The induction of activating transcription factor 3 (ATF3) contributes to anti-cancer activity of Abeliophyllum distichum Nakai in human colorectal cancer cells

Abstract

Background: Recently, Abeliophyllum distichum Nakai (A. distichum) has been reported to exert the inhibitory effect on angiotensin converting enzyme. However, no specific pharmacological effects from A. distichum have been described. We performed in vitro study to evaluate anti-cancer properties of A. distichum and then elucidate the potential mechanisms.

Methods: Cell viability was measured by MTT assay. ATF3 expression level was evaluated by Western blot or RT-PCR and ATF3 transcriptional activity was determined using a dual-luciferase assay kit after the transfection of ATF3 promoter constructs. In addition, ATF3-dependent apoptosis was evaluated by Western blot after ATF3 knockdown using ATF3 siRNA.

Results: Exposure of ethyl acetate fraction from the parts of A. distichum including flower, leaf and branch to human colorectal cancer cells, breast cancer cells and hepatocellular carcinoma reduced the cell viability. The branch extracts from A. distichum (EAFAD-B) increased the expression of activating transcription factor 3 (ATF3) and promoter activity, indicating transcriptional activation of ATF3 gene by EAFAD-B. In addition, our data showed that EAFAD-B-responsible sites might be between -147 and -85 region of the ATF3 promoter. EAFAD-B-induced ATF3 promoter activity was significantly decreased when the CREB site was deleted. However, the deletion of Ftz sites did not affect ATF3 promoter activity by EAFAD-B. We also observed that inhibition of p38MAPK and GSK3β attenuated EAFAD-B-mediated ATF3 promoter activation. Also, EAFAD-B contributes at least in part to increase of ATF3 accumulation.

Conclusion: These findings suggest that the anti-cancer activity of EAFAD-B may be a result of ATF3 promoter activation and subsequent increase of ATF3 expression.

Figure 1

Effect of EAFAD on cell viability in a variety of cancer cells and normal colon cells. (A,B) Human colorectal cancer cells (HCT116 and SW480), breast cancer cells (MCF-7 and MDA-MB-231), hepatocellular carcinoma cells (HepG-2) and (C) normal colon cells (CCD-18co) were plated overnight and then each fraction was treated for 24 h. Cell viability was measured using MTT assay as described in Materials and methods. *P < 0.05 compared to cells without EAFAD treatment.

Figure 2

Effect of EAFAD-B on ATF3 expression and ATF3-mediated apoptosis in human colorectal cancer cells. (A, B, I, J) HCT116 and SW480 cells were plated and then treated with EAFAD-B. Cell lysates were subjected to SDS-PAGE the Western blot was performed using antibodies against ATF3. Actin was used as internal control. (C, D) RT-PCR analysis of ATF3 gene expression, total RNA was prepared after EAFAD-B treatment for 24 h. GAPDH was used as internal control (E, F, G, H) For ATF3 promoter activity, luciferase construct containing -1420 to +34 of human ATF3 promoter region was cotransfected with pRL-null vector and the cells were treated with EAFAD-B and luciferase activity was measured. *P < 0.05 compared to cells without EAFAD-B treatment. (K) ATF3 siRNA was transfected into HCT116 for 48 h and then EAFAD-B was treated for 24 h. Cell lysates were subjected to SDS-PAGE the Western blot was performed using antibodies against PARP. Actin was used as internal control.

Figure 3

Identification of ATF3 promoter sites responsible for EAFAD-B-induced ATF3 activation. (A, B) Each indicated constructs or (C, D) each deletion construct (0.5 μg) of the ATF3 promoter (0.5 μg) was co-transfected with 0.05 μg of pRL-null vector into HCT116 and SW480 cells, and cells were treated with 200 μg/ml of EAFAD-B. Luciferase activity was measured. *P < 0.05 compared to cells without EAFAD-B treatment.

Figure 4

Up-stream signaling pathways affecting EAFAD-B-mediated ATF3 activation. (A, B) Luciferase construct containing -1420 to +34 of human ATF3 promoter region was cotransfected with pRL-null vector. Then, the cells were pretreated with 20 μM of PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor) or SB216763 (GSK3β inhibitor) and then co-treated with 200 μg/ml of EAFAD-B for 24 h. Luciferase activity was measured. *P < 0.05 compared to cells without EAFAD-B treatment. (C, D) HCT116 and SW480 cells were pre-treated with 20 μM of PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor) or SB216763 (GSK3β inhibitor) and then co-treated with 200 μg/ml of EAFAD-B for 24 h. Cell lysates were subjected to SDS-PAGE the Western blot was performed using antibodies against ATF3. Actin was used as internal control. (E) SW480 cells were treated with 200 μg/ml of EAFAD-B for indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-p38, p38, p-ERK1/2, ERK1/2, p-GSK3β or GSK3β. (F) HCT116 cells were pre-treated with 200 μg/ml of EAFAD-B for 12 h and then co-treated with CHX (10 μg/ml) for the indicated times in absence/presence of EFAD-B. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against ATF3. Actin was used as internal control.