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. 2018 May 1:20:10.
doi: 10.1186/s12575-018-0073-x. eCollection 2018.

Blockade of ITGA2 Induces Apoptosis and Inhibits Cell Migration in Gastric Cancer

Yu-Chang Chuang #  1 , Hsin-Yi Wu #  2 , Yu-Ling Lin  1   3 , Shey-Cherng Tzou  1   2 , Cheng-Hsun Chuang  2 , Ting-Yan Jian  2 , Pin-Rong Chen  2 , Yuan-Ching Chang  4 , Chi-Hsin Lin  5 , Tse-Hung Huang  6   7   8 , Chao-Ching Wang  6 , Yi-Lin Chan  9 , Kuang-Wen Liao  1   2   10   11   12
Affiliations

Blockade of ITGA2 Induces Apoptosis and Inhibits Cell Migration in Gastric Cancer

Yu-Chang Chuang et al. Biol Proced Online. .

Abstract

Background: Gastric cancer is currently the fourth leading cause of cancer-related death worldwide. Gastric cancer is often diagnosed at advanced stages and the outcome of the treatment is often poor. Therefore, identifying new therapeutic targets for this cancer is urgently needed. Integrin alpha 2 (ITGA2) subunit and the beta 1 subunit form a heterodimer for a transmembrane receptor for extracellular matrix, is an important molecule involved in tumor cell proliferation, survival and migration. Integrin α2β1 is over-expressed on a variety of cancer cells, but is low or absent in most normal organs and resting endothelial cells.

Results: In this report, we assessed the ITGA2 as the potential therapeutic target with the bioinformatics tools from the TCGA dataset in which composed of 375 gastric cancer tissues and 32 gastric normal tissues. According to the information from the Cancer Cell Line Encyclopedia (CCLE) database, the AGS cell line with ITGA2 high expression and the SUN-1 cell line with low expression were chosen for the further investigation. Interestingly, the anti-ITGA2 antibody (at 3 μg/ml) inhibited approximately 50% survival of the AGS cells (over-expressed ITGA2), but had no effect in SNU-1 cells (ITGA2 negative). The extents of antibody-mediated cancer inhibition positively correlated with the expression levels of the ITGA2. We further showed that the anti-ITGA2 antibody induced apoptosis by up-regulating the RhoA-p38 MAPK signaling to promote the expressions of Bim, Apaf-1 and Caspase-9, whereas the expressions of Ras and Bax/Bcl-2 were not affected. Moreover, blocking ITGA2 by the specific antibody at lower doses also inhibited cell migration of gastric cancer cells. Blockade of ITGA2 by a specific antibody down-regulated the expression of N-WASP, PAK and LIMK to impede actin organization and cell migration of gastric cancer cells.

Conclusions: Here, we showed that the mRNA expression levels of ITGA2 comparing to normal tissues significantly increased. In addition, the results revealed that targeting integrin alpha 2 subunit by antibodies did not only inhibit cell migration, but also induce apoptosis effect on gastric cancer cells. Interestingly, higher expression level of ITGA2 led to significant effects on apoptosis progression during anti-ITGA2 antibody treatment, which indicated that ITGA2 expression levels directly correlate with their functionality. Our findings suggest that ITGA2 is a potential therapeutic target for gastric cancer.

Keywords: Apoptosis; Cell migration; Gastric cancer; ITGA2; Integrin alpha-2; Proliferation; Therapeutic target.

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Conflict of interest statement

Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Analysis of ITGA2 expression levels in gastric cancers. a Data of ITGA2 mRNA levels were extracted from 32 normal gastric tissue and 375 gastric cancer tissue in the TCGA datasets. The result was expressed as mean ± standard deviation (SD), and statistical comparisons were made by Wilcoxon signed rank test. **** p < 0.0001. b The mRNA levels of ITGA2 in 27 available paired gastric normal and tumor tissues were compared, and a positive log2 (tumor/normal) value indicates increased expression, while a negative log2 value indicates decreased expression, of ITGA2 in the gastric tumor tissues. ITGA2 mRNA was significantly overexpressed in gastric tumor tissues as compared with normal tissues (p < 0.01, paired t-test). Result was expressed as mean ± standard deviation (SD). c mRNA expression profiles for ITGA2 across gastric cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) database. Red arrowheads mark the expression levels of AGS and SUN-1 cells. d The expression of ITGA2 mRNA was measured by quantitative real-time PCR in AGS and SNU-1 cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. e The ITGA2 protein expression was semi-quantified by western blot analyses. d and e The densitometric measurements of ITGA2 in AGS and SNU-1 cells were normalized to the internal control (β-actin). f The surface expressions of ITGA2 were determined on PI-negative cells, using flow cytometry. Data are representative of three independent experiments
Fig. 2
Fig. 2
The anti-ITGA2 antibody induced apoptosis in AGS cells but not SUN-1 cells. a Human gastric cancer cell lines AGS and SUN-1 were treated with different concentrations of an anti-ITGA2 antibody for 48 h, and cell survivals were measured by an MTT assay. An isotype control antibody was used as a negative control. b AGS cells were treated with mitomycin C as a positive control, or incubated with 0.3 μg of anti-ITGA2 antibodies or isotype control (negative control) for 48 h. Cells were stained with PE-conjugated anti-active caspase-3 antibodies, and analyzed by flow cytometry. The percentages of caspase-3 positive-staining cells summarized in the right panel, showing mean ± standard deviation (S.D). Statistical comparisons were made by one-way ANOVA or two-way ANOVA with Bonferroni comparisons. * p < 0.05 and **** p < 0.0001. Data are representative of three independent experiments
Fig. 3
Fig. 3
The anti-ITGA2 antibody induced a RhoA-p38 MAPK-mediated apoptotic pathway in AGS cells. a mRNA expression of Ras, RhoA, Bax, p38, Bim, Bcl-2, Apaf-1 and Caspase-9 in AGS cells treated with 0.3 μg of anti-ITGA2 antibodies or isotype control antibodies (negative control) for 48 h were analyzed by RT-PCR. Each analysis was derived from the same experiment and gels were processed in parallel. Cropped gels are displayed to compare gene expressions in different treatment groups. b Densitometric measurements on the intensities of each RT-PCR product was normalized to the mRNA level of GAPDH, and displayed as mean ± standard deviation (S.D). Statistical comparisons were made by two-way ANOVA with Bonferroni comparisons. ** p < 0.01, **** p < 0.0001. Data are representative of three independent experiments. c Summary of the anti-ITGA2 antibody-mediated apoptosis signaling pathway in AGS gastric cancer cells
Fig. 4
Fig. 4
Blockade of ITGA2 reduced migration of AGS cells. a AGS cells were treated with 0.1 μg anti-ITGA2 antibodies or isotype control antibodies (negative control) for 18 h. Cells in the lower face of transwell membranes were stained by PI and imaged (upper panel) and data summarized as mean ± standard deviation (S.D) (lower panel). Statistical comparisons were made by two-way ANOVA with Bonferroni comparisons. *** p < 0.001. b AGS cells were treated with 0.3 μg of the anti-ITGA2 antibody for 12 h. F-actin (green) was stained with FITC-conjugated phalloidin, and nuclei stained by DAPI. Scale bar = 10 μm. Data are representative of three independent experiments
Fig. 5
Fig. 5
Anti-ITGA2 antibody reduced cell migration of AGS cells through down-regulation of Rac1 and CDC42 signaling pathway. a Gene expression of LIMK, Rac1, PAK, CDC42 and N-WASP in AGS after treatment with 0.1 μg anti-ITGA2 antibodies or isotype control antibodies (negative control) for 18 h. Each analysis was derived from the same experiment and gels were processed in parallel. Cropped gels are displayed for comparison between gene expressions in different groups. b Densitometric measurements on the intensities of each RT-PCR product was normalized to the mRNA level of GAPDH, and displayed as mean ± standard deviation (S.D). Statistical comparisons were made by two-way ANOVA with Bonferroni comparisons. ** p < 0.01 and **** p < 0.0001. c Summary of the anti-ITGA2 antibody-mediated migration signaling pathway in AGS gastric cancer cells
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