Effects of ranibizumab and amfenac on the functional abilities and radiosensitivity of uveal melanoma cells / Efeitos do ranibizumabe e do amfenac nas habilidades funcionais e na radiossensibilidade de células de melanoma uveal

ABSTRACT Purpose: To evaluate the effects of ranibizumab and amfenac in human uveal melanoma cell lines and to explore the ability of these compounds to sensitize uveal melanoma cells to radiation therapy. Methods: The 92.1 human uveal melanoma cell line was cultured and subjected to the proposed treatment (ranibizumab, amfenac, and a combination of both). Proliferation, migration, and invasion assays of the 92.1 uveal melanoma cell line were assessed after pretreatment with ranibizumab (125 mg/mL), amfenac (150 nM), or a combination of both. In addition, proliferation rates were assessed after treatment with ranibizumab and amfenac, and the cells were subsequently exposed to various radiation doses (0, 4, and 8 Gy). Results: Proliferation assay: cells treated with a combination of ranibizumab and amfenac had lower proliferation rates than controls (p=0.016) and than those treated with only ranibizumab (p=0.033). Migration assay: a significantly lower migration rate was observed in cells treated with amfenac than the control (p=0.014) and than those treated with ranibizumab (p=0.044). Invasion assay: there were no significant differences among the studied groups. Irradiation exposure: in the 4 Gy dose group, there were no significant differences among any groups. In the 8 Gy dose group, treatment with ranibizumab, amfenac, and their combination prior to application of the 8 Gy radiation led to a marked reduction in proliferation rates (p=0.009, p=0.01, and p=0.034, respectively) compared with controls. Conclusion: Combination of ranibizumab and amfenac reduced the proliferation rate of uveal melanoma cells; however, only amfenac monotherapy significantly decreased cell migration. The radiosensitivity of the 92.1 uveal melanoma cell line increased following the administration of ranibizumab, amfenac, and their combination. Further investigation is warranted to determine if this is a viable pretreatment strategy to render large tumors amenable to radiotherapy.

RESUMO Objetivo: Avaliar os efeitos do ranibizumabe em associação com o amfenac nas células de melanoma uveal humano e explorar a capacidade desses compostos em sensibilizar as células de melanoma uveal à radioterapia. Métodos: Células de melanoma uveal humano do tipo 92.1 foram cultivadas e submetidas ao tratamento proposto (ranibizumabe, amfenac e a combinação de ambos). Ensaios de proliferação, migração e invasão com as células de melanoma uveal do tipo 92.1 foram avaliados após tratamento com ranibizumabe (125 mg/ml), amfenac (150 nM) e a combinação de ambos. Além disso, as taxas de proliferação foram avaliadas após tratamento com ranibizumabe e amfenac com subsequente exposição das células a diferentes doses de radiação (0 Gy, 4 Gy e 8 Gy). Resultados: Ensaio de proliferação: células tratadas com ranibizumabe e amfenac combinados apresentaram taxas de proliferação inferiores em comparação ao grupo controle (p=0,016), do que as tratadas apenas com ranibizumabe (p=0,033). Ensaio de migração: foi observada uma taxa de migração significativamente mais baixa nas células tratadas com amfenac do que no grupo controle (p=0,014) e do que nas tratadas com ranibizumabe (p=0,044). Ensaio de invasão: não houve diferenças significativas entre os grupos estudados. Exposição à irradiação: no grupo da dose de 4 Gy, não houve diferença significante entre os grupos. No grupo da dose de 8 Gy, o tratamento com ranibizumabe, afenac e sua combinação antes da aplicação da radiação de 8 Gy levou a uma redução acentuada nas taxas de proliferação (p=0,009, p=0,01 e p=0,034, respectivamente) em comparação aos grupos controle. Conclusão: A combinação de ranibizumabe e amfenac reduziu a taxa de proliferação das células de melanoma uveal; no entanto, apenas o amfenac diminuiu significativamente a migração celular. A radiossensibilidade das células de melanoma uveal do tipo 92.1 aumentou após a administração de ranibizumabe, amfenac e sua combinação. Mais investigações são necessárias para determinar se esta é uma estratégia de pré-tratamento viável para tornar grandes tumores passíveis de radioterapia.

