The evidence of HeLa cell apoptosis induced with tetraethylammonium using proteomics and various analytical methods

Abstract

Tetraethylammonium (TEA) is a potassium channel (KCh) blocker applied in the functional and pharmacological studies of the KChs. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, a colorimetric assay to quantitatively measure living cells, demonstrated that TEA reduced the HeLa cell viability dose-dependently. Flow cytometry analysis indicated an increased apoptosis rate of the HeLa cell after exposing to TEA. The patch clamp technique revealed that the K(+) current of the HeLa cell was inhibited up to 80% when exposed to TEA. In addition, quantitative real-time PCR approach set up cross-talk among the cytotoxicity of TEA, 4-aminopyridine, and anti-cancer drug such as cisplatin. Using comparative proteomics combined with MALDI-TOF MS/MS, 33 significantly changed proteins were found from TEA treatment group; among these proteins, 12 were up-regulated, and 21 were down-regulated. Here we indicated that these proteins were closely connected with many biological functions such as oxidative stress response, signal transduction, metabolism, protein synthesis, and degradation. Both Western blotting and quantitative real-time PCR approaches further verified these differential proteins. Ingenuity Pathways Analysis software, a tool to analyze "omics" data and model biological system, was applied to analyze the interaction pathways of these proteins. The subcellular locations of the differential proteins are also predicted from Uniprot. All results above can help in our understanding of the mechanism of TEA-induced cytotoxicity and provide potential cancer biomarkers. Various experimental results in this study (like those for cisplatin) indicated that TEA is not only a KCh blocker but also a potential anti-cancer drug.

Keywords: Apoptosis; Bioinformatics; Flow Cytometry; HeLa Cell Line; Patch Clamp Technology; Pharmacology; Proteomics; RT-qPCR; Tetraethylammonium; Western Blotting.

FIGURE 1.

Dose-response curve between TEA concentration and cell viability of HeLa cells. Cells were treated with different concentrations of TEA for 24, 36, and 48 h. Cell viability rates were determined by using MTT assay. The results are expressed as the percentage of the viability rate of control cells. All values are represented as the means ± S.D. of three independent experiments.

FIGURE 2.

Flow cytometry analysis of cell cycle distribution and apoptosis rate by TEA inducement of HeLa cell. A, cell phase analysis of HeLa cells using flow cytometry with PI staining. B, quantitative analysis of the distribution of cells in cell phases in the bar graph (% of cells in each phase relative to the total population). C, assessment of TEA-induced apoptosis and necrosis using flow cytometry with annexin V-FITC/PI staining. Lower left, annexin V−/PI− cells (normal); lower right, annexin V+/PI− cells (early apoptosis); upper right, annexin V+/PI+ cells (necrosis). All values are represented as the means ± S.D. of three independent experiments. * represents p < 0.05, ** represents p < 0.01.

FIGURE 3.

Voltage-activated outward K⁺ currents in HeLa cells and Kv2.1 current in CHO cells blocked by TEA. A, the whole-cell patch clamp was applied in recording HeLa cells of voltage-gated outward K⁺ currents. The current-voltage relation of K⁺ current was set up by depolarizing from −40 to 60 mV with 10-mV increment of each pulse. These outward K⁺ currents in the same cell were inhibited by external TEA (10 mm). B, the whole-cell patch clamp was applied in recording one CHO_kv2.1 cell of Kv2.1 current. These Kv2.1 currents in the same cells were inhibited by external TEA (10 mm). All values are represented as the means ± S.D. of seven independent experiments.

FIGURE 4.

Heatmap visualization of RT-qPCR analysis of mRNA levels in the HeLa cell. RT-qPCR was performed on cDNA using gene-specific primers for differential proteins induced by CDDP of the HepG2 cell. Relative quantification of each gene expression level was normalized according to the β-actin gene expression. First column, TEA (20 mm); second column, 4-AP (5 mm); third column, CDDP (2 μg/ml). Red represents up-regulation, and green represents down-regulation. Color intensity represents the mRNA expression values. Rows, mRNA; column, treatment.

FIGURE 5.

Representative two-dimensional electrophoresis maps indicating protein spots that had significant change after 20 mm TEA exposure in HeLa cells. Whole cell lysate proteins (150 μg) were separated by two-dimensional electrophoresis and visualized using silver staining. Arrows indicate the proteins with a significantly modified expression level after TEA treatment. A, control; B, 20 mm TEA. C, relative quantification of the differential expressed proteins.

FIGURE 6.

Magnified images of significantly different changed protein spots between the control and TEA-treated groups. A, differential proteins found in control group. B, differential proteins found in the TEA group.

FIGURE 7.

Pathway prediction of TEA induced HeLa cell cytotoxicity based on proteomic analysis. Differentially expressed proteins participate in RNA binding, oxidative stress, fatty acid metabolism, ion binding signal transduction, protein synthesis and degradation, and TCA cycle. Subcellular predictions and molecular functions of these proteins were constructed manually using UniProt database. The blue rectangles represent proteins located in cytoplasm, the pink rectangles represent proteins located in nucleus, and the purple rectangles represent proteins located in mitochondrion.

FIGURE 8.

Western blotting and RT-qPCR analysis of representative proteins and their corresponding mRNAs identified by two-dimensional electrophoresis gel. A, Western blotting analysis of GSTO1 and Rho-GDIα expression levels in the HeLa cell. Left panel, 45 μg of protein was loaded onto a 12% SDS-PAGE and probed with antibody against GSTO and Rho-GDIα. β-Actin and α-tubulin were also measured as the loading control and used for data normalization. Right panel, the quantification of the signal has been analyzed with densitometric scanning. All values are represented as the means ± S.D. of three independent experiments. * represents p < 0.05; ** represents p < 0.01. B, RT-qPCR analysis of mRNA level in the HeLa cell. RT-qPCR was performed on cDNA using gene specific primers. Relative quantification of each gene expression level was normalized according to the β-actin gene expression. The PCR data of treatments was calibrated to the control values (control = 1). All the spots show significant difference (p < 0.05) in mRNA expression levels where the TEA concentrations are set at 20 and 5 mm. Spot numbers labeled with black squares are expressed as significantly different between control and TEA treated groups (all in 1, 5, and 20 mm).

FIGURE 9.

Network analysis of differentially expressed proteins performed using the IPA software. The networks describing proteins affecting gene expression, cell death, and cellular development are presented. Proteins are represented as nodes. Nodes in black represent up-regulated proteins, whereas nodes in gray represent down-regulated proteins. No proteins represented by white nodes were observed. Direct interactions or regulation are connected by solid lines, whereas indirect effects mediated by additional proteins are connected by dashed lines.