Alice C. Levine

Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo.

PURPOSE Cyclooxygenase (COX)-2, an inducible enzyme which catalyzes the formation of prostaglandins from arachidonic acid, is expressed in prostate cancer specimens and cell lines. To evaluate the in vivo efficacy of a COX-2 inhibitor in prostate cancer, NS398 was administered to mice inoculated with the PC-3 human prostate cancer cell line. MATERIALS AND METHODS A total of 28 male nude mice were inoculated subcutaneously with 1 million PC-3 cells. Tumors were palpable in all 28 animals 1 week after inoculation and mice were randomized to receive either vehicle (control) or NS398, 3 mg./kg. body weight, intraperitoneally three times weekly for 9 weeks. Tumors were measured at weekly intervals. After a 10-week experimental period, mice were euthanized and tumors were immuno- histochemically assayed for proliferation (PCNA), apoptosis (TUNEL) and microvessel density (MVD) (Factor-VIII-related antigen). Tumor VEGF content was assayed by Western blotting. RESULTS NS398 induced a sustained inhibition of PC-3 tumor cell growth and a regression of existing tumors. Average tumor surface area from control mice was 285 mm.2 as compared with 22 mm.2 from treated mice (93% inhibition, p <0.001). Immunohistochemical analysis revealed that NS398 had no effect on proliferation (PCNA), but induced apoptosis (TUNEL) and decreased MVD (angiogenesis). VEGF expression was also significantly down regulated in the NS398-treated tumors. CONCLUSIONS These results demonstrate that a selective COX-2 inhibitor suppresses PC-3 cell tumor growth in vivo. Tumor growth suppression is achieved by a combination of direct induction of tumor cell apoptosis and down regulation of tumor VEGF with decreased angiogenesis

EXPRESSION OF CYCLOOXYGENASE-1 AND CYCLOOXYGENASE-2 IN THE HUMAN PROSTATE

OBJECTIVES To determine the cell-specific expression of the two major isoforms of cyclooxygenase (COX-1 and COX-2) in human noncancerous and cancerous prostatic tissues. METHODS Thirty-one specimens of prostate carcinoma (CaP) and 10 specimens of benign prostatic hyperplasia (BPH) were stained with mouse antihuman COX-1 and COX-2 monoclonal antibodies. The stained specimens were analyzed both descriptively and in a semiquantitative manner by assigning an immunoreactive intensity score (0 to 4). The averaged results were compared for different histologic tissue types, including luminal and basal epithelium of BPH, the peripheral zone, high-grade prostatic intraepithelial neoplasia (PIN), and CaP of varying Gleason grades. RESULTS COX-1 expression in noncancerous prostatic tissue was seen predominantly in the basal epithelial cells of BPH (90% positive staining). COX-1 expression was minimal in noncancerous luminal epithelial cells (0% to 10%) but was upregulated in CaP (63% of CaP specimens). Strong COX-2 expression was demonstrated in the smooth muscle cells of the prostate. COX-2 was also expressed in the basal epithelial cells (60% BPH, 94% peripheral zone, 75% PIN). Luminal epithelial cells derived from BPH, the peripheral zone, and PIN expressed COX-2 in 0%, 26%, and 86% of samples, respectively. COX-2 expression in CaP was intense and uniform, with 87% of samples demonstrating immunoreactivity. CONCLUSIONS The results of the present study indicate that expression of both COX-1 and COX-2 in human CaP is increased. COX-2 expression is also increased in the basal and luminal epithelial cells of PIN. These data indicate that COX-1 and COX-2 (and/or their prostaglandin products) may play a role in the malignant transformation of the prostate.

Prostaglandin E2 Induces Hypoxia-inducible Factor-1α Stabilization and Nuclear Localization in a Human Prostate Cancer Cell Line

Hypoxia-induced up-regulation of vascular endothelial growth factor (VEGF) expression is a critical event leading to tumor neovascularization. Hypoxia stimulates hypoxia-inducible factor-1α (HIF-1α), a transcriptional activator of VEGF. Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, is also induced by hypoxia. We reported previously that COX-2 inhibition prevents hypoxic up-regulation of VEGF in human prostate cancer cells and that prostaglandin E2 (PGE2) restores hypoxic effects on VEGF. We hypothesized that PGE2 mediates hypoxic effects on VEGF by modulating HIF-1α expression. Addition of PGE2 to PC-3ML human prostate cancer cells had no effect on HIF-1α mRNA levels. However, PGE2 significantly increased HIF-1α protein levels, particularly in the nucleus. This effect of PGE2 largely results from the promotion of HIF-1α translocation from the cytosol to the nucleus. PGE2 addition to PC-3 ML cells transfected with a GFP-HIF-1α vector induced a time-dependent nuclear accumulation of the HIF-1α protein. Two selective COX-2 inhibitors, meloxicam and NS398, decreased HIF-1α levels and nuclear localization, under both normoxic and hypoxic conditions. Of several prostaglandins tested, only PGE2 reversed the effects of a COX-2 inhibitor in hypoxic cells. Finally, PGE2 effects on HIF-1α were specifically inhibited by PD98059 (a MAPK inhibitor). These data demonstrate that PGE2 production via COX-2-catalyzed pathway plays a critical role in HIF-1α regulation by hypoxia and imply that COX-2 inhibitors can prevent hypoxic induction of HIF-mediated gene transcription in cancer cells.

