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Background/aim: Our aim was to investigate the crosstalk between tumor and immune cells (M2 macrophages) and its effects on cyclo-oxygenase-2 (COX2) regulation in canine mammary tumors (CMT)., Materials and Methods: Sh1b CMT cells and human BT474 mammary or HT29 colon cancer cells were co-cultured with canine peripheral blood mononuclear cells (PBMCs) or with macrophage-like differentiated THP1 monocytes (dTHP1). Intracellular COX2 expression by PBMCs, dTHP1 and cancer cells was evaluated by flow cytometry., Results: Co-culturing of Sh1b and canine PBMCs induced COX2 overexpression in CMT cells. In turn, COX2 expression by PBMCs, mostly CD68 + macrophages, was attenuated by co-culture with Sh1b (p=0.0001). In accordance, co-culture with dTHP1 prompted intracellular production of COX2 in both Sh1b CMT cells and HT29 human colon cancer cells and reduced production of COX2 in BT474 human mammary cancer cells. The intracellular COX2 expression from dTHP1 decreased when treated with conditioned medium from cultured Sh1b and HT29 cancer cells., Conclusion: Bidirectional COX2 regulation between cancer and monocytes/macrophages might shape a tolerogenic tumor microenvironment in CMT., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: The present study determined the efficacy of extracts of Astragalus membranaceus (AM) and Curcuma wenyujin (CW), a traditional Chinese medicine herbal mixture, at different tumor stages of an orthotopic nude mouse model of human ovarian cancer expressing red fluorescent protein., Materials and Methods: The tumor-bearing mice were treated with cisplatinum (CDDP), AM, CW, or a combination of AM and CW in each of three tumor stages, using the same regimen. Group 1 received saline as negative control. Group 2 received CDDP i.p. as positive control with a dose of 2 mg/kg, every three days. Group 3 received AM daily via oral gavage, at a dose of 9120 mg/kg. Group 4 received CW daily via oral gavage, at a dose of 4560 mg/kg. Groups 5, 6 and 7 received combinations of AM and CW daily via oral gavage at low (AM, 2280 mg/kg; CW, 1140 mg/kg), medium (AM, 4560 mg/kg; CW 2280 mg/kg), and high (AM, 9120 mg/kg; CW, 4560 mg/kg) doses. The expression of angiogenesis- and apoptosis-related genes in the tumors were analyzed by immunohistochemistry for matrix metalloproteinase 2 (MMP-2), vascular endothelial growth factor (VEGF) fibroblast growth factor 2 (FGF-2), B-cell lymphoma 2 (Bcl-2) and cyclooxygenase 2 (Cox-2), and by polymerase chain reaction for MMP-2, FGF-2 and Bcl-2., Results: CDDP, AM, and its combination with CW-induced significant growth inhibition of Stage I tumors. Strong efficacy of the combination of AM and CW at high dose was observed. Monotherapy with CDDP, AM, CW, and the combination treatments did not significantly inhibit Stage II and III tumors. The expression of MMP-2, VEGF, FGF-2, and Cox-2 was significantly reduced in Stage I tumors treated with AM, CW, and their combination, suggesting a possible role of these angiogenesis- and apoptosis-related genes in the observed efficacy of the agents tested., Conclusion: This study is the first report on the efficacy of anticancer agents at different stages of ovarian cancer in an orthotopic mouse model. As the tumor progressed, it became treatment-resistant, similar to the clinical situation, further demonstrating the utility of the model and the need for agents acrtive in advanced-stage ovarian cancer., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background/aim: The ability of tumors to evade the immune system is one of cancer hallmarks. In breast cancer, it has been demonstrated that the cyclooxygenase-2(+)/ epidermal growth factor receptor(+) (COX-2(+)/EGFR(+)) status might influence tumor microenvironment allowing escape of cancer cells to the immune system. This topic is unknown in canine mammary tumors (CMT). Therefore, the potential relationship between CD3(+) T-lymphocytes and concurrent COX-2/EGFR expression was investigated., Materials and Methods: Formalin-fixed paraffin-embedded malignant CMT samples (n=63) were submitted to immunohistochemical staining to detect CD3, COX-2 and EGFR., Results: Tumoral CD3(+) T-lymphocytes were significantly associated with tubular differentiation grade (p=0.006), tumor necrosis (p=0.025), histological grade of malignancy (p=0.027) and presence of lymph node metastasis (p=0.009). A correlation between COX-2 and EGFR was observed (r=0.741, p<0.0001). The COX-2(+)/EGFR(+) group was associated with tumor size (p=0.002), mitotic index (p=0.019), histological grade of malignancy (p=0.035) and presence of lymph node metastasis (p=0.041). CD3(+) T-lymphocytes and COX-2/EGFR groups were significantly associated (p=0.025) and positively correlated (r=0.399; p=0.003)., Conclusion: The present results suggest that the COX-2(+)/EGFR(+) status may be part of a strategy adopted by tumor cells to evade the cytotoxic tumor-specific immune responses., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Previous clinicopathological studies demonstrated that overexpression of cyclooxygenase-2 (COX-2) is associated with a poor treatment response of esophageal carcinoma. The aim of this study was to elucidate the role of COX-2 overexpression in the chemosensitivity of esophageal carcinoma cells. TE13 human esophageal squamous cell carcinoma cells were transfected with a COX-2 constitutive expression vector, and stable transfectants overexpressing COX-2 were established. COX-2 overexpression in COX-2 transfectants was confirmed with western blotting and prostaglandin-E(2) (PGE(2)) assay. Chemosensitivity testing revealed that sensitivity of COX-2 transfectants to 5-fluorouracil and cisplatin was significantly lower than in control vector-only transfectants, and that sensitivity of COX-2 transfectants was restored by the transfection of COX-2-specific siRNA. In addition, expression of antiapoptotic B-cell lymphoma-extra large (BCL-xL) and myeloid cell leukaemia-1 (MCL-1) was increased in COX-2 transfectants. These results indicate that COX-2 overexpression may reduce the chemosensitivity of esophageal carcinoma cells through up-regulation of the expression of antiapoptotic BCL-2 family proteins.

