RESUMO
Hepatic stellate cells (HSCs) are the main extracellular matrix (ECM)producing cells in liver fibrosis. Activated HSCs stimulate the proliferation and migration of hepatocellular carcinoma (HCC) cells. Cysteinerich 61 (CCN1/Cyr61) is an ECM protein. Our previous studies demonstrated that the expression of CCN1 was significantly higher in benign hepatic cirrhosis tissue and canceradjacent hepatic cirrhosis tissues. CCN1 is a target gene of ßcatenin in HCC and promotes the proliferation of HCC cells. The present study aimed to examine whether CCN1 can activate HSCs and affect the function of activated HSCs in promoting the progression of HCC. CCN1 expression was determined during the progression of liver fibrosis in a mouse model. LX2 cells, which were infected with adenoviruses AdCCN1 or AdRFP, and HepG2 cells were cocultured or subcutaneously coimplanted into in nude mice. MTT assay, Crystal Violet staining, Boyden chamber, matrigel invasion and monolayer scratch assays were used to analyze the proliferation, migration and invasion capability of HepG2 cells. Xenograft sizes were measured and histological analyses were performed by hematoxylin and eosin, immunohistochemical, immunefluorescence and Sirius Red staining. It was demonstrated that the expression of CCN1 was continually increased in liver fibrosis and the that expression may be correlated with the progression of liver fibrosis. CCN1 affected the function of LX2 and enhanced the effect of LX2 on promoting the viability, migration and invasion of HepG2 cells in vitro. CCN1 enhanced the effect of LX2 on promoting the growth of HepG2 xenografts in vivo. CCN1 also affected the function of activated HSCs and regulated the formation of the xenograft microenvironment, including fibrogenesis and angiogenesis, which are beneficial for the progression of HCC. These findings demonstrated that CCN1 may be involved in the progression of the hepatic cirrhosisHCC axis through regulating HSCs.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteína Rica em Cisteína 61/metabolismo , Progressão da Doença , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Animais , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Células Hep G2 , Humanos , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos BALB C , Microvasos/patologia , Invasividade Neoplásica , Tela Subcutânea/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND/AIMS: Joint cartilage defects are difficult to treat due to the limited self-repair capacities of cartilage. Cartilage tissue engineering based on stem cells and gene enhancement is a potential alternative for cartilage repair. Bone morphogenetic protein 2 (BMP2) has been shown to induce chondrogenic differentiation in mesenchymal stem cells (MSCs); however, maintaining the phenotypes of MSCs during cartilage repair since differentiation occurs along the endochondral ossification pathway. In this study, hypoxia inducible factor, or (HIF)-1α, was determined to be a regulator of BMP2-induced chondrogenic differentiation, osteogenic differentiation, and endochondral bone formation. METHODS: BMP2 was used to induce chondrogenic and osteogenic differentiation in stem cells and fetal limb development. After HIF-1α was added to the inducing system, any changes in the differentiation markers were assessed. RESULTS: HIF-1α was found to potentiate BMP2-induced Sox9 and the expression of chondrogenesis by downstream markers, and inhibit Runx2 and the expression of osteogenesis by downstream markers in vitro. In subcutaneous stem cell implantation studies, HIF-1α was shown to potentiate BMP2-induced cartilage formation and inhibit endochondral ossification during ectopic bone/cartilage formation. In the fetal limb culture, HIF-1α and BMP2 synergistically promoted the expansion of the proliferating chondrocyte zone and inhibited chondrocyte hypertrophy and endochondral ossification. CONCLUSION: The results of this study indicated that, when combined with BMP2, HIF-1α induced MSC differentiation could become a new method of maintaining cartilage phenotypes during cartilage tissue engineering.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Condrogênese , Membro Anterior/crescimento & desenvolvimento , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Mesenquimais/fisiologia , Osteogênese , Animais , Proteína Morfogenética Óssea 2/genética , Diferenciação Celular , Células Cultivadas , Membro Anterior/embriologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Cartilagem Hialina/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
BACKGROUND: CCN1 plays distinct roles in various tumor types, but little is known regarding the role of CCN1 in leukemia. METHODS: We analyzed CCN1 protein expression in leukemia cell lines and in AML bone marrow samples. We also evaluated the effects of antibody- or siRNA-mediated inhibition of CCN1 on the growth of two AML cell lines (U937 and Kasumi-1 cells) and on the MEK/ERK pathway, ß-catenin and other related genes. RESULTS: U937 and Kasumi-1 cells had markedly higher CCN1 expression than the 5 other leukemia cell lines, and CCN1 protein expression was higher in the AML bone marrow samples than in the normal bone marrow samples. Blocking CCN1 with an antibody in U937 and Kasumi-1 cells suppressed proliferation, increased apoptosis, down-regulated Bcl-xL and c-Myc expression, up-regulated Bax expression, and had no effect on Survivin. siRNA-mediated down-regulation of CCN1 inhibited the proliferation and colony formation of U937 and Kasumi-1 cells and increased cytarabine-induced apoptosis. Furthermore, CCN1 siRNA reduced MEK and ERK phosphorylation without affecting ß-catenin; the CCN1 antibody similarly affected MEK and ERK phosphorylation. These changes in phosphorylation could influence the expression of Bcl-xL, c-Myc and Bax in AML cells. CONCLUSIONS: The data suggested that CCN1 is a tumor promoter in AML that acts through the MEK/ERK pathway to up-regulate c-Myc and Bcl-xL and to down-regulate Bax.
