Background
Colon cancer is the third most commonly diagnosed cancers in the world [
1], with an estimated 140,000 new cases and over 50,830 deaths in 2013 in the USA, and over 1.2 million new cases and 600,000 deaths worldwide [
2]. Surgery, radiotherapy and chemotherapy are used for treating colon cancer patients [
3]. However, those treatments are not sufficient because of resistance against chemotherapy, toxicity and side-effects. Therefore, it is urgent to develop novel anti-cancer agents with fewer side effects to satisfy the unfulfilled therapeutic demands of patients.
Apoptosis plays an important role in anti-cancer effects of chemotherapeutics [
4]. Apoptosis can be induced by stimulation of death receptor (DRs) [
5]. Binding to their respective ligands, DRs are activated and recruit the intracellular adaptor protein (Fas-associated death domain protein) which results in the activation of caspase-3, caspase-8 and caspase-9 as well as Bax to kill cancer cells [
6]. Chemo-resistances are also related to the expression of DRs in colon cancer cells [
7]. Therefore, DR-mediated apoptosis has emerged as an effective strategy for cancer therapy. Several compounds have demonstrated the anticancer effects through activation of DRs. Baicalein, a naturally occurring flavonoid, enhanced TRAIL-induced apoptosis via increase of DR5 expression [
8]. Casticin, a flavonoid, induced apoptosis through stimulation of the expression of DRs in colon cancer cells [
9]. Nuclear factor-κB (NF-κB) represents a family of eukaryotic transcription factors participating in the regulation of various cellular responses involved in apoptosis [
10]. NF-κB plays a regulatory role in the expression of an array of apoptotic (caspase-3 and Bax), anti-apoptotic (Bcl-2 and IAP family), and cell proliferation gens (cylooxygenase-2 and cyclins) [
11]. NF-κB is constitutively activated in human colorectal carcinoma tissue and colon cancer cells [
12]. From this knowledge, NF-κB can be specifically targeted to prevent colon cancer cell growth. Several of our studies have demonstrated that compounds inhibiting NF-κB have shown to be useful for inhibition of colon cancer cell growth. 4-O-methylhonokiol and inflexinol inhibited colon cancer cell growth through suppression of NF-κB pathway [
13,
14]. Furthermore, the activation of NF-κB in response to chemotherapy is a principal mechanism of inducible chemo-resistance [
15‐
18]. Thus, inactivation of NF-κB is intended as a strategy to eliminate cancerous cells.
Flavonoids are a diverse family of natural phenolic compounds commonly found in fruits and vegetables [
19]. It is classified as flavonols, flavonones, flavans, etc. Flavonoids especially display a wide range of pharmacological properties including anti-inflammatory, anti-mutagenic, anti-carcinogenic and anti-cancer effects [
20]. An epidemiologic study also showed that flavonoids reduce the risk of colon cancer [
21]. Several studies have reported that some flavonoids have direct effects on apoptosis in colon cancer cell. Luteolin, a type flavonoid, has effects on apoptosis of colon cancer cells [
22]. Another flavonoid, chrysin, also suppressed cancer cell growth through inhibition of the expression of NF-κB in colon cancer cells.
Alpinia oxyphylla Miquel is used for treating intestinal disorders, dieresis, uresis, ulceration and diarrhea [
23]. Yakuchinone A and yakuchinone B existed in
Alpinia oxyphylla Miquel (Zingiberaceae) have anti-cancer effects in skin carcinogenesis [
24]. Tectochrysin, another flavonoid compound, is isolated from
Alpinia oxyphylla Miquel
. Our previous study showed that tectochrysin suppressed lung cancer cell growth via inactivation of STAT3 [
25]. Moreover, our preliminary study showed that tectochrysin was found to bind NF-κB. However, the anti-cancer effects and the molecular mechanisms of tectochrysin in colon cancer cells have not yet been reported. Thus, in this study, we investigated whether tectochrysin could inhibit colon cancer cell growth via suppression of NF-κB activity and enhancement of DR expression in
in vitro and
in vivo.