Selective COX-2 Inhibitor (Meloxicam) and Tooth-Supporting Bone Quality. A Histomorphometric Study in Rats

Abstract The effects of the non-steroidal anti-inflammatory drugs (NSAIDs) on bone quantity and quality were investigated for years. However, there is lack of information on the impact of NSAIDs on the quality of tooth-supporting alveolar bone in absence of periodontal inflammation. Thus, the aim of this study was to evaluate histometrically the influence of a selective COX-2 NSAID (Meloxicam) on the inter-radicular bone mineral density in rats. Forty-nine adult male Wistar rats were randomly divided into four experimental groups: Subcutaneous injection of 0.9% sterile saline for 15 days (G1; n=12) and 45 days (G2; n=11); and subcutaneous injection of Meloxicam for 15 days (G3; n=13) and 45 days (G4; n=13). Mineral density was histometrically determined in the inter-radicular area of the 1st mandibular molars and data analysis performed by two-way ANOVA (a=5%). Results showed no interaction between time and treatment (p>0.05) and that meloxicam did not affect the alveolar bone density. In contrast, it was found that inter-radicular alveolar bone density increased with time (91.88±3.08% and 92.86±2.38% for groups 15 and 45 days, respectively) (p<0.05). Within the limits of this study, daily administration of a selective COX-2 inhibitor (Meloxicam) did not affect the quality of the inter-radicular alveolar bone in absence of periodontal infection.

Resumo Os efeitos dos fármacos anti-inflamatórios não esteroidais (AINEs) sobre a quantidade e qualidade óssea tem sido investigados ao longo dos anos.Entretanto, há falta de informação sobre o impacto dos AINEs na qualidade do osso alveolar de suporte na ausência de inflamação periodontal. Assim, o objetivo deste estudo foi avaliar, histometricamente, a influência de um AINE seletivo para COX-2 (Meloxicam) na densidade mineral óssea inter-radicular em ratos. Quarenta e nove ratos Wistar, machos e adultos foram divididos aleatoriamente em quatro grupos experimentais: injeções subcutâneas de 0,9% de solução salina estéril por 15 dias (G1, n=12) e 45 dias (G2, n=11); e injeções subcutâneas de Meloxicam por 15 (G3, n=13) e 45 dias (G4, n=13). A densidade mineral foi determinada histometricamente na área inter-radicular dos primeiros molares mandibulares e a análise dos dados realizada por meio de ANOVA (a=5%). Os resultados mostraram nenhuma interação entre tempo e tratamento (p>0,05) e que o meloxicam não afetou a densidade óssea alveolar. Em contraste, foi encontrado que a densidade óssea alveolar inter-radicular aumentou ao longo do tempo (91,88±3,08% e 92,86±2,38% para os grupos 15 e 45 dias, respectivamente) (p<0,05). Dentro dos limites deste estudo, a administração diária de um inibidor seletivo para COX-2 (Meloxicam) não afetou a qualidade do osso alveolar inter-radicular na ausência de infecção periodontal.

Comparison of pharmacological and genetic inhibition of cyclooxygenase-2: effects on adult neurogenesis in the hippocampal dentate gyrus

Inducible cyclooxygenase-2 (COX-2) has received much attention because of its role in neuro-inflammation and synaptic plasticity. Even though COX-2 levels are high in healthy animals, the function of this factor in adult neurogenesis has not been clearly demonstrated. Therefore, we performed the present study to compare the effects of pharmacological and genetic inhibition of COX-2 on adult hippocampal neurogenesis. Physiological saline or the same volume containing celecoxib was administered perorally every day for 5 weeks using a feeding needle. Compared to the control, pharmacological and genetic inhibition of COX-2 reduced the appearance of nestin-immunoreactive neural stem cells, Ki67-positive nuclei, and doublecortin-immunoreactive neuroblasts in the dentate gyrus. In addition, a decrease in phosphorylated cAMP response element binding protein (pCREB) at Ser133 was observed. Compared to pharmacological inhibition, genetic inhibition of COX-2 resulted in significant reduction of neural stem cells, cell proliferation, and neuroblast differentiation as well as pCREB levels. These results suggest that COX-2 is part of the molecular machinery that regulates neural stem cells, cell proliferation, and neuroblast differentiation during adult hippocampal neurogenesis via pCREB. Additionally, genetic inhibition of COX-2 strongly reduced neural stem cell populations, cell proliferation, and neuroblast differentiation in the dentate gyrus compared to pharmacological inhibition.

