Relationship between survival and edema in malignant gliomas: role of vascular endothelial growth factor and neuronal pentraxin 2.

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent mediator of vascular permeability. VEGF inhibition reduces edema and tumor burden in some patients with malignant glioma, whereas others show no response. The role of VEGF expression in edema production and the relationship to survival is not well understood. EXPERIMENTAL DESIGN: Using DNA microarray analysis, we examined VEGF and related gene expression in 71 newly diagnosed malignant gliomas and analyzed the relationship to edema and survival. RESULTS AND CONCLUSIONS: VEGF expression was predictive of survival in tumors with little or no edema [Cox proportional hazard model, 6.88; 95% confidence interval (95% CI), 2.61-18.1; P<0.0001], but not in tumors with extensive edema. The expression of several proangiogenic genes, including adrenomedullin (correlation coefficient, 0.80), hypoxia-inducible factor-1A (0.51), and angiopoietin-2 (0.44), was correlated with VEGF expression (all with P<0.0001), whereas that of several antiangiogenic genes was inversely correlated. The expression of six genes was increased greater than 3-fold in edematous versus nonedematous tumors in the absence of increased VEGF expression. The most increased, neuronal pentraxin 2 (NPTX2, 7-fold change), was predictive of survival in tumors with the highest levels of edema, in contrast to VEGF (hazard ratio, 2.73; 95% CI, 1.49-5.02; P=0.049). NPTX2 was tightly correlated with expression of the water channel aquaporin-3 (0.74, P<0.0001). These results suggest that there are both VEGF-dependent and VEGF-independent pathways of edema production in gliomas and may explain why edema is not reduced in some patients following anti-VEGF treatment.     

The role of cyclooxygenase-2 in breast cancer: review

There is a growing body of evidence that COX-2 expression s a fundamental step in breast cancer pathogenesis acting through prostaglandin-dependent and independent mechanisms. Epidemiological studies suggest that NSAIDs confer a moderate degree of benefit against breast cancer. However further work is required to establish how this enzyme system can be best manipulated for therapeutic benefit.     

Increased expression of interleukin-1alpha and cyclooxygenase-2 in human gastric cancer: a possible role in tumor progression

BACKGROUND: Cyclooxygenase (COX)-2 mRNA and protein expression have been found to be frequently up-regulated in human gastric cancers and cell lines. COX-2 is the inducible form of COX, and interleukin (IL)-1alpha may be one of the stimulators of COX-2. MATERIALS AND METHODS: The relationship between IL-1alpha and COX-2 expression was examined in human gastric cancer tissues and cell lines. RESULTS: IL-1alpha mRNA was expressed in 19 out of 32 human gastric cancer specimens (60%), while it was not expressed in any of the paired normal gastric mucosa specimens. The incidence of IL-1alpha mRNA expression was significantly higher in patients with pT2-pT4 tumors than in those with pT1 tumors (86% vs. 39%, p<0.05). IL-1alpha mRNA was expressed in 94% of COX-2-positive tumors, while it was expressed in only 25% of COX-2-negative tumors (p<0.0001). When IL-1alpha was added to the medium of gastric cancer cell lines (MKN28 and MKN45), it enhanced the expression of IL-1alpha itself and COX-2 mRNA in both cell lines. Exogenous IL-1alpha stimulated cancer cell growth in both cell lines, while such growth stimulation was suppressed by anti-IL-1alpha antibody or IL-1 receptor antagonist. IL-1alpha-stimulated cell growth was also suppressed by anti-COX-2 antibody. CONCLUSION: Our data demonstrated that IL-1alpha and COX-2 mRNA were frequently co-expressed in human gastric cancer tissues, and suggested that the IL-1alpha-COX-2 pathway might be involved in tumor progression by regulating cancer cell proliferation.     

A novel role for cyclooxygenase-2 in regulating vascular channel formation by human breast cancer cells

Introduction

Cyclo-oxygenase (COX)-2 expression correlates directly with highly aggressive and metastatic breast cancer, but the mechanism underlying this correlation remains obscure. We hypothesized that invasive human breast cancer cells that over-express COX-2 have the unique ability to differentiate into extracellular-matrix-rich vascular channels, also known as vasculogenic mimicry. Vascular channels have been associated with angiogenesis without involvement of endothelial cells, and may serve as another mechanism by which tumor cells obtain nutrients to survive, especially in less vascularized regions of the tumor.

