Effects of Obesity on the Regulation of Macrophage Population in the Prostate Tumor Microenvironment.

Obesity is associated with a greater risk of prostate cancer mortality. However, the mechanisms connecting obesity to the progression of prostate cancer remain unknown. This study determined the impact of obesity on macrophage recruitment and tumor-associated macrophage (TAM) polarization in the prostate tumor microenvironment, since a high concentration of TAMs in tumors has been linked to progression in prostate cancer. We utilized an in vitro model in which pre-adipocytes, prostate cancer cells, and macrophages were exposed to sera from obese or nonobese men, or conditioned media generated under obese or nonobese conditions. Matrigel invasion chambers were used to assess macrophage recruitment in vitro, and immunohistochemical analysis evaluated recruitment in a PTEN knockout mouse model. qPCR was used to measure mRNA levels of CCL2, COX-2, IL-10, TGF-beta, VEGF-A, arginase-1, and MMP-9. PGE2 production was measured by ELISA. Obesity increased macrophage and TAM recruitment, and increased mRNA levels of TAM markers in macrophages. Similarly, obese conditions increased CCL2 and COX-2 expression, as well as PGE2 levels in prostate cancer cells. COX-2 inhibition resulted in lower expression of obesity-induced TAM markers. Our data suggest that obesity promotes macrophage infiltration into the prostate tumor microenvironment, and induces TAM polarization through the COX-2/PGE2 pathway.

Obesity-related systemic factors promote an invasive phenotype in prostate cancer cells.

BACKGROUND: Obesity is associated with larger tumors, shorter time to PSA failure, and higher Gleason scores. However, the mechanism(s) by which obesity promotes aggressive prostate cancer remains unknown. We hypothesize that circulating factors related to obesity promote prostate cancer progression by modulating components of the metastatic cascade. METHODS: Male C57BL/6 mice (6 weeks) were fed an ad libitum diet-induced obesity (60% fat) or control diet (10% fat) for 12 weeks. Serum was collected, metabolic and inflammatory proteins were measured by an antibody array. Sera were used to measure, in vitro, characteristics of a metastatic phenotype. RESULTS: Comparable to obese men, obese sera contained higher levels or leptin, vascular endothelial growth factor, PAI-1, interleukin-6 (IL-6) and lower levels of testosterone. In prostate cells, serum was used to assess: proliferation, invasion, migration, epithelial-mesenchymal-transition (EMT) and matrix metalloproteinase (MMP) activity. LNCaP and PacMetUT1 cells exposed to obese sera increased proliferation, whereas PrEC and DU145 were unaffected. LNCaP, PacMetUT1 and DU145 cancer cells exposed to obese sera resulted in increased invasion, migration and MMP-9 activity. Prostate cancer cells exposed to obese sera showed increased vimentin, dispersion of e-cadherin and ß-catenin from the plasma membrane. CONCLUSION: We report, prostate cancer cells exposed to sera from obese mice increases proliferation, invasion, migration, MMP activity and induces changes in proteins critical for EMT.

Characterization of PacMetUT1, a recently isolated human prostate cancer cell line.

BACKGROUND: Existing prostate cancer cell lines have limitations. METHODS: Cells were characterized using Western blotting, immunohistochemistry, invasion into Matrigel, and by studying xenograft tumors. RESULTS: We describe a cell line (PacMetUT1) isolated from a lymph node of a 57-year-old male with prostate cancer. Compared to existing prostate cancer cell lines, the growth rate of PacMetUT1 xenograft tumors is slower with tumors occurring at injection sites and with metastases to lung and liver. Androgen receptor (AR) was detected in vivo by Western blotting and the cells responded to methyltrienolone (R1881). PacMetUT1 cells are more invasive in Matrigel than DU-145, PC-3, and LNCaP cells, and showed greater anchorage-independent growth in soft agar. The cells do not express prostate specific antigen (PSA) in vitro or in xenografts. However, the green fluorescent protein (GFP) gene was introduced and stably expressed in PacMetUT1 cells, allowing tumor imaging in vivo. Xenograft tumors show epithelial features and are positive for keratin, epithelial membrane antigen, EGF receptor, and E cadherin. In contrast, fibroblast markers vimentin, desmin, and Factor VIII, were negative. Karyotyping showed losses of 6p, 7q, 8p, 18q, and 22q, and gains of 8q and 9q; additional genetic material was observed at 2q and 12p. CONCLUSION: The PacMetUT1 cell line allows metastases to be assessed using a single animal model. Because of its slower growth, PacMetUT1 more closely mimics the human disease. Studies of tumor progression or metastasis can be conducted over a longer period of time.

