Pigmented Sorghum Phenolic Extracts Regulate the Expression of Cancer Development Pathway Genes in HT‐29 and Hypoxia‐Induced CCD 841 CoN Cells
Aduba Collins, Kenneth Chinkwo, Nidhish Francis, Abishek Bommannan Santhakumar, Christopher Blanchard
TL;DR
Pigmented sorghum extracts reduce cancer cell viability and modulate genes linked to cancer development and metabolism.
Contribution
This study reveals how pigmented sorghum polyphenols affect key cancer-related genes in colorectal cancer cells.
Findings
Sorghum extracts significantly reduced cancer cell viability at concentrations of 500 and 2000 μg/mL.
BlackSs extract upregulated APC and TTN genes and downregulated GLUT-1 and HIF-1α/β in cancer cells.
Processed RedBu2 also showed significant upregulation of the TTN gene.
Abstract
Sorghum polyphenols have been shown to inhibit gastrointestinal cancer cell growth by inducing apoptosis and other pathways such as chronic inflammation. However, the impact of sorghum polyphenols on the most frequently mutated genes in the genome including mutation and instability and dysregulated cellular metabolism pathways is unknown. This study evaluated the gene and protein expression levels regulated by raw and fermented‐cooked (processed) sorghum phenolic extracts (BlackSs, BlackSb, and RedBu2) on HT‐29 and hypoxia‐induced CCD 841 CoN cells. Cancer cell viability was measured by Resazurin cytotoxicity assay and the gene and protein expression of APC, KRAS, TTN, HIF‐1α, HIF‐1β, and GLUT‐1 were measured using rtPCR and ELISA. Pigmented sorghum extracts showed a significant reduction in cancer cell viability at 500 and 2000 μg/mL after 12 and 24 h for raw samples but only after 24…
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TopicsPlant Gene Expression Analysis · melanin and skin pigmentation · Microbial Metabolism and Applications
Introduction
1
Cancer is a heterogeneous disease characterized by many forms that can encompass the esophagus, stomach, colon, and other gastrointestinal organs primarily due to poor dietary habits (Kerschbauma and Nüssler 2019). Increasing awareness of the potential health benefits of wholegrain consumption has prompted investigations into the cancer preventative properties of sorghum ( Sorghum bicolor L. Moench) (Collins, Santhakumar, Francis, et al. 2024; Tullio et al. 2020; Xie et al. 2019; Zhu and Sang 2017). Wholegrain sorghum is a recognized abundant source of phenolic compounds, such as anthocyanins and proanthocyanidins, which are primarily found in pigmented varieties (Brantsen et al. 2021; Herrman et al. 2020). These compounds have been reported to contain bioactivity, capable of scavenging free radicals and have demonstrated antiproliferative and antioxidant properties that contribute to the modulation of cancer development pathways (Lee et al. 2020; Chen et al. 2021).
Genome mutation and instability is one of 10 cancer development pathways that involves changes to the genome of a cell affecting its growth and progression (Hanahan 2022). Often linked to apoptosis (programmed cell death), several morphological changes can lead to disease malignancy including cell shrinkage, cell membrane alterations, and DNA fragmentation (Waarts et al. 2022; Lachance et al. 2020). Apoptosis can be triggered through the extrinsic (death receptor‐mediated) or intrinsic (mitochondria‐mediated) pathways, influenced significantly by the tumor suppressor gene APC, crucial in regulating the WNT (Wingless/Integrated) signaling pathway (Zhou et al. 2022; Stefanski and Prosperi 2020). Cancer cells often evade apoptosis by suppressing APC and downregulating proapoptotic proteins like KRAS and TTN that are involved in signaling pathways into the cell (Wang et al. 2021). Studies have reported the chemopreventative potential of sorghum phenolic extracts (Stefoska‐Needham 2024; Gilchrist et al. 2020; Rao et al. 2019), however, their potential effects on APC, KRAS, TTN, and the mechanisms of action have not been elucidated.
Metabolic rewiring, also known as dysregulated cellular metabolism, allows cancer cells to rapidly divide by overriding the metabolic controls typical of normal tissues (Schirrmacher 2020). Crucial to this adaptation is the upregulation of glucose transporters, particularly GLUT‐1, which enables increased glucose uptake into the cytoplasm to meet the increased metabolic demand (Schirrmacher 2020). The metabolic switch from aerobic to anaerobic glycolysis is coordinated by the HIF‐1 oncogenes, HIF‐1α, and HIF‐1β (Figure 1) (Raskov et al. 2023). These genes can reprogram cancer cell metabolism to enhance survival in hypoxic environments. To our knowledge, there are no studies that investigate the effect of sorghum phenolic extracts on HIF‐1α and HIF‐1β and GLUT‐1 involved in these pathways. Therefore, understanding the interaction between nutrition, glycolysis, and the possible changes of cellular metabolism by sorghum phenolic compounds is critical.
