RESUMO
Malignant mesothelioma is an aggressive neoplasm for which effective treatments are lacking. We often encounter mesothelioma cases with a profound desmoplastic reaction, suggesting the involvement of cancerassociated fibroblasts (CAFs) in mesothelioma progression. While the roles of CAFs have been extensively studied in other tumors and have led to the view that the cancer stroma contains heterogeneous populations of CAFs, their roles in mesothelioma remain unknown. We previously showed that connective tissue growth factor (CTGF), a secreted protein, is produced by both mesothelioma cells and fibroblasts and promotes the invasion of mesothelioma cells in vitro. In this study, we examined the clinical relevance of CAFs in mesothelioma. Using surgical specimens of epithelioid malignant pleural mesothelioma, we evaluated the clinicopathological significance of the expression of αsmooth muscle actin (αSMA), the most widely used marker of CAFs, the expression of CTGF, and the extent of fibrosis by immunohistochemistry and ElasticaMasson staining. We also analyzed the expression of mesenchymal stromal cell and fibroblastexpressing Linx paralogue (Meflin; ISLR), a recently reported CAF marker that labels cancerrestraining CAFs and differ from αSMApositive CAFs, by in situ hybridization. The extent of fibrosis and CTGF expression in mesothelioma cells did not correlate with patient prognosis. However, the expression of αSMA and CTGF, but not Meflin, in CAFs correlated with poor prognosis. The data suggest that CTGF+ CAFs are involved in mesothelioma progression and represent a potential molecular target for mesothelioma therapy.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Mesotelioma Maligno/mortalidade , Pleura/patologia , Neoplasias Pleurais/mortalidade , Actinas/análise , Actinas/metabolismo , Idoso , Fator de Crescimento do Tecido Conjuntivo/análise , Progressão da Doença , Feminino , Fibrose , Humanos , Imunoglobulinas/análise , Imunoglobulinas/metabolismo , Estimativa de Kaplan-Meier , Masculino , Mesotelioma Maligno/patologia , Mesotelioma Maligno/terapia , Pessoa de Meia-Idade , Terapia Neoadjuvante , Pleura/cirurgia , Neoplasias Pleurais/patologia , Neoplasias Pleurais/terapia , Taxa de SobrevidaRESUMO
The onset of malignant mesothelioma (MM) is linked to exposure to asbestos fibers. Asbestos fibers are classified as serpentine (chrysotile) or amphibole, which includes the crocidolite, amosite, anthophyllite, tremolite, and actinolite types. Although few studies have been undertaken, anthophyllite has been shown to be associated with mesothelioma, and tremolite, a contaminant in talc and chrysotile, is a risk factor for carcinogenicity. Here, after characterizing the length and width of these fibers by scanning electron microscopy, we explored the cytotoxicity induced by tremolite and anthophyllite in cells from an immortalized human mesothelial cell line (MeT5A), murine macrophages (RAW264.7), and in a rat model. Tremolite and short anthophyllite fibers were phagocytosed and localized to vacuoles, whereas the long anthophyllite fibers were caught on the pseudopod of the MeT5A and Raw 264.7 cells, according to transmission electron microscopy. The results from a 2-day time-lapse study revealed that tremolite was engulfed and damaged the MeT5A and RAW264.7 cells, but anthophyllite was not cytotoxic to these cells. Intraperitoneal injection of tremolite in rats induced diffuse serosal thickening, whereas anthophyllite formed focal fibrosis and granulomas on peritoneal serosal surfaces. Furthermore, the loss of Cdkn2a/2b, which are the most frequently lost foci in human MM, were observed in 8 cases of rat MM (homozygous deletion [5/8] and loss of heterozygosity [3/8]) by array-based comparative genomic hybridization techniques. These results indicate that tremolite initiates mesothelial injury and persistently frustrates phagocytes, causing subsequent peritoneal fibrosis and MM. The possible mechanisms of carcinogenicity based on fiber diameter/length are discussed.
