Red rice bran polyphenols suppress invasive properties of HepG2 cells, possibly through Wnt/β-catenin-mediated EMT reversal

Chemicals and reagents

Ethanol was an analytical grade of RCI Labscan Limited (Thailand); Gallic acid, (±)-Catechin hydrate, dimethylsulfoxide (DMSO), Folin-ciocalteau reagent, sodium carbonate (Na2CO3) were purchased from Sigma-Aldrich (USA); Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), and phosphate buffer solution (PBS) were purchased from Caisson Laboratories (USA); fetal bovine serum (FBS) was purchased from Capricorn Scientific (Germany); resazurin sodium was purchased from Himedia (India); sodium hydroxide, aluminum chloride, and sodium nitrite were obtained from Ajax Finechem Pty Ltd (Australia). All reagents were analytical grade.

Plant materials and preparation

Rice bran (10 kg), Red Jasmine rice or Hawm Dawk Mali Daeng in Thai, from Chiang Mai, Thailand, was macerated with 40% (v/v) ethanol in water at room temperature (RT), and the supernatant was collected after 7 days. The remaining bran was macerated again, and this procedure was repeated twice. Then, the collected supernatants were combined, filtered through Whatman filter paper No.1 (Whatman, UK), and concentrated in a rotary evaporator and a freeze dryer. Red rice bran extract (RRBE) was obtained, weighed, and stored at -20°C until use.

Analysis of anti-cancer effects

Cell culture

The human hepatoma cell line HepG2 and the mouse hepatocyte cell line BNL CL2 were purchased from the American Type Culture Collection (ATCC, USA). HepG2 cells were cultured in MEM (Minimum Essential Medium). BNL CL2 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium). Both media were supplemented with 10% inactivated fetal bovine serum (FBS). Both cell lines were maintained at 37 °C in a 5% CO₂ atmosphere.

Resazurin cell viability assay

HepG2 cells (2x10⁶ cells/well) and BNL CL2 cells (1.2x10⁶ cells/well) were seeded in 6-well plates and incubated overnight at 37 °C in an atmosphere of 5% CO₂. HepG2 cells were treated with 3 mL of RRBE in completed media at concentrations ranging from 62.5 to 375 μg/mL; BNL CL2 cells were treated with 3 mL of RRBE in completed media at concentrations ranging from 62.5 to 500 μg/mL. Both cell lines were further incubated for 72 h (for selective effects). In addition, HepG2 cells at the same density were treated with RRBE at equal concentrations and incubated for 24, 48, and 72 h (for dose- and time-dependent effects). The untreated cells were used as the control group. After the removal of the old media, 2 mL of fresh media containing 0.5 mM resazurin (Himedia laboratories, India) was added and then incubated for 2 h. Fluorescence intensity at 488 nm (excitation) and 570 nm (emission) was measured, and the percentage of cell survival was calculated by the following formula:

T = RFU of treated cells with RRBE

C = RFU of control cells with only media

T₀ = RFU of cells before the addition of RRBE (Day₀ plate)

(RFU is relative fluorescence unit)

Cell cycle distribution analysis

HepG2 cells (2x10⁶ cells/well) were seeded in 6-well plates and incubated overnight at 37 °C in an atmosphere of 5% CO₂. Thereafter, HepG2 cells were treated with 3 mL of 375 μg/mL RRBE and further incubated for 24, 48, and 72 h. The untreated cells were used as the control group. The cells were harvested, pelleted by centrifugation, and permeabilized with 80% ice-cold ethanol at -20 °C. Next, permeabilized cells in a single-cell suspension were stained with propidium iodide (PI)/RNase staining buffer (BD bioscience, USA) at RT for 30 min. These stained cells were collected using a flow cytometer (Becton-Dickinson FACScan, BD Biosciences, SanJose, USA) and analyzed for cell cycle phases with ModFit LT^TM trial version (Verity Software House, USA, available at https://www.vsh.com/products/mflt/). The sub-G1 population was calculated to estimate the apoptotic cell population

Cell migration assay

Scratch assay

HepG2 cells (2x10⁶ cells/well) were seeded in 6-well plates and incubated overnight at 37 °C in an atmosphere of 5% CO₂. Next, cell monolayers were scratched with a sterile 200 μL pipette tip, creating the gap area, and then incubated with 3 mL of RRBE at concentrations of 125 and 167 μg/mL in MEM with 2% FBS. The untreated cells were used as the control group. The gap area was captured at 0, 24, 48, and 72 h and measured in μm² by using the T.bar program (Nikon Eclipse TS100). The migration ability of cells, with or without treatment, was quantified by the change in gap area over time relative to the initial gap area at time point 0 h. The migration ability of untreated cells at 24 hours was used as a reference point with a value of 1, and the migration ability of untreated cells at 48 and 72 h was determined relative to this reference point.