Schematic representation of the potential role of sorghum polyphenols in modulating hypoxia and deregulated cellular metabolism in cancer. Under normal aerobic conditions, cells convert glucose to pyruvate using oxygen, releasing carbon dioxide (CO2) and promoting the degradation of the hypoxia‐inducible factor 1α (HIF‐1α) oncogene. In contrast, cancer cells under hypoxic conditions rely on anaerobic glycolysis, where HIF‐1α recruits HIF‐1β to form a complex that binds to hypoxia response elements (HREs), driving the transcription of genes that support tumor growth and survival—a process known as the Warburg effect. Sorghum polyphenols are proposed to inhibit HIF‐1α, disrupting this pathway and promoting a metabolic switch back to aerobic glycolysis. Adapted from Collins, Santhakumar, Francis, et al. (2024).
This study aims to explore the pro‐apoptotic genes and mechanisms of phenolic extracts from pigmented sorghum wholegrains that induce programmed cell death and regulate cellular metabolism in colorectal cancer cells and hypoxia‐induced normal colon cells. In addition, this study aims to determine the impact of processed sorghum phenolic extracts on the previously mentioned cancer development pathways. These findings could contribute to the evolving understanding of sorghum as a nutraceutical with potential implications for cancer risk reduction.
Materials and Methods
2
Sorghum Sample Collection
2.1
Wholegrain white (WhiteLi_1_ and WhiteLi_2_), red (RedBa_1_, RedBu_1_, RedBa_2_, RedBu_2_), and black (BlackSb and BlackSs) sorghum samples were sourced as previously described (Collins, Francis, Chinkwo, et al. 2024). Briefly, the black samples were obtained from 2021 glasshouse trials conducted in Warwick, Queensland. Red and white sorghum samples were acquired from 2021 field trials conducted in Bellata and Croppa Creek in New South Wales. Preceding experimentation, wholegrain samples were stored at 4°C. Each sample was analyzed in triplicates in each experiment.
Processing and Preparation of Sorghum Extracts
2.2
Using a Retsch Ultra Centrifugal Mill ZM 200 (Haan, North Rhine‐Westphalia, Germany) with a 0.5 mm sieve, wholegrains were finely milled to obtain the resultant sorghum flour. Hexane was added to flour samples (1:20 w/v) three times then evaporated in the fume hood for 24 h to obtain defatted flour samples. Prior to processing, the defatted sorghum flour samples were stored at 4°C. Fermentation‐cooking of sorghum flour samples was performed according to Collins, Francis, Chinkwo, et al. (2024). Briefly, sorghum flour and deionized water were mixed at a ratio of 1:2 then left to ferment at 25°C until pH 4 was reached (approximately 48 h). This mixture was added to 80 mL of boiling water and stirred for 10 min then transferred to a water bath to stop the cooking process. The fermented‐cooked sorghum porridge samples were frozen at −80°C then freeze‐dried (Christ‐Alpha 2–4 LD Plus freeze dryer; Biotech International, Germany) and milled to procure the fermented‐cooked (processed) flour. The preparation of samples was completed in triplicates.
The phenolic extraction procedure was performed according to previous studies conducted by our research group (Rao et al. 2018). The extraction solvent consisted of 70% acetone, 29.5% water and 0.5% acetic acid (v/v/v). This mixture was added to the defatted raw or processed flour at a ratio of 1:10 (w/v) and left to stir at 25°C for 1 h. The mixtures were centrifuged at 4000 rpm for 10 min; the extraction was repeated twice more and the supernatants were pooled together. This step was repeated three times to a total volume of 30 mL per gram of sample. A rotary vacuum evaporation (Rotavapor R‐210 BUCHI Labortechnik, Flawil, Switzerland) was used to remove acetone at 50°C. The remaining liquid in the samples was then frozen at −80°C and freeze‐dried (Christ‐Alpha 2–4 LD Plus freeze dryer; Biotech International, Germany). The resultant phenolic extracts were stored at −20°C until further analysis. A solution of 50% methanol was used to reconstitute sorghum extracts prior to analysis then stored at −20°C. All samples were extracted in triplicates.
