Turturro Francesco, Friday Ellen, Fowler Rocky, Surie Diya, Welbourne Tomas
Department of Medicine, Feist-Weiller Cancer Center, Gene Therapy Program, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130, USA.
Clin Cancer Res. 2004 Oct 15;10(20):7022-30. doi: 10.1158/1078-0432.CCR-04-0879.
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.
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) gamma involvement was assessed using an antagonist and PPARgamma(-/-) NIH3T3 cells.
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 micromol/L; P < 0.001 and P < 0.01 for MCF-7 and MDA-MB-231, respectively) associated with a decreased number of viable cells. TRO-mediated inhibition of proliferation was not reversed by the presence of the PPARgamma inhibitor GW9662 and was demonstrable in PPARgamma(-/-) NIH3T3 cells, consistent with a PPARgamma-independent mechanism.
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 PPARgamma antagonist. Our results support a NHE-mediated action of TRO that exerts its effect independent of PPARgamma.
本研究旨在评估曲格列酮（TRO）是否会通过抑制乳腺癌来源细胞系中的钠/氢交换体（NHE）1诱导细胞酸中毒，如果是，细胞酸中毒是否与增殖减少有关。
使用（2,7）-双羧乙基-5（6）-羧基荧光素检测经TRO处理的MCF-7和MDA-MB-231细胞中外源性酸负荷后的细胞内pH（pH(i)）和酸排出能力。使用放射性标记的胸苷掺入法评估DNA合成。使用拮抗剂和PPARγ(-/-) NIH3T3细胞评估过氧化物酶体增殖物激活受体（PPAR）γ的参与情况。
TRO在12分钟后在MCF-7细胞（从7.54±0.23降至6.77±0.06；P<0.001）和MDA-MB-231细胞（从7.38±0.18降至6.89±0.25；P<0.05）中均诱导迅速（<4分钟）且严重的细胞酸中毒，且不增加酸产生。通过对外源性酸负荷（氯化铵脉冲）的反应评估的酸排出明显减弱（MDA-MB-231细胞，P<0.01）或消除（MCF-7细胞，P<0.001）。长期暴露于TRO导致NHE1活性降低（P<0.05）以及DNA合成呈剂量依赖性减少（在100μmol/L时抑制率<75%；MCF-7细胞和MDA-MB-231细胞分别为P<0.001和P<0.01），并伴有活细胞数量减少。PPARγ抑制剂GW9662的存在并未逆转TRO介导的增殖抑制，且在PPARγ(-/-) NIH3T3细胞中也可观察到，这与不依赖PPARγ的机制一致。
TRO在MCF-7和MDA-MD-231细胞中诱导明显的细胞酸中毒。持续性酸中毒与增殖和生长减少一致，且不受PPARγ拮抗剂逆转。我们的结果支持TRO通过NHE介导的作用，其作用独立于PPARγ。
