Study of theobromine and theanine effects, alone and in combination, on prevention of colon cancer in animal model of rat

Abstract

Background & aim: The presence of a tumor in the large intestine is called colon cancer. This cancer is ranked as fourth in incidence and fifth in mortality among cancers, worldwide. Today, the use of natural compounds to prevent or suppress colon cancer has been considered by numerous researchers. Among them, beneficial effects of theobromine, the predominant methylxanthine of cocoa, and theanine, a unique analog of the amino acids specific to tea, on cancer have been reported in some studies. However, the effects of these two phytochemicals on colon cancer prevention have not been assessed in the previous literature. Therefore, the present study was aimed to investigate the effects of theobromine and theanine, alone and in combination, on the prevention of dimethylhydrazine (DMH)-induced colon cancer by focusing on their impacts on the expression of two oncogenes (protein kinase B1 (AKT1) and Janus Kinase (JAK2)) in an animal model. Methods: Thirty male Wistar rats were were randomly divided into five groups of negative control, positive control, Theobromine (100 mg/kg), Theanine (400 mg/kg), and Theobromine (100 mg/kg)-Theanine (400 mg/kg) combination. Then, the animals of the negative and positive control groups were treated by daily gavage of drinking water and others were gavaged with theobromine, theanine, or a combination of theobromine and theanine, based on the mentioned doses. After two weeks, colon cancer was induced by subcutaneous injection of 30 mg/kg DMH, twice a week for 12 weeks, in groups 2-5. During this period, the animals of the negative control group were injected with saline and all animals were gavaged according to the above instructions. At the end of the 14th week, rats were anesthetized, and their abdominal cavities were opened. Then the incidence rate and multiplicity, number and volume of tumors, number of aberrant crypt foci (ACF, foci of aberrant crypts of Lieberkuhn) and AC (aberrant crypts of Lieberkuhn), and histopathological features (microscopic status of crypts, number of goblet cells, and the presence of hyperplasia and adenoma) were evaluated. The gene expression of AKT1 and JAK2 was also assessed by Real-time quantitative reverse transcriptase PCR. Results: Multiplicity, number and volume of tumors, the total number of ACFs and ACs as well as the severity of histopathological changes were significantly lower in the colon of animals treated with theobromine and theanine, alone or in combination, compared to the positive control group. The expression of AKT1 and JAK2 oncogenes also decreased after gavage of animals with theobromine, theanine, and the combination of theobromine and theanine compared to the positive control group. The inhibitory effect of theobromine and theanine combination on the AKT1 expression was greater than each of them alone, and the theobromine effect was greater than theanine in this regard (p-value<0.05). Tumor incidence was not affected by theobromine and/or theanine interventions. Conclusion: Theobromine and theanine, alone and in combination, reduced severity of DMH-induced tumorigenesis. Probably, some of the observed effects are related to the inhibition of AKT1 and JAK2 expression, which was determined in the present study. Further studies are suggested to investigate the effects of these two phytochemicals on other oncogenic pathways. Keywords: Colon cancer, Theobromine, Theanine, Dimethylhydrazine, Protein kinase B, Janus kinase