Reagents and Chemicals
2.3
The cell culture materials included dimethyl sulphoxide (DMSO), Dulbecco's modified eagle's medium (DMEM), Eagle's minimum essential medium (EMEM), foetal bovine serum (FBS), penicillin–streptomycin, phosphate buffer saline (PBS), trypsin, hydrogen peroxide (H_2_O_2_), and Resazurin red dye were obtained from Sigma‐Aldrich (St. Louis, MI, USA). HT‐29 colorectal cancer cells and normal CCD 841 CoN colon cells were sourced from the ATCC distributor In Vitro Technologies Pty Ltd. (Victoria, Australia). 24‐well and 96‐well cell culture plates were purchased from Corning Incorporated (Corning, New York, USA). Hard‐Shell 96‐well PCR plates (HSP9601) and adhesive plate sealing film (MSB1001) were purchased from Bio‐Rad (Bio‐Rad Laboratories, California, USA). Cobalt (II) Chloride hexahydrate (CoCl_2_•6H_2_O), Folin–Ciocalteu reagent, formic acid, ABTS, Trolox, acetic acid, acetone, hexane, and methanol were acquired from Chem Supply Pty Ltd. (Port Adelaide, South Australia, Australia), and Sigma‐Aldrich (St. Louis, MO, USA). Assay kits used to measure gene expression were purchased from Bio‐Rad (Bio‐Rad Laboratories, California, USA), including an Aurum total RNA extraction kit, iScript cDNA synthesis kit, and an SsoAdvanced Universal SYBR Green Supermix kit. The PrimePCR SYBR Green primers of human APC, KRAS, TTN, HIF‐1α, HIF‐1β (ARNT), GLUT‐1 (SLC2A1), and the housekeeping gene, β‐Actin, were also purchased from Bio‐Rad. To measure protein concentration, human APC and TTN ELISA kits were purchased from the antibodies.com distributor Sapphire Biosciences (Beaconsfield, NSW, Australia).
Cell Culture Treatment
2.4
HT‐29 colorectal cancer cell lines were cultured and maintained as a monolayer in 89% (v/v) DMEM, 10% (v/v) FBS, and 1% (v/v) penicillin–streptomycin. The normal colon cell line CCD 841 CoN was grown in EMEM supplemented with FBS and penicillin–streptomycin at the same volume ratio. For optimal growth, the normal colon cells were used between passages 5 and 9, with experiments performed at 70%–80% confluence. The cells were maintained in an incubator at 37°C in a humidified atmospheric CO_2_ level of 5%. These conditions were sustained when using assays unless otherwise stated.
Resazurin Red Cytotoxicity Assay
2.5
To test the cytotoxicity of sorghum extracts, a time and dose response cytotoxicity assay was conducted according to Rao et al. (2019). The extracts were diluted with cell culture media (DMEM supplemented with 10% FBS and 1% penicillin–streptomycin) to concentrations of 50, 100, 250, 500, 1000, 1500 and 2000 μg/mL prior to testing. HT‐29 colon cancer cells were seeded in 96 well plates at a cell density of 5 × 10^3^ cells per well. The cells were washed with PBS after 24 h of incubation and then incubated with the different extract preparations. A separate plate was prepared (extracts with no cells) to compensate for extract color and used to blank correct the data. A positive control of 10 mM hydrogen peroxide, negative control of fresh media, and 2% DMSO control (the maximum concentration in reconstituted extracts) were also included. The cytotoxicity of the sorghum extracts was also tested on CCD 841 CoN normal colon cells. After 4, 6, 12, and 24 h treatments, extracts were removed, the wells were washed with PBS and 200 μL of 14 mg/L resazurin red dye was added to each well for an additional 4 h incubation. The absorbance was measured at 570 and 600 nm and calculated as percentage viability according to Ataollahi et al. (2015). Experiments and treatment conditions were performed in triplicates.
Experimental Design and Hypoxia Induction
2.6
To determine the impact of sorghum extracts on metabolic genes, the gene expression of HIF‐1α, HIF‐1β, and GLUT‐1 was determined using rtPCR. Briefly, hypoxic conditions were induced by Cobalt (II) Chloride hexahydrate (CoCl_2_•6H_2_O) on normal CCD 841 CoN colon cells as described by Wu and Yotnda (2011). A 25 mM of CoCl_2_ stock solution was freshly prepared to attain a final concentration of 100 μM in EMEM media. A negative control of normoxia (cells without CoCl_2_) and a positive control of hypoxia (hypoxia‐induced cells without extracts) were also prepared. Cells were seeded in 24‐well plates at a density of 2 × 10^−5^ and incubated for 24 h at 37°C and 5% CO_2_. The working solution of CoCl_2_ was added to normal colon cells for 24 h after reaching 90% confluency then supplemented with sorghum extracts at various treatment levels as described in Section 2.5. Following incubation, the extracts were removed, the cells were washed with PBS, then lysed for subsequent rtPCR analysis.
RNA and cDNA Extraction
2.7
The total RNA was extracted using a Bio‐Rad Aurum total RNA extraction kit, according to the manufacturer's instructions. A volume of 2 μL RNA sample was added to a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific, MA, USA) to assess the RNA purity. An iScript cDNA synthesis kit was used with a CFX96 Real‐Time System (Bio‐Rad, CA, USA) to synthesize the cDNA, according to the manufacturer's specifications. The reaction was performed in Hard‐Shell 96‐well PCR plates and consisted of 4 μL 5× iScript reaction mix, 1 μL iScript reverse transcriptase (RT), 10 μL nuclease‐free water and 5 μL RNA template (20 μL total volume per well). The thermal reaction protocol consisted of priming at 25°C for 5 min, RT at 46°C for 20 min, RT inactivation at 95°C for 1 min then hold at 4°C. The reaction was stored at −20°C prior to rtPCR analysis.
