RESUMEN
During apoptosis, myosin light chain phosphorylation induced by ROCK 1, activated by caspase 3-mediated cleavage, results in the formation of membrane blebs. Additionally, actin-myosin-based contraction induced by the activation of ROCK is involved in the apoptotic nuclear disintegration. In previous studies, it was reported that ROCK 1 was only cleaved by caspase 3 in cell death and caspase 7 was involved in truncation of ROCK 1 in in-vitro cell-free conditions. Here we reported that caspase 2 is involved in the truncation of ROCK 1 directly as well as caspase 3 and caspase 7. Utilizing caspase 3-deficient MCF-7, MDA-MB-231 and HeLa cells, we demonstrated that caspase 2 produced an active fragment of approximately 130 kDa of ROCK 1 in cell death. The cleaved active fragment of ROCK 1 is also responsible for the formation of membrane blebbing in cell death. Interestingly, caspase 2-mediated cleavage of ROCK 1 might occur in the region where caspase 3 truncates ROCK 1. Moreover, the presence of an active cleaved form of ROCK 1 in the nuclei implies that this fragment might play a role in the disruption of nuclear integrity. Taken together, it was determined that caspase 2 has a role in the truncation of ROCK 1 in cell death, and a new activation mechanism has been defined for ROCK 1.
Asunto(s)
Caspasas/metabolismo , Neoplasias/metabolismo , Quinasas Asociadas a rho/metabolismo , Antineoplásicos/farmacología , Caspasa 2/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cisteína Endopeptidasas/metabolismo , Células HeLa , Humanos , Células MCF-7 , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteolisis , Quinasas Asociadas a rho/químicaRESUMEN
Recently drug-induced senescence has gained momentum as a new approach in cancer therapy. It is accepted that senescent cells display typical phenotypic features including flattened, enlarged, and multinucleated cell morphology. However, it is not well elucidated how these morphological alterations occur. The current study evaluates the possible role of Rho/Rho kinase pathway in cardiac glycoside-induced senescent cell morphology in HeLa cells. Our results indicate that the administration of cardiac glycosides, ouabain, digoxin, bufalin, to HeLa cells induced cellular senescence leading to an increase in the volume, area and maximum thickness of the cells. Although preincubation of specific Rho kinase inhibitor Y-27632 did not inhibit the occurrence of cardiac glycoside-induced senescence in cells, it reduced the cell area and cell volume. Inhibition of Rho by CT04 produced similar results as seen for the preincubation of Y-27632. In addition, inhibition of Rock caused a decrease in increased actin stress fibers in senescent cells induced by ouabain. Additionally, preincubation of Y-27632 decreased the ouabain-induced the phosphorylation of MYPT and cofilin. In conclusion, Rock inhibition-mediated alteration of senescent cell morphology may be associated with the decreased actin stress fibers formation. Since it is known that secretory activity is accompanied by the changes of cell morphology, these morphological alterations observed by the inhibition of Rho/Rho kinase pathway may also lead to important secretory functions of senescent cells.
Asunto(s)
Glicósidos Cardíacos/farmacología , Tamaño de la Célula/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Amidas/farmacocinética , Células HeLa , Humanos , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Piridinas/farmacocinética , Fibras de Estrés/metabolismo , Fibras de Estrés/patología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
The aim of this study is to formulate and compare the physicochemical properties of negatively charged liposomes and poly(lactide-co-glycolide) (PLGA) nanoparticles loaded with gemcitabine hydrochloride. The influence of the formulation variables on the liposome and nanoparticle properties on particle size, zeta potential, encapsulation efficiency, and drug release was evaluated. Although the PEGylated nanoparticles and PEGylated liposomes were of the same size (â¼200 nm), the encapsulation efficiency was 1.4 times higher for PEGylated liposomes than for PEGylated nanoparticles. The optimized formulation of PEGylated liposomes and PEGylated nanoparticles had 26.1 ± 0.18 and 18.8 ± 1.52% encapsulation efficiency, respectively. The release of drug from the PEGylated liposomes and PEGylated nanoparticles exhibited a biphasic pattern that was characterized by a fast initial release during the first 2 h followed by a slower continuous release. Transmission electron microscopy (TEM) images identified separate circular structures of the liposomes and nanoparticles. The in vitro cytotoxicity of the optimized formulations was assessed in MCF-7 and MDA-MB-231 cells, and the results showed that the cytotoxicity effect of the gemcitabine hydrochloride-loaded liposomes and nanoparticles was more than commercial product Gemko® and gemcitabine hydrochloride solution.