Background: There are no reliable biomarkers for diagnosis, prediction of outcome or treatment effect monitoring for upper tract urothelial carcinoma (UTUC), which is a uniquely prevalent cancer in Taiwan. In the present study, 128 primary UTUC specimens of various grades and primary tumor status were examined for the expression of cyclooxygenase-2 (COX-2) in tumor and stroma tissues aiming to clarify the association of COX-2 expression with clinical outcomes in Taiwanese patients with UTUC., Materials and Methods: Immunohistochemistry was implemented to investigate COX-2 expression levels in 128 paired tumor and stroma specimens. The association of COX-2 expression with tumor progression and prognosis was analyzed., Results: Our data demonstrated that positive COX-2 expression was more frequent in stromal cells (57.0%), than in tumor sites (53.1%), and the up-regulation of COX-2 was strongly associated with higher cancer-specific death and cancer recurrence rates. In COX-2-negative cases, no similar correlation was found., Conclusion: COX-2 expression was up-regulated in both stromal and tumor cells of more than half of the studied UTUC patients and the positive expression of COX-2 in stromal cells may be a potential predictive and prognostic biomarker for UTUC, especially for cancer-specific death and recurrence.

Aim/purpose: We investigated the expression of melanoma-associated antigen gene (MAGE), human synovial sarcoma on X chromosome (SSX) and their clinical implications in sporadic colon cancer., Materials and Methods: Fresh tissue samples from 37 patients with colorectal adenocarcinomas were analyzed for MAGE and SSX mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and their paraffin-embedded tissues were used for immunohistochemistry for cyclooxygenase 2 (COX2), vascular endothelial growth factor (VEGF), and survivin., Results: Expression of MAGE and SSX was not detected in normal tissues. Colon cancer expressed SSX in 32.4% and MAGE in 51.4% of cases. Co-expression of MAGE and SSX was directly correlated with liver metastasis (p=0.024) and also correlated with nuclear expression of survivin (p=0.016), yet did not correlate with expression of COX2 and VEGF. Nuclear survivin expression (83.3%) was found in the cancer tissues exclusively. No significant relationships between the expression of COX2 (73.9%) and VEGF (72.4%) and other clinicopathologic variables were found., Conclusion: Our results suggest that nuclear expression of survivin, lymph node metastasis, vascular and perineural invasion, and co-expression of MAGE and SSX may be associated with the metastasis of colorectal cancer to the liver.

With improvements in systemic control, metastasis to the brain has been more frequently found in patients with breast cancer. In order to gain access to the brain, breast cancer cells must overcome the blood-brain barrier (BBB), a highly selective filter against cellular and soluble substances. Human brain endothelial cells (HBECs) comprise a major element of the BBB, and breast cancer cells first encounter and pass through them for extravasation. To date, however, the precise role of HBECs in metastasis to the brain is unknown. In this study, we examined how HBECs take part in the extravasation process. In an established in vitro model of the BBB, unexpectedly, the transmigration of breast cancer cells was markedly enhanced in the presence of HBECs than in their absence, suggesting that HBECs facilitate the transmigration of breast cancer cells rather than acting as a barrier against them. We then showed that cyclooxygenase (COX-2) induced from HBECs rather than that from breast cancer cells plays a key role in the transmigration. Moreover, expression of matrix metalloproteinase (MMP-2) mediating the transmigration was induced in HBECs by COX-2 after co-culture with breast cancer cells. Taken together, our results suggest that COX-2 and MMP-2 produced from HBECs facilitate the extravasation of breast cancer cells across the BBB.

Prostaglandin metabolism plays a pivotal role in inflammatory processes and has also been demonstrated to have a role in carcinogenesis, tumor differentiation and tumor growth in breast cancer. Cyclooxygenase-2 (COX-2) is the key involved enzyme, as it triggers prostaglandin synthesis. We reviewed the current literature regarding the impact of prostaglandin metabolism on breast cancer and illustrated the current evidence of the COX-2 influence on breast cancer, delineating possible future prophylactic and therapeutic strategies.

Background: Because of clinicopathologic and genetic differences between left-sided colorectal cancer (LSCRC) and right-sided colon cancer (RSCC), cyclooxygenase-2(COX-2) and adenomatous polyposis coli (APC) expression may be of clinical relevance., Materials and Methods: Clinicopathologic information for 72 primary colon tumors, 44 left and 28 right, from 72 patients (34 F, 38 M) were analyzed. COX-2 and wild-type APC (W-APC) immunohistochemical expressions were determined for each case. The data were analyzed using the Chi-square test and exact binomial confidence intervals., Results: Overall, 31 out of 44 (70%) LSCRC were W-COX-2 positive vs. 13 out of 28 (46%) RSCC (p-value=0.042). When evaluated independently of the anatomic location, COX-2 expression showed a borderline statistical correlation with the lack of W-APC protein (p-value=0.054). When considering location of tumors, the inverse correlation between COX-2 and W-APC expression became statistically significant (p-value=0.024)., Conclusion: We report a strong inverse correlation between COX-2 and W-APC expression, with COX-2 being more frequently as expressed in LSCRC. These data may be useful to stratify colorectal cancer patients into right- and left-sided and COX-2 expressor and non-expressor subsets, when evaluating COX-2 inhibitor and other targeted therapies in colon cancer.

The multi-functional apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) DNA repair and redox signaling protein has been shown to have a role in cancer growth and survival, however, little has been investigated concerning its role in inflammation. In this study, an APE1 redox-specific inhibitor (E3330) was used in lypopolysaccharide (LPS)-stimulated macrophages (RAW264.7). E3330 clearly suppressed secretion of inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL-6) and IL-12 and inflammatory mediators nitric oxide (NO) as well as prostaglandin E(2) (PGE(2)) from the LPS-stimulated RAW264.7 cells. These data were supported by the down-regulation of the LPS-dependent expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) genes in the RAW264.7 cells. The effects of E3330 were mediated by the inhibition of transcription factors nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) in the LPS-stimulated macrophages, both known targets of APE1. In conclusion, pharmacological inhibition of APE1 by E3330 suppresses inflammatory response in activated macrophages and can be considered as a novel therapeutic strategy for the inhibition of tumor-associated macrophages.