Real‐Time Polymerase Chain Reaction (rtPCR) Assay
2.8
Real time PCR was performed using an SSoAdvanced Universal SYBR Green Supermix on a CFX96 Real‐Time System. The primers listed in Section 2.3 were diluted in 1:10 (v/v) Tris‐EDTA (TE) buffer. The reaction was performed in Hard‐Shell 96‐well PCR plates and included 10 μL SSoAdvanced Universal SYBR Green Supermix, 2 μL primer, 2 μL cDNA template (diluted in 1:20 v/v TE buffer), and 6 μL nuclease‐free water (20 μL total volume per well). The thermal reaction protocol consisted of denaturation at 95°C for 3 min followed by amplification at 95°C for 15 s and annealing at 60°C for 30 s for 40 cycles. A melt curve analysis was performed from 65°C to 95°C. Each gene was normalized with the housekeeping gene, ß–actin, for their relative abundance. The cycle threshold/quantification cycle (C t/C q) values obtained for each gene target were normalized to the housekeeping gene to obtain the mean relative gene expression.
Enzyme‐Linked Immunosorbent Assay (ELISA)
2.9
To quantify the intracellular protein concentration, human APC, and TTN ELISA kits (antibodies.com, Cambridge, UK) were used due to their rtPCR gene expression levels. Briefly, HT‐29 colon cancer cells were seeded in 24‐well plates then treated with raw and processed BlackSs, BlackSb, and RedBu_2_ sorghum for 12 and 24 h at the concentrations 500 and 2000 μg/mL due to their cytotoxic effects as determined by the Resazurin red cytotoxicity assay in Section 2.5. Following treatment, the extracts were removed, the cells were washed with PBS and lysed with lysis buffer. The cell lysates were added to 96‐well microplates pre‐coated with APC or TTN antibodies and subsequently incubated at 37°C for 2 h according to the manufacturer's instructions. After adding the detection antibody, Streptavidin‐HRP and PBS plate washing several times, the reaction was stopped by addition of a stop solution and a microplate reader was used to measure the absorbance at 450 nm. A standard curve was prepared by adding seven dilutions to each antibody plate to determine the protein concentration.
Data Analysis
2.10
Data are represented as mean ± SD. All data were performed on GraphPad Prism Software (version 10) using an ordinary one‐way analysis of variance (ANOVA) coupled with Tukey's multiple comparison post hoc followed by a two‐way analysis of variance (ANOVA) and an uncorrected Fisher's LSD test. Differences at values were set at p < 0.05 for statistical significance.
Results
3
Cytotoxicity Assay
3.1
The resazurin red assay was used to determine the impact of eight sorghum extracts on HT‐29 colon cancer cells. The cells were incubated for 4, 6, 12, and 24 h with sorghum‐derived phenolic extracts concentrations of 50, 100, 250, 500, 1000, 1500, 2000 μg/mL. The significant (p < 0.05) decline in cancer cell viability commencing at 500 μg/mL after 12 and 24 h is shown in Figure 2A,B,D. Raw white and red sorghum varieties did not display a decline in cancer cell viability. However, the red pericarp sorghum variety RedBu2 exhibited a significant reduction in cell viability at 24 h when the sample was processed via fermentation‐cooking. The black sorghum varieties BlackSs and BlackSb showed a regression in cell growth initially at 500 μg/mL for raw samples, while processed samples of the same varieties demonstrated cell growth inhibition at 2000 μg/mL. Based on the cytotoxic effects of red and black sorghum extracts to reduce cell viability by 50% (IC50), the treatment conditions of 500 and 2000 μg/mL at 12 and 24 h were selected for subsequent experiments. No cytotoxicity was exhibited for the CCD 841 CoN normal colon cell line at the described treatment conditions (data not shown).
Effect of raw [(A) and (B)] and processed [(C) and (D)] sorghum phenolic extracts on the viability of human HT‐29 colon cancer cells (determined by a Resazurin red cytotoxicity assay).
