Background
Colorectal cancer is a serious health problem that has progressively increased to be one of the most common cancers worldwide [
1]. Combined therapeutic approaches, such as surgery, chemotherapy and radiation were applied to patients with colorectal cancer. Enormous progress has been made during last decade using fluorouracil (FU) to treat colorectal cancer, with a doubling in dose intensity or tumor response through combined treatment with leucovorin (LV) or bevacizumab or through continuous intravenous administration of FU instead of a bolus injection [
2]. In recent years, the chemotherapeutic agents (irinotecan (CPT-11) and oxaliplatin) have resulted in significant progress in the treatment of advanced colorectal cancer. Initial treatment with irinotecan infused FU/LV, commonly known as IFL, resulted in significant increases in response rate, time to disease progression and median overall survival [
3,
4]. However, retrospective analyses suggested that the administration of IFL might be limited to patients with a performance status of 0 [
5]. When oxaliplatin infused LVFU-2 regimen was compared with a treatment of oxaliplatin plus LVFU-2, known as FOLFOX4, the latter treatment significantly increased the response rate and the time to progression to 9.0 months. The increase in median survival from 14.7 to 16.2 months did not reach statistical significance [
6]. Unfortunately, the metastasis and recurrence of colorectal cancer after curative surgery or resistance to chemotherapy eventually lead to a half of colon cancer patients still die [
7].
Since cancer is a very complex disease with multiple known and unknown regulatory mechanisms, treating of cancer based on single target mechanism could be less effective than using multi-target strategy [
8,
9]. Combination chemotherapies, commonly known as cocktail therapies, targeting diverse abnormalities of cancer, have shown better treatment outcome from various clinical studies [
10,
11]. In terms of this fact, natural herbal medicines traditionally used for various disease management, could be valuable sources in developing anti-cancer drugs due to their multi-target/multi-component nature. Herbal prescription may amplify the therapeutic efficacies of each herbal component, exhibiting maximum outcome with less side effects [
12]. The traditional usage of these medicinal plants has been successfully performed for preventing asthma, reducing edema, relieving fever, cough, hemorrhages, diarrhea and protecting liver [
12].
For research of tumor biology or evaluation of anticancer drugs, in vivo xenograft models have been performed extensively [
13,
14]. However, most of these in vivo models are based on a limited number of cancer cells previously isolated from tumors and selected prior to implantation in animals. Unfortunately, these in vivo models were difficult to reproduce the tumor microenvironment and cancer cell adaptation to the innate immune system, both of which are essential to architecture of the primary tumors, proliferation and metastasis [
15]. In contrast, patient-derived tumor xenograft (PDTX) model obtained by engraftment of patient biopsies transplanted directly into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, subcutaneously, seems to be able to reduce the biologic differences between the primary patient tumor and the in vivo model [
16]. In recent years, PDTX models that have been characterized for predicting drug response in various cancer types [
17] and used in numerous preclinical studies [
18].
The present study aimed to evaluate the anti-cancer effect of KIOM-CRC#BP3B (BP3B), which is novel herbal prescription of ethanol extract of three medicinal plants on colon cancer using PDTX model. The components of BP3B are
Descurainia sophia seed, Peucedanum radix (
Peucedanum praeruptorum Dunn
.) and
Alnus japonica branch. Several reports including our previous study identified that the seeds of
D. sophia contained several cytotoxic and anti-inflammatory substances to induce the death of various cancer cell lines in vitro [
19‐
21]. We also showed that pyranocoumarins from
P. praeruptorum possessed considerably significant multidrug-resistant reversal activity in multidrug resistant MES-SA/Dx5 cancer cells [
22].
A. japonica as well contained anti-proliferative and pro-apoptotic compounds that kill some cancer cells such as human leukemia and prostate cancer [
23,
24]. Although these three plant materials possessed potential anti-cancer activities, it is very important to measure the anti-cancer activity of herbal mixtures in a reliable in vivo model system. To confirm the anti-tumorigenic activity of BP3B against colon cancer, we investigated the changes of histopathological characteristics, proliferation, angiogenesis and apoptotic cell death in colon tumor tissues.
Methods
Reagents
Antibodies against Pecam-1 (M-20:sc-1506),PARP (H-250:sc-7150) and Tie-2 (H-176:sc-9026) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against p27kip1 (#3688) and Cleaved-Caspase-3 (#9661) were obtained from Cell Signaling (Beverly, MA). Anti-Ki67 antibody was purchased from Vector laboratories (Burlingame, CA). Anti-β-actin (A5441) was purchased from Sigma-Aldrich (St. Louis, MO). The 4’6-Diamino-2-phenylindole dihydrochloride was purchased from Thermo Fisher (Waltham, MA).
