5-Fluorouracil Potentiates the Anti-Cancer Effect of Oxaliplatin on Colo320 Colorectal Adenocarcinoma Cells

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1 5-Fluorouracil Potentiates the Anti-Cancer Effect of Oxaliplatin on Colo320 Colorectal Adenocarcinoma Cells Ioana Berindan-Neagoe 1,2,3, Cornelia Braicu 1,3*, Valentina Pileczki 3,4, Roxana Cojocneanu Petric 3,5, Nicolae Miron 2,6, Ovidiu Balacescu 1, Dana Iancu 7, Tudor Ciuleanu 7 1) Department of Functional Genomics and Experimental Pathology, Oncological Institute Ion Chiricuta 2) Department of Immunology, University of Medicine and Pharmacy Iuliu Hatieganu 3) Research Center for Functional Genomics, Biomedicine and Translational Medicine Iuliu Hatieganu University of Medicine and Pharmacy 4) Faculty of Pharmacy, Iuliu Hatieganu University of Medicine and Pharmacy 5) Faculty of Biology and Geology, Babeş-Bolyai University 6) Clinical Laboratory, IRGH Prof. dr. O. Fodor 7) Department of Medical Oncology, Oncological Institute Ion Chiricuta Cluj, Romania Address for correspondence: Cornelia Braicu Functional Genomics and Experimental Pathology Department, Oncological Institute Ion Chiricuta Republicii Str., Cluj-Napoca, Romania. braicucornelia@yahoo.com Received: Accepted: ABSTRACT Background & Aims. The present study was designed to examine the combined effects of Oxaliplatin (OXA) and 5-Fluorouracil (5-FU) in the Colo320 cell line. Methods. The antiproliferative effects were evaluated using the MTT assay, apoptosis by flow cytometry, and RT-PCR-array technology was used to determine the major effects of the two chemotherapeutic drugs upon the most important genes involved in apoptosis. Results. The antiproliferative effects of the therapeutic agents, as individual therapy or combined, proved to be dose and time-dependent, with increased efficiency for the combined treatment. Flow cytometry data revealed increased apoptotic processes in the case of the combined treatment at 24 hours after administration. The RT-PCR-array data indicated that at 24 hours after OXA treatment, 49 genes were differentially expressed, of which 45 were up-regulated and 4 down-regulated. In the case of the 5-FU treatment, 35 genes were down regulated and 2 up regulated. In the combined treatment of 5-FU and OXA, 19 genes were up-regulated and 15 down-regulated. Conclusions. This study proved that drug resistance could be counteracted by combining OXA with 5-FU to form a tandem that is capable of reducing cell proliferation and to stimulate extrinsic apoptosis pathway by targeting death receptors on the cell surface. Key words: colorectal cancer Colo320 Oxaliplatin 5-Fluorouracil apoptosis. Abbreviations: APAF1 - apoptotic peptidase activating factor 1; BAK 1 - BCL2-antagonist/killer 1; BID - BH3 interacting domain death agonist; CRC Colorectal cancer; DMSO Dimethyl sulfoxide; FBS Fetal Bovine Serum; FITC - Fluorescein isothiocyanate; 5-FU 5 Fluoro-uracil; IC50 half maximal Inhibitory Concentration; KT1 - v-akt murine thymoma viral oncogene homolog 1; LTA - lymphotoxin alpha (TNF superfamily, member 1); LTBR - lymphotoxin beta receptor (TNFR superfamily, member 3); MTT - Dimethyl thiazolyl diphenyl tetrazolium salt; NAIP - NLR family, apoptosis inhibitory protein; OXA Oxaliplatin; PBS Phosphate Buffered Saline; PI - Propidium Iodide; RIN - RNA integrity number; RT-PCR Real-Time Polymerase Chain Reaction; TNF Tumor Necrosis Factor; TRAF2 - TNF receptor-associated factor 2; TRAF3 - TNF receptor-associated factor 3; TRAF5 - TNF receptor-associated factor 5; TRAIL - tumor necrosis factor (ligand) superfamily, member 10; TWEAK - tumor necrosis factor (ligand) superfamily, member 12 INTRODUCTION Colorectal cancer (CRC) is a widespread malignant disease, mostly prevailing in industrialized countries. In its early stages, colorectal cancer is a curable disease, but in all advanced cases, without proper treatment regimen, patients have a poor prognosis [1]. The introduction of platinum derivative drugs, like oxaliplatin (OXA), shows good therapeutic results by increasing survival rates [2, 3]. The molecular mechanism of OXA in tumor cells is not yet clear but the drug presents powerful cytotoxic properties and its function is based on the ability to covalently bind DNA, forming platinum-dna adducts which lead to apoptosis [4]. 