Impact of Raw and Processed Sorghum Extracts on the Expression of Genome Mutation and Instability Cancer Development Pathway Genes
3.2
A quantitative real‐time PCR (rtPCR) was used to elucidate the impact of raw BlackSs and BlackSb on the gene expression of the APC, KRAS, and TTN in HT‐29 colon cancer cells (Figure 3). The results showed a significant increase (p < 0.05) in gene expression with variations in magnitude between treatment groups. Treatment with 500 μg/mL BlackSs at 12 h resulted in an 80% upregulation of the APC gene (Figure 3A), with a further twofold increase at 24 h from 2.75 ± 0.74 to 6.07 ± 1.31 (p < 0.05, Cohen's d = 1.03, 95% CI [1.17, 5.47]), indicating a large effect size. A significant eight‐fold upregulation was observed for the KRAS gene (Figure 3B) at the highest concentration of 2000 μg/mL after 24 h only, suggesting that black sorghum extracts affect cancer cell growth in a time‐dependent manner while the other treatment conditions did not result in significant changes. Gene expression levels of TTN (Figure 3C) significantly increased (p < 0.05) after treatment at 2000 μg/mL from 1.40 ± 0.34 to 6.26 ± 0.76 with a slight increase to 6.77 ± 1.11, signifying that upregulation may occur in a dose‐dependent manner. Supplementation with 500 μg/mL BlackSb also resulted in the upregulation of the APC gene at 12 h by 2‐fold (Figure 3D), however, there was no significant change in gene expression at 24 h. Notably, BlackSb also upregulated the genes KRAS and TTN; however, these were at a lower expression level when cancer cells were treated with 2000 μg/mL for 24 h (Figure 3E) or 12 h (Figure 3F).
*Changes in the RT‐PCR gene expression profile of the genome mutation and instability pathway genes after treatment of HT‐29 cells with BlackSs [APC (A), KRAS (B), and TTN (C)] and BlackSb [APC (D), KRAS (E), and TTN (F)] sorghum extracts. Data is presented as mean ± SD (n = 3). Statistical significance was determined by two‐way ANOVA with Dunnett's post hoc test, adjusting p values for Type I error when comparing multiple treatment groups to a single control. Significance level increases with decreasing p value represented by *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. APC, adenomatous polyposis coli; KRAS, Kirsten rat sarcoma virus; TTN, titin.
Given that the study findings also indicated a reduction in cancer cell viability after treatment with processed BlackSs, BlackSb, and RedBu_2_ sorghum extracts at 24 h, this study further investigated the gene expression of APC, KRAS, and TTN using rtPCR (Figure 3). The supplementation of processed BlackSs at 500 μg/mL significantly (p < 0.05) upregulated APC gene expression by 2‐fold (1.45 ± 0.81–3.53 ± 0.95) when compared to the control (Figure 3A). Notably, the KRAS gene was significantly upregulated at 500 μg/mL from 1.07 ± 0.44 to 2.29 ± 0.84, stabilizing at the higher concentration with no further change (2.33 ± 0.51). Treatment of processed BlackSs did not change the expression of TTN at 500 or 2000 μg/mL. BlackSb revealed similar gene expression levels for APC and TTN, although the KRAS gene showed a dose‐dependent increase by 162% then a further 58% (Figure 3B). Interestingly, the processed red sorghum, RedBu_2_, was the only sample investigated that showed in increase in the expression of the TTN gene (Figure 3C). No changed in gene expression were detected for APC or KRAS for this sample.
Effect of Raw Black Sorghum Extracts on the Expression of Hypoxia‐Induced Cellular Metabolism Cancer Development Pathway Genes
3.3
Hypoxia was induced in CCD 841 CoN normal colon cells to determine the effect of sorghum extracts on the HIF‐1 oncogenes and glucose transporter genes in comparison to normoxia (Figure 4). Treatment of the cells with BlackSs at 2000 μg/mL for 12 h resulted in a significant downregulation of the HIF‐1α gene back to normoxia levels (p < 0.05, Cohen's d = 2.32, 95% CI [0.37, 3.25]; Figure 4A), indicating a very large effect size. The same gene expression level was observed for HIF‐1α at 500 μg/mL for 24 h with a further 3‐fold decrease at the higher concentration, demonstrating dose and time‐dependent activity. Treatment of hypoxia‐induced colon cells with 2000 μg/mL BlackSs showed a 3‐fold decrease in HIF‐1β but not at any of the other treatments (Figure 4B). Interestingly, GLUT‐1 exhibited the greatest difference in expression levels, demonstrating a significant decrease in expression compared to hypoxia levels in all treatment conditions (Figure 4C). The gene expression levels of HIF‐1α in BlackSs were comparable to those observed in BlackSb, where a decrease occurred to normoxia (Figure 4D). A significant upregulation was detected for HIF‐1β post‐BlackSb supplementation at 500 μg/mL for 12 h, with no significant changes at the other treatment conditions (Figure 4E). Similar to BlackSs, the GLUT‐1 gene decreased in expression levels; however, this was only observed in a dose‐dependent manner at 2000 μg/mL (Figure 4F). No changes in HIF‐1α, HIF‐1β, and GLUT‐1 gene expression levels were exhibited by the processed sorghum samples RedBu2, BlackSs, and BlackSb (data not shown).
