Comparative Study of Two-Dimensional (2D) vs. Three-Dimensional (3D) Organotypic Kertatinocyte-Fibroblast Skin Models for Staphylococcus aureus (MRSA) Infection

The invasion of skin tissue by Staphylococcus aureus is mediated by mechanisms that involve sequential breaching of the different stratified layers of the epidermis. Induction of cell death in keratinocytes is a measure of virulence and plays a crucial role in the infection progression. We established a 3D-organotypic keratinocyte-fibroblast co-culture model to evaluate whether a 3D-skin model is more effective in elucidating the differences in the induction of cell death by Methicillin-resistant Staphylococcus aureus (MRSA) than in comparison to 2D-HaCaT monolayers. We investigated the difference in adhesion, internalization, and the apoptotic index in HaCaT monolayers and our 3D-skin model using six strains of MRSA representing different clonal types, namely, ST8, ST30, ST59, ST22, ST45 and ST239. All the six strains exhibited internalization in HaCaT cells. Due to cell detachment, the invasion study was limited up to two and a half hours. TUNEL assay showed no significant difference in the cell death induced by the six MRSA strains in the HaCaT cells. Our 3D-skin model provided a better insight into the interactions between the MRSA strains and the human skin during the infection establishment as we could study the infection of MRSA in our skin model up to 48 h. Immunohistochemical staining together with TUNEL assay in the 3D-skin model showed co-localization of the bacteria with the apoptotic cells demonstrating the induction of apoptosis by the bacteria and revealed the variation in bacterial transmigration among the MRSA strains. The strain representing ST59 showed maximum internalization in HaCaT cells and the maximum cell death as measured by Apoptotic index in the 3D-skin model. Our results show that 3D-skin model might be more likely to imitate the physiological response of skin to MRSA infection than 2D-HaCaT monolayer keratinocyte cultures and will enhance our understanding of the difference in pathogenesis among different MRSA strains.

Idarubicin-induced oxidative stress and apoptosis in cardiomyocytes: An in vitro molecular approach

Idarubicin (IDA) is an anthracycline antibiotic, frequently used for the treatment of various human cancers. In vivo rodent model studies have identified a variety of possible adverse outcomes from IDA including heart effects like increased heart weights, myocardial histopathological injury, electrocardiogram abnormalities, and cardiac dysfunction. Despite significant investigations, the molecular mechanisms responsible for the cardiotoxicity of IDA have not been fully clarified. The aim of the current study was to investigate the effects of IDA on the HL-1 cardiac muscle cell. Different concentrations of IDA (10−6, 10−5, 10−4, and 10−3 M) were used at different time (6, 12, 24, and 48 h) periods, and the Cell Counting Kit-8 (CCK-8); 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe method; and enzyme-linked immunosorbent assay (ELISA) were used to detect the oxidative stress level. In addition, we used network analysis to predict IDA-induced cardiotoxicity. The TUNEL assay, qRT-PCR, ELISA assay, and Western blotting detection of related apoptotic factors including caspase family, Bax, and Bcl-2. Overall, we found that IDA was generally more toxic at high concentrations or extended durations of exposure. At the same time, IDA can increase the content of reactive oxygen species (ROS), malondialdehyde (MDA), and decrease the level of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) in cells, and increase the content of lactate dehydrogenase (LDH) and nitric oxide synthase (NOS) in the medium. Network analysis showed that the apoptosis signaling pathway was activated; specifically, the caspase family was involved in the signal pathway. The results of the TUNEL assay, qRT-PCR, ELISA, and Western blot found that IDA can activate apoptotic factors. The mechanism may be related to the activation of apoptosis signaling pathway. These results indicate that the cardiotoxic effects of IDA are most likely associated with oxidative stress and ROS formation, which finally ends in apoptotic factors’ activation and induction of cell apoptosis.

Protective Effect of Penetratin Analogue-Tagged SOD1 on Cisplatin-Induced Nephrotoxicity through Inhibiting Oxidative Stress and JNK/p38 MAPK Signaling Pathway

