Up-regulation of P-glycoprotein is involved in the increased paclitaxel resistance in human esophageal cancer radioresistant cells.

OBJECTIVE: Development of drug and radiation resistance is one of the major causes of cancer treatment failure with chemoradiotherapy. Whether radiotherapy affects drugs resistance in esophageal cancer cells remain to be determined. The purpose of the study was to investigate the change of drug-sensitivity and P-glycoprotein (P-gp) expression in ionization radiation-induced human esophageal cancer radioresistant cells. MATERIALS AND METHODS: Radioresistant cells were established by means of continuous fractionated gamma-ray irradiation on human esophageal squamous cancer cell line EC9706. The radiosensitivity and drug-sensitivity between established radioresistant cells and parental cells were detected by a colony-forming assay and 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, respectively. The expressions of multidrug resistance type 1 gene (MDR1) mRNA and protein for P-gp were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot methods. The roles of P-gp activity in irradiation-induced drugs resistance were studied by using verapamil, an inhibitor of P-gp activity. RESULTS: The esophageal cancer radioresistant cells showed an increased cisplatin or paclitaxel resistance. Compared with their parental cells, the expressions of MDR1 mRNA and protein for P-gp were increased significantly in radioresistant cells. Verapamil reduced paclitaxel resistance but had no effect on cisplatin resistance in human esophageal cancer radioresistant cells. CONCLUSIONS: These results suggested that up-regulation of P-gp is involved in the increased paclitaxel resistance but not cisplatin resistance in ionization radiation-induced human esophageal squamous cancer radioresistant cells.

Various doses of fractioned irradiation modulates multidrug resistance 1 expression differently through hypoxia-inducible factor 1α in esophageal cancer cells.

To evaluate the effect of different regimen of radiotherapy on multidrug resistance 1 (MDR1) expression and analyze the role hypoxia-inducible factor 1α (HIF1α) played in the whole process. Fifty-four cell lines established from 96 esophageal cancer biopsy samples were given various doses of fractioned irradiation. The mRNA and protein levels of HIF1α and MDR1 post-irradiation were measured by quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. HIF1α-siRNA was used to verify the effect of HIF1α on radiation-mediated MDR1 modulation. In esophageal cancer cells surviving 28 Gy irradiation (2 Gy/f, 14 fractions), MDR1 mRNA expression increased 65.27 ± 5.58%, and HIF1α was elevated by 27.21 ± 2.25%. Interestingly, their expression decreased by 54.38 ± 11.53% and 32.08 ± 4.75% after 7 Gy irradiation (0.5 Gy/f, 14 fractions). HIF1α expression showed a positive correlation with MDR1 expression in the whole process (P < 0.05). Silencing of HIF1α decreased MDR1 expression and blocked changes in MDR1 and HIF1α expression induced by fractioned irradiation. These results indicate that MDR1 is differentially modulated by different doses of fractionated radiation, which should be taken into account when combining radiotherapy and chemotherapy for patients with esophageal cancer.

Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation.

PURPOSE: To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possible role of P-glycoprotein (PGP) and murine multidrug resistance associated protein (Mrp1) in the drug-resistant phenotype of these cells. METHODS AND MATERIALS: Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy). Western blot analysis was performed for determination of PGP and Mrp1, reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semiquantitative RT-PCR for Mrp1 mRNA. The clonogenic assay was applied to investigate sensitivity, whereas the steady-state drug accumulation of daunorubicin (DNR), 3H-vincristine (VCR), and 3H-etoposide (VP16) was measured by spectrofluorometry and scintillation counting, respectively. For determining of ATPase activity, the release of inorganic phosphate from ATP was quantified using a colorimetric method. RESULTS: Compared with EHR2, the irradiated cell line EHR2/irr showed increased expression of PGP (threefold), Mrp1 (eightfold), and Mrp1 mRNA (sixfold), and a slight reduction of mdr1b mRNA, whereas mdr1a was present in EHR2 but could not be detected in EHR2/irr. EHR2/irr developed sixfold resistance to VP16, twofold resistance to vincristine, but remained sensitive to DNR. Addition of the PGP inhibitor, verapamil (VER) or depletion of glutathione by buthionine sulfoximine (BSO) partly reversed the resistance in EHR2/irr. In EHR2/irr, the steady-state accumulation of 3H-VCR and 3H-VP16 was significantly decreased as compared with EHR2, whereas the accumulation of DNR was unchanged. The ATPase activity of plasma membrane vesicles prepared from EHR2/irr cells was similar to that of wild-type EHR2 cells. The ATPase activity was neither stimulated by vinblastine nor VER. CONCLUSION: Irradiation induced a multidrug-resistant phenotype in sensitive tumor cells. This phenotype was characterized by increased expression of Mrp1 mRNA, Mrp1, and PGP but decreased expression of mdr1a + b mRNA. The influence of irradiation on PGP and Mrp1 expression seemed to be different.

Detection of intracellular antigens by flow cytometry: comparison of two chemical methods and microwave heating.

Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFix (OPF) and the FIX&PERM Cell Permeabilization Kit (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 and rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, using positive- and negative-control cell lines and peripheral blood mononuclear cells. Western blotting was performed as a control of protein expression. For the four antigens assessed, cellular morphology, discrimination between intact cells and debris, percentage of positive cells, and mean fluorescence intensity were examined. For this last parameter, the assessment of the MWH technique was performed using SD and a graphical approach inspired by the concepts described by Bland and Altman (Lancet 1986;346: 1085-7) as well as Petersen et al. (Clin Chem 1997;43: 2039-46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equivalent to OPF and F&P. As assessed for some of the most clinically relevant intracytoplasmic and intranuclear antigens, the MWH method appears to be a valuable and inexpensive alternative. It is worth noting that, unlike commercial reagents, MWH altered surface antigens. Interestingly, this feature, which would prevent cell selection on the basis of combined membrane and intracellular epitopes, is associated with a decrease of nonspecific background fluorescence.

Mechanism of action of human P-glycoprotein ATPase activity. Photochemical cleavage during a catalytic transition state using orthovanadate reveals cross-talk between the two ATP sites.

Human P-glycoprotein (P-gp), an ATP-dependent efflux pump responsible for cross-resistance of human cancers to a variety of lipophilic compounds, is composed of two homologous halves, each containing six transmembrane domains and an ATP-binding/utilization domain. To determine whether each site can hydrolyze ATP simultaneously, we used an orthovanadate (Vi)-induced ADP-trapping technique (P-gp.MgADP.Vi). In analogy with other ATPases, a photochemical peptide bond cleavage reaction occurs within the Walker A nucleotide binding domain consensus sequence (GX4GK(T/S)) when the molecule is trapped with Vi in an inhibited catalytic transition state (P-gp.MgADP.Vi) and incubated in the presence of ultraviolet light. Upon reconstitution into proteoliposomes, histidine-tagged purified P-gp from baculovirus-infected insect cells had drug-stimulated ATPase activity. Reconstituted P-gp was incubated with either ATP or 8-azido-ATP in the presence or absence of Vi under ultraviolet (365 nm) light on ice for 60 min. The resultant products were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with seven different human P-gp-specific antibodies covering the entire length of the molecule. Little to no degradation of P-gp was observed in the absence of Vi. In the presence of Vi, products of approximately 28, 47, 94, and 110 kDa were obtained, consistent with predicted molecular weights from cleavage at either of the ATP sites but not both sites. An additional Vi-dependent cleavage site was detected at or near the trypsin site in the linker region of P-gp. These results suggest that both the amino- and carboxyl-terminal ATP sites can hydrolyze ATP. However, there is no evidence that ATP can be hydrolyzed simultaneously by both sites.

P-glycoprotein is a dimer in the kidney and brain capillary membranes: effect of cyclosporin A and SDZ-PSC 833.

Radiation-inactivation studies were performed in order to elucidate the oligomeric nature of P-glycoprotein (P-gp) expressed in brain capillaries and renal brush border membranes (BBMs). Irradiation of renal BBMs resulted in a dose-dependent loss of P-gp, which corresponded to a target size (TS) of 255 and 211 kDa, as detected by Western blot and [125I]arylazidoprazosin labeling, respectively. Similar TSs were determined for P-gp expressed in brain capillaries. These TSs correspond to approximately twice the size (120 kDa) of deglycosylated P-gp. Furthermore, the estimated TS for P-gp was not significantly different when renal BBMs were incubated with SDZ-PSC 833 (PSC) prior and during exposure to ionizing radiation. To confirm these results, the size of P-gp was evaluated from its mobility on blue-native polyacrylamide gels followed by Western blot analysis. Using this method, an apparent molecular size of 334 and 264 kDa was determined for P-gp in brain capillaries and renal BBMs, respectively. This corresponds to approximately twice the size of the glycosylated monomeric subunit of P-gp in brain capillaries (162 kDa) or renal BBMs (140 kDa). P-gp expressed in renal BBMs isolated from rats which had been treated daily with cyclosporin A (CsA) or PSC also migrated as a 264 kDa protein. These results suggest that P-gp exists mainly as a dimer in normal tissues and that resistance modulators such as CsA and PSC do not alter its oligomeric state.

Inhibition of the ATPase activity of P-glycoprotein by porphyrin photosensitization of multidrug-resistant cells in vitro.

The effectiveness of photodynamic therapy against P-glycoprotein ATPase activity in multidrug-resistant cells was studied. Chinese hamster ovary AUXB1 (drug-sensitive) and CR1R12 (multidrug-resistant) cell lines were compared with respect to uptake of 14C-polyhematoporphyrin and porphyrin photosensitization. Phototoxicity of Photofrin was similar in both cell lines, and no major differences in uptake or efflux of 14C-polyhematoporphyrin were observed. Porphyrin photosensitization in vitro of CR1R12 cells or isolated plasma membranes from these cells caused inhibition of P-glycoprotein ATPase activity. Application of porphyrin photosensitization at a sublethal level to CR1R12 cells resulted in a small but significant increase in adriamycin-induced cytotoxicity. The hydrophobic "picket-fence" porphyrin, meso-tetrakis-(o-propionamidophenyl)porphyrin, alpha,alpha,alpha,beta-isomer, was more inhibitory toward P-glycoprotein ATPase activity than the two less hydrophobic porphyrins tetraphenylporphine tetrasulfonate and Photofrin.