Lauri J. Miller

Vascular Endothelial Growth Factor (VEGF) Expression in Human Prostate Cancer: In Situ and in Vitro Expression of VEGF by Human Prostate Cancer Cells

PURPOSE A growing body of literature supports the role of angiogenesis in the development and spread of a variety of human cancers including prostate cancer (Pca). Angiogenesis is controlled by chemical signals known as angiogenic factors (AF) however, little is known about angiogenesis factors in prostate cancer. We evaluated the in situ and in vitro expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, in archival prostate cancer specimens and prostate cancer cell cultures during unstimulated and cytokine stimulated conditions. METHODS Ex-vivo studies involved immunohistochemical analysis for VEGF expression and distribution in 25 archival specimens including, prostate cancer, benign prostatic hyperplasia (BPH) and normal prostate tissue. In-vitro studies utilized prostate cancer cells (DU-145) grown in culture and stimulated with cytokines thought to induce VEGF (i.e. IL-1 alpha, IL-1 beta, TNF-alpha and TNF-beta). Cell culture supernatants were analyzed by ELISA for VEGF levels. RESULTS Immunohistochemical studies demonstrated that in 20 of 25 specimens prostate cancers cells stained positively for VEGF. BPH and normal prostate cells displayed little staining for VEGF. DU-145 prostate cancer cells produced low levels of VEGF in unstimulated conditions. Induction of DU-145 cells with cytokines resulted in differential stimulation whereby TNF was a potent inducer of VEGF, and IL-1 produced lesser but statistically significant increases in VEGF expression. CONCLUSIONS Our immunohistochemical results indicate that significant levels of VEGF are present in prostate cancer, but not in BPH or normal prostate cells in-vivo. In-vitro studies suggest that differential regulation of angiogenesis factor expression by IL-1 and TNF occurs in prostate cancer. Identifying the angiogenesis factors involved in prostate cancer growth and understanding their regulation will lead to the development of anti-angiogenic strategies useful for diagnostic studies and therapeutic interventions.

Angiogenesis and prostate cancer : In vivo and in vitro expression of angiogenesis factors by prostate cancer cells

OBJECTIVES Recently, it was confirmed that angiogenesis is important in the development and spread of a variety of human cancers, including prostate cancer (PCa). Tumor neovascularization is thought to be controlled by chemical signals, known as angiogenic factors (AF). To date, little is known regarding the existence and role of AF in PCa. We previously reported on vascular endothelial growth factor (VEGF) in PCa. Currently, we compare VEGF expression with that of interleukin-8 (IL-8), another putative regulator of angiogenesis. We evaluated the expression of these two important AF in PCa and explored the role of inflammatory cytokines IL-1 and tumor necrosis factor (TNF) in their regulation. METHODS Ex vivo studies involved previously reported immunohistochemical analysis for VEGF and recent evaluation of IL-8 expression and distribution in archival tissue samples of PCa, benign prostatic hyperplasia (BPH), and normal prostate tissue. In vitro studies used PCa cells (DU-145) grown in culture and stimulated with cytokines thought to induce VEGF and IL-8 (ie, IL-1 alpha, IL-1 beta, TNF-alpha, and TNF-beta). After 24 hours, with or without cytokines, cell culture supernatants were analyzed by enzyme-linked immunosorbent assay or radioimmunoassay for VEGF or IL-8 levels. RESULTS Immunohistochemical studies of prostate tissue showed that PCa cells stained positively for VEGF and IL-8. Benign prostatic hyperplasia and normal prostate cells displayed little staining for either AF. Low levels of VEGF and IL-8 were produced by unstimulated DU-145 cells. Induction of DU-145 cells with cytokines resulted in differential stimulation whereby TNF was the predominant inducer of VEGF, whereas IL-1 was the predominant inducer of IL-8. CONCLUSIONS Our results indicate that significant levels of VEGF and IL-8 are present in PCa, but not BPH or normal prostate cells in vivo. In vitro studies suggest that differential regulation of AF expression occurs in PCa. Because IL-1 and TNF are present in the PCa tumor microenvironment, it is likely that differential regulation of AF also occurs in human PCa and contributes to differential tumor growth and metastasis.