Preparation of KIOM-CRC#BP3B
The dried seeds of Descurainia sophia, roots of Peucedanum praeruptorum Dunn. and branches of Alnus japonica were purchased from Kwangmyungdang Medicinal Herbs Co. (Ulsan, Republic of Korea). The identities of each herb material were formally confirmed by Dr. Go Ya Choi, K-Herb Research Center, Korea Institute of Oriental Medicine. All voucher specimens have been deposited at KM-Convergence Research Division, Korea Institute of Oriental Medicine. A whole extract of each herb was separately prepared. In brief, dried plant materials were finely pulverized and immersed in 70% (v/v) ethanol (100 g/L). Then, the solvent extraction was performed by maceration at room temperature (48 h, three times). The extract solutions were filtered through a Whatman filter paper No. 2 (Whatman International, Maidstonem, UK), concentrated using a EYELA rotary evaporation system (Tokyo Rikakikai, Tokyo, Japan) and dried a WiseVen vacuum oven (WOW-70, Daihan Scientific, Seoul, Republic of Korea) to produce a 70% ethanol extract. The dried powder of extract was homogenized and then stored in the dark at 4 °C until use. KIOM-CRC#BP3B was prepared by mixing the three herbal extracts at an equal ratio, 1:1:1 (w/w/w). KIOM-CRC#BP3B was dissolved in 0.5% Na-carboxymethyl cellulose (Na-CMC) solution right before being used in animal experiments.
Generation of colon PDTX model and in vivo drug efficacy test
The 6-8 week old male
nu/nu mice (Orient Bio, Seongnam, Korea) were used for in vivo studies and all experiments using immunodeficient mice were carried out in accordance with the guidelines approved by Institutional Animal Care and Use Committees of Gachon University. Fresh surgical tumor tissues (F
0) were collected immediately after surgery from Gil hospital (Incheon, Korea) and cut into 1 ~ 2 mm
3-sized pieces in antibiotics-containing RPMI medium. Written informed consent was obtained from each patient and the study was approved by the Gil hospital ethics committee. Tumor fragments were implanted into subcutaneous pockets of mice, which were made in each side of the lower back. When the tumor size reached to 100 ~ 200 mm
3, those samples were called F
1, subsequently divided into pieces for passaging in vivo to make F
2 xenograft tumors. When F2 tumor size reached to 100 ~ 200 mm
3, collected and cut into 1 ~ 2 mm
3 sized pieces and implanted into subcutaneous layer on the backs of mice to make F3. When F3 tumor size reached to 100 ~ 200 mm
3, mice were randomly divided into 4 groups with 5 mice per each group. Mutation status of important cancer-related genes and patient information of colon PDTX samples is listed in Table
1.
Table 1
Characteristics of PDTX patient samples
Age | 83 | 50 |
Sex | F | F |
pT (primary status) | 3 | 4a |
pM (distant metastasis) | 0 | 1 |
pN (Lymph node status) | 0 | 2b |
Tumor cell type | Adenocarcinoma, Moderately differentiated | Adenocarcinoma, Moderately differentiated |
Microsatellite instability | Stable | Stable |
KRAS mutation status | G13D | WT |
EGFR overexpression | - | - |
p53 expression | + | + |
One group was treated twice per week with intraperitoneal injection of 5 mg/kg oxaliplatin in PBS and other groups were treated once daily orally with low dose (250 mg/kg) or high dose (500 mg/kg) of BP3B dissolved in 0.5% Na-CMC (w/v). Tumor diameters were serially measured with a digital caliper every 2-3 days and tumor volumes were calculated using the following formula: V = (L × W2)/2, V = volume, L = length and W = width. On day 21, mice were sacrificed and tumor tissues were collected, fixed with 10% formalin and embedded in paraffin. Remaining tissues were kept on -80 °C deepfreezer for isolating protein and RNA.
TUNEL assay
Terminal deoxynuclotidyl transferase-mediated deoxyuridine triphosphate nick-end-labeling (TUNEL) assay was performed to measure nuclear DNA fragmentation in apoptotic cells using DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI), according to the manufacturer’s instruction. In brief, paraffin sections of colon tumor samples were deparaffinized in xylene and rehydrated in a series of graded alcohols and fixed in 4% paraformaldehyde for 30 min and permeabilized with 20 μg/ml proteinase K for 10 min at room temperature. The tissue section were then incubated with TUNEL reaction buffer in a 37 °C humidified chamber for 1 h, rinsed twice with 2xSSC and PBS and then incubated with DAPI for 1 min at room temperature. Stained apoptotic cells were visualized by fluorescence microscopy.