Methods

To determine whether COX-2 regulates vascular channel formation, we assessed whether treatment with celecoxib (a selective COX-2 inhibitor) or silencing COX-2 synthesis by siRNA inhibits vascular channel formation by breast cancer cell lines. Cell lines were selected based on their invasive potential and COX-2 expression. Additionally, gene expression analysis was performed to identify candidate genes involved in COX-2-induced vascular channel formation. Finally, vascular channels were analyzed in surgically resected human breast cancer specimens that expressed varying levels of COX-2.

Results

We found that invasive human breast cancer cells that over-express COX-2 develop vascular channels when plated on three-dimensional matigel cultures, whereas non-invasive cell lines that express low levels of COX-2 did not develop such channels. Similarly, we identified vascular channels in high-grade invasive ductal carcinoma of the breast over-expressing COX-2, but not in low-grade breast tumors. Vascular channel formation was significantly suppressed when cells were treated with celecoxib or COX-2 siRNA. Inhibition of channel formation was abrogated by addition of exogenous prostaglandin E₂. In vitro results were corroborated in vivo in tumor-bearing mice treated with celecoxib. Using gene expression profiling, we identified several genes in the angiogenic and survival pathways that are engaged in vascular channel formation.

Conclusion

Antivascular therapies targeting tumor cell vasculogenic mimicry may be an effective approach to the treatment of patients with highly metastatic breast cancer.     

Melanin as a component of cerebral gliomas: the melanotic cerebral ependymoma.

Even though melanin is found in certain central nervous system neurons and leptomeningeal melanocytes, gliomas rarely possess melanin. A few rare lethal melanotic medulloblastomas found typically in the cerebellar vermis of children have been described, but neuroglial cells have not been observed to contain this pigment. Since glial elements and the melanotic pigmented layer of the retina are derived from the same ciliated epithelium of the embryonic neural tube, recognition of melanin in a cerebral glioma was predictable. Indeed, melanin was observed in a cystic cerebral ependymal glioma from a 30-year-old woman, supporting the potential of glial cells to produce melanin.     

Prognostic role of cyclooxygenase-2 expression in esophageal carcinoma

Cyclooxygenase-2 (COX-2) is involved in esophageal carcinogenesis, but its clinical significance is not clarified yet. The association of several clinicopathological parameters with the immunohistochemical expression of COX-2 was analyzed in paraffin-embedded esophageal cancer specimens. In adenocarcinomas, COX-2 upregulation showed to be a late event and in a multivariate analysis it was associated independently with a reduced survival after surgery compared with low COX-2 expressed tumors (P=0.02). Although, the expression was extensively high in well-differentiated parts of squamous cell carcinoma, it was not related with a poor outcome. This study supports further investigation of selective COX-2 inhibitors in these tumors.     

A role for cyclooxygenase-2 in ultraviolet light-induced skin carcinogenesis

Nonmelanoma skin cancer is the most prevalent cancer in the United States and its incidence is on the rise. These cancers generally arise on sun-exposed areas of the body and the ultraviolet (UV) B spectrum of sunlight has been clearly identified as the major carcinogen responsible for skin cancer development. Besides inducing DNA damage directly, UV exposure of the skin induces the expression of the enzyme cyclooxygenase-2 (COX-2), which catalyzes the first step in the conversion of arachidonic acid to prostaglandins, the primary product in skin being prostaglandin E(2) (PGE(2)). COX-2 has been shown to be overexpressed in premalignant lesions as well as in nonmelanoma skin cancers in both humans and mice chronically exposed to UV. Through the use of COX-2-selective inhibitors and COX-2 knockout mice, it has been shown that UV-induced COX-2 expression plays a major role in UV-induced PGE(2) production, inflammation, edema, keratinocyte proliferation, epidermal hyperplasia, and generation of a pro-oxidant state leading to oxidative DNA damage. Chronic exposure to UV leads to chronic up-regulation of COX-2 expression and chronic inflammation along with the accumulation of DNA damage and mutations, all of which combine to induce malignant changes in epidermal keratinocytes and skin cancers. Both inhibition of COX-2 activity and reduction in COX-2 expression by genetic manipulations significantly reduce, while overexpression of COX-2 in transgenic mice significantly increases UV-induced skin carcinogenesis. Together these studies demonstrate that COX-2 expression/activity is critical to the development of UV-related nonmelanoma skin cancers.     

Osteopontin: possible role in prostate cancer progression.

