Serum-deprived Medium Research Articles

Hydrogen Promotes the Effectiveness of Bone Mesenchymal Stem Cell Transplantation in Rats with Spinal Cord Injury.

Although bone mesenchymal stem cell (BMSC) transplantation has been applied to the treatment of spinal cord injury (SCI), the effect is unsatisfactory due to the specific microenvironment (inflammation and oxidative stress) in the SCI area, which leads to the low survival rate of transplanted cells. Thus, additional strategies are required to improve the efficacy of transplanted cells in the treatment of SCI. Hydrogen possesses antioxidant and anti-inflammatory properties. However, whether hydrogen can enhance the effect of BMSC transplantation in the treatment of SCI has not yet been reported. This study was aimed at investigating whether hydrogen promotes the therapeutic effect of BMSC transplantation in the treatment of SCI in rats. In vitro, BMSCs were cultured in a normal medium and a hydrogen-rich medium to study the effect of hydrogen on the proliferation and migration of BMSCs. BMSCs were treated with a serum-deprived medium (SDM), and the effects of hydrogen on the apoptosis of BMSCs were studied. In vivo, BMSCs were injected into the rat model of SCI. Hydrogen-rich saline (5 ml/kg) and saline (5 ml/kg) were given once a day via intraperitoneal injection. Neurological function was evaluated using the Basso, Beattie, and Bresnahan (BBB) and CatWalk gait analyses. Histopathological analysis, oxidative stress, inflammatory factors (TNF-α, IL-1β, and IL-6), and transplanted cell viability were detected at 3 and 28 days after SCI. Hydrogen can significantly enhance BMSC proliferation and migration and tolerance to SDM. Hydrogen and BMSC codelivery can significantly enhance neurological function recovery by improving the transplant cell survival rate and migration. Hydrogen can enhance the migration and proliferation capacity of BMSCs to repair SCI by reducing the inflammatory response and oxidative stress in the injured area. Hydrogen and BMSC codelivery is an effective method to improve BMSC transplantation in the treatment of SCI.

Pro-Myogenic Environment Promoted by the Synergistic Effect of Conductive Polymer Nanocomposites Combined with Extracellular Zinc Ions.

A new strategy based on the combination of electrically conductive polymer nanocomposites and extracellular Zn2+ ions as a myogenic factor was developed to assess its ability to synergically stimulate myogenic cell response. The conductive nanocomposite was prepared with a polymeric matrix and a small amount of graphene (G) nanosheets (0.7% wt/wt) as conductive filler to produce an electrically conductive surface. The nanocomposites' surface electrical conductivity presented values in the range of human skeletal muscle tissue. The biological evaluation of the cell environment created by the combination of the conductive surface and extracellular Zn2+ ions showed no cytotoxicity and good cell adhesion (murine C2C12 myoblasts). Amazingly, the combined strategy, cell-material interface with conductive properties and Zn bioactive ions, was found to have a pronounced synergistic effect on myoblast proliferation and the early stages of differentiation. The ratio of differentiated myoblasts cultured on the conductive nanocomposites with extracellular Zn2+ ions added in the differentiation medium (serum-deprived medium) was enhanced by more than 170% over that of non-conductive surfaces (only the polymeric matrix), and more than 120% over both conductive substrates (without extracellular Zn2+ ions) and non-conductive substrates with extracellular Zn2+. This synergistic effect was also found to increase myotube density, myotube area and diameter, and multinucleated myotube formation. MyoD-1 gene expression was also enhanced, indicating the positive effect in the early stages of myogenic differentiation. These results demonstrate the great potential of this combined strategy, which stands outs for its simplicity and robustness, for skeletal muscle tissue engineering applications.

Serum deprivation enhanced ethanol-induced toxic responses in A549, lung carcinoma cells

