A robust immune-related gene pairs signature for predicting the overall survival of esophageal cancer.

BACKGROUND: Identifying reliable biomarkers could effectively predict esophagus carcinoma (EC) patients with poor prognosis. In this work, we constructed an immune-related gene pairs (IRGP) signature to evaluate the prognosis of EC. RESULTS: The IRGP signature was trained by the TCGA cohort and validated by three GEO datasets, respectively. Cox regression model together with LASSO was applied to construct the overall survival (OS) associated IRGP. 21 IRGPs consisting of 38 immune-related genes were included in our signature, according to which patients were stratified into high- and low-risk groups. The results of Kaplan-Meier survival analyses indicated that high-risk EC patients had worse OS than low-risk group in the training set, meta-validation set and all independent validation datasets. After adjustment in multivariate Cox analyses, our signature continued to be an independent prognostic factor of EC and the signature-based nomogram could effectively predict the prognosis of EC sufferers. Besides, Gene Ontology analysis revealed this signature is related to immunity. 'CIBERSORT' analysis revealed the infiltration levels of plasma cells and activated CD4 memory T cells in two risk groups were significantly different. Ultimately, we validated the expression levels of six selected genes from IRGP index in KYSE-150 and KYSE-450. CONCLUSIONS: This IRGP signature could be applied to select EC patients with high mortality risk, thereby improving prospects for the treatment of EC.

Cytotoxic Activity of Tropane Alkaloides of Species of Erythroxylum.

Erythroxylaceae is a family composed of four genera, with Erythroxylum being the only one represented in the Neotropical region. Chemical studies indicate the presence of alkaloids, terpenes, flavonoids, and phenolic compounds as main compounds. The incorporation of cytotoxic activity assays of natural products using cell cultures assists in the selection of potential chemotherapeutic agents. In this work, we describe a revision of the cytotoxicity evaluation studies performed with extracts or pure substances obtained from Erythroxylum species through an integrative review. We found studies that evaluated the cytotoxic activity of 21 species of Erythroxylum against 45 different cell lines. The analysis of the chemical composition of these species shows that the metabolites present in each species influence their cytotoxic potential, especially the presence of disubstituted tropane alkaloid species with the highest cytotoxic potential. MTT and Sulforrodamine B assays were the main in vitro tests used for the evaluation of the cytotoxic activities. From the total species, less than 10% of the Erythroxylum species have already been evaluated for cytotoxic activity. Four of them showed high cytotoxic activity according to the criteria of the NCI plant screening program. Thus, this genus represents a potential source of natural products with antitumor activity.

Ocular Cell Lines and Genotoxicity Assessment.

Genotoxicity screening tests aim to evaluate if and to what extent a compound in contact with the human body (e.g., a drug molecule, a compound from the environment) interacts with DNA. The comet assay is a sensitive method used to predict the risk of DNA damage in individual cells, as it quantifies the tape breaks, being the alkaline version (pH > 13) the most commonly used in the laboratory. Epithelial cells serve as biomatrices in genotoxicity assessments. As ca. 80% of solid cancers are of epithelial origin, the quantification of the DNA damage upon exposure of epithelial cells to a drug or drug formulation becomes relevant. Comet assays run in epithelial cells also have clinical applications in human biomonitoring, which assesses whether and to what extent is the human body exposed to environmental genotoxic compounds and how such exposure changes over time. Ocular mucosa is particularly exposed to environmental assaults. This review summarizes the published data on the genotoxicity assessment in estimating DNA damage in epithelial cells with a special focus on ocular cell lines. General comet assay procedures for ex vivo and in vivo epithelium samples are also described.

Cancer/testis antigens expression during cultivation of melanoma and soft tissue sarcoma cells.

