Black rice is a functional food of Indonesia that has several pharmaceutical activities including anti-cancer. This study aimed to evaluate the immunomodulatory potential of aqueous black rice (ABR) extract on mouse breast cancer model. A carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), was applied for breast cancer induction in mice. The study was designed to be seven groups (six mice in each group), including Normal or control group (N), a Cancer (C; DMBA-induced mice) group, Cisplatin (Cis; DMBA-induced mice + Cisplatin) treatment group, and DMBA-induced mice + ABR extract (ABR1; dose 1 or 0.2 g/kg, ABR2; dose 2 or 0.3 g/kg, ABR3; dose 3 or 0.4 g/kg, and ABR4; dose 4 or 0.5 g/kg) treatment group. The cancer-bearing mice were given ABR extract for two weeks, and flow cytometry was used to assess the numbers of CD4⁺ and CD8⁺ T cells, as well as the production of inflammatory cytokines IL-17, TNFα, and IFNγ. The histopathology of breast tissue in mice was analyzed using hematoxylin and eosin staining and microscope observation. The induction of DMBA in mice lower the activation of CD4⁺ and CD8⁺ T cells, and induced IL-17, TNFα, and IFNγ production. The histology of the mammary tissue of the untreated mice demonstrated an infiltration of cancer cells toward stroma, whereas that of the treated groups, especially those under high doses of ABR extract, showed a better prognosis, exhibiting a reduction in cancer cells surrounded by a relatively larger number of adipose tissues. The activation of CD4⁺ and CD8⁺ T cells, as well as the reduction of cytokines (IL-17, TNFα, and IFNγ), were thought to be influenced by ABR extract. The activation of T cells optimized the elimination of cancer cells activity; moreover, the production of pro-tumor cytokines was inhibited.

black rice, breast cancer, DMBA, cytokine, T cell

Based on GLOBOCAN 2018 data, the new cancer cases and deaths in 2018 was about 18.1 and 9.6 million. The leading cause of death among all cancer types was lung cancer (18.4% of total deaths) followed by breast cancer (11.6%), which was mostly found in women. In Indonesia, the most diagnosed cancer type was breast cancer (16.7% in both sexes and 30.9% in females) (Bray et al. 2018). Conventionally, cancer diseases can be treated by chemotherapy that has various side effect such as vomiting (Nurgali et al. 2018), decreased quality of life, and gastrointestinal side effects as well as the radiation therapy and immunotherapy side effects (O’Reilly et al. 2020). However, at present, anti-cancer herbal medicine is being increasingly adopted for treatment. Therefore, a detailed examination of the anti-cancer activity of herbs is both significant and urgently needed (Zheng et al. 2018).

Black rice is a pigmented rice commonly found in Asian countries. Black rice is described as a functional food of several countries including Indonesia, due to its benefits in health (Pratiwi and Purwestri 2017; Thanuja and Parimalavalli 2018). Recently, much attention was received in the use of black rice extract due to the high bioactive compounds content, including tocopherols, oryzanols, polyphenols, B vitamins, and fibers, as well as a wealth of anthocyanins, a group of flavonoids that exhibit antioxidant properties (Dias et al. 2017). Other nutrients and bioactive components found in black rice include essential amino acids, functional lipids, dietary fiber, vitamins, minerals, anthocyanins, phenolic compounds, -oryzanols, tocopherols, tocotrienols, phytosterols, and phytic acid (Ito and Lacerda 2019). Anthocyanin, exhibiting anti-cancer activity, is one of most abundant compounds in black rice and has been well reported. Anthocyanins have anti-cancer properties such as cytotoxicity against breast cancer cell lines (MCF-7, MDA-MB-231, and MDA-MB-453) and the ability to induce apoptosis in MDA-MB-453 by activating its apoptotic intrinsic pathway (Chen et al. 2015). In addition, black rice anthocyanins also involved in the inhibition of MDA-MB-453 cells migration and invasion via the suppression of mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and production of MMP-2 and MMP-9 (Chen et al. 2015). In addition, isolated anthocyanin also exhibit hepatoprotective activity in rats (Saati 2016). Although black rice has been shown to have anti-cancer properties, research on its relationship with cancer immunology is still restricted. Immune system plays an essential role in eliminating cancer especially anti-tumor immune effector such as CD8⁺ T cells (Nafisah et al. 2023). However, immune cells involved in all stages of cancer, to eliminate or promote cancer progression. Pro-inflammatory cytokines tend to increase in cancer and it was associated with tumor development through the induction of tumor proliferation, invasion, and angiogenesis (Ramadhani et al. 2020). Because the immune system plays an important role in cancer treatment, the goal of this study is to evaluate the effect of black rice extract on the immunological profile and histology of a mouse breast cancer model.

