological and neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases, in idiopathic pulmonary fibrosis, in diabetes, and in several forms of cancer. Copper chelating agents are among the most promising tools to keep copper concentration at physiological levels. In this review, we focus on the most relevant compounds experimentally and clinically evaluated for their ability to counteract copper homeostasis deregulation. In particular, we provide a general overview of the main disorders characterized by a pathological increase in copper levels, summarizing the principal copper chelating therapies adopted in clinical trials.

Challenges and Strategies for Improving the Regenerative Effects of Mesenchymal Stromal Cell-Based Therapies.

Cell-based therapies have the potential to revolutionize current treatments for diseases with high prevalence and related economic and social burden. Unfortunately, clinical trials have made only modest improvements in restoring normal function to degenerating tissues. This limitation is due, at least in part, to the death of transplanted cells within a few hours after transplant due to a combination of mechanical, cellular, and host factors. In particular, mechanical stress during implantation, extracellular matrix loss upon delivery, nutrient and oxygen deprivation at the recipient site, and host inflammatory response are detrimental factors limiting long-term transplanted cell survival. The beneficial effect of cell therapy for regenerative medicine ultimately depends on the number of administered cells reaching the target tissue, their viability, and their promotion of tissue regeneration. Therefore, strategies aiming at improving viable cell engraftment are crucial for regenerative medicine. Here we review the major factors that hamper successful cell engraftment and the strategies that have been studied to enhance the beneficial effects of cell therapy. Moreover, we provide a perspective on whether mesenchymal stromal cell-derived extracellular vesicle delivery, as a cell-free regenerative approach, may circumvent current cell therapy limitations.

Promotion of Survival and Engraftment of Transplanted Adipose Tissue-Derived Stromal and Vascular Cells by Overexpression of Manganese Superoxide Dismutase.

Short-term persistence of transplanted cells during early post-implant period limits clinical efficacy of cell therapy. Poor cell survival is mainly due to the harsh hypoxic microenvironment transplanted cells face at the site of implantation and to anoikis, driven by cell adhesion loss. We evaluated the hypothesis that viral-mediated expression of a gene conferring hypoxia resistance to cells before transplant could enhance survival of grafted cells in early stages after implant. We used adipose tissue as cell source because it consistently provides high yields of adipose-tissue-derived stromal and vascular cells (ASCs), suitable for regenerative purposes. Luciferase positive cells were transduced with lentiviral vectors expressing either green fluorescent protein as control or human manganese superoxide dismutase (SOD2). Cells were then exposed in vitro to hypoxic conditions, mimicking cell transplantation into an ischemic site. Cells overexpressing SOD2 displayed survival rates significantly greater compared to mock transduced cells. Similar results were also obtained in vivo after implantation into syngeneic mice and assessment of cell engraftment by in vivo bioluminescent imaging. Taken together, these findings suggest that ex vivo gene transfer of SOD2 into ASCs before implantation confers a cytoprotective effect leading to improved survival and engraftment rates, therefore enhancing cell therapy regenerative potential.

The synergism of SMC1A cohesin gene silencing and bevacizumab against colorectal cancer.

BACKGROUND: SMC1A is a subunit of the cohesin complex that participates in many DNA- and chromosome-related biological processes. Previous studies have established that SMC1A is involved in cancer development and in particular, is overexpressed in chromosomally unstable human colorectal cancer (CRC). This study aimed to investigate whether SMC1A could serve as a therapeutic target for CRC. METHODS: At first, we studied the effects of either SMC1A overexpression or knockdown in vitro. Next, the outcome of SMC1A knocking down (alone or in combination with bevacizumab, a monoclonal antibody against vascular endothelial growth factor) was analyzed in vivo. RESULTS: We found that SMC1A knockdown affects cell proliferation and reduces the ability to grow in anchorage-independent manner. Next, we demonstrated that the silencing of SMC1A and the combo treatment were effective in increasing overall survival in a xenograft mouse model. Functional analyses indicated that both treatments lead to atypical mitotic figures and gene expression dysregulation. Differentially expressed genes were implicated in several pathways including gene transcription regulation, cellular proliferation, and other transformation-associated processes. CONCLUSIONS: These results indicate that SMC1A silencing, in combination with bevacizumab, can represent a promising therapeutic strategy for human CRC.

hMENA isoforms regulate cancer intrinsic type I IFN signaling and extrinsic mechanisms of resistance to immune checkpoint blockade in NSCLC.

