CASP8 Genes Research Articles

Growth Inhibition and Apoptotic Effect of Pine Extract and Abietic Acid on MCF-7 Breast Cancer Cells via Alteration of Multiple Gene Expressions Using In Vitro Approach.

In vitro anti-proliferative activity of Pinus palustris extract and its purified abietic acid was assessed against different human cancer cell lines (HepG-2, MCF-7 and HCT-116) compared to normal WI-38 cell line. Abietic acid showed more promising IC50 values against MCF-7 cells than pine extract (0.06 µg/mL and 0.11 µM, respectively), with insignificant cytotoxicity toward normal fibroblast WI-38 cells. Abietic acid triggered both G2/M cell arrest and subG0-G1 subpopulation in MCF-7, compared to SubG0-G1 subpopulation arrest only for the extract. It also induced overexpression of key apoptotic genes (Fas, FasL, Casp3, Casp8, Cyt-C and Bax) and downregulation of both proliferation (VEGF, IGFR1, TGF-β) and oncogenic (C-myc and NF-κB) genes. Additionally, abietic acid induced overexpression of cytochrome-C protein. Furthermore, it increased levels of total antioxidants to diminish carcinogenesis and chemotherapy resistance. P. palustris is a valuable source of active abietic acid, an antiproliferative agent to MCF-7 cells through induction of apoptosis with promising future anticancer agency in breast cancer therapy.

Protein-coding gene interaction network prediction of bioactive plant compound action against SARS-CoV-2: a novel hypothesis using bioinformatics analysis.

This study aimed to verify the action of bioactive compounds from Brazilian plants on the leader genes involved in the SARS-CoV-2 pathway. The main human genes involved were identified in GeneCards and UNIPROT platforms, and an interaction network between leader genes was established in the STRING database. To design chemo-biology interactome networks and elucidate the interplay between genes related to the disease and bioactive plant compounds, the metasearch engine STITCH 3.1 was used. The analysis revealed that SMAD3 and CASP3 genes are leader genes, suggesting that the mechanism of action of the virus on host cells is associated with the molecular effects of these genes. Furthermore, the bioactive plant compounds, such as ascorbate, benzoquinone, ellagic acid, and resveratrol was identified as a promising adjuvant for the treatment inhibiting CASP3-mediated apoptosis. Bioactive plant compounds were verified as the main pathways enriched with KEGG and related to viral infection, assessments/immune/infections, and cell proliferation, which are potentially used for respiratory viral infections. The best-ranked molecule docked in the CASP3 binding site was rutin, while the SMAD3 binding site was resveratrol. In conclusion, this work identified several bioactive compounds from Brazilian plants showing potential antiviral functions that can directly or indirectly inhibit the new coronavirus.

Impact assessment of cadmium chloride on human cell lines and yeast knockout strains

Background Cadmium (Cd) is a heavy metal that contributes to pollution in the environment. Cd intoxication can lead to diseases in the liver, kidneys, and lungs, among other organs. The liver is the primary organ affected by Cd overdose.Objective To evaluate the cytotoxic and genotoxic responses of cadmium chloride (CdCl2) on three different human cell lines and four different genotypes of yeast knockout strains.Materials and methods The effects of different concentrations of CdCl2 on cell viability in hepatocellular carcinoma (HepG2), lung cancer (A549), and normal lung cell carcinoma (Wi38) were investigated systematically using the MTT method. Moreover, flow cytometry was used to assess cell cycle arrest using propidium iodide (PI) staining and to quantify apoptotic cell death using PI staining and Annexin V/PI staining, respectively. The gene expression of p53, casp3, and Bcl-2 was measured using qRT-PCR. The Comet methodology was used to indicate DNA damage of yeast knockout strains.Results and conclusion The MTT assay revealed that CdCl2 was highly potent against hepatocellular carcinoma (HepG2), lung cancer (A549), and normal lung cell lines (Wi38) (IC50=3.12, 28.81, 191.14 μg/ml). These findings showed that CdCl2 reduced therapeutic efficacy in malignant cells at relatively low concentrations compared with nonmalignant cells, as well as confirmed the antitumor effect of the metal. CdCl2-damaged A549 cells revealed a significant increase in arrest of cell cycle in ‘S’ phases, and then apoptosis increased. Subsequently, when A549 cells were treated with a higher dosage of CdCl2, the gene expression of p53 and casp3 genes was upregulated, whereas Bcl-2 was downregulated. CdCl2 revealed its genotoxic activity at different concentrations of 10, 25, 50 μg/ml. MRE11, CLN1 and ZRC1 genes exhibited marked genotoxic effects, while MMT1 gene generated modret genotoxicity of yeast knockout strains. The Comet assay revealed that yeast cells were more responsive, which was indisputably demonstrated.

