Cell Journal (Yakhteh)
Volume:23 Issue: 6, Nov 2021

This study was designed to fabricate a suitable permanent scaffold for the normal aligned myotube formation and improve the process of myogenic differentiation of selected stem cells.

In this experimental study, an engineered scaffold composed of decellularized human amniotic membrane (DHAM) and electrospun fibers of poly(ε-caprolactone) (PCL) was fabricated and characterized. PCL nanofibers were superimposed on DHAM (PCL-DHAM) in two different patterns, including randomized fibers (Random) and aligned fibers (Aligned). Adipose derived stem cells (ADSCs) were isolated from adult Wistar rats and cultured on designed scaffold and induced to myotube differentiation. Using an MTT assay, the vitality of cells was determined. Then, myogenic cell differentiation was assessed using scan electron microscopy (SEM), immunofluorescence assay, and reverse transcription-polymerase chain reaction (RT-PCR).

The mechanical properties of engineered PCL-DHAM composite improved significantly compared to DHAM as a control. The engineered PCL-DHAM promoted cell growth and high expression of myosin, Mhc2 and myogenin and thus enhanced the myotube formation.

These findings revealed that bio-composite membrane prepared from PCL nanofibers and DHAM, may represent a promising biomaterial as a desirable scaffold for applying in the bioengineered muscle repair.

The interaction of tumor cells with surrounding stem cells such as adipose-derived mesenchymal stem cells (ASCs) would be a crucial mechanism of tumor progression. It has been shown that irradiation can affect tumor microenvironment through different mechanisms. Given that, we aimed to examine the bystander radiation-induced effects of ASCs on different cancer cell lines.

In this experimental study, ASCs were extracted from five healthy donors, cultured and then irradiated with a 5Gy of gamma radiation. Following 72 hours of incubation, irradiated ASCs-conditioned media (IACM) and non-irradiated ASCs-conditioned media (NIACM) were collected. Following incubation of different cell lines, Jurkat, LNCaP, U87-MG, MDA-MB-231 and MCF-7, in different media, DMEM, NIACM, and IACM, ALDEFLUOR assay and wound healing assays, were conducted. Using quantitative real-time polymerase chain reaction (qRT-PCR), the expression of ATP-binding cassette transporter genes, ABCA1 and ABCG2, was measured in these cell lines.

NIACM significantly increased ALDH activity in MDA-MB-231 cell (P=0.02), while IACM was associated with significant decrease in the LNCaP and MCF-7 cell lines, respectively P=0.02, P=0.03, compared to DMEM as the control. The area of the scratch site was significantly reduced in MDA-MB-231 cells cultured with NIACM compared to DMEM (P=0.04). Furthermore, ABCA1 mRNA expression was considerably decreased in IACM- but not in DMEMtreated LNCaP line (P=0.01).

It seems, after exposing to radiation, ASCs modify to prevent tumor development and metastasis through their radiation-induced bystander effects. Therefore, a better understanding of ASCs function in the tumor microenvironment may provide new insights into therapeutic strategies to surmount radio-resistance in cancer treatment.

Destruction of pancreatic beta-cells induces an insulin deficiency and causes type 1 diabetes. The role of autophagy in inducing insulin-secreting cells (ISCs) from adipose-derived mesenchymal stem cells (AMSCs) was investigated in the current study.

In this experimental study, the isolated AMSCs were characterization and exposed to a cocktail differentiation medium (CDM) in the absence or presence of 3-methyladenine (3MA), an autophagy inhibitor. The differentiation of ISCs was confirmed by the evaluation of the expression of beta-cell-specific genes including pancreatic and duodenal homeobox 1 (PDX1), musculoaponeurotic fibrosarcoma oncogene homolog A (MAF-A), Nk class of homeodomain-encoding genes 6.1 and 2.2 (NKX6-1 and NKX2.2), Glucose transporter 2 (GLUT-2) and INSLIN. Using Newport Green (NG), insulin-positive cells were identified. Insulin secretion in response to various glucose concentrations was measured. Autophagy was evaluated by Acridine orange (AO) staining. Also, expression of autophagy-associated genes, including autophagy-related gene 5 (ATG-5), autophagy-related gene 7 (ATG-7), BECLIN-1, and mammalian target of rapamycin (mTOR), was evaluated by Real-time polymerase chain reaction (PCR) method.