Aim: The current study explores the toxic consequences of ethanol on human lung carcinoma cell, A549 in serum-deprived condition. Methodology: Human lung carcinoma cells, A549, were cultured in complete and serum-deprived medium for 6 hr. Subsequently, they were exposed to 50 mM and 100 mM concentrations of ethanol. Cytotoxicity studies linked with cell viability, oxidative stress, cell cycle arrest and micronuclei formation were performed using various toxicological parameters, namely MTT assay, DCFDA based ROS generation, cell cycle analysis and micronuclei formation assay. The cytotoxicity of ethanol in complete and serum deprived medium were compared at similar doses and time duration. Results: The metabolic viability assay demonstrated that 50 mM and 100 mM concentration of ethanol did not induce significant levels of cytotoxic alteration in A549 lung carcinoma cells in complete medium. However, in serum-deprived conditions, 50 mM and 100 mM ethanol concentration significantly altered cell viability. Further, exposure of 50 mM and 100 mM concentration of ethanol enhanced reactive oxygen species levels in A549 cells more significantly in serum-deprived conditions than in complete medium. In addition to cytotoxicity and oxidative stress, 50 mM and 100 mM ethanol also arrested the cells at G0 phase more significantly in serum deprived conditions compared to complete medium. Interpretation: Both 50 mM and 100 mM ethanol concentration enhanced the cell cytotoxicity and reactive oxygen species, cell cycle arrest and micronuclei formation more severely in serum-deprived medium than in complete medium (containing 10% FBS) under similar treatment conditions.

Establishment of Doxorubicin Resistant Stem-like Cell line from MCF-7 Cells

Background Conventional therapy of Breast cancer (BCa) is associated with numerous challenges, relapse being the topmost. It has been shown that disease relapse occurs due to the existence of cancer stem cells (CSCs), residing within the tumor. Objectives To develop a cancer stem cell line model, exploiting the property of stem cells to resist treatment with anti-cancer therapeutic agents and to develop novel therapeutic interventions targeting the elimination of CSCs. Methods Single-cell clones (SCC) from MCF-7 cells were established in 96-well plates using a culture medium containing 5% FBS. Enrichment of cancer stem cells was carried out from established single cell clones which were initially cultured in a serum-deprived nutrient medium for six weeks followed by Doxorubicin treatment. Doxorubicin-resistant clones were established and evaluated for their growth and sphere-forming abilities. Further, these clones were characterized based on the presence of stem-cell markers using a semi-quantitative reverse transcription-polymerase chain reaction and results were compared with the parental MCF-7 cell line. Results In a complete medium, these spheres differentiated and started growing as a monolayer with differential expression levels of genes involved in stemness and cellular differentiation. When these spheres were sub-cultured again in stem-cell medium and detached to give rise to single cells, these clones retained sphere-forming ability. Doxorubicin-resistant clones showed a tendency to grow in spheres in a stem-cell medium with serum-deprived culture conditions. Conclusion Our results provide undeviating evidence of the successful establishment of clones of MCF-7 cell line exhibiting stem cell-like properties. These cell lines have been reprogrammed as stem cell-like models and can be used to delineate recurrent breast cancer attributed to CSCs.

Involvement of autophagy in the maintenance of rat intervertebral disc homeostasis: an in-vitro and in-vivo RNA interference study of Atg5

In the largest avascular low-nutrient intervertebral disc, resident cells would utilize autophagy, a stress-response survival mechanism by self-digestion and recycling wastes. Our goal was to elucidate the involvement of autophagy in disc homeostasis through RNA interference of autophagy-related gene 5 (Atg5). In vitro, small interfering RNAs (siRNAs) targeting autophagy-essential Atg5 were transfected into rat disc cells. Cell viability with levels of autophagy including Atg5 expression, apoptosis, and senescence was assessed under serum starvation and/or pro-inflammatory interleukin-1 beta (IL-1β) stimulation. In vivo, time-course autophagic flux was monitored following Alexa Fluor® 555-labeled Atg5-siRNA injection into rat tail discs. Furthermore, 24-h temporary static compression-induced disruption of Atg5 siRNA-injected discs was observed by radiography, histomorphology, and immunofluorescence. In disc cells, three different Atg5 siRNAs consistently suppressed autophagy with Atg5 protein knockdown (mean 44.4% [95% confidence interval:-51.7,-37.1], 51.5% [-80.5,-22.5], 62.3% [-96.6,-28.2]). Then, Atg5 knockdown reduced cell viability through apoptosis and senescence not in serum-supplemented medium (93.6% [-0.8, 21.4]) but in serum-deprived medium (66.4% [-29.8,-8.6]) further with IL-1β (44.5% [-36.9,-23.5]). In disc tissues, immunofluorescence detected intradiscal signals for the labeled siRNA even at 56-d post-injection. Immunoblotting found 56-d autophagy suppression with prolonged Atg5 knockdown (33.2% [-52.8,-5.3]). With compression, Atg5 siRNA-injected discs presented radiographic height loss ([-43.9,-0.8]), histological damage ([-5.5,-0.2]), and immunofluorescent apoptosis ([2.2, 22.2]) and senescence ([4.1, 19.9]) induction compared to control siRNA-injected discs at 56d. This loss-of-function study suggests Atg5-dependent autophagy-mediated anti-apoptosis and anti-senescence. Autophagy could be a molecular therapeutic target for degenerative disc disease.

