1.
Introduction
Colon cancer is the third leading cause of cancer-induced deaths in both women and men globally, after lung and breast cancer [1]. According to the American Cancer Society, in 2017, 95,520 people contracted colon cancer and 50,260 people died from it in the United States [2]. Several studies have reported that dietary intervention, such as the consumption of functional foods, is one of the main factors for colon cancer prevention. Results of research on the effects of functional food components on colon cancer prevention have been widely reported [3]. Dietary bioactive components, such as phenolic compounds and fibers, have been reported to be potential chemopreventive agents.
Black rice bran (BRB) is well-known for its high composition of antioxidants and fiber. Many bioactive components in BRB contribute to preventing colon cancer. Compared to brown and white rice, BRB has higher levels of anthocyanin and ferulic acid [4]. In addition, the dietary fiber in BRB is known to increase caspase enzyme activity, which marks the occurrence of apoptosis in cancer and induces changes in the morphological characteristics of cells undergoing apoptosis [5,6]. The dietary fiber in the digestive tract traps carcinogens that cause colon cancer and increases the fecal period, facilitating the dissolution of carcinogens in the stool [7].
Colon cancer cell proliferation can be detected by proliferating cell nuclear antigen (PCNA) marker expression. PCNA is a proliferation marker that is an effector in DNA synthesis and is involved in the DNA replication process [8]. During the development of cancer in a tissue, cells undergo a biochemical mechanism aimed at killing cancer cells (apoptosis). The enzymes involved in this apoptotic process in colon cancer are caspase-3, which acts as an effector, and caspase-8z which acts as an initiator. The qRT-PCR analysis will assess PCNA, caspase-3, and caspase-8 markers.
The potential of BRB in inhibiting colon cancer has not been studied in detail. Therefore, an in vivo research study using animal experiments with azoxymethane (AOM) and dextran sodium sulphate (DSS)-induced mice is needed. Studies aim to evaluate the effect of BRB on inhibiting colon cancer development by assessing body weight, organ weights (colon, liver, kidney, and spleen), nodule distribution in the colon, and generally expression of PCNA, caspase-8, and caspase-3.
2.
Materials and methods
2.1. Preparation of BRB
BRB, the main ingredient used in this research, was prepared based on our previous study [9]. The "Cempo Ireng" varieties of BRB were sourced from farmers in Cigudeg, Bogor, Indonesia. It was obtained as paddy and then hulled through milling using the Yanmar Rice Huller type HW-60A (Japan) to obtain brown-black rice. The rice was then polished using Satake polisher type N-70-F (Japan), yielding two products: polished rice and BRB. Thereafter, the BRB was sieved using 20 mesh sieves, dried using a FreeZone 4.5 Liter Benchtop Freeze Dryers Labconco (USA) at −50 ℃ for 24 hours, and stored at −20 ℃ until use.
2.2. Animal studies
All procedures involving the care and use of laboratory animals were performed following approval from the Bimana Indomedical Institutional Animal Care and Use Committee, Bogor, Indonesia (approval number R.02-17-IR). Twenty-four male BALB/C mice (5–8 weeks old) were obtained from Indoani Lab (Bogor, Indonesia), classified into three groups (n = 8 per group), and housed individually in rooms with regulated air and light cycles. During a one-week acclimation, all the mice were fed a standard diet. Afterward, the BRB group alone was further adapted to a modified diet with BRB as a fiber source. Following the third week (pre-induction), the carcinogen-induced group (C+) and BRB mice were injected intraperitoneally with azoxymethane (AOM) and then with dextran sodium sulphate (DSS) (Sigma Aldrich, Merck, St. Louis, MO, USA) at a dose of 10 mg/kg body weight.
The standard diet based on the AIN-93 M standard (Table 1), should contain the following: 14% protein, 4% fat, 5% food fiber, 3.5% minerals, 1% vitamins, 10% sucrose, and carbohydrates [10]. The proximate analysis findings of sodium caseinate and BRB, which include water, lipid, protein, ash, dietary fiber, and carbohydrate content by difference, were used to calculate the ratio.