RESUMO
Imatinib Mesylate is widely used for the treatment of chronic myelogenous leukaemia (CML), and its effects on CML cells are influenced by several signalling proteins. The research is aimed at determining whether Wnt5a affects the effects of Imatinib Mesylate against BCR-ABL positive CML cells (K562 cells and KU812 cells) and which signalling proteins are involved in. The results showed that Wnt5a augmented the effects of Imatinib Mesylate on inhibiting CML cells proliferation and inducing apoptosis in vitro; Wnt5a enhanced the inhibition effect of Imatinib Mesylate on the growth of K562 cells xenograft tumour in an animal model. Furthermore, Wnt5a inhibited ß-catenin and its target gene Survivin, increased the activity of JNK and suppressed γ-catenin expression. When inhibiting the activity of JNK, the influence of Wnt5a on the effects of Imatinib Mesylate was attenuated. Moreover, JNK suppressed ß-catenin and its target gene Survivin, and enhanced the effects of Imatinib Mesylate. These results suggest that Wnt5a can enhance the efficacy of Imatinib Mesylate through JNK/ß-catenin/Survivin and γ-catenin/ß-catenin/Survivin pathways.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Proliferação de Células , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , MAP Quinase Quinase 4/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Pirimidinas/farmacologia , Proteínas Wnt/metabolismo , gama Catenina/metabolismo , Animais , Apoptose , Western Blotting , Citometria de Fluxo , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Células Tumorais Cultivadas , Proteína Wnt-5aRESUMO
γ-catenin plays different roles in different types of tumors, and its role in chronic myeloid leukemia (CML) cells has yet to be identified. In our study, two CML cell lines (K562, KU812) had higher γ-catenin expression levels compared to five types of BCR-ABL-negative leukemia cells. Knockdown of the expression of BCR-ABL resulted in downregulation of γ-catenin. Furthermore, downregulation of γ-catenin by siRNA inhibited the proliferation and colony formation of CML cells and the expression of the c-Myc and cyclin D1 genes; downregulation of γ-catenin also potentiated the effects of imatinib (inhibiting CML cell proliferation and inducing apoptosis) and suppressed the anti-apoptotic genes Bcl-xL and survivin. We also showed that downregulation of γ-catenin suppressed the phosphorylation of STAT5, promoted the phosphorylation of ß-catenin and reduced the translocation of ß-catenin into the nucleus, although there were no effects on the total level of ß-catenin expression in the whole cells. Furthermore, downregulation of γ-catenin was found to promote glycogen synthase kinase-3ß (GSK3ß) and inhibit its phosphorylation. Collectively, our results suggest that γ-catenin is an oncogene protein in CML that can be regulated by BCR-ABL and that suppression of γ-catenin inhibits CML cell growth and potentiates the effects of imatinib on CML cells through inhibition of the activation of STAT5 and suppression of ß-catenin by activating GSK3ß.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Regulação para Baixo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Piperazinas/farmacologia , Pirimidinas/farmacologia , beta Catenina/metabolismo , gama Catenina/genética , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Fosforilação , Fator de Transcrição STAT5/metabolismo , gama Catenina/metabolismoRESUMO
Abnormal activation of the canonical Wnt signaling pathway has been implicated in carcinogenesis. Transcription of Wnt target genes is regulated by nuclear ß-catenin, whose over-expression is observed in Hepatocellular Carcinoma (HCC) tissue. Cyr61, a member of the CCN complex family of multifunctional proteins, is also found over-expressed in many types of tumor and plays dramatically different roles in tumorigenesis. In this study, we investigated the relationship between Cyr61 and ß-catenin in HCC. We found that while Cyr61 protein was not expressed at a detectable level in the liver tissue of healthy individuals, its expression level was elevated in the HCC and HCC adjacent tissues and was markedly increased in cancer-adjacent hepatic cirrhosis tissue. Over-expression of Cyr61 was positively correlated with increased levels of ß-catenin in human HCC samples. Activation of ß-catenin signaling elevated the mRNA level of Cyr61 in HepG2 cells, while inhibition of ß-catenin signaling reduced both mRNA and protein levels of Cyr61. We identified two TCF4-binding elements in the promoter region of human Cyr61 gene and demonstrated that ß-catenin/TCF4 complex specifically bound to the Cyr61 promoter in vivo and directly regulated its promoter activity. Furthermore, we found that over-expression of Cyr61 in HepG2 cells promoted the progression of HCC xenografts in SCID mice. These findings indicate that Cyr61 is a direct target of ß-catenin signaling in HCC and may play an important role in the progression of HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteína Rica em Cisteína 61/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Proteína Rica em Cisteína 61/genética , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Regiões Promotoras Genéticas , Transplante Heterólogo , Via de Sinalização WntRESUMO
