CRISPR/Cas9-mediated knockout of MLL5 enhances apoptotic effect of cisplatin in HeLa cells in vitro

Mixed lineage leukemia 5 (MLL5) transactivates the expression of E6 and E7 oncogenes in cervical cancer cells. In this study, we utilized ‎CRISPR/Cas9 system with the aim to target HPV-E6 and MLL5 to enhance apoptosis efficiency in HPV-18 positive HeLa cells and to improve chemotherapeutic efficacy of Cisplatin as the most common anticancer drug, used for cervical cancer. ‎sgRNAs against MLL5 and E6 were designed and cloned into PX458 plasmid vector. ‎Real-time ‎PCR was used to determine knockout expression of MLL5 and E6 following, ‎transfection with cloned plasmids. Cell viability and apoptosis were evaluated, using ‎Dimethyl-thiazolyl diphenyl tetrazolium bromide (MTT) ‎assay and Annexin V flow cytometry. ‎‏Cellular p‎53 level was measured, using enzyme linked immune sorbent assay (ELISA).‏ Real-time ‎PCR indicated the downregulation of E6 and MLL5 in the transfected cells. ‎A significant increase in the accumulation of P53 was observed due to targeting MLL5 and E6 genes. MTT and ‎apoptosis assays showed a significant decrease in cell viability and enhanced apoptosis rate of ‎transfected cells. Combination therapy showed that targeting E6 and MLL5 enhanced ‎apoptotic effect of Cisplatin in MLL5 knockout cells in a synergistic manner. ‎‏The results suggest that CRISPR/Cas9 targeting of E6 and MLL5 genes can increase‎ apoptotic effects of Cisplatin and can be considered as a ‎‎potential combination therapy for the treatment of HPV-‎related cervical cancer.

DECELLULARIZATION OF LUNG TISSUE AND ANALYSIS OF ITS DIFFERENTIATIVE POTENTIAL ON BONE MARROW MESENCHYMAL STEM CELLS OF RAT

Background: Pulmonary diseases are one of the most common causes of mortality worldwide. In some cases lung transplantation is the only curative treatment. Unfortunately, very few lungs are available for transplantation and also the 5-year survival after lung transplantation is only 50%. Furthermore, transplant recipients require immunosuppressant therapy in that’s time. Currently, production of engineered lung tissue using in vitro stem cells is a promising approach. Extraction of natural ECM or decellularization of lung and application of it in tissue engineering is one of the most important strategies in this regard. Using this technique can preserve the natural characteristics of the ECM and would leads to the removal of the immunogen agents (MHC I, II) and allows reconstruction of graft. Objective: In this study we have decellularized rat lung and cultured bone marrow stem cells on it to evaluate differentiative potential of it. Methods: In this study along with extraction of rat lung, its femur and tibia bones were also isolated for extraction of mesenchymal stem cells. Lung tissue was decellularized using SDS detergent. SEM and H&E staining used to assay decellularization. Finally mesenchymal stem cells were seed on the decellularized tissue sections and immunocytochemistry for CC10 and SPD; as markers for differentiation toward lung cells, performed on these cells after 21 days. Results:In the H&E slides of decellularized tissue there were not any cells. In the electron microscope images of decellularized tissue compared with normal tissue, alveolar structure was well maintained. Also, immunocytochemistry assay showed evidence of differentiation of seeded mesenchymal cells toward lung tissue cells. Conclusion:In this method decellularization take place without any significant change in tissue structure. Decellularized tissue can induce differentiation of bone marrow stem cells toward lung epithelial cells so natural ECM saved relatively

Effects of Two-by-Two Combination Therapy with Valproic Acid, Lithium Chloride, and Celecoxib on the Angiogenesis of the Chicken Chorioallantoic Membrane

