RESUMO
Adverse local tissue reactions (ALTRs) are a prominent cause of hip implant failure. ALTRs are characterized by aseptic necrosis and leukocyte infiltration of synovial tissue. The prevalence of ALTRs in hips with failing metal implants, with highest rates occurring in patients with metal-on-metal articulations, suggests a role for CoCrMo corrosion in ALTR formation. Although hypersensitivity reactions are the most accepted etiology, the precise cellular mechanism driving ALTR pathogenesis remains enigmatic. Here we show that cobalt ions released by failing hip implants induce mitochondrial stress and cytokine secretion by synovial fibroblasts: the presumptive initiators of ALTR pathogenesis. We found that in-vitro treatment of synovial fibroblasts with cobalt, but not chromium, generated gene expression changes indicative of hypoxia and mitophagy responses also observed in ALTRs biopsies. Inflammatory factors secreted by cobalt-exposed synovial fibroblasts were among those most concentrated in ALTR synovial fluid. Furthermore, both conditioned media from cobalt-exposed synovial fibroblasts, and synovial fluid from ALTRs patients, elicit endothelial activation and monocyte migration. Finally, we identify the IL16/CTACK ratio in synovial fluid as a possible diagnostic marker of ALTRs. Our results provide evidence suggesting that metal ions induce cell stress in synovial fibroblasts that promote an inflammatory response consistent with initiating ALTR formation. STATEMENT OF SIGNIFICANCE: We demonstrate that the cytotoxic effects of cobalt ions on the synovial cells (fibroblast) is sufficient to trigger inflammation on hip joints with metal implants. Cobalt ions affect mitochondrial function, leading to the auto phagocytosis of mitochondria and trigger a hypoxic response. The cell's hypoxic response includes secretion of cytokines that are capable of trigger inflammation by activating blood vessels and enhancing leukocyte migration. Among the secreted cytokines is IL-16, which is highly concentrated in the synovial fluid of the patients with adverse local tissue reactions and could be use as diagnostic marker. In conclusion we define the cells of the hip joint as key players in triggering the adverse reactions to hip implants and providing biomarkers for early diagnosis of adverse reactions to hip implants.
Assuntos
Artroplastia de Quadril , Prótese de Quadril , Próteses Articulares Metal-Metal , Quimiocinas , Cromo , Cobalto/toxicidade , Citocinas , Fibroblastos , Prótese de Quadril/efeitos adversos , Humanos , Íons , Desenho de Prótese , Falha de Prótese , Estresse FisiológicoRESUMO
Aberrant cholesterol metabolism is increasingly appreciated to be essential for prostate cancer initiation and progression. Transcript expression of the high-density lipoprotein-cholesterol receptor scavenger receptor B1 (SR-B1) is elevated in primary prostate cancer. Hypothesizing that SR-B1 expression may help facilitate malignant transformation, we document increased SR-B1 protein and transcript expression in prostate cancer relative to normal prostate epithelium that persists in lethal castration-resistant prostate cancer (CRPC) metastasis. As intratumoral steroid synthesis from the precursor cholesterol can drive androgen receptor (AR) pathway activity in CRPC, we screened androgenic benign and cancer cell lines for sensitivity to SR-B1 antagonism. Benign cells were insensitive to SR-B1 antagonism, and cancer line sensitivity inversely correlated with expression levels of full-length and splice variant AR. In androgen-responsive CRPC cell model C4-2, SR-B1 antagonism suppressed cholesterol uptake, de novo steroidogenesis, and AR activity. SR-B1 antagonism also suppressed growth and viability and induced endoplasmic reticulum stress and autophagy. The inability of exogenous steroids to reverse these effects indicates that AR pathway activation is insufficient to overcome cytotoxic stress caused by a decrease in the availability of cholesterol. Furthermore, SR-B1 antagonism decreased cholesterol uptake, growth, and viability of the AR-null CRPC cell model PC-3, and the small-molecule SR-B1 antagonist block lipid transport-1 decreased xenograft growth rate despite poor pharmacologic properties. Overall, our findings show that SR-B1 is upregulated in primary and castration-resistant disease and is essential for cholesterol uptake needed to drive both steroidogenic and nonsteroidogenic biogenic pathways, thus implicating SR-B1 as a novel and potentially actionable target in CRPC. SIGNIFICANCE: These findings highlight SR-B1 as a potential target in primary and castration-resistant prostate cancer that is essential for cholesterol uptake needed to drive steroidogenic and nonsteroidogenic biogenic pathways.
