Over-expression of cyclooxygenase-2 in endoscopic biopsies of ectopic gastric mucosa

Ectopic gastric mucosa (EGM) is considered to be a congenital condition. Rare cases of adenocarcinoma have been described. There are no data justifying regular biopsies or follow-up. Cyclooxygenase-2 (COX-2) is a protein involved in gastrointestinal tumor development by inhibiting apoptosis and regulating angiogenesis. The aim of this prospective study was to evaluate COX-2 expression in EGM and compare it with normal tissue and Barrett's esophagus. We evaluated 1327 patients. Biopsies were taken from the inlet patch for histological evaluation and from the gastric antrum to assess Helicobacter pylori infection. Biopsies taken from normal esophageal, gastric antrum and body mucosa and Barrett's esophagus were retrieved from a tissue bank. EGM biopsies were evaluated with respect to type of epithelium, presence of H. pylori, and inflammation. COX-2 was detected by immunohistochemistry using the avidin-biotin complex. EGM islets were found in 14 patients (1.1%). Histological examination revealed fundic type epithelium in 58.3% of cases, H. pylori was present in 50% and chronic inflammation in 66.7%. Expression of COX-2 was negative in normal distal esophagus, normal gastric antrum and normal gastric body specimens (10 each). In contrast, EGM presented over-expression of COX-2 in 41.7% of cases and Barrett's esophagus in 90% of cases (P = 0.04 and 0.03, respectively). COX-2 immunoexpression in EGM was not related to gender, age, epithelium type, presence of inflammation or intestinal metaplasia, H. pylori infection, or any endoscopic finding. Our results demonstrate up-regulation of COX-2 in EGM, suggesting a possible malignant potential of this so-called harmless mucosa.

Cytotoxic effect of essential oil of thyme (Thymus broussonettii) on the IGR-OV1 tumor cells resistant to chemotherapy

The anti-tumor effect of the Moroccan endemic thyme (Thymus broussonettii) essential oil (EOT) was investigated in vitro using the human ovarian adenocarcinoma IGR-OV1 parental cell line OV1/P and its chemoresistant counterparts OV1/adriamycin (OV1/ADR), OV1/vincristine (OV1/VCR), and OV1/cisplatin (OV1/CDDP). All of these cell lines elicited various degrees of sensitivity to the cytotoxic effect of EOT. The IC50 values (mean ± SEM, v/v) were 0.40 ± 0.02, 0.39 ± 0.02, 0.94 ± 0.05, and 0.65 ± 0.03% for OV1/P, OV1/ADR, OV1/VCR, and OV1/CDDP, respectively. Using the DBA-2/P815 (H2d) mouse model, tumors were developed by subcutaneous grafting of tumor fragments of similar size obtained from P815 (murin mastocytoma cell line) injected in donor mouse. Interestingly, intra-tumoral injection of EOT significantly reduced solid tumor development. Indeed, by the 30th day of repeated EOT treatment, the tumor volumes of the animals were 2.00 ± 0.27, 1.35 ± 0.20, and 0.85 ± 0.18 cm³ after injection with 10, 30, or 50 µL per 72 h (six times), respectively, as opposed to 3.88 ± 0.50 cm³ for the control animals. This tumoricidal effect was associated with a marked decrease of mouse mortality. In fact, in these groups of mice, the recorded mortality by the 30th day of treatment was 30 ± 4, 18 ± 4, and 8 ± 3%, respectively, while the control animals showed 75 ± 10% of mortality. These data indicate that the EOT which contains carvacrol as the major component has an important in vitro cytotoxic activity against tumor cells resistant to chemotherapy as well as a significant antitumor effect in mice. However, our data do not distinguish between carvacrol and the other components of EOT as the active factor.

Comparison of blood neoangiogenesis and lymphatic vascularization in colorectal adenomas from patients with and without concomitant colorectal cancer

Blood and lymphatic vessel proliferation is essential for tumor growth and progression. Most colorectal carcinomas develop from adenomas (adenoma-carcinoma sequence) in a process due to accumulation of molecular genetic alterations. About 5% of adenomatous polyps are expected to become malignant, but data on the differential angiogenic patterns of these lesions in patients with and without concomitant cancer are missing. The aim of the present study is to compare the angiogenic and lymphatic patterns of adenomatous polyps from patients with and without sporadic cancer. Thirty adenomatous polyps (15 from patients with another principal malignant lesion, and 15 from patients without cancer) were submitted to immunohistochemical staining for CD105 (marker for neoangiogenesis) and D2-40 (marker for lymphatic endothelium). Microvessel density and total vascular area were determined by computer image analysis to quantify the immunostained and total areas, and to assess the number of microvessels. Adenomas from patients with carcinoma showed significantly higher values of total vascular area determined by immunostaining for CD105 (cutoff value = 4386 µm²; P = 0.019) and of lymphatic microvessel density determined by immunostaining with D2-40 (cutoff value = 11.5; P = 0.041) when compared with those from patients without cancer. The present data indicate a significant increase in blood microvascular area and in lymphatic microvascular counts in adenomas removed from patients with cancer.

