Nanotherapy silencing the interleukin-8 gene produces regression of prostate cancer by inhibition of angiogenesis.

Interleukin-8 (IL-8) is a pro-angiogenic cytokine associated with aggressive prostate cancer (CaP). We detected high levels of IL-8 in sera from patients with CaP compared with healthy controls and patients with benign prostatic hypertrophy. This study examines the role of IL-8 in the pathogenesis of metastatic prostate cancer. We developed a biocompatible, cationic polylactide (CPLA) nanocarrier to complex with and efficiently deliver IL-8 small interfering RNA (siRNA) to CaP cells in vitro and in vivo. CPLA IL-8 siRNA nanocomplexes (nanoplexes) protect siRNA from rapid degradation, are non-toxic, have a prolonged lifetime in circulation, and their net positive charge facilitates penetration of cell membranes and subsequent intracellular trafficking. Administration of CPLA IL-8 siRNA nanoplexes to immunodeficient mice bearing human CaP tumours produced significant antitumour activities with no adverse effects. Systemic (intravenous) or local intra-tumour administration of IL-8 siRNA nanoplexes resulted in significant inhibition of CaP growth. Magnetic resonance imaging and ultrasonography of experimental animals demonstrated reduction of tumour perfusion in vivo following nanoplex treatment. Staining of tumour sections for CD31 confirmed significant damage to tumour neovasculature after nanoplex therapy. These studies demonstrate the efficacy of IL-8 siRNA nanotherapy for advanced, treatment-resistant human CaP.

Anti-angiogenic activity of PSA-derived peptides.

BACKGROUND: PSA is a biomarker for diagnosis and management of prostate cancer. PSA is known to have anti-tumorigenic activities, however, the physiological role of PSA in prostate tumor progression is not well understood. METHODS: Five candidate peptides identified based upon computer modeling of the PSA crystal structure and hydrophobicity were synthesized at >95% purity. The peptides in a linear form, and a constrained form forced by a di-sulfide bond joining the two ends of the peptide, were investigated for anti-angiogenic activity in HUVEC. RESULTS: None of the five PSA-mimetic peptides exhibited PSA-like serine protease activity. Two of the peptides demonstrated significant anti-angiogenic activity in HUVEC based on (i) inhibition of cell migration and invasion; (ii) inhibition of tube formation in Matrigel; (iii) anti-angiogenic activity in a sprouting assay; and (iv) altered expression of pro- and anti-angiogenic growth factors. Constrained PSA-mimetic peptides had greater anti-angiogenic activity than the corresponding linearized form. Complexing of PSA with ACT eliminated PSA enzymatic activity and reduced anti-angiogenic activity. In contrast, ACT had no effect on the anti-angiogenic effects of the linear or constrained PSA-mimetic peptides. Modeling of the ACT-PSA complex demonstrated ACT sterically blocks the anti-angiogenic activity of the two bioactive peptides. CONCLUSIONS: The interaction of a hydrophilic domain on the surface of the PSA molecule with a target on the cell membrane of prostate endothelial and epithelial cells was responsible for the anti-angiogenic or anti-tumorigenic activity of PSA: enzymatic activity was not associated with anti-angiogenic effects. Furthermore, since PSA and ACT are both expressed within the human prostate tissue microenvironment, the balance of their expression may represent a mechanism for endogenous regulation of tissue angiogenesis.

New serum biomarkers for prostate cancer diagnosis.

BACKGROUND: Prostate-specific antigen (PSA) is currently used as a biomarker for diagnosis and management of prostate cancer (CaP). However, PSA typically lacks the sensitivity and specificity desired of a diagnostic marker. OBJECTIVE: The goal of this study was to identify an additional biomarker or a panel of biomarkers that is more sensitive and specific than PSA in differentiating benign versus malignant prostate disease and/or localized CaP versus metastatic CaP. METHODS: Concurrent measurements of circulating interleukin-8 (IL-8), Tumor necrosis factor-α (TNF-α) and soluble tumor necrosis factor-α receptors 1 (sTNFR1) were obtained from four groups of men: (1) Controls (2) with elevated prostate-specific antigen with a negative prostate biopsy (elPSA_negBx) (3) with clinically localized CaP and (4) with castration resistant prostate cancer. RESULTS: TNF-α Area under the receiver operating characteristic curve (AUC = 0.93) and sTNFR1 (AUC = 0.97) were strong predictors of elPSA_negBx (vs. CaP). The best predictor of elPSA_negBx vs CaP was sTNFR1 and IL-8 combined (AUC = 0.997). The strongest single predictors of localized versus metastatic CaP were TNF-α (AUC = 0.992) and PSA (AUC = 0.963) levels. CONCLUSIONS: The specificity and sensitivity of a PSA-based CaP diagnosis can be significantly enhanced by concurrent serum measurements of IL-8, TNF-α and sTNFR1. In view of the concerns about the ability of PSA to distinguish clinically relevant CaP from indolent disease, assessment of these biomarkers in the larger cohort is warranted.