Immunohistochemistry and immunofluorescence assay
Paraffin sections of colon tumor samples were deparaffinized in xylene and rehydrated in a series of graded alcohols and antigen was retrieved in 0.01 M sodium citrate buffer. Samples were incubated with 3% H2O2 for 10 min and followed by 1 h blocking in 1% bovine serum albumin in PBS. The slides were incubated overnight at 4 °C, followed by incubating sections with secondary antibody using ABC kit (Vector laboratories) for 1 h at room temperature. Then, samples were developed with diaminobezidine (Vector Laboratories) reagent and counterstained with hematoxylin and mounted with permount.
To quantify the immunostaining intensity of TUNEL, Ki67, CD31 or Tie-2-positive cells, we used and ImmunoRatio software by analyzing the control and treatment groups. The percentage of positively stained nuclear area was calculated by using a color deconvolution for separating the staining components (diaminobezidine and hematoxylin) in at least 3 fields per each slide. The results were presented as percentage of treated group compared to control one.
Western blot analysis
Collected colon tumor tissues were lysed in a buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM EDTA and a protease inhibitor cocktail. Protein concentration was determined using the bicinchoninic acid assay. For western blotting, equal amount of proteins were separated by SDS-polyacrylamide gel electrophoresis, followed by transfer to Immobilon®-P PVDF transfer membrane (Millipore, Bedford, MA). After immunoblotting using specific antibodies, proteins were visualized by chemiluminescence, according to the manufacturer’s instructions (Pierce, Rockford, IL).
Statistical analysis
These results were represented as the mean ± SD values. The differences between groups were determined using 2-tailed student’s tests and the differences were considered significant when the P-values were ≤0.05.
Discussion
KIOM-CRC#BP3B is a novel herbal prescription that is composed of three plant materials in equal weighs.
Descurainia sophia (L.) Weeb ex Prantl (Flixweed) is a member of the family Brassicaceae that is widely distributed in northeastern China and its seed has been broadly used in folk medicine as a cure for throat diseases and viral diseases such as measles and smallpox. Several studies of this plant material have reported that it contains diverse secondary metabolites including cardiac glycoside, flavonoids, lactones, lipids, nor-lignans, and coumarins with various biological activities such as cytotoxicity and anti-inflammatory activity [
28,
29]. The dried roots of
Peucedanum praeruptorum Dunn (Family Umbelliferae) are a well-known traditional Chinese medicine. Several phytochemical and pharmacological studies have shown that various coumarins are the major constituents of this plant and these have diverse biological properties such as anti-inflammatory, chemopreventive and neuroprotective effects [
30‐
32].
Alnus japonica is a member of the Family Betulaceae, and the bark of this plant has long been used in traditional medicine in the treatment of fever, hemorrhage, diarrhea, and alcoholism. Several scientific studies revealed that the extract of
A. japonica contains various biological compounds with anti-adipogenic, anti-proliferative, and anti-parasitic activities [
33,
34]. In this study, we showed that BP3B have similar or better therapeutic efficacy compared with oxaliplatin (Fig.
1). Because herbal drugs have been known to exhibit fewer side effects (12), combination therapy with already clinically used drugs, such as oxaliplatin or irrinotecan can increase the anti-cancer activity and show synergistic effect of chemotherapy. Although we did not identify the exact mechanism for anti-tumorigenic activity of BP3B, it will be very valuable information for further study and clinical trial.
Preclinical trial for validation of potential therapeutic targets via in vivo model is regarded as an indispensable procedure for anti-cancer drug development and make the conquest of cancer or other diseases [
35]. Recently, many studies have adopted the PDTX technique to perform the preclinical testing of anti-cancer drugs [
36]. Previous studies also indicated that transplantation of fresh surgical specimens better represents the cellular and clinical phenotypes of human cancers compared with traditional cell line-based preclinical testing [
37,
38]. Histological examination also showed that PDTX model exhibited very similar histology and immunohistochemical phenotypes of patient’s original tumors and maintained the invasive/metastatic features even during serial subtransplantations in vivo. According to their gene expression patterns in breast cancer samples, less than 5% of genes showed variation in expression between PDTX and the homologous primary tumor [
37,
39]. Although we did not present in this paper, we also confirmed that the xenograft tumors (F
3) exhibited similar histologic architecture and pathologic characteristics to that of the original tumors [
38]. Established PDTX models of colon cancer were good systems for evaluating the efficacy of anti-tumor drug (Fig.
1). In addition, these models will provide very important tools for developing the novel drugs against drug-resistant tumors, such as cetuximab, using Ras wild-type and mutant samples.
Acknowledgement
This research was supported by a grant from the Korea Institute of Oriental Medicine (K14062), from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C1364) and from the Gil Medical Center, Gachon University (FRD2014-09) to S. Hong.