metallothionein and Adenocarcinoma-of-Lung

Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomous growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Arsenic; Carcinogens; Cell Line, Tumor; Cells, Cultured; Epithelial Cells; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Metallothionein; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phenotype; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins

Although aberrant DNA methylation has been implicated in the pathophysiology of lung cancer, the role of methylation in multidrug resistance (MDR) of lung cancer has remained unclear. To investigate whether certain distinct DNA methylation pattern is associated with acquired MDR of lung adenocarcinoma, methylated-DNA immunoprecipitation-chromatin immunoprecipitation (MeDIP-ChIP) was utilised to compare the genome-wide promoter methylation of the human lung adenocarcinoma MDR A549/cisplatin (A549/DDP) cells with its progenitor A549 cells. The comparison identified 3617 genes with differentially methylated promoter, of which 1581 were hypermethylated and 2036 were hypomethylated. Then, bisulphite sequencing polymerase chain reaction (PCR) (BSP) and quantitative reverse transcription (RT)-PCR (Q-PCR) were used to validate the promoter methylation of five candidate genes and to determine whether the expression of genes was associated with the promoter methylation. BSP confirmed that the promoter methylation incidence of the hypermethylated genes, G protein-coupled receptor 56 isoform 3 (GPR56), metallothionein 1G (MT1G), and RAS association domain family gene 1 (RASSF1), was significantly higher in A549/DDP cells compared with A549 cells (p<0.001, p=0.0099, and p=0.0165), whereas no significant difference was found in that of the other two genes, CCNL2 and BAD (p=0.0594 and p=0.5546). Additionally, Q-PCR showed that the mRNA expression of the three hypermethylated genes was significantly lower in A549/DDP cells compared with A549 cells (all p<0.001). In conclusion, this study reported for the first time that a distinct promoter methylation pattern is associated with MDR of lung adenocarcinoma A549/DDP cells and suggested that GPR56, MT1G, and RASSF1 might be the potential methylation markers associated with acquired MDR of lung adenocarcinoma.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Cell Line, Tumor; DNA Methylation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Genome-Wide Association Study; Humans; Lung Neoplasms; Metallothionein; Promoter Regions, Genetic; Receptors, G-Protein-Coupled; Tumor Suppressor Proteins