Discussion
Recent evidence indicates that NF-κB signaling pathway is significantly involved in tumor development [
27]. The constitutively activated NF-κB transcription factor has been associated with several aspects of tumorigenesis such as tumor cell growth, anti-apoptosis, metastasis, angiogenesis, resistance against chemotherapeutics, and tumor promotion in many cancer cells including colon cancer [
28]. The NF-κB transcription factor is constitutively activated in human colorectal carcinoma tissue and colon cancer cells to give favorable circumstance for cancer cell growth [
12,
29]. There have been many recent reports demonstrating anti-cancer effects of several compounds through inactivation of NF-κB. Our previous studies also showed that compounds inhibiting NF-κB activity such as 4-O-methylhonokiol and inflexinol inhibited colon cancer cell growth [
30,
31,
14,
13]. Several studies have especially showed that flavonoids inhibit colon cancer growth through inactivation of NF-κB. For example, Methyl 3,5-dicaffeoyl quinate (200 μg/mL), a flavonoid glucoside, induced cell cycle arrest and apoptosis by inhibiting NF-κB activation in HT-29 human colon cancer cells [
32]. Furthermore, the flavonoids inhibited other cancer growth via inactivation of NF-κB. Quercetin, a principal flavonoid compound in onions, inhibits human oral cancer cells through inhibition of NF-κB [
33]. We, in the present study, found that tectochrysin inhibited constitutively activated NF-κB in colon cancer cells and colon cancer tissues in xenograft animal model. In
in vitro, tectochrysin prevented NF-κB target anti-apoptosis gene expression (but increased apoptosis gene expression) and inhibited DNA binding activity of NF-κB. In
in vivo, the immunohistochemical analysis of tumor section by p50 staining revealed that translocation of p50 into nucleus was significantly lowered in the tumor tissues treated with tectochrysin. We also found that tumor tissues treated with tectochrysin inhibited the activated DNA binding activity of NF-κB. Thus, it is possible that alteration in the expression level of NF-κB target anti-apoptotic and pro-apoptotic proteins is likely to influence apoptotic cell death by tectochrysin. These data indicates the possibility that tectochrysin may inhibit NF-κB, thereby inhibit colon cancer cell growth. Thus, tectochrysin, like other flavonoid compounds, could be effective for the treatment of colon cancer cells.
In further chemical target identification studies, the interaction of tectochrysin-epoxy-sepharose 6B beads with NF-κB p50 protein was assessed using a pull-down assay. The interaction of tectochrysin-epoxy-sepharose 6B beads with NF-κB p50 was then detected by immunoblotting with anti-NF-κB p50 antibody. The results indicated that tectochrysin interacted with NF-κB p50. To identify the binding site of tectochrysin to NF-κB p50, we performed computational docking experiments with tectochrysin and NF-κB p50. Tectochrysin forms two hydrogen bonds with Gly365 and Val412 of the p50 subunit on the amide backbone. It is further surrounded by neighboring hydrophobic amino acid residues such Val358, Phe353, Ser363, Val412, Leu437 and Leu440. Many of these residues are from β-sheets and a loop near DNA binding areas, which may interfere with the DNA binding to the dimeric NF-κB. Our previous studies reported that inflexinol and snake venom toxin modifies a cysteine residue and cysteine62 of p50 in NF-κB via direct interaction of these sites, and thus inhibited the DNA binding activity of NF-κB [
14,
34]. Another study also showed that kaurane diterpene inhibited NF-κB directly targeting the DNA-binding activity of cysteine62 in p50 [
35]. Moreover, the abolished effect on growth inhibition and inactivation of NF-κB in colon cancer cells transfected a p50 mutant (Vp50A, Valine412 was substituted with Alanine) were observed after a treatment with tectochrysin. Therefore, these data indicate that tectochrysin inhibits colon cancer cell growth through inactivation of NF-κB by direct binding on Val412 residue of p50.
