RESUMEN
Avocado (Persea americana) fruits are consumed as part of the human diet and extracts have shown growth inhibitory effects in various types of human cancer cells, although the effectiveness of individual components and their underlying mechanism are poorly understood. Using activity-guided fractionation of the flesh of avocado fruits, a chloroform-soluble extract (D003) was identified that exhibited high efficacy towards premalignant and malignant human oral cancer cell lines. From this extract, two aliphatic acetogenins of previously known structure were isolated, compounds 1 [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] and 2 [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate]. In this study, we show for the first time that the growth inhibitory efficacy of this chloroform extract is due to blocking the phosphorylation of EGFR (Tyr1173), c-RAF (Ser338), and ERK1/2 (Thr202/Tyr204) in the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. Compounds 1 and 2 both inhibited phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). Compound 2, but not compound 1, prevented EGF-induced activation of the EGFR (Tyr1173). When compounds 1 and 2 were combined they synergistically inhibited c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204) phosphorylation, and human oral cancer cell proliferation. The present data suggest that the potential anticancer activity of avocado fruits is due to a combination of specific aliphatic acetogenins that target two key components of the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway.
Asunto(s)
Acetogeninas/farmacología , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Ácidos Grasos Insaturados/farmacología , Frutas/química , Neoplasias de la Boca/enzimología , Persea/química , Acetogeninas/química , Antineoplásicos/química , Receptores ErbB/antagonistas & inhibidores , Ácidos Grasos Insaturados/química , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Extractos Vegetales/química , Extractos Vegetales/farmacología , Quinasas raf/antagonistas & inhibidores , Proteínas ras/antagonistas & inhibidoresRESUMEN
Avocados have a high content of phytochemicals with potential chemopreventive activity. Previously we reported that phytochemicals extracted from avocado meat into a chloroform partition (D003) selectively induced apoptosis in cancer but not normal, human oral epithelial cell lines. In the present study, we observed that treatment of human oral cancer cell lines containing high levels of reactive oxygen (ROS) with D003 increased ROS levels twofold to threefold and induced apoptosis. In contrast, ROS levels increased only 1.3-fold, and apoptosis was not induced in the normal cell lines containing much lower levels of basal ROS. When cellular ROS levels in the malignant cell lines were reduced by N-acetyl-l-cysteine (NAC), cells were resistant to D003 induced apoptosis. NAC also delayed the induction of apoptosis in dominant negative FADD-expressing malignant cell lines. D003 increased ROS levels via mitochondrial complex I in the electron transport chain to induce apoptosis. Normal human oral epithelial cell lines transformed with HPV16 E6 or E7 expressed higher basal levels of ROS and became sensitive to D003. These data suggest that perturbing the ROS levels in human oral cancer cell lines may be a key factor in selective apoptosis and molecular targeting for chemoprevention by phytochemicals.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Boca/tratamiento farmacológico , Persea , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Línea Celular Tumoral , Proteína de Dominio de Muerte Asociada a Fas/fisiología , Humanos , Mitocondrias/fisiología , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Oligopéptidos/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
OSU03012, a Celecoxib derivative, has been shown to inhibit proliferation and induce apoptosis in human cancer cell lines. However, its underlying mechanisms are not completely understood. In our study, the relationship between cell cycle inhibition and apoptosis induced by OSU03012 was investigated in human oral cancer cell lines. In the premalignant and malignant cell lines, OSU03012-induced growth inhibition, S-phase arrest, and apoptosis were accompanied by a marked increase in the activity of Erk1/2 and Cdk2/cyclin A. Inhibition of Cdks by roscovitine partially blocked OSU03012-induced growth inhibition and apoptosis. Although the activity of cdc2/cyclin B was reduced, expression of constructively active cdc2AF did not reverse OSU03012-induced S-phase arrest. When Erk1/2 was inhibited by U0126 before addition of OSU03012, growth inhibition and apoptosis induced by OSU03012 were attenuated. The levels of the Cdk2/cyclin A were reduced and cells accumulated in the G(0)/G(1) phase. When cells were allowed to accumulate in S-phase before addition of U0126, apoptosis also was attenuated suggesting that Erk1/2 is required for both progression of cells into the S-phase and apoptosis. Expression of constructively active MEK enhanced OSU03012-induced apoptosis. OSU03012 selectively inhibited the proliferation in premalignant and malignant, but not normal human oral cell lines. In conclusion, we show that OSU03012 has potent anti-proliferative and apoptotic activity against premalignant and malignant human oral cells through activation of Erk1/2, and Cdks. OSU0312 may provide unique opportunities for cancer prevention and sensitization of cancer cells to S-phase modalities.