Assuntos
Amianto/toxicidade , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias Pulmonares/genética , Mesotelioma/genética , Animais , Amiantos Anfibólicos/toxicidade , Asbesto Crocidolita/toxicidade , Asbestos Serpentinas/toxicidade , Hibridização Genômica Comparativa , Homozigoto , Humanos , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/patologia , Mesotelioma/induzido quimicamente , Mesotelioma/patologia , Mesotelioma Maligno , Ratos , Fatores de Risco , Deleção de Sequência/genéticaRESUMO
Malignant mesothelioma is an aggressive neoplasm with no particularly effective treatments. We previously reported that overexpression of connective tissue growth factor (CTGF/CCN2) promotes mesothelioma growth, thus suggesting it as a novel molecular target. A human monoclonal antibody that antagonizes CTGF (FG-3019, pamrevlumab) attenuates malignant properties of different kinds of human cancers and is currently under clinical trial for the treatment of pancreatic cancer. This study reports the effects of FG-3019 on human mesothelioma in vitro and in vivo. We analyzed the effects of FG-3019 on the proliferation, apoptosis, migration/invasion, adhesion and anchorage-independent growth in three human mesothelioma cell lines, among which ACC-MESO-4 was most efficiently blocked with FG-3019 and was chosen for in vivo experiments. We also evaluated the coexistent effects of fibroblasts on mesothelioma in vitro, which are also known to produce CTGF in various pathologic situations. Coexistent fibroblasts in transwell systems remarkably promoted the proliferation and migration/invasion of mesothelioma cells. In orthotopic nude mice model, FG-3019 significantly inhibited mesothelioma growth. Histological analyses revealed that FG-3019 not only inhibited the proliferation but also induced apoptosis in both mesothelioma cells and fibroblasts. Our data suggest that FG-3019 antibody therapy could be a novel additional choice for the treatment of mesothelioma.
RESUMO
Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/ß-catenin signaling by promoting phosphorylation and degradation οf ß-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of ß-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the ß-catenin degradation complex, minimized the suppressive effect of H2 on ß-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of ß-catenin, as well as the ß-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/ß-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating ß-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/ß-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases.
Assuntos
Hidrogênio/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Proteína da Polipose Adenomatosa do Colo/antagonistas & inibidores , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Proteína Axina/metabolismo , Caseína Quinase I/metabolismo , Linhagem Celular , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Gases/química , Quinase 3 da Glicogênio Sintase/metabolismo , Células HCT116 , Células HT29 , Células HeLa , Humanos , Hidrogênio/química , Leupeptinas/farmacologia , Cloreto de Lítio/farmacologia , Masculino , Microscopia de Fluorescência , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/veterinária , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição SOX9/metabolismo , Água/química , Proteína Wnt3A/metabolismoRESUMO
Asbestos was abundantly used in industry during the last century. Currently, asbestos confers a heavy social burden due to an increasing number of patients with malignant mesothelioma (MM), which develops after a long incubation period. Many studies have been conducted on the effects of the asbestos types that were most commonly used for commercial applications. However, there are few studies describing the effects of the less common types, or minor asbestos. We performed a rat carcinogenesis study using Japanese tremolite and Afghan anthophyllite. Whereas more than 50% of tremolite fibers had a diameter of < 500 nm, only a small fraction of anthophyllite fibers had a diameter of < 500 nm. We intraperitoneally injected 1 or 10 mg of asbestos into F1 Fischer-344/Brown-Norway rats. In half of the animals, repeated intraperitoneal injections of nitrilotriacetate (NTA), an iron chelator to promote Fenton reaction, were performed to evaluate the potential involvement of iron overload. Tremolite induced MM with a high incidence (96% with 10 mg; 52% with 1 mg), and males were more susceptible than females. Histology was confirmed using immunohistochemistry, and most MMs were characterized as the sarcomatoid or biphasic subtype. Unexpectedly NTA showed an inhibitory effect in females. In contrast, anthophyllite induced no MM after an observation period of 550 days. The results suggest that the carcinogenicity of anthophyllite is weaker than formerly reported, whereas that of tremolite is as potent as major asbestos as compared with our previous data.
