Gustavo Blanco - Academia.edu (original) (raw)

Papers by Gustavo Blanco

Journal of Pathology, 2007

With an overall 5 year survival rate as low as 15% for non-small cell lung cancer (NSCLC), even w... more With an overall 5 year survival rate as low as 15% for non-small cell lung cancer (NSCLC), even with surgical intervention and the use of newer molecules in adjuvant chemotherapy, there is an urgent need for new biological targets and associated novel anti-cancer agents. The present study was undertaken to evaluate the potential of the Na+/K+-ATPase α1 subunit as a novel target in NSCLC and revealed that α1 expression is markedly higher in a significant proportion of NSCLC clinical samples compared to normal lung tissue. Furthermore, reduction in α1 expression in A549 NSCLC cells by anti-α1 siRNA resulted in markedly impaired proliferation and migration of these cancer cells. Finally, of three cardenolides investigated, UNBS1450, which is known to bind to Na+/K+-ATPase and displays potent anti-tumour activity in vivo in experimental models of human NSCLCs, is the most potent inhibitor of Na+/K+-ATPase isozymes (α1β1, α2β1 and α3β1), most strikingly of α1β1. This was reflected in the compound's more potent anti-proliferative activity in all NSCLC cell lines evaluated (A549, Cal-12T, NCI-H727 and A427); the first three of which over-express α1. The marked impairment in A549 NSCLC cell proliferation and migration, and resulting similar morphology following anti-α1 siRNA or UNBS1450 treatment, was associated with features of abnormal cytokinesis, mediated in the case of UNBS1450 by disorganization of the actin cytoskeleton. Collectively these data strongly suggest that targeting the Na+/K+-ATPase α1 using specific cardenolides could represent a novel means to combat certain NSCLCs. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Seminars in Nephrology, 2005

Proceedings of The National Academy of Sciences, 1994

The Na,K-ATPase is a heterodimer consisting of an alpha and a beta subunit. Both Na,K-ATPase subu... more The Na,K-ATPase is a heterodimer consisting of an alpha and a beta subunit. Both Na,K-ATPase subunits are encoded by multigene families. Several isoforms for the alpha (alpha 1, alpha 2, and alpha 3) and beta (beta 1, beta 2, and beta 3) subunits have been identified. All these isoforms are capable of forming functionally active enzyme. Although there is general agreement that the Na,K-ATPase consists of alpha and beta subunits in equimolar amounts, the quaternary structure of the Na,K-ATPase and its functional significance is unknown. Several studies have demonstrated that the enzyme exists within the plasma membrane as an oligomer of alpha beta dimers. However, because the alpha beta protomer seems to be catalytically competent, the possibility exists that higher oligomers are irrelevant to function. The ability to express different alpha isoforms in insect cells and the availability of isoform-specific antibodies has provided the opportunity to test for the existence of stable and specific associations among alpha subunits. By coexpressing different alpha-subunit isoforms in cultured cells, we demonstrate that the Na,K-ATPase alpha subunits specifically and stably associate into oligomeric complexes. This same association among alpha-subunit isoforms was demonstrated in the native enzyme from rat brain. The interaction between Na,K-ATPase alpha subunits is highly specific. When the Na,K-ATPase alpha subunit is coexpressed with the alpha subunit from the H,K-ATPase, the H,K subunit does not associate with the Na,K subunit. Moreover, expression of the truncated alpha 1T isoform with the full-length alpha subunit demonstrates that the C-terminal portion of the polypeptide is important in the alpha-subunit association. Although these results do not clarify the functional role of alpha alpha associations, they do establish their highly specific nature and suggest that oligomerization of alpha beta protomers may be important to the stability and physiological regulation of the enzyme.

Annals of The New York Academy of Sciences, 1997

Biochemistry, 1995

The coexpression of multiple isoforms of the a and p subunits of the Na,K-ATPase in mammalian tis... more The coexpression of multiple isoforms of the a and p subunits of the Na,K-ATPase in mammalian tissues gives rise to the complex molecular heterogeneity that characterizes the Na pump.

Biochemistry, 1999

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) R and subunits h... more Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) R and subunits have been identified in mammals. The association of the various R and polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase R4 isoform in Sf-9 cells using recombinant baculoviruses. When R4 and the Na pump 1 subunit are coexpressed in the cells, Na,K-ATPase activity is induced. This activity is reflected by a ouabainsensitive hydrolysis of ATP, by a Na + -dependent, K + -sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K + . Furthermore, the activity of R4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase R4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the 1 and 3 subunits. In insect cells, the R4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the R4 1 and R4 3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na + , K + , ATP, and ouabain. The enzymatic properties of R4 1 and R4 3 are, however, distinct from the other Na pump isozymes. A Na,K-ATPase activity with similar properties as the R4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the R4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.

Archives of Biochemistry and Biophysics, 1998

While several studies have investigated the regulation of the Na,K-ATPase consisting of the ␣1 an... more While several studies have investigated the regulation of the Na,K-ATPase consisting of the ␣1 and ␤1 subunits, there is little evidence that intracellular messengers influence the other Na pump isozymes. We studied the effect of different protein kinases and arachidonic acid on the rat Na,K-ATPase isoforms expressed in Sf-9 insect cells. Our results indicate that PKA, PKC, and PKG are able to differentially modify the function of the Na,K-ATPase isozymes. While PKC activation leads to inhibition of all isozymes, PKA activation stimulates the activity of the Na,K-ATPase ␣3␤1 and decreases that of the ␣1␤1 and ␣2␤1 isozymes. In contrast, activation of PKG diminishes the activity of the ␣1␤1 and ␣3␤1 isozymes, without altering that of ␣2␤1. Treatment of cells with arachidonic acid reduced the activities of all the isozymes. The changes in the catalytic capabilities of the Na pump isozymes elicited by PKA and PKC are reflected by changes in the molecular activity of the Na,K-ATPases. One of the mechanisms by which PKA and PKC affect Na pump isozyme activity is through direct phosphorylation of the ␣ subunit. In the insect cells, we found a PKA-and PKC-dependent phosphorylation of the ␣1, ␣2 and ␣3 polypeptides. In conclusion, several intracellular messengers are able to modulate the function of the Na,K-ATPase isozymes and some of them in a specific fashion. Because the Na,K-ATPase isozymes have kinetic properties that are unique, this isozyme-specific regulation may be important in adapting Na pump function to the requirements of each cell.