Background: Prognosis of renal cell carcinoma (RCC) differs within the same stage and grade. Our aim was to investigate the incidence of COX-2 in primary RCC tumors at different stages according to the occurrence of metastasis, and the impact of this biomarker on the survival of RCC patients., Patients and Methods: The cytoplasmic/membranous COX-2 protein expression was examined by immunohistochemistry in RCC tumors from 102 patients. The patients were divided into those with: no metastasis during 7.5 years' follow-up (nm), no metastasis at the time of nephrectomy but who later developed metastases (lm), and those with metastasis at presentation (pm). The immunoreactivity of COX-2 was classified as none (absent/weak intensity in fewer than 10% of the cancer cells), low (weak intensity in over 10% of the cancer cells) or high immunostaining (strong intensity in the majority of the cancer cells). In addition p53 and Ki-67 immunostaining was also assessed in tumors., Results: Percentages of COX-2 reaction were (no/low/high): 78/16/7 in the nm, 53/28/19 in the lm, 92/8/0 in the pm groups (p=0.014). Median metastasis-free survival was shorter in lm patients with COX-2-negative tumors when compared to those with COX-2-positive ones (15 vs. 46 months; p=0.020). Median overall survival was shorter in pm/lm patients with COX-2-negative tumors when compared to those with COX-2-positive ones (28 vs. 94 months; p=0.027), and with COX-2-negative/Ki-67-positive tumors when compared to COX-2-positive/Ki-67-negative ones (19 vs. 97 months; p=0.004). Findings for patients with COX-2-negative/p53-positive tumors were similar, with shorter survival compared to those with COX-2-positive/p53-negative ones (19 vs. 97; p=0.006)., Conclusion: COX-2 protein expression is associated with slow development of metastases, and favourable prognosis in metastatic RCC.

Unlabelled: The aim of this study was to To investigate correlations between serum progesterone and serum estradiol levels and expression of tissue tumor markers in cervical intraepithelial neoplasia (CIN) and normal epithelium., Materials and Methods: Eighty women of fertile ages with cervical biopsies ranging histologically from normal to CIN III were included. Expression of eleven tumor markers was studied. Serum levels of progesterone and estradiol were analyzed. Exclusion criterion was hormonal contraceptive use., Results: In normal epithelium, low progesterone levels correlated to expression of epidermal growth factor receptor (EGFR) and CD4+. In initial analyses of CIN, high progesterone levels correlated with expression of retinoblastoma protein, p16 and cyclooxygenase-2 (COX-2), but after adjustment for CIN grade, only correlation to COX-2 expression remained significant. Expression of COX-2 and CD4(+) correlated to serum estradiol levels in CIN., Conclusion: Serum levels of progesterone and estradiol appear to correlate with increased COX-2 expression in CIN. In addition, the study shows that evaluation of expression of tumor markers must take into account the grade of CIN.

Background: Blocking therapies aimed at COX-2, HSP90 and CD117 have been described recently. The objective of the study was to analyze expression of these antigens and the proliferation rate in metastatic melanomas., Materials and Methods: Fine-needle aspirates from 30 patients were analyzed. Immunocytochemical methods were applied to assess COX-2, HSP-90 and CD117. Cell proliferation was analysed using expression of Ki-67. Findings were compared with histopathological parameters., Results: All cases expressed COX-2 and HSP90. CD117 was expressed in 46% of cases. The proliferation index ranged between 7% and 54%. No correlation was found between histological properties of the primary tumours and expression of CD117, COX-2 and HSP90 in their metastases. An inverse correlation was found between HSP90 and MIB-1 index., Conclusion: A large proportion of metastatic melanomas expressed COX-2 and HSP90. This may have a clinical implication for blocking therapy of the corresponding molecules. Expression of CD117 antigen was observed in only 5/30 melanoma cases. We hypothesize that such melanomas may benefit from targeted therapy with kinase inhibitors.

Background: Celecoxib and other non-steroidal anti-inflammatory drugs (NSAIDs) are being evaluated in the prevention of bladder and other cancers. Here we investigate molecular effects of celecoxib independent of cyclooxygenase (COX)-2 expression levels in urothelial carcinoma of the bladder., Materials and Methods: Low-grade RT-4 and high-grade UM-UC-3 bladder cancer cells were treated with 0-50 muM celecoxib. Growth, cell cycle and apoptosis were measured by crystal violet elution and flow cytometry. Western analysis was performed for COX-2, Rb, cyclin B1/D1, and phospho-cyclin B1/D1. COX-2 induction was achieved with phorbol ester., Results: Celecoxib inhibited growth of RT-4 and UM-UC-3, with G(1) cell cycle arrest and altered cyclin B1/D1 expression in RT-4, whereas Rb up-regulation occurred in UM-UC-3. Apoptosis occurred in both cell lines., Conclusion: Celecoxib induces G(1) cell cycle arrest in low- and high-grade bladder cancer by different pathways. This heterogeneous molecular response supports combination approaches to prevention and treatment.

The mitogenic and anti-apoptotic effects of insulin-like growth factor-I (IGF-I) are regulated by a family of insulin-like growth factor binding proteins (IGFBPs), particularly IGFBP-3. Little is known about the IGF-independent role of IGFBP-3 in breast cancer and the mechanisms regulating its production. The expression of IGFBP-3 in paired malignant and adjacent normal (n=53), and healthy normal (n=17) breast tissue samples was investigated using RT-PCR, immunohistochemistry and ELISA. We compared IGFBP-3 expression with other members of the IGF-I axis, other known tumorigenic genes and clinicopathological parameters. We also developed a novel tissue explant system using fresh normal and malignant breast tissue, with which we examined the in vitro effects of IGFBP-3 alone and in combination with known apoptotic agent, doxorubicin (n=6), on tissue viability and apoptosis. We demonstrated a high level of expression of IGFBP-3 mRNA in all samples. 96% of samples also expressed IGFBP-3 protein. No significant correlation was seen between IGFBP-3 expression and other clinicopathological parameters. The in vitro tissue explant system demonstrated that IGFBP-3 had little effect by itself on apoptosis. However, when used in combination with doxorubicin, increased apoptosis was seen in tumours. In contrast, less apoptosis was seen in normal tissue suggesting a protective effect. These divergent effects suggest a potential novel chemotherapeutic approach in the treatment of breast cancer. These findings suggest that IGFBP-3 may play a role in tumorigenesis, and that IGFBP-3 levels could be used in the future in cancer risk assessment/prevention or as markers of response to cancer treatments.

