The ADAM9/WISP-1 axis cooperates with osteoblasts to stimulate primary prostate tumor growth and metastasis

Background: Metastatic prostate cancer (PCa) predicts a poor prognosis and lower likelihood of survival. Osteoblasts (OBs) are known to be responsible for the synthesis and mineralization of bone, although it is unclear as to whether PCa in the prostate gland cooperates with OBs in bone to promote PCa malignant transformation. We aimed to elucidate how primary PCa cells cooperate with distal OBs and contribute to the vicious cycle that leads to metastatic PCa.Methods: N-cadherin, E-cadherin, and Twist protein expression were measured by Western blot. Twist translocation into the nucleus was detected by the immunofluorescence (IF) assay. Enzyme-linked immunosorbent assay (ELISA) detected protein levels in human serum samples. Levels of candidate protein expression were examined by the human cytokine array. Prostate tumor growth and metastasis were analyzed by orthotopic and metastatic prostate cancer models, respectively. Immunohistochemistry (IHC) staining was used to observe ADAM metallopeptidase domain 9 (ADAM9) and WNT1 inducible signaling pathway protein 1 (WISP-1) expression in tissue.Results: Our in vitro and in vivo analyses have now discovered that primary PCa expressing ADAM9 protein enables the transformation of OBs into PCa-associated osteoblasts (PCa-OBs), inducing WISP-1 secretion from PCa-OBs in the bone microenvironment. The upregulation of WISP-1 in bone provided feedback to primary PCa and promoted PCa cell aggressiveness via epithelial-mesenchymal transition (EMT) activity. Elevated levels of WISP-1 expression were detected in the serum of patients with PCa. ADAM9 levels were overexpressed in tumor tissue from PCa patients; ADAM9 blockade interrupted OB-induced release of WISP-1 and also suppressed primary tumor growth and distal metastasis in orthotopic PCa mouse models.Conclusion: Our study suggests that the ADAM9/WISP-1 axis assists with metastatic PCa progression. Thus, targeting the ADAM9/WISP-1 axis may help to prevent the malignant phenotypes of PCa cells.     

Osteoblastic protein kinase D1 contributes to the prostate cancer cells dormancy via GAS6-circadian clock signaling

Disseminated prostate cancer (PCa) is known to have a strong propensity for bone marrow. These disseminated tumor cells (DTCs) can survive in bone marrow for years without obvious proliferation, while maintaining the ability to develop into metastatic lesions. However, how DTCs kept dormant and recur is still uncertain. Here, we focus on the role of osteoblastic protein kinase D1 (PKD1) in PCa (PC-3 and DU145) dormancy using co-culture experiments. Using flow cytometry, western blotting, and immunofluorescence, we observed that in co-cultures osteoblasts could induce a dormant state in PCa cells, which is manifested by a fewer cell divisions, a decrease Ki-67-positive populations and a lower ERK/p38 ratio. In contrast, silencing of PKD1 gene in osteoblasts impedes co-cultured prostate cancer cell's dormancy ability. Mechanismly, protein kinase D1 (PKD1) in osteoblasts induces PCa dormancy via activating CREB1, which promoting the expression and secretion of growth arrest specific 6 (GAS6). Furthermore, GAS6-induced dormancy signaling significantly increased the expression of core circadian clock molecules in PCa cells, and a negative correlation of circadian clock proteins (BMAL1, CLOCK and DEC2) with recurrence-free survival is observed in metastatic prostate cancer patients. Interestingly, the expression of cell cycle factors (p21, p27, CDK1 and PCNA) which regulated by circadian clock also upregulated in response to GAS6 stimulation. Taken together, we provide evidence that osteoblastic PKD1/CREB1/GAS6 signaling regulates cellular dormancy of PCa cells, and highlights the importance of circadian clock in PCa cells dormancy.     

Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway

Background: M2 macrophages are well accepted to promote cancer progression in the prostate cancer (PCa). Paracrine is the principally studied mode of communication between M2 macrophages and tumor cells. In addition to this, we present here a novel model to demonstrate these cellular communications.Methods: PCa cells were co-cultured with THP-1/ human peripheral blood mononuclear cells derived M2 macrophages in direct contact manner. Cancer cell proliferation and invasion were examined to explain how direct contact communicates. Cell-based findings were validated in two xenograft models and patients samples.Results: M2 macrophage direct contact induced a higher proliferation and invasion in PCa cells when compared with noncontact coculture manner. In direct contact manner, NOTCH1 pathway was greatly activated in PCa cells, induced by elevated γ-secretase activity and higher coactivator MAML2 expression. Additionally, blocking γ-secretase activity and depletion of MAML2 completely abolished M2 macrophage direct contact-mediated PCa cell proliferation and invasion. In vivo, inhibiting NOTCH1 signalling impaired M2 macrophage-mediated PCa tumor growth and lung metastasis. Notably, M2 macrophage infiltration as well as high NOTCH1 signaling in cancer cells indicated more aggressive features and worse survival in PCa patients.Conclusion: Our results demonstrated the cell-cell direct contact pattern is an important way in PCa microenvironment cell communication. In this manner, elevated γ-secretase activity and MAML2 expression induced higher NOTCH1 signalling in PCa cells, which increased tumor cells proliferation and invasion. This potentially provided a therapeutic target for PCa.     

Secretome Analysis of an Osteogenic Prostate Tumor Identifies Complex Signaling Networks Mediating Cross-talk of Cancer and Stromal Cells Within the Tumor Microenvironment

A distinct feature of human prostate cancer (PCa) is the development of osteoblastic (bone-forming) bone metastases. Metastatic growth in the bone is supported by factors secreted by PCa cells that activate signaling networks in the tumor microenvironment that augment tumor growth. To better understand these signaling networks and identify potential targets for therapy of bone metastases, we characterized the secretome of a patient-derived xenograft, MDA-PCa-118b (PCa-118b), generated from osteoblastic bone lesion. PCa-118b induces osteoblastic tumors when implanted either in mouse femurs or subcutaneously. To study signaling molecules critical to these unique tumor/microenvironment-mediated events, we performed mass spectrometry on conditioned media of isolated PCa-118b tumor cells, and identified 26 secretory proteins, such as TGF-β2, GDF15, FGF3, FGF19, CXCL1, galectins, and β2-microglobulin, which represent both novel and previously published secreted proteins. RT-PCR using human versus mouse-specific primers showed that TGFβ2, GDF15, FGF3, FGF19, and CXCL1 were secreted from PCa-118b cells. TGFβ2, GDF15, FGF3, and FGF19 function as both autocrine and paracrine factors on tumor cells and stromal cells, that is, endothelial cells and osteoblasts. In contrast, CXCL1 functions as a paracrine factor through the CXCR2 receptor expressed on endothelial cells and osteoblasts. Thus, our study reveals a complex PCa bone metastasis secretome with paracrine and autocrine signaling functions that mediate cross-talk among multiple cell types within the tumor microenvironment.A distinct feature of human prostate cancer (PCa)¹ with lethal potential is the development of metastases in bone with a bone-forming phenotype (1). This property of PCa bone metastasis suggests that PCa cells have unique interactions with cells in the bone microenvironment. Cells that are known to be present in the bone microenvironment include osteoblasts, osteoclasts, adipocytes, fibroblasts, and endothelial cells. Communication between PCa cells and each of these cells in the microenvironment is known to promote metastatic growth. This communication involves metastatic PCa cells that secrete factors to affect stromal cells in the bone microenvironment. The tumor-modified stromal cells may further alter the properties of the PCa cells to allow them to progress in the bone environment (1). Determining how secretory proteins from the metastatic PCa cells affect the PCa/stromal communication network will lead to the development of strategies to treat bone metastases.Although men with PCa and bone metastasis most frequently present with osteoblastic bone lesions, the commonly-used PCa cell lines to study metastatic properties, for example, PC3 and C4–2B, induce osteolytic or mixed osteoblastic/osteolytic lesions, respectively, when the cells are implanted into mouse femurs or tibia (2). In contrast, the PCa-118b patient-derived xenograft (PDX), generated from an osteoblastic bone lesion of a patient with PCa and bone metastasis, shows phenotypic characteristics similar to the tumor from which it was derived, including induction of a strong osteoblastic response when implanted into femurs (3). Interestingly, PCa-118b cells are also able to induce ectopic bone formation when implanted subcutaneously (3, 4). The capacity of PCa-118b cells to induce bone formation, in which human tumor cells interact with the murine stromal microenvironment, makes this PDX an ideal model system to study tumor-microenvironment signaling pathways that create a bone-like tumor microenvironment conducive to metastatic PCa growth.In this study, we identified secreted factors from the conditioned medium of isolated PCa-118b cells by mass spectrometry. A total of 26 secretory proteins, including cytokines and growth factors, were identified. Human- and mouse-specific PCR probes were used to identify the cells that expressed these factors. Analysis of the receptor for the corresponding secreted factor determined whether the factor exerted activities in a paracrine and/or autocrine manner. The effects of selected factors on PCa cells or stromal cells, including osteoblasts and endothelial cells, were also examined. Our studies showed that PCa-118b cells secreted multiple factors that establish an autocrine or paracrine signaling network that can mediate cross-talk among multiple cell types within the bone microenvironment.     