DRs are the cell surface receptors that are a part of tumor necrosis factor (TNF) members of cytokines. It is well-known that apoptosis can be induced by stimulation of DRs including TNFR1/2, DR3, DR4, DR5, DR6 and Fas by their respective ligands [
36]. Therefore, these receptors emerged as attractive targets for anti-cancer therapeutics. Several compounds induced apoptotic cell death of cancer cells through increasing DR expression. Our previous study showed that snake venom toxin induced apoptosis of HCT116 and HT-29 colon cancer cells via enhancement of DR4 and DR5 expression [
26]. Flavonoid, such as fucoidan, increased apoptosis of human colon cancer cells via increased expression of DR4, DR5 and Fas [
37]. Flavonoid compounds are also known to have an anti-cancer effect through activation of DR-caspase pathways. Caspases play a critical role in apoptosis by DRs [
38]. Hesperetin, a flavonoid from citrus fruits, exhibited a potential anticancer activity against human cervical cancer cell lines through the induction of apoptosis via caspase-3 activation by increasing Fas expression [
39]. Genistein, one of well-known isoflavones, enhanced apoptosis in lung cancer cells induced by increasing the expression of cleaved caspase-3 via up-regulation of TNFR-1 DR signaling [
40]. Other compounds such as grape seed extract inhibited human colon cancer cell growth by increasing caspase-3 [
41]. Similar to these results, our results showed that the expression of DR3, DR4, Fas and cleaved caspase-3 and -9 highly increased by tectochrysin in a concentration-dependent manner. Moreover, knock down of DR3, DR4 and Fas with siRNA abolished the growth inhibitory effect and caspase-3 activation of tectochrysin on colon cancer cells. In a previous study, we found that tectochrysin inhibited lung cancer cell growth via overexpression of DR3 and Fas [
25]. These data demonstrated that depending on the different cancer type, differential DR pathway may be significant. These results indicate that the colon cancer cell growth inhibitory effects of tectochrysin could be related caspase-3 pathway linked to DR3, DR4 and Fas.
NF-κB inhibition could also be effective to overcome chemo-resistance [
42]. Our previous study reported that natural snake venom toxin suppressed TRAIL-resistant HT-29, A549 and HepG2 cells growth via inhibition of NF-κB activity [
26,
43] . Our present results showed that tectochrysin further inhibited TRAIL-inactivated NF-κB activity, as well as expression of p50 and p-IκB in HT-29 (resistant colon cancer cell), A549 (resistant lung cancer cell) and MCF-7 (resistant breast cancer cell). Therefore, tectochrysin inhibits colon cancer cell growth and has the potential to overcome chemotherapy resistance. TRAIL is a potential anticancer agent because of its capacity to kill selectively cancer cells without toxic effects on normal cells, and thus many chemoresistant cancer cells are resistant to TRAIL [
44]. Previous studies have reported that natural compounds enhanced TRAIL induced apoptotic cell death in TRAIL-resistant cancer cells. Curcumin, a natural flavonoid compound, can synergistically induce apoptosis in three TRAIL-resistant breast cancer cell lines [
45]. We found that tectochrysin enhanced TRAIL induced cell growth inhibition up to 65.8 % in HT-29 TRAIL-resistant colon cancer cells. We also demonstrated that the synergistic effects of tectochrysin and TRAIL on the activation of caspase-3 cleavage and the expression of DR4 in the TRAIL-resistant cancer cells (HT-29, A549 and MCF-7). Combination index values of A549 and HT-29 cells were 0.021, and MCF-7 was 0.034. These results indicated that tectochrysin enhances TRAIL-induced apoptotic cell death through the over-expression of DR4 as well as the down-regulation of anti-apoptotic protein expression via inhibiting NF-κB pathways.