BACKGROUND: Understanding how cancer signaling pathways promote an immunosuppressive program which sustains acquired or primary resistance to immune checkpoint blockade (ICB) is a crucial step in improving immunotherapy efficacy. Among the pathways that can affect ICB response is the interferon (IFN) pathway that may be both detrimental and beneficial. The immune sensor retinoic acid-inducible gene I (RIG-I) induces IFN activation and secretion and is activated by actin cytoskeleton disturbance. The actin cytoskeleton regulatory protein hMENA, along with its isoforms, is a key signaling hub in different solid tumors, and recently its role as a regulator of transcription of genes encoding immunomodulatory secretory proteins has been proposed. When hMENA is expressed in tumor cells with low levels of the epithelial specific hMENA11a isoform, identifies non-small cell lung cancer (NSCLC) patients with poor prognosis. Aim was to identify cancer intrinsic and extrinsic pathways regulated by hMENA11a downregulation as determinants of ICB response in NSCLC. Here, we present a potential novel mechanism of ICB resistance driven by hMENA11a downregulation. METHODS: Effects of hMENA11a downregulation were tested by RNA-Seq, ATAC-Seq, flow cytometry and biochemical assays. ICB-treated patient tumor tissues were profiled by Nanostring IO 360 Panel enriched with hMENA custom probes. OAK and POPLAR datasets were used to validate our discovery cohort. RESULTS: Transcriptomic and biochemical analyses demonstrated that the depletion of hMENA11a induces IFN pathway activation, the production of different inflammatory mediators including IFNß via RIG-I, sustains the increase of tumor PD-L1 levels and activates a paracrine loop between tumor cells and a unique macrophage subset favoring an epithelial-mesenchymal transition (EMT). Notably, when we translated our results in a clinical setting of NSCLC ICB-treated patients, transcriptomic analysis revealed that low expression of hMENA11a, high expression of IFN target genes and high macrophage score identify patients resistant to ICB therapy. CONCLUSIONS: Collectively, these data establish a new function for the actin cytoskeleton regulator hMENA11a in modulating cancer cell intrinsic type I IFN signaling and extrinsic mechanisms that promote protumoral macrophages and favor EMT. These data highlight the role of actin cytoskeleton disturbance in activating immune suppressive pathways that may be involved in resistance to ICB in NSCLC.

Strategies for Efficient Targeting of Tumor Collagen for Cancer Therapy.

The tumor stroma, which comprises stromal cells and non-cellular elements, is a critical component of the tumor microenvironment (TME). The dynamic interactions between the tumor cells and the stroma may promote tumor progression and metastasis and dictate resistance to established cancer therapies. Therefore, novel antitumor approaches should combine anticancer and anti-stroma strategies targeting dysregulated tumor extracellular matrix (ECM). ECM remodeling is a hallmark of solid tumors, leading to extensive biochemical and biomechanical changes, affecting cell signaling and tumor tissue three-dimensional architecture. Increased deposition of fibrillar collagen is the most distinctive alteration of the tumor ECM. Consequently, several anticancer therapeutic strategies have been developed to reduce excessive tumor collagen deposition. Herein, we provide an overview of the current advances and challenges of the main approaches aiming at tumor collagen normalization, which include targeted anticancer drug delivery, promotion of degradation, modulation of structure and biosynthesis of collagen, and targeting cancer-associated fibroblasts, which are the major extracellular matrix producers.

HIPK2 Cooperates with KRAS Signaling and Associates with Colorectal Cancer Progression.

Homeodomain-interacting protein kinase 2 (HIPK2) is an evolutionary conserved kinase that has gained attention as a fine tuner of multiple signaling pathways, among which those commonly altered in colorectal cancer. The aim of this study was to evaluate the relationship of HIPK2 expression with progression markers and mutational pattern and gain insights into the contribution of HIPK2 activity in colorectal cancer. We evaluated a retrospective cohort of colorectal cancer samples by IHC for HIPK2 expression and by next-generation sequencing (NGS) for the detection of mutations of cancer associated genes. We show that the percentage of HIPK2-positive cells increases with tumor progression, significantly correlates with tumor-node-metastasis (TNM) staging and associates with a worse outcome. In addition, we observed that high HIPK2 expression significantly associates with KRAS mutations but not with other cancer-related genes. Functional characterization of the link between HIPK2 and KRAS show that activation of the RAS pathway either due to KRAS mutation or via upstream receptor stimulation, increases HIPK2 expression at the protein level. Of note, HIPK2 physically participates in the active RAS complex while HIPK2 depletion impairs ERK phosphorylation and the growth of tumors derived from KRAS mutated colorectal cancer cells. Overall, this study identifies HIPK2 as a prognostic biomarker candidate in patients with colorectal cancer and underscores a previously unknown functional link between HIPK2 and the KRAS signaling pathway. IMPLICATIONS: Our data indicate HIPK2 as a new player in the complex picture of the KRAS signaling network, providing rationales for future clinical studies and new treatment strategies for KRAS mutated colorectal cancer.

