The involvement of apoptotic regulators during in vitro decidualization

Objectives: The uterus responds to an implanting blastocyst by undergoing extensive tissue modification leading to decidualization. This modification includes differentiation and apoptosis of epithelial as well as stromal cell compartments. It is generally accepted that the decidual cell regression pattern is similar to the pattern of initial differentiation, suggesting that decidual cell death is the end point of timed differentiation. However, the molecular mechanisms controlling these events are not understood clearly. Therefore, we aimed to investigate the involvement of apoptotic factors using an in vitro cell culture system. Design: In order to assess the role of apoptotic factors during decidualization, we used a decidual cell line (GG-AD) that had been transformed with a temperature-sensitive SV-40 mutant. At the non-permissive temperature (39°C), these cells showed the characteristics of differentiated decidual cells. They dedifferentiated into stromal cells when the temperature was shifted back to 33°C. Methods: We performed Northern blot analysis for bax, bcl-XL and bcl-2 at both temperatures. The onset of apoptosis was examined by Annexin V staining. The expression of p53 protein was also determined by Western blot. Results: We found an increase in the expression of bax when GG-AD cells were grown at 39°C. We also showed apoptosis with Annexin V staining at 39°C. The p53 protein expression was also similar to that of the animal models, suggesting that the programmed cell death of the decidual cells occurred in a p53-independent manner. Conclusions: These data indicate that a parallelism exists between the increased expression of pro-apoptotic genes and decidual cell death, similar to animal models. Therefore, an in vitro model of GG-AD cells can be used to assess directly the relationship between apoptotic regulators and decidualization and could be used to study the mechanism of decidual cell regression

Redundant expression of canonical Wnt ligands in human breast cancer cell lines

Human breast cancer displays nuclear accumulation of β-catenin and induction of cyclin D1 expression, which suggests that canonical Wnt/β-catenin signaling is activated. In other cancers, the activation of canonical wnt/β-catenin signaling is associated with APC, CTNNB1 or AXIN1 mutations. However, these mutations are rare or absent in breast cancer. In search of alternative mechanisms, we performed comprehensive expression analysis of Wnt signaling molecules, including 19 Wnt ligands, ten Frizzled receptors, two co-receptors and four Lef/TCF transcription factors in immortalized normal human mammary epithelial cells (HMEC) and six breast cancer cell lines. HMEC expressed all Frizzled receptors except FZD9 and FZD10. They also expressed LRP5 and LRP6 co-receptors, as well as four Lef/TCF transcription factors. HMEC cells also expressed many Wnt ligands, including WNT1, WNT2B, WNT3, WNT5A, WNT5B, WNT7B, WNT9A, WNT10B and WNT16 . Redundant expression of Wnt ligands, Frizzled receptors, co-receptors and Lef/TCF transcription factors was maintained in breast cancer cell lines with some exceptions. The most important changes in cancer cell lines concerned Wnt ligand expression. We noticed that most breast cancer cell lines overexpressed WNT3A, WNT4, WNT6, WNT8B, WNT9A and WNT10B. In contrast, the expression of WNT5A, WNT5B and WNT16 was usually down-regulated. It is noteworthy that all six Wnt ligands that are overexpressed in malignant cell lines are known to signal through the canonical Wnt/β-catenin signaling pathway, whereas down-regulated WNT5A and WNT5B ligands signal via the non-canonical pathway. The expression of both canonical Wnt ligands and most Frizzled receptors in breast cancer cell lines suggests that canonical Wnt/β-catenin signaling is activated in these cell lines by an autocrine/paracrine mechanism. In support of this prediction, we observed nuclear β-catenin accumulation and cyclin D1 induction in breast cancer cell lines, but not in HMEC. These results imply that ligand-dependent canonical Wnt/β-catenin signaling is active in human breast cancer

Patterned carbon nanotubes as a new three-dimensional scaffold for mesenchymal stem cells

