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Sialic acid (SA) is a monosaccharide usually linked to the terminus of glycan chains on the cell surface. It plays a crucial role in many biological processes, and hypersialylation is a common feature in cancer. Lectins are widely used to analyze the cell surface expression of SA. However, these protein molecules are usually expensive and easily denatured, which calls for the development of alternative glycan-specific receptors and cell imaging technologies. In this study, SA-imprinted fluorescent core-shell molecularly imprinted polymer particles (SA-MIPs) were employed to recognize SA on the cell surface of cancer cell lines. The SA-MIPs improved suspensibility and scattering properties compared with previously used core-shell SA-MIPs. Although SA-imprinting was performed using SA without preference for the α2,3- and α2,6-SA forms, we screened the cancer cell lines analyzed using the lectins Maackia Amurensis Lectin I (MAL I, α2,3-SA) and Sambucus Nigra Lectin (SNA, α2,6-SA). Our results show that the selected cancer cell lines in this study presented a varied binding behavior with the SA-MIPs. The binding pattern of the lectins was also demonstrated. Moreover, two different pentavalent SA conjugates were used to inhibit the binding of the SA-MIPs to breast, skin, and lung cancer cell lines, demonstrating the specificity of the SA-MIPs in both flow cytometry and confocal fluorescence microscopy. We concluded that the synthesized SA-MIPs might be a powerful future tool in the diagnostic analysis of various cancer cells.
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Fibroblast growth factor receptors (FGFRs) 1-4 are involved in prostate cancer (PCa) regulation, but the role of FGFR-like 1 (FGFRL1) in PCa is unclear. FGFRL1 expression was studied by qRT-PCR and immunohistochemistry of patient tissue microarrays (TMAs) and correlated with clinical patient data. The effects of FGFRL1 knockdown (KD) in PC3M were studied in in vitro culture models and in mouse xenograft tumors. Our results showed that FGFRL1 was significantly upregulated in PCa. The level of membranous FGFRL1 was negatively associated with high Gleason scores (GSs) and Ki67, while increased cytoplasmic and nuclear FGFRL1 showed a positive correlation. Cox regression analysis indicated that nuclear FGFRL1 was an independent prognostic marker for biochemical recurrence after radical prostatectomy. Functional studies indicated that FGFRL1-KD in PC3M cells increases FGFR signaling, whereas FGFRL1 overexpression attenuates it, supporting decoy receptor actions of membrane-localized FGFRL1. In accordance with clinical data, FGFRL1-KD markedly suppressed PC3M xenograft growth. Transcriptomics of FGFRL1-KD cells and xenografts revealed major changes in genes regulating differentiation, ECM turnover, and tumor-stromal interactions associated with decreased growth in FGFRL1-KD xenografts. Our results suggest that FGFRL1 upregulation and altered cellular compartmentalization contribute to PCa progression. The nuclear FGFRL1 could serve as a prognostic marker for PCa patients.
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BACKGROUND: Breast cancer is a very common cancer that can be severe if not discovered early. The current tools to detect breast cancer need improvement. Cancer has a universal tendency to affect glycosylation. The glycosylation of circulating extracellular vesicle-associated glycoproteins, and mucins may offer targets for detection methods and have been only explored in a limited capacity. AIM: Our aim was to develop an approach to detect the aberrant glycosylation of mucins and extracellular vesicle-associated glycoproteins from human sera using fluorescent nanoparticles, and preliminarily evaluate this approach for the differential diagnosis of breast cancer. METHODS AND RESULTS: The assay involved immobilizing glycosylated antigens using monoclonal antibodies and then probing their glycosylation by using lectins and glycan-specific antibodies coated on Eu+3 -doped nanoparticles. Detection of mucin 1 and mucin 16 glycosylation with wheat germ agglutinin, and detection of the extracellular vesicle-associated CD63 were found to have better diagnostic ability for localized breast cancer than the conventional assays for mucin 1 and mucin 16 based tumor markers when the receiver operating characteristics were compared. CONCLUSIONS: These results indicate that successful differential diagnosis of primary breast cancer may be aided by detecting cancer-associated glycosylation of mucin 1 and mucin 16, and total concentration of CD63, in human serum.