Direct CD32 T-cell cytotoxicity: implications for breast cancer prognosis and treatment.

The FcγRII (CD32) ligands are IgFc fragments and pentraxins. The existence of additional ligands is unknown. We engineered T cells with human chimeric receptors resulting from the fusion between CD32 extracellular portion and transmembrane CD8α linked to CD28/ζ chain intracellular moiety (CD32-CR). Transduced T cells recognized three breast cancer (BC) and one colon cancer cell line among 15 tested in the absence of targeting antibodies. Sensitive BC cell conjugation with CD32-CR T cells induced CD32 polarization and down-regulation, CD107a release, mutual elimination, and proinflammatory cytokine production unaffected by human IgGs but enhanced by cetuximab. CD32-CR T cells protected immunodeficient mice from subcutaneous growth of MDA-MB-468 BC cells. RNAseq analysis identified a 42 gene fingerprint predicting BC cell sensitivity and favorable outcomes in advanced BC. ICAM1 was a major regulator of CD32-CR T cell-mediated cytotoxicity. CD32-CR T cells may help identify cell surface CD32 ligand(s) and novel prognostically relevant transcriptomic signatures and develop innovative BC treatments.

Reduction of Cell Proliferation by Acute C2H6O Exposure.

Endogenous acetaldehyde production from the metabolism of ingested alcohol exposes hematopoietic progenitor cells to increased genotoxic risk. To develop possible therapeutic strategies to prevent or reverse alcohol abuse effects, it would be critical to determine the temporal progression of acute ethanol toxicity on progenitor cell numbers and proliferative status. We followed the variation of the cell proliferation rate in bone marrow and spleen in response to acute ethanol intoxication in the MITO-Luc mouse, in which NF-Y-dependent cell proliferation can be assessed in vivo by non-invasive bioluminescent imaging. One week after ethanol administration, bioluminescent signals in bone marrow and spleen decreased below the level corresponding to physiological proliferation, and they progressively resumed to pre-treatment values in approximately 4 weeks. Boosting acetaldehyde catabolism by administration of an aldehyde dehydrogenase activity activator or administration of polyphenols with antioxidant activity partially restored bone marrow cells' physiological proliferation. These results indicate that in this mouse model, bioluminescent alteration reflects the reduction of the physiological proliferation rate of bone marrow progenitor cells due to the toxic effect of aldehydes generated by alcohol oxidation. In summary, this study presents a novel view of the impact of acute alcohol intake on bone marrow cell proliferation in vivo.

Regenerative medicine approaches for the management of respiratory tract fistulas.

Respiratory tract fistulas (or fistulae) are abnormal communications between the respiratory system and the digestive tract or the adjacent organs. The origin can be congenital or, more frequently, iatrogenic and the clinical presentation is heterogeneous. Respiratory tract fistulas can lead to severely reduced health-related quality of life and short survival. Therapy mainly relies on endoscopic surgical interventions but patients often require prolonged hospitalization and may develop complications. Therefore, more conservative regenerative medicine approaches, mainly based on lipotransfer, have also been investigated. Adipose tissue can be delivered either as unprocessed tissue, or after enzymatic treatment to derive the cellular stromal vascular fraction. In the current narrative review, we provide an overview of the main tissue/cell-based clinical studies for the management of various types of respiratory tract fistulas or injuries. Clinical experience is limited, as most of the studies were performed on a small number of patients. Albeit a conclusive proof of efficacy cannot be drawn, the reviewed studies suggest that grafting of adipose tissue-derived material may represent a minimally invasive and conservative treatment option, alternative to more aggressive surgical procedures. Knowledge on safety and tolerability acquired in prior studies can lead to the design of future, larger trials that may exploit innovative procedures for tissue processing to further improve the clinical outcome.

DHA Affects Microtubule Dynamics Through Reduction of Phospho-TCTP Levels and Enhances the Antiproliferative Effect of T-DM1 in Trastuzumab-Resistant HER2-Positive Breast Cancer Cell Lines.