We investigated the cellular adhesive features of mesenchymal stem cells (MSC) on non-coated and collagen coated patterned and vertically aligned carbon nanotube (CNT) structures mimicking the natural extra cellular matrix (ECM). Patterning was achieved using the elasto-capillary induced by water treatment on the CNT arrays. After confirmation with specific markers both at transcript and protein levels, MSCs from different passages were seeded on either collagen coated or non-coated patterned CNTs. Adhesion and growth of MSCs on the patterned CNT arrays were examined using scanning electron microscopy image analysis and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assays. The highest MSC count was observed on the non-coated patterned CNTs at passage zero, while decreasing numbers of MSCs were found at the later passages. Similarly, MTT assay results also revealed a decrease in the viability of the MSCs for the later passages. Overall, the cell count and viability experiments indicated that MSCs were able to better attach to non-coated patterned CNTs compared to those coated with collagen. Therefore, the patterned CNT surfaces can be potentially used as a scaffold mimicking the ECM environment for MSC growth which presents an alternative approach to MSC-based transplantation therapy applications. © 2013 Elsevier B.V

Cloning and expression profile of FLT3 gene during progenitor cell-dependent liver regeneration

Background and Aim: The liver has a unique capacity to regenerate upon exposure to viral infections, toxic reactions and cancer formation. Liver regeneration is a complex phenomenon in which several factors participate during its onset. Cellular proliferation is an important component of this process and the factors that regulate this proliferation have a vital role. FLT3, a well-known hematopoietic stem cell and hepatic lineage surface marker, is involved in proliferative events of hematopoietic stem cells. However, its contribution to liver regeneration is not known. Therefore, the aim of this study was to clone and examine the role of FLT3 during liver regeneration in rats. Methods: Partial cDNA of rat homolog of FLT3 gene was cloned from thymus and the tissue specific expression of this gene at mRNA and protein levels was examined by RT-PCR and Western blot. After treating with 2-AAF and performing hepatectomy in rats to induce progenitor-dependent liver regeneration, the mRNA and protein expression profile of FLT3 was investigated by real-time PCR and Western blot during liver regeneration. In addition, cellular localization of FLT3 protein was determined by immunohistochemistry. Results: The results indicated that rat FLT3 cDNA has high homology with mouse and human FLT3 cDNA. It was also found that FLT3 is expressed in most of the rat tissues and during liver regeneration. In addition, its intracellular localization is altered during the late stages of liver regeneration. Conclusion: The FLT3 receptor is activated at the late stages of liver regeneration and participates in the proliferation response that is observed during progenitor-dependent liver regeneration. © 2006 The Authors

Poly (hydroxyethyl methacrylate-glycidyl methacrylate) films modified with different functional groups: In vitro interactions with platelets and rat stem cells

Copolymerization of 2-hydroxyethylmethacrylate (HEMA) with glycidylmethacrylate (GMA) in the presence of α-α′- azoisobisbutyronitrile (AIBN) resulted in the formation of hydrogel films carrying reactive epoxy groups. Thirteen kinds of different molecules with pendant NH2 group were used for modifications of the p(HEMA-GMA) films. The NH2 group served as anchor binding site for immobilization of functional groups on the hydrogel film via direct epoxy ring opening reaction. The modified hydrogel films were characterized by FTIR, and contact angle studies. In addition, mechanical properties of the hydrogel films were studied, and modified hydrogel films showed improved mechanical properties compared with the non-modified film, but they are less elastic than the non-modified film. The biological activities of these films such as platelet adhesion, red blood cells hemolysis, and swelling behavior were studied. The effect of modified hydrogel films, including NH2, (using different aliphatic CH2 chain lengths) CH3, SO3H, aromatic groups with substituted OH and COOH groups, and amino acids were also investigated on the adhesion, morphology and survival of rat mesenchymal stem cells (MSCs). The MTT colorimetric assay reveals that the p(HEMA-GMA)-GA-AB, p(HEMA-GMA)-GA-Phe, p(HEMA-GMA)-GA-Trp, p(HEMA-GMA)-GA-Glu formulations have an excellent biocompatibility to promote the cell adhesion and growth. We anticipate that the fabricated p(HEMA-GMA) based hydrogel films with controllable surface chemistry and good stable swelling ratio may find extensive applications in future development of tissue engineering scaffold materials, and in various biotechnological areas. © 2012 Elsevier B.V