Proliferation of Mouse Prostate Cancer Cells Inflamed by Trichomonas vaginalis

Our objective was to investigate whether inflammatory microenvironment induced by Trichomonas vaginalis infection can stimulate proliferation of prostate cancer (PCa) cells in vitro and in vivo mouse experiments. The production of CXCL1 and CCL2 increased when cells of the mouse PCa cells (TRAMP-C2 cell line) were infected with live T. vaginalis. T. vaginalis-conditioned medium (TCM) prepared from co-culture of PCa cells and T. vaginalis increased PCa cells migration, proliferation and invasion. The cytokine receptors (CXCR2, CCR2, gp130) were expressed higher on the PCa cells treated with TCM. Pretreatment of PCa cells with antibodies to these cytokine receptors significantly reduced the proliferation, mobility and invasiveness of PCa cells, indicating that TCM has its effect through cytokine-cytokine receptor signaling. In C57BL/6 mice, the prostates injected with T. vaginalis mixed PCa cells were larger than those injected with PCa cells alone after 4 weeks. Expression of epithelial-mesenchymal transition markers and cyclin D1 in the prostate tissue injected with T. vaginalis mixed PCa cells increased than those of PCa cells alone. Collectively, it was suggested that inflammatory reactions by T. vaginalis-stimulated PCa cells increase the proliferation and invasion of PCa cells through cytokine-cytokine receptor signaling pathways.     

Antioxidation and Antiapoptosis Characteristics of Heme Oxygenase-1 Enhance Tumorigenesis of Human Prostate Carcinoma Cells

Heme oxygenase-1 (HO-1) has antiinflammatory and antioxidant properties and is deemed as a tissue protector. However, effects of HO-1 in prostate cancer remain in controversy. We evaluated the role of HO-1 in prostate carcinoma in vitro and in vivo.Overexpression of HO-1 did not affect prostate cell proliferation in the normal condition but enhanced cell proliferation under serum starvation. HO-1 overexpression enhanced cell invasion of PC-3 cells through epithelial–mesenchymal transition (EMT) induction, which was supported by increased Slug, N-cadherin, and vimentin expressions. In the xenograft animal study, HO-1 overexpression enhanced PC-3 cell tumor growth in vivo. HO-1 attenuated reactive oxygen species induced by H₂O₂ or pyocyanin treatment in PC-3 and DU145 cells. HO-1 further reduced PC-3 and DU145 cell apoptosis induced by H₂O₂ or serum starvation. Our results suggested that HO-1 was able to increase prostate carcinoma cell invasion in vitro and tumor growth in vivo. The EMT induction and antioxidant and antiapoptotic effects of HO-1 in the prostate carcinoma cells may be responsible for these findings.     

Prostate cancer-derived exosomes promote osteoblast differentiation and activity through phospholipase D2

Prostate cancer (PCa) is the most frequent cancer in men aged 65 and over. PCa mainly metastasizes in the bone, forming osteosclerotic lesions, inducing pain, fractures, and nerve compression. Cancer cell-derived exosomes participate in the metastatic spread, ranging from oncogenic reprogramming to the formation of pre-metastatic niches. Moreover, exosomes were recently involved in the dialog between PCa cells and the bone metastasis microenvironment. Phospholipase D (PLD) isoforms PLD1/2 catalyze the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA), regulating tumor progression and metastasis. PLD is suspected to play a role in exosomes biogenesis. We aimed to determine whether PCa-derived exosomes, through PLD, interact with the bone microenvironment, especially osteoblasts, during the metastatic process. Here we demonstrate for the first time that PLD2 is present in exosomes of C4-2B and PC-3 cells. C4-2B-derived exosomes activate proliferation and differentiation of osteoblasts models, by stimulating ERK 1/2 phosphorylation, by increasing the tissue-nonspecific alkaline phosphatase activity and the expression of osteogenic differentiation markers. Contrariwise, when C4-2B exosomes are generated in the presence of halopemide, a PLD pan-inhibitor, they lose their ability to stimulate osteoblasts. Furthermore, the number of released exosomes diminishes significantly (−40%). When the PLD product PA is combined with halopemide, exosome secretion is fully restored. Taken together, our results indicate that PLD2 stimulates exosome secretion in PCa cell models as well as their ability to increase osteoblast activity. Thus, PLD2 could be considered as a potent player in the establishment of PCa bone metastasis acting through tumor cell derived-exosomes.     