Akt-induced endocrine therapy resistance is reversed by inhibition of mTOR signaling.

BACKGROUND: Resistance to endocrine therapy is a major impediment in breast cancer therapeutics. The Phosphatidylinositol-3-OH kinase (PI3K)/Protein kinase B (Akt/PKB) kinase signaling pathway has been implicated in altering breast cancer response to multiple therapies. How Akt modulates response is an area of significant clinical relevance. METHODS: We have used an in vitro model to discern the effects of robust Akt activity on breast cancer cellular response to endocrine therapies. RESULTS: High levels of Akt activity confer resistance to the aromatase inhibitor Letrozole (Let) and the selective estrogen receptor (ER) down-regulator Fulvestrant (ICI). Akt-induced resistance is not due to failure of these endocrine agents to inhibit estrogen receptor alpha activity. Instead, resistance is characterized by altered cell cycle and apoptotic response. Cotreatment with low concentrations of the mTOR inhibitor RAD-001 and either Let or ICI restores response of the resistant cells to levels observed in the responsive cells treated with either Let or ICI as a single agent. CONCLUSIONS: Our preliminary findings in experiments with RAD-001 indicate that cotreatment with mTOR inhibitors and either Let or ICI reverses the Akt-mediated resistance and restores responsiveness to antiestrogens. Concurrent ER and mTOR inhibition is therefore an effective strategy to overcome growth factor-induced resistance and bears significant implications for optimal clinical development of these agents in breast cancer treatment.

Urothelial progenitor cells: regional differences in the rat bladder.

OBJECTIVES: Because the trigone is a unique region in the caudal bladder with a higher risk of neoplasia, we hypothesized that this area would have a high proportion of progenitor cells. As yet there is no marker nor methodology to specifically isolate urothelial stem cells, and thus demonstrate multi-potential differentiation and self-renewal. Here, our goal was to evaluate the distribution of progenitor cells that carry two general major attributes of stem cells: clonogenicity and proliferative capacity. MATERIALS AND METHODS: The bladders of Fisher rats were divided into caudal and cephalic segments and primary cultures were established from the harvested urothelial cells. RESULTS: We found that colony-forming efficiency was almost 2-fold higher for cells from the caudal bladder compared to the cephalic bladder. Doubling time was significantly faster for cells harvested from the caudal bladder at initial plating. This suggested that the caudal bladder harbours a higher density of urothelial progenitor cells. With passage to p4, the differences between the upper and lower bladder were lost, suggesting selection of proliferative cells with serial passage. Based on Ki-67 staining, there was no geographical difference in cell proliferation under normal homeostatic in vivo conditions. CONCLUSIONS: These results demonstrate geographical sequestration of urothelial progenitor cells to the area of the bladder that encompasses the bladder neck and trigone, which may be a factor in pathological disparities between the trigone and remaining bladder.

Reduced PTEN expression in breast cancer cells confers susceptibility to inhibitors of the PI3 kinase/Akt pathway.

The PTEN protein is a lipid phosphatase with putative tumor suppressing abilities, including inhibition of the PI3K/Akt signaling pathway. Inactivating mutations or deletions of the PTEN gene, which result in hyper-activation of the PI3K/Akt signaling pathway, are increasingly being reported in human malignancies, including breast cancer, and have been related to features of poor prognosis and resistance to chemotherapy and hormone therapy. Prior studies in different tumor models have shown that, under conditions of PTEN deficiency, the PI3K/Akt signaling pathway becomes a fundamental proliferative and survival pathway, and that pharmacological inhibition of this pathway results in tumor growth inhibition. This study aimed to explore further this hypothesis in breast cancer cells. To this end, we have determined the growth response to inhibition of the PI3K/Akt signaling pathway in a series of breast cancer cell lines with different PTEN levels. The PTEN-negative cell line displayed greater sensitivity to the growth inhibitory effects of the PI3K inhibitor, LY294002 and rapamycin, an inhibitor of the PI3K/Akt downstream mediator mTOR, compared with the PTEN-positive cell lines. To determine whether or not these differences in response are specifically due to effects of PTEN, we developed a series of cell lines with reduced PTEN protein expression compared with the parental cell line. These reduced PTEN cells demonstrated an increased sensitivity to the anti-proliferative effects induced by LY294002 and rapamycin compared with the parental cells, which corresponded to alterations in cell cycle response. These findings indicate that inhibitors of mTOR, some of which are already in clinical development (CCI-779, an ester of rapamycin), have the potential to be effective in the treatment of breast cancer patients with PTEN-negative tumors and should be evaluated in this setting.

