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

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.


Background
Abeliophyllum distichum Nakai (A. distichum) has been known to be a deciduous shrub and native to the south and central areas of Korea [1]. Recently, A. distichum has been reported to exert the inhibitory effect on angiotensin converting enzyme [1]. However, no specific pharmacological effects from A. distichum have been described. Therefore, extensive biological screenings of A. distichum may be necessary to examine the potential use.
Activating transcription factor 3 (ATF3) as a member of the ATF/CREB family is activated under various physiological and pathological stimuli [2], which has been regarded to exert cell-depending effects including cell cycle arrest and apoptosis [3,4]. For the incidence and development of cancer, the roles of ATF3 in cancer progression and anti-cancer therapeutics are complex. There are growing evidences to suggest that ATF3 is expressed in cells treated with serum and growth factor and induces DNA synthesis and expression of cyclin D1 in hepatocytes [5,6]. Furthermore, ATF3 activates the canonical Wnt/β-catenin pathway in human breast cancer cell [7]. However, ATF3 activation promotes a proapoptotic response, as there are evidences to suggest that ATF3 overexpression induced apoptosis in human colorectal cancer cells [8][9][10]. Therefore, it is suggested that ATF3 activation may be a promising cancer preventive and therapeutic target in human colorectal cancer.
In the course of this investigation, we tested the anticancer effect of A. distichum in human cancer cell lines and elucidated the potential anti-cancer mechanism of A. distichum in human colorectal cancer cells.

Sample preparation
The plant parts of A. distichum (voucher number: Park 1001(ANH)) was formally identified by Jae Ho Park as the professor of Jungwon University, Korea. Its flower, leaf and branch were cultivated and collected at Goesan-gun, Chungbuk, Korea. Five hundred grams of fresh parts was extracted with 1,000 ml of 80% methanol with shaking for 24 h. The methanol-soluble fraction was filtered and concentrated to approximately 20 ml volume using a vacuum evaporator and a fraction was placed in a separating funnel. The ethyl acetate fractions from the parts of A. distichum was separated from the mixture, evaporated by a vacuum evaporator and prepared aseptically and kept in a refrigerator until use.

Cell culture and treatment
Human colorectal cancer cell lines (HCT116, SW480 and LoVo cells), human breast cancer cell lines (MCF-7 and MDA-MB-231), hepatocellular carcinoma cells (HepG-2) and colon normal cells (CCD-18co) were purchased Korean Cell Line Bank (Seoul, Korea) and grown in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS), 100 U/ ml penicillin and 100 μg/ml streptomycin. The cells were maintained at 37°C under a humidified atmosphere of 5% CO 2 . Ethyl acetate fractions from A. distichum were dissolved in dimethyl sulfoxide (DMSO) and treated to cells. DMSO was used as a vehicle and the final DMSO concentration was not exceeded 0.1% (v/v).

Cell viability
Cell viability was measured using MTT assay system. Briefly, cells were plated onto 96-well plated and grown overnight. The cells were treated with 0, 50, 100 and 200 μg/ml of EAFAD-B for 24 and 48 h. Then, the cells were incubated with 50 μl of MTT solution (1 mg/ml) for the additional 2 h. The resulting crystals were dissolved in DMSO. The formation of formazan was measured by reading absorbance at a wavelength of 570 nm.

Transient transfections
Transient transfections were performed using the Poly-Jet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) according to the manufacturers' instruction. HCT116 and SW480 cells were plated in 12-well plates at a concentration of 2 × 10 5 cells/well. After growth overnight, plasmid mixtures containing 0.5 μg of ATF3 promoter linked to luciferase and 0.05 μg of pRL-null vector were transfected for 24 h. The transfected cells were cultured in the absence or presence of the ethyl acetate fractions for the indicated times. The cells were then harvested in 1 × luciferase lysis buffer, and luciferase activity was normalized to the pRL-null luciferase activity using a dual-luciferase assay kit (Promega).

Transfection of small interference RNA (siRNA)
The cells were plated in six-well plates and incubated overnight. HCT116 cells were transfected with control siRNA and ATF3 siRNA for 48 h at a concentration of 100 nM using TransIT-TKO transfection reagent (Mirus, Madison, WI) according to the manufacturer's instruction. Then the cells were treated with EAFAD-B (200 μg/ml) for 24 h.

SDS-PAGE and Western blot
After EAFAD-B treatment, cells were washed with 1 × phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma Aldrich) and phosphatase inhibitor cocktail (Sigma Aldrich), and centrifuged at 15,000 × g for 10 min at 4°C. Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). The proteins were separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then incubated with specific primary antibodies in 5% nonfat dry milk at 4°C overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences) and visualized in Polaroid film.

Statistical analysis
Statistical analysis was performed with the Student's unpaired t-test, with statistical significance set at *, P < 0.05.