Effects of the secretome of human Wharton's jelly mesenchymal stem cells on the proliferation and apoptosis gene expression of the retinal pigmented epithelium

Human retinal pigmented epithelium (RPE) can undergo an uncontrolled proliferation in some disorders such as retinal detachment associated with proliferative vitreoretinopathy (PVR). The present study was conducted to evaluate the effect of the conditioned medium secreted by human Wharton's jelly mesenchymal stem cells (WJMSCs-CM) on the proliferation and apoptosis gene expression of the RPE. WJMSCs-CM was collected from WJMSCs after two periods of 24-h and 9-h culture in serum-free medium. RPE cells were cultured in WJMSCs-CM versus serum-deprived media for 24 h. The effect of WJMSCs-CM on RPE cell proliferation was determined using the MTT assay. Relative expression of apoptotic genes (Bcl2, Bax, and IL-1B) was also assessed by real-time PCR. MTT assay demonstrated that RPE cell viability was reduced significantly in WJMSCs-CM treated RPE cells compared to those cultured in serum-deprived medium (64.23 ± 2.44 vs 100.10 ± 5.68; P = 0.006). Moreover, the expression of anti-apoptotic Bcl2 was significantly decreased in WJMSCs-CM compared to serum-deprived medium (0.52 ± 0.06 in WJMSCs-CM vs 1.02 ± 0.2 in serum-free treatment; P = 0.03), while the expression of pro-apoptotic biomarkers of Bax and IL-1B was not significantly different between the two treatments. The represented data showed that WJMSCs-CM can induce apoptosis in RPE cells in vitro through activating apoptosis pathways. This proof-of-the-concept study provides basic evidence for the possible effect of WJMSCs-CM on preventing PVR.

Effect of Stem Cell-Derived Extracellular Vesicles on Damaged Human Corneal Endothelial Cells.

Purpose Human corneal endothelial cells (HCECs) are essential to visual function; however, since they have limited proliferative capacity in vivo, they are prone to corneal endothelial dysfunction. At present, the only treatment is a corneal transplantation from donor cadavers. Also, due to a global shortage of donor corneas, it is important to find alternative strategies. Recent studies highlight that stem cell–derived extracellular vesicles (EVs) play a relevant role in stem cell-induced regeneration by reprogramming injured cells and inducing proregenerative pathways. The aim of this work is to evaluate whether EVs derived from mesenchymal stem cells (MSC-EVs) are able to promote regeneration of damaged HCECs. Methods We isolated HCECs from discarded corneas in patients undergoing corneal transplantation or enucleation (N = 23 patients). Bone marrow mesenchymal stem cells (MSCs) were obtained from Lonza, cultured, and characterized. MSC-EVs were obtained from supernatants of MSCs. In order to establish a valid in vitro damage model to test the regenerative potential of EVs on HCECs, we evaluated the proliferation rate and the apoptosis after exposing the cells to serum-deprived medium at different concentrations for 24 hours. We then evaluated the HCEC migration through a wound healing assay. Results In the selected serum deprivation damage conditions, the treatment with different doses of MSC-EVs resulted in a significantly higher proliferation rate of HCECs at all the tested concentrations of EVs (5‐20 × 103 MSC-EV/cell). MSC-EVs/cell induced a significant decrease in number of total apoptotic cells after 24 hours of serum deprivation. Finally, the wound healing assay showed a significantly faster repair of the wound after HCEC treatment with MSC-EVs. Conclusions Results highlight the already well-known proregenerative potential of MSC-EVs in a totally new biological model, the endothelium of the cornea. MSC-EVs, indeed, induced proliferation and survival of HCECs, promoting the migration of HCECs in vitro.

Protective effect of a novel clinical-grade small molecule necrosis inhibitor against oxidative stress and inflammation during islet transplantation.

