Abstract

The objective of this study was to find the best inhibition of WiDr colon cancer cells growth using Fermented Black Rice Bran (FBRB) extract and the mechanism of colon cancer inhibition by fermented rice bran. In this study, BRB was fermented by Rhizopus oligosporus (FBROLI), Rhizopus oryzae (FBRO), respectively, for 0, 24, 48, 72, and 96 h to analyze changes profile of phenolic content, total flavonoid, and antioxidant activity during fermentation. The result of elected fermentation and non-fermented (0 h) (NFBR) analyzed cytotoxic activity on Vero normal cells and WiDr cells. Genes’ mRNA expression of proliferation and apoptotic markers on WiDr cells was also analyzed that normalized the ACTB housekeeping gene. The results demonstrated that fermentation for 72 h produced the highest phenolic content, total flavonoids, and antioxidants activity with FBROLI extract, which had the highest inhibition of WiDr cells with IC₅₀ value 650.7 μg/mL, while the FBRO with IC₅₀ was 873.9 μg/mL, and NFBR with IC₅₀ was 831.8 μg/mL. The mechanism of inhibition of WiDr cells through suppression of PCNA proliferation gene expression and increased apoptotic gene expression of p53, caspase-8, caspase-9, and caspase-7 with the highest increase of apoptotic gene expression obtained FBROLI extracts.

Keywords:
fermentation; rice byproduct; rhizopus ; WiDr; apoptosis

1 Introduction

Colon cancer is the third highest cause of death globally, reaching as high as 1,148,515 incidents in 2020. It is expected to increase by 2040, with an estimated 1,823,278 incidents around the globe (International Agency for Research on Cancer, 2020International Agency for Research on Cancer – IARC (2020). Cancer tomorrow: Estimated number of new cases from 2020 to 2040, Incidence, Both sexes, age [0-85+]. Retrieved from https://gco.iarc.fr/tomorrow/en/dataviz/tables?cancers=8&single_unit=50000&mode=population.
https://gco.iarc.fr/tomorrow/en/dataviz/... ). Cancer begins with the mutation of DNA (Deoxyribonucleic Acid) in cells known as mutation cells. The mutated cells begin to replicate and uncontrollably divide at a high rate, thus damaging the surrounding tissue. This mutation is due to the damage of tumor suppressor genes and the oncogenic activation (Gamudi & Blundell, 2010Gamudi, D., & Blundell, R. (2010). Tumor suppressor genes. Research Journal of Medical Sciences, 4(4), 280-284. http://dx.doi.org/10.3923/rjmsci.2010.280.284.
http://dx.doi.org/10.3923/rjmsci.2010.28... ). Colon cancer occurs in the inner lining of the large intestine.

Rice bran is a byproduct of the rice polishing process. It contains tricin, γ-oryzanol, ferulic acid, ß-sitosterol, tocopherols, tocotrienols, and phytic acid that might be able to inhibit the growth of colon cancer cells. The inhibition of colon cancer cell growth by bioactive compounds in rice bran is classified as preventive. On the contrary, chemotherapy agents, such as oxaliplatin, irinotecan, leucovorin, UFT, capecitabine, and 5-fluorouracil, are generally used to treat cancer patients, and are classified as curative. The use of these curative agents often disrupts the function of other organs (Focaccetti et al., 2015Focaccetti, C., Bruno, A., Magnani, E., Bartolini, D., Principi, E., Dallaglio, K., Bucci, E. O., Finzi, G., Sessa, F., Noonan, D. M., & Albini, A. (2015). Effects of 5-fluorouracil on morphology, cell cycle, proliferation, apoptosis, autophagy and ROS production in endothelial cells and cardiomyocytes. PLoS One, 10(2), e0115686. http://dx.doi.org/10.1371/journal.pone.0115686. PMid:25671635.
http://dx.doi.org/10.1371/journal.pone.0... ). Therefore, rice bran is likely to be developed as a functional food to prevent colon cancer.

The potential of cancer prevention in plant material is often related to their high antioxidant activity, which is determined by the number of free forms bioactive compounds present (Chen et al., 2015Chen, M., Meng, H., Zhao, Y., Chen, F., & Yu, S. (2015). Antioxidant and in vitro anticancer activities of phenolics isolated from sugar beet molasses. BMC Complementary and Alternative Medicine, 15(1), 313. http://dx.doi.org/10.1186/s12906-015-0847-5. PMid:26347338.
http://dx.doi.org/10.1186/s12906-015-084... ).

One effective technique to increase bioactive compounds in rice bran was found to be the solid-state fermentation technique. Fermentation using R. oryzae and R. oligosporus fungi were reported effectively for increasing bioactive compounds in rice bran (Zulfafamy et al., 2018Zulfafamy, K. E., Ardiansyah, A., & Budijanto, S. (2018). Antioxidative properties and cytotoxic activity against colon cancer cell WiDr of Rhizopus oryzae and Rhizopus oligosporus-fermented black rice bran extract. Current Research in Nutrition and Food Science, 6(1), 23-34. http://dx.doi.org/10.12944/CRNFSJ.6.1.03.
http://dx.doi.org/10.12944/CRNFSJ.6.1.03... ; Razak et al., 2017Razak, D. L., Rashid, N. Y., Jamaluddin, A., Sharifudin, S. A., Kahar, A., & Long, K. (2017). Cosmeceutical potentials and bioactive compounds of rice bran fermented with single and mix culture of Aspergillus oryzae and Rhizopus oryzae. Journal of the Saudi Society of Agricultural Sciences, 16(2), 127-134. http://dx.doi.org/10.1016/j.jssas.2015.04.001.
http://dx.doi.org/10.1016/j.jssas.2015.0... ; Ardiansyah et al., 2019Ardiansyah, David, W., Handoko, D. D., Kusbiantoro, B., Budijanto, S., & Shirakawa, H. (2019). Fermented rice bran extract improves blood pressure and glucose in stroke-prone spontaneously hypertensive rats. Nutrition & Food Science, 49(5), 844-853. http://dx.doi.org/10.1108/NFS-12-2018-0340.
http://dx.doi.org/10.1108/NFS-12-2018-03... ). Martins et al. (2011)Martins, S., Mussatto, S. I., Martínez-Avila, G., Montañez-Saenz, J., Aguilar, C. N., & Teixeira, J. A. (2011). Bioactive phenolic compounds: production and extraction by solid-state fermentation. A review. Biotechnology Advances, 29(3), 365-373. http://dx.doi.org/10.1016/j.biotechadv.2011.01.008. PMid:21291993.
http://dx.doi.org/10.1016/j.biotechadv.2... reported that the increase in the number of bioactive compounds is dependent on the type of inoculum used as well as the fermentation time.

