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1.
Mol Carcinog ; 55(12): 2106-2120, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-26756739

RESUMEN

The p85α subunit of phosphatidylinositol 3-kinase (PI3K) acts as a key regulator of cell proliferation and motility, which mediates signals that confer chemoresistance to many human cancer cells. Using small interfering RNAs against matrix metalloproteinase-2 (MMP-2) and the MMP-2 promoter-driven luciferase assay, we showed that the new synthetic bichalcone analog TSWU-CD4 inhibits the invasion of human cancer cells by down-regulating MMP-2 expression. Treatment with TSWU-CD4 inhibited MMP-2 expression and cell invasion, which were restored by ectopic wild type (wt) p85α or a constitutively active form of MAPK kinase 3 (CA MKK3), CA MKK6, or CA p38α mitogen-activated protein kinase (MAPK). The attenuated formation of lipid raft-associated phospho (p)-p85α-GTP-Rac1 complexes, protein kinase B (Akt) Ser 473 phosphorylation, and cell invasion by TSWU-CD4 was reversed by overexpression of wt p85α or the p85α Brc-homology (BH) domain. The ectopic expression of CA Rac1L61 (but not wt Rac1) could overcome the suppression of Ser 473 phosphorylation, lipid raft association of Akt, the interaction between GTP-bound Rac1 and p85α in lipid rafts, and cell invasion by TSWU-CD4. The involvement of Akt activity in the functions of NF-κB-mediated MMP-2 was further confirmed through the attenuation of Akt phosphorylation signaling using the Akt-specific inhibitor MK-2206 and ectopic expression of NF-κB p65. Collectively, the inhibitory effect of TSWU-CD4 on cancer cell invasion was likely to suppress the p-p85α-GTP-Rac1 interaction in lipid rafts by targeting the p85α BH domain, which resulted in the suppression of MMP-2 expression via the PI3K-Akt-mediated ERK-MKK3/MKK6-p38 MAPK-NF-κB signaling pathway. © 2016 Wiley Periodicals, Inc.


Asunto(s)
Antineoplásicos/farmacología , Chalconas/farmacología , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Microdominios de Membrana/metabolismo , Invasividad Neoplásica/prevención & control , Piperazinas/farmacología , Proteína de Unión al GTP rac1/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo , Guanosina Trifosfato/metabolismo , Humanos , Metaloproteinasa 2 de la Matriz/genética , FN-kappa B/metabolismo , Invasividad Neoplásica/genética , Transducción de Señal/efectos de los fármacos
2.
Apoptosis ; 19(11): 1637-53, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25183449

RESUMEN

Suppression of the activity of pro-apoptotic Bcl-2-family proteins frequently confers chemoresistance to many human cancer cells. Using subcellular fractionation, the ER calcium (Ca(++)) channel inhibitor dantrolene and small interfering RNA (siRNA) against Bax or Bak, we show that the new synthetic bichalcone analog TSWU-CD4 induces apoptosis in human cancer cells by releasing endoplasmic reticulum (ER)-stored Ca(++) through ER/mitochondrial oligomerization of Bax/Bak. Blockade of the protein kinase RNA-like ER kinase or the unfolded protein response regulator glucose-regulated protein 78 expression by siRNA not only suppressed oligomeric Bax/Bak-mediated pro-caspase-12 cleavage and apoptosis but also resulted in an inhibition of Bcl-2 downregulation induced by TSWU-CD4. Induction of the ER oligomerization of Bax/Bak and apoptosis by TSWU-CD4 were suppressed by Bcl-2 overexpression. Inhibition of lipid raft-associated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling by TSWU-CD4 induced ER stress- and oligomeric Bax/Bak-mediated apoptosis, which were substantially reversed by overexpression of the wt PI3K p85α subunit. Taken together, these results suggest that suppression of lipid raft-associated PI3K/Akt signaling is required for the ER stress-mediated apoptotic activity of Bax/Bak, which is responsible for the ability of TSWU-CD4-treated cancer cells to exit the ER-mitochondrial apoptotic cell death pathway.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Chalconas/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Caspasa 12/metabolismo , Línea Celular , Dantroleno/farmacología , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Humanos , Mitocondrias/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal , eIF-2 Quinasa/metabolismo
3.
Mol Carcinog ; 53(5): 360-79, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-23192861