The normal (C−) group was intraperitoneally administered Isotonic NaCl 0.9% (Ecosol NaCl, Braun Pharmaceutical, Karawang, Indonesia) to reduce stress levels. One week after induction, the C+ and BRB groups were given 1% dextran sulfate sodium (DSS) (MP-Bio, Solon, Ohio, USA) in their drinking water ad libitum for four consecutive days to accelerate the occurrence of colon cancer. The cancer formation protocol can be seen in Figure 1. The diet was supplied according to the daily nutrition of mice, which was 15 g/100 g of body weight with diet composition, as shown in Table 1.
Cage cleaning and weighing of mice were done twice a week. At the end of the 20-week study, mice in all groups were euthanized for colon, liver, kidney, and spleen tissue collection. Colonic tissue was visually observed for the presence of masses or nodules. Furthermore, the tissue was frozen at −80 ℃ for molecular evaluation. The analyses included nodule distribution evaluation using ImageJ, histopathology with lesion neoplasia analysis, and evaluation of caspase-3, caspase-8, and PCNA using real-time PCR techniques [11].
2.3. Quantitative RT-PCR analysis
RNA was extracted from colonic tissue that had been frozen at −80 ℃. The colonic tissue was homogenized using a grinding mortar. Then, about 30 mg of the homogenate was transferred into a sterile 1.5 mL micro-centrifugation tube containing Tryzol. The extraction procedure was performed using Direct-Zol RNA Miniprep Plus following standard protocols (Zymo Research, CA, USA). The RNA concentration was measured using Nanodrop spectrophotometer (Thermo Scientific, USA). Reverse transcription of RNA to cDNA was conducted using a standard procedure from SuperScript III Reverse Transcriptase (Invitrogen, Thermo Scientific, USA). Analysis of the relative quantification of gene expression was performed with Real-Time PCR Detection System (iQ5, Bio-Rad Laboratories Inc., Hercules, CA, USA) using SsoFast EvaGreen Supermix (Bio-Rad, CA, USA). The master mix for real-time PCR was prepared for the expression analysis of caspase-3 (CASP3), caspase-8 (CASP8), and PCNA genes, normalized toward the GAPDH gene (Glyceraldehyde 3-phosphate dehydrogenase). The primers used in this study were: CASP3 (Forward 5'-TGGGCCTGAAATACCAAGTC-3'; Reverse 5'-AAATGACCCCTTCAT CACCA-3'), CASP8 (Forward 5'-GATGAGGCAGACTTTCTGCT-3'; Reverse 5'-CATAGTTCAC GCCAGTCAGGAT-3'), PCNA (Forward 5'-CTCACGTCTCCTTGGTACAGCTTAC-3'; Reverse 5'CTTCCTCATCT TCAATCTTGGAG-3'), and GAPDH (Forward 5'GCCATCTACAAGCAGTCACA-3'; Reverse 5'CCCTTTCTTGCGGAG ATTCT-3'). The protocol for real-time PCR amplification included 42 cycles comprising enzyme activation at 98 ℃ for 2 minutes, denaturation at 98 ℃ for 5 seconds, and annealing, extension, and data collection at 55 ℃ for 10 seconds. Evaluating of gene expression was performed using the Livak method or comparison of delta threshold (∆CT) [12].
2.4. Data analysis
This study, which included eight replications, used a completely randomized design (CRD) in three treatments. Data are reported as averages ± SD. Experimental data were processed using MiniTab 17.0 software and displayed in one-way ANOVA to identify differences between treatment levels. Tukey's HSD test continued different results between treatment levels, and the significance value was determined based on the 5% level.
3.
Results
3.1. Animal conditions during cancer development
The study found that the C−, C+, and C+BRB groups exhibited good clinical conditions and behaviors. This finding was evidenced by data showing that the body weight of these groups remained stable. According to the data (Table 2), an increase in body weight was found in the C+BRB group. All three groups were given the same feed intake, meeting the daily nutritional requirement of 15 g/100 g of body weight on average.