In a xenograft nude mouse, tumor weight and volume in mice treated with tectochrysin at 5 mg/kg doses were 57.9 % and 46.4 % of the vehicle group, respectively. The expression of DR3, DR4 and cleaved caspase-3 was also significantly increased in tectochrysin treated mice, but DNA binding activities of NF-κB and translocation of p50 and p65 into the nucleus were clearly lowered in tumor tissues treated with tectochrysin. Silibinin and wogonin, different flavonoids, inhibited HCT116 colon cancer cells with IC
50 value of 75 μg/mL and 42.6 μg/mL [
46,
47]. However, in the present study, tectochrysin inhibited human colon cancer cells growth with IC
50 value of 8.4 μg/mL and 6.3 μg/mL. In
in vivo study, silibinin (200 mg/kg) or aciculatin (30 mg/kg), inhibited human colon tumor growth about 49.1 %, 40 % respectively [
48,
49]. However, 5 mg/kg tectochrysin showed 48.1 % inhibition in HCT116 human colon cancer growth. These data indicate that tectochrysin could be more for chemotherapeutics compared to other flavonoids. Moreover, we also found that tectochrysin could be a well absorbed compound as a high degree of plasma protein binding compound as determined by the ADME prediction program (pre ADME version 1.0.2). Several drug-likeness predictions such as Lipinski’s, Lead-like, CMC-like, 2.91 as sklogP value and WDI-like rules indicate that this compound is suitable to be used as a drug. Toxicity prediction indicated that there is no toxic effect by this compound. In conclusion, the current study showed that tectochrysin exerts its cell growth inhibitory effects through inhibition of NF-κB and enhancement of DR expression in human colon cancer cells, and enhances sensitivity of TRAIL-resistant cancer cells, suggesting that tectochrysin can be a useful agent for the treatment of colon cancer as well as an adjuvant agent for chemo-resistant cancer.
Methods
Chemicals
We subsequently identified the key compound according to activity-guided purification, as described elsewhere [
25]. The active principle was obtained as white amorphous powder with physico-chemical properties of ESI-MS
m/z: 291 [M + Na]
+;
1H-NMR (500 MHz, CDCl
3): ,
13C-NMR (100 MHz, CDCl
3). The structure of tectochrysin was identified by comparison with its physico-chemical and spectroscopic data reported by an investigator [
50] as described elsewhere [
25].
Cell culture
SW480, HCT116, HT-29, A549 and MCF-7 cells were obtained from the American Type Culture Collection (Manassas, VA). SW480, HCT116, HT-29, A549 and MCF-7 cells were cultured in RPMI 1640 and DMEM medium supplemented with 10 % fetal bovine serum (FBS) and penicillin/streptomycin (100 U/mL). Cell cultures were then maintained at 37 °C in a humidified atmosphere with 5 % CO2. The human colon CCD-18co normal cell was also obtained from the Korea Cell Line Bank and were grown in DMEM medium with 10 % fetal bovine serum, 25 mM HEPES and penicillin/streptomycin (100 U/mL) at 37 °C in a humidified atmosphere with 5 % CO2.
MTT assay and evaluation of apoptotic cell death
Each cell line (
\( 1\times {10}^4 \) cells) was incubated in 200 μl of RPMI 1640, DMEM medium with tectochrysin (concentrations ranging from 1, 5, 10 μg/mL) in a 96-well flat-bottomed plate in triplicate. After incubation for 72 h at 37 °C, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St Louis, MO, USA) diluted in RPMI 1640, DMEM medium were added to each well and incubation was carried out for 90 min. The supernatant was then discarded and the crystal products were eluted with DMSO (200 μL/well; Sigma, St Louis, MO, USA). Colorimetric evaluation was performed with a spectrophotometer at 540 nm. The apoptosis assay was first performed by using DAPI staining. SW480 and HCT116 human colon cancer cells were cultured with concentrations of tectochrysin (5 μg/mL), and induction of apoptotic cell death was evaluated after 24 h. Tunel assay was done as described previously [
51].
Western blot analysis and gel electromobility shift assay
Western blot analysis was performed as described previously [
25]. The membranes were immunoblotted with the following primary antibodies: mouse monoclonal antibodies directed against Fas, Bax, p65, p-IκB, cytochrome-C, β-actin, and Histone-H1 (1:1000 dilutions; Santa Cruz Biotechnology), and rabbit polyclonal antibodies directed against DR3, DR4, Bid, p50, and IκB (1:1000 dilutions; Santa Cruz Biotechnology), and against c-IAP1, XIAP, Bcl-2, cleaved caspase-3, -8, -9 and PARP (1:1000 dilutions; Cell Signaling Technology, Beverly, MA). Immunoreactive proteins were detected with the Enhanced Chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech, Inc., Buckinghamshire, UK). Gel electromobility shift assay (EMSA) was done as described previously [
25]. The relative density of the protein bands was scanned by densitometry using My Image and quantified by Labworks 4.0 software (UVP, Inc., California, USA).