The role of cyclooxygenase-2 in prostate cancer.

Cyclooxygenase-2 (COX-2) is the inducible isozyme of COX, a key enzyme in the conversion of arachidonic acid to prostaglandins and other eicosanoids. COX-2 is highly expressed in a number of human cancers and cancer cell lines, including prostate cancer. We studied the immunohistochemical expression of COX-2 in the human prostate gland. The enzyme is strongly expressed in smooth muscle cells of both the normal and cancerous prostate. Its expression in noncancerous epithelial cells is limited to the basal cell layer. In prostatic inflammation, luminal epithelial cells surrounded by lymphocytes are induced to express the enzyme. COX-2 is expressed in the epithelial cells of high-grade prostatic intraepithelial neoplasia and cancer. We have demonstrated that treatment of human prostate-cancer cell lines with a selective COX-2 inhibitor induces apoptosis both in vitro and in vivo. The in vivo results also indicate that the COX-2 inhibitor decreases tumor microvessel density and angiogenesis. COX-2 inhibitors can prevent the hypoxic upregulation of a potent angiogenic factor, vascular endothelial growth factor. These results indicate that COX-2 inhibitors may, therefore, serve as effective chemopreventive and therapeutic agents in cancer of the prostate.

Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line.

Upregulation of vascular endothelial growth factor (VEGF) expression induced by hypoxia is crucial event leading to neovascularization. Cyclooxygenase-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, has been demonstrated to be induced by hypoxia and play role in angiogenesis and metastasis. To investigate the potential effect of COX-2 on hypoxia-induced VEGF expression in prostate cancer. We examined the relationship between COX-2 expression and VEGF induction in response to cobalt chloride (CoCl2)-simulated hypoxia in three human prostate cancer cell lines with differing biological phenotypes. Northern blotting and ELISA revealed that all three tested cell lines constitutively expressed VEGF mRNA, and secreted VEGF protein to different degrees (LNCaP > PC-3 > PC3ML). However, these cell lines differed in the ability to produce VEGF in the presence of CoCl2-simulated hypoxia. CoCl2 treatment resulted in 40% and 75% increases in VEGF mRNA, and 50% and 95% in protein secretion by LNCaP and PC-3 cell lines, respectively. In contrast, PC-3ML cell line, a PC-3 subline with highly invasive, metastatic phenotype, exhibits a dramatic upregulation of VEGF, 5.6-fold in mRNA and 6.3-fold in protein secretion after treatment with CoCl2. The upregulation of VEGF in PC-3ML cells is accompanied by a persistent induction of COX-2 mRNA (6.5-fold) and protein (5-fold). Whereas COX-2 expression is only transiently induced in PC-3 cells and not affected by CoCl2 in LNCaP cells. Moreover, the increases in VEGF mRNA and protein secretion induced by CoCl2 in PC-3ML cells were significantly suppressed following exposure to NS398, a selective COX-2 inhibitor. Finally, the effect of COX-2 inhibition on CoCl2-induced VEGF production was reversed by the treatment with exogenous PGE2. Our data demonstrate that VEGF induction by cobalt chloride-simulated hypoxia is maintained by a concomitant, persistent induction of COX-2 expression and sustained elevation of PGE2 synthesis in a human metastatic prostate cancer cell line, and suggest that COX-2 activity, reflected by PGE2 production, is involved in hypoxia-induced VEGF expression, and thus, modulates prostatic tumor angiogenesis.

Targeted inhibition of the KLF6 splice variant, KLF6 SV1, suppresses prostate cancer cell growth and spread.

Prostate cancer is a leading cause of cancer death in men. Risk prognostication, treatment stratification, and the development of rational therapeutic strategies lag because the molecular mechanisms underlying the initiation and progression from primary to metastatic disease are unknown. Multiple lines of evidence now suggest that KLF6 is a key prostate cancer tumor suppressor gene including loss and/or mutation in prostate cancer tumors and cell lines and decreased KLF6 expression levels in recurrent prostate cancer samples. Most recently, we identified a common KLF6 germ line single nucleotide polymorphism that is associated with an increased relative risk of prostate cancer and the increased production of three alternatively spliced, dominant-negative KLF6 isoforms. Here we show that although wild-type KLF6 (wtKLF6) acts as a classic tumor suppressor, the single nucleotide polymorphism-increased splice isoform, KLF6 SV1, displays a markedly opposite effect on cell proliferation, colony formation, and invasion. In addition, whereas wtKLF6 knockdown increases tumor growth in nude mice >2-fold, short interfering RNA-mediated KLF6 SV1 inhibition reduces growth by approximately 50% and decreases the expression of a number of growth- and angiogenesis-related proteins. Together, these findings begin to highlight a dynamic and functional antagonism between wtKLF6 and its splice variant KLF6 SV1 in tumor growth and dissemination.

