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Alpha-fetoprotein (AFP) is a biomarker that is used to diagnose hepatocellular carcinoma (HCC) and can promote malignancy in HCC. AFP is an important target in the treatment of liver cancer. To obtain enough AFP to screen for AFP inhibitors, we expressed and purified AFP in HEK-293 cells. In the present study, we produced AFP in the cells and harvested highly pure rAFP (or recombinant expression AFP in HEK-293 cells). We also analysed the bioactivity of rAFP and found that rAFP promoted growth of the human HCC cells, antagonize paclitaxel inhibition of HCC cell proliferation, suppress expression of active caspase-3, and promote expression of Ras and survivin. This study provides a method to produce significant amounts of AFP for use in biochemical assays and functional studies and to screen AFP inhibitors for use in HCC therapy.
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Expresión Génica , alfa-Fetoproteínas , Carcinoma Hepatocelular/metabolismo , Caspasa 3/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Survivin , alfa-Fetoproteínas/biosíntesis , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/aislamiento & purificación , alfa-Fetoproteínas/farmacologíaRESUMEN
A high level of serum alpha fetoprotein (AFP) is positively associated with human hepatocellular carcinoma (HCC) carcinogenesis and metastasis; however, the function of AFP in HCC metastasis is unknown. This study has explored the effects of AFP on regulating metastatic and invasive capacity of human HCC cells. Forty-seven clinical patients' liver samples were collected and diagnosed; HCC cells line, Bel 7402 cells (AFP-producing) and liver cancer cell line cells (non-AFP-producing) were selected to analyse the role of AFP in the metastasis of HCC cells. The results indicated that high serum concentration of AFP was positively correlated with HCC intrahepatic, lymph nodes and lung metastasis. Repressed expression of AFP significantly inhibited the capability of migration and invasion of Bel 7402 cells, expression of keratin 19 (K19), epithelial cell adhesion molecule (EpCAM), matrix metalloproteinase 2/9 (MMP2/9) and CXC chemokine receptor 4 (CXCR4) were also down-regulated in Bel 7402 cells; migration and invasion, expression of K19, EpCAM, MMP2/9 and CXCR4 were significantly enhanced when HLE cells were transfected with AFP-expressed vector. The results demonstrated that AFP plays a critical role in promoting metastasis of HCC; AFP promoted HCC cell invasion and metastasis via up-regulating expression of metastasis-related proteins. Thus, AFP may be used as a novel therapeutic target for treating HCC patients.
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Carcinoma Hepatocelular/sangre , Neoplasias Hepáticas/sangre , alfa-Fetoproteínas/fisiología , Adulto , Anciano , Carcinoma Hepatocelular/secundario , Línea Celular Tumoral , Movimiento Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Regulación hacia ArribaRESUMEN
BACKGROUND: Hepatitis B virus (HBV)-X protein(HBx) is a transactivator of host several cellular genes including alpha-fetoprotein(AFP) and AFP receptor(AFPR) which contributes to HBV-associated tumor development. The expression of AFP/AFPR are correlated with hepatocellular carcinoma(HCC)-initial cells. But the role of AFP and AFPR in promoting occurrence of HBV-related HCC were still unclear. METHODS: A total of 71 clinical patients' liver specimens, normal human liver cells L-02 and HCC cell lines, PLC/PRF/5 were selected for analyzing the effects of HBx on expression of AFP, AFPR and Src. The expression of goal proteins were detected by Immunohistochemical stained and Western blotting; HBx-expressed vectors were constructed and transfected into L-02 cells, laser confocal microscopy was applied to observe expression and location of AFP, AFPR and Src in the normal liver cells and HCC cells, soft agar colony formation assay was used to observe colonies formed of the cells. RESULTS: We confirmed HBx gives preference to promote the expression of AFP and AFPR; HBx priors to up-regulate the expression of AFPR and AFP in L-02 cells and in normal liver specimens; AFPR signal been able to stimulate Src expression. The results also indicated that phosphatidylinositol 3-kinase(PI3K) inhibitors Ly294002 and GDC0941 effectively suppress AFPR mediated up-regulation expression of Src in AFPR positive HCC lines. CONCLUSIONS: HBx priors to drive the expression of AFP and AFPR to promote expression of Src in normal liver cells and hepatoma cells; AFP and AFPR maybe play pivotal role in HBV-related hepatocarcinogenesis; Targeting AFPR is an available therapeutic strategy of HCC.
