Gossypol decreased cell viability and down-regulated the expression of a number of genes in human colon cancer cells

Plant polyphenol gossypol has anticancer activities. This may increase cottonseed value by using gossypol as a health intervention agent. It is necessary to understand its molecular mechanisms before human consumption. The aim was to uncover the effects of gossypol on cell viability and gene expression in cancer cells. In this study, human colon cancer cells (COLO 225) were treated with gossypol. MTT assay showed significant inhibitory effect under high concentration and longtime treatment. We analyzed the expression of 55 genes at the mRNA level in the cells; many of them are regulated by gossypol or ZFP36/TTP in cancer cells. BCL2 mRNA was the most stable among the 55 mRNAs analyzed in human colon cancer cells. GAPDH and RPL32 mRNAs were not good qPCR references for the colon cancer cells. Gossypol decreased the mRNA levels of DGAT, GLUT, TTP, IL families and a number of previously reported genes. In particular, gossypol suppressed the expression of genes coding for CLAUDIN1, ELK1, FAS, GAPDH, IL2, IL8 and ZFAND5 mRNAs, but enhanced the expression of the gene coding for GLUT3 mRNA. The results showed that gossypol inhibited cell survival with decreased expression of a number of genes in the colon cancer cells.

Overall effect of gossypol on gene expression in human colon cancer cells. To provide a general idea how these genes were regulated by gossypol, we analyzed the pooled qPCR data using BCL2 mRNA as the internal reference and DMSO treatment as the sample control. As shown in the right column of Table 2, expression of a number of genes was affected by gossypol. Gossypol decreased the expression of six mRNAs with less than 50% of the control and only up-regulated the expression of two mRNAs with at least twofold of the control. The up-regulated mRNAs were IL10 (2.14 fold) and IL12 (4.94 fold) ( Table 2, right column). The down-regulated mRNAs were COX1 (1%), COX2 (43%), CYP19A1 (5%), E2F1 (46%), ELK1 (38%) and PPARR (47%) ( Table 2, right column).
Gossypol decreased the expression of reference GAPDH and RPL32 genes in human colon cancer cells. The expression of the two well-known reference genes was analyzed in the colon cancer cell line under treatment with various concentration of gossypol using internal reference gene BCL2 selected in this study. The qPCR data showed that GAPDH and RPL32 mRNA levels were 39 and 42 fold of BCL2 in the controlled cells (Table 4). High concentrations of gossypol treatment resulted in a remarkable reduction of both GAPDH and RPL32 mRNA levels in the cells (Fig. 3A). Both GAPDH and RPL32 mRNA levels were reduced more than 80% by 40-100 µg/mL of gossypol treatment (Fig. 3A).
Gossypol effect on reported gene expression in human colon cancer cells. The expression of a number of genes was shown previously to be regulated by gossypol in cancer cells 20,33-39 and macrophages 40 .
We analyzed the expression of BNIP3, CYP19A1, FAS, HUA, P53, PPARR and TNFSF10 genes under various concentrations of gossypol in the colon cancer cell line using BCL2 as the internal reference gene. In general, this group of genes was expressed lower than BCL2 control except BINP3 and TNFSF10 (Table 4). The expression of all these genes except PPARR gene was inhibited to a large extent by the highest concentration of gossypol tested at 100 µg/mL (Fig. 3B). It appears that PPARR gene expression was increased but the large standard deviation among the measurements prevented from making such a conclusion (Fig. 3B).

Gossypol effect on DGAT gene expression in human colon cancer cells. Diacylglycerol acyltrans-
ferases (DGATs) esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA and catalyze the rate-limiting step of triacylglycerol biosynthesis in eukaryotic organisms 52 . DGATs are divided into DGAT1 and DGAT2 subfamilies in animals and DGAT3 subfamily are present in plants [52][53][54][55] . DGAT2 mRNA was the major form of DGAT mRNAs in the mouse adipocytes and macrophages 56,57 . Gossypol was shown to be a strong stimulator of DGAT2 gene expression in mouse macrophages 56 . The qPCR data showed here that DGAT1 mRNA was the major form and the two variants of DGAT2 mRNA accounted for only half of the DGAT1 mRNA levels in the human colon cancer cells (Table 4). In contrast to mouse macrophages, we showed here that gossypol inhibited DGAT1 and DGAT2 expression in the human colon cancer cells (Fig. 3C).

Gossypol effect on GLUT gene expression in human colon cancer cells. Glucose transporter
(GLUT) family proteins consist of four isoforms which are responsible for glucose uptake in mammalian cells. GLUT1 mRNA is the major form and GLUT2 mRNA is undetectable in macrophages by TaqMan qPCR 51,58 . In this study, GLUT1 mRNA was also shown to be the major form of GLUT mRNAs but GLUT4 mRNA was barely detected in the colon cancer cells (Table 4). Gossypol treatment at high concentrations significantly decreased GLUT1 mRNA levels but increased GLLUT3 mRNA levels (Fig. 3D).