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Neoplasias de la Mama , Vesículas Extracelulares , Biomarcadores de Tumor , Neoplasias de la Mama/diagnóstico , Antígeno Ca-125 , Femenino , Glicoproteínas , Glicosilación , Humanos , Mucina-1 , MucinasRESUMEN
BACKGROUND: Patient-derived xenografts (PDXs) are considered to better recapitulate the histopathological and molecular heterogeneity of human cancer than other preclinical models. Despite technological advances, PDX models from hormone naïve primary prostate cancer are scarce. We performed a detailed analysis of PDX methodology using a robust subcutaneous model and fresh tissues from patients with primary hormone naïve prostate cancer. METHODS: Clinical prostate tumor specimens (n=26, Gleason score 6-10) were collected from robotic-assisted laparoscopic radical prostatectomies at Turku University Hospital (Turku, Finland), cut into pieces, and implanted subcutaneously into 84 immunodeficient mice. Engraftments and the adjacent material from prostatic surgical specimens were compared using histology, immunohistochemistry and DNA sequencing. RESULTS: The probability of a successful engraftment correlated with the presence of carcinoma in the implanted tissue. Tumor take rate was 41%. Surprisingly, mouse hormone supplementation inhibited tumor take rate, whereas the degree of mouse immunodeficiency did not have an effect. Histologically, the engrafted tumors closely mimicked their parental tumors, and the Gleason grades and copy number variants of the engraftments were similar to those of their primary tumors. Expression levels of androgen receptor, prostate-specific antigen, and keratins were retained in engraftments, and a detailed genomic analysis revealed high fidelity of the engraftments with their corresponding primary tumors. However, in the second or third passage of tumors, the carcinoma areas were almost completely replaced by benign tissue with frequent degenerative or metaplastic changes. CONCLUSIONS: Subcutaneous primary prostate engraftments preserve the phenotypic and genotypic landscape. Thus, they serve a potential model for personalized medicine and preclinical research but their use may be limited to the first passage.
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Advanced breast cancer has a high incidence of bone metastases. In bone, breast cancer cells induce osteolytic or mixed bone lesions by inducing an imbalance in bone formation and resorption. Activated fibroblast growth factor receptors (FGFRs) are important in regulation of tumor growth and bone remodeling. In this study we used FGFR1 and FGFR2 gene amplifications containing human MFM223 breast cancer cells in an experimental xenograft model of breast cancer bone growth using intratibial inoculation technique. This model mimics bone metastases in breast cancer patients. The effects of an FGFR inhibitor, dovitinib dilactic acid (TKI258) on tumor growth and tumor-induced bone changes were evaluated. Cancer-induced bone lesions were smaller in dovitinib-treated mice as evaluated by X-ray imaging. Peripheral quantitative computed tomography imaging showed higher total and cortical bone mineral content and cortical bone mineral density in dovitinib-treated mice, suggesting better preserved bone mass. CatWalk gait analysis indicated that dovitinib-treated mice experienced less cancer-induced bone pain in the tumor-bearing leg. A trend towards decreased tumor growth and metabolic activity was observed in dovitinib-treated mice quantified by positron emission tomography imaging with 2-[18F]fluoro-2-deoxy-D-glucose at the endpoint. We conclude that dovitinib treatment decreased tumor burden, cancer-induced changes in bone, and bone pain. The results suggest that targeting FGFRs could be beneficial in breast cancer patients with bone metastases.