Nephroprotective effect of exercise training in cisplatin-induced renal damage in mice: influence of training protocol.

Cisplatin is an effective antineoplastic agent, but its use is limited by its nephrotoxicity caused by the oxidative stress in tubular epithelium of nephrons. On the other hand, regular exercise provides beneficial adaptations in different tissues and organs. As with many drugs, dosing is extremely important to get the beneficial effects of exercise. Thus, we aimed to investigate the influence of exercise intensity and frequency on cisplatin-induced (20 mg/kg) renal damage in mice. Forty male Swiss mice were divided into five experimental groups (n=8 per group): 1) sedentary; 2) low-intensity forced swimming, three times per week; 3) high-intensity forced swimming, three times per week; 4) low-intensity forced swimming, five times per week; and 5) high-intensity forced swimming, five times per week. Body composition, renal structure, functional indicators (plasma urea), lipid peroxidation, antioxidant enzyme activity, expression of genes related to antioxidant defense, and inflammatory and apoptotic pathways were evaluated. Comparisons considered exercise intensity and frequency. High lipid peroxidation was observed in the sedentary group compared with trained mice, regardless of exercise intensity and frequency. Groups that trained three times per week showed more benefits, as reduced tubular necrosis, plasma urea, expression of CASP3 and Rela (NFkB subunit-p65) genes, and increased total glutathione peroxidase activity. No significant difference in Nfe2l2 (Nrf2) gene expression was observed between groups. Eight weeks of regular exercise training promoted nephroprotection against cisplatin-mediated oxidative injury. Exercise frequency was critical for nephroprotection.

Tomentosin shows anticancer effect on U87 human glioblastoma multiforme cells

Glioblastoma multiforme (GBM) is one of the most common brain tumors. Chemotherapy, radiotherapy and surgical resection are methods used in GBM treatment, however, the investigation of possible anticancer effects of low-toxicity natural products on various cancer cells, including GBM, leads to promising results. In this study, it was aimed to investigate anticancer effect of tomentosin, which is a sesquiterpene lactone, on U87 human GBM cells. The cytotoxic effect of tomentosin was evaluated by XTT assay. The concentration of tomentosin that inhibits 50% cell viability (IC50) was determined by the results from XTT, and, in further analyzes cells were treated with tomentosin at IC50 concentration. Then, total RNA isolation and cDNA synthesis were performed in control and dose groups, and, the possible anticancer effect of tomentosin was determined by evaluating the expression levels of important genes associated with apoptosis and metastasis by qPCR analysis. In addition, the effect of tomentosin on the colony forming capacity of GBM cells was evaluated by colony formation assay. According to our results, IC50 dose of tomentosin was found to be 28.8 µM in U87 cells for 48 hours. When compared to the control group, tomentosin increased expression of BAX, CASP3, CASP8, CASP9, CYCS, FADD, TNF, TNFR1, TNFR2 and TIMP2 genes. And, tomantosin significantly decreased colony forming capacity of U87 cells. In conclusion, it is thought that tomentosin exerts its anticancer effect by changing the expression levels of genes associated with apoptosis and metastasis in U87 GBM cells.

Sorafenib, rapamycin, and venetoclax attenuate doxorubicin-induced senescence and promote apoptosis in HCT116 cells

