Tea samples and preparation
White tea (Silver needle, KWF Food Industries, China), was purchased from the local market. The extract was prepared by placing 2 g of coarse-grounded tea leaves in 100 ml of ethanol at room temperature, and stirring by using a magnetic stirrer for 5 h. The sample (Silver needle white tea in ethanol) was filtered through Whatman filter paper No.1 and the solvent was evaporated under lowered air pressure to collect the extract. The concentrated white tea extract (WTE) was stored at -20°C for further investigations.
Cell Culture
Two human colorectal adenocarcinoma cells (HCT 116 and HT-29), and human dermal fibroblasts-adult (HDF-a) were used in this study. HCT 116 (ATCC® CCL-247™) and HT-29 (ATCC® HTB-38™) cells were purchased from the American Type Culture Collection (ATCC, USA) and cultured in ATCC-formulated McCoy's 5A Medium (ATCC® 30-2007™). HDF-a was purchased from ScienCell Research Laboratories, USA. HDF-a cells were routinely cultured in fibroblast growth medium (ScienCell Research Laboratories, CA, USA). All cells were supplemented with 5 or 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100lg/ml streptomycin (iDNA,South America). Cells were grown at 37°C in a humidified incubator with 5% CO2.
Proliferation Assay
Cells were detached from the culture flask using Trypsin-EDTA and centrifuged at 220xG for 5 minutes. Cells were re-suspended, counted and diluted to appropriate concentration. Cancer cells were seeded into each 96-well plate containing 100µl of growth media. After 24 hours, cells were treated using 20µl of different concentration of extract, while a 10% v/v DMSO solution was used as blank. 48 hour after treatment, 10 µL of 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well and incubated for another 4 hours. Growth media was removed and 100 µL of DMSO was added to dissolve the cell wall and spectrophotometric measurement was taken at 595 nm [15].
Animal studies and ethical issues
The Animal Use Protocol for this study was approved by the Faculty of Medicine, Institutional Animal Care and Use Committee (FOM IACUC) University of Malaya, in accordance with Ethic No.2015-180804/BMS/R/MAA. The animals were cared for according to the criteria of the National Academy of Science’s Guide for the Care and Use of Laboratory Animals [28,29].
Acute toxicity evaluation
In order to demonstrate the safety usage of the extract, acute toxicity of the WTE was determined. Twelve female Sprague Dawley (SD) rats were assigned evenly into groups categorized according to the OECD guideline [16]. The animals were fasted overnight prior to the dosing (free access to water). A single dose of the WTE (300 mg/kg) was administered to the animals by oral gavage. Food was withheld for another 3 to 4 h after dosing. The animals were observed for 30 min and 2, 4, 8, 24 and 48 h following the administration to monitor any onset of clinical or toxicological symptoms. According to the guideline, if 0-1 animal died in the tested dosed (300 mg/kg) the animal would be treated for higher concentration of the extract (2000 mg/kg), but if the 2-3 animal died in the tested dosed (300 mg/kg), a new group (n=3) would be assigned and treated with 50 mg/kg. At this dosed (50 mg/kg) if 0-1 animals died the dose would be categories in ˃50-300, but if 2-3 animals died next group would be treated with 5 mg/kg and the safe dose category would be determined (>0-5). If only 0-1 of the animal administered with 2000 mg/kg of the extract died, the safe value would be exceeded 2000 mg/kg but if 2-3 animals died, then dosing proceeded at 300-2000 mg/kg. Any signs of toxicity, behavioral changes and mortality was recorded over a period of 2 weeks. The animals were sacrificed on day 15. Following sacrificing the rats, the blood and organs (liver and kidney) were collected for serum biochemical analysis and histopathological evaluation for signs of toxicity.
Experimental procedure for evaluation of the chemopreventive effect of WTE against colon cancer
Twenty-four male Sprague Dawley rats were divided randomly into four groups of 6 rats. The animals were anesthetized prior to AOM (carcinogen) injection. Azoxymethane (AOM)-induced aberrant crypt foci (ACF) in animal model is a well-known procedure in colon cancer studies. The rats in cancer groups were injected subcutaneously with the colon-specific carcinogen (AOM) at 15 mg/kg once a week for two weeks. After AOM injection, these four groups were continued (two-month) to be orally administrated with distilled water (vehicle), 200 mg/kg and 400 mg/kg WTE once a day and weekly intravenous injection of 5-Fluorouracil (≤0.5 ml), respectively (table 1).
All animals were given food and water ad libitum and the body weight of rats were recorded weekly until the end of experiment. After ten weeks of experiment, all the animals were sacrificed and the colon tissue was collected and examined histologically for the presence of aberrant crypt foci (ACF). Liquid nitrogen was used to prepare colon tissue samples for tissue homogenization process.
Table 1. The experimental design and specifications.
Groups
|
Description
|
Induction
|
Treatment
|
Group I
|
Cancer Control
|
AOM Injection
|
Oral administration of distilled water
|
Group II
|
Low dose treatment
|
AOM Injection
|
Oral administration of WTE compound (200 mg/kg)
|
Group III
|
High dose treatment
|
AOM Injection
|
Oral administration of WTE compound (400 mg/kg)
|
Group IV
|
Reference control
|
AOM Injection
|
Intravenous injection of 5-Fluorouracil (35 mg/kg)
|
Number of animals in each group is 6 (n=6).
Scoring of aberrant crypts
The animals were euthanized using an overdose of ketamine and xylazine, and colon tissues were separated and flushed with cold phosphate buffered saline (PBS). The colon specimens then were longitudinally opened from anus to rectum and stained with 0.2% methylene blue to observe and record the incidence of aberrant crypts foci (ACF) under dissecting microscope. The numbers of crypts per sample were detailed and the ACF score was calculated based on the number of aberrant crypts foci in each focus.
Histological classification of ACFs
The colon tissues were fixed in Buffered formalin (10%) and proceeded by using tissue-processing machine (Leica, Germany) prior to embedded in paraffin blocks. Then the paraffin blocks were cut into 5-mm sections and stained with hematoxylin and eosin (H&E). The slides were captured under a light microscope (Nikon, Japan) for histological evaluation.
Statistical analysis
Experimental results are presented as means ± SD, and all measurements and analyses were carried out in triplicate. Excel 2007 and SPSS V.18.0 statistical software were used for the statistical and graphical evaluations in this study. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. All p-values < 0.05 were be considered significant.