Therefore, this study aimed to assess the inoculum and fermentation time to obtain rice bran while also examining cancer cells’ highest antioxidant activity and cytotoxic activity. For comparison, the fermented and non-fermented black rice bran was examined for its cytotoxic activity on WiDr cancer cells and normal Vero cells. According to the authors’ knowledge at present, no studies have been conducted to examine the relations of rice bran fermented with gene expression of colon cancer cells. Hence, in this study, the expression of apoptosis marker genes was also identified to determine the inhibitory growth pathways of WiDr cells, including p53, caspase 7, 8, and 9 genes, and PCNA proliferation marker genes. The findings of this study can contribute to increase the production of functional food products produced with black rice bran.

2 Materials and methods

2.1 Chemicals

2,2-diphenyl-1-picrylhydrazyl (DPPH•), quercetin, gallic acid, Fetal Bovine Serum (FBS), Phosphate Buffer Saline (PBS), Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI-1640), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO, USA). Folin-Ciocalteu, sodium carbonate (Na₂CO₃), aluminum chloride (AlCl₃), tartaric acid, trypsin, Dimethyl Sulfoxide (DMSO), 100x antibiotic solution (100,000 U/I of penicillin), and 100,000 mg/L streptomycins were purchased from Merck (Darmstadt, Germany). The caspase genes p53, 9, 8, 7, PCNA, and housekeeping gene ACTB were purchased from Integrated DNA Technologies (Coralville, Lowa, USA). Butterfield’s Phosphate-Buffered (BPB), Plate Count Agar (PCA), Potato Dextrose Agar (PDA), Nutrient Agar (NA), Buffered Pepton Water (BPW), and Violet Red Bile Glucose Agar (VRBGA) were purchased from Oxoid Ltd (Altrincham, CH, England).

2.2 Black rice bran sample

The black rice used in this study was Cempo Ireng variety obtained from the Cigudeg Local Farmers, Bogor regency, Indonesia. It was ground using a husker machine (Yanmar, HW-60A, Japan) and then polished using a rice polisher (type N-70F, Japan) for 90 s/200 g to obtain the bran.

2.3 Black rice bran fermentation

Rice bran was fermented with a single culture of R. oligosporus isolated from commercial Tempe starter culture (RAPRIMA, Indonesia) and R. oryzae IPBCC 88010 (IPB Culture Collection, Bogor, Indonesia). The rice bran fermentation method referred to Zulfafamy et al. (2018)Zulfafamy, K. E., Ardiansyah, A., & Budijanto, S. (2018). Antioxidative properties and cytotoxic activity against colon cancer cell WiDr of Rhizopus oryzae and Rhizopus oligosporus-fermented black rice bran extract. Current Research in Nutrition and Food Science, 6(1), 23-34. http://dx.doi.org/10.12944/CRNFSJ.6.1.03.
http://dx.doi.org/10.12944/CRNFSJ.6.1.03... with modification. The spore suspension used was 15% (10⁶ spores/mL) of the weight of bran. The bran was incubated using an incubator (Heraeus, Jerman) at 30 °C with the fermentation time of 0, 24, 48, 72, and 96 h. After the fermentation was done, the bran was dried using a freeze dryer (Labconco, USA) for 24 h, then stored at -18 °C to be extracted.

2.4 Sample extract preparation

The sample extract preparation was conducted by referring to the method used by Salazar-Aranda et al. (2011)Salazar-Aranda, R., Pérez-Lopez, L. A., Lopez-Arroyo, J., Alanís-Garza, B. A., & Torres, N.W. (2011). Antimicrobial and antioxidant activities of plants from northeast of Mexico. Evidence-Based Complementary and Alternative Medicine, 2011, 536139. http://dx.doi.org/10.1093/ecam/nep127. PMid:19770266.
http://dx.doi.org/10.1093/ecam/nep127... with a few modifications. 20 g of rice bran was weighed, and 120 mL methanol (1 : 6 w/v) was added. Subsequently, it was macerated using a magnetic stirrer for 2 h. The extract was filtered using Whatman filter papers (Grade 1, pore size 11 μm), and the extraction process was repeated twice. The filtrate result was evaporated with a rotary evaporator (IKA, Staufen im Breisgau, BW, Germany) at 38 °C with low pressure. After the evaporation, the filtrate was dried using a freeze dryer and stored in a refrigerator at 4 °C. During the analysis, the extract was dissolved in methanol (1 mg/10 mL).

2.5 Analysis of phenolic content

Phenolic content analysis was performed by referring to the method employed by Wanyo et al. (2014)Wanyo, P., Meeso, N., & Siriamornpun, S. (2014). Effects of different treatments on the antioxidant properties and phenolic compounds of rice bran and rice husk. Food Chemistry, 157, 457-463. http://dx.doi.org/10.1016/j.foodchem.2014.02.061. PMid:24679804.
http://dx.doi.org/10.1016/j.foodchem.201... . A sample extract of 0.3 mL was added with 2.25 mL of a Folin-Ciocalteu reagent that had been diluted with demineralized water (1 : 10) and allowed to stand for 5 min. A 2.25 mL solution of Na₂CO₃ (60 g/L) was subsequently added. The solution was incubated for 90 min at room temperature. The absorbance of the sample was measured with a UV-VIS spectrophotometer (ThermoScientificTM GENESYSTM 150, Waltham, MA, USA) at 725 nm. The total phenolic compound was expressed in milligrams of gallic acid equivalent per gram of dry extract (mg GAE/g extract).

