Troglitazone Acts on Cellular pH and DNA Synthesis through a Peroxisome Proliferator-Activated Receptor γ-Independent Mechanism in Breast Cancer-Derived Cell Lines

Abstract

Purpose: The purpose of this study was to assess whether troglitazone (TRO) would induce cellular acidosis by inhibiting Na ؉ /H ؉ exchanger (NHE) 1 in breast carcinoma-derived cell lines and, if so, whether cellular acidosis would be associated with a reduction in proliferation.Experimental Design: Intracellular pH (pH i ) and acid extrusion capacity after an exogenous acid load were assayed using (2, 7)-biscarboxyethyl-5(6)-carboxyfluorescein in MCF-7 and MDA-MB-231 cells treated with TRO. Radiolabeled thymidine incorporation was used to assess DNA synthesis. Peroxisome proliferator-activated receptor (PPAR) ␥ involvement was assessed using an antagonist and PPAR␥ ؊/؊ NIH3T3 cells. Results: TRO induced a prompt (<4 minute) and severe cellular acidosis in both MCF-7 (7.54 ؎ 0.23 to 6.77 ؎ 0.06; P < 0.001) and MDA-MB-231 cells (7.38 ؎ 0.18 to 6.89 ؎ 0.25; P < 0.05) after 12 minutes, without increasing acid production. Acid extrusion as assessed by the response to an exogenous acid load (NH 4 Cl pulse) was markedly blunted (MDA-MB-231, P < 0.01) or eliminated (MCF-7, P < 0.001). Chronic exposure to TRO resulted in NHE1 activity reduction (P < 0.05) and a dose-dependent decrease in DNA synthesis (<75% inhibition at 100 mol/L; P < 0.001 and P < 0.01 for MCF-7 and MDA-MB-231, respectively) associated with a decreased number of viable cells. TROmediated inhibition of proliferation was not reversed by the presence of the PPAR␥ inhibitor GW9662 and was demonstrable in PPAR␥ ؊/؊ NIH3T3 cells, consistent with a PPAR␥-independent mechanism.Conclusions: TRO induces marked cellular acidosis in MCF-7 and MDA-MD-231 cells. Sustained acidosis is consonant with decreased proliferation and growth that is not reversed by a PPAR␥ antagonist. Our results support a NHE-mediated action of TRO that exerts its effect independent of PPAR␥.

Novel high-affinity PPARγ agonist alone and in combination with paclitaxel inhibits human anaplastic thyroid carcinoma tumor growth via p21WAF1/CIP1

“…We also used a pharmacological approach to test the role of PPARg in Tzd regulated transcription. Using similar micromolar concentrations and conditions as cited in published reports for the irreversible PPARg antagonist, GW9662 (Perez-Ortiz et al, 2004;Rumi et al, 2004;Turturro et al, 2004), a complete blockade of PPARg mediated luciferase transcription was demonstrated for all three Tzds (Figure 3c). Thus, using a siRNA and a pharmacological approach, transcription mediated by functional PPARg was completely blocked.…”

“…At nanomolar concentrations, the ability of RS5444 to antagonize cell proliferation in ATC cells was absolutely dependent upon the presence of functional PPARg as shown by the ability of PPARg siRNA and GW9662 to inhibit cell growth. Our study is one of few that clearly documents PPARg-mediated growth inhibitory effects of a Tzd (Palakurthi et al, 2001;Place et al, 2003;Perez-Ortiz et al, 2004;Rumi et al, 2004;Turturro et al, 2004). Collectively, these data demonstrate that the high-affinity PPARg agonist, RS5444, is completely dependent upon PPARg to mediate inhibition of cell proliferation while the other two Tzds activate PPARg along with other unidentified cell proliferation inhibitory pathways in ATC cells.…”

Cellular adaptations to hypoxia and acidosis during somatic evolution of breast cancer

“…To detect evolution of constitutive upregulation of glycolysis, we stained specimens for GLUT-1 since prior studies have demonstrated a direct correlation between GLUT-1 expression and increased glucose flux as measured by FdG PET (Brown et al, 1996(Brown et al, , 2002. The presence of regional acidosis was defined by increased expression of NHE-1, which is primarily responsible for extrusion of H þ ions in MCF-7 and other breast cancer-derived populations (Bischof et al, 1996;Turturro et al, 2004). Finally, we examined clinical specimens of DCIS and invasive cancer to detect evidence for cellular response to hypoxia and acidosis similar to those predicted in the mathematical models and observed in the spheroids.…”

Role of Glutamate Dehydrogenase in Cancer Growth and Homeostasis

“…Noteworthy overexpression of PIG promotes tumorigenesis [16] presumably by building up extracellular glutamate and suppressing local immune responses [18] . In addition NHE mediated acid extrusion is up-regulated in cancer cells [19,20] importing a Na + load requiring Na+/K+ ATPase -ATP expenditure and ATP regeneration associated with acidogenic aerobic glycolysis(Warburg effect) and by substrate level phosphorylation. Because PIG (GGT/GGTP), NHE, glutamine transporter and glutaminase activities are all up-regulated in rapidly growing tumors, tagging molecular target inhibitors [21][22][23][24] with a ϒ-glutamyl moiety offers a tumor specific vehicle specific for limiting anaplerosis and preventing elevated cell pH, prerequisites for rapid tumor growth.…”

“…Noteworthy troglitazone acutely inhibits GDH flu x ( 0 -3 h r s ) a s t h e r e s u l t o f a f a l l i n mitochondrial membrane potential( Ѱm) requiring accelerated GDH flux(3-24hrs) to fully restore the Ѱm [7], a response that is PPARγ independent [7,25] and possibly mitoNEET [35] dependent. Little recognized is the direct inhibition of NHE [20,34] by both troglitazone and rosiglitazone as well as indirect inhibition mediated through PPARγ suppression of NHE gene expression [20,36]; in contrast, pioglitazone does not inhibit NHE directly [34] rather acts indirectly through PPARγ [36]lowering cell pH [34] and accelerating GDH flux("push" mechanism). In combination, TRO +PIO together exert an additive effect on GDH flux presumably reflecting both TRO's "pull" action and PIO's PPARγ-mediated downregulation of NHE gene expression.…”