RESUMEN
We isolated a novel lectin (AHL) from Artocarpus hypargyreusHance and showed its immunomodulatory activities. In this study, the amino acid sequence of AHL was determined by cDNA sequencing. AHL cDNA (875bp) contains a 456-bp open reading frame (ORF), which encodes a protein with 151 amino acids. AHL is a new member of jacalin-related lectin family (JRLs), which share high sequence similarities to KM+ and Morniga M, and contain the conserved carbohydrate binding domains. The antitumor activity of AHL was also explored using Jurkat T cell lines. AHL exhibits a strong binding affinity to cell membrane, which can be effectively inhibited by methyl-α-D-galactose. AHL inhibits cell proliferation in a time- and dose-dependent manner through apoptosis, evidenced by morphological changes, phosphatidylserine externalization, poly ADP-ribose polymerase (PARP) cleavage, Bad and Bax up-regulation, and caspase-3 activation. We further showed that the activation of ERK and p38 signaling pathways is involved for the pro-apoptotic effect of AHL.
Asunto(s)
Apoptosis , Artocarpus , Lectinas/genética , Artocarpus/genética , Clonación Molecular , ADN Complementario , Humanos , Células JurkatRESUMEN
Paclitaxel is one of the best anticancer agents that has been isolated from plants, but its major disadvantage is its dose-limiting toxicity. In this study, we obtained evidence that the active mutant IPP5 (8-60hIPP5m), the latest member of the inhibitory molecules for protein phosphatase 1, sensitizes human cervix carcinoma cells HeLa more efficiently to the therapeutic effects of paclitaxel. The combination of 8-60hIPP5m with paclitaxel augmented anticancer effects as compared to paclitaxel alone as evidenced by reduced DNA synthesis and increased cytotoxicity in HeLa cells. Furthermore, our results revealed that 8-60hIPP5m enhances paclitaxel- induced G2/M arrest and apoptosis, and augments paclitaxel-induced activation of caspases and release of cytochrome C. Evaluation of signaling pathways indicated that this synergism was in part related to down- regulation of NF-?B activation and serine/threonine kinase Akt pathways. We noted that 8-60hIPP5m down- regulated the paclitaxel-induced NF-?B activation, I?Bα degradation, PI3-K activity and phosphorylation of the serine/threonine kinase Akt, a survival signal which in many instances is regulated by NF-?B. Together, our observations indicate that paclitaxel in combination with 8-60hIPP5m may provide a therapeutic advantage for the treatment of human cervical carcinoma.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Tolerancia a Medicamentos/genética , Mutación/genética , Paclitaxel/farmacología , Proteínas/genética , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , Apoptosis/efectos de los fármacos , Western Blotting , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/patologíaRESUMEN
Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. The active mutant IPP5 (8-60hIPP5(m)), the latest member of the inhibitory molecules for PP1, has been shown to inhibit the growth of human cervix carcinoma cells (HeLa). In order to elucidate the underlying mechanisms, the present study assessed overexpression of 8-60hIPP5(m) in HeLa cells. Flow cytometric and biochemical analyses showed that overexpression of 8-60hIPP5(m) induced G2/M-phase arrest, which was accompanied by the upregulation of cyclin B1 and phosphorylation of G2/M-phase proteins ATM, p53, p21(cip1/waf1) and Cdc2, suggesting that 8-60hIPP5(m) induces G2/M arrest through activation of the ATM/p53/p21(cip1/waf1)/Cdc2/ cyclin B1 pathways. We further showed that overexpression of 8-60hIPP5(m) led to delayed nuclear translocation of cyclin B1. 8-60hIPP5(m) also could translocate to the nucleus in G2/M phase and interact with pp1α and Cdc2 as demonstrated by co-precipitation assay. Taken together, our data demonstrate a novel role for 8-60hIPP5(m) in regulation of cell cycle in HeLa cells, possibly contributing to the development of new therapeutic strategies for cervix carcinoma.
Asunto(s)
Puntos de Control de la Fase M del Ciclo Celular/genética , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteínas/genética , Neoplasias del Cuello Uterino/patología , Transporte Activo de Núcleo Celular , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína Quinasa CDC2 , División Celular/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular/genética , Ciclina B1/biosíntesis , Ciclina B1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Femenino , Células HeLa , Humanos , Fosforilación , Transporte de Proteínas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
OBJECTIVE: To investigate the expression and mutation of PIK3CA gene in hepatocellular carcinomas (HCC). METHODS: HCC samples and the corresponding adjacent tissues were collected from the surgical patients with pathologically verified diagnosis. The exons 1, 9 and 20 of PIK3CA gene were detected by PCR-SSCP and DNA sequencing. Immnohistochemistry was employed to test the expression of PIK3CA gene in these samples. RESULTS: No mutation was found in exons 1, 9 or 20 of PIK3CA gene in the HCC tissue and the adjacent tissues by PCR-SSCP and DNA sequencing, while abnormal superimposed peaks were found on the sequence map of exon 9 in 25 cases of HCC tissue. Immunohistochemistry showed that expression of PIK3CA was higher in the HCC tissue than in the corresponding adjacent tissue (50.81% vs 14.75%). CONCLUSION: PIK3CA gene mutation may exist in HCC in Guangxi, which can be associated with the development of HCC, but the ratio of hotspot mutations is low.
Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Mutación , Fosfatidilinositol 3-Quinasas/genética , Secuencia de Bases , Fosfatidilinositol 3-Quinasa Clase I , Exones/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Análisis de Secuencia de ADNRESUMEN
BACKGROUND & OBJECTIVE: Inhibitor-1 of protein phosphatase I(PPI 1) is one of the regulatory subunits of protein phosphatase 1 (PP1). When phosphorylated by protein kinase A (PKA) at Thr-35, PPI 1 inhibits the activity of PP1. This study was to observe the effects of wild type and activated mutant of PPI 1 (PPI 1/wt and PPI 1/sI 1) on the apoptosis of human cervical carcinoma cell line HeLa, and to explore its possible mechanism. METHODS: Plasmids containing PPI 1/wt or PPI 1/sI 1 were transfected into HeLa cells. Stable cell clones expressing PPI 1 protein (HeLa-PPI 1/wt and HeLa-PPI 1/sI 1) were selected by G418. Untransfected and mock-transfected HeLa cells were used as control. The expression of PPI 1/wt and PPI 1/sI 1 were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Cell clonogenic ability was detected by colony formation assay. The morphologic alterations of the cells were observed with Hoechst 33258 staining. The apoptosis before and after stimulation of tumor-necrosis factor (TNF) was detected by flow cytometry. The expression of apoptosis-related proteins in the cells was detected by Western blot. RESULTS: PPI 1/wt and PPI 1/sI 1 were efficiently expressed in HeLa cells. The clonogenic ability of HeLa-PPI 1/sI 1 cells was inhibited. The apoptosis of HeLa-PPI 1/sI 1 cells were enhanced. The expression of P53, P27 and BAK were up-regulated, while the expression of Bcl-2 and Bcl-x(L) were down-regulated. After TNF stimulation, the apoptosis rate was 19.06% for HeLa-PPI 1/sI 1 cells, 2.67% for HeLa-mock cells, and 1.89% for untransfected HeLa cells. PPI 1/sI 1 enhanced the phosphorylation of JNK, and induced the activation of P38 at 30 min after TNF stimulation. CONCLUSION: PPI 1/sI 1 could induce apoptosis of HeLa cells, and enhance the sensitivity of HeLa cells to TNF stimulation, which might enhance the activation of JNK and p38 signal transduction.