RESUMEN
Calcium electroporation is a novel anti-cancer treatment investigated in clinical trials. We explored cell sensitivity to calcium electroporation and electroporation with bleomycin, using viability assays at different time and temperature points, as well as heat calorimetry, lipidomics, and flow cytometry. Three cell lines: HT29 (colon cancer), MDA-MB231 (breast cancer), and HDF-n (normal fibroblasts) were investigated for; (a) cell survival dependent on time of addition of drug relative to electroporation (1.2 kV/cm, 8 pulses, 99 µs, 1 Hz), at different temperatures (37 °C, 27 °C, 17 °C); (b) heat capacity profiles obtained by differential scanning calorimetry without added calcium; (c) lipid composition by mass spectrometry; (d) phosphatidylserine in the plasma membrane outer leaflet using flow cytometry. Temperature as well as time of drug administration affected treatment efficacy in HT29 and HDF-n cells, but not MDA-MB231 cells. Interestingly the HT29 cell line displayed a higher phase transition temperature (approximately 20 °C) versus 14 °C (HDF-n) and 15 °C (MDA-MB231). Furthermore the HT29 cell membranes had a higher ratio of ethers to esters, and a higher expression of phosphatidylserine in the outer leaflet. In conclusion, lipid composition and heat capacity of the membrane might influence permeabilisation of cells and thereby the effect of calcium electroporation and electrochemotherapy.
Asunto(s)
Neoplasias de la Mama/terapia , Neoplasias del Colon/terapia , Electroquimioterapia/métodos , Electroporación/métodos , Lípidos/análisis , Bleomicina/farmacología , Calcio/farmacología , Calorimetría , Línea Celular Tumoral , Membrana Celular/química , Supervivencia Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Células HT29 , Humanos , Lipidómica , Transición de Fase , Fosfatidilserinas/análisisRESUMEN
Altered pH-regulatory ion transport is characteristic of many cancers; however, the mechanisms and consequences are poorly understood. Here, we investigate how a truncated, constitutively active ErbB2 receptor (DeltaNErbB2) common in breast cancer impacts on the Na(+)/H(+)-exchanger NHE1 and the Na(+),HCO(3)(-)-cotransporter NBCn1 in MCF-7 human breast cancer cells and address the roles of these transporters in chemotherapy resistance. Upon DeltaNErbB2 expression, mRNA and protein levels of NBCn1, yet not of NHE1, increased several-fold, and the localization of both transporters was altered paralleling extensive morphological changes. The rate of pH(i) recovery after acid loading increased by 50% upon DeltaNErbB2 expression. Knockdown and pharmacological inhibition confirmed the involvement of both NHE1 and NBCn1 in acid extrusion. NHE1 inhibition or knockdown sensitized DeltaNErbB2-expressing cells to cisplatin-induced programmed cell death (PCD) in a caspase-, cathepsin-, and reactive oxygen species-dependent manner. NHE1 inhibition augmented cisplatin-induced caspase activity and lysosomal membrane permeability followed by cysteine cathepsin release. In contrast, NBCn1 inhibition attenuated cathepsin release and had no net effect on viability. These findings warrant studies of NHE1 as a potential target in breast cancer and demonstrate that in spite of their similar transport functions, NHE1 and NBCn1 serve different functions in MCF-7 cells.
Asunto(s)
Equilibrio Ácido-Base/genética , Neoplasias de la Mama/genética , Proteínas de Transporte de Catión/fisiología , Resistencia a Antineoplásicos/genética , Receptor ErbB-2/genética , Simportadores de Sodio-Bicarbonato/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Antineoplásicos/uso terapéutico , Transporte Biológico/genética , Transporte Biológico/fisiología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Catepsinas/metabolismo , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Estructura Terciaria de Proteína/genética , ARN Interferente Pequeño/farmacología , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Simportadores de Sodio-Bicarbonato/antagonistas & inhibidores , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/metabolismo , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found that severe hypertonic stress greatly diminished the viability of murine fibrosarcoma cells (WEHI-902) and immortalized murine embryonic fibroblasts (iMEFs). This effect was attenuated markedly by Hsp70 over-expression. To determine whether the protective effect of Hsp70 was mediated via an effect on volume regulatory ion transport, we compared regulatory volume decrease (RVD) and increase (RVI) in control WEHI-902 cells and after increasing Hsp70 levels by heat shock or over-expression (WEHI-912). Hsp70 levels affected neither RVD, RVI nor the relative contributions of the Na(+)/H(+)-exchanger (NHE1) and Na(+),K(+),2Cl(-)-cotransporter (NKCC1) to RVI. Hypertonic stress induced caspase-3 activity in WEHI cells and iMEFs, an effect potentiated by Hsp70 in WEHI cells but inhibited by Hsp70 in iMEFs. Osmotic shrinkage-induced PCD was associated with Hsp70-inhibitable cysteine cathepsin release in iMEFs and attenuated by caspase and cathepsin inhibitors in WEHI cells. Treatment with TNF-alpha or the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl)amiloride (EIPA) reduced the viability of WEHI cells further under isotonic and mildly, but not severely, hypertonic conditions. Thus, it is concluded that shrinkage-induced PCD involves both caspase- and cathepsin-dependent death mechanisms and is potently counteracted by Hsp70.