Inhibition of mitochondrial reactive oxygen species (ROS) and subsequent damage-associated molecular patterns (DAMPs)-induced inflammatory responses could be a novel target in clinical islet transplantation. We investigated the protective effects of NecroX-7, a novel clinical-grade necrosis inhibitor that specifically targets mitochondrial ROS, against primary islet graft failure. Islets from heterozygote human islet amyloid polypeptide transgenic (hIAPP+/- ) mice and nonhuman primates (NHPs) were isolated or cultured with or without NecroX-7 in serum-deprived medium. Supplementation with NecroX-7 during hIAPP+/- mouse islet isolation markedly increased islet viability and adenosine triphosphate content, and attenuated ROS, transcription of c-Jun N-terminal kinases, high mobility group box 1, interleukin-1beta (IL-1 ), IL-6, and tumor necrosis factor-alpha. Supplementation of NecroX-7 during serum-deprived culture also protected hIAPP+/- mouse and NHP islets against impaired viability, serum deprivation-induced ROS, proinflammatory response, and accumulation of toxic IAPP oligomer. Supplementation with NecroX-7 during isolation or serum-deprived culture of hIAPP+/- mouse and NHP islets also improved posttransplant glycemia in the recipient streptozotocin-induced diabetic hIAPP-/- mice and BALB/c-nu/nu mice, respectively. In conclusion, pretransplant administration of NecroX-7 during islet isolation and serum-deprived culture suppressed mitochondrial ROS injury, generation of DAMPs-induced proinflammatory responses, and accumulation of toxic IAPP oligomers ex vivo, and improved posttransplant glycemia in vivo.

P0023RNA-BINDING PROTEIN HUR REGULATES HYPOXIA-INDUCED PARKIN/BNIP3L EXPRESSIONS AND MITOPHAGY IN RENAL TUBULAR CELLS

Abstract Background and Aims Mitochondrial dysfunction contributes to the pathophysiology of acute kidney injury (AKI). Mitophagy selectively degrades damaged mitochondria and thereby regulates cellular function and homeostasis. RNA-binding proteins (RBPs) regulate RNA processing at multiple levels and thereby control cellular function. In this study, we report that RBP, human antigen R (HuR) regulates mitophagy in renal tubular cells (HK-2) under hypoxia condition. Method Human kidney proximal tubular 2 (HK-2) were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium (Gibco, Invitrogen, GmbH, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, GmbH, Germany), 1% penicillin/streptomycin (Gibco, Invitrogen, GmbH, Germany) at 37°C and 5% CO2. The cells were exposed to normoxia (37°C; 5% CO2) or hypoxia (serum and glucose deprived RPMI medium, 37°C, 1% O2) conditions. Mitophagy marker expressions (Parkin, p-Parkin, PINK1, BNIP3L, BNIP3, LC3) were determined by western blot analysis. ROS and mitochondrial membrane potential were determined using DCF-DA and JC-1 dye. Parkin and LC3 localization to mitochondria were performed by confocal laser scanning microscopy. HuR bound Parkin/BNIP3L mRNA expressions were determined by RNA-immunoprecipitation analysis. Results In this study, up-regulated mitophagy markers such as Parkin, PINK1, BNIP3 and BNIP3L protein expressions and mitophagosome formation were found in HK-2 cells under hypoxia condition. Stable HuR knockdown studies revealed that, HuR regulates mitophagy by mitophagosome (LC3 co-localization with mitochondria) and mitolysosome (mitochondria co-localization with lysosome) formation under hypoxia. Further, we show that HuR significantly regulated hypoxia-induced mitophagy by regulating Parkin/BNIP3L protein expressions and their mitochondrial localization. Bioinformatics studies showed that, HuR specifically binds to the AU-rich region in 3’UTR and CDS of Parkin and BNIP3L mRNA sequence. HuR was significantly bound to Parkin and BNIP3L mRNA and thereby regulated their mRNA stability under hypoxic conditions. Conclusion Altogether our data highlight on the functional importance of HuR in regulating mitophagy under hypoxic conditions in renal tubular cells through Parkin/BNIP3L expressions.

Anticlastogenic, antimutagenic, and cytoprotective properties of Orthosiphon stamineus ethanolic leaves extract