Background: Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the steroid receptor superfamily. Liganded PPARgamma can inhibit cancer cell proliferation. The in vitro and in vivo inhibitory effect of the synthetic ligands, ciglitazone (CGZ) and pioglitazone (PGZ), on human colon cancer was investigated., Materials and Methods: Cell proliferation and the expression of PPARgamma, cyclooxygenase (COX)-2 and cyclin D1 were assessed in colon cancer cells treated with CGZ or PGZ. After subcutaneous or splenic inoculation of severe combined immunodeficient (SCID) mice using colon cancer HT-29 and SW480 cells, PGZ was administered orally and tumor growth inhibition was assessed by xenograft volume. The COX-2, cyclin D1 and PPARgamma expression in the HT-29 cells was evaluated., Results: Cultured HT-29 and SW480 cells expressed PPARgamma and proliferation was inhibited by CGZ and PGZ. Oral PGZ inhibited xenograft tumor growth and liver metastases in the SCID mouse and suppressed expression of COX-2 and cyclin D1 in HT-29 cells., Conclusion: PGZ down-regulates COX-2 and cyclin D1 and inhibits colon cancer proliferation and liver metastasis, making PPARgamma a candidate target for the treatment/prevention of colon cancer metastasis.

Background: The primary method of treatment of non-small cell lung cancer (NSCLC) in stage IIIB and IV is chemotherapy. Previous data suggested a correlation between cyclooxygenase-2 (COX-2) expression and the multidrug-resistant phenotype of cancer cells., Materials and Methods: In this prospective study, 32 patients with NSCLC in stage IIIB and IV from 1,078 patients were included. The expression of COX-2 as well as the expression of the ABC transporters MDR1/P-glycoprotein (MDR1/P-gp), BCRP and MRP1 were detected immunohistochemically., Results: Univariate and multivariate analyses demonstrated no prognostic or predictive significance of these proteins. It was merely demonstrated that complete or partial response are favourable factors for prediction of longer progression-free survival time. However, a strong positive correlation between the expression of COX-2, MDR1/P-gp and BCRP was found in NSCLC., Conclusion: These data suggest no clinical impact for the expression of MDR1/P-gp, MRP1, BCRP or COX-2 in NSCLC, but a putative coregulation of COX-2 and MDRI/P-gp and BCRP in NSCLC.

Background: Cycloxygenase (COX)-2 has been associated with proliferation, apoptosis and angiogenesis in urothelial cancer. The prognostic significance of COX-2 in patients who received adjuvant chemotherapy for urothelial cancer was examined., Patients and Methods: Expression of COX-2, p53, ki67, beta-catenin, vascular endothelial growth factor (VEGF) and microvessel density (MVD) were studied retrospectively in 59 patients with urothelial cancer (pT3, pT4, N+) who had undergone surgery. The patients had subsequently received adjuvant chemotherapy., Results: Thirty-eight out of 59 cases (64%) were positive for COX-2. COX-2 was not associated either with progression-free survival (PFS) or overall survival (OS). MVD levels > or =47 were associated with longer median PFS compared with lower levels (not reached vs. 13 months [95% CI: 8-18], p=0.048). The median PFS for patients with beta-catenin nuclear accumulation and COX-2 expression was 6 months (95% CI: 4-7) compared with 19 months (95% CI: 14-23) for neither or only one of these factors (p=0.018)., Conclusion: MVD may be a useful indicator of relapse in high-risk urothelial cancer treated with adjuvant chemotherapy.

Unlabelled: THE AIM of the present study was to investigate the differences in the expression of tumor markers in squamous cell and in adenomatous carcinomas in pre- and postmenopausal women, respectively., Patients and Methods: The study population comprised 53 premenopausal and 107 postmenopausal women. Thirty-four tumors were adenomatous (n=31) or adenosquamous carcinomas (n=3), distributed between 13 premenopausal and 21 postmenopausal women. The remaining 126 squamous cell carcinomas were diagnosed in 40 pre- and 86 postmenopausal women. Expression of ten tumor markers of possible clinical importance in cervical cancer was evaluated., Results: Expression of three tumor markers, p53 (>0% vs. 0%), p27 (> or =20% vs. <20%) and cyclooxygenase-2 (COX-2) (high intensity vs. moderate/none) differed significantly between pre- compared to postmenopausal women with squamous cell (p27; 54% vs. 72%, p=0.009) or adenomatous carcinomas (p53; 8% vs. 63%, p=0.006 and COX-2; 46% vs. 20%, p=0.03). All results were adjusted for clinical cancer stage., Conclusion: The unusual age-specific incidence curve in cervical cancer has rarely been related to expression of tumor markers. Age-related differences in expression of tumor markers could reflect some age-related different biological mechanisms in cervical cancer.