2.6 Analysis of total flavonoid compound

Total flavonoid compound analysis employed the method used by Chahardehi et al. (2010)Chahardehi, A.M., Ibrahim, D., & Sulaiman, S.F. (2010). Antioxidant, antimicrobial activity and toxicity test of Pilea microphylla. International Journal of Microbiology, 2010, 826830. http://dx.doi.org/10.1155/2010/826830. PMid:20652052.
http://dx.doi.org/10.1155/2010/826830... . A sample extract of 2 mL was added to 2 mL of 2% AlCl₃ solution in methanol and incubated for 10 min at room temperature. The absorbance of the sample was measured using a UV-VIS spectrophotometer at 415 nm against a blank sample consisting of a mixture of 2 mL of sample extract and 2 mL of methanol without AlCl₃. The total flavonoid compound was expressed in milligrams of quercetin equivalent per gram of dry extract (mg QE/g extract).

2.7 Analysis of antioxidant activity

Analysis of antioxidant activity used Baba & Malik’s procedure (Baba & Malik, 2015Baba, S. A., & Malik, S. A. (2015). Determination of total phenolic and flavonoid content, antimicrobial and antioxidant activity of a root extract of Arisaema jacquemontii Blume. Journal of Taibah University for Science, 9(4), 449-454. http://dx.doi.org/10.1016/j.jtusci.2014.11.001.
http://dx.doi.org/10.1016/j.jtusci.2014.... ). 0.2 mL sample extract was added with 3.8 mL of DPPH solution in methanol (25 mg/L). The solution was incubated for 60 min at room temperature in dark conditions. The absorbance of the sample was measured using a UV-VIS spectrophotometer at 517 nm. Control absorbance consisted of 0.2 mL methanol and 3.8 mL DPPH. The ability of bran extract antioxidant activity in capturing DPPH free radicals was expressed in percentage, which was calculated through the following equation (Equation 1):

2.8 Culture cell

Vero cells (ATCC CCL-81) and WiDr cells (ATCC CCL-218) were obtained from the Microbiology and Immunology Laboratory, Primate Animal Study Center, IPB University, Bogor, Indonesia. Vero and WiDr cells were cultured in DMEM media equipped with 5% FBS and 1% penicillin 100,000 U/I, streptomycin 100,000 mg/L. The cells were then incubated at 37 °C with 5% CO₂ using a CO₂ incubator (Binder Inc. Johnson Ave, Bohemia, USA).

2.9 Analysis of Vero and WiDr cell cytotoxic activity

Cytotoxic activity of bran methanol extract was measured using MTT colorimetric testing (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to measure the number of living cells. Confluent cells were sub-cultured by removing the media in a flask and replaced with 10 mL PBS and 5 mL trypsin (0.125%), then incubated at 37 °C with 5% CO₂ for 10 min. Cells separated from the flask wall were moved into a 15 mL tube and centrifuged at 1500 RPM for 5 min (Flexpin, Tomy Kogyo Co., Ltd, Tagara, Tokyo, Japan). The cells were then counted using a hemacytometer (Blaubrand, Brand Gmbh + Co. Kg, Wertheim, Germany).

Cells were grown into 96 well plates with a density of 5x10³ cells/well and incubated for 24 h at 37 °C. The media was discarded and replaced with new media containing extracts of NFBR, FBROLI, FBRO as much as 100 µL/well and incubated for 48 h at 37 °C. PBS was added to remove the remaining sample that could disturb the reading, and 100 µL of RPMI 1640 media (5% PBS, 1% Penicillin-Streptomycin, NaHCO₃ (2 g/L)) was incorporated to facilitate reading. The MTT reagent dissolved in PBS (5 mg/mL) was added at 10 µL/well and incubated for 4 h at 37 °C. Following the MTT reagent, 100 µL/well ethanol (96%) was added, and absorbance was measured at 595 nm using iMark™ microplate absorbance reader (Bio-Rad Laboratories Inc, Hercules, California, USA). The following formula refers to Matuszewska et al. (2018Matuszewska, A., Jaszek, M., Stefaniuk, D., Ciszewski, T., & Matuszewski, Ł. (2018). Anticancer, antioxidant, and antibacterial activities of low molecular weight bioactive subfractions isolated from cultures of wood degrading fungus Cerrena unicolor. PLoS One, 13(6), e0197044. http://dx.doi.org/10.1371/journal.pone.0197044. PMid:29874240.
http://dx.doi.org/10.1371/journal.pone.0... ) to calculate the ability to inhibit 50% of cell growth by test extract (Equation 2):

2.10 Analysis of gene expression

WiDr cells were plated on a 6-well plate containing DMEM media with an initial density of 5 x 10⁴ cells/well and incubated for 24 h at 37 °C with 5% CO₂. After the incubation, each cell was treated with a concentration of 1700 µg/mL NFBR test extract, 1400 µg/mL FBRO, and 1100 µg/mL FBROLI. 2-well plate contained only WiDr cells (untreated cell) as negative controls (C-). Subsequently, the cells were incubated at 37 °C with 5% CO₂ for 72 h. WiDr cells were then harvested and stored at -20 °C until the next testing process.

RNA extraction of both treated and untreated WiDr cells employed the Zymo Direct-zolTM RNA MiniPrep Plus (USA) protocol. The purity of RNA was checked by measuring the concentration using NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). RNA was converted to cDNA following the SuperScriptTM III Reverse Transcriptase procedure (Invitrogen, Waltham, MA, USA). Relative quantification of gene expression was conducted using the Bio-Rad iQ5^TM Real-Time PCR (Bio-Rad Laboratories Inc. Hercules, California, USA) equipped with SsoFast^TM EvaGreen Supermix (Bio-Rad, USA), nuclease-free water (Bio-Rad, USA), gene primer of caspase apoptosis 7, 8, 9, p53, PCNA proliferation gene, and ACTB gene for normalization.

