IP₆ treatment showed marked growth inhibition and enhanced apoptotic cell death of human colorectal adenocarcinoma HT-29 cells in a dose- and time-dependent manner. Initially, we proved strong evidence that IP₆ showed an increase of early apoptosis in HT-29 cells using Annexin V assay. IP₆ elicited its growth inhibitory effects by increasing the total number of apoptosis; 9%, 22% and 41% following 9.5, 12.0 or 14.5 μg/mL of IP₆, respectively, relative to the control in a dose-dependent manner after 24 h of incubation [21]. Moreover, incubation with IP₆ (9.5 μg/mL) also markedly increased the total number of apoptoses: 12%, 27% and 38% after 24, 48 or 72 h, respectively, relative to the control in a time-dependent manner [21]. However, the associated signaling pathways and cellular events controlling apoptosis were varied depending on the types of cancer and not well defined. We further examined the molecular mechanism by which IP₆ induces apoptotic cell death in HT-29 cells at mRNA and protein levels.

Apoptosis can be triggered by both internal and external stimuli, causing deregulated mechanisms in different cancers. Induction of apoptosis is important in cancer treatment. To investigate the molecular mechanism of apoptosis induced by IP₆ in HT-29 cells, the mRNA expression levels of pro- and anti-apoptotic genes, Bax and Bcl-xl respectively were examined. The melting curve analysis of the products showed a single peak for each target gene (data not shown). Each gene of interest resulted in an efficiency of 1.9–2.1 (slope of −2.9 to −3.5) over a minimum of three-log concentration range. Furthermore, the similar amplification efficiency of gene of interests with housekeeping genes was confirmed by assuring a slope of less than 0.1 when ΔC_T (the difference between target gene C_T and housekeeping gene C_T) was plotted against log concentration of RNA (data was not shown).

Additionally, 5% agarose gel electrophoresis of the PCR product exhibited the presence of a single product band and the absence of a primer dimer band. The change in mRNA expression was analyzed using ΔΔC_T method with β-actin as housekeeping gene. Figure 1 showed that IP₆ treatment of cells at various doses (9.5, 12.0 or 14.5 μg/mL) for 72 h marked a noticeable alteration in Bax and Bcl-xl mRNA level compared to control (untreated HT-29 cells) (p < 0.05). Notably, IP₆ upregulated the transcript of the pro-apoptotic gene, Bax and downregulated the transcription of the anti-apoptotic gene, Bcl-xl by several fold. The expression level of Bax in HT-29 cells incubated with 9.5, 12.0 or 14.5 μg/mL of IP₆ for 72 h increased by 3.18, 6.78 or 7.76 fold respectively as compared with control (Figure 1A). In contrast, the expression level of Bcl-xl in HT-29 cells incubated with 9.5, 12.0 or 14.5 μg/mL of IP₆ for 72 h reduced by 0.56, 0.34 or 0.18 fold, respectively (Figure 1B). Collectively, these results show a marked increase in Bax and decrease in Bcl-xl expression that were found to be dose-dependent.

Caspase-8 is an initiator, while caspase-3 is an executioner, where activation of both caspases represent important biochemical markers that have been identified in cells favoring apoptosis [23]. Figure 2 summarized the gene expression changes of caspase-8 and -3. It was clearly shown that IP₆ upregulated the transcript of caspase-8 and -3 at mRNA level by several folds. The expression levels of caspase-8 gene in HT-29 cells treated with 9.5, 12.0 or 14.5 μg/mL of IP₆ for 72 h were significantly (p < 0.05) increased by 6.38, 10.5 or 11.4 fold, respectively, as compared to the levels in control (Figure 2A). Similarly, the expression levels of caspase-3 gene in HT-29 cells incubated with 9.5, 12.0 or 14.5 μg/mL of IP₆ for 72 h increased by 4.63, 5.17 or 11.6 fold, respectively as compared to the levels in control (Figure 2B). Together, these data suggest that IP₆ caused a marked increase in apoptosis, which was accompanied by upregulation of caspase-8 and caspase-3 expression at mRNA level.

In order to establish that the changes in mRNA levels monitored by RT-PCR were reflecting changes at the protein level and for further confirmation for translation of apoptosis related genes’ mRNAs into apoptosis related proteins, western blot analysis was performed in parallel. As mentioned previously, Bax was upregulated and Bcl-xl was downregulated at the mRNA level after incubation with IP₆ in HT-29 cells as shown in Figure 1. Likely, similar changes can be observed at the protein level as well. Pro-apoptotic, Bax protein level, which was detected as a 23 kDa band, was significantly (p < 0.05) increased after IP₆ treatment of HT-29 cells in a dose-dependent manner (Figure 3A). The protein expression level of Bax of HT-29 after incubation with 9.5, 12.0 or 14.5 μg/mL for 72 h was increased by 2.33, 3.65 or 4.3 fold respective to the control. Furthermore, Bcl-xl which was detected as a 30 kDa band was reduced by 0.21, 0.19 or 0.17 fold after incubation with 9.5, 12.0 or 14.5 μg/mL of IP₆, respectively, relative to the control (Figure 3B).