RESUMEN

Stromal cell-derived factor-1α (SDF-1α) is a ligand for C-X-C chemokine receptor type 4 (CXCR4), which contributes to the metastasis of cancer cells by promoting cell migration. Here, we show that the SDF-1α/CXCR4 axis can significantly increase invasion of esophageal carcinoma (EC) cells. We accomplished this by examining the effects of CXCR4 knockdown as well as treatment with a CXCR4-neutralizing antibody and the CXCR4-specific inhibitor AMD3100. Curcumin suppressed SDF-1α-induced cell invasion and matrix metalloproteinase-2 (MMP-2) promoter activity, cell surface localization of CXCR4 at lipid rafts, and lipid raft-associated ras-related C3 botulinum toxin substrate 1 (Rac1)/phosphatidylinositol 3-kinase (PI3K) p85α/Akt signaling. Curcumin inhibited SDF-1α-induced cell invasion by suppressing the Rac1-PI3K signaling complex at lipid rafts but did not abrogate lipid raft formation. We further demonstrate that the attenuation of lipid raft-associated Rac1 activity by curcumin was critical for the inhibition of SDF-1α-induced PI3K/Akt/NF-κB activation, cell surface localization of CXCR4 at lipid rafts, MMP-2 promoter activity, and cell invasion. Collectively, our results indicate that curcumin inhibits SDF-1α-induced EC cell invasion by suppressing the formation of the lipid raft-associated Rac1-PI3K-Akt signaling complex, the localization of CXCR4 with lipid rafts at the cell surface, and MMP-2 promoter activity, likely through the inhibition of Rac1 activity.


Asunto(s)
Quimiocina CXCL12/metabolismo , Curcumina/farmacología , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/patología , Microdominios de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Antineoplásicos/farmacología , Apoptosis , Western Blotting , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Movimiento Celular , Proliferación Celular , Quimiocina CXCL12/genética , Neoplasias Esofágicas/metabolismo , Citometría de Flujo , Humanos , Inmunoprecipitación , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Microdominios de Membrana/efectos de los fármacos , Persona de Mediana Edad , FN-kappa B/genética , FN-kappa B/metabolismo , Invasividad Neoplásica , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Transcripción/genética , Células Tumorales Cultivadas , Proteína de Unión al GTP rac1/genética
4.
Mol Med ; 18: 286-96, 2012 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-22160218

RESUMEN

Glycine N-methyltransferase (GNMT) is a tumor suppressor for hepatocellular carcinoma (HCC). High rates of Gnmt knockout mice developed HCC. Epigenetic alteration and dysregulation of several pathways including wingless-type MMTV integration site (Wnt), mitogen-activated protein kinase (MAPK) and Janus kinase and signal transducer and activator of transcription (JAK-STAT) are associated with HCC development in Gnmt knockout mice. We hypothesized that GNMT may regulate signal transduction through interacting with other proteins directly. In this report, we identified a mammalian target of rapamycin (mTOR) inhibitor (DEP domain containing MTOR-interacting protein [DEPDC6/DEPTOR]) as a GNMT-binding protein by using yeast two-hybrid screening. Fluorescence resonance energy transfer assay demonstrated that the C-terminal half of GNMT interact with the PSD-95/Dlg1/ZO-1 (PDZ) domain of DEPDC6/DEPTOR. Immunohistochemical staining showed that 27.5% (14/51) of HCC patients had higher expression levels of DEPDC6/DEPTOR in the tumorous tissues than in tumor-adjacent tissues, especially among HCC patients with hepatitis B viral infection (odds ratio 10.3, 95% confidence interval [CI] 1.05-11.3) or patients with poor prognosis (death hazard ratio 4.51, 95% CI 1.60-12.7). In terms of molecular mechanism, knockdown of DEPDC6/DEPTOR expression in HuH-7 cells caused S6K and 4E-BP activation, but suppressed Akt. Overexpression of DEPDC6/DEPTOR activated Akt and increased survival of HCC cells. Overexpression of GNMT caused activation of mTOR/raptor downstream signaling and delayed G2/M cell cycle progression, which altogether resulted in cellular senescence. Furthermore, GNMT reduced proliferation of HuH-7 cells and sensitized them to rapamycin treatment both in vitro and in vivo. In conclusion, GNMT regulates HCC growth in part through interacting with DEPDC6/DEPTOR and modulating mTOR/raptor signaling pathway. Both GNMT and DEPDC6/DEPTOR are potential targets for developing therapeutics for HCC.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Glicina N-Metiltransferasa/metabolismo , Neoplasias Hepáticas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Adulto , Anciano , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Everolimus , Femenino , Células HEK293 , Hepatitis B/complicaciones , Hepatitis B/metabolismo , Hepatitis C/complicaciones , Hepatitis C/metabolismo , Humanos , Inmunosupresores/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Sirolimus/análogos & derivados , Sirolimus/uso terapéutico , Técnicas del Sistema de Dos Híbridos , Ensayos Antitumor por Modelo de Xenoinjerto
5.
J Cell Biochem ; 112(4): 1176-91, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21308745