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Quinasas Ciclina-Dependientes/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mucosa Bucal/efectos de los fármacos , Neoplasias de la Boca/tratamiento farmacológico , Pirazoles/farmacología , Fase S/efectos de los fármacos , Sulfonamidas/farmacología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Mucosa Bucal/citologíaRESUMEN
OSU03012 is a non-COX inhibiting celecoxib derivative with growth inhibiting and apoptotic activity in many cancer cell lines. To investigate mechanisms related to cell cycle proteins in growth inhibition and apoptosis induced by OSU03012, the primary human oral epithelial cell line, TE1177, was transformed with HPV16 E6 (TE/E6), HPV16 E7 (TE/E7) or empty vector (TE/V). TE/E6 cell lines exhibiting low levels of p53 and undetectable levels of p21(WAF1/CIP1) were sensitized to the growth inhibiting and apoptotic effects of OSU03012. The TE/E7 cell lines expressing low levels of Rb and elevated levels of p53 and p21(WAF1/CIP1) were resistant. OSU03012 reduced the number of cells in the S phase of the TE/E7 and TE/V cell lines with intact p53-p21(WAF1/CIP1) checkpoint, but not in the checkpoint defective TE/E6 cell lines. Treatment with OSU03012 also markedly reduced the levels of cyclin A and Cdk2 in TE/E7 and TE/V, but not in TE/E6 cell lines, which had significantly enhanced basal levels of cyclin A and Cdk2. Consistent with the TE/E6 cell line, p21(WAF1/CIP1)-/- mouse embryo fibroblasts were more sensitive to OSU03012-induced apoptosis as evidenced by PARP and caspase 3 cleavages. These data suggest that p21(WAF1/CIP1) is an important factor in the sensitivity of cells to the growth inhibiting and apoptotic effects of OSU03012.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Mucosa Bucal/efectos de los fármacos , Pirazoles/farmacología , Sulfonamidas/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Western Blotting , Bromodesoxiuridina/metabolismo , Línea Celular , Ciclina A/efectos de los fármacos , Ciclina A/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Embrión de Mamíferos , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Fase G1/efectos de los fármacos , Silenciador del Gen , Humanos , Inmunoprecipitación , Ratones , Mucosa Bucal/citología , Mucosa Bucal/virología , Proteínas Oncogénicas Virales/efectos de los fármacos , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus/metabolismo , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Infecciones Tumorales por Virus/metabolismoRESUMEN
BACKGROUND: In a phase I clinical trial of oxaliplatin (OPT) in combination with paclitaxel (PXL), a pharmacokinetic interaction was observed when OPT was given as a 2-h i.v. infusion followed by a 1-h i.v. infusion of PXL. The purpose of this study was to use a rat model to evaluate whether the pharmacokinetic interaction between OPT and PXL is dosing sequence-dependent. METHODS: One group of rats was given OPT as a 2-h i.v. infusion followed by a 1-h i.v. infusion of PXL formulated in 50% Cremophor EL (CrEL)/50% ethanol (OPT-->fPXL), similar to the current phase I clinical protocol. In a second group of rats, the fPXL was infused first to reach a quasi-steady-state plasma level of PXL, followed by an i.v. bolus dose of OPT (CIfPXL-->OPT). In a third group of rats, fPXL was replaced with the formulation vehicle, CrEL, which was infused in the same manner as in the second group. Each combination was accompanied with a control of either OPT alone or with replacement of PXL with dextrose 5% in water (CID5W-->OPT). The total platinum (Pt) levels in plasma and plasma ultrafiltrate were measured by a validated inductively coupled plasma mass spectrometry (ICPMS) method. The protein binding, red blood cell (RBC) uptake and urinary elimination of Pt were also examined in each group of rats. RESULTS: The concentration-time profiles of plasma Pt and ultrafiltrable Pt followed triexponential decays in all groups of rats. In the rat receiving OPT-->fPXL, the terminal elimination rate constant (gamma) of plasma Pt increased, with essentially no change in the total body clearance (CL) and