BACKGROUND: Autologous dendritic cells (DC) loaded with tumor-associated antigens (TAAs) are a promising approach for anticancer immunotherapy. Polyantigen lysates appear to be an excellent source of TAAs for loading onto the patient's dendritic cells. Cancer/testis antigens (CTA) are expressed by a wide range of tumors, but are minimally expressed on normal tissues, and could serve as a universal target for immunotherapy. However, CTA expression levels can vary significantly in patients with the same tumor type. We proposed that patients who do not respond to DC-based therapy may have distinct features of the CTA expression profile on tumor cells. PATIENTS AND METHODS: We compared the gene expression of the principal families CTA in 22 melanoma and 27 soft tissue and bone sarcomas cell lines (STBS), received from patients and used for DC vaccine preparation. RESULTS: The majority (47 of 49, 95.9%) cell lines showed CTA gene activity. The incidence of gene expression of GAGE, NYESO1, MAGEA1, PRAME's was significantly different (adj. p < 0.05) between melanoma and sarcoma cell lines. The expression of the SCP1 gene was detected neither in melanoma cells nor in the STBS cells. Clustering by the gene expression profile revealed four different expression patterns. We found three main patterns types: hyperexpression of multiple CTA, hyperexpression of one CTA with almost no expression of others, and no expression of CTA. All clusters types exist in melanoma and sarcoma cell lines. We observed dependence of killing efficacy from the PRAME (rho = 0.940, adj. p < 0.01) expression during real-time monitoring with the xCELLigence system of the interaction between melanoma or sarcoma cells with the T-lymphocytes activated by the lysate of selected allogenous melanoma cell lines with high expression of CTA. CONCLUSION: Our results demonstrate that one can use lysates from allogeneic melanoma cell lines as a source of CTA for DC load during the production of anticancer vaccines for the STBS treatment. Patterns of CTA expression should be evaluated as biomarkers of response in prospective clinical trials.

Metalloproteomics analysis in human mammary cell lines treated with inorganic mercury.

The interest in inorganic Hg toxicity and carcinogenicity has been pointed to target organs such as kidney, brain or placenta, but only a few studies have focused on the mammary gland. In this work, analytical combination techniques (SDS-PAGE followed by CV-AFS, and nanoUPLC-ESI-MS/MS) were used to determine proteins that could bind Hg in three human mammary cell lines. Two of them were tumorigenic (MCF-7 and MDA-MB-231) and the other one was the non-tumorigenic cell line (MCF-10A). There are no studies that provide this kind of information in breast cell lines with IHg treatment. Previously, we described the viability, uptake and the subcellular distribution of Hg in human breast cells and analysis of RNA-seq about the genes that encode proteins which are related to cytotoxicity of Hg. This work provides important protein candidates for further studies of Hg toxicity in the mammary gland, thus expanding our understanding of how environmental contaminants might affect tumor progression and contribute with future therapeutic methods.

Biosynthesis of silver nanoparticles from leaf extract of Litchi chinensis and its dynamic biological impact on microbial cells and human cancer cell lines.

Green synthesis of metallic nanoparticles has attracted a great deal of attention from scientific community due to its biocompatibility and environment friendly nature. In the present study, silver nanoparticles were biologically synthesized using leave extracts of Litchi chinensis. Biosynthesized silver nanoparticles were characterized and their applications were observed by different methodologies. Bio-reduction reaction was confirmed by the surface plasmon resonance of silver nanoparticles at 417 nm through UV-VIS spectrophotometer. FTIR analysis revealed that the amine groups present in the leaf extracts were responsible for the reduction of silver ions to silver nanoparticles. X-ray diffraction analysis was used to determine the crystalline nature of silver nanoparticles and their diameter was noted in the range of 41-55 nm by scanning electron microscopy. Antibacterial activity was observed against gram positive and gram negative strains of bacteria. Furthermore, human epithelial type 2 cancer cells (HEp-2) and Human breast adenocarcinoma cells lines (MCF-7) were treated with the biosynthesized silver nanoparticles using MTT assay. The resulting cell death rate was noted up to 40.91+1.99%. This study concludes that plant mediated biosynthesis of nanoparticles is the superior alternative compared to chemical and physical approaches, to utilize them as drug delivery tool and need to conjugate apoptosis inducing biological agents with silver nanoparticles to suppress the uncontrolled division of cancer cells.

Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines.

The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via 'shock and kill' (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to cause selective cell death via the lytic properties of the virus, or the immune system now recognizing the infected cells. Recently, a number of studies have described the therapeutic potential of pharmacologically inhibiting members of the bromodomain and extraterminal (BET) family of human bromodomain proteins (Filippakopoulos et al., 2010; Dawson et al., 2011; Delmore et al., 2011) that include BRD2, BRB3, BRD4 and BRDT. Small-molecule BET inhibitors, such as JQ1 (Filippakopoulos et al., 2010; Delmore et al., 2011), I-BET (Nicodeme et al., 2010), I-Bet151 (Dawson et al., 2011), and MS417 (Zhang et al., 2012) successfully activate HIV transcription and reverse viral latency in clonal cell lines and certain primary T-cell models of latency. To identify the mechanism by which BET proteins regulate HIV-1 latency, we utilized small hairpin RNAs (shRNAs) that target BRD2, BRD4 and Cyclin T1, which is a component of the critical HIV-1 cofactor positive transcription elongation factor b (P-TEFb) and interacts with BRD2, and tested them in the CD4+ J-Lat A2 and A72 cell lines. The following protocol describes a flow cytometry-based method to determine the amount of transcriptional activation of the HIV-1 LTR upon shRNA knockdown. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines.