To examine the translational confirmation of caspase-8 and -3, western blot analysis for protein assay was performed. Interestingly, caspase-8 and -3 protein expression, detected as 55 and 32 kDa bands respectively, were markedly increased after IP₆ treatment of HT-29 cells in a dose-dependent manner as compared to the control (Figure 4A,B). We clearly observed that both caspases were significantly (p < 0.05) upregulated by 2.13, 2.7 or 4.37 fold for caspase-8 and 1.24, 1.28 or 2.66 fold for caspase-3 relative to their respective control following the IP₆ treatments (9.5; 12.0 or 14.5 μg/mL). The results showed that the increased caspase-8 was observed in a dose-dependent manner (Figure 4A). Notably, IP₆ has shown significantly (p < 0.05) increased caspase-3 protein expression only after incubation with the highest dose of IP₆, 14.5 μg/mL (Figure 4B). The activation of caspase protein content was due to the proteolytic cleavage of caspases occurred during apoptosis because these changes were observed after the induction of apoptosis, which in this case triggered after 72 h of IP₆ treatment. To further confirm the enzymatic activity of caspase-8 and -3, the colorimetric measurement of respective substrates of caspases were performed. HT-29 cells were incubated with IP₆ for 72 h at the concentrations of 9.5, 12.0 or 14.5 μg/mL. Notably, we observed a significant (p < 0.05) increase in the activities of both caspase-8 and -3 following the increment of the dose of IP₆, demonstrating a dose-dependent manner (Figure 4C,D). Subsequently, addition of inhibitors of caspase-8 and -3 showed reduced activities were observed. The combination of inhibitor and IP₆ showed diminished activities of caspase-8 and -3. Collectively, these results show an interaction between changes of mRNA and protein levels of caspases after incubation of IP₆ in HT-29 cells and activation of caspases were induced by IP₆ as we clearly observed the increment of initiator caspase-8 and executioner caspase-3.

TRI reagent, 1% agarose gel, tris-borate-EDTA (TBE) buffer and specific primers were purchased from Sigma (St. Louis, MO, USA). Gene Amp Gold RNA PCR Core Kit was bought from Applied Biosystems (Foster City, CA, USA). Agarose gel electrophoresis materials were purchased from 1st Base, Kuala Lumpur, Malaysia. Kapa Sybr Fast qPCR kit (Kapa Biosystems, Boston, MA, USA), AllPrep DNA/RNA/Protein Mini Kit and QIAshredder homogenizer were bought from Qiagen (Dusseldorf, Germany). Western blotting reagents were purchased from BioRAD (Hercules, CA, USA). Dimethylformamide (DMF) was purchased from Fermentas (Vilnius, Lithuania).

Freshly milled raw rice bran samples from mixed local varieties were kindly supplied by the BERNAS Milling Plant (Selangor, Malaysia). Extraction and isolation of IP₆ was initially carried out according to the established methods in our laboratory [20,36].

Cell lines used in this study, HT-29 (human colorectal cancer cell line) was bought from American Type Culture Collection (ATCC) (Manassas, VA, USA) and the cells were grown in DMEM media with the following supplements; 10% (v/v) fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/mL streptomycin. Cells were grown in sterile cell culture flask at 37 °C in the presence of 5% carbon dioxide (CO₂). All steps were performed aseptically in a biosafety hood using sterile equipment.

HT-29 cells were pre-incubated at a density of 1 × 10⁵ cells in 25 cm² culture flasks for 24 h. The culture medium was replaced with fresh aliquots containing IP₆ compounds at three different concentrations based on the IC₅₀ values: (9.5, 12.0 or 14.5 μg/mL) that were initially carried out in our laboratory [21] and incubated for another 72 h. TRI-reagent (Sigma, St. Louis, MO, USA) was used to extract total RNA directly from cultured cells, according to the manufacturer’s instructions. Then 1 μg of the total RNA sample was reverse transcribed using the Gene Amp Gold RNA PCR Core Kit (Applied Biosystem, Foster, CA, USA) in a final reaction volume of 20 μL, according to the manufacturer’s protocol. The reverse transcription reaction was carried out at 42 °C for 30 min in an authorized thermal cycler (Eppendorf, Hamburg, NY, USA), followed by a 10 min step at 99 °C to denature the enzyme and then it was cooled to 4 °C.

The primers used are listed in Table 1, where the nucleotide numbers indicate the primer location in the corresponding sequences of human origin obtained from National Center for Biotechnology Information Gene Bank. The specific primers were subsequently validated for the specificity of amplification, amplification efficiency over a concentration range and consistency with amplification efficiency of housekeeping gene. All primers used for qRT-PCR were commercially obtained from Sigma (St. Louis, MO, USA). The mRNA levels of Bax, Bcl-xl, Caspase-8 and Caspase-3 were assayed using Kapa Sybr Green Fast qPCR kit (Kapa Biosystems, Boston, MA, USA) and was performed in a reaction volume of 20 μL, according to the manufacturer’s instructions. Real-time PCR was run according to the following conditions: (i) PCR activation at 95 °C for 20 s; (ii) denaturation at 95 °C for 3 s and (iii) annealing/extension at 55–60 °C for 20 s. All samples and controls were run in triplicate on a Mastercycler realplex system (Eppendorf, Hamburg, NY, USA). The quantitative RT-PCR data was analyzed using a comparative threshold (C_T) method, and the fold inductions of the samples were compared with the untreated samples. β-actin was used as an internal reference gene to normalize the expression of the target genes.

