Abstract
Cotton plant provides economically important fiber and cottonseed, but cottonseed contributes 20% of the crop value. Cottonseed value could be increased by providing high value bioactive compounds and polyphenolic extracts aimed at improving nutrition and preventing diseases because plant polyphenol extracts have been used as medicinal remedy for various diseases. The objective of this study was to investigate the effects of cottonseed extracts on cell viability and gene expression in human colon cancer cells. COLO 225 cells were treated with ethanol extracts from glanded and glandless cottonseed followed by MTT and qPCR assays. Cottonseed extracts showed minor effects on cell viability. qPCR assay analyzed 55 mRNAs involved in several pathways including DGAT, GLUT, TTP, IL, gossypol-regulated and TTP-mediated pathways. Using BCL2 mRNA as the internal reference, qPCR analysis showed minor effects of ethanol extracts from glanded seed coat and kernel and glandless seed coat on mRNA levels in the cells. However, glandless seed kernel extract significantly reduced mRNA levels of many genes involved in glucose transport, lipid biosynthesis and inflammation. The inhibitory effects of glandless kernel extract on gene expression may provide a useful opportunity for improving nutrition and healthcare associated with colon cancer. This in turn may provide the potential of increasing cottonseed value by using ethanol extract as a nutrition/health intervention agent.
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Introduction
Cotton (Gossypium hirsutum L.) plant provides economically important fiber and cottonseed. Cottonseed contributes to approximately 20% of the crop value. It is either glanded or glandless depending on its seed with or without gossypol glands (Fig. 1A)1,2,3. Glanded cottonseed contains high concentrations of gossypol4, which limits its use primarily to feed ruminants due to its toxicity towards humans and most animals5,6,7,8,9. Glandless cottonseed has only trace levels of gossypol which may be useful as a food for humans or feed for non-ruminant animals10,11,12,13. Glanded and glandless cottonseed contains many other bioactive components including quercetin, gallic acid, 3,4-dihydroxybenzoic acid, flavonoids, cyclopropenoid fatty acids, and peptides. Most of these value-added products possess health promotion and disease prevention potentials14,15. Since plant bioactive products have been used for disease prevention and treatment since ancient history, cottonseed value could be increased by providing high value bioactive compounds and polyphenolic extracts aimed at improving nutrition and preventing diseases.
Cottonseed and ethanol extracts. (A) Glanded and glandless cottonseed section. Glanded seeds are smaller than glandless seeds and contain numerous dark-green-colored gossypol glands. (B) Ethanol extracts. Cottonseed coat or kernel was ground into fine powder and homogenized. The kernel fraction was defatted with chloroform and hexane. The coat fraction was treated with acetic acid followed by autoclave and centrifugation. The defatted materials were extracted with ethanol followed by evaporation to remove acetic acid and ethanol. Ethanol extracts were reconstitution in 100% DMSO (100 mg/mL) and analyzed by HPLC-MS24.
Colon cancer is a serious disease with 4.3% men and 4.0% women developing colorectal cancer during their lifetime according to American Cancer Society’s 2021 estimate (www.cancer.org). According to World Cancer Research Fund International, colorectal cancer is the third most commonly occurring cancer in men and the second most commonly occurring cancer in women. There were over 1.8 million new cases in 2018 (www.wcrf.org).
Plant polyphenols are major bioactive compounds present in most diet with beneficial effects on human health16. They regulate gene expression in numerous studies. Green tea polyphenols affect many gene expression in rats fed a high fructose diet17,18. Cinnamon polyphenols regulate the expression of genes involved in the insulin signaling pathway, inflammatory responses and lipid metabolism19,20,21,22,23. We recently isolated bioactive ethanol extracts from glanded and glandless cottonseed which were shown to be essentially free of gossypol by HPLC–MS analysis (Fig. 1B)24. These bioactive cottonseed extracts affect human cancer cell growth24. They also regulate mouse gene expression coding for diacylglycerol acyltransferase (DGAT), tristetraprolin/zinc finger protein 36 (TTP/ZFP36) family genes and human antigen R (HuR)25,26,27. However, cottonseed extracts on gene expression in cancer cells was unknown.
It is our aim to survey the effects of cottonseed ethanol extracts on regulating the expression of a wide range of genes involved in colon cancer cells. In this study, we analyzed the effects of cottonseed extracts on cell viability and expression of 55 genes which were shown to be regulated by cottonseed-derived gossypol in cancer cells28,29,30,31,32,33,34,35 and macrophages26 or by TTP/ZFP36 in tumor cells36,37,38,39,40,41,42,43,44 and macrophages21,23. The genes selected for analysis are involved in a variety of pathways including lipid biosynthesis (DGATs), glucose transport (GLUTs), anti-inflammation (TTP family), pro-inflammation (TNF, COX, CSF, HUA, ILs, VEGFs), cancer development (BCL2, BNIP3, CYP19A1, FAS, HUA, P53, PPARR and TNFSF10), and TTP-mediated mRNA stability (AHRR1, BCL2L1, CsnK2A1, CXCL1, E2F1, ELK1, HIF1a, HMOX1, ICAM1 and ZFAND5) (Table 1). Cottonseed extracts were used to treat human colon cancer cells (COLO 225) followed by MTT assay and quantitative PCR analysis. COLO 225 (ATCC CCL 222) was selected for the experiments because (1) it is derived from metastatic site with colorectal adenocarcinoma of human, (2) it is loosely attached to the surface of flasks for easy manipulation with trypsinization, and (3) it is widely used in cancer research as a cell model45,46,47,48,49,50,51,52. Our results showed that ethanol extracts from glandless cottonseed kernel significantly reduced the expression of many genes in the colon cancer cells.
Results
Effect of cottonseed ethanol extracts on colon cancer cell viability
Before cottonseed extracts on gene expression were analyzed, we evaluated the effect of the ethanol extracts on colon cancer cell growth. Human colon cancer cells (COLO 225) were treated with 10–100 µg/mL of cottonseed extracts for 2 and 24 h. MTT assay was used to estimate the effect of cottonseed extracts on cell viability. MTT assay did not show significant changes in the viability of colon cancer cells under treatments with various concentrations for 2 or 24 h (Fig. 2). Similar analysis did not show major effect of these cottonseed extracts on the viability of human lung cancer cells (A549 CCL185) (data not shown). Colon cancer cells were selected for gene expression analysis as described below.
Effect of cottonseed extracts on human colon cancer cell viability. (A) Glanded cottonseed coat extract, (B) Glanded cottonseed kernel extract, (C) Glandless cottonseed coat extract, (D) Glandless cottonseed kernel extract. Colon cancer COLO 225 cells were treated with cottonseed extracts for 2 and 24 h. Cell viability was determined by MTT assay. The data represent the mean and standard deviation of three independent samples.
Basal gene expression level in human colon cancer cells
One important factor for relative gene expression evaluation is to get a basic idea about the basal level expression of the genes selected for investigation. The relative mRNA levels of 55 genes (Table 1) were measured in the control cells using the specific qPCR primer pairs as described53. SYBR Green qPCR assay showed that BCL2 mRNA CT (cycle of threshold) was one of the least varied mRNAs (Table 2). BCL2 mRNA CT value was 30 ± 1 (mean ± standard deviation, n = 12) (Table 2). GAPDH and RPL32 mRNA levels were 33 and 51 fold of BCL2 mRNA, respectively. INOS mRNA was undetectable. AHRR1, COX1, CYCLIND1, GLUT4, HUA, ICAM1, IL10, IL12, RAB24, VEGF and ZFP36L2 mRNAs were detected with less than 10% of BCL2 mRNA in the colon cancer cells (Table 2).