The analysis of the mice's body weight revealed significant differences (p < 0.05) in the C+BRB group. The mean body weight of the C+ group (25.66 ± 1.28) was significantly lower than that of the C+BRB group (29.10 ± 3.31) (p < 0.05). The weight of colon organs in the C+ group was significantly higher than in the other groups (p < 0.05). There were no significant differences in the average weight of the liver, kidney, and spleen across all groups of mice, indicating that the induction of AOM/DSS in the test group did not significantly affect the weight of these organs.
3.2. Cancers distribution in colon tissue of BALB/C mice
Macroscopic evaluation of nodules in the colon tissue was performed. Visual analysis using the ImageJ application revealed no nodules or cancers in the C− group, whereas these were found in the C+ and C+BRB groups (Figure 1).
The distribution of cancers in the C+ group was significantly higher (p < 0.05) compared to the C+BRB group (Figure 2), with more than a two-fold difference: 5.73 ± 2.93 and 1.65 ± 0.72, respectively. There was no significant difference between the C− group and the C+ BRB group. Cancer nodules in the C+ group spread to almost all parts of the colon tissue, exhibiting various shapes.
3.3. Histopathology of BALB/C mice colon
Histopathological analysis of the colon tissue revealed morphological differences between the C− group and the AOM/DSS treatment groups (C+ and C+BRB groups) (Figure 3). Histologically, the C− group exhibited normal colonic mucosa characterized by elongated crypts filled with goblet or epithelial cells. In contrast, the C+ group exhibited severe inflammatory cell infiltration in the lamina propia. The crypts in the C+BRB group were no longer apparent or were missing in the multifocal area, but the damaged epithelial cells were still visible on the tissue surface.
3.4. mRNA expression level of PCNA, caspase-3, and caspase-8
Further observation of the effect of BRB on inhibiting the development of colon cancer in BALB/C mice was performed by evaluating PCNA, caspase-3, and caspase-8 markers using qRT-PCR (Table 3). The higher the value of caspase-3 and caspase-8, the higher the level of apoptotic activity in the colon tissue, indicating lower colon cancer activity. PCNA is widely used to detect cancer cells due to its involvement with enzymes in DNA replication.
The observation showed that the C+BRB group had lower PCNA gene expression (0.58 ± 0.09) than the C+ group (5.22 ± 0.80). The administration of fermented BRB extracts up to 0.2% was reported to inhibit colon cancer occurrence through a mechanism that reduces cell proliferation and lesion numbers [11]. At the levels of caspase-3 and caspase-8 apoptosis gene expression, the C+BRB group showed higher levels (0.91 ± 0.20 for caspase-3 and 0.51 ± 0.18 for caspase-8) compared to the C+ group. The C+ group, which received standard feed, was unable to suppress cancer development in colon tissue.
4.
Discussion
4.1. Animal conditions during cancer development
The body weight result shows that the C− and C+BRB groups tend to have higher weights compared to the C+ group. This may be due to the effects of cancer development in the digestive system. This can be seen in the rate of cancer spread in the C+ group, which is more than in the C+BRB group (Figure 1). The presence of cancer in the digestive system, such as in the colon, will inhibit the digestive system from absorbing nutrients into the body, resulting in a decrease in body weight in animals with cancer [13]. This condition is often called cachexia—A condition of malnutrition characterized by weight loss; anemia; metabolic disorders; depression; and changes in acid-base balance, vitamin, and electrolyte levels in the body [14].
The higher average body weight in the C+BRB group is also attributable to the absence of a decrease in their feed intake. Feed modified with BRB can potentially inhibit the development of colon cancer in the digestive system. Therefore, the clinical conditions and body weight of the C+BRB group tend to be better. Some bioactive components in BRB actively contribute to preventing colon cancer, namely ferulic acid, anthocyanin, γ-oryzanol, β-sitosterol, tocotrienol/tocopherol, tricin, ρ-coumaric, sinapic, syringic acid, and phytic acid [15]. These bioactive components can potentially repair cells damaged by the induction of AOM/DSS carcinogens and suppress cancer development. They can also suppress cell damage in the cytoplasm and cancer cell proliferation [16]. This result is consistent with the study on Sprague-Dawley rats conditioned for colon cancer and fed BRB [15].