Overexpression of MMP-9 contributes to invasiveness of prostate cancer cell line LNCaP.

Matrix metallaprotinase-9 (MMP-9) is zinc-containing proteinase whose expression and trafficking are frequently altered in cancer. MMP-9 in the plasma membrane and the secreted forms are thought to contribute to the invasive and metastatic properties of malignant tumors. We have manipulated the expression of MMP-9 in prostate tumor cell line LNCaP and measured their capacity to invade through a basement membrane matrix. Stable expression of human MMP-9 in a poorly metastatic LNCaP prostate cancer cell line produced a 2-3-fold increase in MMP-9 activity and a comparable increase in invasiveness. Transient transfection of LNCaP stable clone expressing MMP-9 with MMP-9 antisense oligonucleotide (ASODN) produced 55-90% less MMP-9 than control cells and were proportionately less invasive. In contrast, manipulating MMP-9 levels had no effect on cell migration across an uncoated membrane. A standard MMP-9 inhibitor at a concentration ranging from 1-10 nM, caused a nearly quantitative inhibition of extracellular MMP-9 activity and had significant effect on basement membrane invasion. Collectively, these results confirm the role of MMP-9 in tissue remodeling associated with prostate tumor invasion.

Enzymatic activity of free-prostate-specific antigen (f-PSA) is not required for some of its physiological activities.

BACKGROUND: Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA. METHODS: Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+ . Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA. RESULTS: Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties. DISCUSSION: Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity.

The dietary bioflavonoid, quercetin, selectively induces apoptosis of prostate cancer cells by down-regulating the expression of heat shock protein 90.

BACKGROUND: Human and animal studies have suggested that diet-derived flavonoids, in particular quercetin may play a beneficial role by preventing or inhibiting oncogenesis, but the underlying mechanism remains unclear. The aim of this study is to evaluate the effect(s) of quercetin on normal and malignant prostate cells and to identify the target(s) of quercetin's action. METHODOLOGY: We addressed this question using cells in culture and investigated whether quercetin affects key biological processes responsible for tumor cell properties such as cell proliferation and apoptosis and also studied the effect of quercetin on the proteome of prostate cancer cells using difference gel electrophoresis (DIGE) to assess changes in the expression of relevant proteins. RESULTS: Our findings demonstrate that quercetin treatment of prostate cancer cells results in decreased cell proliferation and viability. Furthermore, we demonstrate that quercetin promotes cancer cell apoptosis by down-regulating the levels of heat shock protein (Hsp) 90. Depletion of Hsp90 by quercetin results in decreased cell viability, levels of surrogate markers of Hsp90 inhibition (intracellular and secreted), induced apoptosis and activation of caspases in cancer cells but not in normal prostate epithelial cells. Knockdown of Hsp90 by short interfering RNA also resulted in induction apoptosis similar to quercetin in cancer cells as indicated by annexin V staining. CONCLUSION: Our results demonstrate that quercetin down-regulates the expression of Hsp90 which, in turn, induces inhibition of growth and cell death in prostate cancer cells while exerting no quantifiable effect on normal prostate epithelial cells.

Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions.

Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelial cells and prostate cancer cells. We explored "thiophilic-interaction chromatography" (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the "free" form having a molecular weight of 33 kDa. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T-PSA over a wide range of concentrations of Zn(2+) ions (10nM to 50 microM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serine-protease, was affected differently. At higher concentrations of Zn(2+) (10 microM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn(2+) (5 microM and lower), the enzymatic activity was enhanced.

Treatment of viral and neoplastic diseases with double-stranded RNA derivatives and other new agents.

