Abstract
Digoxin, an inhibitor of Na+/K+ ATPase, has been used in the treatment of heart-related diseases (such as congestive heart failure and atrial arrhythmia) for decades. Recently, it was reported that digoxin is also an effective HIF-1α inhibitor. We investigated whether digoxin could suppress tumor cell growth through HIF-1α in non-small cell lung cancer cells (A549 cells) under hypoxic conditions. An MTT assay was used to measure cell viability. RT-PCR and western blotting were performed to analyze the mRNA and protein expression of VEGF, NDRG1, and HIF-1α. HIF-1α nuclear translocation was then determined by EMSA. Digoxin was found to inhibit the proliferation of A549 cells under hypoxic conditions. Our results showed that hypoxia led to the upregulation of VEGF, NDRG1, and HIF-1α both at the mRNA and protein levels. We also found that the hypoxia-induced overexpression of VEGF, NDRG1, and HIF-1α was suppressed by digoxin in a concentration-dependent manner. As expected, our EMSA results demonstrated that under hypoxic conditions HIF-1α nuclear translocation was also markedly reduced by digoxin in a concentration-dependent manner. Our results suggest that digoxin downregulated hypoxia-induced overexpression of VEGF and NDRG1 at the transcriptional level probably through the inhibition of HIF-1α synthesis in A549 cells.
Highlights
Lung cancer is the most commonly diagnosed forms of cancer and is the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) constituting approximately 80% of primary bronchial lung cancers [1,2].The cellular concentration of oxygen plays a critical role in regulating the expression of 50–500 or more genes involved in cell proliferation, angiogenesis, modulation, glucose metabolism, survival, and invasion in solid tumors during tumor progression and metastasis [3,4]
Our MTT results showed that under hypoxic conditions, the viability of A549 cells was reduced by digoxin in a time- and concentration-dependent manner (Figure 1), whereas the cell viabilities under normoxic conditions were markedly higher than those under hypoxia (Figure 1)
Digoxin Attenuates mRNA and Protein Expression of Vascular endothelial growth factor (VEGF) and N-Myc downregulated gene 1 (NDRG1) in A549 Cells mRNA and protein expression levels of VEGF and NDRG1 were evaluated by RT-PCR and western blotting
Summary
Lung cancer is the most commonly diagnosed forms of cancer and is the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) constituting approximately 80% of primary bronchial lung cancers [1,2]. Transcription factor hypoxia-inducible factor-1 (HIF-1) is considered a critical regulator of the adaptation responses of tumor cells [7,8,9]. Under normoxia conditions, HIF-1α is continually degraded though ubiquitinylation by E3 ubiquitin-protein ligases This degradation mechanism is suppressed by hypoxia, resulting in the accumulation of HIF-1α in cell and translocation into the nucleus to bind to HIF-1β, forming the HIF-1α/HIF-1β heterodimer, HIF-1. It has been shown that HIF-1 is an important regulator of VEGF under hypoxic conditions in A549 cells [16]. We hypothesized that the hypoxia-induced overexpression of VEGF and NDRG1 would be downregulated by digoxin through the inhibition of HIF-1α in A549 cells
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