Emerging evidence has shown that the therapy-induced senescent growth arrest in cancer cells is of durable nature whereby a subset of cells can reinstate proliferative capacity. Promising new drugs named senolytics selectively target senescent cells and commit them into apoptosis. Accordingly, senolytics have been proposed as adjuvant cancer treatment to cull senescent tumor cells, and thus, screening for agents that exhibit senolytic properties is highly warranted. Our study aimed to investigate three agents, sorafenib, rapamycin, and venetoclax for their senolytic potential in doxorubicin-induced senescence in HCT116 cells. HCT116 cells were treated with one of the three agents, sorafenib (5 µM), rapamycin (100 nM), or venetoclax (10 µM), in the absence or presence of doxorubicin (1 µM). Senescence was evaluated using microscopy-based and flow cytometry-based Senescence-associated-β-galactosidase staining (SA-β-gal), while apoptosis was assessed using annexin V-FITC/PI, and Muse caspase-3/-7 activity assays. We screened for potential genes through which the three drugs exerted senolytic-like action using the Human Cancer Pathway Finder PCR array. The three agents reduced doxorubicin-induced senescent cell subpopulations and significantly enhanced the apoptotic effect of doxorubicin compared with those treated only with doxorubicin. The senescence genes IGFBP5 and BMI1 and the apoptosis genes CASP7 and CASP9 emerged as candidate genes through which the three drugs exhibited senolytic-like properties. These results suggest that the attenuation of doxorubicin-induced senescence might have shifted HCT116 cells to apoptosis by exposure to the tested pharmacological agents. Our work argues for the use of senolytics to reduce senescence-mediated resistance in tumor cells and to enhance chemotherapy efficacy.

Bioactivity of betulinic acid nanoemulsions on skin carcinogenesis in transgenic mice K14E6

Alternative therapies for cancer treatment have been developed using bioactive compounds such as betulinic acid (BA). The objective of this study was to investigate the bioactivity of BA in its free form and compare it with its nano-encapsulated form under a skin carcinogenesis protocol in a genetically modified murine model. K14E6 and FVB mice were divided into four groups to be treated with free BA and with betulinic acid nanoemulsion (BANE). Lecithin enriched with medium chain fatty acids (MCFAs) was employed as an emulsifier to prepare the nanoemulsions with a mean droplet size of 40 nm. Skin tumors were induced by exposure to DMBA and TPA directly to the transgenic mice. Tumor development was completely inhibited by BANE and by 70% with free BA. This was validated by histological sections and the gene expression of the Cdk4 and Casp8 genes.

Generation of human long-lived plasma cells by developmentally regulated epigenetic imprinting.

Antibody secreting cells (ASCs) circulate after vaccination and infection and migrate to the BM where a subset known as long-lived plasma cells (LLPCs) persists and secrete antibodies for a lifetime. The mechanisms by which circulating ASCs become LLPCs are not well elucidated. Here, we show that human blood ASCs have distinct morphology, transcriptomes, and epigenetics compared with BM LLPCs. Compared with blood ASCs, BM LLPCs have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. LLPCs up-regulate pro-survival genes MCL1, BCL2, and BCL-XL while simultaneously down-regulating pro-apoptotic genes HRK1, CASP3, and CASP8 Consistent with reduced gene expression, the pro-apoptotic gene loci are less accessible in LLPCs. Of the pro-survival genes, only BCL2 is concordant in gene up-regulation and loci accessibility. Using a novel in vitro human BM mimetic, we show that blood ASCs undergo similar morphological and molecular changes that resemble ex vivo BM LLPCs. Overall, our study demonstrates that early-minted blood ASCs in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPCs.

TNF signaling pathway-mediated microglial activation in the PFC underlies acute paradoxical sleep deprivation-induced anxiety-like behaviors in mice

Acute sleep deprivation is a common condition in modern life and increases anxiety symptoms in healthy individuals. The neuroinflammatory response induced by microglial activation could be an important contributing factor, but its underlying molecular mechanisms are still unclear. In the present study, we first found that acute paradoxical sleep deprivation (PSD) induced by the modified multiple platform method (MMPM) for 6 h led to anxiety-like behavior in mice, as verified by the open field test, elevated plus maze test, light–dark box test, and marble burying test. In addition, bioinformatic analysis suggested an important relationship between acute sleep deprivation and brain inflammatory signaling pathways. Key genes enriched in the TNF signaling pathway were confirmed to be altered during acute PSD by qPCR and Western blot analyses, including the upregulation of the prostaglandin-endoperoxide synthase 2 (Ptgs2) and suppressor of cytokine signaling 3 protein (Socs3) genes and the downregulation of the cysteine-aspartic acid protease 3 (Casp3) gene. Furthermore, we found that microglial cells in the prefrontal cortex (PFC) were activated with significant branch structure changes and that the cell body area was increased in the PSD model. Finally, we found that minocycline, a tetracycline with anti-inflammatory properties, may ameliorate the anxiogenic effect and microglial activation. Our study reveals significant correlations of anxiety-like behavior, microglial activation, and inflammation during acute PSD.

Silencing of α-N-acetylgalactosaminidase in the gastric cancer cells amplified cell death and attenuated migration, while the multidrug resistance remained unchanged.