Transwell migration assay

HepG2 cells (2x10⁶ cells/well) were seeded in 6-well plates and incubated overnight at 37 °C in an atmosphere of 5% CO₂. The cells were then treated with 3 mL of RRBE at concentrations of 125 and 167 μg/mL in MEM with 10% FBS for 24 h. The untreated cells were used as the control group. The cells were collected and seeded onto the upper compartment of the transwell inserts (24 mm Transwell with 8.0 μm pore polycarbonate membrane insert sterile, Corning, USA) in MEM with 2% FBS. The lower compartment contained MEM with 10% FBS as chemoattractants. After 24 h incubation, non-migrated cells on the upper surface of the insert membrane were removed. The migrated cells that moved and attached to the lower surface of the insert membrane were fixed with 3% paraformaldehyde for 15 min and stained with crystal violet for 25 min. The total number of migrated cells in each of the random fields within an insert was counted under an inverted microscope (Nikon Eclipse TS100) and averaged.

Western blot analysis

HepG2 cells (2x10⁶ cells/well) were seeded in 6-well plates and incubated overnight at 37 °C in an atmosphere of 5% CO₂. Thereafter, HepG2 cells were treated with 3 mL of RRBE at concentrations of 125 and 167 μg/mL. The untreated cells were used as the control group. After further incubation for 24, 48, and 72 h, the cells were harvested, and total cell lysates were prepared. HepG2 cell lysates containing 50 μg of total protein were separated by SDS-PAGE on 7.5% polyacrylamide gels. After electrophoresis with SDS-PAGE, the separated proteins from the gel were transferred onto the immobilon-FL PVDF membrane (Merck Millipore, USA) and immunoblotted with anti-E-cadherin polyclonal antibody (Merck Millipore, Germany), anti-β-catenin polyclonal antibody (Merck Millipore, Germany), anti-Vimentin polyclonal antibody (Sino Biological, China), and anti-MMP-9 polyclonal antibody (Cell signaling technology, USA). Goat anti-rabbit secondary antibody (LICOR Biotechnologies, USA) was used to detect these target proteins. The signals were then measured using the Odyssey CLx Imaging System (LI-COR Biotechnologies, USA). β-actin was used as a loading control. The anti-beta actin monoclonal antibody (Cell Signaling Technology) and goat anti-mouse secondary antibody (LICOR Biotechnologies, USA) were used.

Analysis of phytochemical contents

Determination of total phenolic content

The total phenolic content (TPC) of RRBE was determined by the Folin-Ciocalteu method. The mixture contained 525 μL of Folin-Ciocalteu reagent, 525 μL of 7.5% (w/v) sodium carbonate, and 70 μL RRBE. After incubation at RT for 30 min, the absorbance of the mixture was measured at 725 nm. Gallic acid was used as the standard curve for the quantitation of TPC. TPC was presented as mg of gallic acid equivalent (GAE) per 1 g of RRBE.

Determination of total flavonoid content

The total flavonoid content (TFC) of RRBE was determined by the aluminum chloride colorimetric assay. The mixture contained 75 μL of 5% (w/v) sodium nitrite, 150 μL of 10% (w/v) aluminum chloride, and 500 μL RRBE. After incubation at RT for 5 min, 500 μL of 1 M sodium hydroxide and 275 μL of DI water were added to the mixture. After further incubation at RT for 30 min, the absorbance of the mixture was measured at 510 nm. Catechin was used as the standard curve for the quantitation of TFC. TFC was presented as mg of catechin equivalent (CE) per 1 g of RRBE.

Statistical analysis

The results were presented as mean ± SD from at least three separate experiments. All statistical analyses were carried out using IBM SPSS Statistics 22.0 software. The one-way analysis of variance (ANOVA) followed by Duncan or Dunnett's T3 for multiple comparisons was used to determine significant differences between groups. A level of significance was set at p-value <0.05. For the anti-proliferation and cytotoxicity assay, the IC₅₀ value was extrapolated from the dose inhibition curve obtained by plotting the percentage of Inhibition versus RRBE concentrations using GraphPad Prism version 5.0 for Windows, serial number license GPS-2690594-LDVW-660EC (GraphPad Software, USA, available at https://www.graphpad.com).

