Monocyte-Induced Prostate Cancer Cell Invasion is Mediated by Chemokine ligand 2 and Nuclear Factor-κB Activity.

STUDY BACKGROUND: The tumor microenvironment contains inflammatory cells which can influence cancer growth and progression; however the mediators of these effects vary with different cancer types. The mechanisms by which prostate cancer cells communicate with monocytes to promote cancer progression are incompletely understood. This study tested prostate cancer cell and monocyte interactions that lead to increased prostate cancer cell invasion. METHODS: We analyzed the prostate cancer cell invasion and NF-κB activity and cytokine expression during interaction with monocyte-lineage cells in co-cultures. The roles of monocyte chemotactic factor (MCP-1/CCL2) and NF-κB activity for co-culture induced prostate cancer invasion were tested. Clinical prostate cancer NF-κB expression was analyzed by immunohistochemistry. RESULTS: In co-cultures of prostate cancer cell lines with monocyte-lineage cells, (C-C motif) ligand 2 (CCL2) levels were significantly increased when compared with monocytes or cancer cells cultured alone. Prostate cancer cell invasion was induced by recombinant CCL2 in a dose dependent manner, similar to co-cultures with monocytes. The monocyte-induced prostate cancer cell invasion was inhibited by CCL2 neutralizing antibodies and by the CCR2 inhibitor, RS102895. Prostate cancer cell invasion and CCL2 expression induced in the co-cultures was inhibited by Lactacystin and Bay11-7082 NF-κB inhibitors. Prostate cancer cell NF-κB DNA binding activity depended on CCL2 dose and was inhibited by CCL2 neutralizing antibodies. Clinical prostate cancer NF-κB expression correlated with tumor grade. CONCLUSIONS: Co-cultures with monocyte-lineage cell lines stimulated increased prostate cancer cell invasion through increased CCL2 expression and increased prostate cancer cell NF-κB activity. CCL2 and NF-κB may be useful therapeutic targets to interfere with inflammation-induced prostate cancer invasion.

Role of monocyte-lineage cells in prostate cancer cell invasion and tissue factor expression.

BACKGROUND: Tissue factor (TF) is a cell surface glycoprotein intricately related to blood coagulation and inflammation. This study was performed to investigate the role of monocyte-lineage cells in prostate cancer cell TF expression and cell invasion. METHODS: Prostate cancer cell invasion was tested with and without added peripheral blood monocytes or human monocyte-lineage cell lines. TF neutralizing antibodies were used to determine the TF requirement for prostate cancer cell invasion activity. Immunohistochemistry was performed to identify prostate tissue CD68 positive monocyte-derived cells and prostate epithelial TF expression. RESULTS: Co-culture of PC-3, DU145, and LNCaP cells with isolated human monocytes significantly stimulated prostate cancer cell invasion activity. TF expression was greater in highly invasive prostate cancer cells and was induced in PC-3, DU145, and LNCaP cells by co-culture with U-937 cells, but not with THP-1 cells. TF neutralizing antibodies inhibited PC-3 cell invasion in co-cultures with monocyte-lineage U-937 or THP-1 cells. Prostate cancer tissues contained more CD68 positive cells in the stroma and epithelium (145 ± 53/mm(2)) than benign prostate (108 ± 31/mm(2)). Samples from advanced stage prostate cancer tended to contain more CD68 positive cells when compared with lower stage lesions. Prostatic adenocarcinoma demonstrated significantly increased TF expression compared with benign prostatic epithelium. CONCLUSIONS: This study shows that co-culture with monocyte-lineage cells induced prostate cancer cell invasion activity. PC-3 invasion and TF expression was induced in co-culture with U-937 cells and partially inhibited with TF neutralizing antibodies.

Lysophosphatidic acid stimulates PC-3 prostate cancer cell Matrigel invasion through activation of RhoA and NF-kappaB activity.

This study was performed to determine the relationship of lysophosphatidic acid (LPA) stimulation and increased Ras homolog A (RhoA) activity to nuclear factor kappa B (NF-kappaB) activity, and the role of these factors in regulating prostate cancer cell invasion. PC-3 high invasive cells demonstrated constitutively increased RhoA, NF-kappaB, and in vitro Matrigel invasion which were further induced by LPA stimulation or transfection with constitutively active RhoA Q63E mutant. LPA treatment rapidly and transiently induced RhoA activity followed by maximally increased DNA binding of NF-kappaB at 1 h and AP-1 at 4 h. The LPA-induced NF-kappaB DNA binding was preceded by transient IkappaBalpha phosphorylation, and decreased total IkappaBalpha levels. Further demonstrating the relationship between RhoA and NF-kappaB activation, PC-3 cells stably transfected with constitutively active RhoA Q63E demonstrated constitutively increased phospho-IkappaBalpha, while PC-3 cells transfected with dominant negative RhoA N19 exhibited decreased phospho-IkappaBalpha levels. The LPA-induced Matrigel invasion and NF-kappaB DNA binding activity were both inhibited by expression of the RhoA inhibitor C3 exoenzyme or dominant negative mutant NF-kappaB inhibitor IkappaBalpha S32/36A. Similarly, transfection with dominant negative IkappaBalpha S32/36A inhibited PC-3 RhoA Q63E cell in vitro invasion. Treatment of PC-3 high invasive and RhoA Q63E cells with sodium salicylate or lactacystin inhibited NF-kappaB and invasion, while pyrrolidine dithiocarbamate (PDTC) treatment of PC-3 high invasive cells inhibited NF-kappaB only. Each inhibitor blocked LPA-induced invasion while PDTC inhibited LPA-induced NF-kappaB and invasion to the greatest extent. These results point to a model where LPA stimulates RhoA and increased PC-3 prostate cancer cell invasion activity through an NF-kappaB-dependent pathway.