The immune system role in killing cancer significantly influences the success of cancer therapy. The involvement of CD4⁺ and CD8⁺ T cells is critical in the response of effective immune anti-tumor activity (Bui and Schreiber 2007). Cancer causes the production of program death-ligand 1 (PD-L1), which can block T cells activation by interacting with programmed cell death protein 1 (PD-1) molecules on surface-activated T cells (Vareki et al. 2017). The activation of T cells, especially CD8⁺ T cells, is crucial and become most powerful effector for eliminating cancer cells. The killing mechanism of CD8⁺ T cells occur through several way such as the production of cytotoxic granules including perforin and granzyme, and death domain FASL activation which lead to DNA fragmentation on cancer cell (Raskov et al. 2021). Thus, the enhancement of CD8⁺ indicated the effect of immunotherapies through the blockade of PD-L1/PD-1 interaction (Nafisah et al. 2022). Phenolic compound was supposed to be the main ingredient of black rice extract and played as immunomodulator (Hartati et al. 2017). Dietary polyphenols can enhance B and T cell proliferation as well as inhibit pro-inflammatory factor through inducing Treg activity (Shakoor et al. 2021). The reduction of CD4⁺ and CD8⁺ T cell activation in the C and Cis groups was linked to increased IFNγ expression, according to our findings. The IFNγ receptor which binds to interferon regulatory factor 1 with the PD-L1 promoter, promoted PD-L1 expression in different cancer cells when IFNγ levels were high (Qian et al. 2018). The IFNγ/PD-L1 axis enhanced the expression of PD-L1, which can eventually bind with PD-1 in T cells, causing T cell dysfunction and apoptosis. So that, the reduction of IFNγ production in the ABR treatment groups (Fig. 2) may be correlated with the induction of CD4⁺ and CD8⁺ T-cell activation (Fig. 1). The activation of CD8⁺ T cells via T-cell receptors that bind to class-1 major histocompatibility complexes (MHCs) can efficiently destroy cancer cells by inducing apoptosis using TNF-related apoptosis-inducing ligands (TRAILs) or the production of perforin and granzyme (Töpfer et al. 2011). This lack of CD8⁺ T cell activation affected the growth and development of cancer, as determined by the histopathological evaluation of the mouse breast tissue (Fig. 3). The histopathological analysis showed a relative reduction in cancer cells that sprouted up only at ductal areas, which indicated a better prognosis in both groups, ABR3 and ABR4.

In this study, the upregulation of IL-17 found in the C group was significantly reduced after treated with Cisplatin and ABR (Fig. 2). It was argued by (Welte and Zhang (2015) that the up regulation of IL-17 found in solid tumors is crucial in the development of cancer. IL-17 can bind to its receptor on cancer cell surfaces, which supports the growth and development of cancer through several mechanisms, including apoptosis suppression in mammary cell carcinoma. Thus, the reduction in IL-17 in the treatment groups seems to be a promising sign for a good prognosis in cancer therapy. It was also reported by Zhao et al. (2020) that the inhibition of IL-17 suppressed cancer metastasis and development. Another study found that IL-17 and TNFα in human prostate and colon cancer can work synergistically to induce PD-L1 expression (Wang et al. 2017). TNFα is a central inflammation mediator that has become an interesting novel therapeutic target in developing cancer therapies. The role of this cytokines was reported to be take a part in the initiation, progression, and metastasis of breast cancer (Waters et al. 2013). TNFα is involved in the epithelial-to-mesenchymal (EMT) transition, which is crucial for cell migration and cancer metastasis (Mercogliano et al. 2020). In addition, pro-inflammatory cytokine IFNγ can promote cancer cell metastasis and contribute to the failure of immunotherapy in cancer (Zaidi 2019; Beziaud et al. 2023). The reduction of these pro-inflammatory cytokines by black rice extract may contributed by anthocyanin which reduce NF-κB signaling activity (Piazza et al. 2022).

This study concluded that ABR extract has immunomodulator effect by increasing T cell activation (CD4 and CD8) and reducing pro-inflammatory cytokines (IL-17, TNFα, and INFγ) production. The enhancement of anti-tumor activity of T cell as well as the reduction of pro-inflammatory cytokines of ABR extract may a promising effect in cancer therapy.