Interactions between Diet,Lifestyle and IL10, IL1B,and PTGS2/COX-2 Gene Polymorphisms in Relation to Risk of Colorectal Cancer in a Prospective Danish Case-Cohort Study

Background & Aims

Diet contributes to colorectal cancer development and may be potentially modified. We wanted to identify the biological mechanisms underlying colorectal carcinogenesis by assessment of diet-gene interactions.

Methods

The polymorphisms IL10 C-592A (rs1800872), C-rs3024505-T, IL1b C-3737T (rs4848306), G-1464C (rs1143623), T-31C (rs1143627) and PTGS2 (encoding COX-2) A-1195G (rs689466), G-765C (rs20417), and T8473C (rs5275) were assessed in relation to risk of colorectal cancer (CRC) and interaction with diet (red meat, fish, fibre, cereals, fruit and vegetables) and lifestyle (non-steroid-anti-inflammatory drug use and smoking status) was assessed in a nested case-cohort study of nine hundred and seventy CRC cases and 1789 randomly selected participants from a prospective study of 57,053 persons.

Results

IL1b C-3737T, G-1464C and PTGS2 T8473C variant genotypes were associated with risk of CRC compared to the homozygous wildtype genotype (IRR=0.81, 95%CI: 0.68-0.97, p=0.02, and IRR=1.22, 95%CI: 1.04-1.44, p=0.02, IRR=0.75, 95%CI: 0.57-0.99, p=0.04, respectively). Interactions were found between diet and IL10 rs3024505 (P-value for interaction (P_int); meat=0.04, fish=0.007, fibre=0.0008, vegetables=0.0005), C-592A (P_int; fibre=0.025), IL1b C-3737T (P_int; vegetables=0.030, NSAID use=0.040) and PTGS2 genotypes G-765C (P_int; meat=0.006, fibre=0.0003, fruit 0.004), and T8473C (P_int; meat 0.049, fruit=0.03) and A-1195G (P_int; meat 0.038, fibre 0.040, fruit=0.059, vegetables=0.025, and current smoking=0.046).

Conclusions

Genetically determined low COX-2 and high IL-1β activity were associated with increased risk of CRC in this northern Caucasian cohort. Furthermore, interactions were found between IL10, IL1b, and PTGS2 and diet and lifestyle factors in relation to CRC. The present study demonstrates that gene-environment interactions may identify genes and environmental factors involved in colorectal carcinogenesis.     

Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE₂) is one of the main catabolic factors involved in OA. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE₂. For both COX and PGES, three isoforms have been described: in cartilage, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. COX-3 (a variant of COX-1) and mPGES-2 have been recently cloned but little is known about their expression and regulation in cartilage, as is also the case for 15-PGDH. We investigated the regulation of the genes encoding COX and PGES isoforms during mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) for 2 to 24 hours. After determination of the amount of PGE₂ released in the media (enzyme immunoassay), mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blotting respectively. Mechanical compression of cartilage explants significantly increased PGE₂ production in a time-dependent manner. This was not due to the synthesis of IL-1, since pretreatment with interleukin 1 receptor antagonist (IL1-Ra) did not alter the PGE₂ synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression, whereas COX-3 and mPGES-2 mRNA expression was not modified. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE₂ synthesis after 18 hours, suggesting that PGE₂ synthesis could be altered by the induction of 15-PGDH expression. We conclude that, along with COX-2, dynamic compression induces mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

The wound-healing effect of 7,3′,4′-trimethoxyflavone through increased levels of prostaglandin E<Subscript>2</Subscript> by 15-hydroxyprostaglandin dehydrogenase inhibition

Objective

To find an inhibitor of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) that rapidly metabolises Prostaglandin E₂ (PGE₂) as a mediator of wound healing, we examined seven flavonoids for this role.

Results

7,3′,4′-Trimethoxyflavone (TMF) had the lowest IC₅₀ value of 0.34 µM for 15-PGDH inhibition but >400 µM for cytotoxicity, indicating a high therapeutic index. TMF elevated PGE₂ levels in a concentration-dependent manner in both A549 lung cancer and HaCaT cells. It also significantly increased mRNA expression of multidrug resistance-associated protein 4 (MRP4) and of prostaglandin transporter (PGT) slightly in HaCaT cells. In addition, TMF facilitated in vitro wound healing in a HaCaT scratch model, which was completely inhibited by adding both 15-PGDH and NAD⁺ as cofactor, confirming the involvement of PGE₂ in its wound healing effect.