NF-kappa B inhibition markedly enhances sensitivity of resistant breast cancer tumor cells to tamoxifen.

Studies show that high Akt activity in breast carcinoma is associated with endocrine therapy resistance. Breast cancer cell lines expressing a constitutively active Akt are able to proliferate under reduced estrogen conditions, and are resistant to the growth inhibitory effects of tamoxifen. Understanding the targets of Akt signaling mediating tamoxifen resistance is of clinical significance. One possible target is nuclear factor kappa B (NF-kappa B), a transcription factor that plays a critical role in resistance to apoptosis and the induction of angiogenesis and invasion. In the present study, we found that Akt activity correlated with phosphorylation of I kappa B (the negative regulator of NF-kappa B), NF-kappa B DNA binding and tamoxifen resistance in vivo. Importantly, we found that co-treatment with the NF-kappa B inhibitor, parthenolide, or overexpression of I kappa B superrepressor restored tamoxifen sensitivity to our refractory Akt MCF-7 cells. These data suggest that activation of NF-kappa B via the PI3K/Akt signaling pathway may be a significant mechanism for development of endocrine therapy resistance in breast cancer, and that inhibition of NF-kappa B may be an effective treatment strategy to limit the progression of this disease.

Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity.

BACKGROUND: Tamoxifen resistance is the underlying cause of treatment failure in a significant number of patients with breast cancer. Activation of Akt, a downstream mediator in the phosphatidylinositol 3-kinase (PI3K) signaling pathway has been implicated as one of the mechanisms involved in tamoxifen resistance. Breast cancers with heightened Akt activity are frequently associated with an aggressive disease and resistance to chemo- and hormone-therapy-induced apoptosis. Inhibition of PI3K restores apoptotic response to tamoxifen in hyperactive Akt cells. Therefore, agents that demonstrate Akt inhibitory properties are attractive therapeutic agents for the treatment of hormone-resistant breast cancer. n-3 fatty acids have proven to be potent and efficacious broad-spectrum protein kinase inhibitors. MATERIALS AND METHODS: In this study we demonstrate that the n-3 fatty acid, eicosapentaenoic acid (EPA), inhibits the kinase activity of Akt. Co-treatment with EPA renders breast cancer cells that overexpress a constitutively active Akt more responsive to the growth inhibitory effects of tamoxifen by approximately 35%. CONCLUSIONS: These findings suggest that EPA may be useful for the treatment of tamoxifen-resistant breast cancer cells with high levels of activated Akt and provide the rationale to test this hypothesis in the clinic.

Wound dressings alter the colony-forming efficiency of keratinocytes in cultured sheet grafts.

Cultured keratinocyte grafts transplanted for skin wound repair are often affixed to a wound dressing to facilitate handling. In this study, the ability of five different types of wound dressings to support cell viability and maintain stem cell populations in the cultured grafts was determined. Postconfluent keratinocyte (NHK) sheets were attached to wound dressings for 24 h and then released by trypsinization. Cell viability was determined and NHKs were assessed for clonogenic capacity by colony-forming efficiency (CFE) assays. CFEs for NHKs exposed to a collagen-bonded, bilaminate membrane and a polyurethane film were significantly less than control. On the other hand, CFEs for NHKs exposed to a collagen/alginate dressing and to petrolatum-impregnated gauze were significantly greater than control. The choice of a wound dressing carrier has implications for maintaining long-term viability of the transplanted sheet of epithelium.

Modulation of human epidermal cell response to 2,3,7,8-tetrachlorodibenzo-p-dioxin by epidermal growth factor.

Cultured human epidermal cells were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of epidermal growth factor (EGF). In both normal keratinocytes and a spontaneously immortalized keratinocyte (SIK) line, TCDD treatment in the absence of EGF induced a marked reduction in colony size and cell number, and it perturbed colony morphology. These effects were largely prevented by EGF, indicating that growth factor action in the cellular microenvironment may considerably modify TCDD action in target cells. Both TCDD and EGF substantially reduced expression of the differentiation markers keratin 1 and keratin 10 in the normal and immortalized cells, and did so in an additive fashion. The cells did not display a general loss of differentiated function, since several other markers, including involucrin, were little affected. EGF dramatically stimulated telomerase activity in SIK cultures, and TCDD prevented this action but not by reducing cell growth. However, EGF did not stimulate telomerase activity in normal human epidermal cells despite an evident increase in their growth. The growth factor stimulation of telomerase in the minimally deviated SIK line suggests that derepression of enzyme activity in normal cells may occur in a stepwise fashion during neoplastic progression. TCDD could act as a late stage tumor promoter by selecting for variants in which telomerase is constitutively active.