Background: Colorectal adenomatous polyps are known as premalignant lesions. Mutations in the mismatch repair (MMR) enzymes hMLH1, hMSH2 and hMSH6 are recognized causes of hereditary non-polyposis colorectal cancer and act by inducing a mutator phenotype characterized by microsatellite instability (MSI). MSI is also detected in sporadic colorectal cancers. Cox-2 is an inducible enzyme that regulates prostaglandin synthesis and it is overexpressed at sites of inflammation, in colorectal adenomatous polyps and cancer. The aim of this study was to evaluate the immunoexpression of hMLH1, hMSH2 and Cox-2 in polyps resected through colonoscopy, and to examine their association with clinicopathological characteristics (age, gender, location, size, histology and grade of dysplasia)., Patients and Methods: One hundred and sixty-seven colonic polyps, 6 normal colonic mucosa samples, and 23 samples of colorectal adenocarcinoma were used in this study. All patients had no family history of colorectal cancer. The samples were prospectively collected and immunostained for hMLH1, hMSH2 and Cox-2 using the ABC-immunohistochemistry technique with amplification by biotinylated tyramide. The mean age was 60.2+/-13.8 years (range 21-90 years) and 77 (55.8%) were men., Results: Tubular adenomas were present in 81.4%, tubulous-villous in 15.9%, serrated in 1.8%, and villous in 0.9%. The majority of the adenomas were located in the rectosigmoid region (63.5%), followed by ascendant in 14.2%, cecum in 7.5%, descendent in 8.2% and transverse in 6.7%. Low-grade dysplasia was detected in 59.6% of the adenomas. Loss of hMLH1 and hMLH2 immunoexpression was observed in 20% and 15.5% of the adenomas, respectively. Cox-2 expression was found in 9% of the adenomas, and in 40% of the adenocarcinomas. Moreover, Cox-2 immunoexpression was associated with the multiplicity of adenomas in the same patient (p=0.001). There was no association between marker immunoexpression and gender, age, location, size, histology or grade of dysplasia., Conclusion: Loss of hMLH1 and hMLH2 immunoexpression in adenomas is relatively frequent in patients without colorectal cancer family history. Cox-2 is overexpressed in colorectal adenomatous polyps and adenocarcinomas, and its positivity in adenomas may indicate a higher risk for multiple lesions.

Background: Celecoxib, a cyclooxygenase-2 inhibitor, has been found to inhibit the proliferation of several kinds of cancer cells; however, the effects of celecoxib on glioma cells are not clear., Materials and Methods: A172 glioma cells were treated with various concentrations of celecoxib for 4, 24 or 48 h. Cytotoxic drug effects were studied by MTT (3-[4,5-dimethylthiazole-2-yl]-2,5- diphenyltetrazolium bromide)-based colorimetric assay, and celecoxib-induced apoptosis of glioma cells was investigated by FACScan. Western blot analysis was used to study celecoxib effects on the expression of mitogen-activated protein kinases (MAPKs), p53, p21, 14-3-4sigma, Bcl-2 and Bax. Caspace-3 activity in glioma cells was analyzed by caspase activity assay., Results: Celecoxib exerted cytotoxic effects upon and induced apoptosis of the A172 glioma cells in a concentration and time-dependent manner (p < 0.05). Celecoxib had no effects on expression of MAPKs, Bax, or p21; however, it increased expression of p53 and 14-3-4sigma, and reduced expression of Bcl-2. Celecoxib also increased the activity of caspace-3 in glioma cells. The apoptotic fraction of A172 cells induced by 24-h treatment with 100 microM celecoxib was reduced from 39% to 23% by pretreatment with caspace-3 inhibitor (DEVD-CHO) (p < 0.001)., Conclusion: The results suggest that celecoxib induced cytotoxicity and apoptosis in this line of glioma cells and that such effects might be related to activation of p53 and 14-3-3sigma, reduced Bcl-2 and Bcl-2/Bax ratio, and increased caspace-3 activity.

Background: Licofelone, a potent antiinflammatory agent has been reported to interfere with the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) signaling pathways with few side-effects. However, the underlying mechanism of licofelone against human cancer is not understood., Materials and Methods: Human and mouse prostate cancer cells were exposed to licofelone in a time- and dose-dependent manner. Cell growth/cell viability, apoptosis, and expression of COX-2 and 5-LOX at the gene and protein levels were investigated., Results: For the first time, it was demonstrated that licofelone inhibited prostate cancer cell growth and significantly down-regulated COX-2 and 5-LOX expression. A weak inhibitory effect on COX-1 protein was also observed., Conclusion: Licofelone inhibited COX-2 and 5-LOX simultaneously and prevented overall cancer cell growth by enhancing apoptosis in both androgen-dependent and androgen-independent prostate cancer cells. Validating the dual role of licofelone in animal models of prostate cancer is critical for promoting its use as a potential chemopreventive or therapeutic agent.

Based on our studies demonstrating first time evidence that the cyclooxygenase-2 (Cox-2) enzyme is abundant within invasive human breast tumors, we developed a clonally derived human breast tumor cell clone designated as MCF-7/Cox-2 Clone 10 by transfection of human Cox-2 cDNA into slow growing, Cox-2 null, non-metastatic MCF-7 human breast tumor cells. The present studies evaluated the biological characteristics of the MCF-7/Cox-2 Clone 10 human breast tumors compared to the characteristics of MCF-7/empty vector control tumors when grown in vivo following injection of 5x10(6) tumor cells into mammary fat pads of ovariectomized female Crl:Nu-Foxn1(nu) mice implanted with slow release 17-beta estradiol pellets. At 60 days after tumor cell injection, MCF-7/Cox-2 Clone 10 human breast tumors were 4-fold greater (p < 0.01) in volume than MCF-7/empty vector control tumors. MCF-7/Cox-2 Clone 10 human breast tumor xenografts were highly angiogenic based on histological observation of large-bore blood vessels, which was confirmed by immunohistochemical staining with anti-CD-31 antibody and quantitation of mean vessel density. MCF-7/Cox-2 Clone 10 human breast tumor cells were present within regional lymph nodes adjacent to mammary fat pads with their local invasion confirmed by Western blotting of Cox-2-protein. This unique Cox-2-dependent breast tumor model rapidly produces large, angiogenic, locally invasive human breast tumor xenografts in mammary fat pads of ovariectomized female Crl:Nu-Foxn1(nu) mice at 42-60 days which recapitulate human breast ductal carcinomas. This unique model may be invaluable as a means to evaluate preclinical safety and efficacy of novel adjuvant therapies for women with metastastic breast cancer including prostanoid receptor antagonists, newly developed anti-angiogenic therapies, as well as other novel approaches for targeting and destruction of human breast tumors and their vasculature.