Human prostate cancer has the propensity to metastasize to the bone where reciprocal cellular interactions between prostate cancer and bone cells are known to occur. Osteopontin (OPN), a noncollagenous bone extracellular matrix, is a secreted adhesive glycoprotein with a functional RGD cell-binding domain that interacts with the alpha(v)beta3 cell surface integrin heterodimer. OPN has been associated with malignant transformation as well as being ligand to the CD44 receptor. Polyclonal antibodies to human OPN (hOPN) were prepared, and specificity was shown by preabsorption with recombinant hOPN. The stimulatory effect of hOPN protein and the inhibitory effect of hOPN antibody on human prostate cancer cell lines LNCaP and C4-2 were assessed by induction or inhibition of anchorage-independent growth, respectively. Expression of hOPN mRNA in prostate cancer cell lines and human prostate cancer tissue specimens were measured by mRNA blot analysis. Protein expression was assessed by immunohistochemistry in human prostate cancer specimens and by Western blot analysis in prostate cancer cell lines. hOPN stimulated anchorage-independent growth of the human prostate cancer cell lines LNCaP and C4-2 in vitro. Antibodies to hOPN inhibited the growth-stimulatory effect by endogenous OPN, which can be overcome by the addition of exogenous hOPN. hOPN mRNA and protein are expressed in human prostate cancer cell lines in vitro and in clinical human prostate cancer specimens. These findings taken together suggest that OPN may act as a paracrine and autocrine mediator of prostate cancer growth and progression.     

Increased cyclooxygenase-2 (COX-2): a potential role in the pathogenesis of lymphoma

B cell lymphomas are a diverse group of clinicopathologic diseases with an increasing incidence. As with other malignancies, the accumulation of genetic abnormalities are required for malignant transformation of human lymphocytes. Cyclooxygenase-2 (COX-2) is a key biosynthetic enzyme in prostaglandin synthesis and has been implicated in the pathogenesis of numerous malignancies including colon, breast, and lung cancer. There is little data on the potential role of COX-2 in lymphoma pathogenesis. In this study, several B lymphoma cell lines and primary B cells obtained from normal volunteer controls were examined for COX-2 protein expression. Immunoblot analysis demonstrated between an approximately 2.2-4.3-fold increase in COX-2 protein expression relative to primary B cells in all lymphoma cell lines examined. Increased COX-2 phosphorylation was found in the BJAB, BL41, and Raji cells whereas the levels in Daudi, Namalwa, and Ramos did not differ from that of primary B cells. Treatment with 25-100 microM celecoxib (CEL) resulted in decreased proliferation as measured by [3H]thymidine in all cell lines examined, and the effect was dose-dependent, and not significantly enhanced by chlorambucil (CHL). The effect of COX-2 inhibition on apoptosis in lymphoma cells was examined and revealed apoptotic induction of greater than 85% in all cell lines examined at 50 microM celecoxib. The pro-apoptotic effect was dose-dependent, and was not significantly enhanced by chlorambucil. Examination of apoptosis-related proteins by immunoblot analysis revealed levels of BCL-2, BCL-X(L), and Bax to be unaffected by celecoxib. In contrast, levels of Akt, MCL-1, and phosphorylated SAP-kinase were all decreased after incubation with 50 microM celecoxib. These findings suggest that increased COX-2 expression and activity, contributes to the pathogenesis of B cell lymphomas and point to a possible role for COX-2 inhibition in their treatment.     

Expression of cyclooxygenase-2 and its correlation with vasogenic brain edema in human intracranial meningiomas

COX-2 expression was evalueted in intracranial meningiomas, relating this molecule to grade, vasculature, VEGF and brain edema. Fifty-six tumors were evaluated for COX-2 and VEGF expression and for microvessel density. In 34/56 cases, the edema was evaluated by CT scan. COX-2 was detected in 46/56 meningiomas (82.14%), and it resulted as being related to histologic grade (t-test: p = 0.006) and to edema (t-test: p = 0.002). No statistical association between COX-2 and VEGF or MVD was found. In conclusion, COX-2 seems to be related to the more aggressive meningiomas and, somehow, to the development of meningioma-associated brain edema.     

