AR-42: A Pan-HDAC Inhibitor with Antitumor and Antiangiogenic Activities in Esophageal Squamous Cell Carcinoma

Abstract

Purpose: Esophageal squamous cell carcinoma (ESCC) is a refractory malignancy with high morbidity and mortality. Thus, there is an urgent need to find effective targets and agents for ESCC treatment. The purpose of this study was to assess the anti-ESCC effects of a pan-histone deacetylase (HDAC) inhibitor AR-42 and its mechanisms of action.

Methods: Immunohistochemical staining was performed to detect HDAC1 expression in ESCC and adjacent tissue samples. MTT assay, Edu cell proliferation test, flow cytometry, and subcutaneous xenograft were used to assess the anti-ESCC effects of AR-42; furthermore, the antiangiogenic activity of AR-42 was evaluated using endothelial cell migration, invasion, and tube formation assays as well as zebrafish angiogenesis assay. Western blot analysis was performed to explore the underlying mechanism of the anti-ESCC activity of AR-42.

Results: HDAC1-positive expression was much higher in ESCC cells than in paracancerous tissues, and the elevated HDAC1 expression was a strong indicator of lymph node metastasis and a more advanced TNM stage of ESCC. Moreover, AR-42 potently suppressed ESCC cell growth through cellular proliferation inhibition and apoptosis induction. Moreover, AR-42 displayed a moderate antiangiogenic activity, and it could significantly inhibit the migration, invasion and tubulogenesis of human umbilical vein endothelial cells as well as intersegmental vessel formation in zebrafish at micromolar concentrations. More importantly, the inhibitory activity of AR-42 on ESCC cells and angiogenesis could also be observed in the TE-1 xenograft model. Further studies showed that AR-42 exerts its anti-ESCC effects mainly by upregulating the expression of p21 and blocking the transduction of multiple signaling cascades related to tumor growth, especially Stat3-mediated signaling.

Conclusion: Overall, AR-42 has significant potency for inhibiting ESCC cell growth and shows moderate effect in suppressing angiogenesis, displaying strong anti-ESCC effects in vitro and in vivo. Thus, AR-42 deserves further evaluation as a potential candidate for ESCC therapy.

Keywords: AR-42; angiogenesis; esophageal squamous cell cancer; histone deacetylase.

Figure 1

HDAC1 expression in human ESCC and ANTT. (A) HDAC1 immunohistochemical staining in ESCC cells and adjacent non-tumor tissues (ANTTs) (20×). (B) Statistical analysis of HDAC1-positive expression in ESCC and ANTT. Abbreviations: HDAC1, histone deacetylase 1; ESCC, esophageal squamous cell carcinoma; ANTT, adjacent non-tumor tissue.

Figure 2

AR-42 inhibits ESCC cell viability in vitro. (A) Chemical structure of AR-42. (B) The sensitivity of ESCC cells to AR-42 was detected using MTT test. Cell viability is presented as mean ± SD (n=3). (C) AR-42 inhibited the formation of Eca109 and TE-1 cell clones. Abbreviation: ESCC, esophageal squamous cell carcinoma.

Figure 3

Anti-proliferation and pro-apoptosis effects of AR-42 in vitro. (A) Cellular proliferation test was performed in ESCC cells after treatment with AR-42 (1 μM) for 18 h. (B) Annexin V/PI double staining was performed in ESCC cells after treatment with the indicated concentrations of AR-42 for 24 h. Apoptotic rate is quantified for statistics. ***P < 0.001 vs vehicle. Abbreviation: ESCC, esophageal squamous cell carcinoma.

Figure 4

Anti-angiogenesis effects of AR-42. (A) Scratch assay was performed to detect HUVEC migration after treatment with the indicated concentrations of AR-42. The migrating cells are quantified for statistics. (B) Transwell chamber test was performed to detect HUVEC invasion after treatment with the indicated concentrations of AR-42. The invading cells are quantified for statistics. (C) Tube formation assay was performed to detect the ability of HUVECs to aggregate into tubes after treatment with AR-42. Vessel branch points are quantified for statistics. (D) Anti-angiogenesis ability of AR-42 and the positive-control sorafenib in transgenic zebrafish model. The length of intersegmental vessels (ISVs) is quantified for statistics. Values are presented as mean ± SD (n=3); *P < 0.05 vs vehicle; **P < 0.01 vs vehicle; ***P < 0.001 vs vehicle. Abbreviations: HUVEC, human umbilical vein endothelial cell; ISV, intersegmental vessel.

Figure 5

Western blot analysis in Eca109 and TE-1 cells treated with AR-42.

Figure 6

In vivo anti-ESCC effects of AR-42. (A) Tumor inhibition curve of AR-42 in the TE-1 xenografts. Tumor volumes are presented as mean ± SD (n=5). (B) Weight monitoring in different treatment groups of TE-1 xenografts. Body weights are presented as mean ± SD (n=5). (C) TE-1 xenografts treated with vehicle and 50 mg/kg AR-42 were harvested for histone H3 (acetyl K9) immunostaining, Ki67 proliferating cell detection and CD31 staining. Scale bars represent 50 μm. (D) TE-1 xenografts treated with vehicle and 50 mg/kg AR-42 were harvested for TUNEL immunostaining. Scale bars represent 50 μm. Abbreviations: ESCC, esophageal squamous cell carcinoma; TUNEL, TdT-mediated dUTP Nick-End Labeling.