Comparative Study for Preventive Effects of Intra-Abdominal Adhesion Using Cyclo-Oxygenase-2 Enzyme (COX-2) Inhibitor, Low Molecular Weight Heparin (LMWH), and Synthetic Barrier

PURPOSE: Postoperative adhesion is the most frequent complication of abdominal surgery. Therefore, we investigated the individual effects of synthetic barrier [hyaluronic acid/carboxymethylcellulose (HA/CMC)] and pharmacologic agents [low molecular weight heparin (LMWH) cyclo-oxygenase-2 inhibitor (COX-2 inhibitor)] using animal model of intra-abdominal adhesion. MATERIALS AND METHODS: The cecum was rubbed with sterile alcohol wet gauze until subserosal haemorrhage and punctate bleeding developed under the general anesthesia. Five animal groups were prepared using the film HA/CMC, gel HA/CMC, LMWH and COX-2 inhibitor. RESULTS: The grade of adhesion by modified Leach method for group I (control), II (film type HA/CMC), III (gel type HA/CMC), IV (LMWH) and V (COX-2 inhibitor) were 5.35+/-1.8, 6.15+/-1.3, 4.23+/-2.6, 5.05+/-0.7 and 5.50+/-0.9, respectively. Group III showed the least grade of adhesion and it is statistically significant in adhesion formation (p=0.028). The numbers of lymphocytes were significantly low in group III and group V compared to the control group (lymphocyte: p=0.004). The mast cell counts were generally low except for the control group (I: 1.05, II: 0.35, III: 0.38, IV: 0.20, V: 0.37), however, it was not statistically significant (p=0.066). CONCLUSION: The gel barriers were shown to be partly efficient in inhibiting the formation of postoperative adhesions and might provide an option for abdominal surgery to reduce postoperative adhesions. The LMWH and COX-2 inhibitor had been known for their inhibitor effect of fibrin formation and anti-angiogenic/anti-fibroblastic activity, respectively. However, their preventive effects of adhesion and fibrosis were found to be obscure.

High-throughput screening assays for cyclooxygenase-2 and 5-lipoxygenase, the targets for inflammatory disorders.

High-throughput screening (HTS) involves testing of compound libraries against validated drug targets using quantitative bioassays to identify ‘hit’ molecules that modulate the activity of target, which forms the starting point of a drug discovery effort. Eicosanoids formed via cyclooxygenase (COX) and lipoxygenase (LOX) pathways are major players in various inflammatory disorders. As the conventional non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit both the constitutive (COX-1) and the inducible (COX-2) isoforms have gastric and renal side effects and the recently developed COX-2 selective anti-inflammatory drugs (COXIBs) have cardiac side effects, efforts are being made to develop more potent and safer anti-inflammatory drugs. Current assay methods for these enzymes, such as oxygraphic, radioisotopic, spectrophotometric etc. are not compatible for screening of large number of compounds as in drug discovery programs. In the present study, HTS-compatible assays for COX-1, COX-2 and 5-LOX were developed for screening of compound libraries with the view to identify potential anti-inflammatory drug candidates. A spectrophotometric assay involving co-oxidation of tetramethyl-p-phenylene diamine (TMPD) during the reduction of prostaglandin G2 (PGG2) to PGH2 was adopted and standardized for screening of compounds against COX-1 and COX-2. Similarly, the HTS-compatible FOX (ferrous oxidation-xylenol orange) based spectrophotometric assay involving the formation of Fe3+/xylenol orange complex showing absorption in the visible range was developed for screening of compounds against 5-LOX.

Celecoxib mitigates cigarette smoke induced oxidative stress in mice.

Cigarette smoke (CS) is a rich source of radicals, predisposing the cell to oxidative stress resulting in inflammation. Chronic inflammation is a recognized risk factor for carcinogenesis. Cyclooxygenase-2 (COX-2) is a mediator of inflammatory pathway and may, therefore, contribute to carcinogenesis. There are several reports that suggest the association between CS and COX-2 associated risk to cancer. In the present study, we examined the role of celecoxib (a selective COX-2 inhibitor) in modulating the oxidative stress caused by CS inhalation in mice. CS exposure for a period of 10 weeks caused oxidative stress in the pulmonary and hepatic tissues, as evident from the increase in lipid peroxidation levels (LPO) and decrease in reduced glutathione (GSH) levels. Celecoxib (125 mg/kg body weight for 8 weeks) administration to CS inhaling mice reduced the oxidative stress by decreasing the LPO levels and enhancing the GSH levels in comparison to the CS-exposed group. CS exposure repressed the enzymatic antioxidant defense system, as evident from the decrease in catalase (CAT) and superoxide dismutase (SOD) activities. Co-adminstration of celecoxib considerably reversed the changes in the enzymatic antioxidant defense system. Histopathological studies of lungs showed that CS exposure induced alveolar wall destruction and air space enlargement. In co-treated group, the alveolar septa were thicker than normal with apparent infiltration of inflammatory cells. In CS-exposed group, hepatic tissue exhibited vacuolization and macrophage infiltration. Co-treatment with celecoxib restored the normal histoarchitechture in hepatic tissues of CS inhaling mice. Thus, the present study demonstrated that celecoxib adminstration reduced the oxidative stress-mediated risk to carcinogenesis, due to its ability to boost the antioxidant defense system.