Although the elevated level of the α-N-acetylgalactosaminidase enzyme (encoded by the NAGA gene) is a well-recognized feature of cancer cells; little research works have been undertaken on the cancer malignancy mechanisms. The effects of NAGA gene downregulation on cancer cells' features such as drug resistance, impaired programmed cell death, and migration were analyzed in this study. The cells grew exponentially with a doubling time of 30 h in an optimal condition. Toxicity of daunorubicin chemotherapy drug on NAGA-transfected EPG85.257RDB cells was evaluated in comparison to control cells and no significant change was recorded. Quantitative transcript analyses and protein levels revealed that the MDR1 pump almost remained unchanged during the study. Moreover, the NAGA gene downregulation enhanced the late apoptosis rate in EPG85.257RDB cells at 24 h posttransfection. The investigated expression level of genes and proteins involved in the TNFR2 signaling pathway, related to cancer cell apoptosis, showed considerable alterations after NAGA silencing as well. MAP3K14 and CASP3 genes were downregulated while IL6, RELA, and TRAF2 experienced an upregulation. Also, NAGA silencing generally diminished the migration ability of EPG85.257RDB cells and the MMP1 gene (as a critical gene in metastasis) expression decreased significantly. The expression of the p-FAK protein, which is located in the downstream of the α2 β1 integrin signaling pathway, was reduced likewise. It could be concluded that despite drug resistance, NAGA silencing resulted in augmentative and regressive effects on cell death and migration.

Effect of 4-Allyl-1-hydroxy-2-methoxybenzene (eugenol) in the expression of genes involved in cellular cycle and apoptotic process in dental pulp fibroblasts

Objective This study sought to evaluate the effect of eugenol on the cell morphology and expression of genes involved in the apoptotic process in human dental pulp fibroblasts (hDPFs) from deciduous teeth. Materials and methods hDPFs were cultured with 4 concentrations of eugenol (0.06 nM, 0.6 nM, 6 nM, 12 nM) and compared with a control group. After a 72 h incubation period, the cytotoxic effect on cell morphology by optical microscopy and gene expression by RT-PCR were evaluated. Results At 0.06 nM and 0.6 nM eugenol concentrations, vacuolisation of the cytoplasm was observed with atypical granulation of the hDPFs, and, at 6 nM and 12 nM cytoplasmic extensions disappeared almost completely. Casp-3, Casp-9, and telomerase genes were not expressed at the concentrations evaluated nor in the control group. The relative expression responses of Bcl-2 and TGF-β genes were overexpressed at the 4 concentrations. MAKP’s 0.06 nM (p < .001), 0.6 nM (p < .05) and 12 nM (p < .05) and Cyclin 1 at 12 nM showed significant difference versus the control group (p < .05). Conclusion Eugenol is capable of causing morphological changes in hDPFs in a dose-dependent manner, higher concentrations may promote overexpression of apoptotic genes.

Increased Chlormethine-Induced DNA Double-Stranded Breaks in Malignant T Cells from Mycosis Fungoides Skin Lesions

Mycosis fungoides (MF) is a type of cutaneous T-cell lymphoma. Chlormethine (CL) is recommended as first-line therapy for MF, with a major purpose to kill tumor cells through DNA alkylation. To study the extent of treatment susceptibility and tumor specificity, we investigated the gene expression of different DNA repair pathways, DNA double-stranded breaks, and tumor cell proliferation of clonal TCR Vβ+ tumor cell populations in cutaneous T-cell lymphoma skin cells on direct exposure to CL. Healthy human T cells were less susceptible to CL exposure than two T-lymphoma cell lines, resulting in higher proportions of viable cells. Interestingly, in T cells from MF lesions, we observed a downregulation of several important DNA repair pathways, even complete silencing of RAD51AP1, FANC1, and BRCA2 involved in homologous recombination repair. In the presence of CL, the double-stranded DNA breaks in malignant MF skin T cells increased significantly as well as the expression of the apoptotic gene CASP3. These data point toward an important effect of targeting CL on MF skin tumor T cells, which support CL use as an early cutaneous lymphoma treatment and can be of synergistic use, especially beneficial in the setting of combination skin-directed therapies for cutaneous T-cell lymphoma.