Background: The synergistic effects of valproic acid (VPA), lithium (Li), and celecoxib (CX) have been shown in combination therapy against the proliferation and metastasis of numerous cancers. Angiogenesis plays a critical role in the pathogenesis of tumor growth and metastasis. The aim of the present study was to evaluate the antiangiogenic effects of VPA, lithium chloride (LiCl), and CX, alone or in 2-by-2 combinations, using the chicken chorioallantoic membrane (CAM) assay. Methods: Fertilized chicken eggs were randomly divided into 10 groups: control, VPA (1.8 and 3.6 µmol/CAM), Li (0.15 and 0.60 µmol/CAM), CX (0.02 and 0.08 µmol/CAM), VPA+Li, VPA+CX, and CX+Li (n=10 per group). A window was made on the eggshells and the CAMs were exposed to a filter disk containing VPA, LiCl, and CX, alone or in 2-by-2 combinations. The control CAMs were treated with distilled water (vehicle). Three days after the treatment, the number of vessel branch points was counted in each CAM. The data were analyzed using SPSS, version 15.One-way ANOVA, followed by the Tukey tests, was used to compare the groups. A P<0.05 was considered a statistically significant difference between the groups. Results: According to the results, all the tested drugs decreased the number of the vessel branch points in a dose-dependent manner compared to the control group (P<0.001). In addition, combinations of the drugs were more effective in decreasing angiogenesis than the use of each drug alone. Conclusion: These findings suggest that 2-by-2 combinations of VPA, CX, and LiCl can be considered an effective antiangiogenesis therapeutic modality

Intrathecal Amylin and Salmon Calcitonin Affect Formalin Induced c-Fos Expression in the Spinal Cord of Rats

Background:Amylin and Salmon Calcitonin belong to the calcitonin family of peptides and have high affinity binding sites in the rat spinal cord. The aim of this study was to characterize receptors for Amylin and Salmon Calcitonin functionally in the spinal cord of rats. We assessed the expression of c-Fos in response to intraplantar formalin in the lumbar regions of the spinal cord in conscious rats. Methods:Amylin (0.05 nmoles) or Salmon Calcitonin (0.005 nmoles) was administered intrathecally (i.t.) 10 minutes before the start of the formalin test. Antagonists were injected intrathecally 10 minutes before the administration of either of the peptides. Results: Two hours after formalin stimulation, rats pretreated intrathecally by either Amylin or Salmon Calcitonin, showed lower numbers of c-Fos immunoreactive nuclei in their lumbar spinal cord as compared to rats pretreated with saline. These effects were reversed upon co-administration of either of the Amylin antagonists AC187 or rat amylin8-37, but not rat α-CGRP8-37. A few cells with c-Fos immunoreactivity were found in the lumbar spinal cord of rats two hours after i.t. injection of saline, Amylin and/or Salmon Calcitonin. However, Fos-like immunoreactivity was increased in the lumbar spinal cord two hours after i.t. treatment of either of the antagonists AC187 and rat amylin8-37,when compared to saline treated rats. Conclusion:Both Amylin and Salmon Calcitonin inhibit formalin induced c-Fos expression in the rat lumbar spinal cord when administered intrathecally. Effects of the two peptides were possibly produced by undefined receptors

Protective effects of nimodipine and lithium against aluminum-induced cell death and oxidative stress in PC12 cells

Objective(s): The role of aluminum (Al) in the pathogenesis of neurodegenerative diseases has been implicated in several studies. However, the exact mechanisms of cytotoxic effects of Al have not been elucidated yet. The aim of this study was to investigate the effect of L-type calcium channel antagonist, nimodipine (NM), and lithium chloride (LiCl) on Al-induced toxicity in PC12 cells. Materials and Methods: PC12 cells were treated with Al-maltolate (Almal) in the presence and absence of different concentrations of NM (50-150 μm) and/or LiCl (0.5-1.0 mm) for 48 hr. Cell viability, apoptosis, and catalase (CAT) activity, a marker of oxidative stress, were then measured using MTT, flow cytometry and enzyme assay, respectively. Results: The results showed that Almal, dose dependently induced cell death, apoptosis and CAT activity in the PC12 cells. NM significantly increased cell viability and decreased apoptosis and CAT activity of Almal-treated cells in a dose dependent mode. LiCl reduced CAT activity and increased cell viability in Almal-treated cells, without significant effect on apoptosis (P=0.74). Conclusion: These findings suggest that NM and Li may have benefits in the prevention of Al-induced cytotoxicity through decreasing oxidative stress

