Proliferation and apoptosis of HeLa cells induced by in vitro stimulation with digitalis

Ramírez-Ortega, Margarita; Maldonado-Lagunas, Vilma; Meléndez-Zajgla, Jorge; Carrillo-Hernandez, Jose F.; Pastelín-Hernández, Gustavo; Picazo-Picazo, Ofir; Ceballos, Guillermo

doi:10.1016/j.ejphar.2006.01.035

Cited by 49 publications

(28 citation statements)

References 23 publications

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“…In smooth muscle, HeLa, and neuroblastoma cells, ouabain induces apoptosis (22,23). In contrast, ouabain has an antiapoptotic effect in LLC-PK1 and kidney cells (24), preventing apoptosis induced through serum deprivation in renal proximal tubular rat cells (24).…”

Section: Resultsmentioning

confidence: 99%

Ouabain modulates epithelial cell tight junction

Larre

Lázaro

Contreras

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

102

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Epithelial cells treated with high concentrations of ouabain (e.g., 1 μM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell-cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K + pumping nor disturb the K + balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (J DEX ). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, J DEX , and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell-cell contacts.claudins | c-Src | ERK1/2 | Madin-Darby canine kidney | occludin S everal lines of evidence, including the high affinity and specificity of ouabain for Na + ,K + -ATPase, suggest that endogenous ouabain analogs might exist. In keeping with this possibility, Hamlyn et al.(1) demonstrated the presence of a substance in plasma that cannot be distinguished from ouabain even by specific antibodies and mass spectrometry (1-4). Endogenous ouabain levels are increased during exercise (5) and in pathological conditions such as arterial hypertension (6-9) and eclampsia (10), raising the possibility that ouabain functions as a hormone and prompting efforts to elucidate physiological role.We have previously demonstrated that ouabain acts on cell-substrate and cell-cell contacts in Madin-Darby canine kidney (MDCK) cells. At 1 μM, ouabain binding to the Na + ,K + -ATPase results in pump inhibition and disassembly of molecules from the tight, adherens, and focal junctions (i.e., a P→A mechanism from pump to adhesion) (11). Consistent with these results, Rajasekaran et al. (12) have observed that 0.5 μM ouabain acts on the tight junction (TJ), decreasing transepithelial electrical resistance (TER) and increasing mannitol and inulin permeability in cultures of human retinal pigment epithelial cells. Furthermore, we have shown that in cocultures of wild-type MDCK and ouabain-resistant MDCK cells, ouabain treatment increases the expression of connexin 32, but not 26 or 43, and increases cell-cell communication via gap junctions to rescue the wild-type cells (13). Although these effects indicate that toxic levels of ouabain affect the structure and function of cell-cell junctions, we do not know how lower levels of ouabain affect adhesive structures.In the present work, we focus on this question and demonstrate that 10 nM ouabain affects neither Na + ,K + -ATPase nor the K + balance of the cells and does not indu...

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Section: Resultsmentioning

confidence: 99%

Ouabain modulates epithelial cell tight junction

Larre

Lázaro

Contreras

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

102

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“…18) High intracellular concentrations of Ca 2ϩ are involved in the double strand breaks of deoxyribonucleic acid (DNA) and induced cell apoptosis. 19) In addition to transporting ions, Na ϩ /K ϩ -ATPase interacts with adjacent membrane proteins and participates in cytosolic cascades of signaling proteins to send messages to intracellular organelles. The signaling pathways that are rapidly activated/elicited by the interaction of CSs (like ouabain) with the Na ϩ /K ϩ -ATPase and which are independent of changes in intracellular Na ϩ and K ϩ concentrations, include the activation of Src kinase, transactivation of the epidermal growth factor receptor (EGFR) by Src, 20,21) modulation of nuclear factor (NF)-kB activity, 22,23) activation of Ras and P42/P44 mitogen-activated protein kinases (MAPKs), and increased generation of reac- -ATPase a a1 siRNA could inhibit HepG2 cell proliferation.…”

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confidence: 99%

Targeting the Na<sup>+</sup>/K<sup>+</sup>-ATPase α1 Subunit of Hepatoma HepG2 Cell Line to Induce Apoptosis and Cell Cycle Arresting

Xu¹,

Wang

Gao³

et al. 2010

Biological & Pharmaceutical Bulletin

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“…4,5,7) Although the proliferation induced by the NKA inhibitors such as ouabain has been known for many years, the mechanism of this action was not well defined. 7,8) Since the vascular endothelium could be a potential target of endogenous ouabain, we investigated the effect of ouabain on proliferation in HUVEC.…”