The Real-Time PCR was performed with 40 cycles consisting of enzyme activity at 95 °C for 30 s, denaturation in 95 °C for 10 s, and annealing for 20 s with optimum annealing temperature adjusted to the condition of the available equipment. Primary sequences and annealing temperatures can be seen in Table 1. The nucleotide sequence and annealing temperature of ACTB, p53, caspace-9, and caspace-7 genes refer to Laila et al. (2020Laila, F., Fardiaz, D., Yuliana, N. D., Damanik, M. R. M., & Dewi, F. N. A. (2020). Methanol extract of Coleus amboinicus (Lour) exhibited antiproliferative activity and induced programmed cell death in colon cancer cell WiDr. International Journal of Food Science, 2020, 9068326. http://dx.doi.org/10.1155/2020/9068326. PMid:32047805.
http://dx.doi.org/10.1155/2020/9068326... ), the caspase-8 gene refers to Borhani et al. (2014Borhani, N., Manoochehri, M., Gargari, S. S., Novin, M. G., Mansouri, A., & Omrani, M. D. (2014). Decreased expression of proapoptotic genes caspase-8 and BCL2-associated agonist of cell death (BAD) in ovarian cancer. Clinical Ovarian and Other Gynecologic Cancer, 7(1-2), 18-23. http://dx.doi.org/10.1016/j.cogc.2014.12.004.
http://dx.doi.org/10.1016/j.cogc.2014.12... ), and the PCNA gene refers to Saigusa et al. (2014).Saigusa, S., Inoue, Y., Tanaka, K., Okugawa, Y., Toiyama, Y., Uchida, K., Mohri, Y., & Kusunoki, M. (2014). Lack of M30 expression correlates with factors reflecting tumor progression in rectal cancer with preoperative chemoradiotherapy. Molecular and Clinical Oncology, 2(1), 99-104. http://dx.doi.org/10.3892/mco.2013.189. PMid:24649315.
http://dx.doi.org/10.3892/mco.2013.189...

Measurement of gene expression used the delta-delta threshold method (2^-ΔΔCT) with the C_T target gene (example p53) normalized with C_T reference (ACTB gene). Sample A was a WiDr cell given rice bran extract, and Sample B was a negative control cell (C-). The following formula refers to Schmittgen & Livak (2008Schmittgen, T. D., & Livak, K. J. (2008). Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3(6), 1101-1108. http://dx.doi.org/10.1038/nprot.2008.73. PMid:18546601.
http://dx.doi.org/10.1038/nprot.2008.73... ) to calculate gene expression (Equation 3):

2.11 Statistical analysis

Data obtained in this experiment were in the form of mean ± standard deviation (SD) for two replications of each sample (n = 2). Data were analyzed using the Generalized Linear Model (GLM) and further tested by Duncan Multiple Range Test (DMRT). The significance value was determined based on a 5% significance level using SPSS 22.0 software (Chicago, IL, USA).

3 Result and discussion

3.1 Phenolic content, flavonoids, and antioxidant activity in fermented black rice bran extract

The phenolic content in rice bran was increased significantly (p < 0.05) at 72 h fermentation time in both R.oligosporus and R. oryzae inoculums (Table 2) by 22.5% and 14.7%, respectively. The increase was caused by enzymes, which were produced by fungi capable of releasing bonds of phenolic compounds in the substrate during the fermentation process. Some fungi enzymes reported to release phenolic in the substrate are β-glucosidase, cellulase, and α-amylase (Chen et al., 2020Chen, Y., Wang, Y., Chen, J., Tang, H., Wang, C., Li, Z., & Xiao, Y. (2020). Bioprocessing of soybeans (Glycine max L.) by solid-state fermentation with Eurotium cristatum YL-1 improves total phenolic content, isoflavone aglycones, and antioxidant activity. RSC Advances, 10(29), 16928-16941. http://dx.doi.org/10.1039/C9RA10344A. PMid:35496929.
http://dx.doi.org/10.1039/C9RA10344A... ).

The phenolic compound in the substrate was known to be bound to glucose or other sugars. Therefore, the production of the β-glucosidase enzyme during the fermentation process by fungus helped in the release of these phenolic bonds-glycons (sugar groups) (Zheng & Shetty, 2000Zheng, Z., & Shetty, K. (2000). Solid-state bioconversion of phenolics from cranberry pomace and role of Lentinus edodes β-glucosidase. Journal of Agricultural and Food Chemistry, 48(3), 895-900. http://dx.doi.org/10.1021/jf990972u. PMid:10725170.
http://dx.doi.org/10.1021/jf990972u... ). The decomposition of these bonds was due to the need for glucose or other sugar groups to support the growth and reproduction of the fungi (Martins et al., 2011Martins, S., Mussatto, S. I., Martínez-Avila, G., Montañez-Saenz, J., Aguilar, C. N., & Teixeira, J. A. (2011). Bioactive phenolic compounds: production and extraction by solid-state fermentation. A review. Biotechnology Advances, 29(3), 365-373. http://dx.doi.org/10.1016/j.biotechadv.2011.01.008. PMid:21291993.
http://dx.doi.org/10.1016/j.biotechadv.2... ; Sánchez, 2009Sánchez, C. (2009). Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnology Advances, 27(2), 185-194. http://dx.doi.org/10.1016/j.biotechadv.2008.11.001. PMid:19100826.
http://dx.doi.org/10.1016/j.biotechadv.2... ). The hydrolysis of these bonds during the fermentation process was also very beneficial to increase the availability of phenolic compounds in the rice bran.