A. distichum induces reduction of cell viability against various cancer cells
The effects of the ethyl acetate fraction from the parts of A. distichum on cell viability were evaluated in human breast cancer cells (MCF-7 and MDA-MB-231), colorectal cancer cells (HCT116 and SW480), hepatocellular carcinoma cells (HepG-2) and colon normal cells (CCD-18co) by MTT assay. As shown in Figure 1A, cell viability of all cancer cells was reduced by the treatment of 100 μg of the ethyl acetate fraction from the parts of A. distichum for 24 h and this effect was highest in the ethyl acetate fraction from the branch of A. distichum. There, we collected the ethyl acetate fraction from the branch of A. distichum (EAFAD-B) for the further studies elucidating the potential mechanism by which A. distichum exerts anti-cancer activity. In a dose-experiment for evaluating the effect of EAFAD-B on cell viability, EAFAD-B dose-dependently reduced the cell viability of human colorectal cancer cells ( Figure 1B). However, EAFAD-B induced minimal reduction of cell viability in CCD-18co (colon normal cells) ( Figure 1C). These data indicate that EAFAD-B's anti-cancer activity may be cancer-specific.

Effect of EAFAD-B on ATF3 expression and ATF3mediated apoptosis in human colorectal cancer cells
There is a growing body of evidence that ATF3 expression could induce apoptosis in human colorectal cancer cells. To test whether EAFAD-B affects ATF3 expression, we measured ATF3 expression in HCT116 and SW480 cells by Western blot and RT-PCR. As shown in Figure 2A and B, ATF3 expression was activated in EAFAD-B-treated HCT116 and SW480 cells. In addition, to elucidate a molecular mechanism by which EAFAD-B increases ATF3 expression in HCT116 and SW480 cells, we first measured the mRNA level of ATF3 to determine whether increase in ATF3 protein expression is involved in transcriptional regulation. ATF3 mRNA was increased in HCT116 and SW480 cells treated with EAFAD-B at the dose-dependent manner ( Figure 2C and D), which was similar to protein expression. In addition, we confirmed the transcriptional regulation of ATF3 gene by EAFAD-B using ATF3 promoter luciferase constructs (pATF3-1420/+34). As shown in Figure 2E and F, EAFAD-B dose-dependently increased ATF3 promoter activity in HCT116 and SW480 cells. In time-course experiment, ATF3 promoter activity was increased at 3 h after EAFAD-B treatment in both HCT116 and SW480 cells ( Figure 2G and H), while, ATF3 protein expression was slightly induced at 1 h after EAFAD-B treatment ( Figure 2I and J).
To see whether ATF3 expression is associated with EAFAD-B-mediated apoptosis, cleaved PARP was measured by Western blot in ATF3 knock downed-HCT116 cells. As shown in Figure 2K, knockdown of ATF3 by ATF3 siRNA reduced the cleavage of PARP, indicating that ATF3 may be one of important genes in apoptosis by EAFAD-B.

ATF3 transcriptional activation by EAFAD-B is dependent on p38 MAPK and GSK3β
To determine the upstream kinase affecting EAFAD-Binduced ATF3 transcriptional activation, ATF3 promoter construct (pATF3-1420/+34) was transfected into HCT116 and SW480 cells and then these cells were pretreated with 20 μM of PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor) or SB216763 (GSK3β) for 2 h and subsequently co-treated with 200 μg/ml of EAFAD-B for 24 h. As shown in Figure 4A and B, inhibitions of p38 MAPK by SB203580 and GSK3β by SB216763 attenuated EAFAD-B-induced ATF3 transcriptional activation. However, inhibition of ERK1/2 by PD98059 did not affect ATF3 activation by EAFAD-B. We also tested that the inhibitions of p38MAPK and GSK3β affect ATF3 protein level. In these experiments, the inhibition of GSK3β by SB216763 inhibited the increase of ATF3 protein level by EAFAD-B, while p38 MAPK inhibition did not affect ATF3 protein level in HCT116 cells ( Figure 4C). However, the inhibitions of p38MAPK and GSK3β reduced EAFAD-B-induced ATF3 protein expression in SW480 cells ( Figure 4D). To see whether EAFAD-B affects the activation of p38, ERK1/2 and GSK3β, we measured the phosphorylation level of these kinases by Western blot and found that EAFAD-B increased the phosphorylation of three kinases from 1 h after the treatment ( Figure 4E).
Interestingly, our findings indicate that inhibition of p38 MAPK differently affect ATF3 transcriptional activation and increase of ATF3 protein in HCT116 cells. Moreover, the time point to increase ATF3 protein level (1 h after EAFAD-B treatment) is earlier than that to start ATF3 transcriptional activation (3 h after EAFAD-B treatment) ( Figure 2G-J). Thus, we hypothesize that EAFAD-Binduced increase of ATF3 protein could be consequence of ATF3 accumulation. To address this question, we investigated the effect of EAFAD-B on ATF3 stability in HCT116 cells. As shown in Figure 4F, the treatment of cycloheximide (CHX) after pre-stimulation of EAFAD-B for 12 h decreased ATF3 protein level. However, co-treatment of EAFAD-B with CHX attenuated CHX-induced decrease of ATF3 protein level. In conclusion, these results demonstrate that EAFAD-B increased the transcription activation 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. and increase of ATF3 protein level through p38 and GSK3β activation and also EAFAD-B contributes at least in part to increase of ATF3 accumulation.

