Arsenite-induced pseudo-hypoxia results in loss of anchorage-dependent growth in BEAS-2B pulmonary epithelial cells.

Epidemiology studies have established a strong link between lung cancer and arsenic exposure. Currently, the role of disturbed cellular energy metabolism in carcinogenesis is a focus of scientific interest. Hypoxia inducible factor-1 alpha (HIF-1A) is a key regulator of energy metabolism, and it has been found to accumulate during arsenite exposure under oxygen-replete conditions. We modeled arsenic-exposed human pulmonary epithelial cells in vitro with BEAS-2B, a non-malignant lung epithelial cell line. Constant exposure to 1 µM arsenite (As) resulted in the early loss of anchorage-dependent growth, measured by soft agar colony formation, beginning at 6 weeks of exposure. This arsenite exposure resulted in HIF-1A accumulation and increased glycolysis, similar to the physiologic response to hypoxia, but in this case under oxygen-replete conditions. This "pseudo-hypoxia" response was necessary for the maximal acquisition of anchorage-independent growth in arsenite-exposed BEAS-2B. The HIF-1A accumulation and induction in glycolysis was sustained throughout a 52 week course of arsenite exposure in BEAS-2B. There was a time-dependent increase in anchorage-independent growth during the exposure to arsenite. When HIF-1A expression was stably suppressed, arsenite-induced glycolysis was abrogated, and the anchorage-independent growth was reduced. These findings establish that arsenite exerts a hypoxia-mimetic effect, which plays an important role in the subsequent gain of malignancy-associated phenotypes

Arsenite-induced phenotypic changes in BEAS-2B.

<p>A) Representative images of soft agar growth over the course of 52 weeks of constant arsenite (1 µM) exposure. B) Colony counts in soft agar. Bars represent mean, 1 standard deviation, from 3 experimental replicates. C) Immunoblot analysis of HIF-1A and E-cadherin (E-cad) in BEAS-2B over the course of 52 weeks of constant arsenite (1 µM) exposure. D) Lactate levels (percent control) in BEAS-2B over the course of 52 weeks of constant arsenite (1 µM) exposure. Absolute lactate production in vector control: 0.733±0.017 µmol/10⁶cells/hr) Bars represent mean +1 standard deviation, from 3 experimental replicates. E) Percentage aneuploid cells in BEAS-2B treated with 1 µM arsenite for 0–52 weeks. Bars represent mean, +1 standard deviation, from 3 experimental replicates. *p<0.05.</p

Effect of suppressed HIF-1A expression on arsenite mediated transformation.

<p>A) Immunoblot analysis of HIF-1A knockdown in BEAS-2B, short immunoblot exposure shown for MG132-treated samples; long immunoblot exposure shown for MG132-untreated samples. B) QPCR for HIF-1A mRNA. Bars represent mean, +1 standard deviation, from 5 experimental replicates. C) Lactate levels (percent control) in arsenite-exposed (denoted “As”, exposed for 8 weeks) and unexposed control (denoted “Ct”) BEAS-2B stably transfected with scrambled control shRNA (denoted “Vector”) or with shRNA targeting HIF1A (denoted “shHIF1A”) expression. Absolute lactate production in vector control: 0.696±0.04 µmol/10⁶cells/hr). Bars represent mean, +1 standard deviation, from 3 experimental replicates. D) Colony count of soft agar assay from BEAS-2B cells treated as described above in panel C. Bars represent mean, +1 standard deviation, from 3 experimental replicates. *p<0.05.</p

Arsenite causes HIF-1A accumulation/translocation in BEAS-2B.

<p>A) Immunoblot analysis of HIF-1A in BEAS-2B treated with 0–8 µM arsenite for 48 hours. B) Immunoblot analysis of HIF-1A in BEAS-2B treated with 1 µM arsenite for 0–48 hours. C) Immunoblot analysis of nuclear and cytosolic fractions of BEAS-2B, control or treated with 1 µM arsenite for 2 weeks, probed for HIF-1A, Lamin A (a nuclear marker) and tubulin (a cytosolic marker). D) Immunofluorescence staining of HIF-1A in BEAS-2B, control or treated with 1 µM arsenite for 2 weeks, arrows show HIF-1A nuclear accumulation. E) QPCR of HIF-1A mRNA in BEAS-2B treated with 1 µM arsenite for 0–4 weeks, bars represent mean, 1 standard deviation. F) Half-life measurement of HIF-1A in BEAS-2B, control or treated with 1 µM arsenite for 2 weeks, protein synthesis blocked with cycloheximide (CHX) for 0–10 min, followed by HIF-1A immunoblot. G) Quantification of HIF-1A protein half-life (t1/2). Densitometry of HIF-1A normalized to Tubulin was used for calculation. Points represent mean, +/− 1 standard deviation, 3 independent replicates. *p<0.05.</p

Glycolysis induction by HIF-1A overexpression in BEAS-2B.

<p>A) Immunoblot analysis of HIF-1A in BEAS-2B, vector control and transiently transfected with degradation-resistant HIF-1A mutant. B) Lactate levels (percent vector control) in cells described in 2A (Absolute lactate production in vector control: 0.729±0.054 µmol/10⁶cells/hr). Bars represent mean, 1 standard deviation, from 3 independent replicates. *p<0.05. C) Intracellular metabolite concentration (percent control BEAS-2B) of 1 µM arsenite-exposed (2 weeks) BEAS-2B cells. Bars represent mean, 1 standard deviation, from 4 experimental replicates. For each metabolite, levels in arsenite-exposed BEAS-2B are significantly different compared to control (p<0.05).</p