Phytophenols such as para-substituted 2-methoxyphenols exhibit antioxidant and anti-inflammatory activities, however, their biological activities are concentration-dependent, possibly due to their dual property of being both antioxidant and prooxidant. Eugenol (2-allyl-2-methoxyphenol) and isoeugenol (4-propenyl-2-methoxyphenol) did not reveal cyclooxygenase-2 (COX-2)-inhibiting activity in macrophages stimulated with lipopolysaccharide (LPS). In contrast, vanillin (2-hydroxy-3-methoxybenzaldehyde) and guaiacol (2-methoxyphenol), especially the former, inhibited LPS-stimulated nuclear factor kappa B (NF-kappaB) activation and cyclooxygenase (COX)-2 gene expression in cells of the RAW 264.7 murine macrophage cell line. Among the 2-methoxyphenols, vanillin demonstrated a potent anti-inflammatory activity. The phenolic O-H bond dissociation enthalpy (BDE) and molecular orbital energies (chemical hardness [eta], electronegativity [chi], and electrophilicity [omega]) were examined to clarify the mechanism responsible for inhibition of COX-2 expression. The BDE, chi, and omega values for vanillin were significantly higher than the corresponding values for the other 2-methoxyphenols. The anti-inflammatory activity of 2-methoxyphenols depended on the BDE and the phenol function was crucial for eliciting this activity. In addition, the anti-inflammatory activity depended on the chi and omega. These findings make vanillin attractive as a candidate therapeutic agent.

Background: Breast cancer is the most common cancer among women, with variable outcomes, justifying a continuous search for new parameters to predict accurate prognosis and indicate suitable adjuvant therapy for patients., Materials and Methods: Fourty-four stage I-III breast cancer specimens were investigated immunohistochemically for the expression of cyclooxygenase-2 enzyme (COX-2), hormone receptors, tumor suppressor gene p53, oncogene HER2 and proliferation marker Ki-67. Additionally, twelve specimens were also investigated for the presence of the phosphorylated extracellular signal-regulated kinase (pERK)., Results: It was demonstrated that expressions of biological markers were related to each other (ER to p53 and Ki-67, COX-2 to ER, PgR, Ki-67 and p53, Ki-67 to p53 and PgR, p53 to PgR)., Conclusion: Our data indicate that concomitant immunohistochemical evaluation of cyclooxygenase-2, hormone receptors, p53 and Ki-67 may be of clinical value in determining an accurate prognosis.

Background: Clinical studies have shown that temozolomide (TMZ) and irinotecan demonstrate activity in high grade astrocytic tumors (HGAT). However, the optimal schedule of administration is unknown., Patients and Methods: In the present study, a total of 45 HGAT patients, 38 with glioblastoma multiforme (GBM) and 7 with anaplastic astrocytoma (AA), were treated with TMZ, 150 mg/m(2) on days 1-5, followed by irinotecan, 150 mg/m(2) on days 6 and 17, every 4 weeks for 6 cycles or until the occurrence of unacceptable toxicity or disease progression. Radiation therapy (60 Gy) was initiated on the first day of treatment., Results: Twenty-two patients completed six cycles of treatment. Most frequently recorded side-effects included neutropenia (37%), nausea/vomiting (66%), diarrhea (31%) and infection (44%). Five episodes of vaso-occlusive disease, all of them fatal, were observed. After a median follow-up of 49.8 months, median progression-free survival for patients with GBM was 7.7 months, while median overall survival was 12.8 months. There were six long-term survivors, three of them with GBM. Two out of the five biomarkers studied, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor-C (VEGF-C), were found to be overexpressed in 74% of the tumors, however they had no predictive value for progression-free or overall survival., Conclusion: The combination of TMZ and irinotecan, as administered in this study, was accompanied by high rates of toxicity, especially myelotoxicity and infection. Further development of this regimen in the treatment of HGAT is not recommended.

Background: Although many tumors arising from epithelial tissues produce mucins, little is known regarding the biological significance of mucins in cancer. Recent researches have revealed that mucins produced by cancer cells play a critical role in the initial induction of cyclooxygenase (COX)-2 in the tumor microenvironment. Moreover, the consequence of abnormal glycosylation and overexpression of mucins includes suppression of the host-immune function., Materials and Methods: The expression of Tn/sialyl-Tn (s-Tn) antigen and its impact on COX-2 expression by tumor cells and infiltration of CD8+ T-cell into the cancer cell nest (nest CD8) in endometrial cancer was evaluated. Tissue specimens from 70 endometrial cancer patients who had undergone a curative resection were evaluated for Tn/s-Tn antigen, COX-2 and CD8 by immunohistochemistry., Results: The overexpression of Tn and s-Tn antigen was significantly correlated with COX-2 overexpression (p <0.005 and p <0.001, respectively). There was a significant association between s-Tn overexpression and low infiltration of nest CD8 (p<0.05). Strong expression of s-Tn antigen was significantly associated with poor prognosis (p < 0. 005). Among several prognostic factors, s- Tn expression remained the strong independent predictor of survival by multivariate analysis (p<0.05)., Conclusion: These results suggested that tumor-produced mucins, which were associated with COX-2 induction and immunosuppression, provided additional prognostic information in endometrial cancer patients.

Butylated hydroxyanisole (BHA; a mixture of 2- and 3-BHA) is widely used as a potent antioxidant, but is reported to have adverse effects, such as carcinogenesis and pro-inflammatory activity, possibly due to the pro-oxidant property of this compound. 2-Methoxyphenol dimers derived from ferulic acid were recently demonstrated to inhibit the expression of lipopolysaccharide-stimulated cyclooxygenase-2 (COX-2) via redox-sensitive transcription factors such as nuclear factor kappa B or activator protein-1 (AP-1), due to a weakening of its pro-oxidant property by dimerization. To develop anti-inflammatory and/or anticancer drugs for the prevention of oral diseases, such as leukoplakia and destructive chronic periodontitis, whether 2-BHA (2-tert-butyl-4-methoxyphenol) and its synthetic ortho dimer, bis-BHA (3,3'-di-tert-butyl-5,5'-dimethoxy-1,1'-biphenyl-2,2'-diol) can inhibit AP-1 transcriptional activity stimulated by Porphyromonas gingivalis fimbriae was examined. The fimbria-stimulated AP-1 activation of RAW 264.7 murine macrophages was markedly inhibited by bis-BHA. However, BHA showed slight inhibition. Furthermore, bis-BHA significantly inhibited fimbria-induced COX-2 gene expression, which is closely involved with inflammation and carcinogenesis. These findings suggest that bis-BHA may possess a potent anti-inflammatory effect against oral diseases.