the AUC value, when compared to those without PXL infusion (CID5W-->OPT). The steady-state volume of distribution (V(ss)) of the ultrafiltrable Pt also showed an increase in the combination group receiving OPT-->fPXL ( P<0.01). These results were similar to those from the clinical trial, although the magnitude of change was less. However, in the CIfPXL-->OPT group, both CL and V(ss) of Pt in plasma and plasma ultrafiltrate decreased, with corresponding increases in AUCs ( P<0.01). The 24-h urinary elimination of total Pt increased in both combination groups, irrespective of the dosing sequence. No difference in protein binding of Pt was observed among the groups. There was a decrease in RBC uptake in the presence of steady-state level of fPXL, but the same was not observed in the OPT-->fPXL group. Additionally, similar results were observed with OPT in combination with CrEL alone. CONCLUSIONS: These results suggest that alterations in the pharmacokinetics of OPT by fPXL are dosing sequence-dependent and mainly caused by the formulation vehicle CrEL. It is suggested that the dosing sequence of fPXL followed by OPT would be more clinically favorable because it would prolong the residence of OPT in systemic circulation. It is further recommended that the use of other formulations of PXL without CrEL or docetaxel would avoid the complication effect of CrEL.
Asunto(s)
Antineoplásicos Fitogénicos/farmacocinética , Antineoplásicos/farmacocinética , Glicerol/análogos & derivados , Compuestos Organoplatinos/farmacocinética , Paclitaxel/farmacocinética , Animales , Área Bajo la Curva , Disponibilidad Biológica , Cromatografía Líquida de Alta Presión , Interacciones Farmacológicas , Glicerol/farmacología , Técnicas In Vitro , Infusiones Intravenosas , Leucocitos/química , Masculino , Oxaliplatino , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
UNLABELLED: Oxaliplatin (OPT), a third-generation platinating agent, is currently being evaluated in a phase II clinical trial in head and neck cancer patients and in a phase I clinical trial in combination with paclitaxel (TXL). PURPOSE: The aim of this study was to investigate the pharmacokinetics and biological correlates of OPT alone as well as the potential pharmacokinetic interaction between OPT and TXL. METHODS: In the phase II study, OPT was given alone as a 2-h i.v. infusion at 60 mg/m(2) weekly for 4 weeks with the cycle repeated after a 2-week rest. In the concurrent phase I combination trial OPT was also given as a 2-h i.v. infusion, but followed by a 1-h i.v. infusion of TXL, weekly for 4 weeks with the cycle repeated after a 2-week rest. The clinical pharmacokinetics of OPT alone and in combination with TXL were investigated in the first cycle of each treatment protocol. The platinum levels in plasma, plasma ultrafiltrate (PUF) and urine were measured by a fully validated inductively coupled plasma mass spectrometry (ICPMS) method. RESULTS: In the ten patients receiving OPT alone, the concentration-time profiles of total platinum exhibited a biexponential decline both in plasma and in PUF. The peak levels of platinum were 2.72+/-0.41 microg/ml in plasma and 1.36+/-0.42 microg/ml in PUF at the end of the OPT infusion, and the platinum levels were still detectable at >10 ng/ml 94 h after the OPT infusion. The mean terminal t(1/2) values of total platinum in plasma and in PUF were 58.9 h and 22.8 h, respectively. The AUC of ultrafilterable platinum represented about 10% of that of the total plasma platinum. The platinum levels in the DNA fraction of peripheral white blood cells (WBC) correlated with the platinum levels in PUF ( r=0.77, P<0.01). In the phase I combination study, the dose level of OPT was escalated from 35 mg/m(2) to 60 mg/m(2). The concentration-time profiles of platinum in the combination trial also showed biexponential decay in plasma and in PUF as exhibited by OPT alone. However, the terminal elimination rate constant (beta) of total plasma platinum increased at all dose levels of OPT when combined with TXL at 45 mg/m(2) ( P<0.05). A concomitant increase in clearance (CL) of total plasma platinum was observed at the OPT dose level of 45 mg/m(2) in combination with TXL at 45 mg/m(2). No statistically significant difference in the 24-h urinary elimination of total platinum was detected between the combination groups and the single-agent group. The AUC values of total platinum in PUF were proportional to OPT doses ranging from 35 to 60 mg/m(2), whether OPT was given alone or in combination with TXL. CONCLUSIONS: OPT clearance may be enhanced by TXL when the two agents are used in combination in patients. The Pt-DNA adduct level in peripheral WBC was found to be a good indicator for oxaliplatin exposure in patients, and should be further exploited for potential tumor drug exposure.