Curcumin induces apoptosis through caspase dependent pathway in human colon carcinoma cells

We investigated the apoptotic effects of curcumin in the colon carcinoma cell line SW480. Cells were treated with 40-200μM curcumin for 24, 48, and 72h, and the IC50 values were determined for each time interval. BrdU, caspase-3, and TUNEL staining results and the gene expression of FADD, CASP8, and CASP3 were evaluated. Curcumin treatments significantly inhibited cell proliferation and significantly induced apoptosis for 24, 48, and 72h. The proportion of BrdU-stained cells in the control groups were 58%, 57% and 61% and 28%, 27%, and 30% in the curcumin treatment groups at 24, 48, and 72h, respectively. The proportion of apoptotic cells was 28%, 29%, and 28% in the control groups and 59%, 61%, and 60% in the curcumin treatment groups at 24, 48, and 72h, respectively. As expected, caspase-3 staining also revealed a higher number of apoptotic cells in curcumin treatment groups at 24, 48, and 72h compared to controls. The proportion of Caspase-3-stained cells in the control groups were 23%, 25%, and 24% and 59%, 60%, and 62% in the curcumin treatment groups at 24, 48, and 72h, respectively. To prove caspase-3 staining results, FADD, CASP8, and CASP3 gene expressions were evaluated by real-time qPCR. Unlike the immunohistochemical results, no statistically significant upregulation was found at 24 and 48h, while relative gene expressions of FADD, CASP8, and CASP3 was significantly upregulated at 72h. The expression level increase was 0.88-, 1.19-, and 2.11-fold for FADD, 1.25-, 1.29-, and 1.59-fold for CASP8, and 1.33-, 1.46-, and 3.00-fold for CASP3 at 24, 48, and 72h, respectively. These results suggest that curcumin may be a potential protective or treatment agent against colon cancer; however, further studies on curcumin-rich diets and curcumin bioavailability are required.

Effects of short‐term temperature changes on the physiology, biochemistry and feeding behaviour of the juvenile yellow seahorse— Hippopotamus kuda Bleeker

In order to study the impact of acute temperature challenge on juvenile hippocampus's growth, the variations of external behavioural indexes and internal physiological, biochemical and genetic indexes were analysed at the same time under acute temperature change. In this paper, the external indexes we selected were the response parameters of feeding behaviour (including feeding reaction time, feeding rate and food intake, etc.). The internal indicators include biochemical indicators (SOD, CAT, AKP, ACP, Trypsin, LPS and AMS enzyme activity and MDA content) and fold change (differentially expression multiples) of differentially expressed gene (Hsp70, Hsp90, Gst, Pyy, mTOR, Bcl-2, Casp9, Casp3, Gadd45α, Fadsd6, Cpt1, Fas, Cyp51, Pdha1, Mdh1, Idh3b and G6pd). The results showed that the juvenile's external indexes had changed significantly at 12 h after short-term temperature challenge exposed. At the same time, the feeding reaction time (s) was extend, and the feeding rate (individuals/min) and food intake (individual) were reduced, suggesting that the growth of juvenile might be suppressed by temperature challenge. At the same time, MDA and AMS showed a significant correlation with the feeding behaviour parameters, and several function genes Gadd45α, Casp3, Idh3b and G6pd also showed a correlation with the behavioural parameters. Our study provide a valuable information for understanding the mechanism of internal and external reaction in juvenile seahorse, which exposed to short-term temperature challenge. It is useful to explore the mechanism of how temperature affects fish growth. And the external behavioural indexes can correctly reflect the excessive fluctuation of environmental temperature that is harmful to the juvenile.

Differential and Common Signatures of miRNA Expression and Methylation in Childhood Central Nervous System Malignancies: An Experimental and Computational Approach.

Simple SummaryEpigenetic mechanisms, that are modifications of the genome without the presence of mutations, are known to play a crucial role in central nervous system (CNS) tumors during childhood. Two well-known epigenetic regulatory mechanisms include methylation and miRNA regulatory mechanisms. Therefore, in the present study we have investigated the presence of methylated genes in childhood CNS tumors, along with miRNA expression. We have searched for correlations between gene methylation and miRNA expression. In addition, we have investigated mRNA expression in order to search for possible miRNA targets. Such approaches could prove useful for the improvement of CNS tumor prognosis, as well as for the discovery of new therapeutic targets.Epigenetic modifications are considered of utmost significance for tumor ontogenesis and progression. Especially, it has been found that miRNA expression, as well as DNA methylation plays a significant role in central nervous system tumors during childhood. A total of 49 resected brain tumors from children were used for further analysis. DNA methylation was identified with methylation-specific MLPA and, in particular, for the tumor suppressor genes CASP8, RASSF1, MGMT, MSH6, GATA5, ATM1, TP53, and CADM1. miRNAs were identified with microarray screening, as well as selected samples, were tested for their mRNA expression levels. CASP8, RASSF1 were the most frequently methylated genes in all tumor samples. Simultaneous methylation of genes manifested significant results with respect to tumor staging, tumor type, and the differentiation of tumor and control samples. There was no significant dependence observed with the methylation of one gene promoter, rather with the simultaneous presence of all detected methylated genes’ promoters. miRNA expression was found to be correlated to gene methylation. Epigenetic regulation appears to be of major importance in tumor progression and pathophysiology, making it an imperative field of study.