We observed a significant increase of beta-cell specific genes expression in the CDM-treated cells (P<0.01 or P<0.001), whereas the expression of these genes was down-regulated in 3MA-exposed cells. Expression of INSULIN and GLUT-2 genes (P<0.01 and P<0.05, respectively), insulin secretion in response to glucose (P<0.01), and percentage of NG-positive cells (P<0.05) in the 3MA-exposed cells were considerably lower than the cells treated with CDM. The percentage of AO-positive cells (P<0.01) and the expression of autophagy-related genes (P<0.001) was significantly enhanced in the CDM group. These events were significantly prevented by the 3MA.

Our data showed that autophagy is necessary for beta-cell differentiation, and preventing autophagy by 3MA causes the reduction of beta-cell differentiation and insulin secretion.

Epstein-Barr virus (EBV) and Human Herpes virus 6 (HHV-6) are believed to involve in multiple sclerosis (MS) pathogenesis. Natural killer (NK) and CD8+ T cells have essential roles in handling viral infections and their phenotypic and functional properties may be influenced following exposure to viral infections. Here, we investigated the association of NK and CD8+ T cells subpopulations frequency with EBV and HHV-6 viral load in MS patients.

In this case-control study, EBV and HHV-6 viral load were evaluated in plasma of newly diagnosed relapsing-remitting MS (RRMS) patients at relapse phase (n=23), who were not on disease-modifying therapy (DMT), and sex- and age-matched healthy controls (n=19) using real-time polymerase chain reaction (PCR). The frequency of NK and CD8+ T cells subsets were assessed by CD27, CD28, CD45RO, CD56, and CD57 markers using flow cytometry.

Despite the increased level of EBV viral load in RRMS patients compared to the control group, there was no statistically significant difference in EBV and HHV-6 copy numbers between the studied groups. In addition, a significant decrease was observed in the percentages of CD56bright CD57- and CD56dim CD57+ CD8low CD45RO- NK cells in RRMS patients in comparison to healthy controls. Analysis of CD8+ T cell subsets showed a substantially high proportion of CD27+ CD28+ CD45RO+ CD57- CD8hi T cells in patients at relapse phase compared to controls. The frequency of NK and T cells subtypes was not associated with EBV and HHV6 plasma viral loads.

These findings further highlight the variation of NK and CD8+ T cells subsets frequency in clinically active RRMS patients. Since the composition of cells was not associated with EBV and HHV-6 viral load, perhaps other viral infections may be involved in altered NK and CD8+ T cells subpopulation. Larger cohort studies are needed to confirm these results.

Pancreatic β cells are recognized as central players in the pathogenesis of types 1 and 2 diabetes. Efficient and robust primary culture methods are required to interrogate β cell biology and screen potential anti-diabetic therapeutics. The aim of this study was to refine monolayer culture of beta cells and to investigate potential inducers of beta cell proliferation.

In this experimental study, we compared different culture methods to optimize conditions required for a monolayer culture of rat pancreatic islet cells in order to facilitate image analysis-based assays. We also used the refined culture method to screen a group of rationally selected candidate small molecules and their combinations to determine their potential proliferative effects on the β cells.

Ham’s F10 medium supplemented with 2% foetal bovine serum (FBS) in the absence of any surface coating provided a superior monolayer β cell culture, while other conditions induced fibroblast-like cell growth or multilayer cell aggregation over two weeks. Evaluation of candidate small molecules showed that a menin inhibitor MI-2 and a combination of transforming growth factor-β (TGF-β) inhibitor SB481542 and protein kinase C (PKC) activator indolactam V (IndV) significantly induced replication of pancreatic β cells.

Overall, our optimized culture condition provided a convenient approach to study the cultured pancreatic islet cells and enabled us to detect the proliferative effect of menin inhibition and combined TGF-β inhibition and PKC activation, which could be considered as potential strategies for inducing β cell proliferation and regeneration.