Selective effects of RRBE on the viability of HepG2 cells compared with BNL CL2 cells

This study assessed how RRBE affected the viability of HepG2 liver cancer cells in comparison to BNL CL2 normal liver cells. The resazurin cell viability assay, conducted after 72 h of incubation, revealed a reduction in the number of viable HepG2 and BNL CL2 cells as the concentrations of RRBE increased, indicating the dose-dependent anti-proliferative effects (see Fig. 1a). Moreover, non-lethal effects were observed in BNL CL2 cells with all concentrations of RRBE, but 250 and 375 μg/mL RRBE induced approximately 25% and 50% reduction in HepG2 cell viability, respectively, suggesting a dose-dependent cytotoxic effects. The IC₅₀ values of RRBE against the two cell lines are depicted (see Fig. 1b). The selectivity index (SI) was based on the IC₅₀ ratio of RRBE in the non-tumor BNL CL2 cell line to the tumor HepG2 cell line, which was found to be > 2. SI values above 2 are considered as high selectivity towards cancer cells. Overall, RRBE exhibited strong anti-proliferative and cytotoxic effects, specifically targeting liver cancer cells.

Figure 1. Effects of RRBE on viability of HepG2 and BNL CL2 cells.

Dose-response viability curves (a). A bar chart showing IC₅₀ values (b). Different letters indicate significant differences (p < 0.05).

Dose- and time-dependent effects of RRBE on the viability of HepG2 cells

After noting the specific impacts of RRBE on anti-proliferative and cytotoxic effects against HepG2 cells, we then investigated the effects of different incubation periods (24, 48, and 72 h). After 24 hours of incubation, the resazurin cell viability assay displayed a decrease in the percentage of viable HepG2 cells as RRBE concentrations increased (see Fig. 2a). After 48 h of incubation, a decrease in viable HepG2 cells and a negative percent viability were noted at 375 μg/mL RRBE, indicating cell death of around 20%. After being exposed to 250 and 375 μg/mL RRBE for up to 72 h, the largest decline in viable cells, along with about 30% and 60% cell death, were recorded, respectively. The overall results demonstrated that RRBE only exerted an anti-proliferative effect that varied with the dosage within a 24-hour period. RRBE caused a complete halt in growth and subsequent cell death in HepG2 cells after 48 and 72 h, indicating a dose and time-related effect. In addition, IC₅₀ values of RRBE decreased progressively over time, with a twofold reduction in an IC₅₀ value at 72 h in comparison to 24 h (see Fig. 2b). The more potent anti-proliferative and cytotoxic effects were associated with lower IC₅₀ values.

Figure 2. Time-dependent effects of RRBE on viability of HepG2 cells.

Dose-response viability curves (a). A bar chart showing IC₅₀ values (b). Different letters indicate significant differences (p < 0.05).

The effect of RRBE on cell cycle distribution of HepG2 cells

Studies conducted before revealed that the growth of HepG2 cells was inhibited, and cell death was induced by 375 μg/mL RRBE in a time-related fashion ( Figure 2a). Consequently, 375 μg/mL RRBE was selected to study how it impacted the cell cycle distribution of HepG2 cells. Flow cytometry showed an elevated proportion of cells in the G2/M phase in the treated group compared to the control group at 24, 48, and 72 h of incubation ( Figure 3). Within the next 24 hours, RRBE treatment caused the G2/M phase population to surge by 50% (see Fig. 3a, 3b). Following 48 and 72 h, there was a gradual decline in the number of G2/M cells, accompanied by a 2 and 3-fold rise in apoptotic cells, respectively, displaying a sub-G1 peak with diminished DNA content, in comparison to the 24-hour period ( Figures 3c, 3d, 3e, and 3f). Before reaching a sub-G1 peak, these arrested cells gradually underwent apoptosis, resulting in varied DNA contents due to variations in DNA degradation levels during apoptosis. By 72 h, a high portion of these cells acquired DNA content resembling that of the S phase, leading to a significant rise in the S phase population.

Figure 3. Effects of RRBE on cell cycle distribution in HepG2 cells.

Representative DNA distribution histograms (a, c, e) and bar graphs (b, d, f ) displaying the percentage of HepG2 cells in different phases after the indicated time points. Asterisks (*) indicate significant differences in relation to the corresponding control (p < 0.05). Different letters indicate significant differences compared to the same phase at different time points (p < 0.05).