Orthosiphon stamineus (O.S) is widely consumed for its medidcinal value including anti-inflammatory, anti-infective, and diuretic properties. The present study evaluates the cytoprotective, anti-mutagenic, and anticlastogenic efficacies of standardized extract of Orthosiphon stamineus. Normal liver cell line (WRL68) exposed to hydrogen peroxide and serum-deprived media as insults to evaluate cytoprotective and glutathione activation activities of (Et. O. s). Salmonella typhimurium TA98 and TA100 exposed to different concentrations of (Et. O. s). The influence of Et. O. s on mitotic, replicative indices as well as chromosomal aberration (CA) and sister chromatid exchange (SCE) induced in human peripheral blood lymphocytes by mitomycin C (MMC). The Et. O.s proved to be a potent scavenger for hydrogen peroxide and other free radicals in serum-depraved media, which showed to stimulate glutathione production in liver cells line. Moreover, it did not induce mutations in S. typhimurium subspecies TA98 and TA100. The standardized extract exhibited powerful antimutagenic activities as verified against both 2-nitrofluorene and sodium azide in S. typhimurium TA98 and TA100 cells, respectively. Cytogenetic tests showed high concentrations of Et. O. s to reduce the values of mitotic and replicative indices without any accompanying side effects, such as chromosomal abnormalities or SCE. To ameliorate MMC effects, pretreatment with the extract proofed to be efficient protocol. These data suggests that O. stamineus extract could be useful as cytoprotective, antimutagenic, and anticlastogenic efficacies, which owes to its potent chemoprevention, antioxidant, and glutathione activation properties.

Resveratrol promotes secretion of wound healing related growth factors of mesenchymal stem cells originated from adult and fetal tissues

Non-healing wounds have demonstrated aberrant regulation of several growth factors, thus using exogenous growth factors and cytokines in the clinical setting may improve the outcomes of non-healing wounds. Mesenchymal stem cells (MSCs) are the source of growth factors that show beneficial effect in promoting impaired wound healing. Certain culture condition should be developed to stimulate growth factor secretion from stem cell. Resveratrol, a small molecule found to increase MSCs therapeutic effectiveness. This study aims to investigate the effect of RV on secretion of wound healing related growth factors. We isolated and characterised MSCs from wharton’s jelly (WJ), amniotic membrane (AM), and adipose tissue. We treated MSCs with serum deprived medium (SDM) supplemented with RV at 0.1 µM, 0.5 µM, 0.8 µM concentration. Our study revealed that RV at 0.1 µM was more effective to increase cell proliferation rate. Resveratrol at 0.1 µM promoted EGF, HGF, PDGF, and TGF-β1 secretion from MSCs. AD-MSCs showed the greatest response to RV stimulation in the term of cell proliferation and growth factors secretion. As conclusion, RV can facilitate cell proliferation and wound healing related growth factors secretion at dosage dependent manner.

Curcumin-Induced Autophagy Augments Its Antitumor Effect against A172 Human Glioblastoma Cells.

Glioblastoma is the most aggressive common brain tumor in adults. Curcumin, from Curcuma longa, is an effective antitumor agent. Although the same proteins control both autophagy and cell death, the molecular connections between them are complicated and autophagy may promote or inhibit cell death. We investigated whether curcumin affects autophagy, which regulates curcumin-mediated tumor cell death in A172 human glioblastoma cells. When A172 cells were incubated with 10 μM curcumin, autophagy increased in a time-dependent manner. Curcumin-induced cell death was reduced by co-incubation with the autophagy inhibitors 3-methyladenine (3-MA), hydroxychloroquine (HCQ), and LY294002. Curcumin-induced cell death was also inhibited by co-incubation with rapamycin, an autophagy inducer. When cells were incubated under serum-deprived medium, LC3-II amount was increased but the basal level of cell viability was reduced, leading to the inhibition of curcumin-induced cell death. Cell death was decreased by inhibiting curcumin-induced autophagy using small interference RNA (siRNA) of Atg5 or Beclin1. Therefore, curcumin-mediated tumor cell death is promoted by curcumin-induced autophagy, but not by an increase in the basal level of autophagy in rapamycin-treated or serum-deprived conditions. This suggests that the antitumor effects of curcumin are influenced differently by curcumin-induced autophagy and the prerequisite basal level of autophagy in cancer cells.

Guided evaluation and standardisation of mesenchymal stem cell culture conditions to generate conditioned medium favourable to cardiac c-kit cell growth.

