Publication: IMMUNOMODULATORY EFFECTS OF TOCOTRIENOL AND SPIRULINA AGAINST A MOUSE BREAST CANCER MODEL: INDIVIDUAL AND COMBINED EFFECTS
Date
2018
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Publisher
International Medical University
Abstract
Breast cancer is the most common cancer and the leading cause of cancer death among women worldwide. The molecular indication of breast cancer includes physiological abnormalities of cellular components, immunomodulatory changes and epigenetic alterations. Tocotrienol and Spirulina have been shown to have potential anti-cancer activities. However, the effects of the combination of tocotrienol and Spirulina against breast cancer have not been reported. The primary aim of this study was to assess the immunomodulatory effects of tocotrienol and Spirulina, and their combination, against breast cancer in a mouse model. The assessment was based on cytotoxicity tests on 4T1 mouse mammary cancer cells as well as immunophenotyping, histopathology study and gene expression analysis in a mouse model of breast cancer.
In vitro studies using various concentrations of tocotrienol and Spirulina were conducted on 4T1 mouse mammary cancer cells for 24-72 h to determine the cytotoxic effects of these substances before proceeding with the animal experiments. The cytotoxic effects of the combination of tocotrienol and Spirulina at their IC50 values (17 and 50 μg/ml respectively) derived from individual testing were assessed. The inhibitory effect of the combination of tocotrienol and Spirulina was not selective as the growth of non-cancerous 3T3 fibroblasT-cell was also inhibited.
The in vivo study was conducted using five-week-old female BALB/c mice fed with Spirulina alone based on treatment and prevention models of breast cancer. In the prevention model, the animals were fed with Spirulina from day 0 - 28 and induced with mammary cancer by injecting 4T1 cells at the mammary fat pad of animals on day 14. In the treatment model, the animals were induced with mammary cancer on day 0 and fed with Spirulina from day 0 - 28. Body weight and tumour volume were recorded every 3 days. Results showed that there were no significant (p>0.05) changes in body weight and tumour volume of the Spirulina fed animals compared to the control group. In the treatment model, immunophenotypic expression of T-cell showed that the population of T helper cells decreased while that of cytotoxic T-cell increased. There was also significant (p<0.05) increase in Regulatory T cell population compared to the control. In comparison, in the prevention model, despite the increase in both T helper cells and cytotoxic T-cell, increase in regulatory T cell population compared to the control was observed. This showed that Spirulina possess both immune protective and immune suppressive effects, based on the immunophenotypic expression of T helper (CD4+, CD4+/CD127+), cytotoxic T (CD8+) cells and regulatory T (CD4+/CD25+, CD4+/CD25+/CD73+) cells, in both treatment and prevention models. Histopathological examination showed that in both models, upon Spirulina treatment, there were abundant areas of necrosis observed in breast tissue. Metastasis was present in the liver but not the lung, heart and kidney. Gene expression studies showed that there were no significant changes (p>0.05) in gene expression upon Spirulina treatment in both the models.
In assessing the combined effects of tocotrienol, the animals were fed with 1 mg/day tocotrienol and 50 mg/kg body weight of Spirulina from day 0 – 56 and were induced with mammary cancer on day 28. Changes in body weight and tumour volume were monitored every 3 days. Results showed that there were no significant (p>0.05) changes in body weight. However, there were significant (p<0.05) decrease in tumour volume on day 37 and 46 upon the feeding of the combination of tocotrienol and Spirulina. The population of T helper cells and T regulatory cells increased while that of cytotoxic T-cell decreased in mice fed tocotrienol and Spirulina compared to the control. This showed that combination of tocotrienol and Spirulina did not possess synergistic effect in enhancing T cell-mediated immune response against breast cancer based on immunophenotypic expression of T helper (CD4+, CD4+/CD127+) cells, cytotoxic T (CD8+) cells and regulatory T-cell (CD4+/CD25+, CD4+/CD25+/CD73+). Histopathological examination showed that there were very few areas of poorly differentiated adenocarcinoma and abundant areas of necrosis in breast tissue upon combined treatment. Metastasis was still present in the liver but was not observed in the lung, heart and kidney tissues. Combination of tocotrienol and Spirulina did not possess synergistic effect in enhancing necrotic effects of tumour cells in breast tissue as tocotrienol alone showed more necrotic cells compared to the combined tocotrienol and Spirulina treatment. Gene regulation showed that there were no significant (p>0.05) differences in gene expression based on Birc5/Survivin, Serpine1/Serbp1/PAI-1, MIG6 and Cadherin 13 expression.
In conclusion, there were no synergistic anti-cancer effects observed upon combination of Spirulina and tocotrienol based on immunophenotypic expression of T-cell (T helper, cytotoxic T and regulatory T), gene expression (Birc5/Survivin, Serpine1/Serbp1/PAI-1, MIG6 and Cadherin 13) studies, and metastasis and tumour growth analysis based on the mouse breast cancer model used in this study.
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Keywords
Breast Neoplasms, Tocotrienols, Spirulina, Gene Expression Profiling, In Vitro Techniques