Asunto(s)
Apoptosis/fisiología , Fibrosarcoma , Proteínas HSP70 de Choque Térmico/metabolismo , Animales , Caspasas/metabolismo , Catepsinas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Línea Celular Tumoral , Tamaño de la Célula , Homeostasis/fisiología , Soluciones Hipertónicas/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Presión Osmótica , Transducción de Señal/fisiología , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Heat shock protein 70 is an antiapoptotic chaperone protein highly expressed in human breast tumors and tumor cell lines. Here, we demonstrate that the mere inhibition of its synthesis by adenoviral transfer or classical transfection of antisense Hsp70 cDNA (asHsp70) results in massive death of human breast cancer cells (MDA-MB-468, MCF-7, BT-549, and SK-BR-3), whereas the survival of nontumorigenic breast epithelial cells (HBL-100) or fibroblasts (WI-38) is not affected. Despite the apoptotic morphology as judged by electron microscopy, the asHsp70-induced death was independent of known caspases and the p53 tumor suppressor protein. Furthermore, Bcl-2 and Bcl-X(L), which protect tumor cells from most forms of apoptosis, failed to rescue breast cancer cells from asHsp70-induced death. These results show that tumorigenic breast cancer cells depend on the constitutive high expression of Hsp70 to suppress a transformation-associated death program. Neutralization of Hsp70 may open new possibilities for treatment of cancers that have acquired resistance to therapies activating the classical apoptosis pathway.
Asunto(s)
Apoptosis , Neoplasias de la Mama/metabolismo , Caspasas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Adenoviridae/genética , Neoplasias de la Mama/terapia , Carcinoma/metabolismo , Carcinoma/terapia , Transformación Celular Neoplásica , ADN Complementario/uso terapéutico , Femenino , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Proteínas HSP70 de Choque Térmico/genética , Humanos , Proteína bcl-XRESUMEN
The major stress-inducible heat shock protein, Hsp70, is a chaperone protein abundantly and preferentially expressed in human tumors and tumor cell lines. Owing to the ability of Hsp70 to protect cells from a wide range of apoptotic and necrotic stimuli, it has been assumed that Hsp70 may confer survival advantage to tumor cells. To investigate this hypothesis in human tumor cell lines, we generated an adenovirus expressing antisense Hsp70 (Ad.asHsp70). The effective and specific depletion of Hsp70 by Ad.asHsp70 resulted in massive cell death of all tumorigenic cell lines tested (carcinomas of breast, colon, prostate and liver as well as glioblastoma). In spite of an effective depletion of Hsp70, Ad.asHsp70 had no effect on the survival or growth of fetal fibroblasts or non-tumorigenic epithelial cells of breast or prostate. Anti-apoptotic proteins Bcl-2, Bcl-XL and CrmA as well as peptide-inhibitors of caspases, DEVD-CHO and zVAD-FMK, failed to rescue tumor cells from Ad.asHsp70-induced cell death. These results indicate that the high expression of Hsp70 is a prerequisite for the survival of human cancer cells of various origins and reveal Hsp70 as the only protein described so far whose expression is specifically needed for the survival of tumorigenic cells.
Asunto(s)
Apoptosis/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Neoplasias/patología , Adenoviridae/genética , Elementos sin Sentido (Genética) , Caspasas/metabolismo , Línea Celular , Supervivencia Celular , Vectores Genéticos , Proteínas HSP70 de Choque Térmico/genética , Humanos , Etiquetado Corte-Fin in Situ , Transducción de Señal , Células Tumorales CultivadasRESUMEN
The pl6INK4a tumor suppressor negatively regulates progression through the G1 phase of the mammalian cell cycle. To mimic the downmodulation of p16INK4a commonly seen in cancer, we designed and characterized a hammerhead ribozyme against exon E1alpha of the murine pl6INK4a transcript. Stable expression of the ribozyme in murine erythroleukemia (MEL) cells reduced the endogenous pl6INK4a protein by more than 70% and significantly accelerated cell cycle progression. The specificity and efficiency of our new ribozyme suggest its possible application in elucidating the role of p16INK4a in fundamental biological processes including homeostatic tissue renewal, protection against oncogenic transformation, and cellular senescence.