Conclusion

TMF with a high therapeutic index can facilitate wound healing through PGE₂ elevation by 15-PGDH inhibition.

     

Intestinal PTGS2 mRNA Levels,PTGS2 Gene Polymorphisms,and Colorectal Carcinogenesis

Background & Aims

Inflammation is a major risk factor for development of colorectal cancer (CRC). Prostaglandin synthase cyclooxygenase-2 (COX-2) encoded by the PTGS2 gene is the rate limiting enzyme in prostaglandin synthesis and therefore plays a distinct role as regulator of inflammation.

Methods

PTGS2 mRNA levels were determined in intestinal tissues from 85 intestinal adenoma cases, 115 CRC cases, and 17 healthy controls. The functional PTGS2 polymorphisms A-1195G (rs689466), G-765C (rs20417), T8473C (rs5275) were assessed in 200 CRC cases, 991 adenoma cases and 399 controls from the Norwegian KAM cohort.

Results

PTGS2 mRNA levels were higher in mild/moderate adenoma tissue compared to morphologically normal tissue from the same individual (P<0.0001) and (P<0.035) and compared to mucosa from healthy individuals (P<0.0039) and (P<0.0027), respectively. In CRC patients, PTGS2 mRNA levels were 8–9 times higher both in morphologically normal tissue and in cancer tissue, compared to healthy individuals (P<0.0001). PTGS2 A-1195G variant allele carriers were at reduced risk of CRC (odds ratio (OR) = 0.52, 95% confidence interval (95% CI): 0.28–0.99, P = 0.047). Homozygous carriers of the haplotype encompassing the A-1195G and G-765C wild type alleles and the T8473C variant allele (PTGS2 AGC) were at increased risk of CRC as compared to homozygous carriers of the PTGS2 AGT (A-1195G, G-765C, T8473C) haplotype (OR = 5.37, 95% CI: 1.40–20.5, P = 0.014). No association between the investigated polymorphisms and PTGS2 mRNA levels could be detected.

Conclusion

High intestinal PTGS2 mRNA level is an early event in colorectal cancer development as it occurs already in mild/moderate dysplasia. PTGS2 polymorphisms that have been associated with altered PTGS2 mRNA levels/COX-2 activity in some studies, although not the present study, were associated with colorectal cancer risk. Thus, both PTGS2 polymorphisms and PTGS2 mRNA levels may provide information regarding CRC risk.     

Berberine Inhibits Invasion and Metastasis of Colorectal Cancer Cells via COX-2/PGE2 Mediated JAK2/STAT3 Signaling Pathway

Berberin, extracted from Chinese herbal medicine Coptis chinensis, has been found to have anti-tumor activities. However, the underlying mechanisms have not been fully elucidated. Our current study demonstrated that berberin inhibited the in vitro and in vivo growth, migration/invasion of CRC cells, via attenuating the expression levels of COX-2/PGE₂, following by reducing the phosphorylation of JAK2 and STAT3, as well as the MMP-2/-9 expression. We further clarified that an increase of COX-2/PGE₂ expression offset the repressive activity of Berberin on JAK2/STAT3 signaling, and a JAK2 inhibitor AZD1480 blocked the effect of COX-2/PGE₂ on MMP-2/-9 expression. In summary, Berberin inhibited CRC invasion and metastasis via down-regulation of COX-2/PGE₂- JAK2/STAT3 signaling pathway.     

