RESUMEN
Platelets are relatively short-lived, anucleated cells that are essential for proper hemostasis. The regulation of platelet survival in the circulation remains poorly understood. The process of platelet activation and senescence in vivo is associated with processes similar to those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, and cell shrinkage. ABT-737, a potent antagonist of Bcl-2, Bcl-X(L), and Bcl-w, induces apoptosis in nucleated cells dependent on these proteins for survival. In vivo, ABT-737 induces a reduction of circulating platelets that is maintained during drug therapy, followed by recovery to normal levels within several days after treatment cessation. Whole body scintography utilizing ([111])Indium-labeled platelets in dogs shows that ABT-737-induced platelet clearance is primarily mediated by the liver. In vitro, ABT-737 treatment leads to activation of key apoptotic processes including cytochrome c release, caspase-3 activation, and PS externalization in isolated platelets. Despite these changes, ABT-737 is ineffective in promoting platelet activation as measured by granule release markers and platelet aggregation. Taken together, these data suggest that ABT-737 induces an apoptosis-like response in platelets that is distinct from platelet activation and results in enhanced clearance in vivo by the reticuloendothelial system.
Asunto(s)
Plaquetas/citología , Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Plaquetas/efectos de los fármacos , Separación Celular , Supervivencia Celular/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Exocitosis/efectos de los fármacos , Citometría de Flujo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Nitrofenoles/farmacología , Fosfatidilserinas/metabolismo , Piperazinas/farmacología , Agregación Plaquetaria/efectos de los fármacos , Recuento de Plaquetas , Sulfonamidas/farmacologíaRESUMEN
Drug resistance is a prevalent problem in the treatment of neoplastic disease, and the effectiveness of many clinically useful drugs is limited by the fact that they are substrates for the efflux pump, P-glycoprotein. Because there is a need for new compounds that are effective in treating drug-resistant tumors, we tested A-204197 (4-[4-acetyl-4,5-dihydro-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]-N,N-dimethylbenzeneamine), a novel oxadiazoline derivative with antiproliferative properties, on cell lines that were either sensitive or resistant to known microtubule inhibitors. Cell lines that were resistant to paclitaxel, vinblastine, or colchicine were equally sensitive to A-204197 (proliferation IC50s ranging from 36 to 48 nM) despite their expression levels of P-glycoprotein. The effect of A-204197 on cell growth was associated with cell cycle arrest in G2-M, increased phosphorylation of select G2-M checkpoint proteins, and apoptosis. In competition-binding assays, A-204197 competed with [3H]-labeled colchicine for binding to tubulin (K(i) = 0.75 microM); however, it did not compete with [3H]-labeled paclitaxel. A-204197 prevented tubulin polymerization in a dose-dependent manner (IC50 = 4.5 microM) in vitro and depolymerized microtubules in a time-dependent manner in cultured cells. These findings indicate A-204197 is a promising new tubulin-binding compound with antimitotic activity that has potential for treating neoplastic diseases with greater efficacy than currently used antimitotic agents.