Asunto(s)
Desoxicitidina/análogos & derivados , Liposomas/química , Nanopartículas/química , Línea Celular Tumoral , Química Farmacéutica/métodos , Desoxicitidina/química , Desoxicitidina/farmacología , Liberación de Fármacos/efectos de los fármacos , Humanos , Ácido Láctico/química , Células MCF-7 , Tamaño de la Partícula , Polietilenglicoles/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , GemcitabinaRESUMEN
In this study, cytotoxic effects of the dichloromethane, ethyl acetate, ethanol, and aqueous extracts of the aerial parts of Cyclotrichium niveum (Boiss.) Manden. & Scheng (Lamiaceae) were evaluated. We tested HeLa, MCF-7 cancer cells, and MRC-5 and MCF-10A normal cells. The ethyl acetate and dichloromethane extracts induced cytotoxicity whereas the ethanol and aqueous extracts had no cytotoxic activity against both cancer cells. IC50 values of the dichloromethane extract were 353.0 ± 84.30 µg/ml, 114.8 ± 40.34 µg/ml, 39 ± 0.56 µg/ml, and 49 ± 13 µg/ml in HeLa, MCF-7, MRC-5, MCF-10A cells, respectively. IC50 values of the ethyl acetate extract were 117.0 ± 36.24 µg/ml in HeLa cells, 156.3 ± 19.86 µg/ml in MCF-7 cells, 1100 ± 340 µg/ml in MRC-5 cells and 7900 ± 1200 µg/ml in MCF-10A cells. Additionally, the ethyl acetate extract showed more selectivity to HeLa and MCF-7 cancer cells than MRC-5 and MCF-10A normal cells. Our results of HPLC analysis showed that apigenin in the ethyl acetate extract (2.2518 ± 0.0005 mg/100 mg extract) might be responsible of that selective cytotoxic effect. In the current work, we have shown for the first time that C. niveum has cytotoxic properties in the cancer cell lines tested.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Lamiaceae/química , Extractos Vegetales/farmacología , Antineoplásicos Fitogénicos/química , Proliferación Celular/efectos de los fármacos , Células HeLa , Humanos , Células MCF-7 , Necrosis/tratamiento farmacológico , Necrosis/fisiopatología , Extractos Vegetales/químicaRESUMEN
Previously, we demonstrated that the Rho/ROCK pathway is involved in ouabain-induced apoptosis in HUVEC. In the current work, we investigated whether the Rho/ROCK pathway is functional during cardiac glycosides-induced cytotoxic effects in cancer cell lines, as well as in non-tumor cells. For that purpose, we evaluated the role of ROCK activation in bleb formation and cell migration over upstream and downstream effectors in addition to ROCK cleavage after cardiac glycosides treatment. All three cardiac glycosides (ouabain, digoxin and bufalin) induced cell death in HeLa and HepG2 cells and increased the formation of blebbing in HeLa cells. In contrast to our previous study, ROCK inhibitor Y27632 did not prevent bleb formation. Observation of ROCK II cleavage after ouabain, digoxin and oxaliplatin treatments in HeLa and/or HepG2 cells suggested that cleavage is independent of cell type and cell death induction. While inhibiting cleavage of ROCK II by the caspase inhibitors z-VAD-fmk, z-VDVAD-fmk and z-DEVD-fmk, evaluation of caspase 2 siRNA ineffectiveness on this truncation indicated that caspase-dependent ROCK II cleavage is differentially regulated in cancer cell lines. In HeLa cells, ouabain induced the activation of ROCK, although it did not induce phosphorylation of ERM, an upstream effector. While Y27632 inhibited the migration of HeLa cells, 10nM ouabain had no effect on cell migration. In conclusion, these findings indicate that the Rho/ROCK pathway is regulated differently in cancer cell lines compared to normal cells during cardiac glycosides-induced cell death.