The mRNA level of a gene at least twofold or less than 50% of BCL2 mRNA could be interpreted as its expression more or less abundant than that of BCL2 mRNA, respectively. By this standard, 14 genes were expressed more abundantly than BCL2 gene (BCL2L1, BNIP3, CSNK2A1, CTSB, GAPDH, GLUT1, GLUT3, HIF1A, HMGR, IL6, MAP1LC3B, RPL32, TNFSF10, and ZFAND5) (Table 2). Similarly, 20 genes were expressed less abundantly than BCL2 gene (AHRR1, COX1, CXCL1, CYCLIND1, DGAT2A, DGAT2B, FAS, GLUT4, HUA, ICAM1, IL2, IL10, IL12, LEPTIN, NFKB, P53, RAB24, TNF, VEGF, and ZFP36L2) (Table 2). TaqMan qPCR assay showed similar trend of SYBR Green qPCR (data not shown). SYBR Green qPCR assay was chosen to conduct gene expression analysis in the following experiments.
Selection of reference gene for qPCR assays in human colon cancer cells
Another important factor for comparing gene expression is to identify reference gene for qPCR analysis. Reference gene mRNA levels for qPCR assays should be minimally variable under experimental treatments. The CT values with smaller standard deviations among the treatments indicate more stable gene expression. The qPCR data from 24 samples (triplicate each of the 8 concentrations: 0, 5, 10, 20, 30, 40, 50 and 100 µg/mL of ethanol extracts) were pooled and calculated for the mean ± standard deviation (Table 2). BCL2 CT value was among the least varied with 29 ± 1, 29 ± 1, 29 ± 1, 28 ± 1 for glanded coat, glanded kernel, glandless coat, and glandless kernel extracts, respectively (mean ± standard deviation, n = 24) (Table 2). GAPDH and RPL32 are well-known reference genes for qPCR assays in mammalian cells. However, their CT values had much larger standard deviations. GAPDH CT value was among the largest variable in the cells with 25 ± 3, 25 ± 3, 25 ± 3, 23 ± 3 for glanded coat, glanded kernel, glandless coat, and glandless kernel extracts, respectively (mean ± standard deviation, n = 24) (Table 2). RPL32 CT value was also among the largest variable in the cells with 24 ± 3, 25 ± 3, 25 ± 3, 23 ± 3 for glanded coat, glanded kernel, glandless coat, and glandless kernel extracts, respectively (mean ± standard deviation, n = 24) (Table 2). Furthermore, GAPDH and RPL32 mRNAs were the most abundant mRNAs among the 55 tested targets in the cells. The relative fold of GAPDH mRNA to BCL2 mRNA was 18, 10, 19, and 39 fold for glanded coat, glanded kernel, glandless coat, and glandless kernel extracts, respectively (n = 24) (Table 2). The relative fold of RPL32 mRNA to BCL2 mRNA was 21, 10, 17, and 30 fold for glanded coat, glanded kernel, glandless coat, and glandless kernel extracts, respectively (n = 24) (Table 2). They were much higher than the other mRNAs (Table 2). These data suggested that GAPDH and RPL32 mRNAs were not suitable internal references for qPCR assays in the human colon cancer cells due to large standard deviations and high expression levels. BCL2 mRNA was selected as the internal reference for our qPCR analyses since BCL2 was widely studied and least regulated gene in colon cancer cells.
There were 10 genes with mRNA levels at least twofold of BCL2 mRNA in the 24 pooled samples treated with glanded coat extract (BCL2L1, BNIP3, CSNK2A1, GAPDH, GLUT3, HIF1A, LEPTIN, MAP1LC3B, RPL32, and ZFAND5) (Table 2). There were 8 genes with mRNA levels at least twofold of BCL2 mRNA in the 24 pooled samples treated with glanded kernel extract (BNIP3, CSNK2A1, GAPDH, GLUT3, MAP1LC3B, RPL32, TNFSF10, and ZFAND5) (Table 2). There were 9 genes with mRNA levels at least twofold of BCL2 mRNA in the 24 pooled samples treated with glandless coat extract (BNIP3, CSNK2A1, GAPDH, GLUT1, GLUT3, MAP1LC3B, RPL32, TNFSF10, and ZFAND5) (Table 2). There were 13 genes with mRNA levels at least twofold of BCL2 mRNA in the 24 pooled samples treated with glandless kernel extract (BCL2L1, BNIP3, CSNK2A1, GAPDH, GLUT1, GLUT3, HIF1A, HMGR, LEPTIN, MAP1LC3B, RPL32, TNFSF10, and ZFAND5) (Table 2).
There were 23 genes with mRNA levels less than 50% of BCL2 mRNA in cells treated with glanded coat extract (AHRR1, CLAUDIN1, COX1, CXCL1, CYCLIND1, DGAT2A, DGAT2B, ELK1, FAS, GLUT4, HUA, ICAM1, INSR, IL2, IL10, IL12, NFKB, P53, RAB24, TNF, VEGF, ZFP36L1, and ZFP36L2) (Table 2). There were 24 genes with mRNA levels less than 50% of BCL2 mRNA in cells treated with glanded kernel extract (AHRR1, CLAUDIN1, COX1, CXCL1, CYCLIND1, DGAT1, DGAT2A, DGAT2B, ELK1, FAS, GLUT4, HMOX1, HUA, ICAM1, INSR, IL2, IL10, IL12, LEPTIN, NFKB, P53, RAB24, TNF, ZFP36L1, and ZFP36L2) (Table 2). There were 22 genes with mRNA levels less than 50% of BCL2 mRNA in cells treated with glandless coat extract (AHRR1, CLAUDIN1, COX1, CXCL1, CYCLIND1, DGAT2A, DGAT2B, ELK1, FAS, GLUT4, HUA, ICAM1, INSR, IL2, IL12, LEPTIN, NFKB, P53, RAB24, VEGF, ZFP36L1, and ZFP36L2) (Table 2). There were 21 genes with mRNA levels less than 50% of BCL2 mRNA in cells treated with glandless kernel extract (AHRR1, COX1, CXCL1, CYCLIND1, DGAT2A, DGAT2B, FAS, GLUT4, HMOX1, HUA, ICAM1, INSR, IL2, IL10, IL12, NFKB, P53, RAB24, TNF, VEGF, and ZFP36L2) (Table 2).
Overall effect of cottonseed ethanol extracts on gene expression in human colon cancer cells
After we analyzed the basal levels of gene expression and identified the reference gene for qPCR analysis as described previously, we evaluated how these genes might be affected by ethanol extracts by using the pooled qPCR data from 24 samples using BCL2 mRNA as the internal reference and DMSO treatment as the sample control. As shown in Table 3, expression of a number of genes was affected by cottonseed ethanol extracts. There were 3 genes with mRNA levels at least twofold of the DMSO control in the cells treated with glanded coat extract (CYCLIND1, CYP19A1, and LEPTIN) (Table 3). There were 2 genes with mRNA levels at least twofold of the DMSO control in the cells treated with glanded kernel extract (CYCLIND1 and CYP19A1) (Table 3). There were 2 genes with mRNA levels at least twofold of the DMSO control in the cells treated with glandless coat extract (CYCLIND1 and CYP19A1) (Table 3). There were 4 genes with mRNA levels at least twofold of the DMSO control in the cells treated with glandless kernel extract (COX2, CYCLIND1, CYP19A1, and LEPTIN) (Table 3).
There were 13 genes with mRNA levels less than 50% of the DMSO control in the cells treated with glanded coat extract (AHRR1, CLAUDIN1, CSNK2A1, CXCL1, DGAT2A, GLUT1, HMGR, ICAM1, INSR, IL16, NFKB, P53, and ZFP36L1) (Table 3). There were 22 genes with mRNA levels less than 50% of the DMSO control in the cells treated with glanded kernel extract (CLAUDIN1, CSNK2A1, CTSB, CXCL1, DGAT1, DGAT2A, DGAT2B, ELK1, FAS, GAPDH, GLUT1, HMGR, ICAM1, INSR, IL16, LEPTIN, MAP1LC3B, NFKB, P53, RPL32, ZFP36, and ZFP36L1) (Table 3). There were 13 genes with mRNA levels less than 50% of the DMSO control in the cells treated with glandless coat extract (AHRR1, CLAUDIN1, CSNK2A1, CTSB, CXCL1, DGAT2A, ELK1, ICAM1, INSR, IL16, LEPTIN, MAP1LC3B, and NFKB) (Table 3). There were 6 genes with mRNA levels less than 50% of the DMSO control in the cells treated with glandless kernel extract (CXCL1, HMOX1, HUA, ICAM1, INSR, and NFKB) (Table 3).