Genetic deletion of mPGES-1 accelerates intestinal tumorigenesis in APC(Min/+) mice

The induced synthesis of bioactive prostanoids downstream of cyclooxygenase-2 (COX-2) and prostaglandin H₂ (PGH₂) exerts a critical event in colorectal carcinogenesis. Here we demonstrate that APC^Min/+ mice with genetic deletion of microsomal prostaglandin E synthase-1 (mPGES-1), which catalyses the terminal conversion of PGH₂ into PGE₂, surprisingly develop more and generally larger intestinal tumors than do mPGES-1 wild type littermates (mean number of tumors/intestine 80 vs. 38, p < 0.0005, mean tumor diameter 1.64 vs. 1.12 mm, p < 0.0005). No deviation regarding the expression of other PGE₂ related enzymes (COX-1, COX-2, mPGES-2, cPGES, and 15-PGDH) or receptors (EP1-4) was obvious among the mPGES-1 deficient mice. PGE₂ levels were suppressed in tumors of mPGES-1 deficient animals, but the concentrations of other PGH₂ derived prostanoids were generally enhanced, being most prominent for TxA₂ and PGD₂. Thus, we hypothesise that a redirected synthesis towards other lipid mediators might (over)compensate for loss of mPGES-1/PGE₂ during intestinal tumorigenesis. Nevertheless, our results question the suitability for mPGES-1 targeting therapy in the treatment or prevention of colorectal cancer.     

Gene Polymorphisms of ADIPOQ +45T>G,UCP2 -866G>A,and FABP2 Ala54Thr on the Risk of Colorectal Cancer: A Matched Case-Control Study

As insulin resistance (IR) is an established risk factor for colorectal cancer (CRC), we explored the association between each of the IR-related gene polymorphisms of adiponectin (ADIPOQ) rs2241766, uncoupling protein 2 (UCP2) rs659366, and fatty acid-binding protein (FABP2) rs1799883 and CRC risk. Genotyping of blood samples and collection of lifestyle and dietary habits were performed for 400 case-control pairs. Unconditional logistic regression (ULR) was applied to assess the effects of the three single nucleotide polymorphisms (SNP), environmental factors. Both ULR and generalized multifactor dimensionality reduction (GMDR) were used to test the gene-gene and gene-environment interactions on CRC risk. Subjects carrying the ADIPOQ rs2241766 TG+GG genotype had a higher CRC risk than those carrying the TT genotype (OR = 1.429, 95% CI 1.069–1.909). The additive and multiplicative interactions between ADIPOQ rs2241766 and FABP2 rs1799883 on CRC were found by ULR (RERI = 0.764, 95%CI 0.218∼1.311, AP = 0.514, 95%CI 0.165∼0.864, S = −1.745, 95%CI is unachievable, and P_multi = 0.017, respectively). Furthermore, the high order gene-gene interaction of the three SNPs were found by GMDR (P = 0.0107). A significant dosage effect with an increasing number of risk genotypes was observed as the risk of CRC increased (P_trend = 0.037). In GMDR, the gene-environment interaction among the three SNPs and red meat consumption on CRC risk was significant (P = 0.0107). Compared with subjects with low red meat consumption and null risk genotypes, those with high-red meat consumption and three risk genotypes had 3.439-fold CRC risk (95% CI 1.410–8.385). In conclusion, the results showed that the ADIPOQ rs2241766 TG+GG genotype increased CRC risk. Given the complexity of the carcinogen for CRC, ADIPOQ rs2241766, UCP2 rs659366, FABP2 rs1799883 and red meat consumption potentially worked together in affecting CRC risk.     

Adenosine and Prostaglandin E2 Cooperate in the Suppression of Immune Responses Mediated by Adaptive Regulatory T Cells