Gossypol effect on TTP gene expression in human colon cancer cells. Tristetraprolin (TTP/
ZFP36/TIS11/G0S24/NUP475) family proteins are post-transcriptional factors controlling cytokine mRNA stability. TTP family proteins exhibit anti-inflammation effects with the potential for controlling inflammationrelated diseases. TTP family proteins have three members in mammals (ZFP36 or TTP, ZFP36L1 or TIS11B, and ZFP36L2 or TIS11D) and fourth member in mouse and rat (ZFP36L3) 59,60 . SYBR Green qPCR showed that TTP/ZFP36 and ZFP36L1 were expressed in similar levels but ZFP36L2 mRNA was barely detectable in the colon cancer cells (Table 4). ZFP36, ZFP36L1 and ZFP36L2 mRNAs were all significantly reduced by high-dose of gossypol treatments (Fig. 4A).  66 . SYBR Green qPCR showed that IL10 and IL12 mRNAs were barely expressed and IL2 mRNA was low, whereas TTP and the other ILs were expressed in similar levels, which were several fold higher than IL2 mRNA in the human colon cancer cells ( Table 4). The qPCR assays showed that gossypol decreased IL2 and IL8 mRNA levels but its effect on IL6 and IL17 was not apparent (Table 3 and Fig. 4B).

Gossypol effect on proinflammatory gene expression in human colon cancer cells. The major
mRNAs destabilized by TTP family proteins are proinflammatory cytokine mRNA molecules. They down-regulate the expression of mRNAs encoding cytokines such as tumor necrosis factor-alpha (TNFα) [67][68][69][70] , granulocytemacrophage colony-stimulating factor/colony-stimulating factor 2 (GM-CSF/CSF2) 71,72 and cyclooxygenase 2/ prostaglandin-endoperoxide synthase 2 (COX2/PTGS2) 48 . TNFα and GM-CSF mRNAs are stabilized in TTP knockout mice cells 69,71 . TTP knockout mice over-express these proinflammatory cytokines which cause a severe systemic inflammatory syndrome 73,74 . TTP over-expression reduces inflammatory responses 75 . These previous studies suggest that TTP is an anti-inflammatory protein. Except TNFSF10 mRNA, all the other proinflammatory mRNAs were expressed much lower than that of TTP and VEGF mRNA was almost undetectable in the colon cancer cells (Table 4). Gossypol did not exhibit significant effects on the mRNA levels of all these proinflammatory gene expression in the human colon cancer cells (Fig. 4C).

Gossypol effect on TTP-targeted other gene expression in human colon cancer cells. A num-
ber of other TTP-mediated mRNAs have been reported in the literature ( Table 1). The basal levels of these mRNAs were either higher than that of TTP mRNA (BCL2L1, CsnK2A1, HIF1a and ZFAND5) or lower than that of TTP mRNA (Ahrr1, CXCL1, E2F1, ELK1, HMOX1 and ICAM1) ( Table 4). Gossypol treatment resulted in a reduction of many of the mRNAs in the colon cancer cells (Fig. 4D).