Effect of Testosterone Administration on Sexual Behavior and Mood in Men with Erectile Dysfunction

This double-blind placebo controlled, cross-over study was carried out to assess the effect of testosterone administration on sexual behavior mood, and psychological symptoms in healthy men with erectile dysfunction. Biweekly injections of 200 mg of testosterone enanthate were given over a period of 6 weeks separated by a washout period of 4 weeks. Blood samples for hormonal assessment, behavioral and psychological ratings were obtained prior to each injection. Luteinizing hormone remained significantly depressed but circulating testosterone had returned to baseline levels by 2 weeks following each hormonal injection. The ejaculatory frequency during the testosterone phase was statistically higher than during the placebo phase. There were marked, although statistically nonsignificant, increases in median frequency of reported sexual desire, masturbation, sexual experiences with partner, and sleep erections during the testosterone period. Testosterone did not have demonstrable effects on ratings of penile rigidity and sexual satisfaction. Mood variables and psychological symptoms did not change following hormonal administration. Results suggest that androgen administration to eugonadal men with erectile dysfunction may activate their sexual behavior without enhancing erectile capacity and without effects on mood and psychological symptoms.

Interactive Effect of Interleukin‐6 and Prostaglandin E2 on Osteoclastogenesis via the OPG/RANKL/RANK System

Abstract:  The OPG/RANKL/RANK system regulates osteoclastogenesis. Both cyclooxygenase‐2 (COX‐2)/prostaglandin E2 (PGE2) and interleukin‐6 (IL‐6) are reported to induce osteoclast differentiation. The mechanisms underlying these signaling pathways on the OPG/RANKL/RANK system are not fully understood. We herein demonstrate that COX‐2 and PGE2 stimulated osteoclastogenesis through inhibition of OPG secretion, stimulation of RANKL production by osteoblasts, and upregulation of RANK expression in osteoclasts. PGE2 also stimulated IL‐6 production, and IL‐6, in turn, increased PGE2 secretion, COX‐2, and EP4/EP2 expression in bone cells. These findings provide evidence of interactive effect of PGE2 and IL‐6 signaling pathways in osteoclastogenesis via effect on the OPG/RANKL/RANK system.

Roles of KLF6 and KLF6-SV1 in Ovarian Cancer Progression and Intraperitoneal Dissemination

Purpose: We investigated the role of the KLF6 tumor suppressor gene and its alternatively spliced isoform KLF6-SV1 in epithelial ovarian cancer (EOC). Experimental Design: We first analyzed tumors from 68 females with EOC for KLF6 gene inactivation using fluorescent loss of heterozygosity (LOH) analysis and direct DNA sequencing and then defined changes in KLF6 and KLF6-SV1 expression levels by quantitative real-time PCR. We then directly tested the effect of KLF6 and KLF6-SV1 inhibition in SKOV-3 stable cell lines on cellular invasion and proliferation in culture and tumor growth, i.p. dissemination, ascites production, and angiogenesis in vivo using BALB/c nu/nu mice. All statistical tests were two sided. Results: LOH was present in 59% of samples in a cell type–specific manner, highest in serous (72%) and endometrioid (75%) subtypes, but absent in clear cell tumors. LOH was significantly correlated with tumor stage and grade. In addition, KLF6 expression was decreased in tumors when compared with ovarian surface epithelial cells. In contrast, KLF6-SV1 expression was increased ∼5-fold and was associated with increased tumor grade regardless of LOH status. Consistent with these findings, KLF6 silencing increased cellular and tumor growth, angiogenesis, and vascular endothelial growth factor expression, i.p. dissemination, and ascites production. Conversely, KLF6-SV1 down-regulation decreased cell proliferation and invasion and completely suppressed in vivo tumor formation. Conclusion: Our results show that KLF6 and KLF6-SV1 are associated with key clinical features of EOC and suggest that their therapeutic targeting may alter ovarian cancer growth, progression, and dissemination.

ACTH protects against glucocorticoid-induced osteonecrosis of bone.

We report that adrenocorticotropic hormone (ACTH) protects against osteonecrosis of the femoral head induced by depot methylprednisolone acetate (depomedrol). This therapeutic response likely arises from enhanced osteoblastic support and the stimulation of VEGF by ACTH; the latter is largely responsible for maintaining the fine vascular network that surrounds highly remodeling bone. We suggest examining the efficacy of ACTH in preventing human osteonecrosis, a devastating complication of glucocorticoid therapy.