The greater weight of the colon in the C+ group is due to the distribution of nodules that spread to colon tissue. Besides being influenced by the original morphological conditions, the weight of an organ can also be affected by abnormalities such as lumps or nodules, as well as the presence of fluid or solids in the organ [17]. Cancer development due to AOM/DSS induction can be observed macroscopically by nodules or lumps in colon organs [11].
4.2. Cancer distribution in colon tissue of BALB/C mice
Nodules or cancers are tissue lumps smaller than 0.5 cm. A nodule may contain inflammation or a mixture of tissue and fluid [18]. Morphologically, in patients with colon cancer, the distribution of nodules/cancers in the colon will be apparent. Generally, nodules or cancers are an early sign of cancerous tissue growth. C+ group cancer nodules spread across almost all parts of the colonic tissue, exhibiting various shapes such as oval or round. BALB/C mice induced by AOM/DSS carcinogen compounds exhibited the presence of nodules or cancers in the colon [11]. The distribution of colonic diarrhea nodules indicates the development of colorectal cancer caused by AOM/DSS carcinogens. The spread of colonic diarrhea nodules is caused by the accumulation of inflammatory cells, resulting in clots or fluid in the colon tissue.
The data obtained from this study shows that the AIN-93M control diet was unable to suppress inflammation and cancer formation induced by the presence of AOM/DSS carcinogens. The cellulose powder component, which is the source of fiber in AIN-93M diet, is believed to be unable to suppress cancer development in mouse colon tissue. This inability may be due to cellulose's relatively low fermentation by bacteria in the colon [19], resulting in lower SCFA production. SCFAs are produced by the fermentation of food fiber by lactic acid bacteria (LAB) in the colon, and they can suppress the development of cancer in the colon [13,20].
On the other hand, this study showed that feed modified with BRB can inhibit the development of colon cancer by suppressing nodule formation in colon tissue, thus inhibiting cancer cell proliferation. Furthermore, bioactive components such as phenolic compounds in BRB can also suppress cancer cell proliferation and DNA damage in colon tissue [21].
4.3. Histopathology of BALB/C mice colon
Histopathological examination of colon tissue in the C+ group reveals cancer development at the dysplasia stage. Dysplasia is a stage of carcinoma characterized by abnormal cell growth, stacked cell nuclei reaching half the thickness of the epithelium, and loss of cell polarity [10]. The dysplasia stage is also characterized by loss of crypts and goblet cells, spread of inflammatory cells in the tissue area, visible damage to epithelial cells, and solidification of the tissue by the cell nucleus [18].
Histopathological examination of the BRB group showed that the cancer development rate was inhibited by substituting fiber with BRB. This is evidenced by the number of nodules in the colon area, indicating that the BRB group has a lower distribution of nodules compared to the C + group. BRB contains dietary fibers, such as β-glucans, that can be fermented by intestinal microbes, such as LAB. This fermentation can later produce SCFA compounds known to be chemopreventive agents in colon cancer. These SCFAs induce several proapoptotic genes such as caspase-3, caspase-9, and bid, while also inhibiting DNA damage in the cell nucleus [22]. It also plays a role in stimulating intestinal epithelial cell multiplication, as these fatty acids are a component of cell membrane phospholipids [21]. In addition to fiber, BRB contains some bioactive components that prevent colon cancer. Anthocyanin, the most significant antioxidant in BRB, binds to the active side of the proliferation protein, PCNA, by stopping the progression of cyclin and CDK in the cell cycle [23]. Furthermore, ferulic acid and tocopherol in BRB reportedly inhibit the proliferation of cancer cells by inducing apoptotic genes [24].