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding.

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.

Update on zona pellucida glycoproteins based contraceptive vaccine.

Zona pellucida (ZP) glycoproteins, due to their critical role in mammalian fertilization, have been proposed as candidate immunogens for development of a contraceptive vaccine. Active immunization studies in a variety of animal species, employing either native or recombinant zona proteins, has established their contraceptive potential. Hence, ZP glycoprotein-based contraceptive vaccines have a very good potential for controlling wild life population. To make it a realistic proposition, additional research inputs are required to develop new potent adjuvants and novel practical strategies for vaccine delivery. The observed ovarian dysfunction, often associated with immunization by ZP glycoproteins, is one of the major obstacles for their application in the control of human population. Ongoing studies to delineate epitopes of ZP glycoproteins that will generate an immune response capable of inhibiting fertility without any untoward effects on ovarian functions will help in determining their feasibility for human use.

Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding.

To minimize ovarian dysfunction subsequent to immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing the antigenic B cell epitopes as immunogens have been proposed. In this study, attempts have been made to clone and express a recombinant chimeric protein encompassing the epitopes corresponding to bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1, amino acid residues 132-147), ZP glycoprotein-2 (bmZP2, amino acid residues 86-113), and ZP glycoprotein-3 (bmZP3, amino acid residues 324-347). The above chimeric recombinant protein (r-bmZP123) was expressed as a polyhistidine fusion protein in Escherichia coli. Immunoblot with murine monoclonal antibody, MA-813, generated against recombinant bmZP1 revealed a major band of approximately 10 kDa. The r-bmZP123 was purified on nickel-nitrilotriacetic acid resin under denaturing conditions. The female rabbits immunized with purified r-bmZP123 conjugated to diphtheria toxoid (DT) generated antibodies that reacted with r-bmZP123 and DT in an ELISA. In addition, the immune sera also reacted with E. coli expressed recombinant bmZP1, bmZP2, and bmZP3. In an indirect immunofluorescence assay, the antibodies against r-bmZP123 recognized native ZP of bonnet monkey as well as human. The immune sera also inhibited, in vitro, the binding of human spermatozoa to the human zona in the hemizona assay (HZA). These studies, for the first time, demonstrate the feasibility of assembling multiple epitopes of different ZP glycoproteins as a recombinant protein that elicit antibodies which are reactive with native zona and also inhibit, in vitro, human sperm-oocyte binding.

Native zona pellucida reactivity and in-vitro effect on human sperm-egg binding with antisera against bonnet monkey ZP1 and ZP3 synthetic peptides.

In mammals, zona pellucida glycoprotein-3 (ZP3) is the putative ligand for primary sperm binding and induces the acrosome reaction. Recent evidence suggests that zona pellucida glycoprotein-1 (ZP1) also play an important role, in some species, during fertilization. In order to identify synthetic peptide immunogens capable of inducing antibodies reactive with native zona and inhibiting sperm-oocyte interaction, peptide encompassing the amino acid (aa) residues 334-343 of bonnet monkey ZP3 (bmZP3) was synthesized co-linearly with a 'promiscuous' T-cell epitope of circumsporozoite protein (CSP, 378-398 aa) of Plasmodium falciparum. In addition, four peptides corresponding to bonnet monkey ZP1 (bmZP1((58-79 aa)), bmZP1((136-153 aa)), bmZP1((212-228 aa)) and bmZP1((251-273 aa))) were synthesized. The synthetic peptides corresponding to bmZP1 were conjugated with diphtheria toxoid. Immunization of female BALB/cJ mice with the above conjugates and CSP-bmZP3((334-343 aa)) peptide led to the generation of an adequate antibody response against the respective zona peptide. Antibodies against bmZP1((251-273 aa)) and CSP-bmZP3((334-343 aa)) recognized bonnet monkey and human zona pellucida in an indirect immunofluorescence assay. Further, these antibodies when tested independently or in combination also significantly inhibited the binding of human spermatozoa to zona pellucida in a hemizona assay. These studies will further help in the design of synthetic peptide immunogens comprising of multiple B cell epitope from different zona proteins for better immunocontraceptive efficacy.

Immunocontraceptive potential of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli and its corresponding synthetic peptide.

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for development of immunocontraceptive vaccines. In this study, the efficacy to block fertility by immunization with recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C (r-bmZPC) expressed in Escherichia coli and its synthetic peptide (P(4): KGDCGTPSHSRRQPHVVSQWSRSA, aa residues 324-347) conjugated to diphtheria toxoid (DT) has been evaluated in a homologous system. Female bonnet monkeys, immunized with P(4)-DT conjugate showed better immunocontraceptive potential as compared to an r-bmZPC-DT immunized group. In spite of high anti-P(4) antibody titres, animals continued to have ovulatory cycles and showed no disturbance in cyclicity (except summer amenorrhoea). No ovarian pathology was observed in the P(4) immunized group. These results suggest that immunization with the P(4) may lead to block in fertility without obvious ovarian dysfunction. However, further inputs are required to identify additional ZP based B-cell epitopes to enhance the contraceptive efficacy.