Many attempts have been made to inhibit viral and neoplastic diseases by targeting the RNA system. The pathophysiologic significance of the microRNA system and the therapeutic potential of its manipulation are discussed. Studies of double-stranded RNA derivatives are reviewed. The therapeutic potential of one of these compounds, polyI:MPC, is emphasized. Studies of other related antiviral and antineoplastic agents are discussed, including 2'-deoxyoligocytidilates and telomerase inhibitors.

Prostate-specific antigen modulates the expression of genes involved in prostate tumor growth.

Prostate-specific antigen (PSA) is a serine protease that is widely used as a surrogate marker in the early diagnosis and management of prostate cancer. The physiological relevance of tissue PSA levels and their role in prostate tumor growth and metastasis are not known. Free-PSA (f-PSA) was purified to homogeneity from human seminal plasma by column chromatography, eliminating hk2 and all known PSA complexes and retaining its protease activity. Confluent monolayers of prostate cancer cell lines, PC-3M and LNCaP, were treated with f-PSA in a series of in vitro experiments to determine the changes in expression of various genes that are known to regulate tumor growth and metastasis. Gene array, quantitative polymerase chain reaction (QPCR), and enzyme-linked immunosorbent assay (ELISA) results show significant changes in the expression of various cancer-related genes in PC-3M and LNCaP cells treated with f-PSA. In a gene array analysis of PC-3M cells treated with 10 muM f-PSA, 136 genes were upregulated and 137 genes were downregulated. In LNCaP cells treated with an identical concentration of f-PSA, a total of 793 genes was regulated. QPCR analysis reveals that the genes for urokinase-type plasminogen activator (uPA), VEGF, and Pim-1 oncogene, known to promote tumor growth, were significantly downregulated, whereas IFN-gamma, known to be a tumor-suppressor gene, was significantly upregulated in f-PSA-treated PC-3M cells. The effect of f-PSA on VEGF and IFN-gamma gene expression and on protein release in PC-3M cells was distinctly dose-dependent. In vivo studies showed a significant reduction (P = .03) in tumor load when f-PSA was administered in the tumor vicinity of PC-3M tumor-bearing BALB/c nude mice. Our data support the hypothesis that f-PSA plays a significant role in prostate tumor growth by regulating various proangiogenic and antiangiogenic growth factors.

Effect of increasing thiolation of the polycytidylic acid strand of poly I:poly C on the alpha, beta and gamma interferon-inducing properties, antiviral and antiproliferative activities.

Double-stranded RNAs induce interferons and cause the development of antiviral and antiproliferative activities. Antiviral activity is related to the production of interferons and other proteins that stimulate various immunologic activities, which appear to contribute to their overall antiproliferative activity. The most active double-stranded RNA, polyI:polyC, was shown to be too toxic for therapeutic use. We conducted selective thiolation of the polyC strand at the five position of the cytosine bases, generating a partially thiolated polyC (MPC) which after annealing with a complimentary unmodified polyI, gave the thiolated double-stranded RNA, pI:MPC. We have explored antiviral and antiproliferative activities at various levels of thiolation and found that optimal responses can be obtained at 7.4% level of thiolation. This compound deserves further study of antiviral and antiproliferative responses in vivo, and eventually clinical exploration. Earlier studies have shown that this and related compounds are active against HIV-1, in human cells, and against DNA polymerases of DNA and RNA tumor viruses.

Two step procedure for purification of enzymatically active prostate-specific antigen from seminal plasma.

The role of prostate-specific antigen (PSA) during the onset of prostate cancer and subsequent tumor growth and metastasis is not well understood. We have developed a simple two step procedure, based on principles of hydrophobic charge-induction chromatography and molecular size chromatography to provide pure free-PSA (f-PSA) preparation that is free from all other known PSA complexes as well as human kallikrein 2 (hK2). The overall recovery of f-PSA is 72%. The isolated f-PSA consists of three known isoforms that corresponds to pI of 6.2, 6.4 and 7.2. f-PSA is enzymatically active and its enzymatic activity can be effectively neutralized by a serine protease inhibitor.

Gene expression of angiogenic factors correlates with metastatic potential of prostate cancer cells.