Prunetin inhibits nitric oxide activity and induces apoptosis in urinary bladder cancer cells via CASP3 and TNF-α genes.

Urinary bladder cancer (UBC) is considered one of the most prevalent malignant tumors worldwide. Complementary and integrative approaches for the treatment of bladder cancer, such as the intake of isoflavonoid phytoestrogens, are of increasing interest due to the risk of mortality and long-term morbidity associated with surgical procedures. The biological effects of prunetin, one of the less-studied phytoestrogens, have not yet been examined in this respect. Therefore, this study aimed to explore the efficacy of prunetin on UBC cells (RT-4). METHODS ANDRESULTS: The cytotoxicity and nitric oxide synthase activities of prunetin were determined in cell cultures. The expression of apoptosis-related genes was determined with RT-PCR. Cell cycle assays were performed using a flow cytometer and cellular apoptotic rate was measured. The results suggested that prunetin has cytotoxic effects at 21.11 µg/mL on RT-4 cells. Flow cytometry analysis showed that prunetin induced apoptosis and arrested th cell cycle in the G0/G1 phase. Prunetin exposure was associated with increases in CASP3 and TNF-α gene expression in RT-4 cells at doses of 21.11 and 42.22 µg/mL, respectively. Strong nitric oxide inhibition was observed at IC50 of 5.18 µg/mL under macrophage mediated inflammatory circumstances. Prunetin possesses anti-cancer properties and may be a candidate compound for the prevention of UBC. This is the first study that evaluated prunetin for its in vitro antitumor activities, clarified its possible apoptotic molecular mechanism and provided novel insights into its anti-inflammatory nature and effects on the expression of related key genes.

Molecular alterations in the culture of lung cancer cells H1299 after exposure to high doses of ionizing radiation

Objective To study the copy number of genes-components of signaling cascades involved in DNA repair, cell cycle regulation and apoptosis under the influence of high doses of ionizing radiation.Material and Мethods The study was carried out on a culture of H1299 non-small cell lung cancer cells. Cell lines were cultured in a Binder incubator (Germany) for 24 h (at 37 °C, 5% CO2 ), and then the groups were divided into therapeutic and control. The first one was irradiated with a NovalisTx, Varian linear accelerator at doses from 18 to 24 Gy, the second was not exposed to radiation. During the study, we monitored cell viability and evaluated apoptotic activity, then each sample was amplified in two iterations. During the study, cell viability was monitored, apoptotic activity was assessed, and then each sample was amplified in two replicates. The relative copy number of genetic loci was determined by Real-Time qPCR (RT-qPCR).Results When comparing the relative copy number in the genetic loci of the H1299 non-small cell lung cancer cell culture after exposure to a high dose of ionizing radiation, a statistically significant decrease in the relative copy number of the CASP3 and RBBP8 genes was found, which may indicate a decrease in the potential of caspase-mediated tumor repopulation and an increase in the radiosensitivity of tumor cells.Conclusion Exposure to high doses of ionizing radiation leads to a detrimental effect on tumor cells and allows to overcome one of the mechanisms of radioresistance – tumor cell repopulation.

Association between Polymorphisms in the IL-1β, TNFRSF11B, CASP1, and IL-6 Genes and Orthodontic-Induced External Apical Root Resorption.