Although down-regulation of WNT5A expression has been reported in some types of leukaemias, the level of WNT5A expression has not been assessed in leukaemia complete remission (CR) cases, the relationship among WNT5A expression level, the status of its promoter methylation, and the curative effect of leukaemia has not been reported, and the effect of WNT5A on cell proliferation has not been assessed. In this study, we analyzed WNT5A expression in various kinds of leukaemia cases, leukaemia CR cases, non-malignant hematopoietic (NMH) cases, as well as in leukemic cell lines and CD34+ cells. The methylation status of the WNT5A promoter and the levels of the Wnt5a protein were also studied. We also investigated the effect of Wnt5a on leukemic cell proliferation. WNT5A expression level was higher in NMH but lower in leukaemia cases compared to that in CR-cases (P<0.01), and was expressed at low level in leukemic cell lines K562, U937 and Jurkat. Wnt5a protein was positive in NMH, CR cases and CD34+, but negative in leukaemia cases. WNT5A promoter was methylated in leukaemia cases and all leukemic cell lines, but not in NMH and CR cases. WNT5A expression was up-regulated after exposure to the demethylating agent 5-Aza-2'-deoxycytidine (Aza) in the K562, U937, Jurkat leukemic cell lines and in 83.3% (10/12) of CR patients after cure, respectively. The increased Wnt5a protein can inhibit K562 malignant proliferation and arrest cell cycle at the G2/M phase after exposure to Aza. These results indicate that WNT5A expression was restored in complete remission cases due to demethylation, and Wnt5a can inhibit leukaemic cell proliferation. We propose that WNT5A can act as a suppressor factor in leukemogenesis and can be used as a potential marker for curative effect assessment in leukaemia.
Assuntos
Proliferação de Células , Metilação de DNA/fisiologia , Leucemia/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Wnt/genética , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/fisiologia , Criança , Regulação para Baixo , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Células Jurkat , Células K562 , Leucemia/diagnóstico , Leucemia/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Células Tumorais Cultivadas , Células U937 , Proteínas Wnt/metabolismo , Proteínas Wnt/fisiologia , Proteína Wnt-5a , Adulto JovemRESUMO
Wnt5a has been shown to be involved in cancer progression in a variety of tumor types, and regulates multiple intracellular signaling cascades; it is a representative ligand that activates a noncanonical Wnt signaling pathway. The mechanism governing how Wnt5a determines the specificity of these pathways and the relationship with tumorigenesis is still unknown. In this study, we aimed to clarify the tumor suppressor role of Wnt5a in leukemogenesis. In particular, we focused on Ror2 functioning as a Wnt5a receptor to mediate noncanonical Wnt signaling, which inhibits canonical Wnt signaling in K562 cells. We found that up-regulation of Wnt5a expression increased Ror2 expression in K562 cells and Wnt5a and Ror2 were co-expressed in the cytoplasm. Also, Wnt5a induced the intrnalization of Ror2. Co-immunoprecipitation experiments were performed to determine whether Ror2 binds to Wnt5a, and inhibits Wnt5a binding with Frizzled4 and LRP5 in Wnt5a treated K562 cells. Wnt5a had no effect on total ß-catenin expression levels, but regulated tyrosine phosphorylation of ß-catenin and translocation of ß-catenin from the cytoplasm to the nucleus. Furthermore, expression of Wnt5a was associated with suppression of ß-catenin/TCF-dependent transcriptional activity and down-regulated the expression of cyclin D1, a downstream target gene of the canonical Wnt signaling pathway. We hypothesize that Wnt5a plays the role of a tumor suppressor in leukemogenesis through the Wnt5a/Ror2 noncanonical signaling pathway that inhibits Wnt canonical signaling.