Diverse Effect of Vitamin C and N-Acetylcysteine on Aluminum-Induced Eryptosis

Purpose. The role of oxidative stress in Aluminum (Al)-induced apoptotic effects has been investigated and suicidal death of erythrocytes, eryptosis, is characterized by cell shrinkage and phosphatidylserine externalization (PSE) at the surface of the erythrocyte cell membrane. Eryptosis is stimulated by an increase in cytosolic Ca2+ concentration and reactive oxygen species (ROS). This ex vivo study was conducted to evaluate the effect of well-known antioxidants including vitamin C (vit C) and N-acetylcysteine (NAC), against Al-induced hemolysis and eryptosis. Methods. Isolated erythrocytes from the healthy volunteers were partitioned into various groups (6 replicates/group) and treated by various concentrations of Al (3–100 µM) in the presence and absence of vit C (0.6 mM) and NAC (1 mM). After 24 hours of treatment, hemolysis was determined from hemoglobin levels in the supernatant. Flowcytometric methods were applied to measure PSE, cell shrinkage, Ca2+ content, and ROS abundance using annexin V-binding, forward scatter, Fluo3-fluorescence, and DCFDA dependent fluorescence, respectively. Reduced glutathione (GSH) was measured by the ELISA method. Results. The results showed that a 24 hours’ exposure of the erythrocytes to Al (10–100 µM) significantly increased hemolysis in a dose and Ca2+dependent manner. Al also dramatically decreased forward scatter. The percentage of PSE cells, Fluo3-fluorescence, and DCFDA fluorescence were increased by Al. Furthermore, cotreatment with NAC inhibited the effect of Al on hemolysis, eryptosis, and ROS production. Vit C decreased Al-induced ROS production. However, increased Al-induced eryptosis. There were no significant changes in glutathione after the ALCL3 treatment. Conclusions. Al-induced eryptosis and hemolysis through triggering oxidative stress, while NAC could diverse this effect. In contrast, vit C might intensify Al-induced eryptosis at particular doses through a less known mechanism

New techniques and methods in the study of the invasion, cell migration and MMPs activity in vitro and in animal models

Background & Objective: Cancer metastasis is the primary cause of cancer morbidity and mortality, it accounts for about 90% of cancer deaths. Cancer treatment has improved significantly, due to early detection and inhibition of cancer growth. The ability to invade and migrate is important in malignant tumor cells. The study of cell migration is valuable in cancer diagnosis, prognosis, drug development and treatment. New methods are available to investigate the invasion, migration and the activity of enzymes involved in the invasion process in the laboratory. The effectiveness and efficacy of various anti-cancer drugs and compounds can be studied using these methods in laboratory and animal models. Conclusion: In this paper, we offer a summary of in-vitro migration assays, including the transwell migration assay, scratch wound assay, microfluidic chamber assay, exclusion zone assay, fence assay, micro carrier bead assay, spheroid migration assay and in-vivo approach, Chick chorioallantoic membrane (CAM) assay. This review also provides an overview of methods, In situ Zymography, ELISA, and FRET based measurement of MMP activity and Substrate Zymography for measuring the level of metalloproteinase enzyme as the major enzyme in the degradation of extracellular matrix

Protective effects of vitamin E and selenium on spermatogenesis in adult male rat insulin-resistant