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Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transactivadores/fisiología , Adulto , Anciano , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Hepatitis B/genética , Hepatitis B/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Hígado/virología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Transducción de Señal , Activación Transcripcional , Proteínas Reguladoras y Accesorias Virales , Adulto Joven , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
OBJECTIVE: To explore the effect of alpha-fetoprotein (AFP) on transduction of the PI3K/ AKT signal in hepatocellular carcinoma cells and the role played by AFP in resistance to cytotoxicity of all-trans retinoic acid (ATRA). METHODS: The effects of ATRA of human liver cancer cells was assessed using the BEL-7402 cell line with the MTT assay (to evaluate proliferation), microscopy (to evaluate morphology), flow cytometry (to evaluate apoptosis), laser confocal microscopy and coimmunoprecipitation (co-IP; to evaluate co-localization and interaction of AFP with PTEN), Western blotting (to evaluate expression of phosphorylated-protein kinase B (pAKT) and Src, and RNA interference (RNAi)-mediated knockdown of AFP. Finally, application of the PI3K-specific inhibitor Ly294002 was used to monitor the influence of AFP in transduction of the PI3K signal pathway. RESULTS: The human hepatoma cell line BEL-7402 were resistant to ATRA cytotoxicity. PTEN and AFP co-localized in the cytoplasm, and co-IP indicated that AFP interacts with PTEN in BEL-7402 cells.RNAi knockdown of AFP expression led to reduced growth of BEL-7402 cells.BEL-7402 cells transfected with AFP-short interfering (si)RNA vectors showed enhanced sensitivity to ATRA and reduced expression of pAKT(Ser473) and Src; Ly294002 reduced the role of AFP in stimulating expression of pAKT(Ser473) and Src. CONCLUSION: AFP can activate transduction of the PI3K/AKT signal, and expression of AFP in hepatoma cells is a pivotal event for resisting ATRA-induced apoptosis.
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Apoptosis , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacología , alfa-Fetoproteínas/metabolismo , Western Blotting , Línea Celular Tumoral , Citoplasma , Humanos , Inmunoprecipitación , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Interferencia de ARN , ARN Interferente Pequeño , TransfecciónRESUMEN
[This corrects the article DOI: 10.7150/jca.13628.].
RESUMEN
OBJECTIVE: To explore the mechanism of Alpha-fetoprotein (AFP) effects on hepatocellular carcinoma cells (HCC) resistances apoptosis induced by tumor necrosis factor-related apoptosis inducing-ligand (TRAIL). METHODS: The expressed alteration of TRAIL receptor-2 (DR5) after the human hepatoma cells line Bel 7402 (AFP-producing) and HLE cells (non-AFP producing) were treated with all trans retinoic acid (ATRA) were determined by Western blot; Interaction of AFP with RAR-beta was analyzed by co-immunoprecipitation (Co-IP); Laser confocal microscopy was used to observe co-localization of AFP and RAR-beta; Short small RNA interfering (RNAi) was applied to knock down the expression of AFP in Bel 7402 cells; The full AFP gene cDNA was inserted into pcDNA3.1 vector and constructed the expressed vector of AFP (named pcDNA3.1-afp); The growth of hepatoma cells was analyzed by MTT. RESULTS: Bel 7402 and HLE cells expressed DR5, lowed dosage of ATRA (40mumol/L) had no influence on the expression of DR5 in Bel 7402 cells, but ATRA (160mumol/L) could inhibit the expression of AFP and promote the expression of DR5 significantly; Co-IP indicated that AFP had a property for interacting with RAR-beta; The results also demonstrated AFP co-localization with RAR-beta in cytoplasm of Bel 7202 cells; The expression of DR5 was enhanced while the expression of AFP was knocked down by RNAi. pcDNA3.1-afp vector was transfected into HLE cells, the growth of HLE cells were stimulated and TRAIL cytotoxicity of HLE cells were reduced. But when the expression of AFP was knocked down the sensitivity of Bel 7402 cells to TRAIL was enhanced. CONCLUSIONS: These data provided that AFP had a capability to interact with RAR-beta and suppressed the expression of DR5. AFP could play pivotal role on hepatoma cells resistance-induced apoptosis by TRAIL.