The above results suggest that cottonseed ethanol extracts affected the expression of many genes in the human colon cancer cells. Therefore, we analyzed the mRNA levels of 55 genes in the human colon cancer cells treated with various concentrations of the four cottonseed extracts as described below.
Effect of glanded coat extract on gene expression
Firstly, we analyzed the effect of glanded coat extract on gene expression. Human colon cancer cells were treated with glanded cottonseed coat extract (0, 5, 10, 20, 30, 40, 50 and 100 µg/ml). SYBR Green qPCR analyzed the expression of all 55 genes with BCL2 mRNA as the internal reference and 1% DMSO treatment as the sample control. The expression of some genes was significantly affected by glanded coat extract (Fig. 3). It appeared that the expression of COX2, GLUT1, LEPTIN, TNF, and TNFSF10 was increased by the glanded coat extract (Fig. 3). Other gene expression was reduced by the coat extract, including BCL22L2, CLUDIN1, CSNK2A1, CTSB, CXC1, DGAT1, GLUT1, HIF1, ZFAND5 and ZFP36 (Fig. 3). The expression of the rest of the 55 genes not mentioned above at mRNA levels was not affected by various concentrations of the glanded kernel extract (data not shown).
Glandless coat extract regulated the expression of genes. Human colon cancer cells (COLO 225) were treated with gossypol for 8 h. The data represent the mean and standard deviation of three independent samples.
Effect of glanded kernel extract on gene expression
Secondly, we analyzed the effect of glanded kernel extract on gene expression. Similarly, human colon cancer cells were treated with glanded cottonseed kernel extract. Gene expression was analyzed by qPCR with BCL2 mRNA as the internal reference and 1% DMSO treatment as the sample control. The expression of ELK1, FAS, and GAPDH genes was increased by the glanded kernel extract (Fig. 4). The expression of other genes at mRNA levels was not affected by various concentrations of the ethanol extract.
Glandless coat extract regulated the expression of genes. Human colon cancer cells (COLO 225) were treated with gossypol for 8 h. The data represent the mean and standard deviation of three independent samples.
Effect of glandless coat extract on gene expression
Thirdly, we analyzed the effect of glandless coat extract on gene expression. Human colon cancer cells were also treated with various concentrations of glandless cottonseed coat extract and analyzed gene expression at the mRNA levels by qPCR using BCL2 mRNA as the internal reference and 1% DMSO treatment as the sample control. The expression of FAS, GAPDH, GLUT1, and ZFP36 was increased by the glandless coat extract (Fig. 5), but only CXC1 expression was reduced by the coat extract (Fig. 5). The expression of the rest of the 55 genes not mentioned above at mRNA levels was not affected by various concentrations of the ethanol extract (data not shown).
Glandless coat extract regulated the expression of genes in human colon cancer cells. Human colon cancer cells (COLO 225) were treated with gossypol for 8 h. The data represent the mean and standard deviation of three independent samples.
Effect of glandless kernel extract on gene expression
Finally, we analyzed the effect of glandless kernel extract on gene expression. Similarly, glandless cottonseed kernel extract treated human colon cancer cells and SYBR Green qPCR analyzed mRNA levels of 55 genes with BCL2 mRNA as the internal reference and 1% DMSO treatment as the sample control. qPCR data indicated that expression of much more genes was affected by the glandless kernel extract. The effect of the glandless kernel extract on gene expression was analyzed in detail according to gene families as described below (Figs. 6, 7, 8).
Glandless kernel extract regulated the expression of genes coded for qPCR reference mRNAs, genes reported to be regulated by gossypol, and genes coded for DGAT and GLUT mRNAs in human colon cancer cells. Human colon cancer cells (COLO 225) were treated with glandless kernel extract for 8 h. The data represent the mean and standard deviation of three independent samples. (A) Genes coded for qPCR reference mRNAs, (B) Genes reported to be regulated by gossypol, (C) Genes coded for DGAT mRNAs, (D) Genes coded for GLUT mRNAs.
Glandless kernel extract regulated the expression of genes coded for TTP family, IL family, TTP-mediated proinflammatory cytokine and other mRNAs in human colon cancer cells. Human colon cancer cells (COLO 225) were treated with glandless kernel extract for 8 h. The data represent the mean and standard deviation of three independent samples. (A) Genes coded for TTP family mRNAs. (B) Genes coded for IL family mRNAs. (C) Genes coded for TTP-mediated proinflammatory cytokine mRNAs. (D) Genes coded for other TTP-mediated mRNAs.
Glandless kernel extract regulated the expression of other genes in human colon cancer cells. Human colon cancer cells (COLO 225) were treated with glandless kernel extract for 8 h. The data represent the mean and standard deviation of three independent samples.
Glandless kernel extract on reference gene expression
The expression of GAPDH and RPL32 genes, the two well-known reference genes in the literature, was analyzed in the colon cancer cells after treatment with various concentration of glandless kernel extract. The qPCR data showed that glandless kernel extract treatment resulted in a large reduction of both GAPDH and RPL32 mRNA levels in the cells (Fig. 6A).
Glandless kernel extract on gossypol-related gene expression
Expression of several genes was regulated by gossypol in cancer cells28,29,30,31,32,33,34,35 and macrophages26. BNIP3, CYP19A1, FAS, HUA, P53, PPARR and TNFSF10 gene expression was analyzed in the colon cancer cells after being treated with glandless kernel extract with various concentrations. The expression of FAS, HUA, P53 and PPARR genes was inhibited to a large extent by the glandless kernel extract (Fig. 6B).
Glandless kernel extract on DGAT gene expression
Diacylglycerol acyltransferases (DGATs) catalyze the rate-limiting step of triacylglycerol biosynthesis in eukaryotes by esterifyingsn-1,2-diacylglycerol with a long-chain fatty acyl-CoA54,55. DGATs are classified with DGAT1 and DGAT2 subfamilies in animals and additional DGAT3 subfamily in plants54,55,56,57 with DGAT2 mRNA being the major form of DGAT mRNAs in mouse adipocytes and macrophages25,58 but DGAT1 as the major one in the colon cancer cells53. The qPCR data showed that glandless kernel extract inhibited DGAT1, 2a and 2b expression in the human colon cancer cells (Fig. 6C).
Glandless kernel extract on GLUT gene expression
Glucose transporter (GLUT) family proteins are responsible for glucose uptake in mammalian cells. Four forms of GLUTs are present in mammalian cells23. The glandless kernel extract treatment only decreased GLUT1 mRNA level without much effect on the other GLUT isoforms (Fig. 6D). GLUT4 mRNA level was very low so that it was difficult to be measured with sufficient confidence (Table 2).
Glandless kernel extract on TTP gene expression
Tristetraprolin (TTP/ZFP36) family proteins regulate mRNA stability59. TTP family genes have anti-inflammatory properties with therapeutic potential for inflammation-related diseases60,61. TTP family proteins consist of three members in mammals (ZFP36 or TTP, ZFP36L1 and ZFP36L2) and the fourth member in mouse and rat but not in humans (ZFP36L3)59,62. SYBR Green qPCR showed that ZFP36 and ZFP36L1 mRNAs were reduced by the glandless kernel extract (Fig. 7A). ZFP36L2 mRNA levels were too low to be assessed reliably (Table 2).