Asunto(s)
Antineoplásicos Fitogénicos/farmacocinética , Antineoplásicos/farmacocinética , Neoplasias/metabolismo , Compuestos Organoplatinos/farmacocinética , Paclitaxel/farmacocinética , Área Bajo la Curva , Aductos de ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Leucocitos/química , Espectrometría de Masas , Oxaliplatino , Platino (Metal)/sangre , Platino (Metal)/orina , Reproducibilidad de los ResultadosRESUMEN
Phytochemicals are recognized as playing an important role in cancer prevention by fruits and vegetables. The avocado is a widely grown and consumed fruit that is high in nutrients and low in calories, sodium, and fats. Studies have shown that phytochemicals extracted from the avocado fruit selectively induce cell cycle arrest, inhibit growth, and induce apoptosis in precancerous and cancer cell lines. Our recent studies indicate that phytochemicals extracted with chloroform from avocado fruits target multiple signaling pathways and increase intracellular reactive oxygen leading to apoptosis. This review summarizes the reported phytochemicals in avocado fruit and discusses their molecular mechanisms and targets. These studies suggest that individual and combinations of phytochemicals from the avocado fruit may offer an advantageous dietary strategy in cancer prevention.
Asunto(s)
Anticarcinógenos/administración & dosificación , Frutas/química , Neoplasias/prevención & control , Persea/química , Anticarcinógenos/análisis , Anticarcinógenos/química , Apoptosis , Proliferación Celular/efectos de los fármacos , Quimioprevención , Humanos , Extractos Vegetales/administración & dosificaciónRESUMEN
Black raspberries are a rich natural source of chemopreventive phytochemicals. Recent studies have shown that freeze-dried black raspberries inhibit the development of oral, esophageal, and colon cancer in rodents, and extracts of black raspberries inhibit benzo(a)pyrene-induced cell transformation of hamster embryo fibroblasts. However, the molecular mechanisms and the active components responsible for black raspberry chemoprevention are unclear. In this study, we found that 2 major chemopreventive components of black raspberries, ferulic acid and beta-sitosterol, and a fraction eluted with ethanol (RO-ET) during silica column chromatography of the organic extract of freeze-dried black raspberries inhibit the growth of premalignant and malignant but not normal human oral epithelial cell lines. Another fraction eluted with CH2Cl2/ethanol (DM:ET) and ellagic acid inhibited the growth of normal as well as premalignant and malignant human oral cell lines. We investigated the molecular mechanisms by which ferulic acid and beta-sitosterol and the RO-ET fraction selectively inhibited the growth of premalignant and malignant oral cells using flow cytometry and Western blotting of cell cycle regulatory proteins. There was no discernable change in the cell cycle distribution following treatment of cells with the RO-ET fraction. Premalignant and malignant cells redistributed to the G2/M phase of the cell cycle following incubation with ferulic acid. beta-sitosterol treated premalignant and malignant cells accumulated in the G0/G1 and G2/M phases, respectively. The RO-ET fraction reduced the levels of cyclin A and cell division cycle gene 2 (cdc2) in premalignant cells and cyclin B1, cyclin D1, and cdc2 in the malignant cell lines. This fraction also elevated the levels of p21waf1/cip1 in the malignant cell line. Ferulic acid treatment led to increased levels of cyclin B1 and cdc2 in both cell lines, and p21waf1/cip1 was induced in the malignant cell line. beta-sitosterol reduced the levels of cyclin B1 and cdc2 while increasing p21waf1/cip1 in both the premalignant and malignant cell lines. These results show for the first time that the growth inhibitory effects of black raspberries on premalignant and malignant human oral cells may reside in specific components that target aberrant signaling pathways regulating cell cycle progression.