Assuntos
Células K562/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Humanos , Células K562/ultraestrutura , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Fosforilação , Proteínas Proto-Oncogênicas/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/genética , Proteína Wnt-5a , beta Catenina/metabolismoRESUMO
This study was aimed to investigate the expression level of Wnt5a gene in some hematologic diseases and leukemic cell lines so as to provide a basis for further research of Wnt5a role and its mechanism in hematologic malignancies. The mononuclear cells of peripheral blood and bone marrow were isolated by human lymphocytic isolation solution. The expression of Wnt5a gene in specimen of 31 cases and three leukemic cell lines (Jurkat, K562, HL-60) were detected by RT-PCR. The results showed that in four out of five AML cases, negative or weak positive expressions were observed and negative expressions were observed also in K562 and HL-60 cells. Only in one AML case with complete remission and Jurkat cells the strong positive expressions were observed. The negative expression was observed in all six CML cases. In three out of four ALL cases, the expression was positive or weak positive and one negative. The expressions in two CLL cases were negative. Out of two MM cases, the expression in one was weak positive and in other was negative. Out of three lymphoma cases, the expression in one case was weak positive and in other two cases were negative. There were positive or weak positive expressions in two cases of AA, two cases of IDA, three cases of ITP, one cases of PV and ET cases. It is concluded that there have obvious down-regulated or lost expression of Wnt5a gene in 31 cases of hematologic disease and myelocytic leukemic cell lines except ALL samples. Nevertheless there have general positive expression of Wnt5a in cases of non-malignant hematologic diseases. These results suggest that the genesis of myelocytic leukemia is related to the down-regulated expression of Wnt5a.
Assuntos
Neoplasias Hematológicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Adolescente , Adulto , Idoso , Criança , Regulação para Baixo , Regulação Leucêmica da Expressão Gênica , Neoplasias Hematológicas/metabolismo , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteína Wnt-5a , Adulto JovemRESUMO
This study was aimed to investigate the effect of exogenous Wnt5a on directional differentiation of K562 cells. Wnt5a and GFP condition mediums were prepared by recombinant adenoviral vector AdWnt5a and AdGFP transfecting CHO cells. K562 cells were treated with Wnt5a and the GFP condition mediums for 1 - 7 days as Wnt5a treated group and control group respectively. The morphological changes of K562 cells were observed by light microscope and electron microscope; the differentiation phenotypes of K562 cells were identified by the cytochemical staining of POX, PAS, alpha-NAE and immunocytochemistry of CD13, CD14, CD68, and the effect of Wnt5a on cell cycle distribution of K562 cells was detected by flow cytometry. The results showed that the morphology and ultrastructure of K562 cells treated by Wnt5a displayed differentiation mature feature; both POX and PAS staining showed higher positive ratio in Wnt5a treated group than that in control group; the alpha-NAE staining also was positive, but positive intensity in Wnt5a treated group could be inhibited up to 70% by NaF. The expressions of monocytic differentiation antigens of CD14, CD68 in Wnt5a treated group were higher than those in control group, but the expression differences of granulocytic differentiation antigen CD13 between Wnt5a treated group and control group were not significant. The cell cycle in treated group was blocked at G2 phase as compared with control group. It is concluded that exogenous Wnt5a can induce K562 cells to differentiate towards mature and K562 cells treated with Wnt5a displays features of differentiation towards monocytic lineage.