Background
Although the detection approaches has been advanced, the incidence of human colorectal cancer with high morbidity and mortality rate remains high [
1]. The annual incidence of human colorectal cancer is estimated to be ~1 million, with ~500,000 mortalities [
2].
In the United States in 2017, about 95,520 cases of human colorectal cancer are expected to be diagnosed and 52,260 cancer deaths are projected to occur due to human colorectal cancer [
3]. Thus, many studies for more effective therapy against human colorectal cancer have been performed. Because long-term treatment using synthetic anti-cancer drugs leads to a lot of side effects, current research in developing a novel anti-cancer agent has been focused to the plant derived chemical compound as a prominent source of new compounds for drug development [
4]. Indeed, many plants have been reported to exert anti-cancer activity [
5‐
9].
Plant by-products have the potential value to food and pharmaceutical products through various phytochemicals and pharmacological properties [
10]. Thus, plant by-products have been focused for the untapped sources of bioactives [
11].
Diospyros kaki Thunb (Persimmon) has been reported to contain a variety of beneficial compounds such as condensed tannin, carotenoids, vitamin C and polyphenols [
12]. In the plant by-products from
Diospyros kaki Thunb such as peels, seeds and calyx, calyx of
Diospyros kaki Thunb (DKC) has been reported to contain high polyphenols and be effective for the treatment of intractable hiccups [
13,
14]. DKC as a traditional medicine in Korea has been treated to relieve asthma, chronic bronchitis, and cough symptoms [
15,
16]. In the study of DKC for the pharmacological properties, DKC has been reported to possess anti-inflammatory effect through suppression of MAP signaling [
17]. In this study, we elucidated anti-cancer activity and potential molecular mechanism of DKC against human colorectal cancer cells. We here reported that 70% ethanol extracts from calyx of
Diospyros kaki Thunb (DKC-E70) suppressed the proliferation of human colorectal cancer cells and downregulated cyclin D1 level through cyclin D1 degradation by T286 phosphorylation dependent on ERK1/2, p38 or GSK3β, and cyclin D1 transcriptional inhibition through Wnt signaling.
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). PD98059, SB203580, LiCl, MG132 and 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies against cyclin D1, phospho-cyclin D1 (T286), HA-tag, β-catenin, TCF4, p-ERK1/2, total-ERK1/2, p-GSK3β, total-GSK3β, p-p38, total-p38 and β-actin were purchased from Cell Signaling (Bervely, MA, USA). All chemicals were purchased from Fisher Scientific, unless otherwise specified.
Calyx of Diospyros kaki Thunberg (DKC) was purchased from Humanherb, Korea and formally identified by Jin Suk Koo as the professor of Andong National University, Korea. Twenty gram of DKC was extracted with 300 ml of 70% ethanol with shaking for 48 h. After 48 h, the ethanol-soluble fraction was filtered and concentrated to approximately 90 ml volume using a vacuum evaporator and then freeze-dried. The ethanol extracts (2 g, yield percentage: 10%) from DKC was kept in a refrigerator until use.
Cell culture and treatment
Human colorectal cancer cell lines such as HCT116, SW480, LoVo and HT-29 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 ethanol extracts from calyx of Diospyros kaki Thunberg (DKC-E70) was 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 proliferation assay
Cell growth was measured using MTT assay system. Briefly, the cells were plated onto 96-well plate and grown overnight. The cells were treated with DKC-E70 for 24 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.
Cell cycle analysis
HCT116 cells were plated in a 6-well plate and grown overnight. The cells were treated with DKC-E70 for 24 h. After then, the cells were dissociated with trypsin, washed in cold PBS and fixed with 70% cold ethanol on ice for 30 min. The suspensions were centrifuged at 1500 rpm for 5 min. The pellets were resuspended in a solution containing 50 μg/ml propidium iodide, 1 mg/ml sodium citrate, 0.3 ml nonidet P-40 and 5 μg/ml RNase A and stayed on ice at least 40 min. Then the pellets were analyzed by a flow cytometer.
Expression vectors
Wild type HA-tagged cyclin D1 and point mutation of T286A of HA-tagged cyclin D1 were provided from Addgene (Cambridge, MA, USA). Transient transfection of the vectors was performed using the PolyJet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) according to the manufacturers’ instruction.
SDS-PAGE and western blot
After DKC-E70 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% 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.