Asunto(s)
Antineoplásicos/farmacología , Microtúbulos/efectos de los fármacos , Oxadiazoles/farmacología , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Sitios de Unión , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Colchicina/metabolismo , Colchicina/farmacología , Interacciones Farmacológicas , Resistencia a Múltiples Medicamentos , Fase G2/efectos de los fármacos , Humanos , Microtúbulos/metabolismo , Mitosis/efectos de los fármacos , Oxadiazoles/metabolismo , Paclitaxel/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Tiempo , Tubulina (Proteína)/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Vinblastina/farmacologíaRESUMEN
A high-throughput screening assay was designed to select compounds that inhibit the growth of cultured mammalian cells. After screening more than 60,000 compounds, A-105972 was identified and selected for further testing. A-105972 is a small molecule that inhibits the growth of breast, central nervous system, colon, liver, lung, and prostate cancer cell lines, including multidrug-resistant cells. The cytotoxic IC50 values of A-105972 were between 20 and 200 nM, depending on the specific cell type. The potency of A-105972 is similar in cells expressing wild-type or mutant p53. A majority of cells treated with A-105972 were trapped in the G2-M phases, suggesting that A-105972 inhibits the progression of the cell cycle. Using [3H]A-105972, we found that A-105972 bound to purified tubulin. Unlabeled A-105972 competed with [3H]A-105972 binding with an IC50 value of 3.6 microL. Colchicine partially inhibited [3H]A-105972 binding with an IC50 value of approximately 90 microM, whereas paclitaxel and vinblastine had no significant effect. Tumor cells treated with A-105972 were observed to contain abnormal microtubule arrangement and apoptotic bodies. DNA ladder studies also indicated that A-105972 induced apoptosis. A-105972 caused a mobility shift of bcl-2 on SDS-PAGE, suggesting that A-105972 induced bcl-2 phosphorylation. A-105972 treatment increased the life span of mice inoculated with B16 melanoma, P388 leukemia, and Adriamycin-resistant P388. These results suggest that A-105972 is a small molecule that interacts with microtubules, arrests cells in G2-M phases, and induces apoptosis in both multidrug resistance-negative and multidrug resistance-positive cancer cells. A-105972 and its analogues may be useful for treating cell proliferative disorders such as cancer.
Asunto(s)
Antineoplásicos/farmacología , Oxadiazoles/farmacología , Animales , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Concentración 50 Inhibidora , Leucemia P388/tratamiento farmacológico , Leucemia P388/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Oxadiazoles/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tubulina (Proteína)/metabolismo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-molecule BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small molecules potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compound A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue experiments. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-molecule MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these molecules as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer.
Asunto(s)
Compuestos de Anilina/farmacología , Apoptosis/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Neoplasias/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonamidas/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Ácidos Carboxílicos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Indoles/farmacología , Proteínas de la Membrana/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is detected in most common human cancers but not in adjacent normal cells. Previous studies suggest that survivin associates with the mitotic spindle and directly inhibits caspase activity. To further investigate the function of survivin, we used a survivin antisense (AS) oligonucleotide to downregulate survivin expression in normal and cancer cells. We found that inhibition of survivin expression increased apoptosis and polyploidy while decreasing colony formation in soft agar. Immunohistochemistry showed that cells without survivin can initiate the cleavage furrow and contractile ring, but cannot complete cytokinesis, thus resulting in multinucleated cells. These findings indicate that survivin plays important roles in a late stage of cytokinesis, as well as in apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Asociadas a Microtúbulos , Neoplasias/terapia , Oligonucleótidos Antisentido/farmacología , Proteínas/fisiología , División Celular , Regulación hacia Abajo , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas de Neoplasias , Neoplasias/patología , Proteínas/antagonistas & inhibidores , Survivin , Células Tumorales CultivadasRESUMEN