We hypothesize that expression of proangiogenic genes correlates with the metastatic potential of prostate cancer cells. LNCaP, DU-145, and PC-3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as we demonstrated by their capacity to invade an extracellular matrix, an established tumor invasion assay. The constitutive gene expression of the proangiogenic factors, vascular endothelial growth factor, intercellular adhesion molecule-1, interleukin-8, and transforming growth factor-beta2, was significantly greater in the more metastatic DU-145 and PC-3 cells as compared with LNCaP cells. Matrix metalloproteinase (MMP)-9 is thought to contribute to the invasive phenotype of tumor cells. PC-3 cells showed increased expression of MMP-9 and membrane type 4-MMP as compared with LNCaP and DU-145. Tissue inhibitors of metalloproteinase 1 and 4 gene expression were elevated in DU-145 and PC-3 cells, but paradoxically, LNCaP cells had undetectable levels of these genes. We transfected and overexpressed MMP-9 in poorly metastatic LNCaP cells and measured their invasive activity. Transient expression of human MMP-9 in LNCaP cells produced a 3-5-fold increase in MMP-9 activity with a comparable increase in invasiveness. Antisense ablation of the expression of MMP-9 in DU-145 and PC-3 cells produced concomitant inhibition of the gene expression of the proangiogenic factors, vascular endothelial growth factor, and intercellular adhesion molecule-1 (ICAM-1). Treatment of DU-145 and PC-3 cells with a selective chemical inhibitor of MMP-9 proteinase activity also inhibited their invasive activity. These results support our hypothesis that metastatic potential of prostate cancer cells correlates with expression of proangiogenic factors.

Interferons and interferon inhibitory activity in disease and therapy.

Interferon (IFN) resistance is an important factor in the pathophysiology of neoplastic disorders, certain viral infections (e.g., AIDS), and autoimmune diseases (e.g., lupus erythematosus and Wegner's granulomatosis). In addition, in some of these disorders, there is also decreased ability to produce IFNs. The capacity of viruses and neoplastic processes to interfere with the IFN system are thought to represent a "virus-against-host" or "cancer-against-host" defense mechanism. Four resistance factors have been identified: 1) release of free IFN-alpha/beta type 1 receptors into the circulation that, at appropriate concentrations, capture and inactivate IFNs; 2) a new IFN inhibitory protein has been isolated and its chemical structure is under study; 3) prostaglandin E2, which is produced by certain tumor cells, inhibits IFN production; and 4) high levels of cAMP phosphodiesterases present, for example in certain tumor cells, reduces cAMP, an important second messenger in IFN synthesis. Studies are under way to reverse these inhibitory effects and to increase endogenous interferon production.

New approaches to the treatment of AIDS with special reference to overcoming interferon resistance.

This is a brief review on studies of attacking HIV through a new angle. In previous studies, we have found that many patients with AIDS are resistant to interferon (IFN) therapy, and some develop resistance during therapy. Four factors were found to be responsible for the resistance of untreated patients: (a). release of free-circulating IFN-alpha/beta type 1 receptors, (b). a newly detected IFN inhibitory protein, (c). high prostaglandin E2, and (d). high levels of cAMP phosphodiesterases, particularly in AIDS-related neoplasms. This may interfere with intrinsic disease resistance and with the efficacy of IFN therapy. In an attempt to overcome this resistance, new compounds were synthesized which increase endogenous production of alpha, beta and gamma IFNs, have anti-template activity against DNA and RNA polymerases, inhibit reverse transcriptases and activates IFN-induced double-stranded RNA (dsRNA)-dependent protein kinase. It is expected that planned nonhuman primate and clinical studies will support preliminary findings. Preliminary in vitro and animal studies suggest that these new compounds may be effective against HIV, including multi-drug resistant strains.

Resistance to interferons.

Extract: Interferons alpha, beta and gamma are a group of structurally and functionally related proteins, produced in response to viruses or double-stranded RNA and defined by their ability to establish an antiviral state in cells. They were originally discovered by Isaacs and Lindenmann (1957). Since their original discovery, several interferon genes and proteins have been identified. Their mechanisms of action were studied and in addition to the antiviral activity, other biological activities were also uncovered including inhibition of cell proliferation and immunomodulation. Human interferon-alpha is a family of at least 23 polypeptides coded by several related genes. The various interferons exhibit a high level of species specificity with some exceptions. For example, there is a high level of activity of human interferon-alpha subtypes on bovine cells. Interferons have been used for the treatment of certain viral infections, multiple sclerosis, certain cancers including hematologic malignancies and Acquired Immune Deficiency Syndrome (AIDS)-related cancerous complications.