Trastuzumab emtansine (T-DM1) is an anti-human epidermal growth factor receptor 2 (HER2) antibody-drug conjugated to the microtubule-targeting agent emtansine (DM1). T-DM1 is an effective agent in the treatment of patients with HER2-positive breast cancer whose disease has progressed on the first-line trastuzumab containing chemotherapy. However, both primary and acquired tumour resistance limit its efficacy. Increased levels of the phosphorylated form of Translationally Controlled Tumour Protein (phospho-TCTP) have been shown to be associated with a poor clinical response to trastuzumab therapy in HER2-positive breast cancer. Here we show that phospho-TCTP is essential for correct mitosis in human mammary epithelial cells. Reduction of phospho-TCTP levels by dihydroartemisinin (DHA) causes mitotic aberration and increases microtubule density in the trastuzumab-resistant breast cancer cells HCC1954 and HCC1569. Combinatorial studies show that T-DM1 when combined with DHA is more effective in killing breast cells compared to the effect induced by any single agent. In an orthotopic breast cancer xenograft model (HCC1954), the growth of the tumour cells resumes after having achieved a complete response to T-DM1 treatment. Conversely, DHA and T-DM1 treatment induces a severe and irreversible cytotoxic effect, even after treatment interruption, thus, improving the long-term efficacy of T-DM1. These results suggest that DHA increases the effect of T-DM1 as poison for microtubules and supports the clinical development of the combination of DHA and T-DM1 for the treatment of aggressive HER2-overexpressing breast cancer.

A ruthenium(II)-curcumin compound modulates NRF2 expression balancing the cancer cell death/survival outcome according to p53 status.

BACKGROUND: Tumor progression and tumor response to anticancer therapies may be affected by activation of oncogenic pathways such as the antioxidant one induced by NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor and the pathways modified by deregulation of oncosuppressor p53. Often, oncogenic pathways may crosstalk between them increasing tumor progression and resistance to anticancer therapies. Therefore, understanding that interplay is critical to improve cancer cell response to therapies. In this study we aimed at evaluating NRF2 and p53 in several cancer cell lines carrying different endogenous p53 status, using a novel curcumin compound since curcumin has been shown to target both NRF2 and p53 and have anti-tumor activity. METHODS: We performed biochemical and molecular studies by using pharmacologic of genetic inhibition of NRF2 to evaluate the effect of curcumin compound in cancer cell lines of different tumor types bearing wild-type (wt) p53, mutant (mut) p53 or p53 null status. RESULTS: We found that the curcumin compound induced a certain degree of cell death in all tested cancer cell lines, independently of the p53 status. At molecular level, the curcumin compound induced NRF2 activation, mutp53 degradation and/or wtp53 activation. Pharmacologic or genetic NRF2 inhibition further increased the curcumin-induced cell death in both mutp53- and wtp53-carrying cancer cell lines while it did not increase cell death in p53 null cells, suggesting a cytoprotective role for NRF2 and a critical role for functional p53 to achieve an efficient cancer cell response to therapy. CONCLUSIONS: These findings underline the prosurvival role of curcumin-induced NRF2 expression in cancer cells even when cells underwent mutp53 downregulation and/or wtp53 activation. Thus, NRF2 inhibition increased cell demise particularly in cancer cells carrying p53 either wild-type or mutant suggesting that p53 is crucial for efficient cancer cell death. These results may represent a paradigm for better understanding the cancer cell response to therapies in order to design more efficient combined anticancer therapies targeting both NRF2 and p53.

Extracellular Vesicles-Encapsulated MicroRNA-125b Produced in Genetically Modified Mesenchymal Stromal Cells Inhibits Hepatocellular Carcinoma Cell Proliferation.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and one of the prominent causes of cancer mortality, leading to approximately 780,000 deaths per year worldwide. Down-regulation of microRNA-125b (miR-125b) is a prognostic indicator in HCC patients. Conversely, over-expression of miR-125b in HCC cells induces cell cycle arrest, inhibits proliferation, migration and invasion. Extracellular vesicles (EVs) function as intercellular messengers transferring proteins, RNAs, DNAs, carbohydrates, and lipids. Since EVs protect their cargo from degradation, delivery of therapeutic bioactive molecules, in particular miRNAs, through EVs represents an innovative avenue for cancer therapy. In this study, we evaluated a replacement strategy for the treatment of HCC via delivery of EVs secreted from human adipose tissue-derived mesenchymal stromal/medicinal signaling cells (ASCs) genetically modified with a lentiviral vector expressing miR-125b with a specific ExoMotif sequence tag to enhance the loading into extracellular vesicles. In particular, we determined that the delivery of miR-125b-loaded EVs produced in engineered ASCs specifically reduces HCC cell proliferation in vitro modulating a series of miR-125b targets, which belong to the p53 signaling pathway. This proof-of-concept study supports the development of innovative therapeutic strategies for HCC via EV-mediated miRNA delivery.