On the contrary, rice bran that was fermented for more than 72 h showed a decrease in phenolic levels. The cause of the suspected decrease was because there was not glucose available in the rice bran. This unavailability of the substrate pushed fungi to degrade the released phenolic compound as a carbon source for its growth. Adetuyi & Ibrahim (2014)Adetuyi, F. O., & Ibrahim, T. A. (2014). Effect of fermentation time on the phenolic, flavonoid and vitamin C contents and antioxidant activities of okra (Abelmoschus esculentus) seeds. Nigerian Food Journal, 32(2), 128-137. http://dx.doi.org/10.1016/S0189-7241(15)30128-4.
http://dx.doi.org/10.1016/S0189-7241(15)... reported that microbes use the available free compounds as a substrate for their growth if the primary substrate for microbial growth is not available.

The phenolic content analysis results also showed that the rice bran fermented by R. oligosporus had an increased content of total phenolic compared to the rice bran fermented by R. oryzae IPBCC 88010. These results were confirmed by the results of the study of Cheng et al. (2013)Cheng, K. C., Wu, J. Y., Lin, J. T., & Liu, W. H. (2013). Enhancements of isoflavone aglycones, total phenolic content, and antioxidant activity of black soybean by solid-state fermentation with Rhizopus spp. European Food Research and Technology, 236(6), 1107-1113. http://dx.doi.org/10.1007/s00217-013-1936-7.
http://dx.doi.org/10.1007/s00217-013-193... , where the highest phenolic content in fermented black soybean was produced from R. oligosporus BCRC 31996 with 2545.3 ± 33.5 (µg/g), whereas R. oryzae showed the result of 2088.3 ± 21.3 (µg/g).

The results of the flavonoid compound analysis demonstrated a significant increase (p < 0.05) at 72 h fermentation time by R. oligosporus from 1.20 mg QE/g (0 h) to 1.54 mg QE/g (72 h) or an increase of 28.3% (Table 2). The bran fermented by R. oryzae did not show a significant increase statistically (p > 0.05) with flavonoid levels ranging from 1.28 mg QE/g (0 h) to 1.30 mg QE/g (72 h). The content difference was suspected due to the fungi type used. Martins et al. (2011)Martins, S., Mussatto, S. I., Martínez-Avila, G., Montañez-Saenz, J., Aguilar, C. N., & Teixeira, J. A. (2011). Bioactive phenolic compounds: production and extraction by solid-state fermentation. A review. Biotechnology Advances, 29(3), 365-373. http://dx.doi.org/10.1016/j.biotechadv.2011.01.008. PMid:21291993.
http://dx.doi.org/10.1016/j.biotechadv.2... reported that the number of bioactive compounds is dependent on the fungi type inoculum, substrate, and fermentation conditions.

In this study, the analysis of antioxidant activity used DPPH as a free radical. The results showed a significant increase (p < 0.05) in antioxidant activity during the fermentation process (Table 2). The percentage increase for rice bran fermented by R. oligosporus was 60.2-72%, whereas rice bran fermented by R. oryzae showed an increase of 59.5-65.8%. Both types of fungi had the same potential to increase antioxidant activity; however, the bran fermented by R. oligosporus showed a higher antioxidant activity. The increase in antioxidant activity was determined by the presence of phenolic acid and flavonoid compounds, as they act as antioxidants by donating hydrogen atoms or transferring electrons (Procházková et al., 2011Procházková, D., Boušová, I., & Wilhelmová, N. (2011). Antioxidant and prooxidant properties of flavonoids. Fitoterapia, 82(4), 513-523. http://dx.doi.org/10.1016/j.fitote.2011.01.018. PMid:21277359.
http://dx.doi.org/10.1016/j.fitote.2011.... ). They showed why fermented rice bran had a higher antioxidant activity (p < 0.05) than non-fermentation based on phenolic content and flavonoid.

3.2 Correlation between phenolic content, total flavonoids, and antioxidant activity in fermented black rice bran extract

The Pearson correlation coefficient value represented the evaluation of the relationship between phenolic content and flavonoids with antioxidant activity in fermented rice bran.

The results showed that phenolic content and antioxidant activity were positively correlated (R = 0.901) with very significant differences (p < 0.01) (Table 3). They also indicated that the range of phenolic content in rice bran played an active role in increasing its antioxidant activity, which was further strengthened by Akoglu’s statement (Akoglu, 2018Akoglu, H. (2018). User’s guide to correlation coefficients. Turkish Journal of Emergency Medicine, 18(3), 91-93. http://dx.doi.org/10.1016/j.tjem.2018.08.001. PMid:30191186.
http://dx.doi.org/10.1016/j.tjem.2018.08... ) that the correlation coefficient of +0.9 and -0.9 interpreted a powerful interplay between the two variables.

In the evaluation results of the flavonoid correlation, the coefficient correlation of flavonoid with antioxidant activity was positively correlated (R = 0.710) with a significant difference (p < 0.05) (Table 3). It indicated that the flavonoid content in fermented rice bran was believed to be 95% able to increase antioxidant activity with a moderate interplay between the two variables. This finding was consistent with Ghasemzadeh & Ghasemzadeh’s statement (Ghasemzadeh & Ghasemzadeh, 2011Ghasemzadeh, A., & Ghasemzadeh, N. (2011). Flavonoids and phenolic acids: role and biochemical activity in plants and human. Journal of Medicinal Plants Research, 5(31), 6697-6703. http://dx.doi.org/10.5897/JMPR11.1404.
http://dx.doi.org/10.5897/JMPR11.1404... ) which said that an increased antioxidant activity was influenced by the number of phenolic and flavonoid content. These compounds significantly contributed to capturing free radicals, reducing agents, and preventing the formation of oxygen singlets.