Expression of vascular endothelial growth factor receptors in human prostate cancer

OBJECTIVES We recently reported the expression and cytokine regulation of vascular endothelial growth factor (VEGF) in human prostate cancer (PCa). VEGF exerts its angiogenic and pro-tumorigenic properties by way of two high affinity receptors, fms-like tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1). We hypothesized that these receptors are expressed and control VEGF functions in the PCa microenvironment. Herein, we evaluate the expression of these receptors in ex vivo PCa tissue, benign prostatic hypertrophy (BPH) tissue, and cultured PCa cell lines. METHODS Ex vivo PCa specimens were obtained from patients undergoing radical retropubic prostatectomy. Specimens were selected to contain both PCa and BPH tissue (n = 15). Immunohistochemical analysis using antihuman FLT-1 and FLK-1 was performed and specimens were analyzed to characterize the expression and distribution of both receptors. Immunocytochemical analysis for FLT-1 and FLK-1 was also performed on cultured PCa cell lines (DU-145 and LNCaP). RESULTS PCa cells expressed the VEGF receptor FLT-1 in 100% of specimens evaluated. Expression of FLK-1 was variable and related to tumor grade; high-grade tumors displayed little or no FLK-1 expression. Vascular endothelial cells (VECs) within areas of PCa consistently expressed both FLT-1 and FLK-1 receptors. FLT-1 and FLK-1 were both expressed in BPH tissue. FLT-1 was expressed in the glandular epithelial cells in BPH, but in most cases FLK-1 was localized specifically to the basal cell layer of hypertrophic glands. FLT-1, but not FLK-1, was expressed by the DU-145 and LNCaP cell lines. CONCLUSIONS Although they are differentially expressed, both FLT-1 and FLK-1 are present in PCa and BPH. Expression of receptors on VECs of tumor vessels supports the well-established role of VEGF in paracrine stimulation of VECs in the tumor microenvironment. The expression of FLT-1 and FLK-1 on tumor cells themselves suggests a potential autocrine function for VEGF (such as regulating tumor cell proliferation). These findings imply that a novel dual role may exist for VEGF, such that it is involved in tumor cell activation (autocrine), in addition to paracrine actions whereby it regulates endothelial cell functions and subsequent neovascular development.

Nerve growth factor and chronic prostatitis/chronic pelvic pain syndrome.

OBJECTIVES To investigate whether the pain experienced by patients with chronic prostatitis/chronic pelvic pain syndrome (CPPS) may be related to the expression of nerve growth factor (NGF), induced by inflammation and tissue injury experienced as a result of chronic inflammation. CPPS is a disease of unknown pathogenesis. METHODS We measured the levels of NGF and the pro-inflammatory cytokine interleukin (IL)-6 and compared these with the levels of IL-8, interferon-gamma, IL-2, and IL-10 in the seminal plasma of 31 patients with CPPS and 14 controls using enzyme-linked immunosorbent assay technology. Results were correlated with health-related quality of life as measured by the multidimensional pain inventory, the McGill pain questionnaire, and the International Prostate Symptom Score. RESULTS The cytokines analyzed were detectable in the seminal plasma from the patients with CPPS and controls. NGF correlated directly with pain severity (P <0.01) and IL-10 levels (P <0.04), and IL-6 correlated inversely with pain severity (P <0.03). CONCLUSIONS These results suggest that NGF and cytokines that regulate inflammation (IL-6 and IL-10) may play a role in the pain symptoms experienced by patients with CPPS.