*Changes in the RT‐PCR gene expression profile of cellular metabolism genes (HIF‐1α, HIF‐1β, and GLUT‐1) following raw BlackSs [HIF‐1α (A), HIF‐1β (B), and GLUT‐1 (C)] and BlackSb [HIF‐1α (D), HIF‐1β (E), and GLUT‐1 (F)] extract treatment on hypoxia‐induced CCD 841 CoN colon cells. Data is presented as mean ± SD (n = 3). Statistical significance was determined by two‐way ANOVA with Dunnett's post hoc test, adjusting p values for Type I error when comparing multiple treatment groups to a single control. Significance level increases with decreasing p value represented by *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. HIF‐1α, hypoxia inducible factor 1 alpha; HIF‐1β, hypoxia inducible factor 1 beta; GLUT‐1, glucose transporter 1.
Effect of Raw and Processed Sorghum Extracts on the Protein Expression of Genome Mutation and Instability Pathway Genes
3.4
ELISA was used to further study the effect of raw and processed BlackSs, BlackSb, and RedBu_2_ extracts on the protein expression of the mutated genes cancer development pathway (Figure 4). The results for the protein expression of APC and TTN closely mirrored those obtained in the rtPCR gene expression (Figures 2 and 3). Two out of three genes were selected for subsequent protein expression analysis (APC and TTN) due to their higher expression levels at both 12 and 24 h. At 500 μg/mL, raw BlackSs significantly increased the protein expression level of APC by 2‐fold in a time‐dependent manner at 12 h with a further twofold increase at 24 h (p < 0.05). The quantitative analysis revealed that after treatment of cells with 2000 μg/mL at 12 and 24 h, the level of APC protein did not show statistically significant changes (p > 0.05). Conversely, supplementation with 500 μg/mL raw BlackSb exhibited a significant 4‐fold increase in APC protein expression level (p < 0.05) at 12 h only, indicating that its effect on protein expression is time and dose‐dependent. Processed sorghum extracts of the RedBu_2_, BlackSs, and BlackSb samples did not have a significant impact on APC protein expression levels.
The treatment of HT‐29 cells with 2000 μg/mL raw BlackSs significantly increased the protein expression level of TTN by 4‐fold at 24 h in a time and dose‐dependent manner (p < 0.05) (Figure 5). Additionally, a slight but significant increase in the protein expression level of TTN was exhibited at 12 and 24 h when treated with 500 μg/mL and at 12 h when treated with 2000 μg/mL. The treatment of cells with 2000 μg/mL raw BlackSb showed a significant 2‐fold increase in the TTN protein expression level at 24 h; however, this increase was only marginal when compared to BlackSs. The other treatment conditions for BlackSb displayed a slight but significant increase in TTN protein expression level. Unexpectedly, the TTN expression levels for processed RedBu_2_ were higher than for BlackSs and BlackSb (Figure 5). At 24 h, this sample significantly increased TTN expression by 2‐fold at 500 and 3‐fold at 2000 μg/mL (p < 0.05). Processed BlackSs exhibited a minor but significant increase in TTN expression at 500 μg/mL; however, the expression level plateaued at the higher concentration. Comparable results were observed for BlackSb; however, the plateau at 2000 μg/mL was not significant.
*APC protein concentration levels in HT‐29 colon cancer cells supplemented with raw (A) and processed (B) sorghum extracts (BlackSs, BlackSb, and RedBu2) and measured by ELISA. Data is represented as mean ± SD (n = 3). Statistical significance was determined by two‐way ANOVA with Dunnett's post hoc test, adjusting p values for Type I error when comparing multiple treatment groups to a single control. Significance level increases with decreasing p value represented by *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. APC, adenomatous polyposis coli.
Discussion
4
In this study, the anticancer properties of raw and processed black and red sorghum extracts (BlackSs, BlackSb, and RedBu2) were investigated using HT‐29 colon cancer and hypoxia‐induced CCD 841 CoN normal colon cells. These pigmented sorghum grain extracts were shown in our prior investigations to exhibit an increase in phenolic accessibility and detection following a fermentation‐cooking process due to elevated total phenolic and antioxidant levels (Collins, Santhakumar, Latif, et al. 2024). In addition, several bioaccessible sorghum polyphenols have been reported, capable of reaching the colon for attributable health benefits through the process of cellular biotransformation (Collins, Francis, Chinkwo, et al. 2024). The results of this study have demonstrated, for the first time, that BlackSs, BlackSb, and RedBu2 exhibited a significant reduction in colon cancer cell viability at 500 or 2000 μg/mL (Figure 1). This was potentially due to the raw and processed black sorghum extracts significantly increasing the gene expression level of pro‐apoptotic genes (Figures 3 and 6) and their corresponding protein concentrations (Figures 5 and 7) in a dose and time‐dependent manner. In addition, BlackSs and BlackSb extracts also downregulated the overexpressed metabolic genes in hypoxia‐induced CCD 841 CoN normal colon cells at different levels across various treatments, thereby exhibiting anticancerous metabolism activity (Figure 4).