Transfection
Colon cancer cells (SW480, HCT116 \( 7\times {10}^3 \) cells/well) were plated in 96-well plates and transiently transfected with 0.4 μg of the empty vector or the constitutively activated 100 nM of negative siRNA, DR3, DR4 or Fas siRNA per well, using a mixture of plasmid and the WelFect-EX PLUS reagent in OPTI-MEM, according to the manufacturer’s specification (WelGENE, Seoul, Korea). The p50 mutant (Vp50A, valine 412 was substituted with Alanine) plasmid was also transfected with welfect-EX plus reagent in OPTI-MEM according to the manufacturer׳s specification (WelGENE, Seoul, Korea). DR3 siRNA seq. 5’-GAAGCCCUAAGUACGGUUAtt; DR4 siRNA seq. 5’-CUCUGAUGCUGUUCUUUGAtt; Fas siRNA seq. 5’ -GAACCCGUGUUUGCAAUCAtt.
Pull-down assay
Western blot analysis was performed as described previously [
25]. The cell lysate or NF-κB (p50) recombinant protein (Abnova, Taipei, Taiwan) were mixed with tectochrysin-conjugated Sepharose 6B or Sepharose 6B at 4 °C for 24 h. The beads were then washed three times with TBST. The bound proteins were eluted with SDS loading buffer. The proteins were then resolved by SDS-PAGE followed by immunoblotting with antibodies against NF-κB p50 (1:1000 dilution, Santa Cruz Biotechnology).
Docking procedure for NF-κB with tectochrysin
Molecular docking studies were performed using Autodock VINA. NF-κB was obtained from the X-ray crystal structure of NF-κB p50/p65 heterodimer complexed to the immunoglobulin κB DNA. (PDB ID: 1VKX). Only p50 of the heterodimer NF-κB structure of p50/p65 was used in the docking experiments and conditioned using AutodockTools by adding all polar hydrogen atoms. The grid box was centered on the p50 and the size of the grid box was adjusted to include the whole monomer. Docking experiments were performed at various exhaustiveness values of the default, 24, and 48. After the best binding mode was chosen, another round of docking experiments were performed with the grid box re-centered at the binding site of the best ligand-binding mode with its grid box size of 30 × 30 × 30.
Antitumor activity study in vivo xenograft animal model
Five-week-old male BALB/c athymic nude mice (n = 10/group) were purchased from Japan SLC, Inc. (Shizuoka, Japan) and housed in clean specific pathogen free (SPF) rooms. All experiments were approved and carried out according to the Guideline for the Care and Use of Animals of the Chungbuk National University Animal Care Committee (CBNU-278-11-01). HCT116 cancer cells were injected subcutaneously (1 × 107 cells/0.1 mL PBS/animal) into the lower right flanks of mice. After 14 days, when the tumors had reached an average volume of 200–300 mm3, the tumor-bearing nude mice were intraperitoneally injected with tectochrysin (5 mg/kg dissolved in 0.1 % DMSO) twice per week for 3 weeks. In in vitro experiments, the IC50 value of 8.4 μg/mL in HCT116 appeared, thus the concentration of the drug (5 mg/kg) was set in animal models. The tumor volumes were measured with vernier calipers and calculated by the following formula: (A × B2)/2, where A is the larger and B is the smaller of the two dimensions.
Immunohistochemistry
All specimens were fixed in formalin and paraffin-enclosed for examination. Sections 4 μm thick were stained with Hematoxylin and Eosin (H&E) and immunohistochemistry as described elsewhere [
14].
Data analysis
The data were analyzed using the GraphPad Prism 4 ver. 4.03 software (GraphPad Software, La Jolla, CA). Data are presented as mean ± SD. The differences in all data were assessed by one-way analysis of variance (ANOVA). When the P value in the ANOVA test indicated statistical significal significance, the differences were assessed by the Dunnett’s test. A value of P < 0.05 was considered to be statistically significant.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JTH and BYH contributed to the design and coordination of the study. JEH performed all experiments. MHP participated in the study design and prepared the manuscript. ESP and HSY helped with image analysis and microscopy. DWS and BKH advised with the in vivo mouse study. SBH revised the manuscript. YWH investigated that binding affinity of tectocrysine. BYH identified tectocrysine. All authors read and approved the final manuscript prior to submission.