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Bisphosphonates are used for prevention of osteoporosis and metastatic bone diseases. Anti-invasive effects on various cancer cells have also been reported, but the mechanisms involved are not well-understood. We investigated the effects of the nitrogen-containing bisphosphonate alendronate (ALN) on the regulation of actin cytoskeleton in PC-3 cells. We analyzed the ALN effect on the organization and the dynamics of actin, and on the cytoskeleton-related regulatory proteins cofilin, p21-associated kinase 2 (PAK2), paxillin and focal adhesion kinase. Immunostainings of cofilin in ALN-treated PC-3 cells and xenografts were performed, and the role of cofilin in ALN-regulated F-actin organization and migration/invasion in PC-3 cells was analyzed using cofilin knockdown and transfection. We demonstrate that disrupted F-actin organization and decreased cell motility in ALN-treated PC-3 cells were associated with decreased levels of total and phosphorylated cofilin. PAK2 levels were also lowered but adhesion-related proteins were not altered. The knockdown of cofilin similarly impaired F-actin organization and decreased invasion of PC-3 cells, whereas in the cells transfected with a cofilin expressing vector, ALN treatment did not decrease cellular cofilin levels and migration as in mock transfected cells. ALN also reduced immunohistochemical staining of cofilin in PC-3 xenografts. Our results suggest that reduction of cofilin has an important role in ALN-induced disruption of the actin cytoskeleton and inhibition of the PC-3 cell motility and invasion. These data also support the idea that the nitrogen-containing bisphosphonates could be efficacious in inhibition of prostate cancer invasion and metastasis, if delivered in a pharmacological formulation accessible to the tumors.
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Signaling via estrogen receptors (ER) is recognized as an essential part of the immune regulation, and ER-mediated signaling is involved in autoimmune reactions. Especially ERα activation in immune cells has been suggested to skew cytokine production toward Th2/M2-type mediators, which can have protective effect on inflammatory diseases and reduce Th1 and Th17 responses. These effects are caused by increased alternative activation of macrophages and changes in the activation of different T cell populations. In humans, hormonal status has been shown to have a major impact on several inflammatory diseases. Selective estrogen receptor modulators (SERMs) are ER ligands that regulate ER actions in a tissue-specific manner mostly lacking the adverse effects of steroid hormones. The impact of SERMs on the immune system is less studied, but it is suggested that certain SERMs may also produce immunoprotective effects. Here, we show that two novel SERMs and raloxifene affect immune cells by promoting M2 macrophage phenotype, alleviating NFκB activity, inhibiting T cell proliferation, and stimulating the production of anti-inflammatory compounds such as IL10 and IL1 receptor antagonist. Thus, these compounds have high potency as drug candidates against autoimmune diseases.
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Inflamación/tratamiento farmacológico , Receptores de Lipopolisacáridos/análisis , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Transducción de Señal/efectos de los fármacos , Adulto , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Humanos , Macrófagos/efectos de los fármacos , Persona de Mediana Edad , FN-kappa B/efectos de los fármacos , Clorhidrato de Raloxifeno/farmacología , Células Th2 , Adulto JovenRESUMEN
BACKGROUND: Despite recent therapeutic and diagnostic advances, prostate cancer remains the second leading cause of cancer-related deaths among men in the Western world. Oncolytic viruses that replicate selectively in tumour cells represent a novel treatment candidate for these malignancies. METHODS: We analysed infectivity of avirulent Semliki Firest virus SFV-VA7 in human prostate cancer cell lines VCaP, LNCaP and 22Rv1 and in nonmalignant prostate epithelial cell line RWPE-1. Therapeutic potency of SFV-VA7 was evaluated in subcutaneous and orthotopic mouse LNCaP xenograft models. RESULTS: SFV-VA7 infected and killed the tested human prostate cancer cell lines irrespective of their hormone response status, while the nonmalignant prostate epithelial cell line RWPE-1 proved highly virus resistant. Notably, a single peritoneal dose of SFV-VA7 was sufficient to eradicate all subcutaneous and orthotopic LNCaP tumours. CONCLUSIONS: Our results indicate that SFV-VA7 is a novel, promising therapeutic virus against prostate cancer warranting further testing in early clinical trials.