AMP kinase/cyclooxygenase-2 pathway regulates proliferation and apoptosis of cancer cells treated with quercetin

AMPK (AMP-activated protein kinase) is highly conserved in eukaryotes, where it functions primarily as a sensor of cellular energy status. Recent studies indicate that AMPK activation strongly suppresses cell proliferation in non-malignant cells as well as in tumor cells. In this study, quercetin activated AMPK in MCF breast cancer cell lines and HT-29 colon cancer cells, and this activation of AMPK seemed to be closely related to a decrease in COX-2 expression. The application of a COX-2 inhibitor or cox-2(-/-) cells supported the idea that AMPK is an upstream signal of COX-2, and is required for the anti-proliferatory and pro-apoptotic effects of quercetin. The suppressive or growth inhibitory effects of quercetin on COX-2 were abolished by treating cancer cells with an AMPK inhibitor Compound C. These results suggest that AMPK is crucial to the anti-cancer effect of quercetin and that the AMPK-COX-2 signaling pathway is important in quercetin-mediated cancer control.

potential renoprotective effect of selective cyclooxygenase inhibitors in experimental diabetic nephropathy

Diabetic nephropathy is a major microvascular complication of diabetes and eventually leads to end-stage renal disease. The present study designed to evaluate the renal effects of selective COX-2 inhibitors on the progression of renal injury in experimental model of diabetic nephropathy. Fifty rats were randomly divided into five equal groups: normal control rats, streptozotocin [STZ]-induced diabetic rats without treatment, STZ-induced diabetic rats treated with celecoxib, STZ-induced diabetic rats treated with enalapril, and STZ-induced diabetic rats treated with combination. Sixteen weeks later, serum glucose, renal functions, and oxidative stress parameters were evaluated. Periodic acid-Schiff [PAS] staining was used to examine the morphological changes by light microscopy. STZ-induced diabetes led to development of diabetic nephropathy associated with increased oxidative stress. Both celecoxib and enalapril produced comparable level of renoprotection manifested by significant decrease of serum creatinine and microalbuminuria, which was accompanied by significant decrease of renal malondialdyehyde content, significant increase of renal reduced glutathione content and superoxide dismutase activity. Glomerulosclerosis seen in untreated-diabetic group were prevented by both celecoxib and enalapril. Combination treatment was superior in renoprotective effects. In conclusion, the selective COX-2 inhibitor celecoxib may prevent or retard the development of diabetic nephropathy

Anti-tumor mechanisms and regulation of survivin by selective cyclooxygenase-2 inhibitor / 대한간학회지

No abstract available.

Comparative QSAR modeling of COX-2 inhibitor 1,2-diarylimidazoles using E-state and physicochemical parameters.

Considering importance of developing selective COX-2 inhibitors, COX-2 binding affinity data of 4-(2-aryl-1-imidazolyl)-phenyl methyl sulfones and sulfonamides (n = 83) have been modeled using electrotopological state (E-state) index as electronic parameter, hydrophobic substituent constant (pi) and molar refractivity (MR) of aryl ring substituents as lipophilic and steric parameters, respectively. Additionally, suitable dummy parameters have been used for the development of multiple regression equations in a stepwise manner. The study suggests that lipophilicity of ortho, meta and para substituents of the aryl ring increases the binding affinity, while molar refractivity (MR) of ortho and meta substituents of the aryl ring decreases the binding affinity. Again, electron-withdrawing substituents at meta and para positions of the aryl ring increase the binding affinity. Additionally, a 4-fluoro substituent on the aryl ring, a trifluoromethyl substituent at R position and simultaneous presence of 3-chloro and 4-methyl groups on the aryl ring are conducive to the binding affinity. Also, an amino substituent is preferred over a methyl group at R2 position suggesting preference of the sulfonamide moiety over the methyl sulfone moiety for the COX-2 binding affinity. Furthermore, importance of E-state values of different atoms in the generated relations suggests the influence of electron density distribution over the 1,2-diarylimidazole nucleus for the binding affinity. For this data set, E-state parameters perform better as electronic parameters in comparison to Hammett sigma parameters. When lipophilic whole molecular descriptor (ClogP) is used, instead of hydrophobic substituent constant (pi), the former performs better than the latter.