Assuntos
Androgênios/metabolismo , Neoplasias Ósseas/secundário , Colesterol/metabolismo , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Depuradores Classe B/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/cirurgia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/cirurgia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/cirurgia , Masculino , Camundongos , Camundongos Nus , Orquiectomia , Prognóstico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/cirurgia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores Depuradores Classe B/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
"Rods and rings" (RR) and loukoumasomes are similarly shaped, subcellular macromolecular structures with as yet unknown function. RR, so named because of their shape, are formed in response to inhibition in the GTP or CTP synthetic pathways and are highly enriched in the two key enzymes of the nucleotide synthetic pathway. Loukoumasomes also occur as linear and toroidal bodies and were initially inferred to be the same as RR, largely due to their shared shape and size and the fact that it was unclear if they shared the same subcomponents. In human retinoblastoma tissue and cells we have observed toroidal, perinuclear, macromolecular structures of similar size and antigenicity to those previously reported in neurons (neuronal-loukoumasomes). To further characterize the subcomponents of the retinal-loukoumasomes, confocal analysis following immunocytochemical staining for alpha-tubulin, beta-III tubulin and detyrosinated tubulin was performed. These studies indicate that retinal-loukoumasomes are enriched for beta-III tubulin and other tubulins associated with microtubules. Immunofluorescence together with the in situ proximity ligation assay (PLA), confirmed that beta-III tubulin colocalized with detyrosinated tubulin within loukoumasomes. Our results indicate that these tissues contain only loukoumasomes because these macromolecular structures are immunoreactive with an anti-tubulin antibody but are not recognized by the prototype anti-RR/inosine monophosphate dehydrogenase (IMPDH) antibody (It2006). To further compare the RR and retinal-loukoumasomes, retinoblastoma cells were exposed to the IMPDH-inhibitor ribavirin, a drug known to induce the formation of RR. In contrast to RR, the production of retinal-loukoumasomes was unaffected. Coimmunostaining of Y79 cells for beta-III tubulin and IMPDH indicate that these cells, when treated with ribavirin, can contain both retinal-loukoumasomes and RR and that these structures are antigenically distinct. Subcellular fractionation studies indicate that ribavirin increased the RR subcomponent, IMPDH, in the nuclear fraction of Y79 cells from 21.3 ± 5.8% (0 mM ribavirin) to 122.8 ± 7.9% (1 mM ribavirin) while the subcellular localization of the retinal-loukoumasome subcomponent tubulin went unaltered. Further characterization of retinal-loukoumasomes in retinoblastoma cells reveals that they are intimately associated with lamin folds within the nuclear envelope. Using immunofluorescence and the in situ PLA in this cell type, we have observed colocalization of beta-III tubulin with MAP2. As MAP2 is a microtubule-associated protein implicated in microtubule crosslinking, this supports a role for microtubule crosslinkers in the formation of retinal-loukoumasomes. Together, these results suggest that loukoumasomes and RR are distinct subcellular macromolecular structures, formed by different cellular processes and that there are other loukoumasome-like structures within retinal tissues and cells.
Assuntos
Estruturas Citoplasmáticas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Retina/citologia , Retina/metabolismo , Neurônios Adrenérgicos/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Laminas/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Retinoblastoma/metabolismo , Ribavirina/farmacologia , Tubulina (Proteína)/metabolismoRESUMO
Anti-integrin-linked kinase (ILK) therapies result in aberrant mitosis including altered mitotic spindle organization, centrosome declustering and mitotic arrest. In contrast to cells that expressed the retinoblastoma tumor suppressor protein Rb, we have shown that in retinoblastoma cell lines that do not express Rb, anti-ILK therapies induced aberrant mitosis that led to the accumulation of temporarily viable multinucleated cells. The present work was undertaken to: 1) determine the ultimate fate of cells that had survived anti-ILK therapies and 2) determine whether or not Rb expression altered the outcome of these cells. Our data indicate that ILK, a chemotherapy drug target is expressed in both well-differentiated, Rb-negative and relatively undifferentiated, Rb-positive retinoblastoma tissue. We show that small molecule targeting of ILK in Rb-positive and Rb-deficient cancer cells results in increased centrosomal declustering, aberrant mitotic spindle formation and multinucleation. However, anti-ILK therapies in vitro have different outcomes in retinoblastoma and glioblastoma cell lines that depend on Rb expression. TUNEL labeling and propidium iodide FACS analysis indicate that Rb-positive cells exposed to anti-ILK therapies are more susceptible to apoptosis and senescence than their Rb-deficient counterparts wherein aberrant mitosis induced by anti-ILK therapies exhibit mitotic arrest instead. These studies are the first to show a role for ILK in chemotherapy-induced senescence in Rb-positive cancer lines. Taken together these results indicate that the oncosuppressive outcomes for anti-ILK therapies in vitro, depend on the expression of the tumor suppressor Rb, a known G1 checkpoint and senescence regulator.