Chemicals
Azoxymethane (CAS number: 25843-45-2) and dextran sulfate sodium salt (CAS number: 9011-18-1) were purchased from Sigma-Aldrich, USA. IRN (Imtus) (CAS number: 136572-09-3) was purchased from Emcure Pharmaceuticals Limited, India, and other test chemicals and stains were purchased from Merck, India. The spectrophotometric kit for alanine transaminase (ALT) and creatinine estimation were purchased from Coral, India. The rest of the chemicals used in the study were of analytical grade purity and obtained locally. GT 400 mg capsules were purchased from Zenith Nutrition India limited and the extract was prepared in distilled water for the treatment.
Extract preparation
GT capsules were used for the treatment along with IRN administration and it was used directly as has been purchased commercially. Four hundred milligrams of GT content was encapsulated in the capsule supplemented with 65% polyphenols, 55% catechins, and 45% epigallocatechin-3-gallate (EGCG), which was dissolved in 10 ml of double-distilled water and was mixed thoroughly overnight at 25–30 °C with vigorous shaking in rotary shaker. The solution was then filtered using Whatman filter paper (number 1). The GT doses of low and high concentration for GT-treated groups were then prepared using the filtrate according to body weight of the animals [12].
Animals and treatment
Male Swiss albino mice (Mus musculus L.) were used during present study. The animals (n = 30) weighing approximately 6 weeks of age were procured from stock animal facility of the institute and randomly divided into six groups (negative control, positive control, IRN, GT, IRN+GTLow, and IRN+GTHigh) containing five mice per group. The animals were acclimatized to the laboratory condition prior to treatment and given food and water ad libitum throughout the experiment period. The test substance was dissolved in double-distilled water and applied through intraperitoneal injections to each animal, except the negative control group animals. The treatment was continued for 12 weeks in total which includes 6 weeks of colon cancer induction period and 6 weeks of treatment period. The experiment was designed in such a way that IRN was administered once a week at a dose of 35 mg/kg in both IRN and IRN+GT groups. On the other hand, GT was administered at a dose of 20 mg/kg in the IRN+GTLow group and 100 mg/kg in GT and IRN+GTHigh group for 5 days in a week consecutively for 6 weeks. After 12 weeks of continuous treatment, animals from all the groups were sacrificed by exsanguinations under Ketamine hydrochloride anesthesia (Fig. 1). Collection of blood was done in Ethylenediaminetetraacetic acid (EDTA) tubes for hematology, ALT, and serum creatinine activity. The body weights of all animals at the start of the experimentation and at the time of termination of experiment (week 12) were recorded weekly [13].
Group I. Distilled water
Group II. Azoxymethane (AOM), 10 mg/kg, 2% dextran sulfate sodium (DSS)
Group IIA. Distilled water
Group IIB. Irinotecan 35 mg/kg
Group IIC. Green tea 100 mg/kg
Group IID. Irinotecan 35 mg/kg + green tea 20 mg/kg
Group IIE. Irinotecan 35 mg/kg + green tea 100 mg/kg
Gross macroscopic morphological observations of colon
Due to the treatment of AOM and DSS in positive control group, tumors are developed in both proximal and distal regions of the colon which can be observed macroscopically. The number of tumors noted from the positive control group and other treated groups and analyzed statistically for the evaluation of treatment efficacy. The length of entire colon is measured and compared with other treated groups for the difference.
Survival
Survival of the individual after treatment of a particular disease signifies effectiveness of the treatment. The more survival percentage, the more effective is the result. Survival percentage of the treatment is analyzed using Kaplan-Meier survival curve in Graphpad Prism 5.0 software.
Hematology
Blood collected in EDTA tubes was used for total count of WBC, neutrophil, serum ALT, and serum creatinine activity. All the hematology procedure was done manually and completed within the same day of blood collection. Total count of WBC and neutrophil was done according to Dacie and Lewis [14]. Counting of WBCs was done in Neubauer hemocytometer subjected to 2 slides per animal per group.
Alanine transaminase (ALT) and creatinine activity
Level of serum ALT was determined using ALT kit by colorimetric method [15]. Samples of serum were incubated with either L-alanine or α ketoglutarate for ALT determination. The pyruvate so formed was then reacted with 2,4-dinitrophenyl hydrazine to form an adduct which absorbs light at 505 nm.
Estimation of creatinine level was performed by alkaline picrate colorimetric method in accordance with creatinine kit where values were expressed as mg% [16]. Picric acid in alkaline medium reacts with creatinine to form an orange colored complex with the alkaline picrate. Intensity of the color formed is directly proportional to the amount of creatinine present in the sample which absorbs maximum light at 520 nm.
Histopathological studies
For histopathological studies, tissues were collected from the colon, liver, and kidney of both control and test animals. The tissues were cut into pieces of adequate size and fixed in 10% neutral-buffered formalin. The tissues were then rehydrated, washed thoroughly in tap water, dehydrated, cleared in xylene, and embedded in melted paraffin following the routine procedure. Sections were cut at 5-μm thickness and stained by routine hematoxylin and eosin (H&E) method.
Measure of tumor volume
Measure of tumor volume in positive control group and other treated groups are important to study the efficacy of GT catechins in combination with IRN in colon cancer model. The size and volume of the tumors are measured using image J software in Leica Microsystems, Germany (DM 2000B).
Statistical analysis
All data were presented as mean ± SEM. Statistical analyses were performed using one-way analysis of variance (ANOVA). A p-value < 0.05 were taken into consideration for determining significance. All statistical procedures were computed using Graphpad Prism 5.0 software.