Orthodontic-induced external apical root resorption (EARR) is a severe condition affecting the roots of the teeth, whose genetic causes have been inconclusive to date. The aim of the present study was to assess the influence of selected single nucleotide polymorphisms (SNPs) IL-1β, TNFRSF11B, CASP1, and IL-6 genes on post-orthodontic EARR. A sample of 101 patients with clearly assessable orthopantomograms and lateral cephalometric radiographs taken before and at the end of the orthodontic treatment was used to evaluate the presence of EARR. The association between genetic polymorphisms and EARR was assessed with the Chi2 test. A binary logistic multi-level model was built to evaluate the ability of patient- and tooth-level variables to predict EARR occurrence. The overall prevalence of EARR resulted to be around 40%. Within the limitations of this study, a significant association was found between EARR presence and the SNP for the IL-1β gene but not for the TNFRSF11B, CASP1, and the IL-6 genes. The final multi-level model demonstrated that the SNP for the IL-1β gene increases the odds of developing EARR by around four times. Since there is currently no accurate method to determine which patients will develop EARR prior to orthodontic treatment, further studies are needed to investigate the predictive ability of further genetic variants on EARR development.

Changes in Methylation Patterns of Tumor Suppressor Genes during Extended Human Embryonic Stem Cell Cultures.

While studies on embryonic stem cells have been actively conducted, little is known about the epigenetic mechanisms in human embryonic stem cells (hESCs) in extended culture systems. Here, we investigated whether CpG island (CGI) methylation patterns of 24 tumor suppressor genes could be maintained during extended hESC cultures. In total, 10 hESC lines were analyzed. For each cell line, genomic DNA was extracted from early and late passages of cell cultures. CGI methylation levels of 24 tumor suppressor genes were analyzed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), pyrosequencing, and real-time polymerase chain reaction (PCR). Different CGI methylation patterns of CASP8, FHIT, and CHFR genes were identified in between early and late passages in some hESC lines. CGI methylation levels of CASP8 significantly increased at late passage in CHA-36, CHA-40, and CHA-42 cell lines compared to those at early passage. The CGI methylation of the FHIT gene was higher at late passage than at early passage in CHA-15, CHA-31, CHA-32, and iPS (FS)-1 cell lines but decreased at the late passage in CHA-20 and H1 cell lines. Different CGI methylation patterns were detected for the CHFR gene only in iPS (FS)-1, and the level significantly increased at late passage. Thus, our findings show that CGI methylation patterns could be altered during prolonged ESC cultures and examining these epigenetic changes is important to assess the maintenance, differentiation, and clinical usage of stem cells.

Influences of cold atmospheric plasma on apoptosis related molecules in osteoblast-like cells in vitro

BackgroundCold atmospheric plasma (CAP) has recently been identified as a novel therapeutic strategy for supporting processes of wound healing. Since CAP is additionally known to kill malignant cells, our study intends to determine the influence of CAP on crucial molecules involved in the molecular mechanism of apoptosis in osteoblast-like cells. MethodsHuman osteoblast-like cells were CAP-treated for 30 and 60 s. CAP effects on critical factors related to apoptosis were studied at transcriptional and protein level using real time-PCR, immunofluorescence staining and western blot. Phalloidin / DAPI staining was used for analyzing the cell morphology. In addition, apoptotic outcomes of CAP were displayed using flow cytometry analysis. For studying intracellular signaling pathways, MAP kinase MEK 1/2 and PI3K were blocked. Finally, the effects of CAP on caspase-3 activity were examined using a caspase-3 assay. ResultsCAP treatment resulted in a significant downregulation of p53 and apoptotic protease activating factor (APAF)-1, caspase (CASP)9, CASP3, BCL2 Antagonist/Killer (BAK)1, and B-Cell Lymphoma (BCL)2 mRNA expression at 1 d. An inhibitory effect of CAP on apoptotic genes was also shown under inflammatory and apoptotic conditions. Nuclear translocation of p53 was determined in CAP treated cells at the early and late stage, after 15 min, 30 min, and 1 h. p53 and APAF-1 protein levels were reduced at 1 d, visualized by immunofluorescence and western blot, respectively. Moreover, a morphological cytoskeleton modification was observed after CAP treatment at 1 d. Further, both CAP-treated and untreated (control) cells remained equally vital as detected by flow cytometry analysis. Interestingly, CAP-associated downregulation of CASP9 and CASP3 mRNA gene expression was also visible after blocking MAP kinase and PI3K. Finally, CAP led to a decrease in CASP3 activity in osteoblast-like cells under normal and apoptotic conditions. ConclusionsOur in vitro-study demonstrated, that CAP decreases apoptosis related molecules in osteoblast-like cells, underlining a beneficial effect on hard-tissue cells.