Glandless kernel extract on IL gene expression
Several interleukins (ILs) are regulated by TTP family proteins which bind to AU-rich elements (ARE) of IL mRNAs and destabilizes the transcripts. TTP-regulated ILs include IL263, IL664, IL865, IL1066, IL1267, IL1642 and IL1768. SYBR Green qPCR showed that glandless kernel extract increased IL12 mRNA level but decreased IL16 mRNA level (Fig. 7B). IL8 and IL10 mRNA levels were difficult to compare due to their low levels in the colon cancer cells (Table 2).
Glandless kernel extract on proinflammatory gene expression
Several proinflammatory cytokine mRNAs are destabilized by TTP family proteins, including tumor necrosis factor-alpha (TNFα)60,69,70,71, granulocyte–macrophage colony-stimulating factor/colony-stimulating factor 2 (GM-CSF/CSF2)72,73 and cyclooxygenase 2/prostaglandin-endoperoxide synthase 2 (COX2/PTGS2)43. TNFα and GM-CSF mRNAs are stabilized in TTP knockout mice and in cells derived from them60,73, resulting in excessive levels of these cytokines causing a severe systemic inflammatory syndrome including arthritis, autoimmunity, and myeloid hyperplasia74,75. Elevated levels of TTP reduce inflammatory responses in macrophages76. These previous studies suggest that TTP is an anti-inflammatory protein. Our results showed that glandless kernel extract decreased COX1, LEPTIN and TNF mRNA levels in the colon cancer cells (Fig. 7C).
Glandless kernel extract on TTP-targeted other gene expression
Other TTP-regulated mRNAs have been reported in the literature (Table 1). SYBR Green qPCR analyzed the mRNA levels of AHRR, BCL22L1, CD36, CLAUDIN1, CSNK2A1, CTSB, CXD1, E2F1, ELK1, HIF1A, HOMX1, ICAMI, PIM1, and ZFAND5 genes. Glandless kernel extract decreased all of these TTP-targeted mRNA levels except CD36 and E2F1 mRNA levels (Fig. 7D).
Glandless kernel extract on other gene expression
A few other gene targets were selected for the analysis of gene expression. The qPCR assays showed that glandless kernel extract decreased the expression of HMGR, INSR, MAPL1C3A, MAPL1C3B, and NFKB mRNA levels (Fig. 8). The effect of glandless kernel extract on ULK2 mRNA level was not much and the effect on CYCLIND1 mRNA level was difficult to assess due to large variation of the results (Fig. 8).
Discussion
Cottonseed accounts for approximately 20% of the crop value. One way to increase cottonseed value is to isolate bioactive materials aimed at improving nutrition and preventing diseases. In this study, we observed that the expression of the majority of genes was significantly reduced by glandless cottonseed kernel extract, although their expression was less affected by three other cottonseed ethanol extracts (glanded cottonseed coat and kernel as well as glandless cottonseed coat extracts).
Cottonseed extracts exhibited only minor effect on the viability of human colon cancer cells under the experimental conditions. Our previous study showed that gossypol strongly inhibited human cancer cell viability24. The current data confirm our HPLC–MS analyses that the cottonseed extracts are essentially free of the toxic compound gossypol24.
Before we examined the effect of cottonseed extracts on gene expression in human colon cancer cells, we evaluated the relative expression levels of 55 genes and selected the internal reference for qPCR analysis since it is important for normalization of gene expression levels77,78,79,80. Our study confirmed that BCL2 mRNA was the most stable among the 55 mRNAs analyzed in human colon cancer cells treated with DMSO vehicle or various concentrations of ethanol extracts (Table 2)53. We also confirmed that GAPDH and RPL32 mRNAs were not good qPCR assay references for the colon cancer cells since they were most abundant mRNAs with large variations under the cell culture conditions53.
Our study showed that expression of many genes in human colon cancer cells was somewhat affected by cottonseed ethanol extracts. Although extracts isolated from glanded seed coat and kernel as well as glandless seed coat showed less effects on gene regulation, the expression of the majority of genes was significantly reduced by glandless seed kernel extract (Figs. 4, 5, 6, 7, 8). qPCR analyses showed that glanded coat extract increased COX2, GLUT2, LEPTIN, TNF, and TNFSF10 but decreased BCL22L2, CLUDIN1, CSNK2A1, CTSB, CXC1, DGAT1, GLUT1, HIF1, ZFAND5 and ZFP36 mRNA levels (Fig. 3). Glanded kernel extract increased ELK1, FAS, and GAPDH mRNA levels (Fig. 4). Glandless coat extract increased FAS, GAPDH, GLUT1, and ZFP36 but decreased CXC1 mRNA levels (Fig. 5).
The most important observation of this study was that glandless kernel extract decreased the mRNA levels of the great majority of the 55 genes tested, including GAPDH involved in the sixth step of breakdown of glucose in glycolysis80 and RPL32, a component of the large 60S subunit of ribosomes involved in protein synthesis77 (Fig. 6A), the genes known to be involved in cancer development, such as BNIP3 involved in the permeability of outer mitochondrial membrane35, CYP19A1 localized to the endoplasmic reticulum and catalyzed the last steps of estrogen biosynthesis30, FAS, a member of TNF-receptor superfamily playing a key role in programmed cell death29, P53 involved in preventing genome mutation28, PPARR, a nuclear receptor involved in gene expression regulation31 and TNFSF10, a TNF super family member functioning as a ligand that induces apoptosis34 (Fig. 6B), the DGAT family members DGAT1, DGAT2a and 2b responsible for the last and rate-limiting step of triacylglycerol biosynthesis54,58 (Fig. 6C), and GLUT1 responsible for glucose transport across the plasma membranes23 (Fig. 6D). In addition, glandless kernel extract reduced ZFP36 mRNA levels in the TTP family which bind to the AU-rich elements of some mRNAs and cause destabilization60,69 (Fig. 7A). It increased IL12, a T-cell stimulating fsctor67 but decreased IL16 functions as a chemoattractant, a modulator of T cell activation, and an inhibitor of HIV replication42 mRNAs levels in the IL family members (Fig. 7B), decreased LEPTIN involved in energy balance81 and TNF, a cytokine promoting inflammation64 mRNA levels (Fig. 7C), and appeared to decrease all of the TTP-targeted mRNAs including AHRR139, BCL2L182, CSNK2A141, CXCL183, HIF1a84, E2F140, ELK137, HMOX185, ICAM186 and ZFAND587 (Fig. 7D). Finally, glandless kernel extract appeared to decrease the expression of HMGR88, INSR21, MAPL1C3A89, MAPL1C3B89, and NFKB90 mRNA levels (Fig. 8).
This study provides valuable information about the effects of cottonseed ethanol extracts on gene expression at the mRNA levels in the human colon cancer cells. Much more investigations need to be conducted in the future. First, it could be a greater addition by confirming the mRNA results with results at the protein levels. Second, the consequence of gene regulation on cellular metabolic levels could be valuable for understanding the molecular mechanism. Finally, additional studies with other cell lines and animals could be required for the potential utilization of cottonseed extracts as viable sources for improving nutrition and preventing diseases.
Conclusions
This study showed that most of the gene expression in human colon cancer cells was not affected by ethanol extracts isolated from glanded cottonseed coat and kernel as well as glandless cottonseed coat, but the expression of the majority of genes was significantly reduced by glandless cottonseed kernel extracts. The inhibitory effects of glandless kernel extract on gene expression in the colon cancer cells may provide a useful opportunity for improving the healthcare associated with colon cancer since it is safe without toxic gossypol contamination and effective in decreasing the expression of so many genes related to cancer development. This in turn may provide the potential of increasing the value of cottonseed by using cottonseed-derived ethanol extracts as a health intervention agent.