Assuntos
Amiantos Anfibólicos/toxicidade , Transformação Celular Neoplásica/induzido quimicamente , Neoplasias Pulmonares/induzido quimicamente , Mesotelioma/induzido quimicamente , Animais , Amiantos Anfibólicos/química , Testes de Carcinogenicidade , Transformação Celular Neoplásica/patologia , Feminino , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/prevenção & controle , Masculino , Mesotelioma/patologia , Mesotelioma/prevenção & controle , Mesotelioma Maligno , Ácido Nitrilotriacético/toxicidade , Tamanho da Partícula , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Medição de Risco , Fatores de Risco , Fatores Sexuais , Fatores de TempoRESUMO
Like many other human cancers, the development of malignant mesothelioma is closely associated with a chronic inflammatory condition. Both macrophages and mesothelial cells play crucial roles in the inflammatory response caused by asbestos exposure. Here, we show that adipocytes can also contribute to asbestos-induced inflammation through dysregulated adipocytokine production. 3T3-L1 preadipocytes were differentiated into mature adipocytes prior to use. These cells took up asbestos fibers (chrysotile, crocidolite and amosite) but were more resistant to asbestos-induced injury than macrophages and mesothelial cells. Expression microarray analysis followed by reverse transcription-PCR revealed that adipocytes respond directly to asbestos exposure with an increased production of proinflammatory adipocytokines [e.g. monocyte chemoattractant protein-1 (MCP-1)], whereas the production of anti-inflammatory adipocytokines (e.g. adiponectin) is suppressed. This was confirmed in epididymal fat pad of mice after intraperitoneal injection of asbestos fibers. Such dysregulated adipocytokine production favors the establishment of a proinflammatory environment. Furthermore, MCP-1 marginally promoted the growth of MeT-5A mesothelial cells and significantly enhanced the wound healing of Y-MESO-8A and Y-MESO-8D human mesothelioma cells. Our results suggest that increased levels of adipocytokines, such as MCP-1, can potentially contribute to the promotion of mesothelial carcinogenesis through the enhanced recruitment of inflammatory cells as well as a direct growth and migration stimulatory effect on mesothelial and mesothelioma cells. Taken together, our findings support a potential cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis.
Assuntos
Adipócitos/patologia , Amianto/toxicidade , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/patologia , Mesotelioma/induzido quimicamente , Mesotelioma/patologia , Células 3T3-L1 , Adipócitos/metabolismo , Adipocinas/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Amianto/farmacocinética , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Mesotelioma Maligno , Camundongos , Camundongos EndogâmicosRESUMO
Multiwalled carbon nanotubes (MWCNTs) have attracted public attention not only for their potential applications in engineering and materials science but also for possible environmental risks. MWCNTs share similar properties with asbestos, a definite human carcinogen causing malignant mesothelioma (MM), in that they are both biopersistent thin fibers with a high aspect ratio. Certain types of MWCNTs do induce MM in animal experiments. Though there are many different types of MWCNTs awaiting use in industry, there is little evidence about what types of MWCNTs present a high risk for MMâ in vivo. We have previously shown that the diameter of MWCNTs is one of the critical factors for mesothelial injury, which eventually leads to MM. Because of the extensive commercial use of MWCNTs, the properties of MWCNTs that determine carcinogenic activity should be clarified. Here we report that a high dose (10 mg) of a tangled form of pristine MWCNT (with a diameter of 15 nm) did not induce MM after intraperitoneal administration in rats, which were followed for up to 3 years after injection. This observation strengthens our previous finding that the rigidity, diameter, length and surface properties of MWCNTs are important factors in MM induction in vivo.