The effects of 26 trihaloacetylazulene derivatives on nitric oxide (NO) production by the mouse macrophage-like Raw 264.7 cells was investigated. The trichloroacetylazulenes [1b-13b] generally showed higher cytotoxicity as compared with the corresponding trifluoroacetylazulenes [1a-13a]. All the compounds inhibited NO production by lipopolysaccharide (LPS)-activated Raw 264.7 cells to various extents. 3-Trifluoroacetylguaiazulene [8a], 1-trifluoroacetyl-4,6,8-trimethylazulene [10a], 3-methyl-l-trichloroacetylazulene [2b] and 3-ethyl-1-trichloroacetylazulene [3b] showed lower cytotoxic activity and most effectively inhibited NO production. Western blot analysis revealed that compounds [8a, 1Oal dose-dependently reduced the intracellular concentration of inducible NO synthase (iNOS), whereas compounds [2b, 3b] only marginally affected the iNOS protein expression. RT-PCR analysis showed that compounds [8a, 2b] reduced the iNOS mRNA expression by approximately 50%. These compounds affected cyclooxygenase-2 protein and mRNA expression, depending on the concentrations. ESR spectroscopy revealed that compounds [8a, 10a, 2b, 3b] neither produced radical, nor scavenged NO, superoxide anion or diphenyl-2-picrylhydrazyl radicals. The present study showed the inhibitory effects of trifluoroacetylazulenes and trichloroacetylazulenes on NO production by activated macrophages.

Calcitriol (1,25-dihydroxyvitamin D3), the active form of vitamin D, promotes growth inhibition and differentiation in prostate cancer (PCa) cells. To unravel the molecular pathways of calcitriol actions, cDNA microarray analysis was used to identify novel calcitriol target genes including two that play key roles in the metabolism of prostaglandins (PGs), known stimulators of PCa growth and progression. Calcitriol significantly decreases the expression of the PG synthesizing cyclooxygenase-2 (COX-2) gene, while increasing that of PG inactivating 15-prostaglandin dehydrogenase (15-PGDH). Calcitriol also inhibits the expression of the PG receptors EP2 and FP. It reduces the levels of biologically active PGs and inhibits PG actions in PCa cells, thereby decreasing the proliferative stimulus of PGs. We postulate that the regulation of the PG pathway contributes to the growth inhibitory actions of calcitriol. We also propose that calcitriol can be combined with non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit COX enzyme activity, as a potential therapeutic strategy in PCa.

An extract of the bark of Khaya senegalensis is commonly used in African traditional medicine for pain and inflammation. Khaya senegalensis bark extract (KSBE) was hypothesized to contain inhibitors of the cyclooxygenase-2 (COX-2) gene and to be useful in the prevention and treatment of colorectal cancer. The diphenyl-2-picrylhydrazyl (DPPH)- free radical activity and the total phenolic content of KSBE were measured, followed by an investigation of cell growth inhibition, COX and prostaglandin E 2 (PGE2) suppression, as well as apoptosis by Western blot analysis and ELISA. Our data clearly showed that KSBE displays anti-proliferative, antiinflammatory and pro-apoptotic effects on HT-29, HCT-15 and HCA-7 cells. Since all three cell lines, irrespective of COX-2 status (HCT-15 is COX-2-deficient), were affected by the treatment, it can be concluded that both COX-dependent and COX-independent pathways are activated by KSBE.

Background: Conjugated linoleic acid (CLA), a naturally occurring linoleic acid isomer found in ruminant-produced foods, has the potential to serve as an effective chemopreventive nutriceutical factor for breast cancer prevention based upon previous published studies. There are several CLA isomers in ruminant-produced food products, among which t10,c12-CLA and c9,t11-CLA are more potent. Expression of cyclooxygenase 2 (COX-2) in mammary tumors has been correlated with poor prognosis. Prostaglandin E2 (PGE2) is a major COX-2 product in various cancers and, as in humans, PGE2 concentrations in canine tumor tissues were frequently elevated. Moreover, a PGE2 receptor subtype, EP2, is highly expressed in mammary tumors. Thus, various studies have implicated the important role of PGE2 and EP2 in COX-2-regulated tumor development., Materials and Methods: Mammary tumor and normal mammary tissues were both collected from a female dog with mammary tumor. Both malignant and normal mammary tissues were subjected to isolation of epithelial and stromal cells. The effects of t10,c12-CLA and c9,t11-CLA on proliferation, as well as COX-2 and EP2 protein expression in canine mammary normal and cancerous cells, were detected by CellTiter 96 AQueous assay and Western blot assay, respectively., Results: Both t10,c12-CLA and c9,t11-CLA not only suppressed malignant mammary cell growth, but also exerted inhibitory effects on tumor-associated non-malignant mammary cells. Similarly, both t10,c12-CLA and c9,t11-CLA suppressed EP2 protein expression in both normal and malignant mammary cells. t10,c12-CLA was more effective in decreasing COX-2 protein expression in malignant mammary cells, while, in contrast, c9,t11-CLA down-regulated COX-2 protein expression in both normal and malignant mammary cells., Conclusion: The results indicate that the dietary component CLA regulates COX-2 and EP2 protein expression in both malignant mammary cells and cells from the tumor-associated stromal compartment. In turn, this may suppress PGE2 signaling, leading to better prognosis. We further speculate that the knowledge obtained from canine studies may also be beneficial to study human breast cancer.