4.4. Proliferation marker (PCNA) and apoptosis markers (caspase-3 and caspase-8)
The result indicates that BRB could reduce the expression of PCNA genes during cancer development. Feed modified with BRB is believed to have an antiproliferative ability by influencing the formation of enzymes that contribute to replication and transcription. This leads to an inhibition of transcription activity in the S phase, leading to cell cycle repair and, ultimately, control of excessive proliferation [25]. Some bioactive components, such as ferulic acid, phytate, and oryzanol, which have the potential for antiproliferative effects in BRB, can be increased in number by fermentation. During fermentation, microbes synthesize enzymes that can break down ester bonds and release bound phenolics, thereby increasing the number of free phenolic compounds [26].
Caspase-3 and caspase-8 genes have not been maximally induced in the metabolism of C+ group mice, preventing suppression of inflammation and DNA damage caused by carcinogens. BRB can potentially suppress cancer occurrence and increase the expression of caspase-3 and caspase-8 apoptotic genes during cancer development in mice. The presence of phytic acid and ferulic acid in BRB has been shown to increase apoptotic genes such as caspase-3 and inhibit cancer proliferation in TRAP mice [27]. Also, it can induce other apoptotic genes, such as pro-caspase 8. Caspase-8, an active form of pro-caspase-8, proteolytically activates caspase-3. Caspase-3 is a caspase effector gene that can alter cell morphology, such as DNA fragmentation, thereby suppressing cancer development. Anthocyanin and ferulic acid in BRB also act as antioxidants that can neutralize free radicals, repairing DNA damage, inducing apoptosis, and suppressing transcription and translation of cancer cells in the proliferation mechanism caused by cancer-forming carcinogens [28]. Phenolic compounds promote apoptosis in cancer cells induced by carcinogenic compounds through intrinsic pathways (mitochondria) [29]. In addition, butyric acid produced from fiber-soluble food fermentation is reported to be one of the agents that can induce apoptosis in cancer cells. Butyric acid can activate apoptotic genes in the intrinsic pathway through mitochondria by activating caspase-9 and caspase-3 enzymes [30].
5.
Conclusions
BRB-based feed can potentially inhibit colon cancer development in BALB/C mice induced with AOM/DSS. The fiber and bioactive components of BRB have been shown to inhibit cancer development by reducing cancer distribution in colon tissue, suppressing cancer cell proliferation by decreasing PCNA gene expression, and inducing apoptosis by increasing caspase-3 and caspase-8 gene expression. In vivo testing is advised on BALB/C mice fed with fermented BRB to inhibit colon cancer, alongside further confirmation of indications of decreased cancer spread and proliferation, as well as induction of apoptosis in the intrinsic pathway. Moreover, some apoptotic genes that work on intrinsic pathways, such as Bcl-2 family members, Apaf-1, and caspase 9, can be further investigated to determine their gene expression levels during colon cancer development inhibition.
Use of AI tools declaration
The authors declare they have not used Artificial Intelligence (AI) tools in the creation of this article.
Acknowledgments
This study was supported from Ministry of Research, Technology and Higher Education of the Republic of Indonesia (Ristekdikti) and grants from the Bilateral Joint Research Projects of the Japan Society for the Promotion of Science (JSPS), Japan, and the JSPS Core-to-Core Program A (Advanced Research Networks) entitled "Establishment of international agricultural immunology research-core for a quantum improvement in food safety".
Conflict of interest
The authors declare no conflict of interest in this research.
Authors contributions
SB, LN, A, and HS: Conceptualization; YK, FNAD, LN, BPP, and US: Methodology; YK: Research; FNAD, US, NDY, S and SB: Analysis and Data Curation; SB, LN, and BPP: Supervision; SB and YK: Writing Draft Preparation; SB, A, and HS: Writing, Review and Editing. All authors have read and agreed to the published version of the manuscript.
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: Shape of nodules in the colon tissue which is marked by the formation of a bulge or lump.
: Inflammation of inflammatory cells: 
: damaged cell.
