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1.
Glycobiology ; 32(8): 701-711, 2022 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-35661210

RESUMO

Interaction of immune cells with the systemic environment is necessary for the coordinated development and execution of immune responses. Monocyte-macrophage lineage cells reside at the junction of innate and adaptive immunity. Previously we reported that the sialyltransferase ST6GAL1 in the extracellular milieu modulates B cell development and IgG production, granulocyte production, and attenuates acute airway inflammation to bacterial challenge in mouse models. Here, we report that extracellular ST6GAL1 also elicits profound responses in monocyte-macrophage lineage cells. We show that recombinant ST6GAL1 adheres to subsets of thioglycolate-elicited inflammatory cells in the mouse peritoneum and to cultured human monocyte THP-1 cells. Exposure of the inflammatory cells to recombinant ST6GAL1 elicited wholesale changes in the gene expression profile of primary mouse myeloid cells; most notable was the striking up-regulation of monocyte-macrophage and monocyte-derived dendritic cell development pathway signature genes and transcription factors PU.1, NFκB and their target genes, driving increased monocyte-macrophage population and survival ex vivo. In the cultured human monocyte cells, the essential cell surface receptor of the monocyte-macrophage lineage, the M-CSF receptor (M-CSF-R, Csfr1) was a target of extracellular ST6GAL1 catalytic activity. Extracellular ST6GAL1 activated the M-CSF-R and initiated intracellular signaling events, namely, the nuclear translocation of NFκB subunit p65, and phosphorylation of ERK 1/2 and AKT. The findings implicate extracellular ST6GAL1 in monocyte development by a mechanism initiated at the cell surface and support an emerging paradigm of an extracellular glycan-modifying enzyme as a central regulator coordinating immune hematopoietic cell development and function.


Assuntos
Fator Estimulador de Colônias de Macrófagos , Monócitos , Animais , Antígenos CD/metabolismo , Diferenciação Celular , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Camundongos , Monócitos/metabolismo , Fosforilação , Sialiltransferases/genética , Sialiltransferases/metabolismo , Transdução de Sinais , Células THP-1
2.
FASEB J ; 34(3): 4293-4310, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32017264

RESUMO

Sphingosine kinase 2 (SphK2) is known to phosphorylate the nuclear sphingolipid metabolite to generate sphingosine-1-phosphate (S1P). Nuclear S1P is involved in epigenetic regulation of gene expression; however, the underlying mechanisms are not well understood. In this work, we have identified the role of nuclear S1P and SphK2 in regulating hypoxia-responsive master transcription factors hypoxia-inducible factor (HIF)-1α/2α, and their functions in breast cancer, with a focus on triple-negative breast cancer (TNBC). We have shown SphK2 is associated with HIF-1α in protein complexes, and is enriched at the promoters of HIF target genes, including vascular endothelial growth factor (VEGF), where it enhances local histone H3 acetylation and transcription. S1P specifically binds to the PAS domains of HIF-1α. SphK2, and HIF-1α expression levels are elevated in metastatic estrogen receptor-positive (ER+) and TNBC clinical tissue specimens compared to healthy breast tissue samples. To determine if S1P formation in the nucleus by SphK2 is a key regulator of HIF functions, we found using a preclinical TNBC xenograft mouse model, and an existing selective SphK2 inhibitor K-145, that nuclear S1P, histone acetylation, HIF-1α expression, and TNBC tumor growth were all reduced in vivo. Our results suggest that S1P and SphK2 in the nucleus are linked to the regulation of HIF-1α/2α functions associated with breast cancer progression, and may provide potential therapeutic targets.


Assuntos
Núcleo Celular/metabolismo , Lisofosfolipídeos/metabolismo , Receptor A2B de Adenosina/metabolismo , Esfingosina/análogos & derivados , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Acetilação , Adenosina/metabolismo , Animais , Pressão Sanguínea/genética , Pressão Sanguínea/fisiologia , Ensaio de Imunoadsorção Enzimática , Epigênese Genética/genética , Epigênese Genética/fisiologia , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Receptor A2B de Adenosina/genética , Esfingosina/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética
3.
Mediators Inflamm ; 2017: 4806541, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29269995