Hydroxyl groups have been reported to play an active role in capturing free radicals or chelating metal ions. Those hydroxyl groups were bound to the phenolic acid and flavonoid aromatic ring structure (Shahidi & Yeo, 2016Shahidi, F., & Yeo, J. (2016). Insoluble-bound phenolics in food. Molecules, 21(9), 1216. http://dx.doi.org/10.3390/molecules21091216. PMid:27626402.
http://dx.doi.org/10.3390/molecules21091... ; Kumar & Pandey, 2013Kumar, S., & Pandey, A. K. (2013). Chemistry and biological activities of flavonoids: an overview. The Scientific World Journal, 2013, 162750. http://dx.doi.org/10.1155/2013/162750. PMid:24470791.
http://dx.doi.org/10.1155/2013/162750... ). The known mechanism states that the hydroxyl group donates hydrogen atoms or electrons to hydroxyl and peroxynitrite radicals to reduce the action of these radicals and produce a more stable flavonoid radical product (Kumar & Pandey, 2013Kumar, S., & Pandey, A. K. (2013). Chemistry and biological activities of flavonoids: an overview. The Scientific World Journal, 2013, 162750. http://dx.doi.org/10.1155/2013/162750. PMid:24470791.
http://dx.doi.org/10.1155/2013/162750... ). Quercetin and isorhamnetin 7-glucoside of flavonoid content were found in rice bran fermented, which synergistically enhance antioxidant activity (Ardiansyah et al., 2021Ardiansyah, Ariffa, A., Astuti, R. M., David, W., Handoko, D. D., Budijanto, S., & Shirakawa, H. (2021). Non-volatile compounds and blood pressure-lowering activity of Inpari 30 and Cempo Ireng fermented and non-fermented rice bran. AIMS Agriculture and Food, 6(1), 337-359. http://dx.doi.org/10.3934/agrfood.2021021.
http://dx.doi.org/10.3934/agrfood.202102... ).

3.3 Analysis of the cytotoxic activity of fermented black rice bran extract

The sample extracts examined for the cytotoxic activity had the highest results of the phenolic content, flavonoids, and antioxidant activity at 72 h fermentation time. The highest cytotoxic activity in WiDr cells was FBROLI extract with IC₅₀ value 650.7 μg/mL, followed by NFBR IC₅₀ extract 831.8 μg/mL, and FBRO IC₅₀ 873.9 μg/mL (Table 4). The lower IC50 value was shown in the FBROLI extract results, which implies that a 650.7 μg/mL concentration could inhibit 50% growth of the WiDr cell culture population.

The high cytotoxic activity at FBROLI extracts due to R. oligosporus fungus released more phenolic and flavonoid content from their bonds into the substrate. The free form of available content was responsible for antioxidants. The amount of free functional compounds was positively correlated with increased antioxidant activity, translated as an increase in anticancer bioactivity (Chen et al., 2015Chen, M., Meng, H., Zhao, Y., Chen, F., & Yu, S. (2015). Antioxidant and in vitro anticancer activities of phenolics isolated from sugar beet molasses. BMC Complementary and Alternative Medicine, 15(1), 313. http://dx.doi.org/10.1186/s12906-015-0847-5. PMid:26347338.
http://dx.doi.org/10.1186/s12906-015-084... ). The effectiveness of phenolic acids and flavonoids contents as anticancer was also determined by the position and number of free hydroxyl groups, aromatic ring structures, and unsaturated fatty acid chain bonds (Anantharaju et al., 2016Anantharaju, P. G., Gowda, P. C., Vimalambike, M. G., & Madhunapantula, S. V. (2016). An overview on the role of dietary phenolics for the treatment of cancers. Nutrition Journal, 15(1), 99. http://dx.doi.org/10.1186/s12937-016-0217-2. PMid:27903278.
http://dx.doi.org/10.1186/s12937-016-021... ).

Cytotoxic activity of rice bran extracts on normal Vero cells showed a lower cytotoxic activity, as seen on the higher IC₅₀ values of Vero cells compared to WiDr cells (Table 4). It indicated that rice bran extract was more effective in inhibiting the proliferation of WiDr cells than Vero cells. In addition, rice bran extract was also proved safe against normal human cells.

In this study, the cytotoxic activity of rice bran extracts on WiDr cells was categorized as weak. The determination of cytotoxic activity was based on the statement of Atjanasuppat et al. (2009)Atjanasuppat, K., Wongkham, W., Meepowpan, P., Kittakoop, P., Sobhon, P., Bartlett, A., & Whitfield, P. J. (2009). In vitro screening for anthelmintic and antitumour activity of ethnomedicinal plants from Thailand. Journal of Ethnopharmacology, 123(3), 475-482. http://dx.doi.org/10.1016/j.jep.2009.03.010. PMid:19473794.
http://dx.doi.org/10.1016/j.jep.2009.03.... that the activity of an extract of natural ingredients was determined from the IC₅₀ value, where ≤ 20 μg/mL was considered as active; > 20-100 μg/mL as moderate; > 100-1000 μg/mL as weak; and > 1000 μg/mL as inactive.

3.4 Gene expression analysis of fermented black rice bran extract

The results depicted an increased p53 gene expression in WiDr cells treated with NFBR, FBRO, and FBROLI extracts (Figure 1A). The highest p53 gene expression was shown in WiDr cells, which were given FBROLI extract. This increase was presumably due to the higher number of phenolic and flavonoid contents available. Thus, it had more potential to have an increased p53 gene expression compared to FBRO and NFBR treatments. The potential of phenolic and flavonoid contents in regulating gene expression associated with tumor control was also widely reported (Xavier et al., 2011Xavier, C. P., Lima, C. F., Rohde, M., & Pereira-Wilson, C. (2011). Quercetin enhances 5-fluorouracil-induced apoptosis in MSI colorectal cancer cells through p53 modulation. Cancer Chemotherapy and Pharmacology, 68(6), 1449-1457. http://dx.doi.org/10.1007/s00280-011-1641-9. PMid:21479885.
http://dx.doi.org/10.1007/s00280-011-164... ; Zhong et al., 2010Zhong, Y., Krisanapun, C., Lee, S. H., Nualsanit, T., Sams, C., Peungvicha, P., & Baek, S. J. (2010). Molecular targets of apigenin in colorectal cancer cells: involvement of p21, NAG-1 and p53. European Journal of Cancer, 46(18), 3365-3374. http://dx.doi.org/10.1016/j.ejca.2010.07.007. PMid:20709524.
http://dx.doi.org/10.1016/j.ejca.2010.07... ).