Discussion
Cottonseed accounts for approximately 20% of the crop value. One way to increase the value of cottonseed is to isolate bioactive compounds aimed to improving nutrition and preventing diseases. The presence of toxic polyphenolic compound gossypol in the seeds limits its use as food and feed source for humans and non-ruminant animals [2][3][4][5][6] . On the other hand, recent studies have shown that gossypol has potential biomedical applications. This may significantly increase cottonseed value by using gossypol from the seeds as a health intervention agent. However, it is necessary to insure safety and effectiveness of gossypol as well as the underlining molecular mechanisms before human consumption. Therefore, we evaluated the effects of gossypol on toxicity and gene expression in human colon cancer cells.
In this study, we observed that gossypol significantly decreased the cell viability of human colon cancer cells (Fig. 2). Our previous study showed that gossypol inhibited breast and pancreas cancer cell viability 76 . The effect of gossypol on decreasing breast cancer cell (MCF-7) viability 76 was in agreement with those using gossypol, gossypol derivative, and gossypol-enriched cottonseed oil 16,17 . Gossypol also decreased pancreatic cancer cell viability after short-term treatment 76 which is in agreement with published reports 20,77-79 .
Before we examined the effect of gossypol 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 Table 3. Gossypol Dosages on Colon Cancer Cell Gene Expression. The data represent the mean and standard deviation of three independent samples. Data with different lowcase letters represent significance between the treatments p < 0.05. ud: undetected. Bold italics: mRNA levels were statistically decreased by gossypol among the treatments with various concentrations. Italics: mRNA levels were statistically increased by gossypol among the treatments with various concentrations. "*" under P value column represents mRNA levels significantly affected by gossypol. Statistical analyses were conducted with Student-Newman-Keuls method for all pairwise multiple comparison. All pairwise multiple comparison was also performed with Tukey test yielding similar results (data not shown).   www.nature.com/scientificreports/ normalization of gene expression levels [80][81][82][83] . Our study showed that BCL2 mRNA was the most stable among the mRNAs from 55 genes analyzed in human colon cancer cells treated with DMSO vehicle or various concentrations of gossypol (Table 2). This result is in consistent with a previous report showing that the effect of gossypol analog on BCL2 gene expression was minimal at the mRNA level 31 . Our results showed that GAPDH and RPL32 (60S ribosomal protein 32) 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 conditions. This is in agreement with a previous report showing that GAPDH and 18s RNA mRNAs are not reliable references for qPCR assays in pharmacogenomics and toxicogenomics studies 83 . In contrast, RPL32 mRNA was shown to be a good reference for qPCR assays in mouse, rat and human post-infarction heart failure 80 and mouse adipocytes and macrophages 57,82 . These results suggest that internal reference mRNA is probably different among the various tissues/cells tested. Our study showed that most of the gene expression in human colon cancer cells was suppressed by high concentrations of gossypol ( Table 3). Some of the p values were less than 5% threshold, suggesting their expression levels were statistically significant. By this standard, gossypol significantly decreased the expression of genes coding for mRNAs of CLAUDIN1, ELK1, FAS, GAPDH, IL2, IL8 and ZFAND5, but increased the expression of the gene coding for GLUT3 mRNA. These genes code for proteins involved in various biological processes and cancer development. CLAUDIN1 is a tight junction protein which is highly upregulated in colon cancer 49 . ELK1 is a transcription factor playing an important role in immunological response, which belongs to ETS protein family with the evolutionary conserved ETS domain stabilized by three key tryptophan residues interacting with DNA 42 . FAS is involved in the apoptotic system which is upregulated in human lung cancer cells (A549) by gossypol treatment for 12 h at 0.5 µmol/L (~ 0.26 µg/mL) 34 . GAPDH catalyzes the sixth step of glycolysis and serves to break down glucose for energy and carbon demands 83 . IL2 is an autocrine and paracrine growth factor involved in clonal T cell expansion, influences the magnitude and duration of an immune response, and contributes to the regulation of programmed cell death in T cells which is down-regulated by TTP through ARE-mediated mRNA decay 61 . IL8 induces chemotaxis in target cells, causes them to migrate toward the site of infection, and stimulates phagocytosis once they have arrived 63 . ZFAND5 enhances ARE-containing mRNA stability by competing with TTP for mRNA binding 84 . GLUT3 expressed specifically in neurons facilitates the transport of glucose across the plasma membranes of mammalian cells 85 .
Among the tested 55 genes, gossypol treatment inhibited the expression of well-known reference genes coding for GAPDH and RPL32 mRNAs (Fig. 3A). Among the genes reported to be regulated by gossypol in cancer www.nature.com/scientificreports/ cells 20,33-39 and macrophages 40 , gossypol decreased the mRNA levels of BNIP3, CYP19A1, FAS, HUA, P53, PPARR and TNFSF10 genes in the human colon cancer cells (Fig. 3B). Gossypol decreased the mRNA levels of almost all of the DAGT, GLUT, TTP and IL gene families except GLUT3 in the cancer cells (Fig. 3C, D, 4A, B). The effect of gossypol on COX2, TNF and other proinflammatory cytokine mRNAs was not apparent (Fig. 4C), although it decreased the levels of a number of other TTP-regulated mRNAs coding for various functional proteins (Fig. 4D). In our previous studies, gossypol significantly increased the expression of DGAT and HuR mRNAs in mouse RAW264.7 macrophages 40,56 . However, DGAT and HuR mRNAs were decreased by gossypol in the human colon cancer cells. Similarly, gossypol decreased FAS mRNA in colon cancer cells reported here but increased its expression in human lung cancer cells reported previously 34 . Finally, the mRNA levels of TTP family genes were shown here to be decreased in the colon cancer cells but reported to be increased in mouse macrophages 86 . These discrepancies might reflect the different responses of normal mouse macrophages and different human cancer cells to gossypol treatment.