RESUMEN

Using short hairpin RNA against p53, transient ectopic expression of wild-type p53 or mutant p53 (R248W or R175H), and a p53- and p21-dependent luciferase reporter assay, we demonstrated that growth arrest and apoptosis of FaDu (human pharyngeal squamous cell carcinoma), Hep3B (hepatoma), and MG-63 (osteosarcoma) cells induced by aloe-emodin (AE) are p53-independent. Co-immunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE caused S-phase cell cycle arrest by inducing the formation of cyclin A-Cdk2-p21 complexes through extracellular signal-regulated kinase (ERK) activation. Ectopic expression of Bcl-X(L) and siRNA-mediated Bax attenuation significantly inhibited apoptosis induced by AE. Cyclosporin A or the caspase-8 inhibitor Z-IETD-FMK blocked AE-induced loss of mitochondrial membrane potential and prevented increases in reactive oxygen species and Ca(++). Z-IETD-FMK inhibited AE-induced apoptosis, Bax expression, Bid cleavage, translocation of tBid to mitochondria, ERK phosphorylation, caspase-9 activation, and the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G from mitochondria. The stability of the mRNAs encoding caspase-8 and -10-associated RING proteins (CARPs) 1 and 2 was affected by AE, whereas CARP1 or 2 overexpression inhibited caspase-8 activation and apoptosis induced by AE. Collectively, our data indicate AE induces caspase-8-mediated activation of mitochondrial death pathways by decreasing the stability of CARP mRNAs in a p53-independent manner.


Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Portadoras/metabolismo , Caspasa 8/metabolismo , Emodina/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Western Blotting , Proteínas Portadoras/genética , Caspasa 8/genética , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Línea Celular , Línea Celular Tumoral , Ciclina A/genética , Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genética
6.
J Metab Bariatr Surg ; 10(1): 32-41, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-36687754

RESUMEN

Purpose: Both primary and revisional bariatric surgery are on the rise due to global obesity pandemic. This study aimed to assess the indications for revision after one-anastomosis gastric bypass (OAGB) and the outcomes after laparoscopic conversion of OAGB to roux-en-y gastric bypass (RYGB). Materials and Methods: Retrospective review on patients that had undergone conversion of OAGB to RYGB between June 2007-June 2019 in a tertiary bariatric center, followed by literature review. Results: Out of 386 revisional bariatric surgery, a total of 14 patients underwent laparoscopic conversion of OAGB to RYGB. The mean age was 44.7 with 71% female. The mean pre-revision BMI was 29.2 kg/m2. The primary indications for revision were bile reflux (n=7), marginal ulcer (n=3), inadequate weight loss or weight regain (IWL/WR) (n=3) and protein-calorie malnutrition (n=1). Conversion of OAGB to RYGB was completed laparoscopically in all cases. The mean length of stay was 4.1 days. There was no intraoperative or early post-operative complication. The mean total weight loss (rTWL%) after revision at year one, year three and year five post-revision were 11.5%, 18.1% and 29.1%, respectively. All patients achieved resolution of bile reflux and marginal ulcer. There was no mortality in this cohort. Conclusion: Bile reflux, marginal ulcer, IWL/WR and malnutrition were the main indications for revision after OAGB in this study. In concordance with the available evidence, laparoscopic conversion of OAGB to RYGB was safe and effective in dealing with late complications of OAGB.

