PFKFB3 gene silencing decreases glycolysis, induces cell‐cycle delay and inhibits anchorage‐independent growth in HeLa cells

Abstract

The high rate of glycolysis despite the presence of oxygen in tumor cells (Warburg effect) suggests an important role for this process in cell division. The glycolytic rate is dependent on the cellular concentration of fructose 2,6-bisphosphate (Fru-2,6-P 2 ), which, in turn, is controlled by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). The ubiquitous PFK-2 isoenzyme (uPFK-2, alternatively named UBI2K5 or ACG) coded by the pfkfb3 gene is induced by different stimuli (serum, progesterone, insulin, hypoxia, etc.) and has the highest kinase/phosphatase activity ratio amongst all PFK-2 isoenzymes discovered to date, which is consistent with its role as a powerful activator of glycolysis. uPFK-2 is expressed in brain, placenta, transformed cells and proliferating cells. In the present work, we analyze the impact of small interfering RNA (siRNA)-induced silencing of uPFK-2 on the inhibition of cell proliferation. HeLa cells treated with uPFK-2 siRNA showed a decrease in uPFK-2 RNA levels measured at 24 h. uPFK-2 protein levels were severely depleted at 48-72 h when compared with cells treated with an unrelated siRNA, correlating with decreased glycolytic activity, Fru-2,6-P 2 , lactate and ATP concentrations. These metabolic changes led to reduced viability, cell-cycle delay and an increase in the population of apoptotic cells. Moreover, uPFK-2 suppression inhibited anchorage-independent growth. The results obtained highlight the importance of uPFK-2 on the regulation of glycolysis, on cell viability and proliferation and also on anchorage-independent growth. These data underscore the potential for uPFK-2 as an effective tumor therapeutic target.

TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown results in radiosensitization of glioma cells

“…Cell number was determined by crystal violet staining as in Calvo et al [21]. Cells attached to the culture plate were stained with crystal violet and, after dissolving the dye with 1% SDS, the absorbance read at 550 nm was plotted as proportional to the cell number.…”

Analysis of fructose and mannose – regulatory peptides signaling pathway in porcine epithelial oviductal cells (OECs) primary cultured long-term in vitro

“…Fructose and mannose metabolism pathways are linked with glucose oxidation via β-D-fructose-6P, obtained inter alia by the activity of PFKFB3. In cancer cells, PFK-2 is one of the factors, just like hexokinase, responsible for the Warburg effect [15]. Thus, over expression of PFK-FB3 is observed in numerous cancers [16][17].…”

PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3)

“…The expression of these genes is dependent on tissue and on development stage (Goren et al, 2000). Importantly, tissue-and cell-specific isoenzymes are not totally exclusive and several cells express more than one isoenzyme (Calvo et al, 2006;Minchenko et al, 2005a;Minchenko et al, 2005b;Telang et al, 2006). This pattern of expression suggests that each isoenzyme plays a key role under different physiological conditions or in response to different stimuli.…”

“…The PFKFB3 gene product is present in proliferating tissues (Duran et al, 2008a;Duran et al, 2009;Goren et al, 2000;Manzano et al, 1998;Sakai et al, 1996) and transformed cells (Calvo et al, 2006;Chesney et al, 1999;Hamilton et al, 1997;Minchenko et al, 2002;Novellasdemunt et al, 2012;Obach et al, 2004;Riera et al, 2002) and various tumors (Atsumi et al, 2002;Fleischer et al, 2011;Kessler et al, 2008). The high kinase/ bisphosphatase activity ratio of this isoenzyme can explain the high Fru-2,6-P 2 found in the cells where it is present, which in turn sustains high glycolytic rates and it is crucial in supporting growing and proliferant cell metabolism.…”

“…PFKFB3 gene was cloned from a fetal brain library (Manzano et al, 1998;Ventura et al, 1995), human placenta (Sakai et al, 1996) and breast cancer cells (Hamilton et al, 1997). PFKFB3 is expressed ubiquitously and it is present in proliferating tissues, transformed cells and in tumours (Almeida et al, 2010;Atsumi et al, 2002;Bando et al, 2005;Calvo et al, 2006;Chesney et al, 1999;Duran et al, 2008a;Duran et al, 2009;Kessler et al, 2008;Novellasdemunt et al, 2012;Riera et al, 2002;Yalcin et al, 2009b). An inducible PFK-2/FBPase-2 (iPFK-2) with proto-oncogenic features was cloned from cancer cell lines (Chesney et al, 1999).…”