Beta(beta)-tubulin isotype variation has recently been implicated in the modulation of resistance to paclitaxel in human lung cancer cells and in primary human ovarian tumour samples. Whether alpha-tubulin is involved in drug resistance has not been reported. We have generated a paclitaxel-resistant cell line (H460/T800) from the sensitive human lung carcinoma parental cell line NCI-H460. The resistant cells are more than 1000-fold resistant to taxol and overexpress P-glycoprotein. Interestingly, H460/T800 cells also overexpress alpha- and beta-tubulin as detected by Western blot analysis. From Northern blot analysis, the mechanism of tubulin overexpression appears to be post-transcriptional. To understand whether alpha-tubulin plays a role in drug resistance, we transfected antisense human kalpha1 cDNA construct into the H460/T800 paclitaxel-resistant cells. The antisense clones displayed a reduced alpha-tubulin expression, and the cells were 45-51% more sensitive to paclitaxel and other known antimitotic drugs, compared with vector transfected controls. Complementary experiments of transfecting the sense kalpha1 cDNA into H460 cells conferred a 1.8- to 3.3-fold increase in the IC(50) of several antimitotic agents. Our study suggests that alpha-tubulin is one of the factors that contributes to drug resistance.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Paclitaxel/uso terapéutico , Tubulina (Proteína)/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Northern Blotting , Western Blotting , Ciclo Celular , División Celular , ADN sin Sentido/genética , ADN Complementario/genética , Resistencia a Antineoplásicos , Citometría de Flujo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Transfección , Tubulina (Proteína)/metabolismo , Células Tumorales CultivadasRESUMEN
Ras oncogenes can contribute to tumour development by stimulating vascular endothelial growth factor (VEGF)-dependent angiogenesis. The effect of Ras on angiogenesis may be affected by farnesyltransferase inhibitors (FTI) since farnesylation of Ras is required for its biological activity. In this paper we evaluated the effect of A-170634, a novel and potent CAAX FTI on angiogenesis. Human umbilical vein endothelial cell (HUVEC) tube formation and VEGF secretion were used to assess the effect of A-170634 on angiogenesis in vitro. In vivo, nude mice were injected with the K-ras mutant colon carcinoma cell line HCT116 and treated subcutaneously with A-170634 using osmotic minipump infusion for 10 days. The effect of A-170634 on corneal angiogenesis in vivo was assessed using pellets containing hydron, VEGF, A-170634 or vehicle. In vitro, A-170634 selectively inhibited farnesyltransferase activity over the closely related geranylgeranyltransferase I, inhibited Ras processing, blocked anchorage-dependent and -independent growth of HCT116 K-ras mutated cells, decreased HUVEC capillary structure formation, decreased VEGF secretion from tumour cells and HUVEC growth stimulating activity in a dose-dependent manner. In vivo, tumour growth was decreased by 30% and vascularisation in and around the tumours was reduced by 41% following drug-treatment with no apparent toxicity to the animals. VEGF-induced corneal neovascularisation was reduced by 80% following A-170634 treatment for 7 days. The data presented here demonstrated that A-170634 was a potent and selective peptidomimetic CAAX FTI with anti-angiogenic properties. These results implied that A-170634 may affect tumour growth in vivo by one or more antitumour pathways.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Neoplasias del Colon/tratamiento farmacológico , Animales , Neoplasias del Colon/irrigación sanguínea , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular , Farnesiltransferasa , Humanos , Linfocinas/metabolismo , Metionina/análogos & derivados , Metionina/uso terapéutico , Ratones , Ratones Desnudos , Neovascularización Patológica/prevención & control , Piridinas/uso terapéutico , Ratas , Células Tumorales Cultivadas , Venas Umbilicales , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Farnesylation of Ras is required for its transforming activity in human cancer and the reaction is catalysed by the enzyme farnesyltransferase. Recently, we discovered a novel chemical series of potent farnesyl pyrophosphate (FPP) analogues which selectively inhibited farnesyltransferase. Our most potent compound to date in this series, A-176120, selectively inhibited farnesyltransferase activity (IC(50) 1.2+/-0.3 nM) over the closely related enzymes geranylgeranyltransferase I (GGTaseI) (IC(50) 423+/-1.8 nM), geranylgeranyltransferase II (GGTaseII) (IC(50) 3000 nM) and squalene synthase (SSase) (IC(50)>10000 nM). A-176120 inhibited ras processing in H-ras-transformed NIH3T3 cells and HCT116 K-ras-mutated cells (ED(50) 1.6 and 0.5 microM, respectively). The anti-angiogenic potential of A-176120 was demonstrated by a decrease in Ras processing, cell proliferation and capillary structure formation of human umbilical vein endothelial cells (HUVEC), and a decrease in the secretion of vascular endothelial growth factor (VEGF) from HCT116 cells. In vivo, A-176120 reduced H-ras NIH3T3 tumour growth and extended the lifespan of nude mice inoculated with H- or K-ras-transformed NIH3T3 cells. A-176120 also had an additive effect in combination with cyclophosphamide in nude mice inoculated with K-ras NIH3T3 transformed cells. Overall, our results demonstrate that A-176120 is a potent FPP mimetic with both antitumour and anti-angiogenic properties.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fosfatos de Poliisoprenilo/farmacología , Animales , División Celular , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/citología , Farnesil Difosfato Farnesil Transferasa/antagonistas & inhibidores , Farnesiltransferasa , Genes ras/genética , Humanos , Linfocinas/metabolismo , Masculino , Ratones , Ratones Desnudos , Mutación/genética , Trasplante de Neoplasias , Neovascularización Patológica , Sesquiterpenos , Trasplante Heterólogo , Células Tumorales Cultivadas , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tubulin is incubated first with unlabeled compound and radiolabeled ligand. Streptavidin-labeled SPA beads are added, and the radiolabel associated with tubulin is directly counted with no separation steps. Under our experimental conditions, the dissociation constant of binding (Kd) for colchicine to tubulin was determined to be 1.4 microM, which was consistent with previously reported values. Assay validation was performed by competitively inhibiting [3H]colchicine binding to tubulin with known microtubule inhibitors and comparing their inhibition constants (Ki). Our SPA bead method is a powerful tool since it overcomes the disadvantage of traditional filtration techniques, as there are no separation steps. It is extremely easy to set up, multiple samples can be assayed and supply and labor costs are reduced because of the minimal volume and test reagents used.
Asunto(s)
Biotinilación , Colchicina/metabolismo , Conteo por Cintilación , Estilbenos , Tubulina (Proteína)/metabolismo , Aminofenoles/metabolismo , Bibencilos/metabolismo , Sitios de Unión , Unión Competitiva , Demecolcina/metabolismo , Mebendazol/metabolismo , Microesferas , Podofilotoxina/metabolismo , Solventes , Estreptavidina , Sulfonamidas/metabolismo , Tritio , ItrioRESUMEN
BACKGROUND: Chkl is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates Cdc25C at the late G2 phase. The inactivation of Cdc25C and consequently, the inactivation of Cdc2, are required for the G2 arrest induced by DNA damage. METHODS: We treated 184B5 cell line and its E6 transformed cell lines with adriamycin in the presence of staurosporine or UCNO1 and examined G2 arrest and cell death. RESULTS: We found that adriamycin induced a p53 and p21 response as well as a G1 arrest in 184B5 cells, but not in its E6 transformed cells. Staurosporine or UCNO1 abrogated the G2 arrest induced by adriamycin in both cell lines. In addition, staurosporine or UCNO1 specifically sensitized p53 incompetent cells to adriamycin. CONCLUSION: G2/M checkpoint abrogators can potentially enhance the cytotoxic effect of conventional chemotherapeutic reagents specifically to tumor cells.
Asunto(s)
Antineoplásicos/farmacología , Daño del ADN/efectos de los fármacos , Doxorrubicina/farmacología , Neoplasias/patología , Proteína p53 Supresora de Tumor/metabolismo , Alcaloides/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Transformada , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Inhibidores Enzimáticos/farmacología , Fase G2 , Humanos , Neoplasias/metabolismo , Proteínas Quinasas/metabolismo , Estaurosporina/farmacología , Células Tumorales CultivadasRESUMEN
Cannabinoids are known to affect a number of cellular systems and functions, but the basis for their action is unclear. In this paper we review the current evidence describing cannabinoid effects on various levels of cellular structure and activity and we present our current studies on the influence of delta-9-tetrahydrocannabinol, cannabidiol and cannabinol on one cellular system, the cytoskeleton. The organization of two cytoskeletal structures, microtubules and microfilaments, were examined and the mRNA levels of tubulin and actin, the major protein components of microtubules and microfilaments, respectively, were analysed.