Tumour-Derived Reg3A Educates Dendritic Cells to Promote Pancreatic Cancer Progression

As a pancreatic inflammatory marker, regenerating islet-derived protein 3A (Reg3A) plays a key role in inflammation-associated pancreatic carcinogenesis by promoting cell proliferation, inhibiting apoptosis, and regulating cancer cell migration and invasion. This study aimed to reveal a novel immuno-regulatory mechanism by which Reg3A modulates tumour-promoting responses during pancreatic cancer (PC) progression. In an in vitro Transwell system that allowed the direct co-culture of human peripheral blood-derived dendritic cells (DCs) and Reg3A-overexpressing/ silenced human PC cells, PC cell-derived Reg3A was found to downregulate CD80, CD83 and CD86 expression on educated DCs, increase DC endocytic function, inhibit DC-induced T lymphocyte proliferation, reduce IL-12p70 production, and enhance IL-23 production by DCs. The positive effect of tumour-derived Reg3A-educated human DCs on PC progression was demonstrated in vivo by intraperitoneally transferring them into PC-implanted severe combined immunodeficiency (SCID) mice reconstituted with human T cells. A Reg3A-JAK2/STAT3 positive feedback loop was identified in DCs educated with Reg3A. In conclusion, as a tumour-derived factor, Reg3A acted to block the differentiation and maturation of the most important antigen-presenting cells, DCs, causing them to limit their potential anti-tumour responses, thus facilitating PC escape and progression.     

Somatostatin Derivate (smsDX) Attenuates the TAM-Stimulated Proliferation,Migration and Invasion of Prostate Cancer via NF-κB Regulation

Tumor development and progression are influenced by macrophages of the surrounding microenvironment. To investigate the influences of an inflammatory tumor microenvironment on the growth and metastasis of prostate cancer, the present study used a co-culture model of prostate cancer (PCa) cells with tumor-associated macrophage (TAM)-conditioned medium (MCM). MCM promoted PCa cell (LNCaP, DU145 and PC-3) growth, and a xenograft model in nude mice consistently demonstrated that MCM could promote tumor growth. MCM also stimulated migration and invasion in vitro. Somatostatin derivate (smsDX) significantly attenuated the TAM-stimulated proliferation, migration and invasion of prostate cancer. Immunohistochemistry revealed that NF-κB was over-expressed in PCa and BPH with chronic inflammatory tissue specimens and was positively correlated with macrophage infiltration. Further investigation into the underlying mechanism revealed that NF-κB played an important role in macrophage infiltration. SmsDX inhibited the paracrine loop between TAM and PCa cells and may represent a potential therapeutic agent for PCa.     

NGF from pancreatic stellate cells induces pancreatic cancer proliferation and invasion by PI3K/AKT/GSK signal pathway

Pancreatic cancer (PC) is a continuously high lethal disease, and the tumour microenvironment plays a pivotal role during PC progression. Herein, we focus on that the Nerve growth factor (NGF)/Tropomyosin-related kinase A (TrkA), in pancreatic stellate cells-pancreatic cancer cells (PSCs-PC cells) co-culture system, influences PC proliferation and invasion. The model of PC cells and PSCs was directly co-cultured in a no-touch manner, using the Transwell as the co-culture system. NGF and TrkA expression was measured in cultured system by real-time PCR, immunofluorescence, Western blotting analysis or ELISA. Small interfering RNA transfection was used to regulate the expression of TrkA in PC cells. The promotion of cancer invasion was investigated using Matrigel Transwell assay. In our study, NGF/TrkA is overexpressed in PSCs-PC cells co-culture system and promotes the invasion and proliferation of PC cells. And the epithelial-mesenchymal transition-related genes are influenced by si-TrkA. What's more, NGF/TrkA regulates the PC cell proliferation and invasion via activation of PI3K/AKT/GSK signalling. The present study demonstrated NGF/TrkA promoted the PC cell proliferation and invasion in the co-culture system by the activation of the PI3K/AKT/GSK signal cascade, providing a potential therapeutic target for PC patients.     

