To investigate the effects of tumor protein p53 (p53 or TP53) α gene on the chemosensitivity of the H1299 human lung adenocarcinoma cell line, the recombinant vector pEGFP-p53α was constructed. The vector pEGFP-p53α was transfected into the cultured p53-null H1299 cells using Lipofectamine 2000. The G418-resistant cells were then selected. The expression of the p53α gene in these cells was examined using reverse transcription-polymerase chain reaction, and TP53 protein expression was examined using western blot analysis and immunocytochemistry. An MTT assay and colony formation assay were used to analyze the response of the transfected cells to cisplatin (CDDP). DAPI staining was used to determine the level of apoptosis of the transfected cells. The transfected H1299 human lung adenocarcinoma cells stably expressed TP53 protein. The MTT assay demonstrated that the 50% inhibitory concentrations for the H1299, H1299/pEGFP-N1 and H1299/pEGFP-p53α cells were 28, 24 and 18 µmol/l, respectively. The survival rate of H1299/pEGFP-p53α cells was significantly reduced compared with that of H1299 and H1299/pEGFP-N1 cells (P<0.05). The colony formation assay and DAPI staining identified that the colony formation rate and the number of apoptotic cells of H1299/pEGFP-p53α were significantly reduced, compared with those of the H1299 and H1299/pEGFP-N1 cells (P<0.05). Therefor, the present study demonstrated that the transfection of H1299 cells with the p53α gene resulted in an increase in sensitivity to CDDP chemotherapy. The combination of CDDP and gene therapy for H1299 lung adenocarcinoma cell line provides an experimental basis for clinical research.
Abstract. Aberrant promoter hypermethylation resulting in the epigenetic silencing of apoptosis-associated genes is a key process in the chemotherapeutic treatment of cancer. The nucleoside analog, 5-aza-2'deoxycytidine (DAC), inhibits the activity of DNA methyltransferase enzymes and is able to restore the expression levels of genes that have been silenced by aberrant DNA methylation. The aim of the present study was to investigate the effect of combined treatment with DAC and cisplatin (CDDP) on the lung adenocarcinoma cell line, P15. Growth inhibition was examined using a clone formation assay and growth inhibitory activities by cell counting during treatment with DAC alone, CDDP alone or DAC followed by CDDP. In addition, changes in the mRNA expression levels of various apoptosis-associated genes following treatment with increasing concentrations of DAC were determined using reverse transcription-polymerase chain reaction. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis was used to detect the number of apoptotic P15 tumor cells following treatment with DAC and/or CDDP. The results indicated that DAC treatment alone restored the mRNA expression levels of p73, p16INK4a , B-cell lymphoma (Bcl)-2-associated agonist of cell death and Bcl-2-associated X protein. In addition, combined therapy with DAC and CDDP was found to significantly suppress the growth of P15 tumor cells compared with DAC or CDDP treatment alone. In conclusion, DAC may enhance the chemosensitivity of the P15 cell line to treatment with CDDP. IntroductionLung cancer is a leading cause of cancer-associated mortality worldwide and the most common type is non-small cell lung cancer (NSCLC) (1). Despite progress in the development of molecular-targeted therapeutic agents and surgical approaches, chemotherapy remains an important strategy for the treatment of NSCLC. A combination of a next-generation cytotoxic agents and a platinum compound is the standard treatment regimen for patients with advanced NSCLC (2,3). The cytotoxicity of cisplatin (CDDP), a platinum compound used in chemotherapy, is predominantly due to its interaction with DNA, forming DNA adducts that result in the activation of a number of apoptosis signaling pathways (4). However, the resistance of NSCLC cells to CDDP, which is currently a barrier in the treatment of patients with NSCLC, is associated with inactivation of these apoptosis signaling pathways (5,6).In tumor cells, numerous apoptosis-associated genes appear to have methylated CpG islands. DNA methylation is a crucial regulator in various biological processes; however, aberrant CpG island hypermethylation in gene promoter regions may impact the cell cycle, proliferation, apoptosis, metastasis, drug resistance and intracellular signaling. Therefore, the occurrence of this aberrant hypermethylation is important in carcinogenesis and cancer treatment. DNA hypermethylation is an epigenetic modification that leads to the transcriptional silencing of regulatory genes in various...
IntroductionGetah virus (GETV) has become a growing potential threat to the global livestock industry and public health. However, little is known about the viral pathogenesis and immune escape mechanisms, leading to ineffective control measures.MethodsIn this study, the antiviral activity of exogenous interferons (IFNs) was assessed by using western blotting (WB), real-time quantitative PCR (RT-qPCR) and indirect immunofluorescence assay (IFA). The comparative transcriptomics among mock- and GETV-infected (MOI = 0.1) ST cells with or without IFN-γ was performed by RNA-seq, and then the transcriptome profiling of GETV-infected ST cells and key pathways and putative factors involved in inhibitory effect of IFN-γ on GETV replication were analyzed by bioinformatics methods and RT-qPCR.ResultsThe results showed that treatment with IFN-γ could suppress GETV replication, and the inhibitory effect lasted for at least 48 h, while the exogenous IFN-α/ω and IFN-λ3 treatments failed to inhibit the viral infection and early replication in vitro. Furthermore, the blueprint of virus-host interaction was plotted by RNA-seq and RT-qPCR, showing systemic activation of inflammatory, apoptotic, and antiviral pathways in response to GETV infection, indicating viral hijacking and inhibition of innate host immunity such as IFN-I/III responses. Last and most importantly, activation of the JAK-STAT signaling pathway and complement and coagulation cascades may be a primary driver for IFN-γ-mediated inhibition of GETV replication.DiscussionThese findings revealed that GETV possessed the capability of viral immune escape and indicated that IFN-γ aided in the prevention and control of GETV, implying the potential molecular mechanism of suppression of GETV by IFN-γ, all of which warrant emphasis or further clarification.
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