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Viroterapia Oncolítica/métodos , Neoplasias de la Próstata/terapia , Virus de los Bosques Semliki , Animales , Western Blotting , Línea Celular Tumoral , Supervivencia Celular , Humanos , Inmunohistoquímica , Masculino , Ratones , Trasplante de Neoplasias , Virus Oncolíticos , Próstata , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: An increased breast cancer incidence and poor survival have been reported for women with neurofibromatosis 1 (NF1). To explain the poor survival, we aimed to link the histopathology and clinical characteristics of NF1-associated breast cancers. METHODS: The Finnish Cancer Registry and the Finnish NF Registry were cross-referenced to identify the NF1 patients with breast cancer. Archival NF1 breast cancer specimens were retrieved for histopathological typing and compared with matched controls. RESULTS: A total of 32 breast cancers were diagnosed in 1404 NF1 patients during the follow-up. Women with NF1 had an estimated lifetime risk of 18.0% for breast cancer, and this is nearly two-fold compared with that of the general Finnish female population (9.74%). The 26 successfully retrieved archival NF1 breast tumours were more often associated with unfavourable prognostic factors, such as oestrogen and progesterone receptor negativity and HER2 amplification. However, survival was worse in the NF1 group (P=0.053) even when compared with the control group matched for age, diagnosis year, gender and oestrogen receptor status. Scrutiny of The Cancer Genome Atlas data set showed that NF1 mutations and deletions were associated with similar characteristics in the breast cancers of the general population. CONCLUSIONS: These results emphasise the role of the NF1 gene in the pathogenesis of breast cancer and a need for active follow-up for breast cancer in women with NF1.
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Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/epidemiología , Neurofibromatosis 1/diagnóstico , Neurofibromatosis 1/epidemiología , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/genética , Neoplasias de la Mama Masculina/diagnóstico , Neoplasias de la Mama Masculina/epidemiología , Neoplasias de la Mama Masculina/genética , Estudios de Casos y Controles , Femenino , Finlandia/epidemiología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Neurofibromatosis 1/genética , Pronóstico , Sistema de Registros , Factores de RiesgoRESUMEN
Bisphosphonates are standard treatments for bone metastases. When given in the adjuvant setting, they reduce breast cancer mortality and recurrence in bone but only among post-menopausal patients. Optimal drug use would require biomarker-based patient selection. Such biomarkers are not yet in clinical use. Based on the similarities in inflammatory responses to bisphosphonates and Toll-like receptor (TLR) agonists, we hypothesized that TLR9 expression may affect bisphosphonate responses in cells. We compared bisphosphonate effects in breast cancer cell lines with low or high TLR9 expression. We discovered that cells with decreased TLR9 expression are significantly more sensitive to the growth-inhibitory effects of bisphosphonates in vitro and in vivo. Furthermore, cancer growth-promoting effects seen with some bisphosphonates in some control shRNA cells were not detected in TLR9 shRNA cells. These differences were not associated with inhibition of Rap1A prenylation or p38 phosphorylation, which are known markers for bisphosphonate activity. However, TLR9 shRNA cells exhibited increased sensitivity to ApppI, a metabolite that accumulates in cells after bisphosphonate treatment. We conclude that decreased TLR9-expression sensitizes breast cancer cells to the growth inhibitory effects of bisphosphonates. Our results suggest that TLR9 should be studied as a potential biomarker for adjuvant bisphosphonate sensitivity among breast cancer patients.