Even a small fragment from the body of planarian can regenerate an entire animal, implying that the different fragments from this flatworm eventually reach the same solution. In this study, our aim was to reveal the differences and similarities in mechanisms between different regenerating fragments from this worm.

In this experimental study, we profiled the dynamic proteome of regenerating head and tail to reveal the differences and similarities between different regenerating fragments using 2-DE combined with MALDITOF/TOF MS.

Proteomic profiles of head and tail regeneration identified a total of 516 differential expressed proteins (DEPs) and showed a great difference in quantity and fold changes of proteome profiles between the two scenarios. Briefly, out of the 516 DEPs, 314 were identified to be specific for anterior regeneration, while 165 were specific for posterior regeneration. Bioinformatics analysis showed a wide discrepancy in biological activities between two regenerative processes; especially, differentiation & development and signal transduction in head regeneration were much more complex than that in tail regeneration. Protein functional analysis combined with protein-protein interaction (PPI) analysis showed a significant contribution of both Wnt and BMP signaling pathways to head regeneration not but tail regeneration. Additionally, several novel proteins showed completely opposite expression between head and tail regeneration.

Proteomic profiles of head and tail regeneration identified a total of 516 differential expressed proteins (DEPs) and showed a great difference in quantity and fold changes of proteome profiles between the two scenarios. Briefly, out of the 516 DEPs, 314 were identified to be specific for anterior regeneration, while 165 were specific for posterior regeneration. Bioinformatics analysis showed a wide discrepancy in biological activities between two regenerative processes; especially, differentiation & development and signal transduction in head regeneration were much more complex than that in tail regeneration. Protein functional analysis combined with protein-protein interaction (PPI) analysis showed a significant contribution of both Wnt and BMP signaling pathways to head regeneration not but tail regeneration. Additionally, several novel proteins showed completely opposite expression between head and tail regeneration.

The clinical studies of acute myeloid leukaemia (AML) revealed that antigen escaping variants cause cancer recurrence even after treatment with chimeric antigen receptor (CAR)-T cells that target a single tumour antigen. Due to the heterogeneous expression of antigens on leukaemia blasts, we hypothesized that a novel bispecific CAR, directed to the folate receptor beta (FRβ)-binding single-chain variable fragment (scFv) and an IL3α-binding receptor (CD123) that has more expression in AML blasts, can decrease CAR-T cell exhaustion and increase the efficacy of CAR-T cells to prevent antigen escaping and consequent recurrence of AML.

In this experimental study, the survival, proliferation, and cytolysis of CAR-T cells remains suboptimal even with a costimulatory endodomain. Hence, we designed and constructed a tandem CAR that joins an FRβ and CD123 in the second generation retroviral vector to generate a bispecific tandem CAR (TanCAR-T cell).

TanCAR FRβ-CD123 T cells showed distinct binding to FRβ or CD123 expressing cells. They could lyse the leukaemia cell lines (66.1 ± 11%) comparable to the single CAR-T cells against these determinants. TanCAR FRβCD123 T cells simultaneously engaged FRβ and CD123, which promoted T cell activation in targeting and lysis of the examined leukaemia cell lines. TanCAR-T cell significantly induced interferon gamma (IFNγ) and interleukin 2 (IL-2) production more than single CAR-T cells, which produced a synergistic enhancement of TanCAR FRβ-CD123 T cell function when dual antigens faced simultaneously.

Dual-specific TanCAR FRβ-CD123 T cells showed therapeutic potential to improve AML control by coengaging FRβ and CD123 molecules in a robust, divalent immune system. This strategy may be a useful therapeutic approach in patients with relapsed B-cell malignancies.

The use of animal or plant exosomes in cancer treatment is promising because of their easy access and low cost. Freshwater crabs are used in traditional Iranian medicine to treat cancer. This study aims to determine the anti-cancer properties of exosomes removed from freshwater crabs on a breast cancer cell line (4T1) compared to bone marrow mesenchymal stem cells (BMSCs).