5-Fluorouracil (5-FU) exerts its anticancer effects by repressing the thymidylate synthase and incorporating its metabolites into RNA and DNA, and has been extensively used in the treatment of solid tumors [5, 6]. Recent experimental data suggest that this drug combination activates the apoptotic pathways [7, 8]. This study was designed to examine the efficiency of each cytostatic drug in comparison with the combined effects of both OXA and 5-FU in Colo320 cells. The antiproliferative effects, either as individual therapy or combined, were evaluated using the MTT assay, apoptosis was evaluated by flow cytometry,

2 38 Berindan-Neagoe et al and RT-PCR-array was used to assess apoptotic genes, for a dose equivalent to the IC 50 (the dose that produces halfmaximal inhibition). 5-FU may further enhance cytotoxicity by sensitizing cancer cells to OXA-induced apoptosis [2]. Our current results from in vitro studies on colorectal cancer cells have demonstrated that the combined therapy works together to inhibit cell proliferation and induce apoptosis, but not in a synergistic manner. MATERIAL AND METHODS Cell cultures. Colo320 cell line (human colon adenocarcinoma established, ATCC catalogue: CCL 220) is maintained in culture in RPMI-1460 medium, 10% FBS (Sigma-Aldrich), 100 U/ml penicillin and 2mM glutamine (Sigma-Aldrich). Cell treatment. For the MTT test, we treated cells with different concentrations of OXA, 5-FU and OXA+5-FU, ranging between µm, and determined the IC 50. For the apoptosis and RT-PCR-array evaluations we used a single dose of 10 µm for OXA and 5 µm for 5-FU, either independently or in tandem. Evaluations were made 24 and 48 hours after treatment for cell proliferation, and 24 hours after treatment for RT-PCR-array analysis. Cell proliferation assay evaluation using MTT test. 1x10 4 Colo320 cells were plated in 96-well plates and treated with the doses previously mentioned for 24 and 48 hours. After the incubation period, the culture medium was removed, and the cells were washed with PBS (Phosphate buffered saline, Sigma-Aldrich), incubated for 2 hours with 455 µg/ml MTT in Hanks salt, then was removed and replaced by 200 µl of DMSO (Dimethyl sulfoxide, Merck) to solubilize the resulted formazan. The absorbance at 490 nm and the IC50 values were determined using GraphPad Prism free trial software. Apoptosis-Flow Cytometry. Each of the triplicate samples from the 4 groups of cells (control and OXA, 5-FU, OXA+5-FU) were resuspended in 100 μl of binding buffer with 2 μl of Annexin V-FITC and 1 μg PI, then incubated 10 minutes at room temperature, in the dark. After washing, each sample was diluted using 250 µl binding buffer and evaluated using a FACSCantoII flow cytometer equipped with BD-FACSDiva software for cell acquisition and data analysis. The RNA extraction and quality control. From each of the 4 groups we extracted the total RNA using TriReagent (Sigma-Aldrich), in accordance with the producer s protocol. DNA contamination was eliminated using DNAse (Qiagen) and the RNA was purified using RNeasy Mini Kit (Qiagen). RNA concentrations were evaluated using NanoDrop-1100, and its quality was appraised with Agilent Bioanalyzer The RIN values for all the measured samples were above 8. RT 2 Profiler TM PCR Array technology. The experiment was performed using the Human Apoptosis RT²Profiler PCR Array 96-well plate from SABiosciences. For this we used the RT 2 First Strand Kit (SABiosciences) to reverse transcribe 400ng of RNA. The cdna was pipetted onto the PCR Array plate, after being combined with the RT 2 SYBRGreen qpcr Master Mix (SABiosciences). The plate was run on a LightCycler 480 (Roche), using the following steps: 10 min at 95 C in order to activate the enzyme, then 