RESUMO

Inflammation is part of our body's response to tissue injury and pathogens. It helps to recruit various immune cells to the site of inflammation and activates the production of mediators to mobilize systemic protective processes. However, chronic inflammation can increase the risk of diseases like cancer. Apart from cytokines and chemokines, lipid mediators, particularly sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), contribute to inflammation and cancer. S1P is an important player in inflammation-associated colon cancer progression. On the other hand, C1P has been recognized to be involved in cancer cell growth, migration, survival, and inflammation. However, whether C1P is involved in inflammation-associated cancer is not yet established. In contrast, few studies have also suggested that S1P and C1P are involved in anti-inflammatory pathways regulated in certain cell types. Ceramide is the substrate for ceramide kinase (CERK) to yield C1P, and sphingosine is phosphorylated to S1P by sphingosine kinases (SphKs). Biological functions of sphingolipid metabolites have been studied extensively. Ceramide is associated with cell growth inhibition and enhancement of apoptosis while S1P and C1P are associated with enhancement of cell growth and survival. Altogether, S1P and C1P are important regulators of ceramide level and cell fate. This review focuses on S1P and C1P involvement in inflammation and cancer with emphasis on recent progress in the field.


Assuntos
Ceramidas/metabolismo , Inflamação/metabolismo , Lisofosfolipídeos/metabolismo , Neoplasias/metabolismo , Esfingosina/análogos & derivados , Animais , Biomarcadores Tumorais/metabolismo , Humanos , Inflamação/etiologia , Mediadores da Inflamação/metabolismo , Modelos Biológicos , Neoplasias/etiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais , Esfingosina/metabolismo
4.
BMC Cell Biol ; 15: 11, 2014 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-24661536

RESUMO

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is the predominant cause of bone infection. Toll like receptors (TLRs) are an important segments of host response to infection and are expressed by a variety of cells including human mesenchymal stem cells (hMSCs). The active form of Vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has potent immunoregulatory properties, but the mechanism remains poorly understood. The genomic action of 1,25(OH)2D3 is mediated by vitamin D receptor (VDR), hormone-regulated transcription factor. VDR interacts with co-activators and co-repressors are associated with chromatin histone modifications and transcriptional regulation. The aim of our study is to explore MRSA-induced TLRs-mediated pro-inflammatory cytokines expression in hMSCs. Further, we hypothesized that 1,25(OH)2D3 inhibits MRSA-induced cytokines synthesis in hMSCs via inhibition of NF-кB transcription factor. Finally, we explored the regulatory role of 1,25(OH)2D3 in MRSA-mediated global epigenetic histone H3 mark, such as, trimethylated histone H3 lysine 9 (H3K9me3), which is linked to gene silencing. RESULTS: Quantitative PCR data revealed that MRSA-infection predominantly induced expression of TLRs 1, 2, 6, NR4A2, and inflammatory cytokines IL-8, IL-6, TNFα in hMSCs. MRSA-mediated TLR ligands reduced osteoblast differentiation and increased hMSCs proliferation, indicating the disrupted multipotency function of hMSCs. Pretreatment of 1,25(OH)2D3 followed by MRSA co-culture inhibited nuclear translocation of NF-кB-p65, reduced expression of NR4A2 and pro-inflammatory cytokines IL-8, IL-6, and TNFα in hMSCs. Further, NF-κB-p65, VDR, and NR4A2 were present in the same nuclear protein complex, indicating that VDR is an active part of the nuclear protein complexes for transcriptional regulation. Finally, 1,25(OH)2D3 activated VDR, restores the global level of H3K9me3, to repress MRSA-stimulated inflammatory cytokine IL-8 expression. Pretreatment of 5-dAZA, DNA methylatransferases (Dnmts) inhibitor, dramatically re-expresses 1,25(OH)2D3-MRSA-mediated silenced IL-8 gene. CONCLUSIONS: This data indicates that TLR 1, 2, and 6 can be used as markers for localized S. aureus bone infection. 1,25(OH)2D3-VDR may exhibits its anti-inflammatory properties in MRSA-stimulated infection by inhibiting nuclear translocation of NF-kB-p65 and transcripts of IL-8, IL-6, TNFα, and NR4A2 in hMSCs. Finally, 1,25(OH)2D3-activated VDR, acting as an epigenetic regulator, inhibits synthesis of cytokines in MRSA-stimulated infection by restoring the global level of H3K9me3, a histone H3 mark for gene silencing.