Cytotoxic effect of a novel naphthylchalcone against multiple cancer cells focusing on hematologic malignancies.

Chalcones are natural compounds described in the literature by its several properties including cytotoxic activity against several tumor types. Considering that the search for new chemotherapeutic agents is still necessary, the aim of this study was to investigate the cytotoxic mechanisms involved in cell death induced by a synthetic chalcone (A23) on different tumor cells. Chalcone A23 reduced the cell viability of twelve tumor cell lines in a concentration and time dependent manner and it was more cytotoxic against acute leukemia cells. Interestingly, the compound was non cytotoxic to normal cells and non-hemolytic to normal red blood cells. Chalcone A23 decreased the expression of cell proliferation marker KI-67 and blocked the G2/M phase in both K562 and Jurkat cell lines. Cells treated with A23 showed morphological features suggestive of apoptosis, the "latter pattern" in agarose gel, the externalization of phosphatidylserine and caspase-3 and PARP cleavage. Chalcone A23 significantly reduced the mitochondrial membrane potential, decreased the expression of anti-apoptotic proteins Bcl-2 and survivin and increased the expression of pro-apoptotic protein Bax, confirming the involvement of the intrinsic pathway. The increased mitochondrial permeability resulted in the release of AIF, cytochrome c and endonuclease G from the mitochondria to the cytosol. In addition, chalcone A23 increased the expression of FasR and induced Bid cleavage, showing the involvement of the extrinsic pathway. Finally, chalcone A23 seems to have a synergic effect with the chemotherapy drugs cytarabine and vincristine. These results suggest that A23 is an interesting compound with strong and selective anti-tumor activity.

Cytotoxicity of nickel zinc ferrite nanoparticles on cancer cells of epithelial origin.

In this study, in vitro cytotoxicity of nickel zinc (NiZn) ferrite nanoparticles against human colon cancer HT29, breast cancer MCF7, and liver cancer HepG2 cells was examined. The morphology, homogeneity, and elemental composition of NiZn ferrite nanoparticles were investigated by scanning electron microscopy, transmission electron microscopy, and energy dispersive X-ray spectroscopy, respectively. The exposure of cancer cells to NiZn ferrite nanoparticles (15.6-1,000 µg/mL; 72 hours) has resulted in a dose-dependent inhibition of cell growth determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The quantification of caspase-3 and -9 activities and DNA fragmentation to assess the cell death pathway of the treated cells showed that both were stimulated when exposed to NiZn ferrite nanoparticles. Light microscopy examination of the cells exposed to NiZn ferrite nanoparticles demonstrated significant changes in cellular morphology. The HepG2 cells were most prone to apoptosis among the three cells lines examined, as the result of treatment with NiZn nanoparticles. In conclusion, NiZn ferrite nanoparticles are suggested to have potential cytotoxicity against cancer cells.

Atorvastatin Reduces the LPS-induced COX-2 Expression in Cultured Human Pulmonary Epithelial Cells / 中国医师杂志

Objective To assess the effect of atorvastatin on the Lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2) expression in cultured human pulmonary epithelial cells (A549). Methods A549 cells were incubated in the medium containing LPS and different concentrations of atorvastatin(0,10,15,20?M/ml, respectively) for 12h. Then the total cellular RNA and proteins from the cells treated with different experimental conditions were extracted for RT-PCR and western blot analysis,respectively. Results In cultured human pulmonary epithelial cells, atorvastatin reduced the expression of COX-2 mRNA and protein induced by LPS in a dose-dependent manner. Conclusion Atorvastatin may down-regulate LPS-induced COX-2 expression in cultured human pulmonary epithelial cells.

Triton X-100 induces apoptosis in human hepatoma cell lines

The detergent Triton X-100 was used to establish a model for apoptosis in hepatoma cell lines. The electrophoresis of DNA extracted from 0.01% Triton X-100 treated hepatoma cell lines showed DNA ladder formation, a hallmark of apoptosis. The DNA fragmentation appeared within less than 60 min of the Triton X-100 treatment. Chromatin condensation and apoptotic bodies were observed by hematoxylin and eosin (H & E) stain, and fragmented nucleosome was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) test. Apoptosis was semi-quantitated by measuring the lactate dehydrogenase (LDH) level for cytotoxity. It was found that apoptosis had been induced in more than 90% of the cells treated with Triton X-100 for 150 min. These data show that Triton X-100 efficiently induces the apoptotic cell death in hepatoma cell lines.