Mesenchymal stem cells (MSCs) are known to secrete cardioprotective paracrine factors that can potentially activate endogenous cardiac c-kit cells (CCs). This study aims to optimise MSC growth conditions and medium formulation for generating the conditioned medium (CdM) to facilitate CC growth and expansion in vitro. The quality of MSC-CdM after optimisation of seeding density during MSC stabilisation and medium formulation used during MSC stimulation including glucose, ascorbic acid, serum and oxygen levels and the effects of treatment concentration and repeated CdM harvesting were assessed based on CC viability in vitro under growth factor- and serum-deprived condition. Our data showed that functional CdM can be produced from MSCs with a density of 20,000 cells/cm2, which were stimulated using high glucose (25mM), ascorbic acid supplemented, serum-free medium under normoxic condition. The generated CdM, when applied to growth factor- and serum-deprived medium at 1:1 ratio, improved CC viability, migration and proliferation in vitro. Such an effect could further be augmented by generating CdM concentrates without compromising CC gene and protein expressions, while retaining its capability to undergo differentiation to form endothelial, smooth muscle and cardiomyocytes. Nevertheless, CdM could not be repeatedly harvested from the same MSC culture, as the protein content and its effect on CC viability deteriorated after the first harvest. In conclusion, this study provides a proof-of-concept strategy to standardise the production of CdM from MSCs based on rapid, stepwise assessment of CC viability, thus enabling production of CdM favourable to CC growth for in vitro or clinical applications.

Evaluating Wharton's Jelly-Derived Mesenchymal Stem Cell's Survival, Migration, and Expression of Wound Repair Markers under Conditions of Ischemia-Like Stress.

The efficacy of mesenchymal stem cell (MSC) therapy is currently limited by low retention and poor survival of transplanted cells as demonstrated by clinical studies. This is mainly due to the harsh microenvironment created by oxygen and nutrient deprivation and inflammation at the injured sites. The choice of MSC source could be critical in determining fate and cellular function of MSCs under stress. Our objective here was to investigate the influence of ischemia-like stress on Wharton's jelly MSCs (WJ-MSCs) from human umbilical cord to assess their therapeutic relevance in ischemic diseases. We simulated conditions of ischemia in vitro by culturing WJ-MSCs in 2% oxygen in serum deprived and low glucose medium. Under these conditions, WJ-MSCs retained viable population of greater than 80%. They expressed the characteristic MSC surface antigens at levels comparable to the control WJ-MSCs and were negative for the expression of costimulatory molecules. An upregulation of many ECM and adhesion molecules and growth and angiogenic factors contributing to wound healing and regeneration was noted in the ischemic WJ-MSC population by a PCR array. Their migration ability, however, got impaired. Our findings provide evidence that WJ-MSCs might be therapeutically beneficial and potent in healing wounds under ischemic conditions.

Distinct Cell Stress Responses Induced by ATP Restriction in Quiescent Human Fibroblasts.

Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly little is known about how quiescent cells respond to energetic and metabolic challenges. To better understand compensatory responses of quiescent cells to metabolic stress, we established, in human primary dermal fibroblasts, an experimental ‘energy restriction’ model. Quiescence was achieved by short-term culture in serum-deprived media and ATP supply restricted using a combination of glucose transport inhibitors and mitochondrial uncouplers. In aggregate, these measures led to markedly reduced intracellular ATP levels while not compromising cell viability over the observation period of 48 h. Analysis of the transcription factor (TF) landscape induced by this treatment revealed alterations in several signal transduction nodes beyond the expected biosynthetic adaptations. These included increased abundance of NF-κB regulated TFs and altered TF subsets regulated by Akt and p53. The observed changes in gene regulation and corresponding alterations in key signaling nodes are likely to contribute to cell survival at intracellular ATP concentrations substantially below those achieved by growth factor deprivation alone. This experimental model provides a benchmark for the investigation of cell survival pathways and related molecular targets that are associated with restricted energy supply associated with biological aging and metabolic diseases.

An Optimized Approach to Recover Secreted Proteins from Fibroblast Conditioned-Media for Secretomic Analysis.

The proteins secreted by a particular type of cell, the secretome, play important roles in the regulation of many physiological processes via paracrine/autocrine mechanisms, and they are of increasing interest to help understanding rare diseases and to identify potential biomarkers and therapeutic targets. To facilitate ongoing research involving secreted proteins, we revisited cell culture protocols and whole secreted protein enrichment protocols. A reliable method for culturing and precipitating secreted protein from patient-derived fibroblast conditioned-medium was established. The method is based on the optimization of cell confluency and incubation time conditions. The well-established carrier-based TCA-DOC protein precipitation method was consistently found to give higher protein recovery yield. According to our results, we therefore propose that protein enrichment should be performed by TCA-DOC precipitation method after 48 h at 95% of confluence in a serum-deprived culture medium. Given the importance of secreted proteins as a source to elucidate the pathogenesis of rare diseases, especially neurological disorders, this approach may help to discover novel candidate biomarkers with potential clinical significance.