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Apoptosis , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , alfa-Fetoproteínas/metabolismo , Línea Celular Tumoral/metabolismo , Humanos , Receptores de Ácido Retinoico/metabolismo , Tretinoina/farmacologíaRESUMEN
Hepatocellular carcinoma (HCC) cell resistance to the effects of paclitaxel has not been adequately addressed. In this study, we found that paclitaxel significantly inhibited the viability of HLE, Bel 7402 and L-02 cells in a dose- and time-dependent manner. HLE cells and L-02 cells resisted the cytotoxicity of paclitaxel when transfected with pcDNA3.1-afp vectors. However, Bel 7402 cell sensitivity to paclitaxel was increased when transfected with alpha fetoprotein (AFP)-siRNA. Bel 7402 cell resistance to paclitaxel was associated with the expression of the "stemness" markers CD44 and CD133. Paclitaxel significantly inhibited growth and promoted apoptosis in HLE cells and L-02 cells by inducing fragmentation of caspase-3 and inhibiting the expression of Ras and Survivin, but pcDNA3.1-afp vectors prevented these effects. However, paclitaxel could not significantly promote the cleavage of caspase-3 or suppress the expression of Ras and Survivin in Bel 7402 cells. Silenced expression of AFP may be synergistic with paclitaxel to restrain proliferation and induce apoptosis, enhance cleavage of caspase-3, and suppress the expression of Ras and Survivin. Taken together, AFP may be an important molecule acting against paclitaxel-inhibited proliferation and induced apoptosis in HCC cells via repressing the activity of caspase-3 and stimulating the expression of Ras and Survivin. Targeted inhibition of AFP expression after treatment with paclitaxel is an available strategy for the therapy of patients with HCC.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos , Paclitaxel/farmacología , alfa-Fetoproteínas/fisiología , Apoptosomas/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Survivin , Proteínas ras/metabolismoRESUMEN
BACKGROUND: The infection of Hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma(HCC), HBV-X protein(HBx) is able to induce expression of alpha-fetoprotein(AFP) in normal liver cells, and AFP harbors a function to promote malignant transformation of normal liver cells, but the role AFP playing in malignant behaviors of HCC cells is still unclear. METHODS: Fifty-six liver tissue samples were collected from the clinical patients through hepatectomy(include normal liver tissues, HBV-related hepatitis liver tissues and HBV-related HCC tissues), and diagnosis of these tissues by pathology section, expression of AFP, Ras and CXCR4 were evidenced by immunohisochemical staining and Western blotting; The proliferation of human normal liver cells line L-02 cells and human hepatoma cells line, HLE cells(non AFP-producing) were performed by MTT method; Repaired capacity of L-02 and HLE cells were compared by wound healing assay; Migration and invasion of these cells were analyzed by Transwell chamber assay; HBx expressed vectors(pcDNA3.1-HBx) were constructed and transfected into L-02 and HLE cells, effects of pcDNA3.1-HBx on the malignant behaviors were also detected by MTT, Transwell chamber assay and the expression of AFP, Ras and CXCR4 were evidenced by Western blotting. RESULTS: we found that expression of AFP, Ras and CXCR4 in HBV-related HCC and lymph nodes metastasis tissues were significantly elevated compared with HBV-related HCC, non metastasis tissues and HBV-related hepatitis tissues; Expression of AFP, Ras and CXCR4 in HBV-related hepatitis tissues were significantly enhanced compared with normal liver tissues; The growth ratio, migratory and invasive ability, expression of AFP, Ras and CXCR4 of the cells were outstanding promoted while L-02 and HLE cells were transfected with pcDNA3.1-HBx vectors. The proliferation ratio, migration and invasion ability, and expression of Ras and CXCR4 were significantly inhibited while L-02-X and HLE-X cells(stably transfected with pcDNA3.1-HBx) were silenced AFP expression by AFP-siRNA. CONCLUSIONS: HBx through stimulating expression of AFP to promote malignant behaviors of human normal liver cells and HCC cells; AFP maybe used as a novel biotarget for therapeutics of HCC patients.
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The hepatitis B virus (HBV)-X protein (HBx) induces malignant transformation of liver cells, and elevated expression of alpha-fetoprotein (AFP) is a significant biomarker of hepatocarcinogenesis. However, the role of AFP in HBV-related hepatocarcinogenesis is unclear. In this study, we investigated the regulatory impact of AFP expression on HBx-mediated malignant transformation of human hepatocytes. We found that HBV induced the expression of AFP before that of oncogenes, e.g., Src, Ras and chemokine (C-X-C motif) receptor 4 (CXCR4), and AFP activated protein kinase B (AKT) and mammalian target of rapamycin (mTOR) in HBV-related HCC tissues and in human liver cells transfected with HBx. Cytoplasmic AFP interacted with and inhibited phosphatase and tensin homolog deleted on chromosome 10 (PTEN), activating the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway and promoting mTOR-mediated stimulation of the transcription factor hypoxia inducible factor-1α (HIF-1α), and therefore led to the activation of the promoters of Src, CXCR4, and Ras genes. On the contrary, reduced expression of AFP by siRNA resulted in the repression of p-mTOR, pAKT, Src, CXCR4, and Ras in human malignant liver cells. Taken together, for the first time our study indicates that HBx-induced AFP expression critically promote malignant transformation in liver cells through the activation of PI3K/mTOR signaling.