This study provides evidence for the toxic effects of gossypol on cell viability and its effects on down-regulation of many gene expression at the mRNA level in the human colon cancer cells. However, there are a few limitations involved in the study which should be addressed in future study. First, the findings were derived from one colon cancer cell line (COLO225). It could be valuable to expand the scope of research with multiple cancer cell lines. Second, the general pattern of gossypol on decreasing mRNA levels of numerous genes was evident. However, the dosage effect of gossypol on mRNA levels was not strong and the standard deviations were large in many cases, probably due to extremely sensitive qPCR assays and many factors affecting the results from extracellular gossypol application, cell harvest, RNA extraction, cDNA synthesis to qPCR analysis. Third, it was a firm conclusion that gossypol negatively regulated many gene expression at the mRNA levels in the colon cancer cells. However, it could be great addition to confirm mRNA data at the protein level. Even though positive correlations between mRNA levels and protein levels are not guaranteed as shown in many publications, it is still valuable to perform experiments at the protein levels. Finally, this study provides evidence for gossypol's toxic effects on cell viability and gene expression at the mRNA level in the human colon cancer cells. However, there is no functional analysis of gossypol affecting any intermediate steps between mRNA changes and cell viability. All these aspects are all worth of further investigation.
In conclusion, this study showed that gossypol significantly reduced the viability of human colon cancer cells. We further showed that BCL2 mRNA was the most stable among the 55 mRNAs in human colon cancer cells. Gossypol decreased the mRNA levels of DGAT, GLUT, TTP, ILs families and a number of previously reported genes. In particular, gossypol significantly suppressed the expression of the genes coding for CLAUDIN1, ELK1, FAS, GAPDH, IL2, IL8 and ZFAND5 mRNAs, but enhanced the expression of the gene coding for GLUT3 mRNA. This study provided evidence for potentially increasing the value of cottonseed by using cottonseedderived gossypol as a health intervention agent. Chemicals, reagents and equipment. The chemicals, reagents and equipment were described essentially as previously 56 . Gossypol (molar mass: 518.56 g/mol) was purified from cottonseed by HPLC and purchased from Sigma (St. Louis, MO). Gossypol stock was prepared in 100% DMSO at 10 mg/mL (approximately 19.2 mM). Cell cytotoxicity reagent (MTT based-In Vitro Toxicology Assay Kit) and DMSO were from Sigma. Tissue culture reagents (RPMI-1640, fetal bovine serum, penicillin, streptomycin, L-glutamine) were from Gibco BRL (Thermo Fisher). Tissue culture incubator was water jacket CO 2 incubator, Forma Series II, Model 3100 Series (Thermo Fisher). Tissue culture workstation was Logic + A2 hood (Labconco, Kansas City, MO). Tissue culture plastic ware (flasks, plates, cell scraper) 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).
RNA isolation and cDNA synthesis. The methods for RNA isolation and cDNA synthesis were essentially as described 56 . Human colon cancer cells in 24-well plates (triplicate) treated with various concentrations of gossypol for 8 h, a treatment showing significant reduction on cell viability (Fig. 2). The dishes were washed twice with 1 mL 0.9% NaCl and lysed directly with 1 mL of TRI ZOL reagent (Invitrogen, Carlsbad, CA, USA). RNA was isolated according to the manufacturer's instructions without DNase treatment and stored in -80 °C freezer. 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 (20 μL) contained 5 μg total RNA, 2.4 μg oligo(dT) 12-18 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 (Life Technologies, Carlsbad, CA). 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 followed the MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments 90 . The qPCR assays were described in details previously 54,82,91,92 . SYBR Green qPCR reaction mixture (12.5 μL) contained 5 ng of total RNA-derived cDNA, 200 nM each of the forward primer and reverse primer, and 1 × iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA). The reactions in 96-well clear plates sealed by adhesives were performed with CFX96 realtime system-C1000 Thermal Cycler (Bio-Rad Laboratories). The thermal cycle conditions were as follows: 3 min at 95 °C, followed by 40 cycles at 95 °C for 10 s, 65 °C for 30 s and 72 °C for 30 s. BCL2 mRNA was selected as the internal reference based on its minimal variation of gene expression among the 55 genes tested in the colon cancer cells (see "Results" for details). Ribosome protein 32 (RPL32) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were widely used as the reference mRNAs in the qPCR analyses 87 but they were not suitable for qPCR analysis for this cell type (see "Results" for details). TaqMan qPCR assay was used to confirm some SBYR Green qPCR assays using identical conditions as described previously 82 .
Data analysis and statistics. The 2 −ΔCT and 2 −ΔΔCT method of relative quantification was used to determine the fold change in expression 93 Table 3 (n = 3). They were analyzed by statistical analysis using ANOVA with SigmaStat 3.1 software (Systat Software). Multiple comparisons among the treatments with different concentrations of gossypol were performed with Student-Newman-Keuls method and Tukey test 87 .