Discussion
Cancer chemoprevention has received attention as the most effective approach to reduce colorectal cancer related mortality. Abeliophyllum distichum Nakai (A. distichum) has been known to be a deciduous shrub and native to the south and central areas of Korea [1]. Recently, A. distichum has been reported to exert the inhibitory effect on angiotensin converting enzyme [1]. However, no specific pharmacological effects from A. distichum have been described. In search for approaches to augment the potential therapeutic efficacy of A. distichum for human colorectal cancer, we investigated the anticancer activity of A. distichum and the role of ATF3 in apoptosis by A. distichum in human colorectal cancer cells. In this study, we for the first time provide evidence that A. distichum exerts anti-cancer effect, which especially is associated with ATF3 activation in human colorectal cancer cells.
The activation of activating transcription factor 3 (ATF3) has been known to exert a proapoptotic effect in human colorectal cancer [8][9][10] and ATF3 is a major target of some anticancer drugs including cisplatin [11] and bortezomib [12]. In addition, there is a growing body of evidences to suggest that antitumorigenic phytochemicals induce ATF3-mediated apoptosis [13][14][15]. This indicates that ATF3 may be an important molecular target of cancer chemoprevention against human colorectal cancer. Our data show that the ethyl acetate fraction from the branch of A. distichum (EAFAD-B) increased ATF3 expression and ATF3 knockdown by siRNA attenuated apoptosis by EAFAD-B, which indicates that ATF3 may be one of the molecular targets for anti-cancer activity of EAFAD-B in human colorectal cancer cells.
ATF3 is both tumor suppressive in early stage tumorigenesis and oncogenic in late stage tumorigenesis in breast cancer cell lines [16]. Although ATF3 has been well known to promote metastasis in the breast cancer [17], ATF3 expression can induce apoptosis in human breast cancer cells (MCF-7 and MDA-MB-231) [18,19]. In addition, ATF3 has been reported to mediate the suppression of cell viability in human hepatocellular carcinoma cells (HepG-2) [20]. In this study, EAFAD-B reduced the cell viability in MCF-7, MDA-MB-231 and HepG-2 cells. Thus, the further elucidation of mechanisms for ATF3 role involved in EAFAD-B-induced apoptosis may be needed in human breast cancer and hepatocellular carcinoma cells.
The level of ATF3 can be regulated by multiple mechanisms. One is through transcriptional regulation. In this study, our data shows that EAFAD-B activated the expression of ATF3 mRNA through increasing promoter activity, which indicates upregulation of ATF3 by EAFAD-B may be dependent on transcription. Another mechanism to regulating ATF3 level is through suppression of proteasomal degradation although it is generally believed that ATF3 induction is activated mainly through transcriptional regulation [21]. There is growing evidence that MDM2 induces ubiquitination and subsequent proteasomal degradation of ATF3 [22]. In this study, we found two 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.
evidences that EAFAD-B may affect ATF3 stability. One is that EAFAD-B increased the protein level of ATF3 at earlier time than mRNA level. Another evidence is that p38 inhibition attenuated ATF3 promoter activation by EAFAD-B, while did not affect ATF3 protein level. Furthermore, we found that CHX treatment time-dependently decreased ATF3 protein level upregulated by the pretreatment of EAFAD-B, while co-treatment of CHX and EAFAD-B attenuated the reduction of ATF3 protein.
These findings indicate that EAFAD-B may inhibit ATF3 proteasomal degradation.
It has been reported that ATF3 expression is regulated by MAPK signaling [15,25] and GSK3β [14]. So, we examined whether EAFAD-B-mediated ATF3 activation is associated with the activation of ERK1/2, p38 or GSK3β and found that EAFAD-B induced the phosphorylation of ERK1/2, p38 or GSK3β and inhibition of p38 or GSK3β attenuated ATF3 promoter activity by EAFAD-B in HCT116 and SW480 cells. However, ERK1/2 inhibition did not affect ATF3 promoter activity. These findings indicate that ATF3 activation by EAFAD-B may be dependent on p38 or GSK3β but not ERK1/2. However, we do not excluded that the EAFAD-B-induced ATF3 activation may be overcome by ERK1/2 inhibitor.

Conclusions
These findings highlight that EAFAD-B increased the transcription activation and increase of ATF3 protein level through p38 and GSK3β activation and also EAFAD-B contributes at least in part to increase of ATF3 accumulation.
In conclusion, the current study provides information on the anti-cancer activity and the potential molecular mechanism of A. distichum.