40 cycles of 15 seconds at 95 C, and in the end, 1 min at 60 C. PCR Array Data Analysis. The data obtained after RT-PCRarray analysis were entered in web-based data analysis software from SABiosciences ( com/pcr/arrayanalysis.php), which uses the ΔΔCt method (p-values 0.05, and fold change ±1.25) RESULTS The combined treatment of OXA and 5-FU induces inhibition of cell proliferation Preliminary studies were done in order to evaluate the antiproliferative effect of OXA, 5-FU and OXA+5-FU on the Colo320 cells. The MTT values 24 and 48h post-treatment were represented as log of concentration (μm), plotted against % of control (Fig. 1). The IC 50 values for OXA, 5-FU, and OXA+5-FU are presented in Table I. The effects are time and dose-dependent, with an increased efficiency for the combined treatment. The results for OXA+5-FU indicate that the combined therapy is more efficient than each compound administered alone. Apoptosis/necrosis evaluation as a response to single or combined treatment with OXA and 5-FU in Colo320 cells Annexin V-FITC/PI staining is presented in Fig. 2, and allows the differentiation between apoptotic and necrotic cells. Fig. 1. The antiproliferative effect measured using MTT assay after 24 and 48 hours post treatment with concentrations between 0 and 100 μm of OXA, 5-FU, alone or combined, on Colo320 cell line; log (conc, μm) = log[concentration drug, μm] (mean ± SD, n = 6).

3 5-fluorouracil and oxaliplatin effect on Colo320 colorectal adenocarcinoma cells 39 Table I. Overview of IC 50 values for Colo-320 for OXA and 5-FU, alone or combined Time Compound IC 50 Slope R2 OXA FU OXA+5-FU OXA FU OXA+5-FU cell death signaling. After treatment with OXA, 49 genes showed statistically significant values, 45 overexpressed and 4 downregulated (online supplementary Table SI), most genes being inducers of apoptosis. In the case of 5-FU we obtained statistically significant results for 37 genes, of which 35 were downregulated and only 2 were overexpressed. For the combined treatment of 5-FU and OXA, 34 genes were statistically significant, of which 19 were overexpressed and 15 downregulated (Table S1). Most of the genes that were overexpressed encode for caspase induced apoptosis and inhibition of the genes that modulate cell proliferation pathways. In the case of the OXA treatment, we have observed an activation of the p53 gene, while 5-FU causes the reduction of intracellular apoptotic molecules. Ingenuity Pathway Analysis allows us to present the major functional networks modulated by the different treatment variants (Fig 3, Table II). The combined treatment may reduce intrinsic and/or acquired resistance to the individual therapeutic agents, leading to the modulation of death receptors, Myc mediated apoptosis and TWEAK Signaling. Hierarchical cluster analysis showed that the OXA treatment leads to the up-regulation of genes involved in apoptosis pathways, as presented in the supplementary Fig. S1. This study demonstrated that each treatment has a specific modulator effect on each group of genes. TNF receptor and ligand family and death effectors domain family are generally activated by OXA treatment, while 5-FU inhibits these groups of genes. Several genes with anti-apoptotic role are activated by OXA, potentially leading to treatment resistance. The apoptotic genes are down-regulated by 5FU, while for the combined treatment 5 of the genes remain activate and 9 are down-regulated (Table S1). This finding is an additional argument that the combined treatment of OXA and 5-FU is a more effective therapeutic design in colorectal cancer, since the main signal transduction pathway that controls apoptosis and cell death appears more intensely activated. Fig. 2. Effect of OXA, 5-FU, OXA+5-FU on Colo320 apoptosis in vitro measured by Annexin V-FITC and PI binding. Colo320 cells (1 106 cells) were incubated in the absence (control cell samples) or presence of 10 μm of OXA, 5 μm of 5-FU, and respectively 10 μm of OXA and 5 μm of 5-FU at 24 and 48 hours post treatment. In the case of the combined treatment, the yield of necrotic cells is the same or lower than the individual treatment at 24 hours post-treatment. The results are expressed as % of apoptotic cells; an increase of these values was observed, particularly 48 h post treatment. OXA treatment revealed that 24.4% and 33.2% of cells undergo apoptosis at 24 and 48 h, respectively. 