*Impact of processed BlackSs (A), BlackSb (B), and RedBu2 (C) on the RT‐PCR gene expression of APC, KRAS, and TTN on HT‐29 cells. Data is presented as mean ± SD (n = 3). Statistical significance was determined by two‐way ANOVA with Dunnett's post hoc test, adjusting p values for Type I error when comparing multiple treatment groups to a single control. Significance level increases with decreasing p value represented by *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. APC, adenomatous polyposis coli; KRAS, Kirsten rat sarcoma virus; TTN, titin.
*TTN protein concentration levels in HT‐29 colon cancer cells supplemented with raw (A) and processed (B) sorghum extracts (BlackSs, BlackSb, and RedBu2) and measured by ELISA. Data is represented as mean ± SD (n = 3). Statistical significance was determined by two‐way ANOVA with Dunnett's post hoc test, adjusting p values for Type I error when comparing multiple treatment groups to a single control. Significance level increases with decreasing p value represented by *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. Nonsignificant differences are not indicated on the graph. TTN, titin.
Cancer cell survival is dependent on antiapoptotic genome modifications. For instance, the BCL‐2 family of genes is commonly overexpressed in cancer cells due to a gene mutation in the “guardian of the genome”—p53 (Feroz and Sheikh 2020). The tumor suppressor gene, APC, has also been reported as a frequently mutated gene with an 80% mutation frequency in colon cancer cells (Hinoi 2021). This gene was upregulated by the raw black sorghum extracts, particularly BlackSs, in both the mRNA and protein levels, showcasing evidence for its role in promoting apoptosis. The mechanism of action may be through modulation of the Wnt/β–catenin signaling pathway: sorghum polyphenols can facilitate the binding of APC to Axin and promote phosphorylation of β–catenin (Raji et al. 2018). Interestingly, the processed black sorghum extracts also demonstrated a significant increase in APC gene expression; however, the protein was not detected when measured by ELISA. This discrepancy may be due to posttranscriptional variations or intracellular mechanisms inhibiting the translation and stability of the protein product (Rani et al. 2020). A study reported that the APC gene was upregulated by the chloroform extracts of the flowering plant, Peucedanum chenur, in HCT‐116 colorectal cancer cells at a half maximal inhibitory concentration (IC50) of 120 μg/mL (Fadaei et al. 2022). Similar to our study, this suggests inhibition of the Wnt/β‐catenin signaling pathway in a dose‐dependent manner. Additionally, the black tea polyphenols theaflavins and thearubigins have been shown to reduce U937 and K562 leukemia cell viability by inhibiting the Akt and Wnt/β–catenin signaling pathway (Halder et al. 2012).
The KRAS and TTN genes, primarily involved in cellular signaling and growth activation, have been reported as having a mutation frequency of 50% and 40% respectively (Wang et al. 2021). Our findings indicate that both genes increased in gene expression, with BlackSs showing a greater upregulation than BlackSb at the supplementation concentration of 2000 μg/mL. BlackSs showed a higher KRAS gene expression level than for BlackSb in raw samples; however, the processed samples showed the opposite trend. This finding suggests that raw BlackSs and processed BlackSb may activate the MAPK/ERK and PI3K/Akt signaling pathways (Sever and Brugge 2015). BlackSb induced a slight but significant increase in TTN gene expression compared to BlackSs but stabilized at the higher concentration (Figures 3F and 7B). This stabilization suggests that the hypoxia‐induced CCD 841 CoN cells may have reached a maximum capacity for TTN protein synthesis for BlackSb or that feedback mechanisms may have been activated to regulate further expression, thereby reducing TTN transcription efficacy (Rani et al. 2020). To our knowledge, our study is the first report of processed sorghum bioactivity for the TTN gene. Notably, processed RedBu2 exhibited superior gene expression levels for TTN compared to BlackSs and BlackSb (Figures 6C and 7B). This distinction may be attributed to differences in phenolic composition or regulatory factors post‐processing that may influence gene expression and concentration levels. In our previous study, we demonstrated that RedBu2 increased in antioxidant activity by 25% post processing via fermentation‐cooking (Collins, Santhakumar, Latif, et al. 2024). This sample contained several elevated antioxidant compounds such as 4‐acetylbutyric acid, caffeic acid, trans‐piceid, and trans‐resveratroloside that may have contributed to the upregulation in TTN in the present study. These compounds, among others, have been shown to play a vital role in the downstream regulatory signaling pathways associated with the stability of cell structures, thereby exerting their effects on the TTN protein (Karadas et al. 2024; Ulanova et al. 2019).
