Arsenic sulfide inhibits cell migration and invasion of gastric cancer in vitro and in vivo

Abstract

Background: We previously showed that arsenic sulfide (As4S4) induced cell cycle arrest and apoptosis in several human solid tumor cell lines, including those of gastric cancer. In this study, we investigated the effect of As4S4 on the migration and invasion of gastric cancer cells both in vitro and in vivo.

Methods: The human gastric cancer cell lines AGS and MGC803 were selected as in vitro models. Wound-healing migration assay and Transwell invasion assay were carried out to determine the effects of As4S4 on cell migration and invasion. The expressions of E-cadherin, β-catenin, Sp1, KLF4, and VEGF were measured by Western blotting analysis. The activities of matrix metalloproteinase (MMP)-2 and MMP-9 in MGC803 cells were demonstrated by zymography assay. A mouse xenograft model was established by inoculation with MGC803 cells, then intraperitoneal injected with As4S4 for 3 weeks and monitored for body weight and tumor changes. Finally, the inhibition rate of tumor growth was calculated, and the expression of proteins and genes associated with tumor invasion and metastasis in tumor tissues were measured by immunohistochemistry, Western blotting, and real-time polymerase chain reaction assay.

Results: As4S4 significantly inhibited the migration and invasion of gastric cancer cell lines. The expression of E-cadherin and KLF4 was upregulated, while the expressions of β-catenin, VEGF, and Sp1 were downregulated following treatment with As4S4. Moreover, the protease activities of MMP-2 and MMP-9 were suppressed by As4S4 in MGC803 cells. Meanwhile, As4S4 effectively suppressed the abilities of tumor growth and invasion in the xenograft tumor model. We found that As4S4 upregulated the expression of E-cadherin and downregulated the expression of β-catenin, Sp1, VEGF, and CD34 in mouse tumor tissues, consistent with the results in vitro.

Conclusion: As4S4 inhibited the migration and invasion of gastric cancer cells by blocking tumor cell adhesion, decreasing the ability of tumor cells to destroy the basement membrane, and therefore suppressing their angiogenesis.

Keywords: As4S4; E-cadherin; MMPs; VEGF; realgar; xenograft.

Figure 1

The effect of As₄S₄ on gastric cancer cell migration. Notes: (A) MGC803 cells were scratched and treated with 0, 0.25, 0.5, and 1 μM As₄S₄ for 0, 24, and 48 hours. The migration was observed under a phase-contrast microscope at a magnification of 50×. (B) The area of MGC803 cells migration into the scratched area after treatment with As₄S₄ was calculated as a percentage of the wound area at 0 hours. (C) The migration analysis of MGC803 cells after treatment with As₄S₄ for 48 hours. (D) AGS cells were scratched and treated with 0, 0.25, 0.5, and 1 μM As₄S₄ for 0 and 24 hours. The migration was observed under a phase-contrast microscope at a magnification of 50×. (E) The migration analysis of AGS cells after treatment with As₄S₄ for 24 hours. Data represent the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Figure 2

The effect of As₄S₄ on gastric cancer cell invasion. Notes: (A) MGC803 cells were cultured in the upper part of a Transwell chamber coated with Matrigel. After treatment with 0, 0.5, and 1 μM As₄S₄ for 12 and 24 hours, cells at the bottom of the chamber were observed under a phase-contrast microscope at a magnification of 200×. (B) The number of MGC803 cells that invaded the underside of the porous polycarbonate after treatment with As₄S₄ for 12 and 24 hours was calculated as a percentage of the invasion at 0 μM. Data represent the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Figure 3

Effect of As₄S₄ on the activity of MMP-2/9 and the expression of proteins associated with invasion and metastasis in gastric cancer cells. Notes: (A) Gelatin zymography assay results about the activity of MMP-2 and MMP-9 secreted by MGC803 cells when treated with 0, 0.25, 0.5, 1, 2, and 4 μM As₄S₄ for 24 hours. (B) Western blotting results of MGC803 cells treated with 0, 0.25, 0.5, 1, and 2 μM As₄S₄ for 24 hours. β-actin was used as a loading control. (C) Protein quantification of the Western blotting results shown in (B). These data are representative of at least three independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Figure 4

Effect of As₄S₄ on the expression of proteins associated with invasion and metastasis in xenograft mice. Notes: (A) Total protein from the tumor was extracted, and the expressions of E-cadherin, VEGF, and Sp1 protein were detected by Western blotting. β-actin was used as a loading control. (B) Protein quantification of the Western blotting results shown in (A). (C) Relative expressions of mRNA levels of E-cadherin, β-catenin, MMP-2, MMP-9, Sp1, VEGF, and CD34 in tumor tissues. (D) The expression of E-cadherin, β-catenin, VEGF, and CD34 in tumor tissues was detected by immunohistochemical assay under a light microscope at a magnification of 200×. These data are representative of at least three independent experiments. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: CTX, cyclophosphamide; NS, normal saline.

Figure 4

Effect of As₄S₄ on the expression of proteins associated with invasion and metastasis in xenograft mice. Notes: (A) Total protein from the tumor was extracted, and the expressions of E-cadherin, VEGF, and Sp1 protein were detected by Western blotting. β-actin was used as a loading control. (B) Protein quantification of the Western blotting results shown in (A). (C) Relative expressions of mRNA levels of E-cadherin, β-catenin, MMP-2, MMP-9, Sp1, VEGF, and CD34 in tumor tissues. (D) The expression of E-cadherin, β-catenin, VEGF, and CD34 in tumor tissues was detected by immunohistochemical assay under a light microscope at a magnification of 200×. These data are representative of at least three independent experiments. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: CTX, cyclophosphamide; NS, normal saline.

Figure 4

Effect of As₄S₄ on the expression of proteins associated with invasion and metastasis in xenograft mice. Notes: (A) Total protein from the tumor was extracted, and the expressions of E-cadherin, VEGF, and Sp1 protein were detected by Western blotting. β-actin was used as a loading control. (B) Protein quantification of the Western blotting results shown in (A). (C) Relative expressions of mRNA levels of E-cadherin, β-catenin, MMP-2, MMP-9, Sp1, VEGF, and CD34 in tumor tissues. (D) The expression of E-cadherin, β-catenin, VEGF, and CD34 in tumor tissues was detected by immunohistochemical assay under a light microscope at a magnification of 200×. These data are representative of at least three independent experiments. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: CTX, cyclophosphamide; NS, normal saline.