Effects of Copper Chelation on BRAFV600E Positive Colon Carcinoma Cells.

High affinity copper binding to mitogen-activated protein kinase kinase 1 (MAP2K1, also known as MEK1) allosterically promotes the kinase activity of MEK1/2 on extracellular signal regulated kinases 1 and 2 (ERK1/2). Consequently, copper-dependent activation of the mitogen-activated (MAP) kinase pathway has a role in promoting tumor growth. Conversely, copper chelation may represent a possible therapeutic approach for a specific subset of tumors characterized by activating mutations in the serine/threonine protein kinase V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF), such as the V600E, occurring within the kinase domain (BRAFV600E). Tetrathiomolybdate (TM) is a specific copper chelating agent currently used for the treatment of Wilson's disease and in preclinical studies for the management of metastatic cancers owing to its anti-angiogenic and anti-inflammatory properties. We evaluated in vitro and in vivo the effects of copper depletion achieved by pharmacological treatment with TM in human colorectal cells bearing the BRAFV600E mutation in comparison with BRAF wild type cells. We provide evidence that selective copper chelation differentially affects proliferation, survival and migration of colon cancer cells bearing the BRAFV600E mutation compared to BRAFwt acting via differential phosphorylation levels of ERK1/2. Moreover, tetrathiomolybdate treatment was also effective in reducing the clonogenic potential of colon cancer BRAFV600E cells resistant to BRAF pharmacological inhibition. In conclusion, these results support further assessment of copper chelation therapy as an adjuvant therapy for inhibiting the progression of colon cancers containing the BRAFV600E mutation.

Intraoperative Strategies for Minimal Manipulation of Autologous Adipose Tissue for Cell- and Tissue-Based Therapies: Concise Review.

The stromal vascular fraction (SVF) is a heterogeneous population of stem/stromal cells isolated from perivascular and extracellular matrix (ECM) of adipose tissue complex (ATC). Administration of SVF holds a strong therapeutic potential for regenerative and wound healing medicine applications aimed at functional restoration of tissues damaged by injuries or chronic diseases. SVF is commonly divided into cellular stromal vascular fraction (cSVF) and tissue stromal vascular fraction (tSVF). Cellular SVF is obtained from ATC by collagenase digestion, incubation/isolation, and pelletized by centrifugation. Enzymatic disaggregation may alter the relevant biological characteristics of adipose tissue, while providing release of complex, multiattachment of cell-to-cell and cell-to-matrix, effectively eliminating the bioactive ECM and periadventitial attachments. In many countries, the isolation of cellular elements is considered as a "more than minimal" manipulation, and is most often limited to controlled clinical trials and subject to regulatory review. Several alternative, nonenzymatic methods of adipose tissue processing have been developed to obtain via minimal mechanical manipulation an autologous tSVF product intended for delivery, reducing the procedure duration, lowering production costs, decreasing regulatory burden, and shortening the translation into the clinical setting. Ideally, these procedures might allow for the integration of harvesting and processing of adipose tissue for ease of injection, in a single procedure utilizing a nonexpanded cellular product at the point of care, while permitting intraoperative autologous cellular and tissue-based therapies. Here, we review and discuss the options, advantages, and limitations of the major strategies alternative to enzymatic processing currently developed for minimal manipulation of adipose tissue. Stem Cells Translational Medicine 2019;8:1265&1271.

Overexpression of the cohesin-core subunit SMC1A contributes to colorectal cancer development.