Effect of cyhalothrin on Ehrlich tumor growth and macrophage activity in mice

Cyhalothrin, a pyrethroid insecticide, induces stress-like symptoms, increases c-fos immunoreactivity in the paraventricular nucleus of the hypothalamus, and decreases innate immune responses in laboratory animals. Macrophages are key elements in cellular immune responses and operate at the tumor-host interface. This study investigated the relationship among cyhalothrin effects on Ehrlich tumor growth, serum corticosterone levels and peritoneal macrophage activity in mice. Three experiments were done with 10 experimental (single gavage administration of 3.0 mg/kg cyhalothrin daily for 7 days) and 10 control (single gavage administration of 1.0 mL/kg vehicle of cyhalothrin preparation daily for 7 days) isogenic BALB/c mice in each experiment. Cyhalothrin i) increased Ehrlich ascitic tumor growth after ip administration of 5.0 x 106 tumor cells, i.e., ascitic fluid volume (control = 1.97 ± 0.39 mL and experimental = 2.71 ± 0.92 mL; P < 0.05), concentration of tumor cells/mL in the ascitic fluid (control = 111.95 ± 16.73 x 106 and experimental = 144.60 ± 33.18 x 106; P < 0.05), and total number of tumor cells in the ascitic fluid (control = 226.91 ± 43.22 x 106 and experimental = 349.40 ± 106.38 x 106; P < 0.05); ii) increased serum corticosterone levels (control = 200.0 ± 48.3 ng/mL and experimental = 420.0 ± 75.5 ng/mL; P < 0.05), and iii) decreased the intensity of macrophage phagocytosis (control = 132.3 ± 19.7 and experimental = 116.2 ± 4.6; P < 0.05) and oxidative burst (control = 173.7 ± 40.8 and experimental= 99.58 ± 41.7; P < 0.05) in vitro in the presence of Staphylococcus aureus. These data provide evidence that cyhalothrin simultaneously alters host resistance to Ehrlich tumor growth, hypothalamic-pituitary-adrenocortical (HPA) axis function, and peritoneal macrophage activity. The results are discussed in terms of data suggesting a link between stress, HPA axis activation and resistance to tumor growth.

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

Genetic variability of vascular endothelial growth factor and prognosis of head and neck cancer in a Brazilian population

Vascular endothelial growth factor (VEGF) is one of the most potent endothelial cell mitogens and plays a critical role in angiogenesis. Polymorphisms in this gene have been evaluated in patients with several types of cancer. The objectives of this study were to determine if there was an association of the -1154G/A polymorphism of the VEGF gene with head and neck cancer and the interaction of this polymorphism with lifestyle and demographic factors. Additionally, the distribution of the VEGF genotype was investigated with respect to the clinicopathological features of head and neck cancer patients. The study included 100 patients with histopathological diagnosis of head and neck squamous cell carcinoma. Patients with treated tumors were excluded. A total of 176 individuals 40 years or older were included in the control group and individuals with a family history of neoplasias were excluded. Analysis was performed after extraction of genomic DNA using the real-time PCR technique. No statistically significant differences between allelic and genotype frequencies of -1154G/A VEGF polymorphism were identified between healthy individuals and patients. The real-time PCR analyses showed a G allele frequency of 0.72 and 0.74 for patients and the control group, respectively. The A allele showed a frequency of 0.28 for head and neck cancer patients and 0.26 for the control group. However, analysis of the clinicopathological features showed a decreased frequency of the A allele polymorphism in patients with advanced (T3 and T4) tumors (OR = 0.36; 95%CI = 0.14-0.93; P = 0.0345). The -1154A allele of the VEGF gene may decrease the risk of tumor growth and be a possible biomarker for head and neck cancer. This polymorphism is associated with increased VEGF production and may have a prognostic importance.

Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells

The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-α) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-α (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-α treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-α decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-α did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-α increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

Increased expression of tissue factor and protease-activated receptor-1 does not correlate with thrombosis in human lung adenocarcinoma

A correlation between cancer and hypercoagulability has been described for more than a century. Patients with cancer are at increased risk for thrombotic complications and the clotting initiator protein, tissue factor (TF), is possibly involved in this process. Moreover, TF may promote angiogenesis and tumor growth. In addition to TF, thrombin seems to play a relevant role in tumor biology, mainly through activation of protease-activated receptor-1 (PAR-1). In the present study, we prospectively studied 39 lung adenocarcinoma patients in relation to the tumor expression levels of TF and PAR-1 and their correlation with thrombosis outcome and survival. Immunohistochemical analysis showed TF positivity in 22 patients (56%), most of them in advanced stages (III and IV). Expression of PAR-1 was found in 15 patients (39%), most of them also in advanced stages (III and IV). Remarkably, no correlation was observed between the expression of TF or PAR-1 and risk for thrombosis development. On the other hand, patients who were positive for TF or PAR-1 tended to have decreased long-term survival. We conclude that immunolocalization of either TF or PAR-1 in lung adenocarcinoma may predict a poor prognosis although lacking correlation with thrombosis outcome.

Classification of brain tumor extracts by high resolution ¹H MRS using partial least squares discriminant analysis

High resolution proton nuclear magnetic resonance spectroscopy (¹H MRS) can be used to detect biochemical changes in vitro caused by distinct pathologies. It can reveal distinct metabolic profiles of brain tumors although the accurate analysis and classification of different spectra remains a challenge. In this study, the pattern recognition method partial least squares discriminant analysis (PLS-DA) was used to classify 11.7 T ¹H MRS spectra of brain tissue extracts from patients with brain tumors into four classes (high-grade neuroglial, low-grade neuroglial, non-neuroglial, and metastasis) and a group of control brain tissue. PLS-DA revealed 9 metabolites as the most important in group differentiation: γ-aminobutyric acid, acetoacetate, alanine, creatine, glutamate/glutamine, glycine, myo-inositol, N-acetylaspartate, and choline compounds. Leave-one-out cross-validation showed that PLS-DA was efficient in group characterization. The metabolic patterns detected can be explained on the basis of previous multimodal studies of tumor metabolism and are consistent with neoplastic cell abnormalities possibly related to high turnover, resistance to apoptosis, osmotic stress and tumor tendency to use alternative energetic pathways such as glycolysis and ketogenesis.

Enhanced anti-tumor effect of a gene gun-delivered DNA vaccine encoding the human papillomavirus type 16 oncoproteins genetically fused to the herpes simplex virus glycoprotein D

Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5) expressing three proteins (E7, E6, and E5) of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id) route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose) induced a strong activation of E7-specific interferon-γ (INF-γ)-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70% of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50% of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.

A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth

Doxorubicin (DOX) was conjugated to a single-chain variable fragment (scFv) against human midkine (MK), and the conjugate (scFv-DOX) was used to target the chemotherapeutic agent to a mouse solid tumor model in which the tumor cells expressed high levels of human MK. The His-tagged recombinant scFv was expressed in bacteria, purified by metal affinity chromatography, and then conjugated to DOX using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was scFv(Dex)1.3(DOX)20. In vitro apoptosis assays showed that the scFv-DOX conjugate was more cytotoxic against MK-transfected human adenocarcinoma cells (BGC823-MK) than untransfected cells (55.3 ± 2.4 vs 22.4 ± 3.8%) for three independent experiments. Nude mice bearing BGC823-MK solid tumors received scFv-DOX or equivalent doses of scFv + DOX for 2 weeks and tumor growth was more effectively inhibited by the scFv-DOX conjugate than by scFv + DOX (51.83% inhibition vs 40.81%). Histological analysis of the tumor tissues revealed that the highest levels of DOX accumulated in tumors from mice treated with scFv-DOX and this resulted in more extensive tumor cell death than in animals treated with the equivalent dose of scFv + DOX. These results show that the scFv-DOX conjugate effectively inhibited tumor growth in vivo and suggest that antigen-specific scFv may be competent drug-carriers.

Participation of the NO/cGMP/K+ATP pathway in the antinociception induced by Walker tumor bearing in rats