Assuntos
Células da Medula Óssea/citologia , Calcitriol/farmacologia , Citocinas/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/imunologia , RNA Mensageiro/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/microbiologia , Diferenciação Celular , Células Cultivadas , Citocinas/genética , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Decitabina , Inativação Gênica/efeitos dos fármacos , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/microbiologia , NF-kappa B/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
5.
World J Oncol ; 15(2): 181-191, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38545475

RESUMO

Background: Spinster homologue 2 (SPNS2) is a transporter of sphingosine-1-phosphate (S1P), a bioactive lipid linked to cancer progression. We studied the link between SPNS2 gene expression, tumor aggressiveness, and outcomes in patients with hepatocellular carcinoma (HCC). Methods: Gene expression in patients with HCC was analyzed from the Cancer Genome Atlas (TCGA) (n = 350) and GSE76427 (n = 115) as a validation cohort, as well as liver tissue cohort GSE6764 (n = 75). Results: High-SPNS2 HCC was significantly associated with high level of lymph-angiogenesis-related factors. SPNS2 expression was significantly higher in normal liver and early HCC versus advanced HCC (P < 0.02). High SPNS2 levels enriched immune response-related gene sets; inflammatory, interferon (IFN)-α, IFN-γ responses, and tumor necrosis factor (TNF)-α, interleukin (IL)-6/Janus kinase/signal transducer and activator of transcription (JAK/STAT3) signaling, complement and allograft rejection, but did not significantly infiltrate specific immune cells nor cytolytic activity score. High-SPNS2 HCC enriched tumor aggravating pathway gene sets such as KRAS (Kirsten rat sarcoma virus) signaling, but inversely correlated with Nottingham histological grade, MKI67 (marker of proliferation Ki-67) expression, and cell proliferation-related gene sets. Further, high-SPNS2 HCC had significantly high infiltration of stromal cells, showing that low-SPNS2 HCC is highly proliferative. Finally, high-SPNS2 HCC was associated with better disease-free, disease-specific, and overall survival (P = 0.031, 0.046, and 0.040, respectively). Conclusions: Although SPNS2 expression correlated with lymph-angiogenesis and other cancer-promoting pathways, it also enriched immune response. SPNS2 levels were higher in normal liver compared to HCC, and inversely correlated with cancer cell proliferation and better survival. SPNS2 expression may be beneficial in HCC patients despite detrimental in-vitro effects.

6.
Cancer Gene Ther ; 29(11): 1662-1675, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35676533

RESUMO

The sialyltransferase ST6GAL1 that adds α2-6 linked sialic acids to N-glycans of cell surface and secreted glycoproteins is prominently associated with many human cancers. Tumor-native ST6GAL1 promotes tumor cell behaviors such as invasion and resistance to cell stress and chemo- and radio-treatments. Canonically, ST6GAL1 resides in the intracellular secretory apparatus and glycosylates nascent glycoproteins in biosynthetic transit. However, ST6GAL1 is also released into the extracellular milieu and extracellularly remodels cell surface and secreted glycans. The impact of this non-canonical extrinsic mechanism of ST6GAL1 on tumor cell pathobiology is not known. We hypothesize that ST6GAL1 action is the combined effect of natively expressed sialyltransferase acting cell-autonomously within the ER-Golgi complex and sialyltransferase from extracellular origins acting extrinsically to remodel cell-surface glycans. We found that shRNA knockdown of intrinsic ST6GAL1 expression resulted in decreased ST6GAL1 cargo in the exosome-like vesicles as well as decreased breast tumor cell growth and invasive behavior in 3D in vitro cultures. Extracellular ST6GAL1, present in cancer exosomes or the freely soluble recombinant sialyltransferase, compensates for insufficient intrinsic ST6GAL1 by boosting cancer cell proliferation and increasing invasiveness. Moreover, we present evidence supporting the existence novel but yet uncharacterized cofactors in the exosome-like particles that potently amplify extrinsic ST6GAL1 action, highlighting a previously unknown mechanism linking this enzyme and cancer pathobiology. Our data indicate that extracellular ST6GAL1 from remote sources can compensate for cellular ST6GAL1-mediated aggressive tumor cell proliferation and invasive behavior and has great clinical potential for extracellular ST6GAL1 as these molecules are in the extracellular space should be easily accessible targets.