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Neoplasias de la Mama/tratamiento farmacológico , Difosfonatos/uso terapéutico , Receptor Toll-Like 9/fisiología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Difosfonatos/farmacología , Femenino , Humanos , Ratones , ARN Interferente Pequeño/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
OBJECTIVE: Ospemifene (Osp) is a novel selective estrogen-receptor modulator (SERM) accepted for the treatment of dyspareunia, a symptom of postmenopausal vulvovaginal atrophy. We aimed to analyze the effects of Osp on human breast tissue (HBT), in comparison with the clinically established SERMs raloxifene (Ral) and tamoxifen (Tam), using ex vivo explant cultures. METHODS: HBT samples were obtained from postmenopausal women undergoing mammoplasty and cultured with or without Osp, Ral, Tam, or 17ß-estradiol (E2) for 7 and 14 days, and studied for morphology, proliferation, and apoptosis. The expression of epithelial markers, the estrogen-receptor alpha (ERα), the androgen receptor (AR), TFF1, and apolipoprotein D was evaluated using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. The PvuII polymorphism of ERS1 was determined. RESULTS: Osp, similar to Ral and Tam, decreased the number of proliferating cells in a concentration-dependent manner (at 100ânM, Pâ<â0.01) and strongly opposed 10ânM E2-stimulated proliferation (Pâ<â0.001). Corresponding effects were observed in the proportions of cells expressing ERα and TFF1 (Pâ<â0.001). At 14 days apoptosis was increased by 100ânM SERMs (Pâ<â0.01), but, notably, decreased by 1ânM Osp and Ral at day 7 (Pâ<â0.05). The SERMs exerted ER-agonist effects on AR-positive cell populations at 1ânM (Pâ<â0.05), but not at 100ânM concentrations. The effects on proliferation and ERα expressing cell numbers were associated with the ERS1 PvuII genotype. CONCLUSIONS: In summary, Osp inhibited proliferation and opposed E2 stimulation in normal HBT in an efficacious, but less potent way than Ral and Tam. The ESR1 PvuII polymorphisms may influence the responsiveness of HBT to E2 and SERMs.
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Mama/efectos de los fármacos , Posmenopausia/efectos de los fármacos , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/análogos & derivados , Anciano , Apolipoproteínas D/metabolismo , Apoptosis/efectos de los fármacos , Mama/metabolismo , Mama/patología , Proliferación Celular/efectos de los fármacos , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Polimorfismo Genético , Clorhidrato de Raloxifeno/farmacología , Receptores Androgénicos/metabolismo , Tamoxifeno/farmacología , Factor Trefoil-1/metabolismoRESUMEN
Fam3c, a cytokine-like growth factor, has been suggested to have a role in epithelial-to-mesenchymal transition (EMT), tumor growth and metastasis. A single-nucleotide polymorphism affecting bone mineral density has been found in the first intron of the Fam3c gene in a study analyzing an Asian population cohort. Other independent studies on different population cohorts have found the fam3c locus to be associated with bone mineral density and fractures. In order to investigate the role of Fam3c in bone biology, we have generated a Fam3c knock-out (KO) mouse strain. The Fam3c KO mice were found to have normal appearance, behavior and fertility, but small changes in bone morphology and content were also observed. Micro-CT analysis of tibiae of the female mice revealed decreased number of trabeculae. In male mice the changes in the bone phenotype were smaller, but hematological changes were observed. Furthermore, there was a negative correlation between body weight and tibial trabecular and cortical bone volume in the male KO mice. There was a small increase in cortical bone mineral density, but in the lateral direction of tibiae the breaking strength was reduced. Fam3c KO bone marrow cells showed accelerated osteogenic differentiation and mineralization in vitro. The reduced number of bone trabeculae in Fam3c KO mice and the stimulated osteogenic differentiation indicate a role for Fam3c in osteoblast differentiation and bone homeostasis.
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LuCaP serially transplantable patient-derived xenografts (PDXs) are valuable preclinical models of locally advanced or metastatic prostate cancer. Using spheroid culture methodology, we recently established cell lines from several LuCaP PDXs. Here, we characterized in depth the features of xenografts derived from LuCaP 136 spheroid cultures and found faithful retention of the phenotype of the original PDX. In vitro culture enabled luciferase transfection into LuCaP 136 spheroids, facilitating in vivo imaging. We showed that LuCaP 136 spheroids formed intratibial, orthotopic, and subcutaneous tumors when re-introduced into mice. Intratibial tumors responded to castration and were highly osteosclerotic. LuCaP 136 is a realistic in vitro-in vivo preclinical model of a subtype of bone metastatic prostate cancer.
