Down-regulation of P-glycoprotein expression by sustained intracellular acidification in K562/Dox cells.
ABSTRACT We have investigated the involvement of intracellular pH (pH(i)) in the regulation of P-glycoprotein (P-gp) in K562/DOX cells. The selective Na(+)/H(+) exchanger1 (NHE1) inhibitor cariporide and the "high K(+)" buffer were used to induce the sustained intracellular acidification of the K562/DOX cells that exhibited more alkaline pH(i) than the K562 cells. The acidification resulted in the decreased P-gp activity with increased Rhodamine 123 (Rh123) accumulation in K562/DOX cells, which could be blocked by the P-gp inhibitor verapamil. Moreover, the acidification decreased MDR1 mRNA and P-gp expression, and promoted the accumulation and distribution of doxorubicin into the cell nucleus. Interestingly, these processes were all pH(i) and time-dependent. Furthermore, the change of the P-gp expression was reversible with the pH(i) recovery. These data indicate that the tumor multidrug resistance (MDR) mediated by P-gp could be reversed by sustained intracellular acidification through down-regulating the P-gp expression and activity, and there is a regulative link between the pH(i) and P-gp in K562/DOX cells.
- SourceAvailable from: PubMed Central[show abstract] [hide abstract]
ABSTRACT: Cells within the acidic extracellular environment of solid tumours maintain their intracellular pH (pHi) through the activity of membrane-based ion exchange mechanisms including the Na(+)/H(+) antiport and the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger. Inhibition of these regulatory mechanisms has been proposed as an approach to tumour therapy. Previously available inhibitors of these exchangers were toxic (e.g. 4,4-diisothiocyanstilbene-2,2-disulphonic acid), and/or non-specific (e.g. 5-N-ethyl-N-isopropyl amiloride). Using two human (MCF7, MDA-MB231) and one murine (EMT6) breast cancer cell lines, we evaluated the influence of two new agents, cariporide (an inhibitor of the Na(+)/H(+) antiport) and S3705 (an inhibitor of the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger) on the regulation of intracellular pH (pHi). The cytotoxicity of the two agents was assessed by using clonogenic assays. Our results suggest that cariporide has similar efficacy and potency to 5-N-ethyl-N-isopropyl amiloride for inhibition of Na(+)/H(+) exchange while S3705 is more potent and efficient than 4,4-diisothiocyanstilbene-2,2-disulphonic acid in inhibiting Na+-dependent Cl(-)/HCO3(-) exchange. The agents inhibited the growth of tumour cells when they were incubated at low pHe (7.0-6.8), but were non-toxic to cells grown at doses that inhibited the regulation of pHi. Our results indicate that cariporide and S3705 are selective cytostatic agents under in vitro conditions that reflect the slightly acidic microenvironment found in solid tumours.British Journal of Cancer 08/2002; 87(2):238-45. · 5.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Aims: Inhibition of the Na+/H+-exchanger (NHE) preserves myocardial morphology and function in rat and mouse models of hypertrophy and failure. The mechanism(s) involved in such cardioprotective effects remain(s) unclear, but might involve blockade of increased protein kinase activity as observed in untreated hearts. Methods and results: We investigated the functional, morphological and biochemical consequences of NHE-inhibition with BIIB722 in rabbits with pacing-induced heart failure (HF). In sham rabbits treated with placebo (n = 9) or BIIB722 (30 mg/kg/day po, n = 9), LV end-diastolic diameter (LVEDD) and systolic fractional shortening (FS, %) remained unchanged. In HF rabbits (n = 9), LVEDD increased and FS decreased from 31.5 +/- 1.4 to 8.1 +/- 0.9 (p < 0.05) at 3 weeks of LV pacing (400 bpm). Apoptosis, fibrosis and myocyte cross-sectional area as well as p38MAPK phosphorylation and iNOS protein expression were significantly increased in HF compared to sham rabbits. The activity of the 90 kDa NHE-kinase was greater in HF than in sham rabbits. In HF rabbits receiving BIIB722 prior to (18.1 +/- 2.2, n = 9) or following 1 week (15.5 +/- 1.6, n = 7) of pacing, FS at 3 weeks was better preserved than in untreated HF rabbits (p < 0.05). Apoptosis, fibrosis, myocyte cross-sectional area, p38MAPK phosphorylation and iNOS protein expression were significantly reduced in HF rabbits receiving BIIB722. Conclusion: NHE-inhibition attenuates the functional, morphological and biochemical derangements of pacing-induced HF in rabbits.Cardiovascular Research 08/2004; 63(2):273-82. · 5.94 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: To identify important amino acid residues involved in intracellular pH (pH(i)) sensing of Na(+)/H(+) exchanger 1, we produced single-residue substitution mutants in the region of the exchanger encompassing the putative 11th transmembrane segment (TM11) and its adjacent intracellular (intracellular loop (IL) 5) and extracellular loops (extracellular loop 6). Substitution of Arg(440) in IL5 with other residues except positively charged Lys caused a large shift in pH(i) dependence of (22)Na(+) uptake to an acidic side, whereas substitution of Gly(455) or Gly(456) within the highly conserved glycine-rich sequence of TM11 shifted pH(i) dependence to an alkaline side. The observed alkaline shift was larger with substitution of Gly(455) with residues with increasing sizes, suggesting the involvement of the steric effect. Interestingly, mutation of Arg(440) (R440D) abolished the ATP depletion-induced acidic shift in pH(i) dependence of (22)Na(+) uptake as well as the cytoplasmic alkalinization induced by various extracellular stimuli, whereas with that of Gly(455) (G455Q) these functions were preserved. These mutant exchangers did not alter apparent affinities for extracellular transport substrates Na(+) and H(+) and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride. These results suggest that positive charge at Arg(440) is required for normal pH(i) sensing, whereas mutation-induced perturbation of the TM11 structure may be involved in the effects of Gly mutations. Thus, both Arg(440) in IL5 and Gly residues in the conserved segment of TM11 appear to constitute important elements for proper functioning of the putative "pH(i) sensor" of Na(+)/H(+) exchanger 1.Journal of Biological Chemistry 05/2003; 278(14):11828-35. · 4.65 Impact Factor