7.
Obes Surg ; 31(8): 3707-3714, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34033013

RESUMEN

BACKGROUND: Taking advantage of isomeric form of vitamin E in the supplement, adherence to supplement could be evaluated by changes in circulating α- and γ-tocopherol concentrations. Accordingly, effects of supplementation on postoperative nutrition and bone metabolism were studied in terms of adherence. METHODS: Thirty-eight SG patients were all prescribed a postoperative nutritional supplement containing a low dose of vitamin D (600 IU) and calcium (200 mg). Blood samples were collected prior to (M0) and 6 months after (M6) surgery and concentrations of nutrients and C-terminal telopeptide of type I collage (CTX), a marker of bone resorption, were measured. Adherence and non-adherence were stratified according to change (△, M6-M0) in serum α-tocopherol concentrations (> 0 vs. ≤ 0, respectively). RESULTS: When M0 and M6 were compared, there were significant increases in serum concentrations of 25(OH)D, α-tocopherol and selenium, whereas there were reductions in parathyroid hormone, ferritin, and γ-tocopherol. At M6, the prevalence of vitamin D insufficiency (25(OH)D < 30 ng/mL) and high CTX were 72 and 26%, respectively. When comparison was made between adherence and non-adherence, only △25(OH)D concentrations, but no other nutrients nor postoperative CTX differed. Multiple linear regression demonstrated that postoperative vitamin D status was independently associated with its preoperative concentrations (ß = 0.85, p < 0.001) and adherence (ß = 0.52, p < 0.05). CONCLUSION: SG patients' adherence to supplementation, even with a low dose of vitamin D and calcium, determined vitamin D status but not bone resorption marker concentrations, at least within 6 months after surgery.


Asunto(s)
Resorción Ósea , Obesidad Mórbida , Deficiencia de Vitamina D , Suplementos Dietéticos , Gastrectomía , Humanos , Obesidad Mórbida/cirugía , Hormona Paratiroidea , Vitamina D
8.
Mol Carcinog ; 49(9): 783-97, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20607722

RESUMEN

Aloe-emodin (AE), extracted from the rhizome of Rheum palmatum, has an anti-proliferative effect on different human cancer cell lines. Nonetheless, the underlying mechanism by which AE inhibits nasopharyngeal carcinoma (NPC) cell invasion is still unclear. The results of this study show that treatment of NPC cells with growth suppressive concentrations of AE caused cell cycle arrest at the S-G(2)/M phase. Coimmunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE-induced cell cycle arrest in NPC cells was associated with increasing levels of cyclin B1 bound to cyclin-dependent kinase 1. The inhibition of NPC cell invasion by AE was evidenced through the suppression of matrix metalloproteinases-2 (MMP-2) expression. MMP-2 promoter activity and cell invasion were inhibited by p38 mitogen-activated protein kinase (MAPK) siRNA, inhibitor 4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1H-imidazole (SB203580), and AE, but not by JNK siRNA and inhibitor 1,9-pyrazoloanthrone. Treatment with AE, SB203580, NF-kappaB inhibitors N-p-tosyl-(L)-phenylalanine chloromethyl ketone (TPCK) and pyrrolidine dithiocarbamate (PDTC) or transfection with p38 MAPK siRNA significantly inhibited NF-kappaB transcriptional activity. In addition, TPCK and PDTC treatment inhibited the expression and promoter activity of MMP-2 and thereby significantly inhibited cell invasion activity. The involvement of p38 MAPK activity in NF-kappaB-mediated MMP-2 function was further confirmed through the attenuation of p38 MAPK by SB203580 and NF-kappaB ectopic expression. Collectively, our results indicate that AE inhibits invasion of NPC cells by suppressing the expression of MMP-2 via the p38 MAPK-NF-kappaB signaling pathway.