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Neoplasias Óseas/patología , Metástasis de la Neoplasia , Neoplasias de la Próstata/patología , Esferoides Celulares/patología , Animales , Neoplasias Óseas/secundario , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fibroblast growth factor homologous factors (FHFs) belong to the fibroblast growth factor (FGF) superfamily, which plays an important role in prostate cancer (PCa). Mining of public database suggests that FGF13 (FHF2) mRNA expression is altered in over 30% of PCa cases. This study examined the FGF13 expression pattern in human PCa specimens and evaluated its potential as a biomarker for patient outcome after radical prostatectomy (RP). Immunohistochemistry (IHC) showed that FGF13 was detectable in the majority of human PCa samples, and FGF13 IHC scores were higher in high-grade prostatic intraepithelial neoplasia, in primary PCa and in metastatic PCa than in benign prostatic tissue. There was a significant association between high cytoplasmic FGF13 staining and a risk of biochemical recurrence (BCR) after RP. This was also evident in the intermediate to high-risk patient groups. In contrast, positive nuclear FGF13 staining along with low cytoplasmic FGF13 group showed a decreased BCR risk. Multivariate regression analysis indicated that high cytoplasmic FGF13 staining was associated with BCR and that this could serve as an independent prognostic marker in PCa. Several PCa cell lines showed increased FGF13 expression at the mRNA and protein levels compared to the immortalized prostate epithelial cell line PNT1a. Analysis of co-labeled cells suggested a possible interaction of FGF13 with α-tubulin and the voltage-gated sodium channel proteins (Na(V)s/VGSCs). Our data indicate that, for PCa patients after RP, FGF13 serves as a potential novel prognostic marker that improves prediction of BCR-free survival, in particular if combined with other clinical parameters.
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Biomarcadores de Tumor/biosíntesis , Factores de Crecimiento de Fibroblastos/biosíntesis , Recurrencia Local de Neoplasia/genética , Neoplasias de la Próstata/genética , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Supervivencia sin Enfermedad , Factores de Crecimiento de Fibroblastos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Recurrencia Local de Neoplasia/patología , Pronóstico , Prostatectomía/efectos adversos , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , ARN Mensajero/biosíntesis , Análisis de Matrices TisularesRESUMEN
Explant tissue culture provides a model for studying the direct effects of steroid hormones, their analogs, and novel hormonally active compounds on normal freshly isolated human breast tissues (HBTs). For this purpose, pre- and postmenopausal HBTs can be maintained in this culture system. The results demonstrate that the morphological integrity of HBT explants can be maintained in tissue culture up to 2 weeks and expression of differentiation markers, steroid hormone receptors, proliferation and apoptosis ratios can be evaluated as a response to hormonal stimulation. This chapter describes an ex vivo culture model that we have applied to study the effects of various hormonally active substances, including 17ß-estradiol and selective estrogen receptor modulators (SERMs), on normal human breast tissues.