Anti-proliferative activity and target cell catabolism of the vitamin D analog 1 alpha,24(S)-(OH)2D2 in normal and immortalized human epidermal cells.

Vitamin D analogs represent valuable new agents for the suppression of proliferation of a variety of cell types, including those of the skin. One such analog is the vitamin D2 metabolite, 1 alpha,24(S)-dihydroxyvitamin D2, which binds strongly to the vitamin D receptor and induces vitamin D-dependent gene expression in vitro. In the work described here, we studied the anti-proliferative activity and target cell metabolism of 1 alpha,24(S)-dihydroxyvitamin D2 in cells of human epidermal origin. We found this analog to be equally potent in its anti-proliferative effect to the hormone 1 alpha,25-dihydroxyvitamin D3. Furthermore, 1 alpha,24(S)-dihydroxyvitamin D2 was metabolized by the human keratinocyte cell line HPK1A-ras at a slower rate than either 1 alpha,25-dihydroxyvitamin D3 or calcipotriol, a drug used effectively in the treatment of psoriasis. We characterized the metabolic products of 1 alpha,24(S)-dihydroxyvitamin D2 as a mixture of side-chain truncated and hydroxylated products. The main product was identified by GC-MS and NMR techniques as 1 alpha,24(S),26-trihydroxyvitamin D2. The biological activity of this main product was determined in a vitamin D-dependent, growth-hormone reporter gene expression system to be lower than that of the parent molecule. We conclude from these data that 1 alpha,24(S)-dihydroxyvitamin D2 is a valuable new anti-proliferative agent with a slower rate of catabolism by cells of epidermal origin. Preliminary evidence suggests that the parent molecule, and not its products, is responsible for this biological activity in vitro.

Cellular expression of ceruloplasmin in baboon and mouse lung during development and inflammation.

Ceruloplasmin (CP) is an important extracellular antioxidant and free radical scavenger. Although CP is expressed mainly in the liver, recent studies have identified the lung as another major site of CP synthesis. The sites and cell types that are responsible for CP expression in baboon and mouse lung are described. CP mRNA is detected in primordial bronchial epithelium in baboon fetuses by 60 days of gestation. At 140 days of gestation and thereafter, CP mRNA is found in airway epithelium and in the ductal cells of the submucosal glands. In developing and mature mice, CP mRNA is present in epithelial cells throughout the airway. In endotoxin-treated mice, the amount of CP mRNA increases several-fold in large airways but increases only moderately in small airways. This suggests that the high concentration of CP in the mucus lining of the upper airway, which serves to filter harmful substances, is particularly important during stressful conditions. Endotoxin treatment in mice also results in the induction of high levels of CP mRNA in a subset of alveolar wall cells. The data suggest that the airway epithelial cells are the major source of CP in the lung fluid and support ceruloplasmin's critical role in host defense against oxidative damage and infection in the lung.

Cell type-specific and inflammatory-induced expression of haptoglobin gene in lung.

BACKGROUND: Haptoglobin (HP) is a hemoglobin-binding protein and a major acute phase reactant. Recently, HP has been shown to possess antioxidant and angiogenic properties. HP is known to be produced mainly in the liver. Expression of HP in specific cells of nonhepatic origin including lung cells has not been studied before. The presence of extracellular plasma proteins in lung epithelial fluid has been assumed to be of blood serum origin. EXPERIMENTAL DESIGN: To investigate the expression of the HP gene in lung, the presence of HP mRNA and the production of HP protein in the lung were examined by Northern blot analysis and immunoprecipitation, respectively. Cellular expression of HP during development and inflammation were studied by in situ hybridization with lung tissues derived from different gestational stages from baboons and mice and from mice treated with lipopolysaccharide. RESULTS: Northern blot and in situ hybridization analyses established a high level of expression of HP in fetal and adult lung tissues, which were confined to the epithelial lining of the airways in mouse and baboon. After inflammation had been induced in vivo, expression of the HP gene rose fourfold in lung, an increase compatible with that observed in normal mouse liver. However, HP mRNA level was not significantly altered in airway epithelium. Instead, HP expression in alveolar epithelial cells, most likely type 2 cells, was strongly increased. CONCLUSIONS: Our data suggest that locally synthesized HP provides a major source of antioxidant and/or antimicrobial activity in the mucus blanket as well as in the alveolar fluid in the lung. The regulation and cell type-specific expression of HP during development and inflammation indicate a protective role for HP in lung and confirm recent reports that HP plays important roles in protecting against infection and in repairing injured tissues.