Asunto(s)
FN-kappa B/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Antraquinonas , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteína Quinasa CDC2/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Ciclina B1/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Imidazoles/farmacología , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/farmacología , FN-kappa B/genética , FN-kappa B/farmacología , Neoplasias Nasofaríngeas/genética , Piridinas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genética
9.
Obes Surg ; 30(10): 3940-3946, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32638247

RESUMEN

BACKGROUND: This is the first report from Taiwan using laboratory tests to assess nutritional status of patients with obesity before bariatric-metabolic surgery. Moreover, the 25(OH)D threshold for maximal suppression of parathyroid hormone (PTH) was evaluated to offer a reference value for preoperative nutritional care. METHODS: Inclusion criteria were Taiwanese, 18-65 years old, and with BMI ≥ 27.5 kg/m2 awaiting bariatric-metabolic surgery. Anthropometric data and blood samples were collected before surgery. Serum concentrations of protein; vitamins B1, B12, folate, A, D, and E; calcium; iron; zinc; copper; selenium; PTH; and erythrocyte glutathione reductase activity coefficient (vitamin B2 status) were measured. RESULTS: For 52 participants with a mean BMI 37.6 ± 6.4 kg/m2, vitamin D deficiency (25(OH)D < 20 ng/mL) and insufficiency (20 < 25(OH)D < 30 ng/mL) were at 73 and 22% prevalence, respectively. Secondary hyperparathyroidism (PTH â‰§ 65 pg/mL) was 24% and hypocalcemia was 50% (ionized Ca < 4.5 mg/dL). Deficiency of other nutrients was sporadic (< 10%) or nil. When participants were stratified according to 25(OH)D concentrations (< 10, 10-15, 15-20, and ≥ 20 ng/mL), PTH increased at 25(OH)D < 10 ng/mL (ß = 48.34, p = 0.001) after adjusting for age, gender, and BMI. CONCLUSION: For patients with obesity before bariatric-metabolic surgery, vitamin D/calcium deficiency was the only nutritional issue that needs to be addressed in Taiwan. However, a lower cutoff point of 25(OH)D, i.e., 10 ng/mL, for vitamin D deficiency may be considered for patients before surgery. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03915158.


Asunto(s)
Cirugía Bariátrica , Obesidad Mórbida , Deficiencia de Vitamina D , Adolescente , Adulto , Anciano , Humanos , Persona de Mediana Edad , Estado Nutricional , Obesidad/complicaciones , Obesidad/cirugía , Obesidad Mórbida/cirugía , Hormona Paratiroidea , Taiwán/epidemiología , Vitamina D/análogos & derivados , Deficiencia de Vitamina D/complicaciones , Deficiencia de Vitamina D/epidemiología , Adulto Joven
10.
Biochem J ; 402(3): 483-90, 2007 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-17134374

RESUMEN

In the present study, we report the development of a sensitive and selective assay based on LC (liquid chromatography)-MS/MS (tandem MS) to simultaneously measure N7-MeG (N7-methylguanine) and N7-EtG (N7-ethylguanine) in DNA hydrolysates. With the use of isotope internal standards (15N5-N7-MeG and 15N5-N7-EtG) and on-line SPE (solid-phase extraction), the detection limit of this method was estimated as 0.42 fmol and 0.17 fmol for N7-MeG and N7-EtG respectively. The high sensitivity achieved here makes this method applicable to small experimental animals. This method was applied to measure N7-alkylguanines in liver DNA from mosquito fish (Gambusia affinis) that were exposed to NDMA (N-nitrosodimethylamine) and NDEA (N-nitrosodiethylamine) alone or their combination over a wide range of concentrations (1-100 mg/l). Results showed that the background level of N7-MeG in liver of control fish was 7.89+/-1.38 mmol/mol of guanine, while N7-EtG was detectable in most of the control fish with a range of 0.05-0.19 mmol/mol of guanine. N7-MeG and N7-EtG were significantly induced by NDMA and NDEA respectively, at a concentration as low as 1 mg/l and increased in a dose-dependent manner. Taken together, this LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of alkylated DNA lesions in small animal models of cancer induced by alkylating agents.