Regulation and Methylation of Tumor Suppressor MiR-124 by Androgen Receptor in Prostate Cancer Cells

Prostate cancer (PCa) is the most frequently diagnosed cancer for men in the developed world. Androgen receptor signaling pathway plays an important role in prostate cancer progression. Recent studies show that microRNA miR-124 exerts a tumor suppressive function in prostate cancer. However, the relationship between AR and miR-124 is unclear. In the present study, we found a negative feedback loop between AR and miR-124 expression. On one hand, miR-124 was a positively regulated target gene of the AR, on the other hand, overexpression of miR-124 inhibited the expression of AR. In addition, we found that miR-124-2 and miR-124-3 promoters were hypermethylated in AR-negative PCa cells. Furthermore, overexpression of miR-124 inhibited proliferation rates and invasiveness capacity of PCa cells in vitro, and suppressed xenograft tumor growth in vivo. Taken together, our results support a negative feedback loop between AR and miR-124 expression. Methylation of miR-124-2 and miR-124-3 may serve as a biomarker for AR-negative PCa cells, and overexpression of miR-124 might be of potential therapeutic value for the treatment of PCa.     

Differential Utilization of Dietary Fatty Acids in Benign and Malignant Cells of the Prostate

Tumor cells adapt via metabolic reprogramming to meet elevated energy demands due to continuous proliferation, for example by switching to alternative energy sources. Nutrients such as glucose, fatty acids, ketone bodies and amino acids may be utilized as preferred substrates to fulfill increased energy requirements. In this study we investigated the metabolic characteristics of benign and cancer cells of the prostate with respect to their utilization of medium chain (MCTs) and long chain triglycerides (LCTs) under standard and glucose-starved culture conditions by assessing cell viability, glycolytic activity, mitochondrial respiration, the expression of genes encoding key metabolic enzymes as well as mitochondrial mass and mtDNA content. We report that BE prostate cells (RWPE-1) have a higher competence to utilize fatty acids as energy source than PCa cells (LNCaP, ABL, PC3) as shown not only by increased cell viability upon fatty acid supplementation but also by an increased ß-oxidation of fatty acids, although the base-line respiration was 2-fold higher in prostate cancer cells. Moreover, BE RWPE-1 cells were found to compensate for glucose starvation in the presence of fatty acids. Of notice, these findings were confirmed in vivo by showing that PCa tissue has a lower capacity in oxidizing fatty acids than benign prostate. Collectively, these metabolic differences between benign and prostate cancer cells and especially their differential utilization of fatty acids could be exploited to establish novel diagnostic and therapeutic strategies.     

Microenvironment Modulates Osteogenic Cell Lineage Commitment in Differentiated Embryonic Stem Cells

Background

Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored.

Methodology/Principal Findings

ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.

Conclusions/Significance

We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/differentiation conditions based upon the influence of microenvironment.     

Sonic Hedgehog Regulates Osteoblast Function by Focal Adhesion Kinase Signaling in the Process of Fracture Healing

Several biological studies have indicated that hedgehog signaling plays an important role in osteoblast proliferation and differentiation, and sonic hedgehog (SHH) expression is positively correlated with phosphorylated focal adhesion kinase (FAK) Tyr³⁹⁷. However, the relationship between them and their role in the process of normal fracture repair has not been clarified yet. Immunohistochemical analysis revealed that SHH and pFAK Tyr³⁹⁷ were expressed in bone marrow cells and that pFAK Tyr³⁹⁷ was also detected in ALP-positive osteoblasts near the TRAP-positive osteoclasts in the fracture site in the ribs of mice on day 5 after fracture. SHH and pFAK Tyr³⁹⁷ were detectable in osteoblasts near the hypertrophic chondrocytes on day 14. In vitro analysis showed that SHH up-regulated the expression of FAK mRNA and pFAK Tyr³⁹⁷ time dependently in osteoblastic MC3T3-E1 cells. Functional analysis revealed that 5 lentivirus encoding short hairpin FAK RNAs (shFAK)-infected MC3T3-E1 cell groups displayed a round morphology and decreased proliferation, adhesion, migration, and differentiation. SHH stimulated the proliferation and differentiation of MC3T3-E1 cells, but had no effect on the shFAK-infected cells. SHH also stimulated osteoclast formation in a co-culture system containing MC3T3-E1 and murine CD11b⁺ bone marrow cells, but did not affect the shFAK-infected MC3T3-E1 co-culture group. These data suggest that SHH signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture and regulated their proliferation and differentiation, as well as osteoclast formation, via FAK signaling.     

Microencapsulation of Neuroblastoma Cells and Mesenchymal Stromal Cells in Collagen Microspheres: A 3D Model for Cancer Cell Niche Study

There is a growing trend for researchers to use in vitro 3D models in cancer studies, as they can better recapitulate the complex in vivo situation. And the fact that the progression and development of tumor are closely associated to its stromal microenvironment has been increasingly recognized. The establishment of such tumor supportive niche is vital in understanding tumor progress and metastasis. The mesenchymal origin of many cells residing in the cancer niche provides the rationale to include MSCs in mimicking the niche in neuroblastoma. Here we co-encapsulate and co-culture NBCs and MSCs in a 3D in vitro model and investigate the morphology, growth kinetics and matrix remodeling in the reconstituted stromal environment. Results showed that the incorporation of MSCs in the model lead to accelerated growth of cancer cells as well as recapitulation of at least partially the tumor microenvironment in vivo. The current study therefore demonstrates the feasibility for the collagen microsphere to act as a 3D in vitro cancer model for various topics in cancer studies.     

Inhibition of vascular smooth muscle cell proliferation by chronic hemin treatment

Hemin, an oxidized form of heme, is an essential regulator of gene expression and cell cycle progression. Our laboratory previously reported (34) that chronic hemin treatment of spontaneously hypertensive rats reversed the eutrophic inward remodeling of small peripheral arteries. Whether long-term treatment of cultured vascular smooth muscle cells (VSMCs) with hemin alters the proliferation status of these cells has been unknown. In the present study, hemin treatment at 5 muM for 4, 7, 14, and 21 days significantly inhibited the proliferation of cultured rat aortic VSMCs (A-10 cells) by arresting cells at G0/G1 phases so as to decelerate cell cycle progression. Heme oxygenase (HO) activity and inducible HO-1 protein expression were significantly increased by hemin treatment. HO inhibitor tin protoporphyrin IX (SnPP) abolished the effects of hemin on cell proliferation and HO activity. Interestingly, hemin-induced HO-1 expression was further increased in the presence of SnPP. Hemin treatment had no significant effect on the expression of constitutive HO-2. Expression of p21 protein and the level of reactive oxygen species (ROS) were decreased by hemin treatment, which was reversed by application of SnPP. After removal of hemin from culture medium, inhibited cell proliferation and increased HO-1 expression in VSMCs were returned to control level within 1 wk. Transfection with HO-1 small interfering RNA significantly knocked down HO-1 expression and decreased HO activity, but had no effect on HO-2 expression, in cells treated with or without hemin for 7 days. The inhibitory effect of hemin on cell proliferation was abolished in HO-1 silenced cells. It is concluded that induction of HO-1 and, consequently, increased HO activity are responsible for the chronic inhibitory effect of hemin on VSMC proliferation. Changes in the levels of p21 and ROS might also participate in the cellular effects of hemin.     

Involvement of Macrophages in Proliferation of Prostate Cancer Cells Infected with Trichomonas vaginalis

Macrophages play a key role in chronic inflammation, and are the most abundant immune cells in the tumor microenvironment. We investigated whether an interaction between inflamed prostate cancer cells stimulated with Trichomonas vaginalis and macrophages stimulates the proliferation of the cancer cells. Conditioned medium was prepared from T. vaginalis-infected (TCM) and uninfected (CM) mouse prostate cancer (PCa) cell line (TRAMP-C2 cells). Thereafter conditioned medium was prepared from macrophages (J774A.1 cell line) after incubation with CM (MCM) or TCM (MTCM). When TRAMP-C2 cells were stimulated with T. vaginalis, protein and mRNA levels of CXCL1 and CCL2 increased, and migration of macrophages toward TCM was more extensive than towards CM. Macrophages stimulated with TCM produced higher levels of CCL2, IL-6, TNF-α, their mRNAs than macrophages stimulated with CM. MTCM stimulated the proliferation and invasiveness of TRAMP-C2 cells as well as the expression of cytokine receptors (CCR2, GP130, CXCR2). Importantly, blocking of each cytokine receptors with anti-cytokine receptor antibody significantly reduced the proliferation and invasiveness of TRAMP-C2 cells. We conclude that inflammatory mediators released by TRAMP-C2 cells in response to infection by T. vaginalis stimulate the migration and activation of macrophages and the activated macrophages stimulate the proliferation and invasiveness of the TRAMP-C2 cells via cytokine-cytokine receptor binding. Our results therefore suggested that macrophages contribute to the exacerbation of PCa due to inflammation of prostate cancer cells reacted with T. vaginalis.