In this experimental study, crab haemolymph exosomes were isolated via the precipitation method and characterised by electron microscopy, dynamic light scattering (DLS), and Western blot analysis. The protein concentration and total antioxidant capacity of these exosomes were determined by bicinchoninic acid (BCA) and cupric reducing antioxidant capacity (CUPRAC). The 4T1 cells and BMSCs were treated with exosomes and we assessed the cell survival by the resazurin and MTT assays. The level of nitric oxide (NO) secretion from the 4T1 cells was determined after treatment with the exosomes.

Electron microscopy, DLS and Western blot for CD63 confirmed that the isolated exosomes were <100 nm in size and expressed CD63. The total antioxidant capacity in these exosomes was 1.003 µM/ml and the protein concentration was 650 mg/ml. Resazurin and MTT assay results showed a decrease in survival of the 4T1 cells (P≤0.001) after treatment with the exosomes compared to cell growth in the exosome-treated BMSCs.

Crab haemolymph contains protein-rich exosomes with antioxidant activities that can have anti-cancer effects on 4T1 cells. These exosomes may be proposed for breast cancer therapeutics.

Colorectal cancer (CRC) imposes great health burdens worldwide. Growth factors contribute to cell growth, differentiation, angiogenesis and, most importantly, tumour formation in many types of cancers. Natural antisense transcripts (NATs) are inclusively predicted to play a major role in cancer progression. The present study aims to evaluate the relationship of fibroblast growth factor 10 (FGF10) and novel long noncoding RNA (lncRNA) antisense FGF10 (FGF10AS) expression with clinicopathologic features in CRC progression to designate a biomarker for CRC early detection

This cross-sectional study was conducted on 100 CRC tumour and parallel adjacent normal tissues. We added 30 normal cases to enhance accuracy of the test. The expression levels of FGF10 and FGF10AS were evaluated by real-time polymerase chain reaction (PCR). The findings were validated by measuring expression levels in the HT29 and SW480 cell lines. Immunohistochemistry analysis was performed systematically to evaluate FGF10 protein expression. The Mann-Whitney U test with Cox regression analysis were applied. P<0.05 were designated as significant.

A significant increase in expression was observed in FGF10 (P<0.001) along with a significant decrease in FGF10AS (P<0.02) in the tumour tissues in comparison with the adjacent normal tissues. Upregulation of FGF10 and downregulation of FGF10AS expression were strongly correlated with the Tumour, Node, Metastasis (TNM) stage (P<0.007 and P<0.004), vascular invasion (P<0.03 and P<0.01), lymph invasion (P<0.02 and P<0.04), and differentiation (P<0.01 and P<0.02), respectively. Moreover, the area under the receiver operating characteristic (ROC) curve for the prognostic value of FGF10 was about 0.84 (95% confidence interval [CI]: 0.771-0.912). Linear regression analysis confirmed a negative correlation between FGF10 expression and its antisense transcript (r=-0.02).

The relationship between the expression levels of FGF10 and FGF10AS in tumour tissues and adjacent normal tissues indicated that sense and antisense FGF RNAs could be remarkable prognostic biomarkers for achieving effective and primitive treatment

Alimta (Pemetrexed) as an antifolate drug has been approved for the treatment of lung cancer. The aim of the present study was to investigate the combination effect of 5-Azacytidine (5-aza) and Alimta on the miR-34a and its target genes expression and induction of apoptotic cell death in non-small lung cancer A549 cells.

In this experimental study, lung cancer A549 cells were treated with various concentrations of Alimta alone and combined with 5-Aza. Then, viability was assessed by trypan blue and MTT assays. mRNA expressions were performed by real time-polymerase chain reaction (PCR) and western blot. Flow cytometry used to detect apoptotic/ necrotic cells and cell cycle arrest.

Alimta alone reduced viability of the cells in a dose dependent manner with the half-maximal inhibitory concentration (IC50) value of 12 µM. Pretreatment of the cells with 5-aza (5 µM) induced a synergistic cytotoxic effect with IC50 of 3 µM. Sequential exposure of the cells to 5-aza and Alimta enhanced miR-34a expression and significantly downregulated HMGB1, HMGA2 and BCL-2 expressions. Also, it was associated with reduction of nuclear HMGB1 and HMGA2 content. Caspase-3 activation, HMGB1 release into extracellular space and staining of the cells with annexine V/PI suggested that 5-aza reduced late apoptotic/necrotic cell death induced by Alimta. In addition, combination of 5-aza and Alimta arrested the cells at S and sub-G1 phases and inhibited colony formation.