More than 50% of cancer patients are marked by mutations in the p53 gene (Basu, 2018Basu, A. K. (2018). DNA damage, mutagenesis and cancer. International Journal of Molecular Sciences, 19(4), 970. http://dx.doi.org/10.3390/ijms19040970. PMid:29570697.
http://dx.doi.org/10.3390/ijms19040970... ). It supported the hypothesis of the reason why the p53 gene in WiDr colon cancer cells shown by negative control (C-) (Figure 1A) was relatively lower than other treatments, albeit it was not statistically significant (p = 0.113). p53 gene expression testing is often done to detect cancer cells as it plays a vital role in stopping the development of progressive cancer.

The increased p53 gene expression positively affected another apoptotic marking gene expression, namely caspase-9 (Figure 1B), although it was not statistically significant (p = 0.153). The increase in caspase-9 expression was due to active p53 inducing the transcription of puma and noxa BH3 proteins, thereby activating Bax, another pro-apoptotic family (Ashkenazi, 2008Ashkenazi, A. (2008). Targeting the extrinsic apoptosis pathway in cancer. Cytokine & Growth Factor Reviews, 19(3-4), 325-331. http://dx.doi.org/10.1016/j.cytogfr.2008.04.001. PMid:18495520.
http://dx.doi.org/10.1016/j.cytogfr.2008... ). Bax in the cytoplasm enters the mitochondrial membrane by damaging the membrane, resulting in membrane permeability changes and promoting the release of cytochromes c from the mitochondria. The release of cytochrome c factors from mitochondria into the cytoplasm are able to activate caspase-9 by collaborating cytochromes c factors with Apaf-1 apoptotic proteins (Ashkenazi, 2008Ashkenazi, A. (2008). Targeting the extrinsic apoptosis pathway in cancer. Cytokine & Growth Factor Reviews, 19(3-4), 325-331. http://dx.doi.org/10.1016/j.cytogfr.2008.04.001. PMid:18495520.
http://dx.doi.org/10.1016/j.cytogfr.2008... ). Caspase-9 is commonly used to detect cancer inhibitory mechanisms of intrinsic pathways and are known as mitochondrial pathways.

The results also showed increased caspase-8 gene expression in the NFBR, FBRO, and FBROLI treatments (Figure 1C). Caspase-8 is an apoptotic marker involved in the extrinsic pathway apoptotic cascade. The increase of caspase-8 gene expression was presumably due to the return of increased sensitivity of the WiDr cells that were resistant to TRAIL resulting in apoptosis. A factor believed to be the cause of cancer cell resistance to TRAIL was the deficiency of mortality receptors on the cell surface (Jin et al., 2004Jin, Z., McDonald, E. R. III, Dicker, D. T., & El-Deiry, W. S. (2004). Deficient tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor transport to the cell surface in human colon cancer cells selected for resistance to TRAIL-induced apoptosis. The Journal of Biological Chemistry, 279(34), 35829-35839. http://dx.doi.org/10.1074/jbc.M405538200. PMid:15155747.
http://dx.doi.org/10.1074/jbc.M405538200... ) or the low expression of DR4 and DR5 mortality receptors, and loss of function as a receptor of mortality due to mutations (Van Geelen et al., 2004Van Geelen, C. M., Vries, E. G., & Jong, S. (2004). Lessons from TRAIL-resistance mechanisms in colorectal cancer cells: paving the road to patient-tailored therapy. Drug Resistance Updates, 7(6), 345-358. http://dx.doi.org/10.1016/j.drup.2004.11.002. PMid:15790545.
http://dx.doi.org/10.1016/j.drup.2004.11... ; Zhang & Fang, 2005Zhang, L., & Fang, B. (2005). Mechanisms of resistance to TRAIL-induced apoptosis in cancer. Cancer Gene Therapy, 12(3), 228-237. http://dx.doi.org/10.1038/sj.cgt.7700792. PMid:15550937.
http://dx.doi.org/10.1038/sj.cgt.7700792... ). WiDr cells were reported to be resistant to TRAIL-induced apoptosis. One of the causes was the low expression of TRAIL receptors (Zhang et al., 2019Zhang, B., Liu, B., Chen, D., Setroikromo, R., Haisma, H. J., & Quax, W. J. (2019). Histone deacetylase inhibitors sensitize TRAIL-induced apoptosis in colon cancer cells. Cancers, 11(5), 645. http://dx.doi.org/10.3390/cancers11050645. PMid:31083396.
http://dx.doi.org/10.3390/cancers1105064... ). It strengthens the suspicion why the result in C- (Figure 1C) showed a low caspase-8 gene expression compared to other treatments, even though the difference was not statistically significant (p = 0.175).

The return of increased sensitivity was believed to be the active role of the bioactive compounds present in rice bran. It supported the result of Kong et al. (2009)Kong, C. K., Lam, W. S., Chiu, L. C., Ooi, V. E., Sun, S. S., & Wong, Y. S. (2009). A rice bran polyphenol, cycloartenyl ferulate, elicits apoptosis in human colorectal adenocarcinoma SW480 and sensitizes metastatic SW620 cells to TRAIL-induced apoptosis. Biochemical Pharmacology, 77(9), 1487-1496. http://dx.doi.org/10.1016/j.bcp.2009.02.008. PMid:19426686.
http://dx.doi.org/10.1016/j.bcp.2009.02.... , where the administration of cycloartenyl ferulate, a bioactive compound of rice bran, could increase the sensitivity of colon cancer cells SW480 to TRAIL-induced apoptosis. It was proved by the increase in the number of DR4 and DR5 mortality receptors and the activity of caspase-8 and 10, thus increasing effector caspase 3,6 and 7.