Free interferon-alpha/beta receptors in the circulation of patients with adenocarcinoma.

BACKGROUND: Many viral and neoplastic diseases are resistant to interferon-alpha/beta (IFN-alpha/beta) therapy or develop resistance during the course of IFN treatment. In patients with viral diseases, the authors identified four IFN inhibitors, of which the most important, most likely is a free IFN receptor of type 1 appearing in the circulation that captures and neutralizes IFN-alpha/beta. METHODS: Ninety-one cancer patients and 25 healthy individuals were studied. Free circulating IFN receptor-alpha/beta type 1 was studied. The patients were ages 35-75 years. The diagnoses were 24 cases of colon carcinoma, 7 cases of prostate carcinoma, 16 cases of breast carcinoma, 8 cases of ovarian carcinoma, 9 cases of uterine carcinoma, 5 cases of lung carcinoma, 3 cases of astrocytoma, 4 cases of transitional cell carcinoma of the bladder, 1 case of osteosarcoma, 3 cases of multiple myeloma, 4 cases of Hodgkin disease, 2 cases of non-Hodgkin lymphoma, 3 cases of myelodysplastic syndrome, and 2 disseminated tumors of unknown origin. RESULTS: All patients were found to have increased free IFN receptor-alpha/beta type 1 in the circulation, with the highest levels reported in patients with adenocarcinoma. CONCLUSIONS: High IFN inhibitory activity in patients with cancer may be a significant factor in their increased susceptibility to progressive disease, infectious complications, and resistance to IFN therapy. Ongoing studies are being performed with the objective of overcoming this inhibitory activity.

Serum interferon inhibitor declines in patients with HIV-1 infection after a change in antiretroviral therapy.

An inhibitor of interferon antiviral activity, which is absent in healthy HIV-seronegative persons, was detected in the sera of all 29 HIV-seropositive study participants. The relationship of the level of interferon inhibitor to CD4 count and HIV-RNA copy number was statistically significant in distinct models. Levels of interferon inhibitor declined by an average of 41-60% in patients who underwent a change in anti-retroviral therapy. Interferon inhibitor levels appear to decline as CD4 cell count rises and HIV-RNA levels fall. This suggests that interferon inhibitor may have a significant role in the host immune response to HIV infection.

The flavonoid, quercetin, differentially regulates Th-1 (IFNgamma) and Th-2 (IL4) cytokine gene expression by normal peripheral blood mononuclear cells.

Flavonoids are plant metabolites that are dietary antioxidants and exert significant anti-tumor, anti-allergic, anti-inflammatory and anti-viral effects. It is generally accepted that Th-1 derived cytokines such as IL-2, IFNgamma and IL-12 promote cellular immunity while Th-2 derived cytokines such as IL-4, IL-5, IL-6 exert negative immunoregulatory effects on cellular immunity while upregulating humoral immunity. The molecular mechanisms underlying the biological activities of flavonoids have not been elucidated. We hypothesize that the flavonoid, quercetin, exert significant anti-viral and anti-tumor effects possibly by modulating the production of Th-1 and Th-2 derived cytokines. Peripheral blood mononuclear cells (PBMC, 1 x 10(6) cells/ml) from normal subjects were cultured with different concentrations of quercetin (0.5-50 microM) for 24-72 h and supernates were quantitated for IFN-gamma and IL-4 by ELISA and antiviral activity of IFNgamma by bioassay. FACS analysis was done to determine the number of IFN-gamma and IL-4 positive cells and RT-PCR was done to quantitate gene expression. Quercetin significantly induces the gene expression as well as the production of Th-1 derived IFNgamma and the downregulates Th-2 derived IL-4 by normal PBMC. Further, quercetin treatment increased the phenotypic expression of IFNgamma cells and decreased IL-4 positive cells by FACS analysis, which corroborate with protein secretion and gene expression studies. These results suggest that the beneficial immuno-stimulatory effects of quercetin may be mediated through the induction of Th-1 derived cytokine, IFNgamma, and inhibition of Th-2 derived cytokine, IL-4.