P53 and HIC1 in Brain Cancer Gliomas

Hypermethylated in Cancer 1 (HIC1) is a novel tumor suppressor gene (TSG) which acts as a transcriptional enhancer and growth obliterator, identified by Baylin et al. more than 10 years back. HIC1 is found to be methylated in varieties of human cancers as seen in ductal carcinoma of the breast wherein promoter methylation and allelic loss of 17p13.3 chromosomal locus has been reported without any significant mutation in p53 gene. To determine the targets, Zhang et al. reported that Ephrin-A1 is one potential target for the TSG role of HIC1 in breast cancer cells. The region 17p13.3 consists of Variable Number of Tandem Repeats (VNTR) microsatellite markers such as YNZ22/D17S5/D17S30 and show intermittent DNA hypermethylation in neural cancer, colon cancer and lung cancer. Epigenetic silencing also is very commonly observed in liver cancer, breast cancer, gastric cancer, prostate cancer and is also seen in human male nonseminomatous germ cell tumors. The central coding domain of HIC1 exhibits elevated and eminent level of methylation in acute myeloid leukemia. Our laboratory had showed that 17p13.3 was preferentially affected in high grade primary human gliomas and the same was associated with increased p53 immunopositivity and proliferation within a particular tumor grade. Both HIC1 and p53 are catalogued as tumor repressors, HIC1 practically comply with p53 in cells cultures in laboratory as well as in genetically modified transgenic animals to repress/supress tumors and have binding sites on their promoters. Since P53 has binding site onto the promoter of HIC1 it transcriptionally up-regulates its expression. In corroboration, our result did showed an increased expression of HIC1 with wild type p53 containing U87MG cells whereas less or no expression with mutated cell lines (U373MG) or null p53 (SaOS-2) (Sanjay et al.). The ambiguity was previously reported that wild type P53 continuously being formed and degraded by ubiquitination and proteasomal mediated pathway in absence of stress signal. On contrary, mutant forms of P53 are usually stable and escape degradation from ubiquitination pathway. We observed almost no expression of p53 in U87MG and SaOS-2 cells whereas U373MG showed high level of mutated p53. Conventionally HIC1 is a tumour suppressor gene (TSG), hence the over expression of this gene leads to arrest of cell cycle. Since HIC1 plays the role of TSG, the activities like cell cycle regulation and p53 dependency is not known. We have done a research on the activity of HIC1 in cell cycle as well as during glioma cell line (U87MG) proliferation, that had the wild type p53 gene in both serum containing and serum deprived medium. Reduction in number of cells and cell proliferation was revealed during HIC1 knocking as compared to control siRNA of cells without any treatment with serum containing medium and serum free medium. Flow cytometry and FacsDiva software verified the arrest of the cell cycle at the G2-M phase without any significant increase in the number of cell death i.e., cell apoptosis in both the medium. An increase in the expression of p53 gene associated with the knockdown of the HIC1 was seen. Moreover, we observed an increase in p21, p27 along with p53 gene when compared to the controls. The expression level of another cell cycle regulatory protein p27 found to be very significantly high comparing to controls. This could be the cause of arresting the cell cycle even after knocking down a tumor suppressor gene like HIC1, which was quite unexpected and beyond the convention. The results revealed the significance of HIC1 in the normal progression of cell cycle and also its activity the at molecular level, affecting the homeostasis of p53 gene as well as a number of genes, which are directly or indirectly related to p53, taking part in the cell cycle process.

Influence of vascular endothelial growth factor stimulation and serum deprivation on gene activation patterns of human adipose tissue-derived stromal cells.