2,3,7,8-Tetrachlorodibenzo-p-dioxin sensitization of cultured human epidermal cells to carcinogenic heterocyclic amine toxicity.

Treatment of cultures of spontaneously immortalized human epidermal cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) sensitized them to carcinogen toxicity. While the tryptophan pyrolysis product 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and the mycotoxin sterigmatocystin were highly toxic to the cultures at moderate concentration (1 microgram/ml), the potency of each agent was increased > or = 10-fold in the presence of TCDD. A toxicity increase was also evident in the several-fold stimulation by TCDD of protein and DNA adducts formed by Trp-P-1. In contrast, the cells were insensitive to toxicity from 3-amino-1-methyl-5H-pyrido[4,3-b]indole. DNA damage mediated by Trp-P-1 was capable of producing inheritable effects, as judged by the induction of hprt mutants in a TCDD-stimulated fashion. Northern blotting showed that TCDD strongly stimulated expression of P4501A1 and 1B1 in the cells, enzymes important for xenobiotic metabolism. These findings demonstrate the potential usefulness of SIK cultures as a model for studying keratinocyte responses to carcinogens activated by TCDD-induced cytochromes P450.

Predictive value of liver slices for metabolism and toxicity in vivo: use of acetaminophen as a model hepatotoxicant.

To establish the usefulness of liver slices as predictive models for in vivo metabolism and toxicity, acetaminophen was used as a model hepatotoxicant for which the role of metabolism in toxicity is well documented. Acetaminophen was incubated with liver slices prepared from the rat and hamster since these species differ in susceptibility to acetaminophen-induced hepatotoxicity. Formation of acetaminophen metabolites (sulfate, glucuronide, and glutathione conjugate), slice glutathione levels, and slice histopathology were assessed. Acetaminophen (0.5, 1, and 2 mM) induced a dose-dependent depletion of glutathione in hamster slices (sensitive species) but not rat slices (insensitive species). Formation of the acetaminophen toxic metabolite, as measured by glutathione conjugate formation, was much lower in rat slices compared with hamster slices. Rat slices also showed greater activity of the nontoxic sulfation and glucuronidation pathways. These data predicted an 11-fold greater susceptibility to toxicity in the hamster based on the proportion of the dose metabolized to the reactive species. In vivo data, using hepatic glutathione depletion as an indicator of toxic metabolite formation, would predict a similar difference (13.3-fold) between the hamster and the rat. To ascertain if the characteristic centrilobular lesion induced by acetaminophen in vivo could be reproduced in vitro, liver slice histopathology was assessed 6 and 12 hr after a 2-hr treatment with acetaminophen (2 mM). Discrete damage to the centrilobular regions of the liver were noted in hamster but not rat slices. Overall, based on metabolite formation, glutathione depletion, and slice histopathology, the liver slices were excellent predictors of acetaminophen hepatotoxicity in vivo.

Elevation of cell cycle control proteins during spontaneous immortalization of human keratinocytes.

A human line of spontaneously immortalized keratinocytes (SIK cells) has been derived from ostensibly normal epidermis and has proven useful in dissecting molecular changes associated with immortalization. The original cultures had a normal karyotype and a colony forming efficiency of approximately 3% through 10 passages. At passage 15, after which normal strains ordinarily senesce, these cells continued vigorous growth and gradually increased in colony forming efficiency, stabilizing at approximately 30% by passage 40. During the early stage of increasing colony forming efficiency, the cells acquired a single i(6p) chromosomal aberration and 5- to 10-fold increases in expression of the cell-cycle control proteins cyclin A, cyclin B, and p34cdc2. Additional chromosomal aberrations accumulated at later passages (i(8q) and +7), but the i(6p) and the increased expression of cell-cycle proteins were maintained, raising the possibility that these features were important for immortalization. Regulation of cell growth and differentiation in the cultures appeared minimally altered compared with normal keratinocytes as judged by their microscopic appearance and generation of abortive colonies, sensitivity to growth suppression by transforming growth factor-beta and tetradecanoylphorbol acetate, and dependence upon epidermal growth factor for progressive growth.