Regional measurements of blood-to-tissue transport in experimental RG-2 rat gliomas

Regional measurements of blood-to-tissue transport were performed in transplanted RG-2 rat gliomas using [alpha- 14C]aminoisobutyric acid (AIB), quantitative autoradiography, and equations to express a unidirectional transfer constant. Thirty-eight intracranial tumors in ten rats were analyzed according to location; 23 intraparenchymal tumors, eight meningeal tumors, six fourth-ventricular tumors, and one third-ventricular tumor were studied. Except for the small third-ventricular tumor, the transfer constant (K) for AIB was similar in all groups and ranged from 0.031 to 0.038 ml/g/min. Within individual tumors, regional variation of K was also small, although some local variation could be correlated with histological features. The K for AIB decreased in brain around tumor and, at a distance of 300 microns from tumor edge, had returned to values similar to those of normal cortex (0.002 ml/g/min). An average extraction fraction (E) of 0.09 was calculated for AIB in the RG-2 tumors. The low E suggests that delivery of water-soluble chemotherapeutic drugs to RG-2 tumors should be limited more by capillary permeability or surface area than by blood flow. RG-2 is an ideal experimental tumor with which to test drug delivery and the methods that attempt to increase drug delivery in brain tumors.     

Regional measurements of blood flow in experimental RG-2 rat gliomas

Regional measurements of blood flow (F) were performed in transplanted intracerebral RG-2 rat gliomas using [14C]iodoantipyrine, Kety-Schmidt blood flow equations, and quantitative autoradiography. Twenty-nine intracranial tumors in ten rats were analyzed by location; 18 intraparenchymal, seven meningeal, two third-ventricular, and two fourth-ventricular tumors were studied. For all tumors, averaged mean F was 91 +/- 33 (S.D.) ml/hg/min. In all but one tumor, mean F was intermediate between normal cortex and corpus callosum values. There was moderate regional variation: averaged mean F was lower in tumor center (78 +/- 47 ml/hg/min) than in tumor periphery (93 +/- 30 ml/hg/min). Within individual tumors, F showed moderate variation which correlated to some extent with histological features; a regional F of less than 10 ml/hg/min was observed in only one tumor within an area of necrosis. F in regions of brain immediately surrounding the tumor was higher than in tumor periphery. Blood flow to RG-2 tumors seems unlikely to limit drug delivery any more than to normal brain, and the consistent levels from tumor to tumor and within individual tumors make the RG-2 model an excellent one with which to study drug delivery in experimental brain tumors.     

Expression of cyclooxygenase-2 in Hodgkin's lymphoma: its role in cell proliferation and angiogenesis

Although many studies have revealed the association between cyclooxygenase-2 (COX-2) and carcinogenesis, the association between COX-2 and Hodgkin's lymphoma (HL) remains unknown. We examined the immunohistochemical expression of COX-2, p53, bcl-2, and Ki-67 in 33 patients with HL, and counted microvessels stained with CD34. Hodgkin and Reed - Sternberg (HRS) cells with COX-2 expression were scored as 0 = no staining; 1 = <25% of cells staining; 2 = 25-49%; 3 = 50-75%; and 4 = > or =75%. COX-2 expression was observed in 15 cases of classical HL. Nevertheless, neither accumulation of p53 nor bcl-2 expression was associated with COX-2 expression. The percentage of Ki-67 positive-HRS cells and microvessel density in COX-2 score groups 2-4 were significantly higher than those in score group 0, respectively. We show that COX-2 expression is associated with cell proliferation and angiogenesis in HL. These findings suggest that COX-2 may be a target for therapy in HL.     

A possible role of cytokines in the formation of peritoneal dissemination

The earliest event in the formation of peritoneal dissemination is considered through the process of the attachment of intraperitoneal free cancer cells to the submesothelial basement membrane, exposed after contraction of mesothelial cells. We studied the mechanisms of the contraction of mesothelial cells using a. highly metastatic sell line (MKN-45-P) to the peritoneum. Four hours after intraperitoneal inoculation of MKN-45-P, mouse mesothelial cells began to contract, and submesothelial basement membrane was widely exposed after 24 h. The same changes developed four hours after i.p. injection of IL-6, TNF-alpha and IL-8, and were most prominently observed in mice treated with IL-8. However, no significant changes were observed after treatment of HGF, EGF and TGF-beta. Furthermore, IL-1 alpha, IL-6, IL-8, TNF and EGF increased the number of intercellular gaps of a human mesothelial cell monolayer, which was incubated on Matrigel coated dishes. Normal mesothelial cells form a contiguous monolayer of closely apposed polygonal cells, each of which had prominent and peripheral bands of F-actin. After incubation with IL-1 alpha, IL-6, IL-8, TNF and EGF, peripheral actin bands became indistinct and the central stress fibers became numerous. However, no significant changes were found in mesothelial cells, which were treated with TGF-beta and HGF. In addition, the number of attached MKN-45-P cells on a mesothelial cell monolayer after treatment of IL-1 alpha (0.1-1 ng/ml), IL-8 (10-100 ng/ml), and TNF-alpha (100 ng/ml) was significantly larger than that of control and TGF-beta significantly reduced the number of attached cells. Concentration of IL-8 in the serum-free medium of MKN-45-P cells was high (3.4 ng/ml), but IL-1 alpha, IL-6, TNF-alpha, TGF-beta, EGF and HGF could not be detected. None of these cytokines were detected in the conditioning medium of human mesothelial cells. Based on these results, mesothelial cell contraction may be mediated by IL-1 alpha, IL-6, IL-8, TNF-alpha, and EGF, and these cytokines may be produced from cancer cells and/or intraperitoneal inflammatory cells. In contrast, TGF-beta have an inhibitory effect on the mesothelial cell contraction and attachment of cancer cells to a mesothelial monolayer. The attachment of free cancer cells on the peritoneum may be controlled with these cytokines.     