Agarose gel (1% (w/v)) was prepared by adding 0.5 g of agarose to 50 mL of 1× TBE buffer (121.1 g/L Tris Base, 55.0 g/L Boric acid, 500 mM of EDTA (pH 8)). 10 μL of each sample was mixed with 1.0 μL of loading dye [10 mM Tris-HCl (pH 7.6), 0.03% bromophenol blue, 0.03% xylene cyanol, 60% glycerol, 60 mM EDTA]. 10 μL of the appropriate marker was loaded into the first well, and the electrophoresis unit was run for 1 h at 120 V and 300 mA. The gel was stained in 0.2 μg/L of ethidium bromide solution for 20 min and destained in distilled water for 5 min. The gel was viewed and photographed under transluminent UV light in a chemiluminescence imager at 302 nm wavelength.

Briefly, HT-29 cells were grown in a monolayer in cell culture flasks for 24 h. The culture medium was replaced with fresh aliquots containing IP₆ compounds at three different concentrations; (9.5, 12.0 or 14.5 μg/mL) and incubated for another 72 h. After treatment, cells were collected and transferred to a new RNase-free tube and centrifuged at 1500 rpm for 5 min. Then extraction of total protein from human cells was performed using AllPrep DNA/RNA/Protein Mini Kit according to the manufacturer’s protocols (Qiagen, Duesseldorf, Germany). Protein concentration was determined by the Bradford assay, according to the manufacturer’s protocol (BioRAD, Berkeley, CA, USA). The protein (50 μg) was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a piece of PVDF membrane using transfer buffer (25 mM Tris-base, 190 mM glycine, 20% (v/v) methanol; pH 8.3). After transfer, the PVDF membrane was blocked at room temperature with blocking solution (25 mM Tris-base, 0.3 M NaCl, 5% Milk Diluent) (BioRAD, Berkeley, CA, USA) for 30 min. After blocking, the membrane was incubated for overnight with primary antibodies, followed by 2 h with secondary antibodies in Tris-buffered saline (TBS) and 0.5% Tween. Mouse anti-human Bax, Bcl-xl, caspase-8, caspase-3 and β-actin antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) were used at a 1:1000 dilution as the primary antibodies, while alkaline phosphatase-labeled goat anti-mouse antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used at a 1:10,000 dilution as secondary antibody. The membrane was then exposed and protein bands were detected using developing solution for alkaline phosphatase conjugated antibodies consisted of 10 mL alkaline phosphatase buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl₂; pH 9.5), 33 μL BCIP (0.5 g 5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt (Fermentas, Lithuania, EU) in 10 mL of 100% (v/v) dimethylformamide (DMF)), and 66 μL NBT (0.75 g nitroblue tetrazolium chloride (Fermentas, Lithuania, EU) in 10 mL of 70% (v/v) DMF). The reaction was stopped when the desired protein band appeared. Densitometric analysis of band intensities obtained from Western blotting experiments were carried out using ImageJ Software (National Institute of Health, NIH, Bethesda, MD, USA).

The protease activity of caspases-3 and 8, in HT-29 cells, was assessed using a colorimetric assay kit (Sigma Aldrich, St. Louis, MO, USA) based on spectrophotometric detection of the caspase enzymes after cleavage from the labeled substrate. About 3 × 10⁶ HT-29 cells were treated with IP₆ at the concentrations of (9.5, 12.0 or 14.5 μg/mL) and incubated for 72 h. Then, the cells were centrifuged for 5 min at 2000 rpm to remove the media. The cells were then washed two times with PBS and centrifuged at 2000 rpm for 5 min. The cell pellets were lysed by the addition of 50 μL cold prepared lysis buffer containing 0.5 μL DTT and 0.25 μL PMSF, mixed well, and incubated on ice for exactly 1 h. During this time, tubes were vortexed with vibration 3–4 times for 10 s each time. The resulting cell lysate was centrifuged for 1 min at 10,000 rpm at 4 °C, and the supernatant was collected. Briefly, the reaction mixture (total volume, 100 μL) containing 30 μL of cell lysate and 10 μL of the acetyl-Ile-Glu-Thr-Asp-p-nitroaniline (caspase 8 substrate) and acetyl-Asp-Glu-Val-Asp-p-nitroanilide (caspae 3 substrate) (final concentration, 200 μM) in assay buffer, and the assay was carried out in a 96-well plate. The mixtures were incubated for 90 min at 37 °C and the absorbance was read at 405 nm using a Universal Microplate Reader (Bio-TEK. Instrument. Inc., Winooski, VT, USA).

Data were expressed as the mean ± standard deviation (SD) and statistically analyzed using SPSS version 20 (SPSS Inc., Chicago, IL, USA), with a one-way ANOVA with Tukey’s test [37,38] and a significance level of p < 0.05.