Assuntos
Neoplasias da Mama , Sialiltransferases , Humanos , Feminino , Sialiltransferases/genética , Sialiltransferases/metabolismo , Neoplasias da Mama/genética , Glicoproteínas , Polissacarídeos/metabolismo , Proliferação de Células , Antígenos CD/genética
7.
J Cancer ; 12(4): 954-964, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33442395

RESUMO

Brain metastases represent a substantial amount of morbidity and mortality in breast cancer (BC). Metastatic breast tumor cells committed to brain metastases are unique because they escape immune surveillance, can penetrate the blood-brain barrier, and also adapt to the brain tissue microenvironment (TME) for colonization and outgrowth. In addition, dynamic intracellular interactions between metastatic cancer cells and neighboring astrocytes in the brain are thought to play essential roles in brain tumor progression. A better understanding of the above mechanisms will lead to developing more effective therapies for brain metastases. Growing literature suggests autophagy, a conserved lysosomal degradation pathway involved in cellular homeostasis under stressful conditions, plays essential roles in breast tumor metastatic transformation and brain metastases. Cancer cells must adapt under various microenvironmental stresses, such as hypoxia, and nutrient (glucose) deprivation, in order to survive and progress. Clinical studies reveal that tumoral expression of autophagy-related proteins is higher in brain metastasis compared to primary breast tumors. In this review, we outline the molecular mechanisms underlying autophagy-mediated BC cell survival and metastasis to the brain.

8.
Am J Cancer Res ; 11(9): 4394-4407, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34659894

RESUMO

Sphingosine-1-Phosphate (S1P) is produced by Sphingosine Kinase 1 (SphK1) in the cell and is transported out of the cells by ABCC1 transporter. S1P induces inflammation, angiogenesis and modulates tumor immune microenvironment (TIME) in autocrine and paracrine manner. We hypothesized that high S1P export is associated with hepatocellular carcinoma (HCC) progression and worse survival. Transcriptome linked with clinical data were obtained from a total of 533 patients from TCGA (The Cancer Genome Atlas)-HCC (n = 350), GSE6764 (n = 75), and GSE89377 (n = 108) cohorts. Both SphK1 and ABCC1 were expressed higher in aggressive HCC than normal liver or cirrhosis and correlated with MKi67 expression. High S1P export by high expression of both SphK1 and ABCC1 enriched gene sets related with cell proliferation (E2F targets, G2M checkpoint, MYC targets), inflammation (Inflammatory response, TNFα, IL6), angiogenesis, metastasis (TGF-ß, epithelial-mesenchymal transition), and immune response (allograft rejection, complement, interferon-gamma) in gene set enrichment analysis. High S1P export was associated with elevation of HGF, HSP90AA1, TRAF2, and AKR1B10. It was also associated with high intratumor heterogeneity, leucocyte fraction, macrophage regulation and lymphocyte infiltration, as well as T helper type2 cells, macrophages, dendritic cells, CD4+ T memory activated cells, B-cells and cytolytic activity score in TIME. High S1P export was associated with significantly worse disease specific survival (P = 0.034) and overall survival (P = 0.004) compared to low S1P export group. In conclusion, simultaneous high expression of SphK1 and ABCC1 that reflect S1P export is associated with enhancement of both HCC progression and immune response. Given that S1P export was also associated with worse survival, we cannot help but speculate that pro-cancer pathways activated by S1P may overwhelm the anti-cancer immune response mediated by S1P.

9.
Cancers (Basel) ; 13(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072157

RESUMO

Heterogeneity is the characteristic of breast tumors, making it difficult to understand the molecular mechanism. Alteration of gene expression in the primary tumor versus the metastatic lesion remains challenging for getting any specific targeted therapy. To better understand how gene expression profile changes during metastasis, we compare the primary tumor and distant metastatic tumor gene expression using primary breast tumors compared with its metastatic variant in animal models. Our RNA sequencing data from cells revealed that parental cell and the metastatic variant cell are different in gene expression while gene signature significantly altered during metastasis to distant organs than primary breast tumors. We found that secreted mediators encoding genes (ANGPTL7, MMP3, LCN2, S100A8, and ESM1) are correlated with poor prognosis in the clinical setting as divulged from METABRIC and TCGA-BRCA cohort data analysis.

10.
Cancers (Basel) ; 12(2)2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32074948

RESUMO

The vast majority of mortality in breast cancer results from distant metastasis. Brain metastases occur in as many as 30% of patients with advanced breast cancer, and the 1-year survival rate of these patients is around 20%. Pre-clinical animal models that reliably reflect the biology of breast cancer brain metastasis are needed to develop and test new treatments for this deadly condition. The patient-derived xenograft (PDX) model maintains many features of a donor tumor, such as intra-tumor heterogeneity, and permits the testing of individualized treatments. However, the establishment of orthotopic PDXs of brain metastasis is procedurally difficult. We have developed a method for generating such PDXs with high tumor engraftment and growth rates. Here, we describe this method and identify variables that affect its outcomes. We also compare the brain-orthotopic PDXs with ectopic PDXs grown in mammary pads of mice, and show that the responsiveness of PDXs to chemotherapeutic reagents can be dramatically affected by the site that they are in.