5-aza synergistically enhances Alimta induced apoptotic cell death through HMG proteins regulation, MIR34A gene expression and intrinsic apoptosis mechanism, providing a promising combination therapy in clinical lung cancer therapy.

Reportedly, circular RNAs (circRNAs) exert a crucial regulatory role in cancer. Circ_0001073 is derived from exons 3-5 of ACVR2A gene, which inhibits cancer progression. However, the role and mechanism of circ_0001073 in non-small cell lung cancer (NSCLC) are unclear. This study aimed to explore the role and mechanism of circ_0001073 in the development of NSCLC.

In this experimental study, microarray analysis was employed to filter differential expressed circRNAs in NSCLC tissues. Also, circ_0001073, microRNA-582-3p (miR-582-3p), and repulsive guidance molecule B (RGMB) mRNA expressions were examined by quantitative real-time polymerase chain reaction (qRT-PCR). NSCLC cell multiplication was measured by the cell counting kit-8 (CCK-8) assay. Scratch healing experiment and Transwell experiment were performed to assess cell migration and invasion, respectively. Flow cytometry was applied to analyze the apoptosis of NSCLC cells. Western blot was employed to assess RGMB protein expression. Additionally, dualluciferase reporter gene experiment and RNA immunoprecipitation (RIP) experiment were applied to probe the binding sites between miR-582-3p and circ_0001073 or RGMB.

Circ_0001073 was remarkably under-expressed in NSCLC tissues and cells. Circ_0001073 overexpression impeded the multiplication, migration, and invasion and enhanced the apoptosis of NSCLC cells in vitro. Circ_0001073 directly bound to miR-582-3p and acted as a miRNA sponge to regulate RGMB expression. Besides, miR-582-3p overexpression or knockdown of RGMB remarkably reversed the malignant phenotypes of NSCLC cells induced by the up-regulation of circ_0001073 expression.

Circ_0001073 up-regulates RGMB expression through adsorbing miR-582-3p to inhibit NSCLC progression, suggesting its potential as a novel therapeutic target in NSCLC

Sulforaphane (SFN) is a natural free radical scavenger that can reduce oxidative stress (OS) through mediating nuclear factor (erythroid-derived 2)-like 2 (NF-E2-related factor 2 or NRF2)/antioxidant response element (ARE) signaling pathway and the downstream antioxidant enzymes. Here, we intended to study the role of SFN in OSinduced human granulosa cells (GCs) by investigating the intracellular levels of reactive oxygen species (ROS), cell death, and NRF2-ARE pathway.

This experimental study was conducted on GCs of 12 healthy women who had normal menstrual cycles with no history of polycystic ovary syndrome (PCOS), endometriosis, menstrual disorders, hyperprolactinemia, or hormonal therapy. After isolation of GCs, the MTT assay was performed to explore GCs viability after treatment with SFN in the presence or absence of H2 O2 . Flow cytometry was utilized to determine the intracellular ROS production and the apoptosis rate. Evaluation of the mRNA and protein expression levels of NRF2 and phase II enzymes including superoxide dismutase (SOD) and catalase (CAT) was performed by quantitative real-time polymerase chain reaction (PCR) and western blotting. Finally, the data were analyzed by SPSS software using One-way ANOVA and the suitable post-hoc test. Significance level was considered as P<0.05.

Pretreatment of GCs with SFN attenuated intracellular ROS production and apoptosis rate in the H2 O2 -exposed cells. Moreover, SFN treatment increased the mRNA expression level of NRF2, SOD, and CAT. Higher expression of NRF2 and SOD was also observed at the protein level.

Our study demonstrated that SFN protects human GCs against H2 O2 induced-OS by reducing the intracellular ROS production and the following apoptosis through a mechanism by which NRF2 increases the antioxidant enzymes such as SOD and CAT. This result may have a potential application in assisted reproduction cycles by improving the quality of GCs and the embedded oocyte, especially in PCOS patients.