Materials and methods
Cottonseed
The cottonseeds used in the study were provided by Drs. Michael Dowd and Rick Byler (USDA-ARS) and Tom Wedegaertner (Cotton, Inc.). The experiments were performed in accordance with national/institutional guidelines and regulations.
Cancer cell line
Human colon cancer cells (COLO 205) and A549 lung cancer cells (CCL185) (ATCC, Manassas, VA) were kept under liquid nitrogen vapor. The cells were maintained in a humidified incubator at 37 °C with 5% CO2 in RPMI-1640 (COLO 205) and F-12K (CCL185) medium, respectively, supplemented with 10% (v:v) fetal bovine serum, 0.1 million units/L penicillin, 100 mg/L streptomycin, and 2 mmol/L L-glutamine (Gibco, Life Technologies).
Chemicals, reagents and equipment
Cell cytotoxicity reagent (MTT based-In Vitro Toxicology Assay Kit) and DMSO were from Sigma. Tissue culture reagents were from Gibco BRL (Thermo Fisher). Tissue culture incubator was water jacket CO2 incubator (Thermo Fisher). Tissue culture workstation was Logic + A2 hood (Labconco, Kansas City, MO). Tissue culture plastic ware was from CytoOne (USA Scientific, Ocala, FL). Cell counting reagent (trypsin blue dye), slides (dual chamber), counter (TC20 Automatic Cell Counter) and microscope (Zoe Florescent Cell Imager) were from Bio-Rad (Hercules, CA). Microplate spectrophotometer (Epoch) was from BioTek Instruments (Winooski, VT).
Cottonseed extracts
Seed kernel extracts were isolated by fractionation, defatting, and ethanol extraction, and seed coat extracts were isolated by fractionation, defatting, acetic acid extraction, and ethanol extraction24 (Fig. 1). Briefly, cottonseed coat or kernel was ground into fine powder and homogenized. The kernel fraction was defatted with chloroform and hexane. The coat fraction was treated with acetic acid followed by autoclave and centrifugation. The defatted materials were extracted with ethanol followed by evaporation to remove acetic acid and ethanol. Ethanol extracts were reconstitution in 100% DMSO (100 mg/mL) and analyzed by HPLC–MS. The ethanol extracts contained trace amount of gossypol (0.82 ng gossypol/mg extract in glanded seed coat, 0.03 ng gossypol/mg extract in glanded seed kernel, 0.37 ng gossypol/mg extract in glandless seed coat and 0 ng gossypol/mg extract in glandless seed kernel)24.
Cell culture and chemical treatment
Cell culture was according to previous procedures19,23,69. Cancer cells were dissociated from flasks with 0.25% (w/v) trypsin-0.53 mM EDTA solution, stained with 0.2% trypsin blue dye and counted the number of live cells with a TC20 Automatic Cell Counter. Cells were subcultured at ~ 1 × 105 cells/mL density in 24-well plates (0.5 mL). The cancer cells were routinely observed under a Zoe Florescent Cell Imager. Cancer cells were treated with 0, 5, 10, 20, 30, 40, 50 and 100 µg/mL of ethanol extracts for 2, 4, 8 and 24 h (“0” treatment as the vehicle control corresponding to 1% DMSO present in all of the culture medium).
Cell viability assay
MTT based-In Vitro Toxicology Assay Kit was used to determine cell cytotoxicity24. Cancer cells in 96-well plates (12 wells/treatment) were treated with ethanol extracts and incubated at 37 °C, 5% CO2 for 2 and 24 h. The cell media were added with 50 µL of MTT assay reagent (thiazolyl blue tetrazolium bromide) and incubated at 37 °C, 5% CO2 for 2 h before adding 500 µL MTT solubilization solution to each well, shaken at room temperature overnight. The color density in the wells was recorded by Epoch microplate spectrophotometer at A570.
Real-time qPCR primers and probes
Fifty-five genes were selected for qPCR analysis of their expression in the colon cancer cells as described previously53. These genes were shown to be regulated by cottonseed-derived gossypol in cancer cells and macrophages or regulated by ZFP36/TTP in tumor cells and macrophages (Table 2). RNA sequences were obtained from NCBI’s non-redundant protein sequence databases (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The qPCR primers were designed with Applied Biosystems’ Primer Express software (Foster City, CA) and synthesized by Biosearch Technologies, Inc. (Navato, CA).
RNA isolation and cDNA synthesis
RNA isolation and cDNA synthesis were essentially as described25. Human colon cancer cells were treated with various concentrations of cottonseed ethanol extracts for 8 h (triplicate). The cells were lysed directly in the washed dishes with 1 mL of TRIZOL reagent. RNA was isolated according to the manufacturer’s instructions without DNase treatment. RNA concentrations were quantified with an Implen NanoPhotometer (Munchen, Germany). The cDNAs were synthesized from total RNA using SuperScript II reverse transcriptase. The cDNA synthesis mixture contained 5 μg total RNA, 2.4 μg oligo(dT)12,13,14,15,16,17,18 primer, 0.1 μg random primers, 500 μM dNTPs, 10 mM DTT, 40 u RNaseOUT and 200 u SuperScript II reverse transcriptase in 1X first-strand synthesis buffer (20 μL). The cDNA synthesis reaction was performed at 42 °C for 50 min. The cDNA was stored in − 80 °C freezer and diluted with water to 1 ng/µL before qPCR analyses.
Quantitative real-time PCR analysis
The qPCR assays were described56,78,79,91 and performed according to the MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments92. The qPCR assay mixture contained 5 ng of RNA-derived cDNA, 200 nM of forward and reverse primers, and 1 × iQ SYBR Green Supermix. Thermal cycle conditions were 3 min at 95 °C, 40 cycles at 95 °C for 10 s, 65 °C for 30 s and 72 °C for 30 s. BCL2 mRNA was used as the internal reference because it had the minimal variation of gene expression among the 55 genes tested (see “Results” for details). Ribosome protein 32 (RPL32) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were not suitable for qPCR analysis for this cell type due to variations (see “Results” for details) although they were widely used as the reference mRNAs in qPCR analyses19. TaqMan qPCR assay confirmed some of the SBYR Green qPCR assays using the same conditions as described78.
Data analysis and statistics
The relative expression in fold was determined with 2−ΔCT or 2−ΔΔCT equations93. The first step was to normalize the threshold cycle (CT) values of the target mRNAs to the CT values of the internal control BCL2 mRNA (ΔCT = CTTarget − CTBcl2). The second step was to normalize treatment ΔCT values with DMSO control ΔCT values (ΔΔCT = ΔCTCottonseed − ΔCTDMSO). Finally, the fold change in expression was calculated. The data in the figures and tables represent the mean and standard deviation of three and 24 independent samples, respectively. These data were subjected to statistical analysis using ANOVA with SigmaStat 3.1 software (Systat Software). Student–Newman–Keuls method and Tukey test were used to perform multiple comparisons among the treatments with different concentrations of cottonseed extracts19.
References
Luo, P., Wang, Y. H., Wang, G. D., Essenberg, M. & Chen, X. Y. Molecular cloning and functional identification of (+)-delta-cadinene-8-hydroxylase, a cytochrome P450 mono-oxygenase (CYP706B1) of cotton sesquiterpene biosynthesis. Plant J. 28, 95–104 (2001).
Ma, D. et al. Genetic basis for glandular trichome formation in cotton. Nat. Commun. 7, 10456. https://doi.org/10.1038/ncomms10456 (2016).
Wang, X., Howell, C. P., Chen, F., Yin, J. & Jiang, Y. Gossypol–a polyphenolic compound from cotton plant. Adv. Food Nutr. Res. 58, 215–263. https://doi.org/10.1016/S1043-4526(09)58006-0 (2009).
He, Z., Zhang, H. & Olk, D. C. Chemical composition of defatted cottonseed and soy meal products. PLoS ONE 10, e0129933. https://doi.org/10.1371/journal.pone.0129933 (2015).