Adaptive regulatory T cells (Tr1) are induced in the periphery upon encountering cognate antigens. In cancer, their frequency is increased; however, Tr1-mediated suppression mechanisms are not yet defined. Here, we evaluate the simultaneous involvement of ectonucleotidases (CD39/CD73) and cyclooxygenase 2 (COX-2) in Tr1-mediated suppression. Human Tr1 cells were generated from peripheral blood mononuclear cell-derived, sorted CD4⁺CD25⁻ T cells and incubated with autologous immature dendritic cells, irradiated COX-2⁺ or COX-2⁻ tumor cells, and IL-2, IL-10, and IL-15 (each at 10–15 IU/ml) for 10 days as described (Bergmann, C., Strauss, L., Zeidler, R., Lang, S., and Whiteside, T. L. (2007) Cancer Immunol. Immunother. 56, 1429–1442). Tr1 were phenotyped by multicolor flow cytometry, and suppression of proliferating responder cells was assessed in carboxyfluorescein diacetate succinimidyl ester-based assays. ATP hydrolysis was measured using a luciferase detection assay, and levels of adenosine or prostaglandin E₂ (PGE₂) in cell supernatants were analyzed by mass spectrometry or ELISA, respectively. Intracellular cAMP levels were measured by enzyme immunoassay. The COX-2⁺ tumor induced a greater number of Tr1 than COX-2⁻ tumor (p < 0.05). Tr1 induced by COX-2⁺ tumor were more suppressive, hydrolyzed more exogenous ATP (p < 0.05), and produced higher levels of adenosine and PGE₂ (p < 0.05) than Tr1 induced by COX-2⁻ tumor. Inhibitors of ectonucleotidase activity, A_2A and EP₂ receptor antagonists, or an inhibitor of the PKA type I decreased Tr1-mediated suppression (p < 0.05), whereas rolipram, a PDE₄ inhibitor, increased the intracellular cAMP level in responder cells and their susceptibility to Tr1-mediated suppression. Tr1 present in tumors or the peripheral blood of head and neck squamous cell carcinoma patients co-expressed COX-2, CD39, and CD73. A concomitant inhibition of PGE₂ and adenosine via the common intracellular cAMP pathway might be a novel approach for improving results of immune therapies for cancer.     

The prostaglandin transporter PGT transports PGH2

Prostaglandin H₂ not only serves as the common precursor of all other PGs, but also directly triggers signals (e.g. platelet aggregation), depending on its location and translocation. The prostaglandin carrier PGT mediates the transport of several prostanoids, such as PGE₂, and PGF_2α. Here we used PGT in the plasma membrane as a model system to test the hypothesis that PGT also transports PGH₂. Using wild-type and PGT-expressing MDCK cells, we show that PGH₂ uptake is mediated both by simple diffusion and by PGT. The PGH₂ influx permeability coefficient for diffusion is (5.66 ± 0.63) × 10⁻⁶ cm/s. The kinetic parameters of PGH₂ transport by PGT are K_m = 376 ± 34 nM and V_max = 210.2 ± 11.4 fmol/mg protein/s. PGH₂ transport by PGT can be inhibited by excess PGE₂ or by a PGT inhibitor. We conclude that PGT may play a role in transporting PGH₂ across cellular membranes.     

Limited effect of anti-rheumatic treatment on 15-prostaglandin dehydrogenase in rheumatoid arthritis synovial tissue

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which prostaglandin E₂ (PGE₂) displays an important pathogenic role. The enzymes involved in its synthesis are highly expressed in the inflamed synovium, while little is known about 15- prostaglandin dehydrogenase (15-PGDH) that metabolizes PGE₂. Here we aimed to evaluate the localization of 15-PGDH in the synovial tissue of healthy individuals or patients with inflammatory arthritis and determine the influence of common RA therapy on its expression.

Methods

Synovial tissue specimens from healthy individuals, psoriatic arthritis, ostheoarthritis and RA patients were immunohistochemically stained to describe the expression pattern of 15-PGDH. In addition, the degree of enzyme staining was evaluated by computer analysis on stained synovial biopsies from two groups of RA patients, before and after RA specific treatment with either intra-articular glucocorticoids or oral methotrexate therapy. Prostaglandins derived from the cyclooxygenase (COX) pathway were determined by liquid-chromatography mass spectrometry in supernatants from interleukin (IL) 1β-activated fibroblast-like synoviocytes (FLS) treated with methotrexate.

Results

15-PGDH was present in healthy and inflamed synovial tissue, mainly in lining macrophages, fibroblasts and vessels. Intra-articular glucocorticoids showed a trend towards reduced 15-PGDH expression in RA synovium (p = 0.08) while methotrexate treatment left the PGE₂ pathway unaltered both in biopsies ex vivo and in cultured FLS.

Conclusions

Early methotrexate therapy has little influence on the expression of 15-PGDH and on any of the PGE₂ synthesizing enzymes or COX-derived metabolites. Thus therapeutic strategies involving blocking induced PGE₂ synthesis may find a rationale in additionally reducing local inflammatory mediators.     

