AT101 exerts a synergetic efficacy in gastric cancer patients with 5-FU based treatment through promoting apoptosis and autophagy

Abstract

Gastric cancer remains a disease with a high mortality rate despite of multiple therapeutic strategies. So far, it is very important to develop new treatment approaches to improve current therapeutic efficacy in gastric cancer. Apurinic/apyrimidinic endonuclease (APE1) involves in DNA base excision repair (BER) during DNA damage pathway. APE1 was found to be associated with poor overall survival with gastric cancer patients. In the in vitro experiment, we tested APE1 inhibitor-AT101 could potently inhibit gastric cancer cell growth and further induce cancer cell apoptosis and autophagy through p53-dependent pathway. Downregulation of APE1 by AT101 has ability to suppress gastric cancer cell migration and renewal through inhibition of CD133, Nanog and LC3expression. Based on findings that Her-2 positive expression cases has poor prognosis from our dataset and TCGA database, we investigated the role of AT101 in synergetic efficacy with 5-FU treatment in Her-2 overexpression gastric cancer in vivo, indicating that AT101 is able to enhance 5-FU in the shrinkage of xenograft mice tumor and induction of cell apoptosis. In summary, the data obtained from our study showed APE1 is guided as a potential therapeutic target for gastric cancer. AT101 could be regarded as a potent inhibitor to promote chemotherapeutic sensitivity in patients with gastric cancer.

Keywords: 5-FU; APE1; AT101; Her-2 positive; gastric cancer.

Figure 1. APE1 and Her-2 overexpression associated with poor prognosis of patients with gastric cancer

(A) and (B) The Kaplan-Meier plot analyzed that APE1 and Her-2 positive expression associated with poor overall survival in patients with gastric cancer. (t test; *, P < 0.05) (C). The samples of patients with gastric adenocarcinoma were stained by Hemotoxylin & Eosin (HE) and immunohistochemistry. The expression of APE1 and Her-2 showed in the nucleus, cytoplasm and membrane (brown stain) in gastric cancer cells.

Figure 2. AT101, as an inhibitor, contributes to gastric cancer cells suppression in vitro.

(A) Chemical structure of AT101 (B) The MTT assay was performed on the viability of AGS and NCI-N87 cells by AT101 at different concentrations (0.5–50 μM) and IC₅₀ value was analyzed as indicated on the plot. (t test; *, P < 0.05). (C and D) Colony formation assays indicated that AT101 enables to inhibit AGS and NCI-N87 colonies formation (viability of cells) with increasing concentrations of AT101 (0–5 μM).

Figure 3. Inhibition of APE1 by AT101 promotes apoptosis and autophagy of gastric cancer cells

AGS and NCI-N87 cells treated with AT101 at different concentrations of 0.5, 2.5, and 5 μM for 48 hours. (A–C) The apoptosis of cells was quantitated on the graph using the Annexin V: PE apoptosis detection kit and a flow cytometer. (t test; *, P < 0.05) (D, E) The markers of BCL-2, P53, Phosphate-activated P53 (Ser15) and NF-κB were detected by western blot assay. (F–H) The autophagic cells was detected by the Cyto-IDr autophagy detection kit and analyzed using the green (FL1) channel of the flow cytometer. (I) The cellular autophagy induced by the concentration of AT101 (5 μM) and APE1 siRNA was examined by confocal microscopy with the application of Cyto-IDr autophagy detection kit.

Figure 3. Inhibition of APE1 by AT101 promotes apoptosis and autophagy of gastric cancer cells

AGS and NCI-N87 cells treated with AT101 at different concentrations of 0.5, 2.5, and 5 μM for 48 hours. (A–C) The apoptosis of cells was quantitated on the graph using the Annexin V: PE apoptosis detection kit and a flow cytometer. (t test; *, P < 0.05) (D, E) The markers of BCL-2, P53, Phosphate-activated P53 (Ser15) and NF-κB were detected by western blot assay. (F–H) The autophagic cells was detected by the Cyto-IDr autophagy detection kit and analyzed using the green (FL1) channel of the flow cytometer. (I) The cellular autophagy induced by the concentration of AT101 (5 μM) and APE1 siRNA was examined by confocal microscopy with the application of Cyto-IDr autophagy detection kit.

Figure 4. Suppression of APE1 enables to inhibition of gastric cancer cells migration and renewal features

(A) AGS and NCI-N87 cells treated with the concentration of AT101 (5 μM) and APE1 siRNA for observing migration determined by transwell assays with matrigel. (B) and (C) The statistical data was shown on the plot (t test; *, P < 0.05). (D) and (E) Several stem-cell like markers (CD133, Nanog and LC3 (lower band shown)) was detected in AGS and NCI-N87 cells with incubation of AT101 (0.5, 2.5, and 5 μM) for 48 hours by western blot.

Figure 5. The role of AT101 in the treatment of Her-2 positive gastric cancer with 5-FU based therapy in vivo

(A) Kaplan-Meier plot of 75 patients with Her-2 positive gastric cancer following 5-FU treatment showing low APE1 (n = 32) expression associated with shorter overall survival. (B) Kaplan-Meier plot of 75 patients with Her-2 positive gastric cancer following 5-FU treatment showing low APE1 (n = 39) expression associated with poorer relapse free survival. (C) and (D) The tumor weight and volume of xenograft mice model in 5-FU sequential treatment with AT101 became smaller than those with 5-FU treatment alone. (E) The downregulated of BCL-2 and Bcl-XL and increased caspase 3 expression were shown in a subset of tumors after 5-FU and 5-FU sequential AT101 treatment.