In the caspase activation, both intrinsic and extrinsic pathways end at effector caspase-7. Caspase-7 is significant as an executor of apoptosis due to its involvement in the fragmentation of DNA chromosomes, cross-linking of proteins, and degradation of core and cytoskeleton proteins (Elmore, 2007Elmore, S. (2007). Apoptosis: a review of programmed cell death. Toxicologic Pathology, 35(4), 495-516. http://dx.doi.org/10.1080/01926230701320337. PMid:17562483.
http://dx.doi.org/10.1080/01926230701320... ). The results showed increased caspase-7 gene expression in the NFBR, FBRO, and FBROLI treatments (Figure 1D). However, it did not show statistically significant differences with controls (p = 0.646). This result demonstrated that one of the mechanisms inhibiting the development of WiDr colon cancer cells by rice bran was with the help of the apoptotic pathway.

The activation of caspase-7 originates from a linear pathway. Cytochrome c with Apaf-1 activates caspase-9, in turn activates the caspase-7, and caspase 8 releases molecules in the cytoplasm, resulting in cleaving and ultimately stimulates the effector caspase-7 (Ashkenazi, 2008Ashkenazi, A. (2008). Targeting the extrinsic apoptosis pathway in cancer. Cytokine & Growth Factor Reviews, 19(3-4), 325-331. http://dx.doi.org/10.1016/j.cytogfr.2008.04.001. PMid:18495520.
http://dx.doi.org/10.1016/j.cytogfr.2008... ). This linear pathway strengthened the reason why WiDr cells that were given extracts of NFBR, FBRO, and FBROLI also showed an increase in expression of the caspase-7 gene. This result was in accordance with the evaluation results in both genes of caspase-9 and caspase-8, which also showed increased expression. It was confirmed by the research results Lim et al. (2007)Lim, D. Y., Jeong, Y., Tyner, A. L., & Park, J. H. (2007). Induction of cell cycle arrest and apoptosis in HT-29 human colon cancer cells by the dietary compound luteolin. American Journal of Physiology-Gastrointestinal and Liver Physiology, 292(1), G66-G75. http://dx.doi.org/10.1152/ajpgi.00248.2006. PMid:16901994.
http://dx.doi.org/10.1152/ajpgi.00248.20... , where it was found that the flavonoid content of the luteolin compound did stimulate not only the activation of the initiator caspase-9, but also the effector caspase-3 and caspase-7 on HT-29 cells. Turktekin et al. (2011)Turktekin, M., Konac, E., Onen, H. I., Alp, E., Yilmaz, A., & Menevse, S. (2011). Evaluation of the effects of the flavonoid apigenin on apoptotic pathway gene expression on the colon cancer cell line (HT29). Journal of Medicinal Food, 14(10), 1107-1117. http://dx.doi.org/10.1089/jmf.2010.0208. PMid:21548803.
http://dx.doi.org/10.1089/jmf.2010.0208... also reported that the administration of the flavonoid content of apigenin to colon HT-29 cancer cells induced apoptosis by regulating the increased expression of caspase-8 and 3.

In this study, activation of effector caspases on WiDr cells might be due to bran bioactive compounds' involvement in reactivating functions key of apoptotic regulators. PCNA (Proliferating Cell Nuclear Antigen) is widely used to detect cancer because of its involvement in replicating DNA. The PCNA activity was found to be higher in colorectal carcinoma patients. The PCNA activity could be expressed 3 to 4 times higher than the normal tissue (Poosarla et al., 2015Poosarla, C., Ramesh, M., Ramesh, K., Gudiseva, S., Bala, S., & Sundar, M. (2015). Proliferating cell nuclear antigen in premalignancy and oral squamous cell carcinoma. Journal of Clinical and Diagnostic Research, 9(6), ZC39-ZC41. PMid:26266215.). The higher the stage of cancer, the higher the PCNA activity. The resulting study showed that the PCNA gene was expressed significantly higher (p < 0.05) in C- compared to NFBR, FBRO, and FBROLI (Figure 1E).

On the other hand, the evaluation results on WiDr cells treated with NFBR, FBRO, and FBROLI extracts showed relatively lower PCNA gene expression than C- (Figure 1E). Decreased expression of PCNA genes was likely due to the binding of PCNA by p21 (cyclin-dependent kinase inhibitor) in its operational condition. Consequently, DNA replication was stopped, and cell cycle progression was inhibited (Singh et al., 2017Singh, S. K., Banerjee, S., Acosta, E. P., Lillard, J. W., & Singh, R. (2017). Resveratrol induces cell cycle arrest and apoptosis with docetaxel in prostate cancer cells via a p53/p21WAF1/CIP1 and p27KIP1 pathway. Oncotarget, 8(10), 17216-17228. http://dx.doi.org/10.18632/oncotarget.15303. PMid:28212547.
http://dx.doi.org/10.18632/oncotarget.15... ). The inhibition of PCNA activity was very beneficial as it inhibited growth in cancer cells. Some researchers widely reported the activity of this p21 protein, regulated by the p53 tumor suppressor gene. Hence, PCNA was freely regulated by an activated p53 gene (Singh et al., 2017Singh, S. K., Banerjee, S., Acosta, E. P., Lillard, J. W., & Singh, R. (2017). Resveratrol induces cell cycle arrest and apoptosis with docetaxel in prostate cancer cells via a p53/p21WAF1/CIP1 and p27KIP1 pathway. Oncotarget, 8(10), 17216-17228. http://dx.doi.org/10.18632/oncotarget.15303. PMid:28212547.
http://dx.doi.org/10.18632/oncotarget.15... ), and bioactive compounds in rice bran were believed to take over in restoring the normal function of p53.

4 Conclusions

The increase in the bioactive compound was influenced by the type of inoculum and the fermentation time. The best results with the highest phenol content, flavonoids, and antioxidant activity were obtained in the fermented FBROLI extract within 72 h. It also had the highest inhibition of WiDr colon cancer cell growth. Its inhibitory mechanism was observed, including suppression of PCNA proliferation gene expression and increased expression of apoptotic genes through extrinsic and intrinsic pathways. The highest increase in apoptotic gene expression was obtained from FBROLI extracts.

References

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