11.
Int J Oncol ; 55(1): 116-130, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059004

RESUMO

Triple­negative breast cancer (TNBC) cells form angiogenesis­independent vessel­like structures to survive, known as vasculogenic mimicry (VM), contributing to a poor prognosis for cancer patients. Nuclear localized class I histone deacetylases (HDACs) enzymes, particularly HDACs 1, 2, 3 deacetylate chromatin histones, are overexpressed in cancers and epigenetically regulate the expression of genes involved in cancer initiation and progression. The specific HDAC inhibitor, entinostat, has been shown to attenuate tumor progression and metastasis in TNBC. In this study, we hypothesized that entinostat would enhance the expression of anti­angiogenic and tumor suppressor genes and would thus suppress VM structures in TNBC cells in a 3D Matrigel cell culture preclinical model. Our data indicated that invasive triple­negative MDA­MB­231, LM2­4 and BT­549 breast cancer cells, but not poorly invasive luminal MCF­7 cells, efficiently underwent matrix­associated VM formation. Approximately 80% of TNBC cells with the stem cell phenotype potential formed vessel­like structures when mixed with Matrigel and cultured in the low attachment tissue culture plate. The molecular mechanisms of VM formation are rather complex, while angiogenesis inhibitor genes are downregulated and pro­angiogenesis genes are upregulated in VM­forming cells. Our data revealed that treatment of the TNBC VM phenotype cells with entinostat epigenetically led to the re­expression of the anti­angiogenic genes, serpin family F member 1 (SERPINF1) and thrombospondin 2 (THBS2), and to that of the tumor suppressor genes, phosphatase and tensin homolog (PTEN) and p21, and reduced VM structures. We also found that treatment of the TNBC VM phenotype cells with entinostat downregulated the expression of vascular endothelial growth factor A (VEGF­A), and that of the epithelial­mesenchymal transition (EMT)­related genes, Vimentin and ß­catenin. METABIRC and TCGA breast cancer cohort mRNA expression data analysis revealed that a high expression of the anti­angiogenesis­associated genes, THBS2, SERPINF1 and serpin family B member 5 (SERPINB5), and of the tumor suppressor gene, PTEN, was associated with a better overall survival (OS) of breast cancer patients. Taken together, the findings of this study demonstrate that HDACs 1, 2, 3 partly contribute to VM formation in TNBC cells; thus, HDACs may be an important therapeutic target for TNBC.


Assuntos
Inibidores da Angiogênese/genética , Benzamidas/farmacologia , Genes Supressores de Tumor/efeitos dos fármacos , Histona Desacetilase 1/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Piridinas/farmacologia , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Epigênese Genética , Proteínas do Olho/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Fatores de Crescimento Neural/genética , Serpinas/genética , Análise de Sobrevida , Trombospondinas/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico
12.
Arch Biochem Biophys ; 477(1): 33-42, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18593564

RESUMO

In this study, we utilized murine renal proximal (MPCT-G) and distal (DKC-8) tubular epithelial cell lines to compare the gene expressions and promoter activities of 1,25(OH)(2)D(3) receptor (VDR) and 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1) in response to 50 nM of parathyroid hormone (PTH) and changes in extracellular calcium (Ca(2+)) concentration. In MPCT-G cells, VDR gene expression was suppressed by PTH, whereas CYP27B1 gene expression was elevated in response to PTH. In DKC-8 cells, treatment of PTH significantly increased the relative gene expression of VDR by 6.5-fold while CYP27B1 gene expression was unchanged. High Ca(2+) exposure stimulated VDR gene expression and repressed CYP27B1 gene expression in both dose and time-dependent fashion in MPCT-G but not DKC-8 cells. The analysis of promoter activities and VDR protein levels corresponded with the gene expression data. We conclude that PTH-mediated decrease in VDR and increase in renal CYP27B1 is proximal cell-specific.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/metabolismo , Receptores de Calcitriol/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Animais , Western Blotting , Linhagem Celular , Imuno-Histoquímica , Túbulos Renais Distais/citologia , Túbulos Renais Distais/enzimologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/enzimologia , Camundongos , Regiões Promotoras Genéticas , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Esteroide Hidroxilases/metabolismo , Vitamina D3 24-Hidroxilase
13.
Mol Cancer Res ; 16(6): 1059-1070, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29523764