Regulatory T cells in colorectal cancer patients suppress anti-tumor immune activity in a COX-2 dependent manner

Objective Naturally occurring regulatory T (T_R) cells suppress autoreactive T cells whereas adaptive T_R cells, induced in the periphery, play an important role in chronic viral diseases and cancer. Several studies indicate that cyclooxygenase (COX) inhibitors prevent cancer development of colon adenomas and delay disease progression in patients with colorectal cancer (CRC). We have shown that adaptive T_R cells express COX-2 and produce PGE₂ that suppress effector T cells in a manner that is reversed by COX-inhibitors. Methods and results Here we demonstrate that CRC patients have elevated levels of PGE₂ in peripheral blood, and CRC tissue samples and draining lymph nodes display increased numbers of FOXP3+ T_R cells. Depletion of T_R cells from PBMC enhanced anti-tumor T-cell responses to peptides from carcinoembryonic antigen. Furthermore, the COX inhibitor indomethacin and the PKA type I antagonist Rp-8-Br-cAMPS significantly improved the anti-tumor immune activity. Conclusion We suggest that adaptive T_R cells contribute to an immunosuppressive microenvironment in CRC and inhibit effector T cells by a COX-2–PGE₂-dependent mechanism and thereby facilitate tumor growth. Therapeutic strategies targeting T_R cells and the PGE₂–cAMP pathway may be interesting to pursue to enhance anti-tumor immune activity in CRC patients.     

Inflammation and MiR-21 Pathways Functionally Interact to Downregulate PDCD4 in Colorectal Cancer

Inflammation plays a direct role in colorectal cancer (CRC) progression; however the molecular mechanisms responsible for this effect are unclear. The inflammation induced cyclooxygenase 2 (COX-2) enzyme required for the production of Prostaglandin E2 (PGE₂), can promote colorectal cancer by decreasing expression of the tumour suppressor gene Programmed Cell Death 4 (PDCD4). As PDCD4 is also a direct target of the oncogene microRNA-21 (miR-21) we investigated the relationship between the COX-2 and miR-21 pathways in colorectal cancer progression. Gene expression profile in tumour and paired normal mucosa from 45 CRC patients demonstrated that up-regulation of COX-2 and miR-21 in tumour tissue correlates with worse Dukes'' stage. In vitro studies in colonic adenocarcinoma cells revealed that treatment with the selective COX-2 inhibitor NS398 significantly decreased miR-21 levels (p = 0.0067) and increased PDCD4 protein levels (p<0.001), whilst treatment with PGE₂ up-regulated miR-21 expression (p = 0.019) and down-regulated PDCD4 protein (p<0.05). These findings indicate that miR-21 is a component of the COX-2 inflammation pathway and that this pathway promotes worsening of disease stage in colorectal cancer by inducing accumulation of PGE₂ and increasing expression of miR-21 with consequent downregulation of the tumour suppressor gene PDCD4.     

Control of the intracellular levels of prostaglandin E2 through inhibition of the 15-hydroxyprostaglandin dehydrogenase for wound healing

Excessive scar formation is an aberrant form of wound healing and is an indication of an exaggerated function of fibroblasts and excess accumulation of extracellular matrix during wound healing. Much experimental data suggests that prostaglandin E₂ (PGE₂) plays a role in the prevention of excessive scarring. However, it has a very short half-live in blood, its oxidization to 15-ketoprostaglandins is catalyzed by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Previously, we reported that 15-PGDH inhibitors significantly increased PGE₂ levels in A549 cells. In our continuing attempts to develop highly potent 15-PGDH inhibitors, we newly synthesized various thiazolidine-2,4-dione derivatives. Compound 27, 28, 29, and 30 demonstrated IC₅₀ values of 0.048, 0.020, 0.038 and 0.048 μM, respectively. They also increased levels of PGE₂ in A549 cells. Especially, compound 28 significantly increased level of PGE₂ at 260 pg/mL, which was approximately fivefold higher than that of control. Scratch wounds were analyzed in confluent monolayers of HaCaT cells. Cells exposed to compound 28 showed significantly improved wound healing with respect to control.     