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Mama/efectos de los fármacos , Receptor alfa de Estrógeno/efectos de los fármacos , Clorhidrato de Raloxifeno/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Mama/metabolismo , Mama/patología , Mama/cirugía , Proliferación Celular/efectos de los fármacos , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Inmunohistoquímica , Adhesión en Parafina , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Fijación del TejidoRESUMEN
BACKGROUND AND METHODS: Pim family proteins are oncogenic kinases implicated in several types of cancer and involved in regulation of cell proliferation, survival as well as motility. Here we have investigated the ability of Pim kinases to promote metastatic growth of prostate cancer cells in two xenograft models for human prostate cancer. We have also evaluated the efficacy of Pim-selective inhibitors to antagonize these effects. RESULTS: We show here that tumorigenic growth of both subcutaneously and orthotopically inoculated prostate cancer xenografts is enhanced by stable overexpression of either Pim-1 or Pim-3. Moreover, Pim-overexpressing orthotopic prostate tumors are highly invasive and able to migrate not only to the nearby prostate-draining lymph nodes, but also into the lungs to form metastases. When the xenografted mice are daily treated with the Pim-selective inhibitor DHPCC-9, both the volumes as well as the metastatic capacity of the tumors are drastically decreased. Interestingly, the Pim-promoted metastatic growth of the orthotopic xenografts is associated with enhanced angiogenesis and lymphangiogenesis. Furthermore, forced Pim expression also increases phosphorylation of the CXCR4 chemokine receptor, which may enable the tumor cells to migrate towards tissues such as the lungs that express the CXCL12 chemokine ligand. CONCLUSIONS: Our results indicate that Pim overexpression enhances the invasive properties of prostate cancer cells in vivo. These effects can be reduced by the Pim-selective inhibitor DHPCC-9, which can reach tumor tissues without serious side effects. Thus, Pim-targeting therapies with DHPCC-9-like compounds may help to prevent progression of local prostate carcinomas to fatally metastatic malignancies.
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Metástasis de la Neoplasia/patología , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Quimiocina CXCL12/metabolismo , Xenoinjertos , Humanos , Linfangiogénesis/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Neovascularización Patológica/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-pim-1/biosíntesis , Receptores CXCR4/metabolismo , Trasplante Heterólogo , Pez CebraRESUMEN
We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for 1-3days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds.
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Fibroblastos/citología , Holografía/métodos , Microscopía/métodos , Neoplasias de la Próstata/patología , Animales , Muerte Celular/efectos de los fármacos , Línea Celular , Proliferación Celular , Tamaño de la Célula , Supervivencia Celular , Etopósido/farmacología , Humanos , Masculino , Ratones , Neoplasias de la Próstata/tratamiento farmacológicoRESUMEN
UNLABELLED: Extracellular adenosine mediates diverse anti-inflammatory, angiogenic, and other signaling effects via binding to adenosine receptors, and it also regulates cell proliferation and death via activation of the intrinsic signaling pathways. Given the emerging role of adenosine and other purines in tumor growth and metastasis, this study evaluated the effects of adenosine on the invasion of metastatic prostate and breast cancer cells. Treatment with low micromolar concentrations of adenosine, but not other nucleosides or adenosine receptor agonists, inhibited subsequent cell invasion and migration through Matrigel- and laminin-coated inserts. These inhibitory effects occurred via intrinsic receptor-independent mechanisms, despite the abundant expression of A2B adenosine receptors (ADORA2B). Extracellular nucleotides and adenosine were shown to be rapidly metabolized on tumor cell surfaces via sequential ecto-5'-nucleotidase (CD73/NT5E) and adenosine deaminase reactions with subsequent cellular uptake of nucleoside metabolites and their intracellular interconversion into ADP/ATP. This was accompanied by concurrent inhibition of AMP-activated protein kinase and other signaling pathways. No differences in the proliferation rates, cytoskeleton assembly, expression of major adhesion molecules [integrin-1ß (ITGB1), CD44, focal adhesion kinase], and secretion of matrix metalloproteinases were detected between the control and treated cells, thus excluding the contribution of these components of invasion cascade to the inhibitory effects of adenosine. These data provide a novel insight into the ability of adenosine to dampen immune responses and prevent tumor invasion via two different, adenosine receptor-dependent and -independent mechanisms. IMPLICATIONS: This study suggests that the combined targeting of adenosine receptors and modulation of intracellular purine levels can affect tumor growth and metastasis phenotypes.
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Adenosina/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Neoplasias de la Próstata/patología , Neoplasias de la Mama/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Invasividad Neoplásica , Neoplasias de la Próstata/metabolismo , Agonistas del Receptor Purinérgico P1/farmacología , Receptores Purinérgicos P1/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy.