The effect of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) on cell viability under hypoxia - PubMed (original) (raw)

. 2013 Nov 16:19:2260-73.

eCollection 2013.

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The effect of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) on cell viability under hypoxia

Leo Tsui et al. Mol Vis. 2013.

Abstract

Purpose: The synthetic compound 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) reduces the protein stability of hypoxia-inducible factor (HIF)-1α and can serve as a potential anticancer agent. Our previous study elucidated that YC-1 decreased the protein level of HIF-1α and inhibited cell proliferation under normoxic conditions. In the present study, we explored the inhibitory effect of YC-1 on the regulation of HIF-1α and cell survival under hypoxia.

Methods: Chemical and physical hypoxia using cobalt chloride and an anaerobic incubator, respectively, was induced in the photoreceptor cell line 661W. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and morphological observation were used to analyze cell survival. Flow cytometry with a LIVE/DEAD cell viability assay and annexin V was used to determine the number of live and dead cells or cell apoptosis, respectively. Cell proliferation was analyzed with high-content screening of MKI67 (K(i)-67) immunofluorescent staining. Immunoblotting and a quantitative reverse-transcription PCR were used to assess the protein and mRNA levels, respectively.

Results: Our results showed that 661W cells exposed to YC-1 decreased cell survival through the induction of cell apoptosis and cell-cycle arrest under hypoxia. We also found that YC-1 reduced the HIF-1α protein level after 2 h of hypoxia, but the mRNA level of HIF-1α was not affected. In addition, YC-1 significantly increased levels of p53, the proapoptotic gene BCL2-associated X protein (Bax), and cell proliferation-related gene, cyclin-dependent kinase inhibitor 1A (p21) mRNAs under hypoxia.

Conclusions: Unlike normoxia, YC-1 not only inhibited cell proliferation but also induced cell death under hypoxia. We also found that YC-1 inhibited hypoxia-induced HIF-1α and partially affected hypoxia-regulated gene expression.

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Figures

Figure 1

Figure 1

3-(5′-hydroxymethyl-2’-furyl)-1-benzylindazole (YC-1) decreased cell survival in a dose- and time-related manner under chemical hypoxia. A: 661W cells were treated with 25, 50, 100, 200, and 400 μM CoCl2 for 24 h, and cell viability was measured with an MTT assay (n=4). B: The viability of 661W cells in the presence of 0.066% DMSO or 20 μM YC-1 for 24 h during chemically induced hypoxia (by 200 μM CoCl2) was evaluated using an MTT assay (n=4). C: Morphological changes in cell density were observed with light microscopy. The black arrows indicate the cell debris. D: Concentration-dependent reduction in cell survival by YC-1 during chemical hypoxia. 661W cells were exposed to 5, 10, 20, and 40 μM YC-1 for 5 min, followed by 200 μM CoCl2 for 24 h (n=4). E: Time-dependent curves of cell survival in response to YC-1 in hypoxia. 661W cells were incubated with 200 μM CoCl2 in the absence or presence of 20 μM YC-1 for 1, 4, 12, and 24 h (n=4). * Indicates p<0.05 compared to the control group or the DMSO vehicle group; # indicates p<0.05 compared to the chemically induced hypoxic group.

Figure 2

Figure 2

LIVE/DEAD cell viability assay of YC-1-treated cells under chemical hypoxia. A: After treatment with 200 μM CoCl2 in the absence or presence of 20 μM YC-1 for 24 h, 661W cells were stained with calcein AM (green for live cells), EthD-1 (red for dead cells), and Hoechst 33,342 (blue for nuclei). Representative fluorescence staining shows the cell density and composition. B: 661W cells were exposed to 0.066% DMSO, 20 μM YC-1, 200 μM CoCl2, or both YC-1 and CoCl2 for 24 h. After fluorescence staining, cells was measured and analyzed with flow cytometry. C: Quantitative data of flow cytometric analysis are from three independent experiments (n=3). * Indicates p<0.05 compared to the control group or DMSO vehicle group; # indicates p<0.05 compared to the chemically induced hypoxic group.

Figure 3

Figure 3

High-content screening (HCS) of YC-1-treated cells under chemical hypoxia. 661W cells were exposed to 0.066% DMSO, 20 μM YC-1, 200 μM CoCl2, or both YC-1 and CoCl2 for 24 h. After fixation and immunofluorescence staining, cells were detected and analyzed using a Cellomics ArrayScan VTI HCS Reader. A: Representative fluorescent photographs show the distribution of Ki-67 (green) and Hoechst-labeled nuclei (blue) using the Columbus Image Data Storage and Analysis System. Panels (B) and (C) show quantified HCS results for the number and ratio of Ki-67-stained nuclei (n=4). * Indicates p<0.05 compared to the control group or the DMSO vehicle group; # indicates p<0.05 compared to the chemically induced hypoxic group.

Figure 4

Figure 4

YC-1 increased cell apoptosis under chemical hypoxia. A: After treatment with 0.066% DMSO or 200 µM CoCl2 in the absence or presence of 20 µM YC-1 for 24 h, 661W cells were stained with annexin V (for apoptotic cells) and propidium iodide (for necrotic cells) and measured and analyzed by flow cytometry. B: The frequency of apoptosis was quantified from three independent experiments (n=3). * Indicates p<0.05 compared to DMSO vehicle group; # indicates p<0.05 compared to the chemically induced hypoxic group.

Figure 5

Figure 5

Effect of YC-1 on HIF-1α protein expression under chemical hypoxia. Total proteins were extracted from 661W cells incubated with 0.066% DMSO, 20 µM YC-1, 200 µM CoCl2, or both YC-1 and CoCl2 for 2 h (A) and 24 h (B). Expressions of HIF-1α and β-actin (which served as the internal control) were determined with immunoblotting.

Figure 6

Figure 6

Quantitative reverse-transcription PCR (qRT-PCR) analysis of related genes in response to YC-1 under chemical hypoxia. 661W cells were incubated with 0.066% DMSO, 20 μM YC-1, 200 μM CoCl2, or both YC-1 and CoCl2 for 2 h or 24 h. After mRNA was extracted and reverse transcription was performed, relative HIF-1α (A), VEGF (B), glucose transporter 1 (GLUT1; C), carbonic anhydrase 9 (Car9; D), NF-κB (E), p53 (F), Bax (G), Bcl2 (H), Apaf1 (I), and cyclin-dependent kinase inhibitor 1A (p21; J) mRNAs were measured and compared to β-actin mRNA with qRT-PCR (n=3). * Indicates p<0.05 compared to the control group or the DMSO vehicle group; # indicates p<0.05 compared to the chemically induced hypoxic group; + indicates p<0.05 compared to the YC-1-treated group. D, DMSO; Y, YC-1; C, CoCl2; Y+C, both YC-1 and CoCl2.

Figure 7

Figure 7

YC-1 reduced cell survival under physical hypoxia. A: 661W cells were exposed to 0.066% DMSO or 20 μM YC-1 for 24 h under normoxia or hypoxia (0.5% O2, 5% CO2). Morphological changes in cell density were observed with light microscopy. Panel (B) is the quantified cell viability determined with an MTT assay (n=4). Panel (C) shows a concentration-dependent cell survival curve of YC-1 during hypoxia (n=4). 661W cells were incubated with 5, 10, 20, and 40 μM YC-1 for 5 min, followed by exposure to physical hypoxia for 24 h. * Indicates p<0.05 compared to the control group or the DMSO vehicle group; # indicates p<0.05 compared to the hypoxic group.

Figure 8

Figure 8

Summary of the effects of YC-1 on 661W cell viability under hypoxia.

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