Background
Cancer is a major problem of public health in many other parts of the world including the United States [
1]. Among the kinds of cancers, human colorectal cancer is the third leading cause of cancer-related death in both male and females in the United States [
1]. Although surgery and chemotherapy have been the most common treatment for colorectal cancer, cancer chemoprevention with dietary factors has received attention as the most effective approach to reduce colorectal cancer-related mortality. Thus, for the last two decades, many researchers have tested and reported anti-cancer activities of natural products in dietary factors such as fruits, vegetables and teas [
2,
3].
Gingers as perennial herbs belonging to the family Zingiberaceae have been widely used as spices, condiments and herbal medicine for treatment of cold, fever, headache, nausea and digestive problems [
4]. Ginger and its general compounds such as gingerols, shogaols, paradols and zingerone exert immuno-modulatory, anti-apoptotic, anti-tumourigenic, anti-inflammatory, anti-hyperglycaemic, anti-hyperlipidaemic, antioxidant and anti-emetic activities [
4]. Ginger leaves have also been used for food flavouring and traditional medicine [
5]. Past pharmacological studies of ginger were confined to rhizomes. Thus, in light of the pharmacological actions of ginger leaves, this study was performed to investigate the anti-cancer activity and elucidate the potential mechanism by which ginger leaves induces the reduction of cell viability and apoptosis in human colorectal cancer cells. Here, for the first time, we report that ginger leaves leads to transcriptional activation of activating transcription factor 3 (ATF3) which may be associated with the reduction of cell viability and induction of apoptosis in human colorectal cancer cells.
Methods
Materials
Cell culture media, Dulbecco’s Modified Eagle medium (DMEM)/F-12 1:1 Modified medium (DMEM/F-12) was purchased from Lonza (Walkersville, MD, USA). The 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and SP600125 was purchased from Sigma Aldrich (St. Louis, MO, USA). SB203580 and PD98059 were purchased from Calbiochem (San Diego, CA, USA). ATF3 antibody and ATF3 siRNA were provided from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Antibodies against β-actin and poly (ADP-ribose) polymerase (PARP), and control siRNA were purchased from Cell Signaling (Bervely, MA, USA). ATF3 promoter constructs (-1420/+34, -718/+34, -514/+34, -318/+34, -147/+34 and -84/+34) were kindly provided by Dr. S-H Lee (University of Maryland College Park, MD, USA). All chemicals were purchased from Fisher Scientific, unless otherwise specified.
Sample preparation
The leaves of ginger (Zingiber officinale, voucher number: PARK1003(ANH)) were kindly provided by the Bonghwa Alpine Medicinal Plant Experiment Station, Korea. One kilogram of ginger leaf was extracted with 1000 ml of 80% methanol with shaking for 24 h. After 24 h, the methanol-soluble fraction was filtered and concentrated to approximately 20 ml volume using a vacuum evaporator and then fractionated with petroleum ether and ethyl acetate in a separating funnel. The ethyl acetate fraction was separated from the mixture, evaporated by a vacuum evaporator, and prepared aseptically and kept in a refrigerator.
Cell culture and treatment
Human colorectal cancer cell lines (HCT116, SW480 and LoVo), human breast cancer cell lines (MCF-7 and MDA-MB231) and human hepatocellular carcinoma (HepG-2) were purchased from Korean Cell Line Bank (Seoul, Korea) and grown in DMEM/F-12 supplemented with 10% fatal 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% CO2. The extracts of ginger leaf (GL) were dissolved in dimethyl sulfoxide (DMSO) and treated to cells. DMSO was used as a vehicle and the final DMSO concentration did not exceed 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 GL for 24 and 48 h. Then, the cells were incubated with 50 μl of MTT solution (1 mg/ml) for an 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.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was prepared using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA (1 μg) was reverse-transcribed using a Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer’s protocol for cDNA synthesis. PCR was carried out using PCR Master Mix Kit (Promega, Madison, WI, USA) with primers for human ATF3 and human GAPDH as follows: human ATF3: 5′-gtttgaggattttgctaacctgac-3′, and reverse 5′-agctgcaatcttatttctttctcgt-3′; huaman GAPDH: forward 5′-acccagaagactgtggatgg-3′ and reverse 5′-ttctagacggcaggtcaggt-3′.
Transient transfections
Transient transfections were performed using the PolyJet 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 × 105 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 GL 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, Madison, WI, USA).
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, USA) according to the manufacturer’s instruction. Then the cells were treated with GL (100 μg/ml) for 24 h.
Cell death assay
Cell death was performed using Cell Death Detection ELISAPLUS Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instruction. Briefly, HCT116 and SW480 cells were seeded in 12-well plate. After 24 h, cells were treated with 0, 25, 50 and 100 μM of GL for 24 h. Cytosol was prepared using Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA). Equal amounts of cytosolic extracts, immunoreagent containing anti-histone-biotin, and anti-DNA-POD were added to microplate well and incubated for 2 h under shaking. After washing, the ABTS solution was added to each well for 20 min and then the ABTS stop solution was added. The absorbance was recorded at 405 nm and 490.
SDS-PAGE and Western blot
After GL 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, St. Louis, MD. USA) 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% non-fat 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% non-fat 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, Piscataway, NJ, USA) 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.