RESUMO

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid mediator, has been implicated in regulation of many processes important for breast cancer progression. Previously, we observed that S1P is exported out of human breast cancer cells by ATP-binding cassette (ABC) transporter ABCC1, but not by ABCB1, both known multidrug resistance proteins that efflux chemotherapeutic agents. However, the pathologic consequences of these events to breast cancer progression and metastasis have not been elucidated. Here, it is demonstrated that high expression of ABCC1, but not ABCB1, is associated with poor prognosis in breast cancer patients. Overexpression of ABCC1, but not ABCB1, in human MCF7 and murine 4T1 breast cancer cells enhanced S1P secretion, proliferation, and migration of breast cancer cells. Implantation of breast cancer cells overexpressing ABCC1, but not ABCB1, into the mammary fat pad markedly enhanced tumor growth, angiogenesis, and lymphangiogenesis with a concomitant increase in lymph node and lung metastases as well as shorter survival of mice. Interestingly, S1P exported via ABCC1 from breast cancer cells upregulated transcription of sphingosine kinase 1 (SPHK1), thus promoting more S1P formation. Finally, patients with breast cancers that express both activated SPHK1 and ABCC1 have significantly shorter disease-free survival. These findings suggest that export of S1P via ABCC1 functions in a malicious feed-forward manner to amplify the S1P axis involved in breast cancer progression and metastasis, which has important implications for prognosis of breast cancer patients and for potential therapeutic targets.Implication: Multidrug resistant transporter ABCC1 and activation of SPHK1 in breast cancer worsen patient's survival by export of S1P to the tumor microenvironment to enhance key processes involved in cancer progression. Mol Cancer Res; 16(6); 1059-70. ©2018 AACR.


Assuntos
Neoplasias da Mama/genética , Lisofosfolipídeos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Animais , Neoplasias da Mama/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Esfingosina/metabolismo , Análise de Sobrevida
14.
J Steroid Biochem Mol Biol ; 103(3-5): 504-8, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17204417

RESUMO

In renal proximal tubules, VDR is transiently decreased by parathyroid hormone (PTH) during times of hypocalcemia and returns to normal levels with the rise in serum calcium (Ca). In this study we tested the hypothesis that elevated extracellular Ca induces VDR in a human renal proximal cell line (HK-2G) stably expressing PTH receptor type I. Exposure of HK-2G cells to increasing Ca concentration, up to 3mM, induced the expression of VDR. The increase in VDR occurred within 1h and was sustained over 24h. The increase in VDR was also dose-dependently increased using 20-100 nM gadolinium, suggesting the induction of VDR is regulated via the extracellular Ca sensing receptor (CaSR) with is naturally expressed in HK-2G cells. In conclusion, an extracellular Ca concentration in the physiological range is capable of direct increase of renal proximal VDR expression, and the induction mechanism represents a strategy the body may use to counterbalance effects of PTH on renal Vitamin D metabolism.


Assuntos
Cálcio/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Túbulos Renais Proximais/metabolismo , Hormônio Paratireóideo/farmacologia , Receptores de Calcitriol/metabolismo , Vitamina D/metabolismo , Linhagem Celular , Regulação para Baixo , Gadolínio/farmacologia , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores de Detecção de Cálcio/agonistas , Receptores de Detecção de Cálcio/metabolismo , Fatores de Tempo
15.
J Bone Joint Surg Am ; 89(4): 841-8, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17403809