Examination of fabrication conditions of acrylate-based hydrogel formulations for doxorubicin release and efficacy test for hepatocellular carcinoma cell

The objective of the present study was to develop 2-hydroxypropyl methacrylate-co-polyethylene methacrylate [p(HPMA-co-PEG-MEMA)] hydrogels that are able to efficiently entrap doxorubicin for the application of loco-regional control of the cancer disease. Systemic chemotherapy provides low clinical benefit while localized chemotherapy might provide a therapeutic advantage. In this study, effects of hydrogel properties such as PEG chains length, cross-linking density, biocompatibility, drug loading efficiency, and drug release kinetics were evaluated in vitro for targeted and controlled drug delivery. In addition, the characterization of the hydrogel formulations was conducted with swelling experiments, permeability tests, Fourier transform infrared, SEM, and contact angle studies. In these drug-hydrogel systems, doxorubicin contains amine group that can be expected a strong Lewis acid-base interaction between drug and polar groups of PEG chains, thus the drug was released in a timely fashion with an electrostatic interaction mechanism. It was observed that doxorubicin release from the hydrogel formulations decreased when the density of cross-linking, and drug/polymer ratio were increased while an increase in the PEG chains length of the macro-monomer (i.e. PEG-MEMA) in the hydrogel system was associated with an increase in water content and doxorubicin release. The biocompatibility of the hydrogel formulations has been investigated using two measures: cytotoxicity test (using lactate dehydrogenase assay) and major serum proteins adsorption studies. Antitumor activity of the released doxorubicin was assessed using a human SNU398 human hepatocellular carcinoma cell line. It was observed that doxorubicin released from all of our hydrogel formulations which remained biologically active and had the capability to kill the tested cancer cells. © 2014 Taylor and Francis

Alternatively spliced Robo2 isoforms in zebrafish and rat

Robo2, a member of the robo gene family, functions as a repulsive axon guidance receptor as well as a regulator of cell migration and tissue morphogenesis in different taxa. In this study, a novel isoform of the zebrafish robo2 (robo2_tv2), which included an otherwise alternatively spliced exon (CAE), has been characterized. Robo2_tv2 is expressed differentially in most non-neuronal tissues of adult zebrafish whereas robo2_tv1 expression to a great extent is restricted to the brain and eye. In zebrafish, robo2_tv2 exhibits a very-low-level basal expression starting from 1 day post fertilization until the mid-larval stages, at which time its expression increases dramatically and could be detected throughout adulthood. Our findings demonstrate that the amino acid sequence coded by CAE of the robo2 gene is highly conserved between zebrafish and mammals, and also contains conserved motifs shared with robo1 and robo4 but not with robo3. Furthermore, we provide an account of differential transcription of the CAE homolog in various tissues of the adult rat. These results suggest that the alternatively spliced robo2 isoforms may exhibit tissue specificity. © Springer-Verlag 2006

Transforming growth factor-beta induces senescence in hepatocellular carcinoma cells and inhibits tumor growth

Senescence induction could be used as an effective treatment for hepatocellular carcinoma (HCC). However, major senescence inducers (p53 and p16Ink4a) are frequently inactivated in these cancers.We tested whether transforming growth factor-β (TGF-β) could serve as a potential senescence inducer in HCC. First, we screened for HCC cell lines with intact TGF-β signaling that leads to small mothers against decapentaplegic (Smad)-targeted gene activation. Five cell lines met this condition, and all of them displayed a strong senescence response to TGF-β1 (1-5 ng/mL) treatment. Upon treatment, c-myc was down-regulated, p21Cip1 and p15Ink4b were up-regulated, and cells were arrested at G1. The expression of p16Ink4a was not induced, and the senescence response was independent of p53 status. A short exposure of less than 1 minute was sufficient for a robust senescence response. Forced expression of p21 Cip1 and p15Ink4b recapitulated TGF-β1 effects. Senescence response was associated with reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) induction and intracellular reactive oxygen species (ROS) accumulation. The treatment of cells with the ROS scavenger N-acetyl-L-cysteine, or silencing of the NOX4 gene, rescued p21Cip1 and p15Ink4b accumulation as well as the growth arrest in response to TGF-β. Human HCC tumors raised in immunodeficient mice also displayed TGF-β1-induced senescence. More importantly, peritumoral injection of TGF-β1 (2 ng) at 4-day intervals reduced tumor growth by more than 75%. In contrast, the deletion of TGF-β receptor 2 abolished in vitro senescence response and greatly accelerated in vivo tumor growth. Conclusion: TGF-β induces p53-independent and p16Ink4a-independent, but Nox4-dependent, p21Cip1-dependent, p15Ink4b-dependent, and ROS-dependent senescence arrest in well-differentiated HCC cells. Moreover, TGF-β-induced senescence in vivo is associated with a strong antitumor response against HCC. Copyright © 2010 by the American Association for the Study of Liver Diseases