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 human primers for cyclin D1 and GAPDH as followed: cyclin D1: forward 5′-aactacctggaccgcttcct-3′ and reverse 5′-ccacttgagcttgttcacca-3′, GAPDH: forward 5′-acccagaagactgtggatgg-3′ and reverse 5′-ttctagacggcaggtcaggt-3′.
Transient transfection and luciferase activity
Transient transfection was performed using the PolyJet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) according to the manufacturers’ instruction. Cells were plated in 12-well plates at a concentration of 2 × 105 cells/well. After growth overnight, plasmid mixtures containing 1 μg of TOP-FLASH or FOP-FLASH luciferase constructs (Addgene, Cambridge, MA, USA) and 0.1 μg of pRL-null vector were transfected for 24 h. The transfected cells were treated with DKC-E70 for 24 h. 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).
Statistical analysis
All the data are shown as mean ± SEM (standard error of mean). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test. Differences with *P < 0.05 were considered statistically significant.
Discussion
In cancer development and progression, much attention has been focused on the cyclin D1 as one of the oncogenes associated with the regulation of cell cycle [
26]. In cell cycle, cyclin D1 has been reported to induce G1 to S-phase cell cycle transition, which promotes cell proliferation and plays a major role in oncogenesis [
27]. Actually, the overexpression of cyclin D1 was observed in many human cancers such as endometrial [
28], thyroid [
29], urothelial bladder [
30], breast [
31], brain gliomas [
32], esophageal [
33] and colorectal cancers [
34]. Thus, the regulation of cyclin D1 protein level may be useful for the prevention and treatment of cancer.
Although cyclin D1 overexpression has been regraded to be a common event in the variety of cancer, cyclin D1 overexpression does not occur solely as a consequence of gene amplification. For example,
CCDN1 amplification and cyclin D1 overexpression have been reported to account for 2.5% and 55% in human colorectal cancer, respectively [
35]. Indeed, there is growing evidence that the upregulation of cyclin D1 protein level frequently is attributed to its defective regulation at the post-translational level [
36,
37]. Because cyclin D1 degradation by many anti-cancer agents has been observed in human cancer cells [
38‐
40], cyclin D1 degradation has been regarded as a useful treatment for anti-cancer. In this study, we suggested two evidences related to the induction of cyclin D1 degradation by DKC-E70. Firstly, decreased level of cyclin D1 protein by DKC-E70 rapidly occurred compared to that of cyclin D1 mRNA. Secondly, MG132 treatment as a proteasome inhibitor abolished DKC-E70-mediated downregulation of cyclin D1 protein level. Lastly, DKC-E70 decreased half-life of cyclin D1 protein in the cells exposed to CHX. These findings indicate that decreased level of cyclin D1 by DKC-E70 may result from its degradation.
Cyclin D1 degradation can be regulated by RxxL motif, and phosphorylation of threonine-286 and -288 [
24]. The RxxL motif is associated with anaphase promoting complex-dependent degradation by genotoxic insult [
41]. And, cyclin D1 degradation by phosphorylation of threonine-288 has been reported to be mediated by the mirk/Dyrk 1b kinase [
42]. In addition, the cyclin D1 stability has been shown to be regulated by threonine-286 (T286) phosphorylation induced by ERK1/2, p38 and GSK3β [
21‐
23,
43,
44]. In this study, we found that DKC-E70 phosphorylated T286 of cyclin D1 and T286A transfection blocked cyclin D1 downregulation by DKC-E70. In addition, it was observed that the inhibition of ERK1/2, p38 and GSK3β associated with T286 phosphorylation attenuated DKC-E70-mediated T286 phosphorylation and subsequent decrease of cyclin D1 protein level. These findings indicate that DKC-E70-mediated cyclin D1 degradation may be attributed to Thr286 phosphorylation dependent on ERK1/2, p38 and GSK3β.
Cyclin D1 overexpression can be regulated by gene amplification through transcriptional activation mediated by Wnt signaling [
25]. Interestingly, we observed that DKC-E70 downregulates cyclin D1 mRNA indicating that DKC-E70 may suppress cyclin D1 transcriptional activity. In addition, DKC-E70 downregulated the levels of β-catenin and TCF4, and β –catenin/TCF-dependent luciferase activity. These data indicate that DKC-E70-mediated downregulation of cyclin D1 protein level may be a consequence of the inhibition of gene amplification through suppressing Wnt signaling.