Asunto(s)
Cannabis , Animales , División Celular/efectos de los fármacos , Células/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Humanos , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismoRESUMEN
A reduction in tubulin mRNA levels up to 35-40 min following pressure (10,000 psi for 5 min) was shown by cDNA hybridization in log growth phase and deciliated Tetrahymena. The level of tubulin mRNA increased to a maximum 1 hour after pressure release. Poly(A+) mRNA derived from pressure-treated and atmospheric control cells following deciliation was translated in vitro. The profile for tubulin synthesized in vitro closely resembled the tubulin mRNA profiles. In vivo tubulin synthesis measured by 35S methionine incorporation was suppressed 60%, 40 min following pressure release. The data supports the hypothesis that both transcription and post transcriptional events are sensitive to hydrostatic pressure.
Asunto(s)
Tetrahymena/fisiología , Tubulina (Proteína)/fisiología , Animales , Regulación de la Expresión Génica , Presión Hidrostática , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Fluorescence staining with rhodamine phalloidin specific for F-actin was employed to examine the effects of delta-9-tetrahydrocannabinol (THC) on the distribution of microfilaments in kangaroo rat epithelial cells (PtK2) and rabbit aortic endothelial cells (RAE). PtK2 cells were more sensitive to THC treatment than RAE cells. Exposure of PtK2 cells to 10 microM THC for 2 h disrupted the microfilament network. After treatment with 20 microM THC for 2 h there was a loss of cell-to-cell contact between PtK2 cells, and at 30 microM THC, the cells started to detach from the substratum. In contrast, microfilament disorganization but not cell detachment was observed in RAE cells at THC concentrations of 80 and 100 microM. The possible mechanisms which may account for the changes in the microfilament system are discussed.
Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Dronabinol/farmacología , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Microscopía Fluorescente , ConejosRESUMEN
Confocal microscopy in association with three-dimensional reconstruction was used to examine the changes in the microtubules and microfilaments following cannabinoid treatment of PC12 cells. Microtubules and microfilaments were disrupted in a dose-dependent manner following treatment with 10-30 microM delta 9-tetrahydrocannabinol (THC). A disruption of microtubules and microfilaments was observed following treatment with 30 microM cannabidiol and cannabinol. The amount of microtubules and microfilaments was reduced in a dose-dependent manner following treatment with 10 and 20 microM THC. Cannabidiol and cannabinol reduced the amount of microtubules and microfilaments; however, the reduction was less than that observed with THC treatment. Following the addition of nerve growth factor, differentiated PC12 cells were generally more sensitive to cannabinoid treatments than undifferentiated cells. The possible mechanisms that may account for the changes in microtubules and microfilaments following cannabinoid treatment are discussed.
Asunto(s)
Cannabinoides/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto de Actina/efectos de los fármacos , Animales , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoesqueleto/ultraestructura , Procesamiento de Imagen Asistido por Computador , Microtúbulos/efectos de los fármacos , Células PC12 , RatasRESUMEN
A recurring problem with cancer therapies is the development of drug resistance. While investigating the protein profile of cells resistant to a novel antimitotic compound (A204197), we discovered an increase in annexin IV expression. When we examined the annexin IV protein expression level in a paclitaxel-resistant cell line (H460/T800), we found that annexin IV was also overexpressed. Interestingly a closely related protein, annexin II, was not overexpressed in H460/T800 cells. Immunostaining with either annexin II or IV antibody revealed that annexin IV was primarily located in the nucleus of paclitaxel-resistant H460/T800 cells. Short-term treatment of H460 cells with 10 nM paclitaxel for up to 4 days resulted in induction of annexin IV, but not annexin II expression. In addition, there was an increase in annexin IV staining in the nucleus starting at day 1. Furthermore, cells pretreated with 10 nM paclitaxel for 4 days resulted in cells becoming approximately fivefold more resistant to paclitaxel. Transfection of annexin IV cDNA into 293T cells revealed that there was a threefold increase in paclitaxel resistance. Thus our results indicate that annexin IV plays a role in paclitaxel resistance in this cell line and it is among one of the earliest proteins that is induced in cells in response to cytotoxic stress such as antimitotic drug treatment.