COX-2 Inhibitors in Inflammatory Bowel Disease: Friends or Foes? / 대한소화기학회지

The cyclooxygenase (COX) is a key enzyme in the coversion of arachidonic acid to prostaglandins. COX-1 is constitutively expressed and is a critical housekeeping gene, whereas COX-2 is rapidly upregulated by growth factors and cytokines and thus responsible for inflammation. COX-2 is frequently overexpressed in colonic adenoma and carcinoma. Specific inhibitors of COX-2 have been shown to induce apoptosis in tumor cells and to inhibit tumor growth in animal models and in humans. Long-standing IBD patients have increased risk of developing colorectal cancer compared to general population. IBD-associated colorectal carcinogenesis is probably promoted by chronic inflammation and closely related to COX-2. In a recent study, powerful chemopreventive ability of selective COX-2 inhibitor was observed in colitis-related colon carcinogenesis in mouse model. But it was reported that even selective COX inhibitors aggravated the DSS-induced colonic inflammation. It is assumed that endogenous PGs are involved in the mucosal defense against DSS-induced colonic ulcerations which are produced by COX-1 at early phase and by COX-2 at late phase. Long-term use of COX-2 inhibitors for the chemoprevention of colitic cancer is needed to define their mechanism of action, that reduce side effects and have specific tumor target.

Cyclooxygenase-2 promotes cell proliferation, migration and invasion in U2OS human osteosarcoma cells

Osteosarcoma is the most common primary bone tumor, but the pathogenesis is not well understood. While cyclooxygeanse-2 (COX-2) is known to be closely associated with tumor growth and metastasis in several kinds of human tumors, the function of COX-2 in osteosarcoma is unclear. Therefore, to investigate the function of COX-2 in osteosarcoma, we established stable cell lines overexpressing COX-2 in U2OS human osteosarcoma cells. COX-2 overexpression as well as prostaglandin E(2) treatment promoted proliferation of U2OS cells. In addition, COX-2 overexpression enhanced mobility and invasiveness of U2OS cells, which was accompanied by increases of matrix metalloproteinase-2 and -9 (MMP-2 and -9) activities. Selective COX-2 inhibitors, NS-398 and celecoxib, inhibited cell proliferation and abrogated the enhanced mobility, invasiveness and MMP activities induced by COX-2 overexpression. These results suggest that COX-2 is directly associated with cell proliferation, migration and invasion in human osteosarcoma cells, and the therapeutic value of COX-2 inhibitors should be evaluated continuously.

Inhibitory effects of Celecoxib and Sc-58125 on proliferation of human carcinoma of larynx Hep-2 in vitro / 华中科技大学学报（医学）（英德文版）

The inhibitory effects of two kinds of selective cyclooxygenase-2 inhibitors on the proliferation of human carcinoma of larynx Hep-2 in vitro and their corresponding mechanisms were investigated. Hep-2 cells were cultured with two kinds of selective cyclooxygenase-2 inhibitors (Sc-58125 and Celecoxib) at various concentrations for 24 h. Morphological changes were observed under the phase microscopy and the growth suppression was detected by using MTT colorimetric assay. Apoptotic DNA fragments were observed by agarose gel electrophoresis, and the cell cycle and apoptotic rate were detected by flow cytometry (FCM) respectively. Hep-2 cells became rounded and detached from the culture dish after being treated with Celecoxib for 24 h, however, they remained morphologically unchanged with Sc-58125. Sc-58125 could increase G2 phase cells, whereas, Celecoxib rose G1 phase cells. Both of the two effects were dose-dependent. Moreover, the Hep-2 cells cultured with 50 micromol/L and 100 micromol/L Celecoxib showed obvious apoptosis, with the nuclear DNA of cells exhibiting characteristic DNA ladder. So Sc-58125 could inhibit the proliferation of Hep-2 cells by altering the G2 phase cells. However, Celecoxib had the same effect by changing the G1 phase cells and inducing apoptosis at higher concentration.