5-FU treatment caused 22.9% and 25.5% of cells to undergo apoptosis at 24 and 48 h, respectively. The impact of the combined therapy was greater after 48 hours of treatment, as 29.9% and 40.9% of cells underwent apoptosis at 24 and 48h, respectively. Apoptotic and cell death signals evaluation at 24 hours post treatment using RT-PCR-array RT-PCR-array technology simultaneously evaluates the level of expression of 84 genes involved in apoptosis and Table II. The main Canonical Pathways modulated by the selected treatment variants in Colo320 cell line Drug Name p-value Ratio Apoptosis Signaling 2.46E-31 19/96 (0.198) TWEAK Signaling 2.65E-30 15/39 (0.385) OXA Death Receptor 5.42E-26 15/65 (0.231) Signaling Molecular Mechanisms 3.93E-21 20/378 (0.053) of Cancer Death Receptor 4.95E-33 17/65 (0.262) Signaling 5-FU TWEAK Signaling 1.58E-24 12/39 (0.308) Apoptosis Signaling 4.85E-23 14/96 (0.146) TNFR1 Signaling 3.91E-20 11/53 (0.208) Apoptosis Signaling 2.42E-19 12/96 (0.125) Death Receptor 3.01E-17 10/65 (0.154) OXA+5-FU Signaling Myc Mediated Apoptosis 4.06E-13 8/61 (0.131) Signaling TWEAK Signaling 5.95E-13 7/39 (0.179)

4 40 Berindan-Neagoe et al As we can see in Fig. 3A and Table S1, caspases are the principal genes leading to treatment response to OXA, and our results indicate that caspase inhibitor activation is correlated with the modulation of NAIP, BAK1 and LTA genes, which are known to be involved in the response to therapy [9]. For 5-FU, the central molecule is FADD, related to TNSFR10 and TRAF2, which are markers involved in the response to therapy. In the case of the combined treatment (Fig. 3C), the central molecule Fig. 3 (continued on page 41)

5 5-fluorouracil and oxaliplatin effect on Colo320 colorectal adenocarcinoma cells 41 Fig. 3. (continued) The canonical network related with the genes implicated in apoptosis mediated by (A) OXA, (B) 5-FU, (C) OXA+ 5-FU. is TRAF2, linked to BAK1 and LTA, playing important roles in increased treatment efficiency. Apoptosis is promoted by TRAF3 and TRAF5 via LTBR. DISCUSSION Surgery is the generally applied course of treatment for early stage CRC, while chemotherapy is the first therapeutic option for advanced CRC, when tumor lesions are usually not completely resectable [10]. Controlling cell proliferation using chemotherapy is mainly based on apoptosis but resistance to therapy is observed in all cytostatic drug treatments, and OXA and/or 5-FU do not make an exception [11-13]. The treatment with 5-FU did not show a clear synergistic effect with OXA in Colo320 cells, while in a similar study a synergic effect was observed [14]. OXA and 5-FU together displayed a more reduced number of genes involved in the resistance to treatment than each drug administered individually (Table SI). Restoration of the signaling pathways that are involved in apoptosis induction, based on targeted multitherapy, may result in improved response to treatment, which translates into decreased resistance to cancer therapy [15-17], by stimulation of the intrinsic pathway that enhance TRAIL receptor targeted therapies [18]. The overexpression of the caspases responsible for apoptosis execution (CASP3, CASP4 CASP6, CASP9) and cell death is confirmed by flow cytometry data (Fig. 2). This is supported by a similar study for CASP3 mrna expression leading to the promotion of apoptosis by OXA [18]. Studies revealed that p53 induced tumor cell resistance to OXA, so TP53 can be used as a therapeutic target in modulating the response to therapy [19]. Our previous data showed that p53 and NFκB were upregulated after treatment with OXA [20]. Other recent studies on OXA using p53 wild-type