IntroductionStimulation of mesenchymal stromal cells and adipose tissue-derived stromal cells (ASCs) with vascular endothelial growth factor (VEGF) has been used in multiple animal studies and clinical trials for regenerative purposes. VEGF stimulation is believed to promote angiogenesis and VEGF stimulation is usually performed under serum deprivation. Potential regenerative molecular mechanisms are numerous and the role of contributing factors is uncertain. The aim of the current study was to investigate the effect of in vitro serum deprivation and VEGF stimulation on gene expression patterns of ASCs.MethodsGene expressions of ASCs cultured in complete medium, ASCs cultured in serum-deprived medium and ASCs stimulated with VEGF in serum-deprived medium were compared. ASC characteristics according to criteria set by the International Society of Cellular Therapy were confirmed by flow cytometry. Microarray gene expressions were obtained using the Affymetrix HT HG-U133+ GeneChip®. Gene set enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes and gene ontology terms. Transcription of selected genes of interest was confirmed by quantitative PCR.ResultsCompared to ASCs in complete medium, 190 and 108 genes were significantly altered by serum deprivation and serum deprivation combined with VEGF, respectively. No significant differences in gene expression patterns between serum-deprived ASCs and serum-deprived ASCs combined with VEGF stimulation were found. Genes most prominently and significantly upregulated by both conditions were growth factors (IGF1, BMP6, PDGFD, FGF9), adhesion molecule CLSTN2, extracellular matrix-related proteins such as matricellular proteins SMOC2, SPON1 and ADAMTS12, and inhibitors of proliferation (JAG1). The most significantly downregulated genes included matrix metalloproteinases (MMP3, MMP1), and proliferation markers (CDKN3) and GREM2 (a BMP6 antagonist).ConclusionThe decisive factor for the observed change in ASC gene expression proves to be serum starvation rather than VEGF stimulation. Changes in expression of growth factors, matricellular proteins and matrix metalloproteinases in concert, diverge from direct pro-angiogenic paracrine mechanisms as a primary consequence of the used protocol. In vitro serum starvation (with or without VEGF present) appears to favour cardioprotection, extracellular matrix remodelling and blood vessel maturation relevant for the late maturation phase in infarct healing.

Polyphenols rich fraction of Dicranopteris linearis promotes fibroblast cell migration and proliferation in vitro

Ethnopharmacological relevanceDicranopteris linearis is a fern used traditionally for the treatment of skin afflictions such as external wounds, boils and ulcers. However, there are no scientific studies to date to demonstrate its ability to induce wound recovery. The objective of the present study was to explore the wound healing properties of an active fraction of D. linearis through several in vitro assays and to determine its chemical profile. Materials and methodsThe antioxidant activities were evaluated by DPPH and FRAP assays. The ability of the active fraction to induce fibroblast cell proliferation in serum deprived medium or under the challenge of exogenous hydrogen peroxide was evaluated by MTT assay. Cell migration was investigated using the scratch-wound assay. Chemical constituents of the active fraction were isolated and their structures were determined by NMR and MS spectroscopic techniques. Chemical profiling was carried out by the HPLC–UV method. ResultsThe fifth subfraction of the methanol extract of D. linearis (sF5) showed prominent antioxidant activities. It was non-cytotoxic at the concentration tested and was able to induce cell proliferation of selected fibroblast cells under serum deprived conditions. In addition, sF5 was found to enhance cell migration and promote cellular repair following oxidative damage caused by hydrogen peroxide through a mechanism which was independent of its hydroxyl radical scavenging capability. Six phenolic glycosides were identified in sF5 where three were known, two were new and one phenolic glycoside first time reported from the plant. ConclusionThe active fraction of D. linearis which was rich in polyphenolic substances exhibited substantial antioxidant activities, induced cellular repair and contributed positively to fibroblasts proliferation and migration in vitro. Hence, it may have potential application in inducing wound recovery and supports its ethnopharmacological use for treating skin problems.

Contribution of the autophagy-lysosomal and ubiquitin-proteasomal proteolytic systems to total proteolysis in rainbow trout (Oncorhynchus mykiss) myotubes.

The ubiquitin-proteasome system (UPS) is recognized as the major contributor to total proteolysis in mammalian skeletal muscle, responsible for 50% or more of total protein degradation in skeletal muscle, whereas the autophagic-lysosome system (ALS) plays a more minor role. While the relative contribution of these systems to muscle loss is well documented in mammals, little is known in fish species. The current study uses myotubes derived from rainbow trout myogenic precursor cells as an in vitro model of white muscle tissue. Cells were incubated in complete or serum-deprived media or media supplemented with insulin-like growth factor-1 (IGF-1) and exposed to selective proteolytic inhibitors to determine the relative contribution of the ALS and UPS to total protein degradation in myotubes in different culture conditions. Results indicate that the ALS is responsible for 30-34% and 50% of total protein degradation in myotubes in complete and serum-deprived media, respectively. The UPS appears to contribute much less to total protein degradation at almost 4% in cells in complete media to nearly 17% in serum-deprived cells. IGF-1 decreases activity of both systems, as it inhibited the upregulation of both proteolytic systems induced by serum deprivation. The combined inhibition of both the ALS and UPS reduced degradation by a maximum of 55% in serum-deprived cells, suggesting an important contribution of other proteolytic systems to total protein degradation. Collectively, these data identify the ALS as a potential target for strategies aimed at improving muscle protein retention and fillet yield through reductions in protein degradation.