Copper/zinc superoxide dismutase (SOD1) can clear cisplatin- (CP-) induced excessive reactive oxygen species (ROS), but exogenous SOD1 cannot enter cells because of its low biomembrane permeability. Cell-penetrating peptides (CPPs) can rapidly cross plasma membranes. This study is aimed at identifying an efficient and stable CPP-SOD1 and investigating its effects on CP-induced nephrotoxicity. We recombined SOD1 with 14 different CPPs and purified them using an NTA-Ni2+ column. In in vitro experiments, CPPs-SOD1 cell membrane penetration ability and JNK/p38 MAPK signaling pathway were evaluated using Western blotting. ROS production, mitochondrial membrane potential (MMP), and cell apoptosis were determined using flow cytometry and immunofluorescence staining in VERO and HK-2 cells. For in vivo experiments, mice were administered PSF-SOD1 for 2 h before cotreatment with a single CP injection for an additional 4 days. Blood and kidney samples were collected for renal function assessment (creatinine, urea nitrogen, histopathology, TUNEL assay, and JNK/p38 MAPK signaling pathway). Compared with TAT-SOD1, we found that PSF-SOD1 is more efficient at crossing the cell membrane and is stable after transduction into cells. Pretreatment with PSF-SOD1 inhibited CP-induced apoptosis, ROS generation, and JNK/p38 MAPK activation and restored CP-induced MMP loss in VERO and HK-2 kidney cells. Treatment of mice with PSF-SOD1 inhibited CP-induced serum creatinine, blood urea nitrogen elevation, and JNK/p38 MAPK activation. H&E staining and TUNEL assay indicated that kidney tissue damage was alleviated following PSF-SOD1 pretreatment. Overall, PSF-SOD1 ameliorated CP-induced renal damage by partially reducing oxidative stress and cell apoptosis by regulating JNK/p38 MAPK signaling pathway and might be a better cytoprotective agent than TAT-SOD1.

ERK knockdown suppress cell biological activities via regulation CD59 in breast cancer

IntroductionIt has been unclear that ERK play the effects and relative mechanism in breast cancer development. The purpose of this work was to discuss the ERK play the effect in breast cancer and relative mechanisms.Material and methodsEvaluating ERK and CD59 proteins expression in difference tissue from patients by IHC assay. Using MCF-7 and MDA-MB-231 cell lines which were breast cancer cell lines as target cell lines in our study. In vitro study, evaluating cell biological activities including proliferation, apoptosis, cell cycle, invasion, adherent and migration by MTT, clone test, TUNEL assay, flow cytometry and wound healing. And measuring relative proteins expressions by WB assay. In vivo study, measuring tumor weight and volume, the apoptosis cell number were evaluated by TUNEL assay and relative proteins expressions by IHC assay.ResultsCompared with adjacent normal tissue, the ERK and CD59 proteins expression were significantly increased in breast cancer tissues (P＜0.001, respectively).In vitro and vivo studies, with ERK knockdown, the cell biological activities were significantly depressed with CD59 suppressing (P＜0.001, respectively). And the relative proteins including CD59, PKD, P53, E-cadherin and Vimentin were significantly differences (P＜0.001, respectively).ConclusionsERK play an oncology gene in breast cancer development, ERK inhibitor had effects to suppress breast cancer biological via regulation CD59 in vitro and vivo study.

Antimelanoma activities of chimeric thiazole–androstenone derivatives

The discovery of chimeric anti-melanoma agents is reported. These molecules are potent growth suppressors of melanoma cells in vitro with growth inhibition of 50% (GI 50 ) values as low as 1.32 µM. Compounds were more toxic to melanoma cells in vitro than commonly used anti-melanoma agent dacarbazine as measured by TUNEL assay. They induced both caspase-independent apoptosis evident by colocalization of TUNEL with endonuclease G (EndoG) and caspase-mediated apoptosis measured by colocalization of TUNEL with caspase-activated DNase (CAD). In addition, compounds 3 and 5 strongly induced oxidative injury to melanoma cells as measured by TUNEL colocalization with heme oxygenase-1 (HO1). Dacarbazine induced only caspase-independent apoptosis, which may explain why it is less cytotoxic to melanoma cells than compounds 3 , 4 and 5 .

Effects of Berberine on Diabetes and Cognitive Impairment in an Animal Model: The Mechanisms of Action

Diabetes is a group of metabolic disorders with an increased risk of developing cognitive impairment and dementia. The hippocampus in the forebrain contains an abundance of insulin receptors related to cognitive function and plays an important role in the pathophysiology of neurodegenerative disorders. Berberine from traditional Chinese medicine has been used to treat diabetes and diabetic cognitive impairment, although its related mechanisms are largely unknown. In this study, a STZ diabetes rat model feeding with a high-fat diet was used to test the effects of berberine compared with metformin. Oral glucose tolerance and hyperinsulinemic-euglycemic clamp were used for glucose metabolism and insulin resistance. The Morris water maze was used to observe the compound effects on cognitive impairment. Serum and hippocampal [Formula: see text]-amyloid peptide (A[Formula: see text], Tau and phosphorylated Tau protein deposition in the hippocampi were measured. The TUNEL assay was used to detect the neuronal apoptosis, supported by histomorphological changes and transmissional electron microscopy (TEM) image. Our data showed that the diabetic rats had a significantly cognitive impairment. In addition to improving glucose metabolism and reducing insulin resistance, berberine significantly improved the cognitive function in the rat. Berberine also effectively decreased the expression of hippocampal tau protein, phosphorylated Tau, and increased insulin receptor antibodies. Moreover, berberine downregulated the abnormal phosphorylation of A[Formula: see text] and Tau protein and improved hippocampal insulin signaling. The TUNEL assay confirmed that berberine reduced hippocampal neuronal apoptosis supported by TEM. Thus, berberine significantly improved the cognitive function in diabetic rats by changing the peripheral and central insulin resistance. The reduction of neuronal injury, A[Formula: see text] deposition, abnormal phosphorylation of Tau protein, and neuronal apoptosis in the hippocampus were observed as the related mechanisms of action.