Curcumin (diferuloylmethane) is a chemical derived from several Curcuma species (turmeric), possessing anti-inflammatory and antioxidant properties and which, thus, may be a potential anticancer drug. However, its mechanism of action is not fully understood. Our previous studies had shown that curcumin induced cytotoxicity, cell cycle arrest and apoptosis in human colon cancer colo 205 cells. In this study, curcumin affected the levels of NF-kappaB/ p65 in a time-dependent manner but did not affect NF-kappaB/ p50, based on Western blotting methods. In vitro experiments revealed that curcumin inhibited Cox-2 levels, but promoted those of Cox-1 in colo 205 cells. Curcumin also inhibited MMP-2 levels and promoted MMP-9 levels, but did not affect MMP-7 levels, based on Western blotting assays. These effects were also confirmed by cDNA microarray. Remarkably, curcumin not only exerted its effect on the protein levels of NF-kappaB, Cox-1 and -2, MMP-2 and -7, but also directly inhibited their mRNA levels. Curcumin was also found to significantly repress the in vitro invasion of colo 205 cells.

Background: Heparin has been widely used to prevent cancer-associated thromboembolism in cancer patients. Recent evidence reveals that heparin could modulate cell proliferation in the stomach. The effect of heparin on gastric cancer growth, however, is unknown. The effect of heparin on the proliferation of a human gastric adenocarcinoma cell line, BGC-823, was investigated., Materials and Methods: Cell proliferation was assessed by [3H]-thymidine incorporation. The expressions of several growth-related genes were determined by RT-PCR and Western blot., Results: Heparin significantly increased cell proliferation in BGC-823 cancer cells by 15.5% at the dose of 0.2 microg/ml. Heparin also up-regulated c-Myc protein expression by 14.4%. In contrast, mRNA and protein levels of epidermal growth factor receptor (EGFR) were, respectively, down-regulated by 12.7% and 8.2% with no effect on cyclooxygenase-2 mRNA or protein expression., Conclusion: Our results suggest that heparin can promote the proliferation and up-regulation of c-Myc protein expression in gastric cancer cells.

Background: Cyclooxygenase-2 (COX-2), a critical enzyme in the conversion of arachidonic acid to prostaglandin E2, influences the biological behavior of human tumors, being involved in carcinogenesis, tumor progression, reduced apoptosis and differentiation. The aim of the present study was to investigate the role of COX-2 protein expression in urothelial carcinoma (UC) of the urinary bladder in relation to clinicopathological data and indices of apoptotic potential., Materials and Methods: Immunohistochemistry was applied to 134 paraffin-embedded specimens of UC for the detection of COX-2, p53, bcl-2, caspase-3, bax protein, MLH1 and hTERT., Results: Ninety-four UCs (70.1%) had an enhanced expression of COX-2. The COX-2 semi-quantitative expression was unrelated to tumor grade and local invasion, but it was positively linked with caspase-3 (CPP32) and bax protein semi-quantitative immunoreactivity (p = 0.007 and p = 0.026), as well as with the quantitative expression of MLH1 (p = 0.019). COX-2 was also found to be inversely correlated with the nuclear localization of the catalyst component of the telomerase complex, hTERT (p = 0.009). Multivariate statistical analysis showed that COX-2 immunopositivity was independently associated with worse prognosis of patients with non muscle-invasive UCs (p = 0.002)., Conclusion: COX-2 overexpression, being possibly a subsequence of apoptosis activation, is associated with an unfavorable overall survival of patients with pTa-T1 UCs.

Cytotoxici and alpha-diisoeugenol were investigated. The cytotoxicity of curcumin and a-diisoeugenol against human promyelocytic leukemia cells (HL-60 cells) and human submandibular cancer cells (HSG cells) was similar (CC50 1-3 microM). However, curcumin induced much more apoptosis, particularly in HL-60 cells compared with HSG cells, as revealed by measurement of the sub-G1/G0 DNA fraction in flow cytometric histograms. Treatment with 15 microM curcumin increased the number of cells with a sub-G1/G0 DNA fraction from control levels of <5% to 55% in HL-60 cells and 30% in HSG cells. Flow cytometry, after staining with annexin V-FITC/PI (the exposure of phosphatidylserine (PS) on the surface of apoptotic cells), showed a dose-dependent induction of early apoptosis by curcumin, which reached about 65% in HL-60 cells and about 20% in HSG cells after treatment with 10 microM curcumin. In contrast, alpha-diisoeugenol failed to induce apoptosis in either cell type. For both cell types, the proportion of late apoptotic/necrotic cells increased rapidly at concentrations of curcumin and a-diisoeugenol greater than 10 microM. The generation of intracellular reactive oxygen species (ROS) in curcumin-treated HL-60 cells was greater than that in HSG cells, as judged by CDFH-DA staining. In both cell types, ROS generation by a-diisoeugenol was at control levels. ROS generation by curcumin was suppressed by antioxidants such as N-acetyl-L-cysteine (NAC) and glutathione (GSH) and by scavengers of hydroxy radicals such as mannitol, but, conversely, was promoted by prooxidants such as the transition metal ions Cu(II) and Zn(II). ROS generation may play a part in the exposure of PS. Curcumin, but not a-diisoeugenol, at 10 microM inhibited LPS (lipopolysaccharide)-induced COX-2 gene expression in RAW 264.7 cells. Semiempirical PM 3 calculations suggested that this activity of curcumin, in which it behaves as a non-steroidal anti-inflammatory drug (NSAID)-like compound, is dependent on its phenolic function, which is more pronounced than that of alpha-diisoeugenol. Taken together, our results suggest that the bioactivity of curcumin is a result of its ability to act as both a prooxidant and an antioxidant.

Cyclooxygenase-2 (COX-2) plays a critical role not only in maintenance of the endometrium during the menstrual cycle, but also in the progression of endometrial cancer. The role of COX-2 in tumor growth and development is complex and multifaceted. This review presents evidence for the expression of COX-2 being linked to angiogenesis, tumor cell apoptosis, tumor cell growth, metastasis and local immunosuppression in cancer progression. It also outlines the clinicopathological and prognostic significance of COX-2 in endometrial cancer.