In spinal cord injury (SCI), the primary mechanical damage leads to a neuroinflammatory response and the secondary neuronal injury occurs in response to the release of reactive oxygen species (ROS). In addition to the suppression of inflammation, autophagy plays a significant role in the survival of neurons during secondary SCI. The present study aimed to examine the anti-inflammatory and autophagic effects of agmatine and rapamycin in SCI and to compare the results with methylprednisolone (MP) used in the clinic.

In this animal-based experimental study, thirty adult male Sprague-Dawley rats were randomly divided into five groups as sham-control, injury, injury+MP, injury+rapamycin, injury+agmatine groups. SCI was induced by compressing the T7-8-9 segments of the spinal cord, using an aneurysm clip for one minute, and then rats were treated daily for 7 days. Seven days post-treatment, damaged spinal cord tissues of sacrificed rats were collected for microscopic and biochemical examinations using histopathologic and transmission electron microscope (TEM) scores. Malondialdehyde (MDA) and glutathione peroxidase (GPx) levels were spectrophotometrically measured.

The results of this study showed that the damaged area was smaller in the rapamycin group when compared to the MP group. Many autophagic vacuoles and macrophages were observed in the rapamycin group. Degeneration of axon, myelin, and wide edema was observed in SCI by electron microscopic observations. Fragmented myelin lamellae and contracted axons were also noted. While MDA and GPx levels were increased in the injury group, MDA levels were significantly decreased in the agmatine and MP groups, and GPx levels were decreased in the rapamycin group.

The results of our study confirmed that rapamycin and agmatine can be an effective treatment for secondary injury of SCI.

The current research was designed to analyze the effect of moderate-intensity exercise training (MEXT), solely and simultaneous with insulin, on the network between oxidative stress and Hsp70 and Hsp90 chaperones after experimental type I diabetes (DM) induction in rats.

In the experimental study, 36 mature Wistar rats were assigned into control and experimental type I DM-induced groups, and then the diabetic animals were categorized to sedentary type I DM-induced (SDM), exercise training-sole without DM (E), exercise training DM-induced (EDM), insulin-treated sedentary DM-induced (ISDM), and exercise training insulin-treated DM-induced (EIDM) groups. After 6 weeks, Johnson’s score was evaluated to analyze the spermatogenesis ratio.

The Hsp70 and Hsp90 expression levels, testicular total antioxidant capacity (TAC), protein peroxidation ratio, testicular DNA fragmentation ratio, and mRNA damage were investigated. The animals in EDM and EIDM groups (solely and simultaneously) represented a significant (P<0.05) improvement in Johnson’s score, spermatogenesis, and TAC ratios versus SDM animals. Moreover, the DM-induced DNA and mRNA damage and protein peroxidation ratio were significantly (P<0.05) recovered in EDM and ISDM groups, which was more remarkable in the EIDM group. The EDM and EIDM groups exhibited significant (P<0.05) increment in Hsp70 and Hsp90 expression levels versus the control and SEDT1 animals. However, the EIDM group exhibited no significant changes compared to the control animals.

The EX could ameliorate the EDT1-induced detrimental impact by up-regulating Hsp70 and Hsp90 expressions. Meanwhile, it exerts potentially more effective impact, when it is considered simultaneously with insulin therapy.

Placental alkaline phosphatase (PLAP) is a membrane enzyme mainly expressed in the placenta. PLAP is shown to be expressed in ovarian cancer (OV), however, there is little known about its expression in other cancers. Using gene and protein expression deposited data, we surveyed PLAP expression across malignant and normal human tissues to explore the potential of PLAP as an immunotherapy target. We detected more than two-fold increased PLAP expression in multiple solid tumors including ovarian cancer, testicular germ cell tumors (TGCT), and uterine corpus endometrial carcinoma (UCEC) compared with matched normal tissues. We also showed association of PLAP expression with high mortality pancreatic adenocarcinoma (PAAD). Altogether, our results suggest that PLAP can be a promising target for immunotherapy of multiple cancers, especially OV, TGCT, and UCEC.