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

“…4,6) However, experimental evidence has been put forward showing that the effect of ouabain on cell survival and proliferation may be either independent of or dependent on the inhibition of cation transport. 7,8) All of these data indicate that there maybe exist other target sites of digitalis in different cell lines besides NKA, and these target proteins play important roles in the regulation of cell growth and transformation with or without interaction with NKA.In this study we focused on the effect of low doses of ouabain on proliferation in endothelial cells and did the differential proteomic analysis of human umbilical vein endothelial cells (HUVEC) in response to low-dose ouabain. We compared the different protein expression of control cells and ouabain-treated cells and identified thirty-two proteins, in comparison with controls cells, the differential proteomic analysis of HUVEC treated by ouabain further revealed the variation of eight proteins regulated by ouabain treatment and expression of five of the protein spots increased and that of three proteins decreased.…”

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Proteomics Analysis of the Proliferative Effect of Low-Dose Ouabain on Human Endothelial Cells

Qiu

Gao

Zhou

et al. 2007

Biological & Pharmaceutical Bulletin

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Digitalis has played a prominent role in the therapy of congestive heart failure since William Withering codified its use in 1785, the mechanisms of action of digitalis have been under extensive investigation for nearly 50 years, yielding one of the most specific mechanisms thus far defined for any agent so extensively used. Its ability to bind to and inhibit the Na-K-ATPase (NKA) has been well established, as has the resulting increase in cellular [Ca 2ϩ ] responsible for its positive inotropic action and its toxicity as well. However, recent observations suggest that digitalis has additional effects, such as the positive inotropic effect on heart muscle, and the inductive effect on cell growth, either hypertrophic on heart muscle cells or proliferative on vascular smooth muscle cells.1) The inductive effect of cell proliferation by nontoxic concentrations of digitalis has been observed by Christen and Dornand in studies on cell growth inducers for lymphocytes since 1975, 2,3) and reports have published that ouabain exerts dual effects (proliferation and death) on cell growth at different concentration in different cell lines. 4,5) Some researchers proposed the mechanism for ouabain's proliferative inductive effect is its interaction with NKA; this interaction occurred at concentrations that did not induce enzyme activity inhibition and that transformed it into a signal transducer system. 4,6) However, experimental evidence has been put forward showing that the effect of ouabain on cell survival and proliferation may be either independent of or dependent on the inhibition of cation transport. 7,8) All of these data indicate that there maybe exist other target sites of digitalis in different cell lines besides NKA, and these target proteins play important roles in the regulation of cell growth and transformation with or without interaction with NKA.In this study we focused on the effect of low doses of ouabain on proliferation in endothelial cells and did the differential proteomic analysis of human umbilical vein endothelial cells (HUVEC) in response to low-dose ouabain. We compared the different protein expression of control cells and ouabain-treated cells and identified thirty-two proteins, in comparison with controls cells, the differential proteomic analysis of HUVEC treated by ouabain further revealed the variation of eight proteins regulated by ouabain treatment and expression of five of the protein spots increased and that of three proteins decreased. This new information gives new sights into the mechanisms for the proliferative effect of digitalis on HUVEC and provides potential avenues for the development of future therapeutic interventions for the treatment of cardiovascular diseases. MATERIALS AND METHODS MaterialsOuabain and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (U.S.A.), Human umbilical vein endothelial cells (HUVEC) were obtained from American Type Culture Collection (U.S.A.), cell culture medium was from Gibco (U.S.A.), 2-DE reagents ...

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Proliferation and apoptosis of HeLa cells induced by in vitro stimulation with digitalis

Cited by 49 publications

References 23 publications

Ouabain modulates epithelial cell tight junction

Ouabain modulates epithelial cell tight junction

Targeting the Na<sup>+</sup>/K<sup>+</sup>-ATPase α1 Subunit of Hepatoma HepG2 Cell Line to Induce Apoptosis and Cell Cycle Arresting

Proteomics Analysis of the Proliferative Effect of Low-Dose Ouabain on Human Endothelial Cells

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Proliferation and apoptosis of HeLa cells induced by in vitro stimulation with digitalis

Cited by 49 publications

References 23 publications

Ouabain modulates epithelial cell tight junction

Ouabain modulates epithelial cell tight junction

Targeting the Na<sup>+</sup>/K<sup>+</sup>-ATPase &alpha;1 Subunit of Hepatoma HepG2 Cell Line to Induce Apoptosis and Cell Cycle Arresting

Proteomics Analysis of the Proliferative Effect of Low-Dose Ouabain on Human Endothelial Cells

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Targeting the Na<sup>+</sup>/K<sup>+</sup>-ATPase α1 Subunit of Hepatoma HepG2 Cell Line to Induce Apoptosis and Cell Cycle Arresting