RESUMO

BACKGROUND: Aseptic loosening is often mentioned as the primary reason for costly revision of total joint arthroplasties. Receptor activator of nuclear factor-kappaB ligand (RANKL) appears to be a major factor in the bone resorption observed in periprosthetic osteolysis. RANKL plays an essential role in the recruitment, differentiation, and survival of the osteoclasts implicated in periprosthetic osteolysis. This study was performed in an effort to identify the cell type in the periprosthetic membrane responsible for expression of RANKL. METHODS: Tissues harvested from osteolytic lesions in nine patients undergoing total joint revision were serially sectioned for immunohistochemical analysis. Intercellular adhesion molecule-1 (ICAM-1) and prolyl 4-hydroxylase (5B5) antibodies were used to detect fibroblasts, and anti-CD-163 (Ber-MAC3) was used to detect macrophages. In addition, antibodies to osteoprotegerin (OPG), RANKL, and receptor activator of nuclear factor-kappaB (RANK) were utilized. The binding pattern of these antibodies was then viewed with confocal microscopy with the use of only secondary antibodies as method controls. RESULTS: Histological analysis was confined to areas of the membrane where cells were detected with use of Hoechst 34580 nuclear stain. In the membrane specimens from all nine patients, diffuse RANKL staining was localized to areas lacking cells and more intense staining was seen in areas containing nucleated cells. There was strong colocalization between RANKL and OPG, and there was weak but specific colocalization between RANKL and both 5B5 and ICAM-1. In contrast, there was complete separation of antibody staining of Ber-MAC3 and RANKL, indicating only generalized overlap of the myeloid markers with the RANKL. CONCLUSIONS: RANKL expression was localized to cells that stained positively for fibroblast markers. The data also indicated that there is an intact RANKL/RANK/OPG system in the periprosthetic membrane that could regulate focalized bone resorption in osteolysis. CLINICAL RELEVANCE: Identifying the cell types responsible for RANKL production is critical to the development of a strategy to prevent periprosthetic osteolysis.


Assuntos
Fibroblastos/metabolismo , Osteólise/metabolismo , Ligante RANK/biossíntese , Idoso , Idoso de 80 Anos ou mais , Artroplastia de Quadril/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteólise/etiologia
16.
Cell Signal ; 32: 85-92, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28108260

RESUMO

About 40,000 American women die from metastatic breast cancer each year despite advancements in treatment. Approximately, 15% of breast cancers are triple-negative for estrogen receptor, progesterone receptor, and HER2. Triple-negative cancer is characterized by more aggressive, harder to treat with conventional approaches and having a greater possibility of recurrence. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid signaling mediator has emerged as a key regulatory molecule in breast cancer progression. Therefore, we investigated whether cytosolic sphingosine kinase type 1 (SphK1) and nuclear sphingosine kinase type 2 (SphK2), the enzymes that make S1P are critical for growth and PI3K/AKT, ERK-MAP kinase mediated survival signaling of lung metastatic variant LM2-4 breast cancer cells, generated from the parental triple-negative MDA-MB-231 human breast cancer cell line. Similar with previous report, SphKs/S1P signaling is critical for the growth and survival of estrogen receptor positive MCF-7 human breast cancer cells, was used as our study control. MDA-MB-231 did not show a significant effect of SphKs/S1P signaling on AKT, ERK, and p38 pathways. In contrast, LM2-4 cells that gained lung metastatic phenotype from primary MDA-MB-231 cells show a significant effect of SphKs/S1P signaling requirement on cell growth, survival, and cell motility. PF-543, a selective potent inhibitor of SphK1, attenuated epidermal growth factor (EGF)-mediated cell growth and survival signaling through inhibition of AKT, ERK, and p38 MAP kinase pathways mainly in LM2-4 cells but not in parental MDA-MB-231 human breast cancer cells. Moreover, K-145, a selective inhibitor of SphK2, markedly attenuated EGF-mediated cell growth and survival of LM2-4 cells. We believe this study highlights the importance of SphKs/S1P signaling in metastatic triple-negative breast cancers and targeted therapies.


Assuntos
Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Humanos , Metástase Neoplásica , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/metabolismo
17.
J Bone Joint Surg Am ; 96(20): 1692-8, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25320195