Assuntos
Transformação Celular Neoplásica , Mesotelioma/patologia , Nanotubos de Carbono , Animais , Amianto/efeitos adversos , Transformação Celular Neoplásica/química , Modelos Animais de Doenças , Saúde Ambiental , Feminino , Injeções Intraperitoneais , Masculino , Mesotelioma/induzido quimicamente , Nanotubos de Carbono/efeitos adversos , Ratos , Ratos Endogâmicos F344RESUMO
Asbestos was used worldwide in huge quantities in the past century. However, because of the unexpected carcinogenicity to mesothelial cells with an extremely long incubation period, many countries face this long-lasting social problem. Mesothelioma is often diagnosed in an advanced stage, for which no effective therapeutic protocols are yet established. We previously reported on the basis of animal experiments that the major pathology in asbestos-induced mesothelial carcinogenesis is local iron overload. Here, we undertook to find an effective strategy to prevent, delay, or lower the malignant potential of mesothelioma during asbestos-induced carcinogenesis. We used intraperitoneal injections of crocidolite to rats. We carried out a 16-week study to seek the maximal-tolerated intervention for iron reduction via oral deferasirox administration or intensive phlebotomy. Splenic iron deposition was significantly decreased with either method, and we found that Perls' iron staining in spleen is a good indicator for iron reduction. We injected a total of 10 mg crocidolite at the age of six weeks, and the preventive measures were via repeated oral administration of 25 to 50 mg/kg/d deferasirox or weekly to bimonthly phlebotomy of 4 to 10 mL/kg/d. The animals were observed until 110 weeks. Deferasirox administration significantly increased the fraction of less malignant epithelioid subtype. Although we found a slightly prolonged survival in deferasirox-treated female rats, larger sample size and refinement of the current protocol are necessary to deduce the cancer-preventive effects of deferasirox. Still, our results suggest deferasirox serves as a potential preventive strategy in people already exposed to asbestos via iron reduction.
Assuntos
Asbesto Crocidolita/toxicidade , Benzoatos/toxicidade , Transformação Celular Neoplásica/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Quelantes de Ferro/toxicidade , Ferro/metabolismo , Mesotelioma/patologia , Triazóis/toxicidade , Animais , Transformação Celular Neoplásica/efeitos dos fármacos , Deferasirox , Feminino , Masculino , Mesotelioma/induzido quimicamente , Flebotomia , Ratos , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologiaRESUMO
Exposure to asbestos fibers increases the risk of mesothelioma in humans. One hypothetical carcinogenic mechanism is that asbestos fibers may directly induce mutations in mesothelial cells. Although the uptake of asbestos fibers by mesothelial cells is recognized, methods for the quantification of the uptake have not been well established. In the present study, we evaluated two distinct methods, using crocidolite fibers and MeT5A mesothelial cells. One method is histological evaluation using the cell-block technique, which allows for the direct cross-sectional observation of cells and fibers. We found the bright field observation with ×1000 magnification (oil-immersion) of the sample with Kernechtrot staining was most suitable for this purpose. The other method is flow cytometric analysis, which permits the evaluation of a much larger number of cells. We observed that the side scatter (SSC) increased with the intracellular fibers, and that the "mean SSC ratio (treated/control)" was useful for quantification. We could collect the cells with abundant internalized crocidolite fibers by sorting. Results of the two methodologies were correlated well in the experiments. The quantities of internalized fibers increased with incubation time and loaded dosage, but they were inversely associated with cellular density in culture.
RESUMO
Multiwalled carbon nanotubes (MWCNTs) have the potential for widespread applications in engineering and materials science. However, because of their needle-like shape and high durability, concerns have been raised that MWCNTs may induce asbestos-like pathogenicity. Although recent studies have demonstrated that MWCNTs induce various types of reactivities, the physicochemical features of MWCNTs that determine their cytotoxicity and carcinogenicity in mesothelial cells remain unclear. Here, we showed that the deleterious effects of nonfunctionalized MWCNTs on human mesothelial cells were associated with their diameter-dependent piercing of the cell membrane. Thin MWCNTs (diameter â¼ 50 nm) with high crystallinity showed mesothelial cell membrane piercing and cytotoxicity in vitro and subsequent inflammogenicity and mesotheliomagenicity in vivo. In contrast, thick (diameter â¼ 150 nm) or tangled (diameter â¼ 2-20 nm) MWCNTs were less toxic, inflammogenic, and carcinogenic. Thin and thick MWCNTs similarly affected macrophages. Mesotheliomas induced by MWCNTs shared homozygous deletion of Cdkn2a/2b tumor suppressor genes, similar to mesotheliomas induced by asbestos. Thus, we propose that different degrees of direct mesothelial injury by thin and thick MWCNTs are responsible for the extent of inflammogenicity and carcinogenicity. This work suggests that control of the diameter of MWCNTs could reduce the potential hazard to human health.