Discussion
Because many dietary factors exert anti-cancer activities, cancer chomoprevention with dietary factors has received attention as the most effective approach to reduce colorectal cancer-related mortality. Ginger leaves have been used as the dietary factors such a vegetable and tea, and the herbal medicine [
5]. However, pharmacological actions of ginger leaves have not been studied. Here, we evaluated the anti-cancer activity of ginger leaves and elucidated its potential mechanism. In this study, we, for the first time, report that ginger leaves showed an anti-cancer activity associated with ATF3 activation in colorectal cancer cells,
ATF3, an ATF/CREB subfamily member, contains the basic-leucine zipper (b-ZIP) DNA binding domain [
7]. ATF3 is dramatically expressed in response to several stresses in many different tissues and exerts diverse biological effects [
7]. In cancer development, ATF3 exerts pro-or anti-apoptotic activities dependent on cell or tissue context [
8,
9]. ATF3 expression was suppressed in human colorectal cancer [
10] and expression of ATF3 induced apoptosis, growth arrest of colorectal cancer cells and
Ras-stimulated tumourigenesis [
11‐
13]. On the other hand, ATF3 induces DNA synthesis and expression of cyclin D1 in hepatocellular carcinoma cells [
14] and enhances cancer cell-initiating features in breast cancer [
15]. Although ATF3 has dual effects in cancer development, ATF3 has been regarded as a major target of cancer chemoprevention in colorectal cancer. Our data indicate that GL increased ATF3 expression in both protein and mRNA level in a time- and dose-dependent manner through the activation of ATF3 promoter. In addition, it was reported that anti-cancer agents such as indole-3-carbinol [
16], conjugated linoleic acid [
17], epicatechin gallate [
18], tolfenamic acid [
19] and PI3 kinase inhibitor [
13] induce ATF3-dependent apoptosis in colorectal cancer cells. In our study, GL increased the PARP cleavage and reduced the viability of colorectal cancer cells, indicating that increased apoptosis and reduction of cell viability may be mediated by activation of ATF3 expression in GL-treated cells.
There is a growing body of evidence to suggest that MAPK signaling is an important pathway regulating ATF3 expression [
18,
20]. Therefore, we examined whether GL-mediated ATF3 activation is associated with the activation of ERK1/2, p38 and JNK. ERK1/2 inhibition by PD98059 attenuated GL-induced activation of ATF3 promoter and ATF3 expression but not in inhibition of p38 and JNK by SB203580 and bySP600125, indicating that ERK1/2 activation may contribute to GL-induced ATF3 activation. In addition, inhibition of ERK1/2 ameliorated GL-mediated apoptosis. In Western blot analysis for phosphorylation of ERK1/2, we found that GL induced a prolonged activation of ERK1/2. Why would late phase ERK activation correlate with proliferation and apoptosis remains to be understood. However, there is one hypothesis that prolonged activation of ERK1/2 can promote accumulation of p21
cip1 resulting in cell cycle arrest and apoptosis [
21]. Similarly, several anti-cancer agents have been reported to induce a prolonged activation of ERK1/2, which results in promoting apoptosis [
22‐
25].
Interestingly, we found that GL-responsible sites for ATF3 activation might be between -318 and -85 region of the ATF3 promoter. ATF3 promoter includes various response elements such as AP-1, ATF/CRE, NF-κB, E2F and Myc/Max binding sites [
26] and especially, EGR-1, CRE and Ftz are cis-acting elements in ATF3 promoter (-318/-85) [
27] from which our data showed that GL-induced ATF3 promoter activity was significantly decreased when the CREB site was deleted. These data indicated that CREB is an important region in GL-induced ATF3 expression.
There is a report that ginger leaves has various bioactive compounds including quercetin, rutin, epicatechin, catechin, kaempferol, naringenin, salicylic acid, cinnamic acid, flavonoids and phenolics [
28]. Among the bioactive compounds, quercetin has been reported to induce ATF3 expression in human colorectal cancer cells, Caco-2 [
29]. Interestingly, we found that ginger leaves had more quercetin (2.124 mg/g dry weight) than ginger rhizoma (1.105 mg/g dry weight), which is similar to the previous report [
28]. According to the previous study [
30,
31] quercetin can be efficiently hydrolysed and absorbed in the intestinal lumen and plasma. Therefore, it is thought that quercetin may be responsible for ATF3-mediated apoptosis by ginger leaves in human colorectal cancer cells and quercetin of GL may be absorbed in the intestinal lumen and plasma of animal.
Acknowledgements
We thank Dr. Seong-Ho Lee (University of Maryland College Park, MD, USA) for providing ATF3 promoter (pATF3-1420/+34, pATF3- 718/+ 34, pATF3- 514/+ 34, pATF3- 318/+ 34, pATF3-147/+ 34, and pATF3- 84/+34). This work was supported by the BK21 PLUS program of Ministry of Education, the Ministry of Education, Science and Technology (2011-0025272), and by a grant from 2014 Research Fund of Andong National University (2014-0169).
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JBJ directed and GHP, JHP & HMS designed the study. JHP undertook the formal identification of the leaves of ginger (Zingiber officinale). GHP, JHP, HMS, HJE, MKK, JWL, MHL, K-HC, JRL and HJC performed the experiments. GHP, JHP and HMS drafted manuscript. HJE, MKK, JWL, MHL, K-HC, JRL, HJC and JBJ corrected the manuscript. All authors read and approved the final manuscript.