Asunto(s)
Anexina A4/fisiología , Neoplasias del Colon/patología , Resistencia a Antineoplásicos , Neoplasias Pulmonares/patología , Proteínas de Neoplasias/fisiología , Paclitaxel/farmacología , Anexina A4/biosíntesis , Anexina A4/genética , Western Blotting , Colchicina/farmacología , Neoplasias del Colon/metabolismo , ADN Complementario/genética , Resistencia a Múltiples Medicamentos , Electroforesis en Gel Bidimensional , Regulación Neoplásica de la Expresión Génica , Inhibidores de Crecimiento/farmacología , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Nocodazol/farmacología , Proteínas Recombinantes de Fusión/biosíntesis , Transfección , Moduladores de Tubulina , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
The negative and positive enantiomers of 7-hydroxy-delta 6-tetrahydrocannabinol-dimethylheptyl (designated HU-210 and HU-211 respectively) differentially affect undifferentiated and differentiating cultured pheochromocytoma cells (PC-12 cells). In general, cell viability and cell proliferation were suppressed to a much greater extent with HU-210 than with HU-211 in differentiating cells. The effects of these synthetic cannabinoids on the cytoskeleton of PC-12 cells were examined by epifluorescence and confocal microscopy. In both undifferentiated and differentiating PC-12 cells, HU-211 has little effect on the cytoarchitecture whereas HU-210 disrupts the distribution of microtubules and microfilaments. Vacuoles (2-4 microns) were evident in the cytoplasm of HU-210-treated cells but not in the cytoplasm of HU-211-treated cells or in vehicle controls. Tubulin and actin mRNA levels were reduced to 5 and 40%, respectively (relative to untreated controls) in 10 microns HU-210-treated cells whereas the same concentration of HU-211 reduced tubulin and actin mRNA levels to 90 and 95%, respectively. A comparison of the effects of the paired enantiomers and delta 1-THC on the cellular parameters studied reveals that in differentiating cells the action of delta 1-THC is intermediate between that of HU-210 and HU-211. This study demonstrates that compared to HU-210 and delta 1-THC the positive enantiomer HU-211 has little cellular activity.
Asunto(s)
Citoesqueleto/efectos de los fármacos , Dronabinol/análogos & derivados , Citoesqueleto de Actina/efectos de los fármacos , Actinas/genética , Animales , Diferenciación Celular , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dronabinol/química , Dronabinol/farmacología , Microtúbulos/efectos de los fármacos , Células PC12 , Ratas , Estereoisomerismo , Tubulina (Proteína)/genéticaRESUMEN
Antiestrogen tamoxifen (Tam) is the most prescribed drug for the treatment of estrogen receptor (ER)-positive breast cancers. It is also used in long-term clinical trials with encouraging preliminary results as a chemopreventive agent for breast cancer. The effect of Tam on ER-negative cancers, however, is unclear. Here we reported that paclitaxel and 4-hydroxytamoxifen (4-HT) have a synergistic cytotoxic effect on the ER-negative colon cancer cell line HCT15, which is refractory to paclitaxel alone. Our results showed that 4-HT at submicromolar concentrations effectively enhanced the antiproliferative effect of paclitaxel. In addition, at 1/10 of the paclitaxel concentrations used for HCT15, 4-HT and paclitaxel also showed synergistic effect on NCI H460, an ER-negative lung cancer cell line. For both cell lines, the effective concentration for paclitaxel to inhibit cell growth was 1 log lower in the combination treatment than the concentration used in the single treatment. Cell cycle analysis showed that the combination of paclitaxel and 4-HT increased the G2/M population and resulted in the increase of apoptosis in both cell lines. Enhanced early release of cytochrome c from mitochondria may be the apoptotic pathway activated in the combination treatment in HCT15 cells.