Asunto(s)
Frutas , Neoplasias de la Boca/patología , Extractos Vegetales/farmacología , Lesiones Precancerosas/patología , Western Blotting/métodos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Quimioprevención/métodos , Ácidos Cumáricos/farmacología , Relación Dosis-Respuesta a Droga , Ácido Elágico/farmacología , Etanol , Citometría de Flujo/métodos , Humanos , Técnicas In Vitro , Modelos Biológicos , Mucosa Bucal/citología , Mucosa Bucal/efectos de los fármacos , Sitoesteroles/farmacología , Células Tumorales CultivadasRESUMEN
Celecoxib is a potent nonsteroid antiinflammatory drug (NSAID) that has shown great promise in cancer chemoprevention and treatment. The tumor suppression activity of celecoxib and other NSAIDs have been related to the induction of apoptosis in many cancer cell lines and animal models. While celecoxib is a specific inhibitor of cyclooxygenase (COX)-2, recent data indicate that its apoptotic properties may also be mediated through COX-independent pathways. In our study, we evaluated second generation celecoxib derivatives, lacking COX-2 inhibitory activity, in a premalignant and malignant human oral cell culture model to determine their potential anticancer effect and mechanisms responsible for the COX-independent apoptotic activity. Celecoxib and its derivatives delayed the progression of cells through the G(2)/M phase and induced apoptosis. The derivatives with apolar substituents at the terminal phenyl moiety of celecoxib greatly enhanced apoptosis and cell cycle delay. Apoptosis and cell cycle arrest appeared to be independent of derivative induced inhibition of PDK1 and phosphorylation of Akt and Erk1/2. Derivatives induced apoptosis was mediated by the cleavage and activation of caspase-9 and caspase-3, but not caspase 8, implicating the mitochondrial pathway for apoptosis induction. Inhibitors of caspase-3 and caspase-9 and cyclosporin A, a mitochondrial membrane potential stabilizer, attenuated derivative induced apoptosis. Inhibition of caspase-3 prevented the activation of caspase 8, while the inhibition of caspase-9 inhibitor blocked activation of both caspase 3 and 8 by the derivatives. Apoptosis was independent of Bcl-2. These results indicate that the second generation celecoxib derivatives induce apoptosis in human oral cancer lines by the disruption of mitochondrial membrane potential activating caspase 9 and downstream caspase 3 and 8. This suggests that the modification of the celecoxib structure can lead to highly effective COX-independent growth inhibitory and apoptotic agents in chemoprevention and therapy.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Activación Enzimática/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Neoplasias de la Boca/metabolismo , Pirazoles/farmacología , Sulfonamidas/farmacología , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Caspasa 9 , Celecoxib , División Celular/efectos de los fármacos , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Fase G2/efectos de los fármacos , Humanos , Isoenzimas/antagonistas & inhibidores , Proteínas de la Membrana , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosforilación , Prostaglandina-Endoperóxido Sintasas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Studies have shown that nonsteroidal antiinflammatory drugs (NSAIDs) reduce the risk of and mortality from a variety of cancers. Although cyclooxygenase (COX)-dependent and -independent pathways may be involved, the mechanisms responsible for these effects remain unknown. In our study, we found that piroxicam inhibited cell growth in premalignant and malignant, but not normal, human oral epithelial cell lines in a concentration- and time-dependent manner. After 6 days of exposure, the concentration that inhibited growth by 50% was 181 and 211 microM for premalignant and malignant cells, respectively. Piroxicam did not induce apoptosis. The growth inhibitory effect was COX and PGE2 independent. Adding PGE2 or infecting cells with a COX-1 transgene did not abrogate piroxicam-induced growth inhibition. After treatment of the premalignant and malignant cell lines with piroxicam, cells accumulated in the S phase of the cell cycle. Upon removal of piroxicam, cells entered the G2 phase. The S phase block was accompanied by a reduction in the protein levels of cyclin A, cyclin B1, cyclin D1, cdc2, PCNA and the c-jun AP-1 component. Therefore, piroxicam may exert its growth inhibitory effects selectively on the premalignant and malignant human oral epithelial cells lines via signaling pathways regulating the progression of cells through the S phase of the cell cycle.