Asunto(s)
Cromatografía Liquida/métodos , Ciprinodontiformes , ADN/análisis , ADN/química , Guanina/análisis , Guanina/química , Espectrometría de Masas en Tándem/métodos , Alquilación , Animales , Automatización , Etilenos/química , Técnicas de Dilución del Indicador , Isótopos , Hígado/metabolismo , Extractos Hepáticos/metabolismo , Modelos Animales
11.
Anticancer Res ; 27(5A): 3313-22, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17970076

RESUMEN

Apoptosis is a physiological mechanism for eliminating malignant cells, including cancer cells, without eliciting damage to normal cells or surrounding tissues. Here, we report that rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), a major constituent in the rhizome of rhubarb, induced apoptosis of human nasopharyngeal carcinoma (NPC) cells. Rhein induced apoptosis in NPC cells as demonstrated by increased nuclear condensation and DNA fragmentation. Moreover, for the first time in NPC cells it was demonstrated that the pathway involved in rhein-induced apoptosis is caspase-dependent, presumably through the endoplasmic reticulum (ER) stress pathway, as shown by an increase in the levels of glucose-regulated protein 78 (GRP 78), PKR-like ER kinase (PERK), activating transcription factor 6 (A TF6) and CCAA TIenhancer-binding protein homologous protein (CHOP) as well as the activation of caspase-3, -8, -9 and -12. This increased susceptibility to ER stress-induced apoptosis may be due to an increased accumulation of reactive oxygen species (ROS). Rapid accumulation of calcium (Ca2+) and a decrease in the mitochondrial membrane potential (MMP) were also observed. Cytochrome c and apoptosis-inducing factor (AIF) were released upon treatment with rhein. Taken together, these results suggest that ER stress and Ca2+-dependent mitochondrial death pathway may be involved in rhein-induced apoptosis in NPC cells.


Asunto(s)
Antraquinonas/farmacología , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neoplasias Nasofaríngeas/tratamiento farmacológico , Apoptosis/fisiología , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/fisiología , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Factor de Transcripción CHOP/biosíntesis
12.
Anticancer Res ; 36(2): 697-705, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26851027

RESUMEN

Release of calcium (Ca(++)) from the endoplasmic reticulum (ER) has been proposed to be involved in induction of apoptosis by oxidative stress. Using inhibitor of ER Ca(++) release dantrolene and inhibitor of mitochondrial Ca(++) uptake Ru-360, we demonstrated that Ca(++) release from the ER was associated with generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, and apoptosis of human oral cancer (OC) cells induced by gallic acid (GA). Small interfering RNA-mediated suppression of protein kinase RNA-like endoplasmic reticulum kinase inhibited tunicamycin-induced induction of 78 kDa glucose-regulated protein, C/EBP homologous protein, pro-caspase-12 cleavage, cytosolic Ca(++) increase and apoptosis, but did not attenuate the increase in cytosolic Ca(++) level and apoptosis induced by GA. Ataxia telangiectasia mutated (ATM)-mediated c-Jun N-terminal kinase (JNK) phosphorylation and apoptosis by GA was blocked by dantrolene. The specificity of ROS-mediated ATM-JNK activation was confirmed by treatment with N-acetylcysteine, a ROS scavenger. Blockade of ATM activation by specific inhibitor KU55933, short hairpin RNA, or kinase-dead ATM overexpression suppressed JNK phosphorylation but did not completely inhibit cytosolic ROS production, mitochondrial cytochrome c release, pro-caspase-3 cleavage, and apoptosis induced by GA. Taken together, these results indicate that GA induces OC cell apoptosis by inducing the activation of mitochondrial apoptotic and ATM-JNK signal pathways, likely through ER Ca(++)-mediated ROS production.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Señalización del Calcio/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Ácido Gálico/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Mitocondrias/efectos de los fármacos , Neoplasias de la Boca/tratamiento farmacológico , Antioxidantes/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Línea Celular Tumoral , Citosol/metabolismo , Retículo Endoplásmico/enzimología , Activación Enzimática , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/patología , Neoplasias de la Boca/enzimología , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Transfección
13.
Cancer Lett ; 291(1): 46-58, 2010 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-19942342