Implantation of Walker 256 tumor decreases acute systemic inflammation in rats. Inflammatory hyperalgesia is one of the most important events of acute inflammation. The L-arginine/NO/cGMP/K+ATP pathway has been proposed as the mechanism of peripheral antinociception mediated by several drugs and physical exercise. The objective of this study was to investigate a possible involvement of the NO/cGMP/K+ATP pathway in antinociception induced in Walker 256 tumor-bearing male Wistar rats (180-220 g). The groups consisted of 5-6 animals. Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. Walker tumor (4th and 7th day post-implantation) reduced prostaglandin E2- (PGE2, 400 ng/paw; 50 µL; intraplantar injection) and carrageenan-induced hypernociception (500 µg/paw; 100 µL; intraplantar injection). Walker tumor-induced analgesia was reversed (99.3% for carrageenan and 77.2% for PGE2) by a selective inhibitor of nitric oxide synthase (L-NAME; 90 mg/kg, ip) and L-arginine (200 mg/kg, ip), which prevented (80% for carrageenan and 65% for PGE2) the effect of L-NAME. Treatment with the soluble guanylyl cyclase inhibitor ODQ (100% for carrageenan and 95% for PGE2; 8 µg/paw) and the ATP-sensitive K+ channel (KATP) blocker glibenclamide (87.5% for carrageenan and 100% for PGE2; 160 µg/paw) reversed the antinociceptive effect of tumor bearing in a statistically significant manner (P < 0.05). The present study confirmed an intrinsic peripheral antinociceptive effect of Walker tumor bearing in rats. This antinociceptive effect seemed to be mediated by activation of the NO/cGMP pathway followed by the opening of KATP channels.

RNAi-mediated knockdown of pituitary tumor- transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.

Tumor growth reduction is regulated at the gene level in Walker 256 tumor-bearing rats supplemented with fish oil rich in EPA and DHA

We investigated the effect of fish oil (FO) supplementation on tumor growth, cyclooxygenase 2 (COX-2), peroxisome proliferator-activated receptor gamma (PPARγ), and RelA gene and protein expression in Walker 256 tumor-bearing rats. Male Wistar rats (70 days old) were fed with regular chow (group W) or chow supplemented with 1 g/kg body weight FO daily (group WFO) until they reached 100 days of age. Both groups were then inoculated with a suspension of Walker 256 ascitic tumor cells (3×107 cells/mL). After 14 days the rats were killed, total RNA was isolated from the tumor tissue, and relative mRNA expression was measured using the 2-ΔΔCT method. FO significantly decreased tumor growth (W=13.18±1.58 vsWFO=5.40±0.88 g, P<0.05). FO supplementation also resulted in a significant decrease in COX-2 (W=100.1±1.62 vsWFO=59.39±5.53, P<0.001) and PPARγ (W=100.4±1.04vs WFO=88.22±1.46, P<0.05) protein expression. Relative mRNA expression was W=1.06±0.022 vsWFO=0.31±0.04 (P<0.001) for COX-2, W=1.08±0.02vs WFO=0.52±0.08 (P<0.001) for PPARγ, and W=1.04±0.02 vs WFO=0.82±0.04 (P<0.05) for RelA. FO reduced tumor growth by attenuating inflammatory gene expression associated with carcinogenesis.

Putative role of ischemic postconditioning in a rat model of limb ischemia and reperfusion: involvement of hypoxia-inducible factor-1α expression

Hypoxia-inducible factor-1α (HIF-1α) is one of the most potent angiogenic growth factors. It improves angiogenesis and tissue perfusion in ischemic skeletal muscle. In the present study, we tested the hypothesis that ischemic postconditioning is effective for salvaging ischemic skeletal muscle resulting from limb ischemia-reperfusion injury, and that the mechanism involves expression of HIF-1α. Wistar rats were randomly divided into three groups (n=36 each): sham-operated (group S), hindlimb ischemia-reperfusion (group IR), and ischemic postconditioning (group IPO). Each group was divided into subgroups (n=6) according to reperfusion time: immediate (0 h, T0), 1 h (T1), 3 h (T3), 6 h (T6), 12 h (T12), and 24 h (T24). In the IPO group, three cycles of 30-s reperfusion and 30-s femoral aortic reocclusion were carried out before reperfusion. At all reperfusion times (T0-T24), serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities, as well as interleukin (IL)-6, IL-10, and tumor necrosis factor-α (TNF-α) concentrations, were measured in rats after they were killed. Histological and immunohistochemical methods were used to assess the skeletal muscle damage and HIF-1α expression in skeletal muscle ischemia. In groups IR and IPO, serum LDH and CK activities and TNF-α, IL-6, and IL-10 concentrations were all significantly increased compared to group S, and HIF-1α expression was up-regulated (P<0.05 or P<0.01). In group IPO, serum LDH and CK activities and TNF-α and IL-6 concentrations were significantly decreased, IL-10 concentration was increased, HlF-1α expression was down-regulated (P<0.05 or P<0.01), and the pathological changes were reduced compared to group IR. The present study suggests that ischemic postconditioning can reduce skeletal muscle damage caused by limb ischemia-reperfusion and that its mechanisms may be related to the involvement of HlF-1α in the limb ischemia-reperfusion injury-triggered inflammatory response.