Tetrahydropalmatine reduces cell death and improves functional recovery after traumatic spinal cord injury in ratsTetrahydropalmatine reduces cell death and improves functional recovery after traumatic spinal cord injury in rats

Purpose: To investigate the effectiveness of tetrahydropalmatine (THP) in the treatment of spinal cord injury (SCI) in rats. Methods: Adult Sprague Dawley rats were divided into 3 groups: normal control group, SCI group, and SCI group treated with THP (100 mg kg-1). The effect of THP on spinal cord water content, levels of inflammatory mediators, oxidative stress and apoptosis were determined. Locomotor activity in rats was measured using Basso, Beattie and Bresnahan (BBB) scores. Various oxidative stress markers as well as cytokine levels (NF-ĸB, IL-6, IL-1β and TNF-α were determined. Apoptotic index was measured using TUNEL assay. Results: After 72 h of treatment with THP, BBB scores in SCI group of rats significantly increased from 4.19 ± 0.41 to 8.89 ± 0.47 (p < 0.05). Tunel assay revealed a higher apoptotic index (42.50 ± 3.19) in the tissues of SCI rats than in SCI rats treated with THP (31.48 ± 1.19, p < 0.01). Expressions of inflammatory cytokines were significantly upregulated in SCI rats. However, THP administration resulted in significant downregulation of the expressions (p < 0.01). Conclusion: These results indicate that THP attenuates traumatic SCI in rats via modulation of the levels of anti-inflammatory mediators. Thus, THP has a promising potential for the management of SCI.

Linalool Inhibits MCF-7 Tumor Growth in a Xenograft Model by Apoptosis Induction and Immune Modulation

In this study, the anti-cancer activity of linalool was investigated in MCF-7 breast cancer-bearing mice. Natural killer (NK) and B cell populations in peripheral blood were studied by flow cytometry. The expressions of proliferating cell nuclear antigen (PCNA) and Ki-67 in xenograft tumors were evaluated by immunohistochemistry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to investigate apoptosis induction in an in vivo model. The results indicated that linalool possesses an inhibitory effect on breast cancer growth in the xenograft model. Linalool reduced B cell counts, but increased NK cell counts in mice peripheral blood. The immunosignals of PCNA and Ki-67 were significantly lower in the linalool treatment group than those of the control group. The TUNEL assay showed that linalool significantly induced apoptosis compared to the control group. The findings of this study provide insight and evidence on the antiproliferative activity of linalool on human breast cancer.

Establishment of a new valid animal model for the evaluation of hyperthermic intraperitoneal chemotherapy (HIPEC) in pediatric rhabdomyosarcoma

Background: Cytoreductive surgery in combination with hyperthermic intraperitoneal chemotherapy has been established as a novel treatment approach for peritoneal sarcomatosis. Despite promising clinical reports, there is still a lack of knowledge regarding optimal drug usage and local effects. Therefore, we intended to establish a murine animal model for further evaluation. Procedure: Alveolar rhabdomyosarcoma cells were xenotransplanted into NOD/LtSz-scid IL2Rγnullmice (n=100). The mice received a continuous intraperitoneal lavage with isotonic saline solution as control- or with cisplatin (30 or 60 mg/m2) as treatment group for 60 minutes at 37 or 42 °C (6 subgroups, each n=16). Tumor spread was documented by an adapted peritoneal cancer index and MRI (n=4). Tumor and tissue samples, harvested at the end of the perfusion, were evaluated regarding morphology, proliferation and apoptosis (H&E-, Ki-67-, Cleaved Caspase 3-staining, TUNEL-assay). Results: Extensive peritoneal sarcomatosis in over 91% of the cases was observed. HIPEC was feasible without acute side effects. Ki-67 staining revealed concentration- or temperature-dependent effects of cisplatin-based HIPEC on the tumors. While Cleaved Caspase-3 showed only sporadic apoptotic effects. TUNEL-assay detected concentration- or temperature-dependent apoptotic effects at the outer tumor surface. MRI scans confirmed the observed tumor dissemination. Conclusion: This is the first animal model for evaluation of HIPEC in pediatric RMS in mice. Cisplatin-based HIPEC had early effects on the proliferation whereas circumscribed apoptotic effects could be detected at the tumor surface. This model allows further insights on the possible efficiency of HIPEC in RMS. Further studies using other drug combinations and treatment will follow.