HCT116 colon cancer cells have demonstrated that p53 and Bax genes play an important role in cell apoptosis [21], data confirmed by the present study. The molecular basis of resistance to OXAinduced cytotoxicity is complex and multifactorial. Previous studies have shown that OXA enhances 5-FU cytotoxicity [22] by activating TRAIL induced apoptosis in several models [23-25, 36]. Renewing the susceptibility of adenocarcinoma cells to TRAIL is beneficial to patients with this neoplasia [26, 35]. The present study reveals that the upregulation of CARD6 may inhibit the activation of NFκB pathway [27, 28]. 5-Fluorouracil induces the inhibition of death receptors, whose alterations are involved in cancer development [29, 30]. Also, by modulating the expression of the Bcl-2 family of genes, 5-FU displays a certain degree of pro-apoptotic activity [31]. As shown in Fig 3, the main molecules involved in apoptosis belong to the NFκB complex for 5-FU [14]; death receptors are also blocked by 5-FU, leading to the inhibition of the cell survival pathway controlled by NFκB [32]. The fact that 48 hours after 5-FU treatment the IC 50 is higher that at 24 h can be explained by the overexpression of apoptosis inhibitory gene NAIP [33]. In previous in vitro and in vivo studies it was observed that NAIP is an important

6 42 Berindan-Neagoe et al member of the IAPs that are involved in the resistance to different chemotherapeutics agents [34]. For this reason, the choice for postoperative treatment of colon cancer is often the combination therapy consisting of 5-FU and OXA [35], drugs which are effective in preventing TRAIL drug-resistance, as was demonstrated in a similar study on HT-29 and RKO cells [36], or by their effects on decreasing the expression of IAPs genes, in particular XIAP [36]. Oxaliplalin seems to modulate the activation of TNF-α in a rapid and transient manner through the TWEAK canonical pathway, less active in the case of OXA+5-FU. Like other TNF family members, TWEAK pathway is capable of enhancing proliferation, cell migration, expression and secretion of proinflammatory molecules [37], and displays proangiogenic activity [38-40]. TWEAK pathway is modulated by AKT1 and by the recruitment of TRAF protein family members. In addition, the inhibition of AKT1 expression stimulates apoptosis and intensifies the sensitiveness of gastric cancer to chemotherapy in many mammalian cells [41]. The activation of these pathways leads to the inhibition of the proapoptotic genes BID and APAF1, thus being a connection point between NFκB activation through TWEAK pathways that induce apoptosis signaling as a response to treatment by mediating apoptosis and inflammasome activation [14, 42]. CONCLUSIONS Our study proved that the drug resistance in Colo320 colorectal adenocarcinoma cells could be counteracted by combining OXA with 5-FU to form a tandem that is capable of reducing cell proliferation and stimulate apoptosis by inhibiting the expression of death receptors. The reduction of cell proliferation and the activation of apoptosis are important indicators for the response to therapy in colorectal cancer; the precise molecular mechanism can be described by identifying the genes that are involved in resistance to therapy. This study highlights the importance of in vitro assays in developing novel therapeutic strategies that bring benefit to the patient. Conflicts of interest: The authors confirm that there is no conflict of interest to declare. Acknowledgements: This work was supported by the PN-II-ID-PCE UEFISCDI grant. REFERENCES 1. Alberts SR, Wagman LD. Chemotherapy for colorectal cancer liver metastases. Oncologist 2008; 13: Board RE, Valle JW. Metastatic colorectal cancer: current systemic treatment options. Drugs 2007; 67: Wang X, Li M, Wanga J, et al. The BH3-only protein, PUMA, is involved in oxaliplatin-induced apoptosis in colon cancer cells. Biochem Pharmacol 2006; 71: Hato SV, de Vries IJ, Lesterhuis WJ. 