A Novel Transporter of SLC22 Family Specifically Transports Prostaglandins and Co-localizes with 15-Hydroxyprostaglandin Dehydrogenase in Renal Proximal Tubules

We identified a novel prostaglandin (PG)-specific organic anion transporter (OAT) in the OAT group of the SLC22 family. The transporter designated OAT-PG from mouse kidney exhibited Na⁺-independent and saturable transport of PGE₂ when expressed in a proximal tubule cell line (S₂). Unusual for OAT members, OAT-PG showed narrow substrate selectivity and high affinity for a specific subset of PGs, including PGE₂, PGF_2α, and PGD₂. Similar to PGE₂ receptor and PGT, a structurally distinct PG transporter, OAT-PG requires for its substrates an α-carboxyl group, with a double bond between C13 and C14 as well as a (S)-hydroxyl group at C15. Unlike the PGE₂ receptor, however, the hydroxyl group at C11 in a cyclopentane ring is not essential for OAT-PG substrates. Addition of a hydroxyl group at C19 or C20 impairs the interaction with OAT-PG, whereas an ethyl group at C20 enhances the interaction, suggesting the importance of hydrophobicity around the ω-tail tip forming a “hydrophobic core” accompanied by a negative charge, which is essential for substrates of OAT members. OAT-PG-mediated transport is concentrative in nature, although OAT-PG mediates both facilitative and exchange transport. OAT-PG is kidney-specific and localized on the basolateral membrane of proximal tubules where a PG-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase, is expressed. Because of the fact that 15-keto-PGE₂, the metabolite of PGE₂ produced by 15-hydroxyprostaglandin dehydrogenase, is not a substrate of OAT-PG, the transport-metabolism coupling would make unidirectional PGE₂ transport more efficient. By removing extracellular PGE₂, OAT-PG is proposed to be involved in the local PGE₂ clearance and metabolism for the inactivation of PG signals in the kidney cortex.     

Bothrops moojeni snake venom induces an inflammatory response in preadipocytes: Insights into a new aspect of envenomation

Bothrops envenomation is a public health problem in Brazil. Despite the advances in the knowledge of the pathogenesis of systemic and local effects induced by Bothrops venom, the target tissues to this venom are not completely characterised. As preadipocytes are important cells of the adipose tissue and synthesize inflammatory mediators, we investigated the ability of B. moojeni snake venom (Bmv) to stimulate an inflammatory response in 3T3-L1 preadipocytes in vitro, focusing on (1) the release of PGE₂, IL-6, TNF-α, MCP-1, KC, leptin and adiponectin; (2) the mechanisms involved in PGE₂ release and (3) differentiation of these cells. Cytotoxicity of Bmv was determined by MTT assay. The concentrations of PGE₂, cytokines and adipokines were quantified by EIA. Participation of the COX-1 and COX-2 enzymes, NF-κB and PGE₂ receptors (EP1-4) was assessed using a pharmacological approach, and protein expression of the COX enzymes and P-NF-κB was analysed by western blotting. Preadipocyte differentiation was quantified by Oil Red O staining. Bmv (1 μg/mL) induced release of PGE₂, IL-6 and KC and increased expression of COX-2 in preadipocytes. Basal levels of TNF-α, MCP-1, leptin and adiponectin were not modified. Treatment of cells with SC560 (COX-1 inhibitor) and NS398 (COX-2 inhibitor) inhibited Bmv-induced PGE₂ release. Bmv induced phosphorylation of NF-κB, and treatment of the cells with TPCK and SN50, which inhibit distinct NF-κB domains, significantly reduced Bmv-induced PGE₂ release, as did the treatment with an antagonist of PGE₂ receptor EP1, unlike treatment with antagonists of EP2, EP3 or EP4. Bmv also induced lipid accumulation in differentiating cells. These results demonstrate that Bmv can activate an inflammatory response in preadipocytes by inducing the release of inflammatory mediators; that PGE₂ production is mediated by the COX-1, COX-2 and NF-κB pathways; and that engagement of EP1 potentiates PGE₂ synthesis via a positive feedback mechanism. Our findings highlight the role of the adipose tissue as another target for Bmv and suggest that it contributes to Bothrops envenomation by producing inflammatory mediators.