Targeted Overexpression of NDRG2 using Survivin Promoter Reduces Viability and Invasiveness of A549 Cell Line.

Background: Anti-tumor effects of N-myc Downstream Regulated Gene2 (NDRG2) have been demonstrated in many tumors. In the present study, NDRG2 was specifically overexpressed in lung cancer cell line using Survivin Promoter (Sur-P). Then, the effects of NDRG2 overexpression on viability, apoptosis, migration, and invasion of A549 cells were evaluated. Methods: Recombinant pAdenoVator-Sur-P-NDRG2-IRES-GFP plasmid harboring NDRG2 gene under transcriptional control of Sur-P and mock plasmid were constructed. A549 lung tumor cells and LX-2 cells (non-tumor cell line) were transfected with pAdenoVator-Sur-P-NDRG2-IRES-GFP, pAdenoVator-CMV-NDRG2-IRES-GFP, or mock plasmids. Tumor specificity of Sur-P was evaluated using fluorescent microscopy for GFP expression. The effects of NDRG2 overexpression on cell viability, apoptosis, and migration of A549 cells were measured using MTT, annexinV/7-AAD flow cytometry, and transwell migration assay, respectively. NDRG2 and matrix metalloproteinase-2 (MMP-2) expression were measured using real time-PCR. Results: pAdenoVator-Sur-P-NDRG2-IRES-GFP transfection resulted in a huge GFP expression in A549 cells, but not in LX-2 cells. The results of real time-PCR analysis also showed that pAdenoVator-Sur-P-NDRG2-IRES-GFP transfection led to an abundant NDRG2 expression in A549 cells. NDRG2 overexpression decreased A549 cell viability through increasing cell apoptosis. In addition, migration, invasion, and MMP-2 expression decreased following NDRG2 overexpression in A549 cells. Conclusion: The findings indicate that the targeted overexpression of NDRG2 using Sur-P can reduce the viability and invasiveness of A549 cells, suggesting possible benefits of this approach in lung cancer therapy.

The Evaluation of tLyP-1-Bound Mda-7/IL-24 Killing Activity on a Liver Tumor Cell Line.

Background: The melanoma differentiation-associated gene-7 (Mda-7)/interleukin-24 (IL-24) is a tumor killing cytokine, the bystander effect of which can be enhanced through tethering to tumor homing peptides (THPs). Materials and Methods: After fusing tLyP-1, RGR, and buforin as THPs to Mda-7/IL-24, enzyme-linked immunosorbent assay (ELISA) was used to determine the secretion potency of the recombinant proteins. The killing potency of plasmids expressing IL-24, IL-24.tLyP1, IL-24.RGR, and buf.IL-24 were assessed, using MTT, Annexin/PI staining assays, as well as measuring the expression level of GADD-153 and BCL2-associated X (BAX) on Huh-7 cells. Three-dimensional structural analysis and protein-receptor interaction were also evaluated by modeling. Results: The ELISA result showed that contrary to IL-24.RGR and buf.IL-24, IL-24.tLyP-1 retained the secretion potency, similar to the native form. The viability assessments showed that IL-24 and IL-24.tLyP-1 had the most growth suppressive effects in comparison with the control group (p < 0.0001). Furthermore, IL-24 and IL-24.tLyP-1 had the highest apoptotic activity and significant upregulatory effect on the GADD-153 and BAX genes (p < 0.0003). The modeling showed that peptide modifications left no detrimental effect on IL-24 attachment to the cognate receptor. Conclusion: IL-24 can tolerate tLyP-1 peptide modification by retaining its secretion potency. Tethering tLyP-1 to IL-24 can induce more apoptosis than its modified versions by RGR or buforin.

The Impact of Long-term Exposure to Low Levels of Inorganic Arsenic on the Hypomethylation of SEPT9 Promoter in Epithelial-Mesenchymal Transformed Colorectal Cancer Cell Lines.

Inorganic arsenicals are worldwide environmental contaminants that affect molecular characteristics in biological systems and lead to genomic and epigenomic instability as well as epithelial mesenchymal transition (EMT). In this study, we aimed to investigate whether low levels of sodium arsenite (iAsIII) can influence EMT and genomic instability through microsatellite analysis. We have also determined epigenomic instability by investigating the methylation status of SEPT9 tumor marker in colorectal cancer (CRC) cell lines, Caco2 and HCT116, which were treated with iAsIII to assess IC50s. Short-term and long-term exposure to low concentrations (1 µM and 0.1 µM) of iAsIII in two separate experiments was implemented to analyze EMT, microsatellite status and the methylation pattern of SEPT9 promoter. As expected, after 20 days of exposure to iAsIII, the expression of CDH1 was significantly decreased while the expression of CDH2, FIB1 and VIM was increased in Caco2 and HCT116, a finding that confirmed EMT induction. However, there was no detectable alteration in the size of microsatellites. As for the methylation pattern, SEPT9 promoter was hypomethylated as a result of long-term exposure to 0.1 µM iAsIII in Caco2. Long-term exposure of HCT116 to both concentrations could induce hypomethylation of SEPT9 promoter. Our findings indicate no linkage between EMT induction and microsatellite status in iAsIII-treated CRC cell lines. For the first time, the current study has shown that the induction of EMT by iAsIII is linked with SEPT9 promoter hypomethylation in Caco2 and HCT116 in a concentration- and time-dependent pattern.

Impact of sodium arsenite on chromosomal aberrations with respect to polymorphisms of detoxification and DNA repair genes.

Arsenic compounds can increase production of reactive oxygen species. Reactive oxygen species can induce double-strand breaks in DNA, which is a cause of chromosome aberrations (CAs). This study was conducted to determine the association between arsenic exposure and polymorphisms of genes involved in detoxification (glutathione S-transferase T1 [GSTT1], glutathione S-transferase M1 [GSTM1], glutathione S-transferase O2 [GSTO2], catalase [CAT], and NAD(P)H quinone oxidoreductase1 [NQO1]) as well as nonhomologous end joining DNA repair genes (XRCC4, XRCC5, and XRCC6) with induction of chromosomal aberrations. The participants consisted of 123 healthy males who were genotyped using polymerase chain reaction-based methods. Primary cultures of whole blood were treated with sodium arsenite (NaAsO(2); iAs(III); at final concentration 1 µmol/L), mitomycin C (at final concentration 60 ηg/mL; as positive control), or untreated. For each culture, mitotic index (MI), chromatid breaks (CBs), CAs, and total percentage of aberrant cells were determined. The levels of CB and percentage of aberrant cells were significantly higher in the TT genotype of CAT (C-262T polymorphism) than the CC genotype. The CB value in samples with GSTM1 active genotype was significantly higher than the null genotype. The MI in samples with TT genotype of NQO1 (C609T polymorphism) was significantly higher than MI in samples having CC and CT genotypes. There was no association between MI, CB, CA, and percentage of aberrant cells and polymorphisms of XRCC4, XRCC5, and XRCC6.