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Hígado/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , alfa-Fetoproteínas/metabolismo , Línea Celular Tumoral , Humanos , Fosforilación , Transducción de Señal , Transfección , Proteínas Reguladoras y Accesorias ViralesRESUMEN
CXCR4, stromal cell-derived factor-1α(SDF 1α) receptor, stimulates growth and metastasis of hepatocellular carcinoma (HCC). Alpha-fetoprotein(AFP) governs the expression of some metastasis-related genes. Here we report that AFP and CXCR4 levels correlated in HCC tissues. AFP-expressing vectors induced CXCR4. In agreement, AFP depletion by siRNA decreased CXCR4. AFP co-localized and interacted with PTEN, thus inducing CXCR4 by activating AKT(Ser473) phosphorylation. In turn, phospho-mTOR(Ser2448) entered the nucleus and bound the CXCR4 gene promoter. Thus, AFP promoted migration of HCC cells. In concusion, AFP induced CXCR4 by activating the AKT/mTOR signal pathway.
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We examined the effect of endogenous and exogenous nitric oxide (NO) on protein kinase C (PKC) activity induced by angiotensin II (Ang II) in cultured neonatal rat cardiomyocytes. The results are as follows. The activity of PKC was increased by Ang II (0.01-10 micromol/L) in a dose-dependent manner, but decreased by NO precursor L-arginine (L-Arg) (10 micromol/L-10 mmol/L) in a dose-dependent manner in cultured neonatal rat cardiomyocytes. Pretreatment with L-Arg (100 micromol/L) decreased significantly Ang II -activated PKC activity and PKC activity induced by phorbol 12-myristate 13-acetate (PMA) ( 10 micromol/L), a PKC activator. Pretreatment with N(G)-nitro-L-argingie methyl ester (L-NAME), a nitric oxide synthase (NOS) blocker, may inhibit significantly the role of L-Arg on Ang II - and PMA-activated PKC activity. The activity of PKC was also decreased by NO donor sodium nitroprusside (SNP) (10 micromol/L-1 mmol/L) in a dose-dependent manner in cultured neonatal rat cardiomyocytes. Pretreatment with SNP (10 micromol/L) decreased significantly Ang II - and PMA-activated PKC activity. These results indicate that PKC was controlled by both NO and Ang II. PKC may be a cross talk between Ang II and NO in cardiomyocytes. NO abolished the activity of PKC and impaired PKC downstream signaling transduction pathway cascades.
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Angiotensina II/fisiología , Miocitos Cardíacos/enzimología , Óxido Nítrico/fisiología , Proteína Quinasa C/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Femenino , Masculino , Miocitos Cardíacos/citología , Ratas , Ratas Sprague-DawleyRESUMEN
The distribution of Y-chromosome specific microsatellite DYS19, in 289 males of three ethnic populations in Hainan Island was studied with the method of PCR followed by denatured polyacrylamide gel electrophoresis and silver staining. The three ethnic populations are Bendi Li, Qi Li and Ha Li. The results showed that four alleles (190bp, 194bp, 198bp, 202bp designated as alleles B, C, D and E respectively), were observed in DYS19, the frequency ranged from 0.660-0.854, and was predominant in three Li subgroups. The pairwise comparison with Fisher's Exact test reveals that there exists significant difference (P<0.01) in DYS19 phenotype distribution between Qi Li and Ha Li populations. The polymorphism of the Y-chromosome specific Alu insert sequence DYS287 was investigated also in three Li populations. The results demonstrated that with 150 bp product in all Bendi-Li, Qi-li and Ha-li, YAP element is absent. The preliminary study provides reference populations for detecting male migration events and for reconstructing paternal history.
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AFP and its receptor (AFPR) are early indicators of HBx-driven hepatocarcinogenesis. Clinical specimens, normal human liver cells L-02 and HCC cell lines were selected for analyzing the effects of HBx on expression of AFP, AFPR, Src, CXCR4, and Ras. Results showed that AFPR localized in the membranes of HCC samples. HBx upregulated the expression of AFPR and AFP prior to expression of Src, CXCR4, and Ras in L-02 cells and in liver specimens; Target-labeled AFPR was able to reveal the location and metastatic status of HCC in vivo. In this way, actuated expression of AFPR served as an indicator suitable for use in the early diagnosis of HBx-driven malignant transformation of hepatocytes. Labeled AFPR may be applied to trace primary and metastatic HCC.