Dysregulated glucose metabolism as a cancer development pathway is driven by the preference for anaerobic glycolysis over oxidative phosphorylation to supply the high energy demand of rapidly proliferating cells (Isa 2022). This metabolic switch is primarily regulated by the HIF‐1 oncogenes, HIF‐1α and HIF‐1ß, that bind to the hypoxia response element and induce cancerous DNA transcription (Nagao et al. 2019). In the current study, we report that BlackSs and BlackSb sorghum extracts can potentially reestablish normal cellular metabolism by inhibiting the HIF‐1α gene in hypoxia‐induced CCD 841 CoN normal colon cells (Figure 4). The mechanism of action was likely due to the inactivation of nuclear‐translocated HIF‐1α from phenolic A and B ring structural activity interactions. A similar study by Ansó et al. (2010) demonstrated that flavonoids apigenin, luteolin, and quercetin (all found in our sorghum samples) reduced HIF‐1α expression by inhibiting Akt phosphorylation in the PI3K/Akt pathway. However, no recent studies have explored these findings. HIF‐1ß expression is considered to remain relatively constant under hypoxic conditions (Mandl and Depping 2014). Similarly, in our study, this gene showed no significant changes to expression levels post hypoxia induction on normal colon cells. Nonetheless, BlackSs downregulated HIF‐1ß at the higher concentration, and no significant changes were observed following supplementation with BlackSb except at 500 μg/mL where the gene was upregulated (Figure 4B,E).
GLUT‐1, a cell‐surface transporter protein, plays a vital role in the “Warburg effect” by increasing the uptake of glucose to meet the metabolic demand in cancerous cells (Vaupel and Multhoff 2021). This study determined that black sorghum extracts decrease overexpressed GLUT‐1 in hypoxia‐induced CCD 841 CON cells. BlackSs reached normoxia at 500 μg/mL compared to BlackSb reaching aerobic glycolysis at the higher concentration (Figure 4C,F). The mechanism of action may be due to sorghum phenolic binding to the GLUT‐1 receptor site thereby blocking access for fuel into the cell (Darvin et al. 2015). Similarly, in cancer cells, the glucose levels may diminish due to phenolic‐receptor interactions, resulting in the starvation of malignant cells and apoptosis induction (Hinoi 2021). Prasad et al. (2015) have reported that sorghum contains a significantly lower glycaemic index (GI) in comparison to other cereals. It was determined that processed sorghum, including pasta, flakes and porridge, had significantly lower GI than wheat and rice. According to Miyazaki et al. (2024), the consumption of sorghum porridge in a double‐blind crossover study significantly reduced the blood glucose levels of individuals. These studies support the hypothesis that low GI sorghum reduces postprandial blood glucose levels by potentially downregulating GLUT‐1, resulting in better metabolic health and reducing the risk of associated chronic diseases. Additional studies, particularly human feeding trials, are required to determine the amount of consumed sorghum needed to deliver bioactive polyphenols to neutralize overexpressed GLUT‐1. Furthermore, studies incorporating in vivo validation are essential to confirm the physiological relevance and potential therapeutic effects determined by the in vitro assays used.
Conclusion
5
The current study has demonstrated that pigmented sorghum phenolic extracts can induce apoptosis by regulating the expression of genes in the genome mutation and instability and dysregulated cellular metabolism cancer development pathways. The phenolic extracts from black sorghum, particularly BlackSs, showed a high affinity for APC, KRAS, and TTN upregulation compared to BlackSb. Processed RedBu2 demonstrated superior mRNA and protein expression levels than both black sorghum samples, indicating the increased anticancer activity of the polyphenols in this sample. Additionally, the hypoxia inducible factor oncogenes (HIF‐1α and HIF‐1β) were inhibited by BlackSs, likely due to the high concentration of antioxidant polyphenols in the black samples compared to the red. Furthermore, GLUT‐1 was also modulated by BlackSs, suggesting a reversal of the altered metabolic state associated with tumorigenesis. Studies using in vivo models are required to elucidate the effects of sorghum phenolic extracts on these genes in solid tumors. The findings could potentially indicate that sorghum phenolic extracts are a promising therapeutic strategy for restoring normal gene expression and metabolic balance in cancerous cells.
Author Contributions
Aduba Collins: conceptualization, data curation, investigation, methodology, formal analysis, writing – review editing, writing – original draft. Kenneth Chinkwo: conceptualization, visualization, writing – review editing, supervision. Nidhish Francis: conceptualization, writing – review editing, visualization, supervision. Abishek Bommannan Santhakumar: conceptualization, writing – review editing, supervision. Christopher Blanchard: conceptualization, writing – review editing, visualization, supervision.
Conflicts of Interest
The authors declare no conflicts of interest.
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