RESUMO

BACKGROUND: Toll-like receptors (TLRs) 1 and 6 are consistent molecular indicators of the host inflammatory response against bacterial infection. Our aims were to determine whether TLR elevation could be detected in infected periprosthetic tissues and to assess the utility of these biomarkers as tests for detecting a periprosthetic joint infection. METHODS: Fifty-nine patients undergoing revision total joint arthroplasty (twenty-seven hips and thirty-two knees) were prospectively evaluated for periprosthetic joint infection according to currently recommended diagnostic criteria. Nine patients were excluded because of insufficient work-up, leaving fifty available for study. Of these, twenty-one were categorized as infected and twenty-nine as noninfected. Periprosthetic tissues were collected intraoperatively, and total RNA was extracted by standard techniques. Expression of TLR messenger RNAs was assessed by first-strand complementary DNA synthesis from 1 µg of total RNA followed by real-time PCR (polymerase chain reaction). Results were normalized relative to the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Expression of TLRs 1, 6, and 10 in the infected and noninfected groups was compared with use of the Student t test. The receiver operating characteristic curve, area under the curve (AUC), sensitivity, specificity, positive likelihood ratio (LR+), and negative likelihood ratio (LR-) were calculated to determine the accuracy of each TLR for predicting periprosthetic joint infection at its optimal diagnostic threshold. RESULTS: Mean TLR1 mRNA expression was significantly elevated in infected compared with noninfected samples (0.600 compared with 0.005, p = 0.0003); the same was true of TLR6 (0.208 compared with 0.0165, p = 0.0059) but not of TLR10 (0.00019 compared with 0.00014, p = 0.6238). The AUC was 0.995 for TLR1, 0.883 for TLR6, and 0.546 for TLR10. The optimal threshold for diagnosing periprosthetic joint infection was 0.0924 for TLR1 (sensitivity = 95.2%, specificity = 100%, LR+ = 13.80, LR- = 0.91) and 0.0215 for TLR6 (sensitivity = 85.7%, specificity = 82.8%, LR+ = 4.98, LR- = 0.83). CONCLUSIONS: In our pilot study, TLR1 expression in periprosthetic tissues most accurately predicted periprosthetic joint infection. This measure of the host response may be particularly helpful in detecting culture-negative infections and avoiding false positives resulting from contamination. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.


Assuntos
Artrite Infecciosa/metabolismo , Infecções Relacionadas à Prótese/metabolismo , Receptor 1 Toll-Like/biossíntese , Artroplastia de Substituição , Biomarcadores/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Pessoa de Meia-Idade , Falha de Prótese , RNA Mensageiro/biossíntese , Reoperação , Receptor 10 Toll-Like/biossíntese , Receptor 6 Toll-Like/biossíntese
18.
Autophagy ; 8(5): 739-53, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22498493

RESUMO

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D 3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D 3 enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D 3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D 3 was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D 3 with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D 3). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.


Assuntos
Autofagia/efeitos da radiação , Neoplasias da Mama/patologia , Colecalciferol/farmacologia , Citoproteção/efeitos dos fármacos , Citoproteção/efeitos da radiação , Tolerância a Radiação/efeitos dos fármacos , Radiação Ionizante , Autofagia/efeitos dos fármacos , Autofagia/genética , Neoplasias da Mama/genética , Calcitriol/análogos & derivados , Calcitriol/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Inativação Gênica/efeitos dos fármacos , Humanos , Fagossomos/efeitos dos fármacos , Fagossomos/efeitos da radiação , Fagossomos/ultraestrutura , Tolerância a Radiação/efeitos da radiação , Receptor ErbB-2/metabolismo , Transfecção , Vacúolos/efeitos dos fármacos , Vacúolos/efeitos da radiação , Vacúolos/ultraestrutura
19.
J Biol Chem ; 283(1): 175-183, 2008 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-17974568

RESUMO

In hypocalcaemia, elevated parathyroid hormone transitorily down-regulates the kidney vitamin D receptor, which returns to normal levels with the rise in serum extracellular calcium [Ca(2+)](e). In this study, we investigated the mechanism that underlies VDR increase in kidney in association with elevated [Ca(2+)](e). Examination of MAP kinase signals in a proximal tubule human kidney (HK-2G) epithelial cell line showed that treatment of [Ca(2+)](e) in the culture medium elevated phosphorylation of both ERK and p38 MAPKs. Blockade of p38 phosphorylation with SB203580 or SB202190 in turn abolished [Ca(2+)](e)-mediated VDR protein increase, while treatment with PD98059 and U0126, specifically blocked ERK phosphorylation, but had no effect on VDR stimulation by [Ca(2+)](e). Furthermore, SB203580 treatment potently repressed [Ca(2+)](e)-mediated activation of VDR promoter. We also demonstrate that si-RNA knock down of p38alpha completely diminished high [Ca(2+)](e)-mediated VDR induction. Direct CaSR involvement was demonstrated by using an si-RNA of CaSR that impeded [Ca(2+)](e)-mediated induction of VDR. In conclusion, a high extracellular [Ca(2+)](e) concentration in the physiological range is capable of directly increasing renal proximal VDR expression, and the induction mechanism requires activation of the CaSR and signal mediation by the p38alpha MAP kinase pathway.


Assuntos
Espaço Extracelular/metabolismo , Túbulos Renais Proximais/metabolismo , Receptores de Calcitriol/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Butadienos/farmacologia , Cálcio/farmacologia , Linhagem Celular , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Imunoprecipitação , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , RNA Interferente Pequeno/genética , Receptores de Detecção de Cálcio/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/genética
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