Camara, A. C. et al. Toxicity of gossypol from cottonseed cake to sheep ovarian follicles. PLoS ONE 10, e0143708. https://doi.org/10.1371/journal.pone.0143708 (2015).
Coutinho, E. M. Gossypol: A contraceptive for men. Contraception 65, 259–263 (2002).
Gadelha, I. C., Fonseca, N. B., Oloris, S. C., Melo, M. M. & Soto-Blanco, B. Gossypol toxicity from cottonseed products. Sci. World J. 2014, 231635. https://doi.org/10.1155/2014/231635 (2014).
Randel, R. D., Chase, C. C. Jr. & Wyse, S. J. Effects of gossypol and cottonseed products on reproduction of mammals. J. Anim. Sci. 70, 1628–1638 (1992).
Zeng, Q. F. et al. Effects of dietary gossypol concentration on growth performance, blood profiles, and hepatic histopathology in meat ducks. Poult. Sci 93, 2000–2009. https://doi.org/10.3382/ps.2013-03841 (2014).
Cornu, A., Delpeuch, F. & Favier, J. C. Utilization of gossypol-free cottonseed and its by-products as human food. Ann. Nutr. Aliment. 31, 349–364 (1977).
Sneed, S. M., Thomas, M. R. & Alford, B. B. Effects of a glandless cottonseed protein diet on fasting plasma amino acid levels in college women. Am. J. Clin. Nutr. 33, 287–292 (1980).
Thomas, M. R., Ashby, J., Sneed, S. M. & O’Rear, L. M. Minimum nitrogen requirement from glandless cottonseed protein for nitrogen balance in college women. J. Nutr. 109, 397–405 (1979).
Lusas, E. W. & Jividen, G. M. Glandless cottonseed: A review of the first 25 years of processing and utilization research. J. Am. Oil Chem. Soc. 64, 839–854 (1987).
Cao, H. in Proceedings of the 2019 Beltwide Cotton Conferences, New Orleans, Louisiana, pp. 559–571 (National Cotton Council of America, 2019).
Cao, H. & Sethumadhavan, K. in Proceedings of the 2020 Beltwide Cotton Conferences, Austin, Texas, pp. 270–281 (National Cotton Council of America, 2020).
Prior, R. L. & Gu, L. Occurrence and biological significance of proanthocyanidins in the American diet. Phytochemistry 66, 2264–2280. https://doi.org/10.1016/j.phytochem.2005.03.025 (2005).
Cao, H. et al. Green tea polyphenol extract regulates the expression of genes involved in glucose uptake and insulin signaling in rats fed a high fructose diet. J. Agric. Food Chem. 55, 6372–6378 (2007).
Cao, H. et al. Green tea increases anti-inflammatory tristetraprolin and decreases pro-inflammatory tumor necrosis factor mRNA levels in rats. J. Inflamm. (Lond) 4, 1 (2007).
Cao, H. & Anderson, R. A. Cinnamon polyphenol extract regulates tristetraprolin and related gene expression in mouse adipocytes. J. Agric. Food Chem. 59, 2739–2744. https://doi.org/10.1021/jf103527x (2011).
Cao, H., Graves, D. J. & Anderson, R. A. Cinnamon extract regulates glucose transporter and insulin-signaling gene expression in mouse adipocytes. Phytomedicine 17, 1027–1032. https://doi.org/10.1016/j.phymed.2010.03.023 (2010).
Cao, H., Polansky, M. M. & Anderson, R. A. Cinnamon extract and polyphenols affect the expression of tristetraprolin, insulin receptor, and glucose transporter 4 in mouse 3T3-L1 adipocytes. Arch. Biochem. Biophys. 459, 214–222 (2007).
Cao, H., Sethumadhavan, K., Li, K., Boue, S. M. & Anderson, R. A. Cinnamon polyphenol extract and insulin regulate diacylglycerol acyltransferase gene expression in mouse adipocytes and macrophages. Plant Foods Hum. Nutr. 74, 115–121. https://doi.org/10.1007/s11130-018-0709-7 (2019).
Cao, H., Urban, J. F. Jr. & Anderson, R. A. Cinnamon polyphenol extract affects immune responses by regulating anti- and proinflammatory and glucose transporter gene expression in mouse macrophages. J. Nutr. 138, 833–840 (2008).
Cao, H., Sethumadhavan, K. & Bland, J. M. Isolation of cottonseed extracts that affect human cancer cell growth. Sci. Rep. 8, 10458. https://doi.org/10.1038/s41598-018-28773-4 (2018).
Cao, H. & Sethumadhavan, K. Cottonseed extracts and gossypol regulate diacylglycerol acyltransferase gene expression in mouse macrophages. J. Agric. Food Chem. 66, 6022–6030. https://doi.org/10.1021/acs.jafc.8b01240 (2018).
Cao, H. & Sethumadhavan, K. Gossypol but not cottonseed extracts or lipopolysaccharides stimulates HuR gene expression in mouse cells. J. Func. Foods 59, 25–29 (2019).
Cao, H. & Sethumadhavan, K. Regulation of cell viability and anti-inflammatory tristetraprolin family gene expression in mouse macrophages by cottonseed extracts. Sci. Rep. 10, 775. https://doi.org/10.1038/s41598-020-57584-9 (2020).
Barba-Barajas, M. et al. Gossypol induced apoptosis of polymorphonuclear leukocytes and monocytes: Involvement of mitochondrial pathway and reactive oxygen species. Immunopharmacol. Immunotoxicol. 31, 320–330. https://doi.org/10.1080/08923970902718049 (2009).
Chang, J. S., Hsu, Y. L., Kuo, P. L., Chiang, L. C. & Lin, C. C. Upregulation of Fas/Fas ligand-mediated apoptosis by gossypol in an immortalized human alveolar lung cancer cell line. Clin. Exp. Pharmacol. Physiol 31, 716–722. https://doi.org/10.1111/j.1440-1681.2004.04078.x (2004).
Dong, Y. et al. Gossypol enantiomers potently inhibit human placental 3beta-hydroxysteroid dehydrogenase 1 and aromatase activities. Fitoterapia 109, 132–137. https://doi.org/10.1016/j.fitote.2015.12.014 (2015).
Huang, Y. W. et al. Molecular mechanisms of (-)-gossypol-induced apoptosis in human prostate cancer cells. Anticancer Res. 26, 1925–1933 (2006).
Kitada, S. et al. Bcl-2 antagonist apogossypol (NSC736630) displays single-agent activity in Bcl-2-transgenic mice and has superior efficacy with less toxicity compared with gossypol (NSC19048). Blood 111, 3211–3219. https://doi.org/10.1182/blood-2007-09-113647 (2008).
Ligueros, M. et al. Gossypol inhibition of mitosis, cyclin D1 and Rb protein in human mammary cancer cells and cyclin-D1 transfected human fibrosarcoma cells. Br. J. Cancer 76, 21–28 (1997).
Yeow, W. S. et al. Gossypol, a phytochemical with BH3-mimetic property, sensitizes cultured thoracic cancer cells to Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand. J. Thorac. Cardiovasc. Surg. 132, 1356–1362. https://doi.org/10.1016/j.jtcvs.2006.07.025 (2006).
Yuan, Y. et al. Gossypol and an HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells. Cell Death. Dis. 4, e690. https://doi.org/10.1038/cddis.2013.191 (2013).
Essafi-Benkhadir, K., Onesto, C., Stebe, E., Moroni, C. & Pages, G. Tristetraprolin inhibits ras-dependent tumor vascularization by inducing VEGF mRNA degradation. Mol. Biol. Cell 18, 4648–4658 (2007).
Florkowska, M. et al. EGF activates TTP expression by activation of ELK-1 and EGR-1 transcription factors. BMC Mol. Biol. 13, 8. https://doi.org/10.1186/1471-2199-13-8 (2012).
Kim, H. K. et al. Expression of proviral integration site for moloney murine leukemia virus 1 (Pim-1) is post-transcriptionally regulated by tristetraprolin in cancer cells. J. Biol. Chem. 287, 28770–28778. https://doi.org/10.1074/jbc.M112.376483 (2012).
Lee, H. H. et al. Tristetraprolin suppresses AHRR expression through mRNA destabilization. FEBS Lett. 587, 1518–1523. https://doi.org/10.1016/j.febslet.2013.03.031 (2013).
Lee, H. H., Lee, S. R. & Leem, S. H. Tristetraprolin regulates prostate cancer cell growth through suppression of E2F1. J. Microbiol. Biotechnol. 24, 287–294. https://doi.org/10.4014/jmb.1309.09070 (2014).
Lee, W. H. et al. Casein kinase 2 regulates the mRNA-destabilizing activity of tristetraprolin. J Biol. Chem 286, 21577–21587. https://doi.org/10.1074/jbc.M110.201137 (2011).
Milke, L. et al. Depletion of tristetraprolin in breast cancer cells increases interleukin-16 expression and promotes tumor infiltration with monocytes/macrophages. Carcinogenesis 34, 850–857. https://doi.org/10.1093/carcin/bgs387 (2013).
Sawaoka, H., Dixon, D. A., Oates, J. A. & Boutaud, O. Tristetraprolin binds to the 3’-untranslated region of cyclooxygenase-2 mRNA. A polyadenylation variant in a cancer cell line lacks the binding site. J. Biol. Chem. 278, 13928–13935 (2003).
Sharma, A., Bhat, A. A., Krishnan, M., Singh, A. B. & Dhawan, P. Trichostatin-A modulates claudin-1 mRNA stability through the modulation of Hu antigen R and tristetraprolin in colon cancer cells. Carcinogenesis 34, 2610–2621. https://doi.org/10.1093/carcin/bgt207 (2013).
Giacomini, P., Natali, P. & Ferrone, S. Analysis of the interaction between a human high molecular weight melanoma-associated antigen and the monoclonal antibodies to three distinct antigenic determinants. J. Immunol. 135, 696–702 (1985).
Kusama, M., Kageshita, T., Tsujisaki, M., Perosa, F. & Ferrone, S. Syngeneic antiidiotypic antisera to murine antihuman high-molecular-weight melanoma-associated antigen monoclonal antibodies. Cancer Res 47, 4312–4317 (1987).
Joshi, S. S., Jackson, J. D. & Sharp, J. G. Comparison of the growth and metastasis of four human intestinal tumor cell line xenografts. Tumour Biol. 10, 117–125. https://doi.org/10.1159/000217607 (1989).
Gilani, A. H. & Janbaz, K. H. Evaluation of the liver protective potential of Cichorium intybus seed extract on acetaminophen and CCl(4)-induced damage. Phytomedicine 1, 193–197. https://doi.org/10.1016/S0944-7113(11)80064-4 (1994).
Corti, A. et al. Tumor targeting with biotinylated tumor necrosis factor alpha: Structure-activity relationships and mechanism of action on avidin pretargeted tumor cells. Cancer Res 58, 3866–3872 (1998).
Zhang, L., Fogg, D. K. & Waisman, D. M. RNA interference-mediated silencing of the S100A10 gene attenuates plasmin generation and invasiveness of Colo 222 colorectal cancer cells. J. Biol. Chem. 279, 2053–2062. https://doi.org/10.1074/jbc.M310357200 (2004).
Lien, H. M. et al. Study of the anti-proliferative activity of 5-substituted 4,7-dimethoxy-1,3-benzodioxole derivatives of SY-1 from antrodia camphorata on human COLO 205 colon cancer cells. Evid. Based Complement. Alternat. Med. 2011, 450529. https://doi.org/10.1093/ecam/nep230 (2011).
Kus, C., Ozer, E., Korkmaz, Y., Yurtcu, E. & Dagalp, R. Benzamide and benzamidine compounds as new inhibitors of urokinasetype plasminogen activators. Mini. Rev. Med. Chem. 18, 1753–1758. https://doi.org/10.2174/1389557518666180816110740 (2018).
Cao, H., Sethumadhavan, K., Cao, F. & Wang, T. T. Y. Gossypol decreased cell viability and down-regulated the expression of a number of genes in human colon cancer cells. Sci. Rep. 11, 5922. https://doi.org/10.1038/s41598-021-84970-8 (2021).
Cao, H. Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms. BMC Res. Notes 4, 249. https://doi.org/10.1186/1756-0500-4-249 (2011).
Liu, Q., Siloto, R. M., Lehner, R., Stone, S. J. & Weselake, R. J. Acyl-CoA:diacylglycerol acyltransferase: Molecular biology, biochemistry and biotechnology. Prog. Lipid Res. 51, 350–377. https://doi.org/10.1016/j.plipres.2012.06.001 (2012).
Cao, H., Shockey, J. M., Klasson, K. T., Mason, C. B. & Scheffler, B. E. Developmental regulation of diacylglycerol acyltransferase family gene expression in tung tree tissues. PLoS ONE 8, e76946. https://doi.org/10.1371/journal.pone.0076946 (2013).
Shockey, J. M. et al. Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum. Plant Cell 18, 2294–2313 (2006).
Cao, H. Identification of the major diacylglycerol acyltransferase mRNA in mouse adipocytes and macrophages. BMC Biochem. 19, 1–11. https://doi.org/10.1186/s12858-018-0103-y (2018).
Blackshear, P. J. Tristetraprolin and other CCCH tandem zinc-finger proteins in the regulation of mRNA turnover. Biochem. Soc. Trans. 30, 945–952 (2002).
Carballo, E., Lai, W. S. & Blackshear, P. J. Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin. Science 281, 1001–1005 (1998).
Lai, W. S., Stumpo, D. J. & Blackshear, P. J. Rapid insulin-stimulated accumulation of an mRNA encoding a proline-rich protein. J. Biol. Chem. 265, 16556–16563 (1990).
Blackshear, P. J. et al. Zfp36l3, a rodent X chromosome gene encoding a placenta-specific member of the tristetraprolin family of CCCH tandem zinc finger proteins. Biol. Reprod. 73, 297–307 (2005).
Ogilvie, R. L. et al. Tristetraprolin down-regulates IL-2 gene expression through AU-rich element-mediated mRNA decay. J. Immunol. 174, 953–961 (2005).
Hochdorfer, T. et al. LPS-induced production of TNF-alpha and IL-6 in mast cells is dependent on p38 but independent of TTP. Cell Signal. 25, 1339–1347. https://doi.org/10.1016/j.cellsig.2013.02.022 (2013).
Balakathiresan, N. S. et al. Tristetraprolin regulates IL-8 mRNA stability in cystic fibrosis lung epithelial cells. Am. J. Physiol. Lung Cell Mol. Physiol. 296, 1012–1018 (2009).
Gaba, A. et al. Cutting edge: IL-10-mediated tristetraprolin induction is part of a feedback loop that controls macrophage STAT3 activation and cytokine production. J. Immunol. 189, 2089–2093 (2012).
Gu, L. et al. Suppression of IL-12 production by tristetraprolin through blocking NF-kcyB nuclear translocation. J. Immunol. 191, 3922–3930. https://doi.org/10.4049/jimmunol.1300126 (2013).
Datta, S. et al. IL-17 regulates CXCL1 mRNA stability via an AUUUA/tristetraprolin-independent sequence. J. Immunol. 184, 1484–1491 (2010).
Cao, H. Expression, purification, and biochemical characterization of the antiinflammatory tristetraprolin: A zinc-dependent mRNA binding protein affected by posttranslational modifications. Biochemistry 43, 13724–13738 (2004).
Cao, H., Dzineku, F. & Blackshear, P. J. Expression and purification of recombinant tristetraprolin that can bind to tumor necrosis factor-alpha mRNA and serve as a substrate for mitogen-activated protein kinases. Arch. Biochem. Biophys. 412, 106–120 (2003).
Lai, W. S. et al. Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. Mol. Cell. Biol. 19, 4311–4323 (1999).
Carballo, E. et al. Decreased sensitivity of tristetraprolin-deficient cells to p38 inhibitors suggests the involvement of tristetraprolin in the p38 signaling pathway. J. Biol. Chem. 276, 42580–42587 (2001).
Carballo, E., Lai, W. S. & Blackshear, P. J. Evidence that tristetraprolin is a physiological regulator of granulocyte-macrophage colony-stimulating factor messenger RNA deadenylation and stability. Blood 95, 1891–1899 (2000).
Phillips, K., Kedersha, N., Shen, L., Blackshear, P. J. & Anderson, P. Arthritis suppressor genes TIA-1 and TTP dampen the expression of tumor necrosis factor alpha, cyclooxygenase 2, and inflammatory arthritis. Proc. Natl. Acad. Sci. USA 101, 2011–2016 (2004).
Taylor, G. A. et al. A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity 4, 445–454 (1996).
Sauer, I. et al. Interferons limit inflammatory responses by induction of tristetraprolin. Blood 107, 4790–4797 (2006).
Brattelid, T. et al. Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies. BMC Mol. Biol. 11, 22. https://doi.org/10.1186/1471-2199-11-22 (2010).
Cao, H., Cao, F., Roussel, A. M. & Anderson, R. A. Quantitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and macrophages. In Vitro Cell Dev. Biol. Anim. 49, 759–770. https://doi.org/10.1007/s11626-013-9671-8 (2013).
Cao, H. & Shockey, J. M. Comparison of TaqMan and SYBR green qPCR methods for quantitative gene expression in tung tree tissues. J. Agric. Food Chem. 60, 12296–12303. https://doi.org/10.1021/jf304690e (2012).
Chen, D., Pan, X., Xiao, P., Farwell, M. A. & Zhang, B. Evaluation and identification of reliable reference genes for pharmacogenomics, toxicogenomics, and small RNA expression analysis. J. Cell Physiol. 226, 2469–2477. https://doi.org/10.1002/jcp.22725 (2011).
Xu, P. et al. Leptin and zeranol up-regulate cyclin D1 expression in primary cultured normal human breast pre-adipocytes. Mol. Med. Rep. 3, 983–990. https://doi.org/10.3892/mmr.2010.370 (2010).
Frevel, M. A. et al. p38 Mitogen-activated protein kinase-dependent and -independent signaling of mRNA stability of AU-rich element-containing transcripts. Mol. Cell. Biol. 23, 425–436 (2003).
Datta, S. et al. Tristetraprolin regulates CXCL1 (KC) mRNA stability. J. Immunol. 180, 2545–2552 (2008).
Fahling, M. et al. Multilevel regulation of HIF-1 signaling by TTP. Mol. Biol. Cell 23, 4129–4141. https://doi.org/10.1091/mbc.E11-11-0949 (2012).
Jamal, U. M. et al. A functional link between heme oxygenase-1 and tristetraprolin in the anti-inflammatory effects of nicotine. Free Radic. Biol. Med. 65C, 1331–1339. https://doi.org/10.1016/j.freeradbiomed.2013.09.027 (2013).
Shi, J. X. et al. CNOT7/hCAF1 is involved in ICAM-1 and IL-8 regulation by tristetraprolin. Cell Signal. 26, 2390–2396. https://doi.org/10.1016/j.cellsig.2014.07.020 (2014).
He, G., Sun, D., Ou, Z. & Ding, A. The protein Zfand5 binds and stabilizes mRNAs with AU-rich elements in their 3’-untranslated regions. J. Biol. Chem. 287, 24967–24977. https://doi.org/10.1074/jbc.M112.362020 (2012).
Kagami, S. et al. HMG-CoA reductase inhibitor simvastatin inhibits proinflammatory cytokine production from murine mast cells. Int. Arch. Allergy Immunol. 146(Suppl 1), 61–66 (2008).
Voss, A. K., Thomas, T. & Gruss, P. Compensation for a gene trap mutation in the murine microtubule-associated protein 4 locus by alternative polyadenylation and alternative splicing. Dev Dyn 212, 258–266 (1998).
Jiang, J., Slivova, V., Jedinak, A. & Sliva, D. Gossypol inhibits growth, invasiveness, and angiogenesis in human prostate cancer cells by modulating NF-kappaB/AP-1 dependent- and independent-signaling. Clin. Exp. Metastasis 29, 165–178. https://doi.org/10.1007/s10585-011-9439-z (2012).
Cao, H., Cao, F. & Klasson, K. T. Characterization of reference gene expression in tung tree (Vernicia fordii). Ind. Crops Prod. 50, 248–255 (2013).
Bustin, S. A. et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622. https://doi.org/10.1373/clinchem.2008.112797 (2009).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408. https://doi.org/10.1006/meth.2001.1262 (2001).
Xu, L. et al. Tristetraprolin induces cell cycle arrest in breast tumor cells through targeting AP-1/c-Jun and NF-kappaB pathway. Oncotarget 6, 41679–41691. https://doi.org/10.18632/oncotarget.6149 (2015).
Warzych, E., Wolc, A., Cieslak, A. & Lechniak-Cieslak, D. 217 transcript abundance of cathepsin genes in cumulus cells as a marker of cattle oocyte quality. Reprod. Fertil. Dev. 25, 257. https://doi.org/10.1071/RDv25n1Ab217 (2012).
Fuhrmann, D. C. et al. Inactivation of tristetraprolin in chronic hypoxia provokes the expression of cathepsin B. Mol. Cell Biol 35, 619–630. https://doi.org/10.1128/MCB.01034-14 (2015).
Ludwig, E. H. et al. DGAT1 promoter polymorphism associated with alterations in body mass index, high density lipoprotein levels and blood pressure in Turkish women. Clin. Genet. 62, 68–73 (2002).
Dey, P., Chakraborty, M., Kamdar, M. R. & Maiti, M. K. Functional characterization of two structurally novel diacylglycerol acyltransferase2 isozymes responsible for the enhanced production of stearate-rich storage lipid in Candida tropicalis SY005. PLoS ONE 9, e94472. https://doi.org/10.1371/journal.pone.0094472 (2014).
Su, N. Y., Tsai, P. S. & Huang, C. J. Clonidine-induced enhancement of iNOS expression involves NF-kappaB. J. Surg. Res. 149, 131–137 (2008).
Militello, R. D. et al. Rab24 is required for normal cell division. Traffic 14, 502–518 (2013).
Gao, W., Shen, Z., Shang, L. & Wang, X. Upregulation of human autophagy-initiation kinase ULK1 by tumor suppressor p53 contributes to DNA-damage-induced cell death. Cell Death. Differ. 18, 1598–1607. https://doi.org/10.1038/cdd.2011.33 (2011).
Acknowledgements
We thank Drs. K. Thomas Klasson and Michael Dowd, Rick Byler and Tom Wedegaertner for their valuable discussion and providing cottonseed used in this study.
Funding
This work was supported by the USDA-ARS Quality and Utilization of Agricultural Products National Program [project numbers CRIS 6054-41000-103-00-D and CRIS 6054-41000-113-00-D]. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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H.C. designed the experiments. H.C., K.S., X.W. and X.Z. performed the experiments. L.Z. provided research resources. H.C. wrote the manuscript. All authors reviewed the manuscript.
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Cao, H., Sethumadhavan, K., Wu, X. et al. Cottonseed extracts regulate gene expression in human colon cancer cells. Sci Rep 12, 1039 (2022). https://doi.org/10.1038/s41598-022-05030-3
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DOI: https://doi.org/10.1038/s41598-022-05030-3
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