Anti-migratory effect of RRBE on HepG2 cells using the scratch assay

The previous findings demonstrated that RRBE at concentrations of 125 and 167 μg/mL exhibited a mild to moderate ability to inhibit cell proliferation. As a result, the two doses were employed to assess the anti-migration effect of RRBE on HepG2 cells, excluding its influence on cell proliferation. In the scratch assay, HepG2 cells were exposed to 125 and 167 μg/mL RRBE in a medium containing 2% FBS rather than the standard 10% for durations of 24, 48, and 72 h, as opposed to control cells that were left untreated. Of note, 1-2% FBS is used to sustain cells, not to stimulate their proliferation. The results demonstrated that untreated HepG2 cells in a medium with 2% FBS could migrate into an artificial gap on a cell monolayer, and their relative migration ability to fill the gap increased by 4-fold at 48 hours and 9-fold at 72 hours compared to 24 h (see Fig. 4a, 4b). In comparison to the untreated cells, the ability of HepG2 cells to migrate was fully blocked after 24 h of exposure to 125 and 167 μg/mL RRBE.

Figure 4. Effects of RRBE on HepG2 cell migration.

Representative images of the scratch assay after the specified doses and time points (a). A bar chart displaying the relative ability of HepG2 cells to migrate over time compared to the migration ability of control cells at 24 h, which was marked as 1 (b). Asterisks (*) indicate significant differences compared to the corresponding control at the same time (p < 0.05). Different letters in the control groups at different time points indicate significant differences relative to the control at 24 h (p < 0.05).

Anti-migratory effect of RRBE on HepG2 cells using the transwell migration assay

In contrast to the scratch assay, the transwell migration assay quantifies cell motility towards a chemoattractant gradient set up across the insert membrane with different FBS concentrations in the upper and lower chambers. After analyzing the scratch assay data, we opted to expose HepG2 cells to 125 and 167 μg/mL RRBE for a duration of 24 h while the control cells remained untreated. Next, we harvested the cells and placed them on the upper compartment of the transwell inserts that held MEM with 2% FBS. Concurrently, MEM with 10% FBS was added into the lower compartment of the chamber to function as chemoattractants. The transwell migration assay was performed for an additional 24 h. The results revealed that about 100 untreated cells per field, on average, moved through pores and reached the underside of the insert membrane (see Fig. 5a, 5b). In contrast, cells treated with RRBE at 125 and 167 μg/mL failed to migrate through transwell inserts, indicating the anti-migratory effect (see Fig. 5a, 5b).

Figure 5. Effects of RRBE on HepG2 cell migration.

Representative images of the transwell assay after the specified doses (a). A bar chart depicting the cell number per field of HepG2 cells at 24 h (b). Asterisks (*) denote significant differences from the control.

Anti-invasive effect of RRBE on HepG2 cells

Upon identifying the anti-migration effect of RRBE on HepG2 cells, we subsequently tested its ability to inhibit invasion. The Western blot technique was utilized to examine changes in the levels of E-cadherin, β-catenin, vimentin, and MMP-9 proteins in HepG2 cells following treatment with RRBE for 24, 48, and 72 h. The results showed that E-cadherin, vimentin, β-catenin, and MMP-9 proteins were undetectable in HepG2 cells after 24 hours, with or without RRBE. However, a clear indication of β-actin protein as a loading control was detected (data not shown). These results suggested that up to 50 μg of total protein lysates was sufficient for detecting only β-actin protein but not the proteins of interest, likely because of their low expression levels and/or the limited sensitivity of antibodies in this context. After 48 h, RRBE induced a dose-dependent increase in the E-cadherin levels, although this effect did not amplify with time (see Fig. 6a, 6 b). Conversely, RRBE resulted in a dose-dependent reduction in the levels of β-catenin, vimentin, and MMP-9 proteins when compared to their respective controls (see Fig. 6a, 6b). In this context, vimentin expression levels were time-dependently inhibited by a lower RRBE dosage, and MMP-9 protein levels were time-dependently suppressed by a higher RRBE dosage. The results indicated that HepG2 cells exhibited epithelial features and lost the mesenchymal phenotypes following RRBE treatments.

Figure 6. Effects of RRBE on the protein expression levels of EMT markers in HepG2 cells.

Representative western blot images (a) and bar charts (b) showing EMT marker levels in HepG2 cells after the specified doses and time points. Asterisks (*) indicate significant differences compared to the corresponding control. For each protein, different letters indicate significant differences compared to the same and different time points (p < 0.05).

The yield and total contents of polyphenols and flavonoids

A high yield of RRBE was achieved, and 1 g of RRBE contained high amounts of polyphenols and flavonoids (see Table 1). These bioactive compounds are likely significant contributors to the anti-cancer effects of RRBE.