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8 Table S1. Genes differentially expressed in the case of OXA, 5-FU and OXA+5-FU treatment versus control cells at 24 h posttreatment Gene name Unigene Gene symbol Fold change OXA 5-FU OXA+5-FU V-akt murine thymoma viral oncogene homolog 1 Hs AKT Apoptotic peptidase activating factor 1 Hs APAF BCL2-associated agonist of cell death Hs BAD BCL2-associated athanogene 3 Hs BAG BCL2-associated athanogene 4 Hs BAG BCL2-antagonist/killer 1 Hs BAK BCL2-associated X protein Hs BAX B-cell CLL/lymphoma 10 Hs BCL B-cell CLL/lymphoma 2 Hs BCL BCL2-related protein A1 Hs BCL2A BCL2-like 10 (apoptosis facilitator) Hs BCL2L BCL2-like 11 (apoptosis facilitator) Hs BCL2L BCL2-like 2 Hs BCL2L BCL2-associated transcription factor 1 Hs BCLAF Bifunctional apoptosis regulator Hs BFAR BH3 interacting domain death agonist Hs BID BCL2-interacting killer (apoptosis-inducing) Hs BIK NLR family, apoptosis inhibitory protein Hs NAIP Baculoviral IAP repeat containing 2 Hs BIRC Baculoviral IAP repeat containing 3 Hs BIRC X-linked inhibitor of apoptosis Hs XIAP Baculoviral IAP repeat containing 6 Hs BIRC Baculoviral IAP repeat containing 8 Hs BIRC BCL2/adenovirus E1B 19kDa interacting protein 1 Hs BNIP BCL2/adenovirus E1B 19kDa interacting protein 3 Hs BNIP BCL2/adenovirus E1B 19kDa interacting protein 3-like Hs BNIP3L V-raf murine sarcoma viral oncogene homolog B1 Hs BRAF Nucleotide-binding oligomerization domain containing 1 Hs NOD Caspase recruitment domain family, member 6 Hs CARD Caspase recruitment domain family, member 8 Hs CARD Caspase 10, apoptosis-related cysteine peptidase Hs.5353 CASP Caspase 14, apoptosis-related cysteine peptidase Hs CASP Caspase 2, apoptosis-related cysteine peptidase Hs CASP Caspase 3, apoptosis-related cysteine peptidase Hs CASP Caspase 4, apoptosis-related cysteine peptidase Hs CASP Caspase 5, apoptosis-related cysteine peptidase Hs CASP Caspase 6, apoptosis-related cysteine peptidase Hs CASP Caspase 7, apoptosis-related cysteine peptidase Hs.9216 CASP Caspase 8, apoptosis-related cysteine peptidase Hs CASP Caspase 9, apoptosis-related cysteine peptidase Hs CASP CD40 molecule, TNF receptor superfamily member 5 Hs CD CD40 ligand Hs CD40LG CASP8 and FADD-like apoptosis regulator Hs CFLAR Cell death-inducing DFFA-like effector a Hs CIDEA Cell death-inducing DFFA-like effector b Hs CIDEB CASP2 and RIPK1 domain containing adaptor with death domain Hs CRADD DNA fragmentation factor, 45kDa, alpha polypeptide Hs DFFA Fas (TNFRSF6)-associated via death domain Hs FADD

9 2 Berindan-Neagoe et al Fas (TNF receptor superfamily, member 6) Hs FAS Fas ligand (TNF superfamily, member 6) Hs.2007 FASLG Growth arrest and DNA-damage-inducible, alpha Hs GADD45A Harakiri, BCL2 interacting protein (contains only BH3 domain) Hs HRK Insulin-like growth factor 1 receptor Hs IGF1R Lymphotoxin alpha (TNF superfamily, member 1) Hs.36 LTA Lymphotoxin beta receptor (TNFR superfamily, member 3) Hs.1116 LTBR Myeloid cell leukemia sequence 1 (BCL2-related) Hs MCL Nucleolar protein 3 (apoptosis repressor with CARD domain) Hs NOL PYD and CARD domain containing Hs PYCARD Receptor-interacting serine-threonine kinase 2 Hs RIPK Tumor necrosis factor Hs TNF Tumor necrosis factor receptor superfamily, member 10b Hs TNFRSF10B Tumor necrosis factor receptor superfamily, member 1A Hs TNFRSF1A Tumor necrosis factor receptor superfamily, member 21 Hs TNFRSF Tumor necrosis factor receptor superfamily, member 25 Hs TNFRSF CD27 molecule Hs CD Tumor necrosis factor receptor superfamily, member 9 Hs TNFRSF CD70 molecule Hs CD Tumor necrosis factor (ligand) superfamily, member 8 Hs TNFSF Tumor protein p53 Hs TP Tumor protein p53 binding protein, 2 Hs TP53BP Tumor protein p73 Hs TP TNF receptor-associated factor 2 Hs TRAF TNF receptor-associated factor 3 Hs TRAF TNF receptor-associated factor 4 Hs.8375 TRAF

10 5-fluorouracil and oxaliplatin effect on Colo320 colorectal adenocarcinoma cells 3 Fig.. S1. The clustergram for OXA, 5-FU, OXA+ 5-FU treated samples versus control samples. P <0.05, genes with 1.25 or 1.25 fold change are shown.