Resveratrol affects histone 3 lysine 27 methylation of vessels and blood biomarkers in DOCA salt-induced hypertension

Hypertension is a risk factor for the cardiovascular diseases. Although, several drugs are used to treat hypertension, the success of the antihypertensive therapy is limited. Resveratrol decreases blood pressure in animal models of hypertension. This study researched the mechanisms behind the effects of resveratrol on hypertension. Hypertension was induced by using the deoxycorticosterone acetate (DOCA)-induced (15 mg/kg twice per week, subcutaneously) salt-sensitive hypertension model of Wistar rats. Hypertension caused a decrease in endothelium-dependent relaxations of the isolated thoracic aorta. Resveratrol treatment (50 mg/l in drinking water) prevented DOCA salt-induced hypertension, but did not improve endothelial dysfunction. Plasma nitric oxide (NO), asymmetric dimethylarginine (ADMA), total antioxidant capacity (TAC) and hydrogen sulfide (H2S) levels were not changed by DOCA salt application. However, treatment of resveratrol significantly decreased ADMA and increased TAC and H2S levels. NO level in circulation was not significantly changed by resveratrol. DOCA salt application and resveratrol treatment also caused an alteration in the epigenetic modification of vessels. Staining pattern of histone 3 lysine 27 methylation (H3K27me3) in the aorta and renal artery sections was changed. These results show that preventive effect of resveratrol on DOCA salt-induced hypertension might due to its action on the production of some blood biomarkers and the epigenetic modification of vessels that would focus upon new aspect of hypertension prevention and treatment. © 2014, Springer Science+Business Media Dordrecht

The effect of telomerase template antagonist GRN163L on Bone-Marrow-Derived rat mMesenchymal stem cells is reversible and associated with altered expression of cyclin d1, cdk4 and cdk6

Telomerase activity is essential for the continued growth and survival of malignant cells, therefore inhibition of this activity presents an attractive target for anti-cancer therapy. The telomerase inhibitor GRN163L, was shown to inhibit the growth of cancer cells both in vitro and in vivo. Mesenchymal stem cells (MSCs) also show telomerase activity in maintaining their self-renewal; therefore the effects of telomerase inhibitors on MSCs may be an issue of concern. MSCs are multipotent cells and are important for the homeostasis of the organism. In this study, we sought to demonstrate in vitro effects of GRN163L on rat MSCs. When MSCs were treated with 1 μM GRN163L, their phenotype changed from spindle-shaped cells to rounded ones and detached from the plate surface, similar to cancer cells. Quantitative-RT-PCR and immunoblotting results revealed that GRN163L holds MSCs at the G1 state of the cell cycle, with a drastic decrease in mRNA and protein levels of cyclin D1 and its cdk counterparts, cdk4 and cdk6. This effect was not observed when MSCs were treated with a mismatch control oligonucleotide. One week after GRN163L was removed, mRNA and protein expressions of the genes, as well as the phenotype of MSCs returned to those of untreated cells. Therefore, we concluded that GRN163L does not interfere with the self-renewal and differentiation of MSCs under short term in vitro culture conditions. Our study provides additional support for treating cancers by administrating GRN163L without depleting the body's stem cell pools. © 2010 Springer Science+Business Media, LLC