INTERLEUKIN-10 LEVELS IN SEMINAL PLASMA: IMPLICATIONS FOR CHRONIC PROSTATITIS-CHRONIC PELVIC PAIN SYNDROME

PURPOSE Chronic prostatitis-chronic pelvic pain syndrome is a disease of unknown pathogenesis. We investigated whether the chronic inflammation and pain experienced by patients may be caused by an imbalance of proinflammatory versus anti-inflammatory cytokines within the prostate, namely interleukin (IL)-8, interferon-gamma and IL-2 versus IL-10. We measured these inflammatory cytokines in seminal plasma as a reflection of the prostate environment. MATERIALS AND METHODS We measured levels of IL-8, interferon-gamma, IL-2 and IL-10 in the seminal plasma of 31 patients with chronic prostatitis-chronic pelvic pain syndrome and 14 controls using enzyme-linked immunosorbent assay. Results were correlated with health related quality of life, as measured by the Multidimensional Pain Inventory, McGill pain questionnaire and International Prostate Symptom Score. RESULTS The cytokines analyzed were detectable in the seminal plasma of patients with chronic prostatitis-chronic pelvic pain syndrome and controls. Interferon-gamma, IL-2 and IL-10 levels were statistically greater in patients. Furthermore, IL-10 levels correlated directly with measures of life interference (r = 0.52, p <0.01) and pain severity (r = 0.53, p <0 0.01), and inversely with spousal support (r = -0.39, p <0.04). CONCLUSIONS Our study suggests that IL-10 may have a role in the pain symptoms experienced by patients with chronic prostatitis-chronic pelvic pain syndrome.

Interleukin-1 Family Expression in Human Breast Cancer: Interleukin- 1 Receptor Antagonist

Abstract We hypothesize that interleukin-1α, β, and receptor antagonist (IL-1α, IL-1β, and IL-1ra, respectively) are present and tumor cell associated in human breast cancer (HBC). We believe the levels of these cytokines in breast tumor homogenates relate to other known prognosticators of patient survival (i.e., estrogen receptor [ER] status). Our results demonstrated that, immunohistochemically, tumor cells express IL-1α, IL-1β, and IL-1ra in most specimens tested. In breast tissue homogenates, IL-1α levels correlated inversely with ER levels (p < 0.06), whereas IL-1ra levels correlated directly with both ER levels (p < 0.009) and IL-1β levels (p < 0.06). When analyzing cytokine levels for the ER (-) versus ER (+) patient groups, we found that in many instances these groups showed a different cytokine profile. These studies suggest that the IL-1 family of cytokines may be important in regulating protumorigenic activities within the HBC tumor microenvironment.

Angiogenesis and neuroblastomas: interleukin-8 and interleukin-8 receptor expression in human neuroblastoma.

PURPOSE Studies have demonstrated that the pro-angiogenic cytokine interleukin-8 (IL-8) and the IL-8 receptors likely have a role in the growth and metastasis of various solid tumors. We hypothesized that in vivo neuroblastoma expresses IL-8 and the IL-8 receptors A and B, and that factors known to regulate IL-8 expression are present and active in the neuroblastoma microenvironment. MATERIALS AND METHODS To confirm the presence of IL-8/IL-8 receptors in neuroblastoma, immunohistochemical analysis for IL-8 and its receptors was performed on 10 archival specimens, including benign adrenal and well to poorly differentiated neuroblastoma samples. Immunohistochemical analysis was also performed for interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha. Cultured neuroblastoma cells SK-N-MC and SK-N-SH were stimulated with 10 ng./ml. IL-1beta or tumor necrosis factor-alpha and control media (15 each). Cell culture supernatants were analyzed with enzyme-linked immunosorbant assay for IL-8 levels at 24 and 48 hours. RESULTS Minimal expression of IL-8 was noted in benign adrenal tissue but expression for IL-8 was present in all neuroblastoma specimens. Microvessel staining was present in 30% of the specimens. All tumor specimens expressed IL-8 receptor B, and both receptors were expressed in the tumor microvasculature. Immunohistochemical analysis confirmed the presence of IL-1beta and tumor necrosis factor in the neuroblastoma microenvironment. In vitro studies demonstrated that SK-N-MC and SK-N-SH cells express low levels of IL-8 under normal conditions and that IL-1beta and tumor necrosis factor-alpha significantly increased expression of IL-8 at 24 and 48 hours. CONCLUSIONS Our results indicate that IL-8 and its receptors are expressed in neuroblastoma tumor specimens. In addition, the fact that IL-1beta and tumor necrosis factor-alpha are expressed in the neuroblastoma microenvironment combined with our in vitro results suggests that these cytokines may be involved in in vivo regulation of IL-8 in human neuroblastoma. Understanding the angiogenic factors and regulatory cascade promoting angiogensis in neuroblastoma may lead to the development of effective anti-angiogenic strategies.