Background & Objective: Diabetes mellitus is a metabolic disease and is a multifactorial disorder characterized by chronic hyperglycemia resulting from impaired insulin secretion and insulin factional or both. In this study, the protective role of vitamin E and sodium selenite in preventing the harmful effects of insulin resistance (diabetes type 2) on spermatogenesis was studied.   Materials & Methods: Male adults (180-200 g) of Wistar rats were divided into five groups, each containing 7 rats (control, sham, and three experimental groups). The rats were fed daily with water-soluble fructose (10%), mg/kg 200 of vitamin E (gavage), and 5/0 mg/kg of sodium selenite (intraperitoneal injection) or both for 110 days. Subsequently, sperm parameters, levels of testosterone, LH, and daily sperm production (DSP) were checked. Additionally, testicular histopathology and malondialdehyde (MDA) in the testis were examined.   Results: Sperm count, sperm motility and viability, and insulin resistance in the rats decreased DSP. A significant decrease was observed in the number of Leydig cells, spermatogonia, spermatogenesis, and spermatozoa in the testis of the insulin-resistant animals, whereas MDA and testosterone rose in the insulin-resistant rats. Vitamin E and sodium selenite intake reduced the levels of MDA and harmful effects of fructose on testicles, as well as sperm parameters and testicular pathology. A simultaneous intake of vitamin E and sodium selenite conferred the highest level of protection.   Conclusion: These findings suggest that vitamin E and sodium selenite can have a protective role in the testes of rats against oxidative stress induced by diabetes type 2

The Effects of NDRG2 Overexpression on Cell Proliferation and Invasiveness of SW48 Colorectal Cancer Cell Line

Background: Colorectal cancer (CRC) is one of the most common causes of cancer-related death in the world. The expression of N-myc downstream-regulated gene 2 (NDRG2) is down-regulated in CRC. The aim of this study was to investigate the effect of NDRG2 overexpression on cell proliferation and invasive potential of SW48 cells. Methods: SW48 cells were transfected with a plasmid overexpressing NDRG2. After stable transfection, the effect of NDRG2 overexpression on cell proliferation was evaluated by MTT assay. The effects of NDRG2 overexpression on cell migration, invasion and cell motility and matrix metalloproteinase 9 (MMP9) activities were also investigated using matrigel transwell assay, wound healing assay and gelatin zymography, respectively. Results: MTT assay showed that overexpression of NDRG2 caused attenuation of SW48 cell proliferation. Transwell and wound healing assay revealed that NDRG2 overexpression led to inhibition of migration, invasion, and motility of SW48 cells. The overexpression of NDRG2 also reduced the activity of secreted MMP-9. Conclusions: The results of this study suggest that NDRG2 overexpression inhibits proliferation and invasive potential of SW48 cells, which likely occurs via suppression of MMP-9 activity

Protective Effect of Vitamins E and C on Endosulfan-Induced Reproductive Toxicity in Male Rats

Background: The role of oxidative stress in endosulfan-induced reproductive toxicity has been implicated. This study was performed to evaluate the possible protective effect of vitamins E and C, against endosulfan-induced reproductive toxicity in rats.Methods: Fifty adult male Sprague–Dawley rats were randomly divided into five groups (n=10 each). The groups included a control receiving vehicle, a group treated with endosulfan (10 mg/kg/day) alone, and three endosulfan-treated group receiving vitamin C (20 mg/kg/day), vitamin E (200 mg/kg/day), or vitamine C+vitamin E at the same doses. After 10 days of treatment, sperm parameters, plasma lactate dehydrogenase (LDH), plasma testosterone and malondialdehyde (MDA) levels in the testis were determined. Results: Oral administration of endosulfan caused a reduction in the sperm motility, viability, daily sperm production (DSP) and increased the number of sperm with abnormal chromatin condensation. Endosulfan administration increased testis MDA and plasma LDH. Supplementation of vitamin C and vitamin E to endosulfan-treated rats reduced the toxic effect of endosulfan on sperm parameters and lipid peroxidation in the testis. Vitamin E was more protective than vitamin C in reducing the adverse effects of the endosulfan.Conclusion: The findings data suggest that administration of vitamins C and E ameliorated the endosulfan-induced oxidative stress and sperm toxicity in rat. The effect of vitamin E in preventing endosulfan-induced sperm toxicity was superior to that of vitamin C