BACKGROUND: Cancer cells are characterized by chromosomal instability (CIN) and it is thought that errors in pathways involved in faithful chromosome segregation play a pivotal role in the genesis of CIN. Cohesin forms a large protein ring that binds DNA strands by encircling them. In addition to this central role in chromosome segregation, cohesin is also needed for DNA repair, gene transcription regulation and chromatin architecture. Though mutations in both cohesin and cohesin-regulator genes have been identified in many human cancers, the contribution of cohesin to cancer development is still under debate. METHODS: Normal mucosa, early adenoma, and carcinoma samples deriving from 16 subjects affected by colorectal cancer (CRC) were analyzed by OncoScan for scoring both chromosome gains and losses (CNVs) and loss of heterozygosity (LOH). Then the expression of SMC1A was analyzed by immunochemistry in 66 subjects affected by CRC. The effects of SMC1A overexpression and mutated SMC1A were analyzed in vivo using immunocompromised mouse models. Finally, we measured global gene expression profiles in induced-tumors by RNA-seq. RESULTS: Here we showed that SMC1A cohesin core gene was present as extra-copies, mutated, and overexpressed in human colorectal carcinomas. We then demonstrated that cohesin overexpression led to the development of aggressive cancers in immunocompromised mice through gene expression dysregulation. CONCLUSION: Collectively, these results support a role of defective cohesin in the development of human colorectal cancer.

Protein disulfide isomerase as a prosurvival factor in cell therapy for muscular and vascular diseases.

BACKGROUND: Cell therapy for degenerative diseases aims at rescuing tissue damage by delivery of precursor cells. Thus far, this strategy has been mostly unsuccessful due to massive loss of donor cells shortly after transplantation. Several strategies have been applied to increase transplanted cell survival but only with limited success. The endoplasmic reticulum (ER) is an organelle involved in protein folding, calcium homeostasis, and lipid biosynthesis. Protein disulfide isomerase (PDI) is a molecular chaperone induced and activated by ER stress. PDI is induced by hypoxia in neuronal, cardiac, and endothelial cells, supporting increased cell survival to hypoxic stress and protection from apoptosis in response to ischemia. METHODS: We achieved ex vivo PDI gene transfer into luciferase-expressing myoblasts and endothelial cells. We assessed cell engraftment upon intramuscular transplantation into a mouse model of Duchenne muscular dystrophy (mdx mouse) and into a mouse model of ischemic disease. RESULTS: We observed that loss of full-length dystrophin expression in mdx mice muscle leads to an increase of PDI expression, possibly in response to augmented ER protein folding load. Moreover, we determined that overexpression of PDI confers a survival advantage for muscle cells in vitro and in vivo to human myoblasts injected into murine dystrophic muscle and to endothelial cells administered upon hindlimb ischemia damage, improving the therapeutic outcome of the cell therapy treatment. CONCLUSIONS: Collectively, these results suggest that overexpression of PDI may protect transplanted cells from hypoxia and other possibly occurring ER stresses, and consequently enhance their regenerative properties.

p53-Dependent PUMA to DRAM antagonistic interplay as a key molecular switch in cell-fate decision in normal/high glucose conditions.

BACKGROUND: As an important cellular stress sensor phosphoprotein p53 can trigger cell cycle arrest and apoptosis and regulate autophagy. The p53 activity mainly depends on its transactivating function, however, how p53 can select one or another biological outcome is still a matter of profound studies. Our previous findings indicate that switching cancer cells in high glucose (HG) impairs p53 apoptotic function and the transcription of target gene PUMA. METHODS AND RESULTS: Here we report that, in response to drug adriamycin (ADR) in HG, p53 efficiently induced the expression of DRAM (damage-regulated autophagy modulator), a p53 target gene and a stress-induced regulator of autophagy. We found that ADR treatment of cancer cells in HG increased autophagy, as displayed by greater LC3II accumulation and p62 degradation compared to ADR-treated cells in low glucose. The increased autophagy in HG was in part dependent on p53-induced DRAM; indeed DRAM knockdown with specific siRNA reversed the expression of the autophagic markers in HG. A similar outcome was achieved by inhibiting p53 transcriptional activity with pifithrin-α. DRAM knockdown restored the ADR-induced cell death in HG to the levels obtained in low glucose. A similar outcome was achieved by inhibition of autophagy with cloroquine (CQ) or with silencing of autophagy gene ATG5. DRAM knockdown or inhibition of autophagy were both able to re-induce PUMA transcription in response to ADR, underlining a reciprocal interplay between PUMA to DRAM to unbalance p53 apoptotic activity in HG. Xenograft tumors transplanted in normoglycemic mice displayed growth delay after ADR treatment compared to those transplanted in diabetics mice and such different in vivo response correlated with PUMA to DRAM gene expression. CONCLUSIONS: Altogether, these findings suggest that in normal/high glucose condition a mutual unbalance between p53-dependent apoptosis (PUMA) and autophagy (DRAM) gene occurred, modifying the ADR-induced cancer cell death in HG both in vitro and in vivo.

Hyperglycemia triggers HIPK2 protein degradation.

Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies.