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. 2012 Dec;130(2):269-80.
doi: 10.1093/toxsci/kfs242. Epub 2012 Aug 7.

Ethanol enhances tumor angiogenesis in vitro induced by low-dose arsenic in colon cancer cells through hypoxia-inducible factor 1 alpha pathway

Lei Wang  1 , Young-Ok Son, Songze Ding, Xin Wang, John Andrew Hitron, Amit Budhraja, Jeong-Chae Lee, Qinchen Lin, Pratheeshkumar Poyil, Zhuo Zhang, Jia Luo, Xianglin Shi
Affiliations

Ethanol enhances tumor angiogenesis in vitro induced by low-dose arsenic in colon cancer cells through hypoxia-inducible factor 1 alpha pathway

Lei Wang et al. Toxicol Sci. 2012 Dec.

Retraction in

Abstract

Health effects due to environmental exposure to arsenic are a major global health concern. Arsenic has been known to induce carcinogenesis and enhance tumor development via complex and unclear mechanism. Ethanol is also a well-established risk factor for many malignancies. However, little is known about the effects of coexposure to arsenic and ethanol in tumor development. In this study, we investigate the signaling and angiogenic effect of coexposure of arsenic and ethanol on different colon cancer cell lines. Results show that ethanol markedly enhanced arsenic-induced tumor angiogenesis in vitro. These responses are related to intracellular reactive oxygen species (ROS) generation, NADPH oxidase activation, and upregulation of PI3K/Akt and hypoxia-inducible factor 1 alpha (HIF-1α) signaling. We have also found that ethanol increases the arsenic-induced expression and secretion of angiogenic signaling molecules such as vascular endothelial growth factor, which further confirmed the above observation. Antioxidant enzymes inhibited arsenic/ethanol-induced tumor angiogenesis, demonstrating that the responsive signaling pathways of coexposure to arsenic and ethanol are related to ROS generation. We conclude that ethanol is able to enhance arsenic-induced tumor angiogenesis in colorectal cancer cells via the HIF-1α pathway. These results indicate that alcohol consumption should be taken into consideration in the investigation of arsenic-induced carcinogenesis in arsenic-exposed populations.

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Figures

FIG. 1.
FIG. 1.
Exposure to low concentrations of arsenic combined with ethanol exhibits low toxicity in colon cancer cells but high toxicity in normal colon cells. (A–C) DLD-1, HCT116, and CRL-1807 cells were exposed to arsenic (As), 0.4% ethanol (EtOH), or arsenic combined with ethanol at indicated concentrations for 24h, after which an MTT assay was performed. (D) Colony formation assays were carried out with DLD-1 cells exposed to arsenic and/or ethanol for 6 days at the indicated concentrations. The quantitative results were shown by manual counting of colonies.
FIG. 2.
FIG. 2.
Low-dose arsenic combined with ethanol induces ROS generation in colon cancer cells. (A) DLD-1 cells were exposed to arsenic and/or ethanol at indicated concentrations for 24h and then stained with 10μM H2DCFDA or 5μM DHE, respectively for 30min. Cells were imaged by fluorescence microscopy. (B). Cells were incubated with H2DCFDA or oxidized DCFDA. The fluorescence in cells was measured with a fluorescence microplate reader. The ratios of H2DCFDA/DCFDA were calculated between different exposure groups. (C) DLD-1 cells were exposed to arsenic and/or ethanol at the indicated concentrations with or without 500U/ml SOD for 24h and then stained with 5μM DHE. Cells were imaged by fluorescence microscopy. (D) Cells were exposed to arsenic and/or ethanol with and without 500U/ml catalase, then incubated with H2DCFDA or oxidized DCFDA. The fluorescence in cells was measured with fluorescence microplate reader.
FIG. 3.
FIG. 3.
Low-dose arsenic in combination with ethanol induces NADPH oxidase in colon cancer cells. DLD-1 and HCT116 cells were exposed to arsenic and/or ethanol at indicated concentrations for 24h. Western blot analysis was carried out using anti-p22phox, anti-p47phox, anti-p67phox, anti-NOX1, anti-NOX2, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies.
FIG. 4.
FIG. 4.
Low-dose arsenic in combination with ethanol activates PI3K/Akt signaling in colon cancer cells. (A) DLD-1 cells were exposed to arsenic or ethanol either alone or combined at the indicated concentrations in a time course, after which Western blot assays were carried out using anti-PI3K (Y458), anti-PI3K, anti-Akt (Y308), anti-Akt (S473), anti-Akt, anti-GSK3β (S9), anti-GSK3β and anti-GAPDH antibodies. (B) Densitometry analysis of expression level from Western blot as in (A). (C) DLD-1 cells were treated as described in (A) for 24h. Western blot assays were performed. (D). Densitometry analysis of expression level from Western blot as in (C).
FIG. 5.
FIG. 5.
Low-dose arsenic in combination with ethanol induces HIF-1α activity and enhances the expression of its target genes. (A) pHIF-1α-luciferase vector, pNF-κB-luciferase vector, and sup-Top-luciferase vector each were transfected individually into DLD-1 cells. The luciferase activity of cell lysates was measured. (B) Western blot assays were performed on cytoplasmic and nuclear extracts from treated DLD-1 cells to show HIF-1α translocation. (C) Western blot analysis was carried out using anti-VEGF, anti-MMP2, anti-Cox-2, and anti-REDD antibodies. (D) VEGF secretion in medium collected from treated DLD-1 cells was detected by ELISA.
FIG. 6.
FIG. 6.
Low-dose arsenic in combination with ethanol enhances tumor angiogenesis. (A) Arsenic and/or ethanol exposed DLD-1 medium induced HUVEC migration in Boyden chamber assay. (B) Cells migrated through the membrane as shown in (A) were quantified by manual counting. (C) HUVEC migration in wound healing migration assay. (D) Migrated HUVEC shown in (C) were quantified by manual counting. (E) Medium from DLD-1 cells treated with arsenic and/or ethanol induced tube formation of HUVEC. (F) Branch points in (E) were quantified by manual counting. (G) Catalase or SOD suppresses arsenic and/or ethanol-induced tube formation of HUVEC. (H). Branch points in (G) were quantified by manual counting. (I) MnTBAP or HIF-1 inhibitor LW6 suppresses arsenic and/or ethanol-induced tube formation of HUVEC. (J) Branch points in (I) were quantified by manual counting. (K) In vivo Matrigel plug assays using medium from DLD-1 cells treated with arsenic and/or ethanol.
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