Variations in catechol O-methyltransferase activity in rodent tissues: possible role in estrogen carcinogenicity

Catechol-O-methyltransferase (COMT) [EC 2.1.1.6 [EC] ] is a ubiquitouscytosolic enzyme which has a pertinent role in the inactivationof both natural and synthetic catechol estrogens in mammaliantissues. We have compared the COMT activity in mouse, hamsterand rat kidney, liver and red blood cells and examined the kineticcharacteristics of this enzyme in the latter two species usingvarious catechol estrogens as substrates. Results presentedhere indicate that the ratios of COMT activity in the kidneyversus the liver of the rat and mouse are nearly identical,0.48–0.52, whereas there is a 29-fold ratio between thelevel of COMT activity in these two tissues in the hamster.In red blood cells, the level of COMT activity is 4- and 12-foldlower in the hamster compared to mouse and rat, respectively.When the kinetic characteristics of this enzyme were assessedin the hamster and rat kidney and liver, except for 2-hydroxymoxestrolwhich had an apparent K_m value of 15–48 µM, theother catechol estrogen substrates exhibited K_m values rangingfrom 1–10 µM. Generally, the V_max values were markedlyhigher in the rat kidney and liver than those observed in correspondinghamster tissues. The significantly lower COMT activity in thehamster liver and red blood cells suggests that under chronicestrogen treatment at high doses, the concentration of catecholestrogens in these tissues may exceed the capacity of COMT toeffectively catalyse their O-methylation into inactive metabolites.The resulting accumulation of catechol estrogens may contributeto the estrogen carcinogenicity observed in the hamster liverand kidney. Additionally, when 2-hydroxy-estrone was used asa substrate, the estrogen-induced renal carcinoma exhibitedonly 8.6% of the COMT activity found in the normal kidney.     

Prognostic role of the ratio between cyclooxygenase-2 in tumor and stroma compartments in cervical cancer.

PURPOSE: The aim of this study was to analyze the clinical role of cyclooxygenase (COX)-2 in a large series of 175 cervical cancer patients. EXPERIMENTAL DESIGN: Immunohistochemistry was performed on paraffin-embedded sections by using rabbit antiserum against COX-2. The tumor:stroma (T/S) ratio of COX-2 expression was used to define the overall COX-2 content in the tumor. RESULTS: The T/S COX-2 ratio values ranged from 0.03 to 48.2 (mean +/- SE, 3.7 +/- 0.5). A total of 95 of 175 patients (54.3%) were scored as having a high (>1) T/S COX-2 ratio. In locally advanced cervical cancer patients who underwent neoadjuvant treatment, the percentage of cases showing a high T/S COX-2 ratio was greater in patients who did not respond to treatment (26 of 29 patients, 89.7%) than in patients with a partial (32 of 50 patients, 64.0%) or complete (19 of 44 patients, 43.2%) response (P = 0.0003). When logistic regression was applied, International Federation of Gynecologists and Obstetricians (FIGO) stage (chi(2) = 11.3; P = 0.0008) and T/S COX-2 ratio (chi(2) = 5.3; P = 0.021) retained an independent role in predicting a poor chance of response. Cases with a high T/S COX-2 ratio had a shorter overall survival (OS) [2-year OS, 61%(95% confidence interval 750-83)] than cases with a low T/S COX-2 ratio (2-year OS, 90%; 95% confidence interval, 81-99; P = 0.0001). In multivariate analysis, the status of T/S COX-2 IDV ratio, together with advanced stage, retained an independent negative prognostic role for OS. CONCLUSIONS: The assessment of COX-2 status in both tumor and stroma compartment could provide valuable information to identify cervical cancer patients endowed with a very poor chance of response to neoadjuvant treatment and unfavorable prognosis.