MOLECULAR MECHANISMS OF TOCOTRIENOLS ADJUVANTED DENDRITIC CELL USING A MOUSE MODEL OF BREAST CANCER

Abstract

There are several types of vaccines used for prevention of infectious diseases such as attenuated microorganisms, recombinant proteins and DNA vaccines. Currently, studies are being carried out in developing vaccines for certain tumours. The cell-mediated arm of the immune system is the main arm involved in providing the host with the ability to defend, recover from viral infections and to prevent recurrent infections by the same virus. This type of immune response is also crucial in protecting the host against the onset, development and spread of tumour. Dendritic cells (DC) vaccine is a potent form of cancer immunotherapy that is currently being explored as a form of immunotherapy for various types of cancer. The potency of the DC vaccines is due to the ability of the DC to process and present antigens to T-lymphocytesas well as to induce antigen-specific immune responses. Development of DC vaccines against cancers has been hampered by poor immunogenicity of some of the tumour peptides. It has been proposed that induction of tumour-specific immune responses by DC vaccines could be boosted with the use ofasuitable adjuvants, which can help to induce a higher immune response to tumours as well as to confer protection against the said tumours. In addition, a few key elements of immune system such as metastatic tumour biology, cytokines productions, and antigen-specific lymphocyte activation need further understanding in order to develop an effective therapeutic agent to combat cancer diseases. The aim of this study is to examine the effectiveness of daily supplementation of tocotrienol-rich fraction (TRF) in enhancing the immunisation using DC vaccines against tumour antigens. Tocotrienol-rich fraction is a non-toxic natural micronutrient isolated from palm oil. There are several studies on the immune-enhancing effects of TRF and in this study we evaluate the use of TRF as an adjuvant to enhance tumour-specific immune response induced by DC-based cancer vaccines to prevent tumour growth and metastasis in a mouse model of breast cancer. In this model, 4T1 cells, which are mouse mammary cancer cells derived from BALB/c mice was used to induce breast cancer in female BALB/c mice. The results from thein-vitro work show that TRF markedly (p<0.05) inhibited proliferation of the 4T1 mouse mammary cancer cells. The 4T1 cells were more susceptible to TRF when compared toDC or mitogen-stimulated murine splenocytes. The IC50 value of TRF on 4T1 cells following 72 hours of culturewas found to be 8g/ml. In contrast, there was an overall increase in the viability of DCs and murine splenocytes treated with 20 to 25g/ml TRF for 72 hours. In thein-vivostudy, we found that the combination treatment of using DC pulsed with tumour lysate (TL) from 4T1 cells and supplemented daily with TRF (DC+TL+TRF) markedly (p<0.05) inhibited tumour growth and metastasis in mice with pre-established tumours. Systemic administration of 1 mg TRF daily was found to be capable of mediating significant increases in DC-based immunisations. In addition, TRF supplementation also was also efficacious in increasing cell-mediated immunity to tumour challenge as evident by the increased cytotoxic T-lymphocyte (CTL) activity observed in mice from the DC+TL+TRF group. Furthermore, interferon-gamma (IFN-) and interleukin-12 (IL-12) production was increased (p<0.05) in mice from the DC+TL+TRFtreated group compared to the other groups. From the analysis of the microarray data, we found several genes that were differentially regulated in the DC exposed to different treatments. From this list of genes,we chose to carry out further work on one interesting gene, which is known as the “Special AT rich binding protein-1” (SATB1) gene. The SATB1 gene was reported to have dual functions in different cells. It addition, it was reported to have the ability to induce growth of aggressive breast cancer cells. The expression of the SATB1 gene was stably silenced in the 4T1 cells (4T1SATB1-). When the SATB1-silenced 4T1 cells were used to induce tumour in female BALB/c mice, we observed that tumour growth and incidence was lower in mice injected with the 4T1SATB1-cells and treated with DC+TL and supplemented with TRF (DC+TL+TRF) when compared to corresponding group where tumour was induced using the wild-type 4T1 (4T1-WT) cells. In conclusion, in this study we show that TRF can be used as an adjuvant to enhance tumour-specific immune response induced by DC-based vaccines in a syngeneic mouse model of breast cancer. In addition, we also show that TRF may exert its anti-tumour activity by down regulating the expression of the SATB1 gene in the tumour cells as well as by activating tumour-specific cell-mediated (TH1) immune responses in the animal as evident by increased production of IFN- and IL-12 and increased CTL activity. Hence, DC-based vaccines together with TRF as adjuvant is useful in controlling growth and metastasis of pre-established tumours and therefore may be clinically useful as a new immunotherapeutic approach towards In conclusion, in this study we show that TRF can be used as an adjuvant to enhance tumour-specific immune response induced by DC-based vaccines in a syngeneic mouse model of breast cancer. In addition, we also show that TRF may exert its anti-tumour activity by down regulating the expression of the SATB1 gene in the tumour cells as well as by activating tumour-specific cell-mediated (TH1) immune responses in the animal as evident by increased production of IFN- and IL-12 and increased CTL activity. Hence, DC-based vaccines together with TRF as adjuvant is useful in controlling growth and metastasis of pre-established tumours and therefore may be clinically useful as a new immunotherapeutic approach towards cancers.