RESUMEN

Aloe-emodin (AE), a natural, biologically active compound from the rhizome of Rheum palmatum, has been shown to induce apoptosis in several cancer cell lines in vitro. However, its molecular mechanism of action in the apoptosis induction of human nasopharyngeal carcinoma (NPC) cells has not been explored. This study shows that AE induced G(2)/M phase arrest by increasing levels of cyclin B1 bound to Cdc2, and also caused an increase in apoptosis of NPC cells, which was characterized by morphological changes, nuclear condensation, DNA fragmentation, caspase-3 activation, cleavage of poly (ADP-ribose) polymerase (PARP) and increased sub-G(1) population. Treatment of NPC cells with AE also resulted in a decrease in Bcl-X(L) and an increase in Bax expression. Ectopic expression of Bcl-X(L) but not Bcl-2 or small interfering RNA (siRNA)-mediated attenuation of Bax suppressed AE-induced apoptotic cell death. AE-induced loss of mitochondrial membrane potential (MMP) and increase in cellular Ca(++) content, reactive oxygen species (ROS) and apoptotic cell death were suppressed by the treatment of cyclosporin A (CsA) or caspase-8 inhibitor Z-IETD-FMK. Co-treatment with caspase-9 inhibitor Z-LEHD-FMK could inhibit AE-induced cell death and the activation of caspase-3 and -9. In addition, suppression of caspase-8 with the specific inhibitor Z-IETD-FMK inhibited AE-induced the activation of Bax, the cleavage of Bid, the translocation of tBid to the mitochondria and the release of cytochrome c, apoptosis-inducing factor (AIF) and Endo G from the mitochondria and subsequent apoptosis. Taken together, these results indicate that the caspase-8-mediated activation of the mitochondrial death pathway plays a critical role in AE-induced apoptosis of NPC cells.


Asunto(s)
Antraquinonas/farmacología , Apoptosis/efectos de los fármacos , Caspasa 8/fisiología , Mitocondrias/efectos de los fármacos , Neoplasias Nasofaríngeas/tratamiento farmacológico , Proteína Quinasa CDC2 , Caspasa 3/fisiología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ciclina B/metabolismo , Ciclina B1/metabolismo , Quinasas Ciclina-Dependientes , Humanos , Mitocondrias/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Especies Reactivas de Oxígeno/metabolismo , Proteína X Asociada a bcl-2/fisiología , Proteína bcl-X/fisiología
14.
Oral Oncol ; 45(6): 531-7, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18804415

RESUMEN

Progression of cancer invasion is believed to be dependent on the remodeling of extracellular matrix induced by tumor cells. Rhein has been shown to inhibit the growth and proliferation of human nasopharyngeal carcinoma (NPC) cells. However, the molecular mechanism underlying rhein-induced inhibition of cancer invasion has not been explored. Herein, we show that rhein could inhibit the invasion and migration of NPC cells in vitro. Rhein inhibits invasion by reducing the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). Moreover, we demonstrate that the pathway involved in rhein-inhibited invasion is presumably through the growth factor receptor bound protein 2/son of sevenless-Ras-mitogen-activated protein kinase (GRB2/SOS-Ras-MAPK) pathway, as shown by an decrease in the expression levels of GRB2, SOS-1 and Ras as well as led to suppression of the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK. Further study has shown that rhein also inhibited activation of transcription factor nuclear factor kappaB (NF-kappaB), which is known to implicate the regulation of MMP-9 and VEGF gene expression in cancer invasion. Our findings suggest that rhein inhibits the invasion of NPC cells may be mediated in part through the suppression of MMP-9 and VEGF expression via the modulation of NF-kappaB signaling pathway.


Asunto(s)
Antraquinonas/farmacología , Inhibidores Enzimáticos/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , FN-kappa B/antagonistas & inhibidores , Neoplasias Nasofaríngeas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo , Proteína Adaptadora GRB2/metabolismo , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias Nasofaríngeas/patología , Invasividad Neoplásica , Proteínas Son Of Sevenless/metabolismo , Proteínas ras/metabolismo
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