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In this study, cepharanthine hydrochloride (CH) was tested for its potential ability to modulate the expression and function of P-glycoprotein (P-gp) in the multidrug-resistant human chronic myelogenous leukemia cell line K562/ADR. Cytotoxicity of adriamycin (ADR) alone or in combination with CH or verapamil (VER) in K562 and K562/ADR cells was determined by MTT assay. Based on flow cytometric technology, the effect of CH or VER on the uptake and efflux of rhodamine123 (Rho123) and the accumulation of ADR in these cells was detected by measuring Rho123 or ADR-associated mean fluorescence intensity (MFI). The effects of CH and VER on P-glycoprotein (P-gp) expression in K562 and K562/ADR cells were also measured using a flow cytometry with PE-conjugated P-glycoprotein antibody. The results show that CH significantly enhanced the sensitivity of K562/ADR cells to ADR, 4 micromol x L(-1) of CH enhanced the sensitivity of K562/ADR cells to ADR by 7.43 folds, the reversal activity was 3.19 times higher than that of verapamil. However, CH had no effect on drug-sensitive K562 cells (P < 0.05). CH increased Rho123 and ADR accumulation in a concentration-dependent manner (2-8 micromol x L(-1)) and inhibited the efflux of Rho123 from these cells, but did not affect the accumulation and efflux of Rho123 from the wild-type drug-sensitive K562 cells. The inhibition effect of CH on P-gp expression in K562/ADR cells is in a time- and concentration-dependent manner. The reversal activity of CH is possibility related to inhibition of P-gp function and expression, which lead to an increased intracellular accumulation of anticancer drugs.

An in vitro P-glycoprotein mediated drug biliary excretion model (B-Clear model) was developed and validated using sandwich-cultured rat hepatocytes (SCRH) and a model substrate rhodamine 123 (Rh123). SCRH formed functional bile canalicular networks after 5 days of culture. Rh123 (10 micromol x L(-1)) was then incubated with the SCRH in standard Ca+ Hanks buffer or Ca(2+)-free buffer. The cumulative cell uptake and canalicular efflux of Rh123 under Ca2+ and Ca(2+)-free conditions were measured with a LC-MS/MS method. The biliary excretion index (BEI) and instinct biliary clearance (CL(bile, int)) were calculated. To assess the effect of known P-gp inhibitors on the efflux of Rh123, cyclosporine A (CyA), tariquidar (TQD) or quinidine (QND) (10, 50 and 100 micromol x L(-1)) was pre-incubated separately with SCRH for 30 min, then co-incubated with Rh123. The BEI and CL(bile, int) of Rh123 obtained from the SCRH model were (17.8 +/- 1.3) % and (10.7 +/- 0.9) mL x min(-1) x kg(-1), respectively. All the three P-gp inhibitors showed a dose-dependent inhibition on the bile clearance of Rh123, indicating that the B-Clear model with SCRH was functional properly. The biliary excretion of loperamide (LPAD) and the role of P-gp were further investigated with this validated model. The BEI and CL(bile, int) for LPAD (20 micromol x L(-1)) were obtained after it was incubated with SCRH for 30 min, and found to be (12.9 +/- 1.2)% and (6.1 +/- 0.3) mL x min(-1) x kg(-1) respectively. The dose-dependent inhibition on LPAD biliary excretion by CyA, TQD or QND confirmed the major role of P-gp in LPAD canalicular efflux. The results suggested that the B-Clear model with SCRH would be a useful tool for evaluation of P-gp mediated efflux and drug-drug interaction.

Objective: To analyze the relationship between P-glycoprotein function in peripheral blood cells and primary multidrug resistance in breast carcinoma., Methods: P-gp function was investigated by flow cytometry in NK cells of 16 breast cancer patients treated with anthracyclines and taxanes. Among all the patients, 8 were in chemotherapy-sensitive group and 8 in chemotherapy-resistant group. P-gp function was determined by rhodamine 123 (Rh123)-ejection test. Mathematical model was established by a regression of the fluorescence-time curve. The efflux rate constants of the chemotherapy-sensitive and -resistant groups were compared., Results: There was no significant difference of Rh123 accumulation, retention or efflux between the two groups. The mathematical model of F(t) = F(0) · e(-kt) was established. K was the efflux rate constant, which was significantly different between the chemotherapy-sensitive and -resistant groups (P = 0.025). When k > 3.9 was used as diagnostic criterium for primary resistance, the sensitivity, specificity and accuracy were 75.0%, 100% and 87.5%, respectively., Conclusion: P-glycoprotein function in peripheral blood cells is associated with primary multidrug resistance in breast carcinoma. The efflux rate constant may be a good predictor for chemotherapy sensitivity.

Objective: To investigate the effect of liquorice in functional modulation of intestinal P-glycoprotein (P-gp) in rats., Methods: An in vitro diffusion chamber system (Ussing chamber) was used to examine the direct effect of liquorice decoction on rhodamine 123 (a subtrate of P-gp) transport and evaluate the permeability of rhodamine 123 or fluorescein sodium through rat jejunum membranes after oral administration of liquorice decoction., Results: Direct application of liquorice decoction did not obviously affect rhodamine 123 transport across the intestinal mucosa. Oral administration of liquorice decoction (10 g/kg, twice daily for a week) significantly increased the absorption of rhodamine 123 and also enhanced rhodamine 123 secretion across the jejunum mucosa. Liquorice had no obvious effect on the transport of CF across the jejunum mucosa., Conclusion: Liquorice may slightly inhibit P-gp function in the intestinal mucosa to increase the intestinal absorption of rhodamine 123.

Aim: To investigate the reversion of multidrug resistance of drug-resistant hepatocellular carcinoma (HCC) cell line BEL-7402/FU by sorafenib and the possible mechanisms., Methods: Flow cytometry was applied to detect the expression of rhodamine 123 (Rho123), immunohistochemistry was applied to observe the expression of P-glycoprotein (P-gp) on the cell membrane, the fluorescence quantitative PCR assay was applied to determine the changes of the multidrug-resistant gene mdr1 mRNA level and the Western blot assay was applied to test the changes of the expression level of the mdr1 gene product P-gp., Results: After 4 micromol/L treatment by sorafenib, Rho123 accumulation was increased by BEL-7402/FU and exocytosis was slowed down, which evidently reduced the expression of cell surface P-gp; the mdr1 mRNA level was reduced by 35.4% compared to its level prior to drug administration; the expression level of the mdr1 gene product P-gp was significantly suppressed, 14.3% fewer than that of BEL-7402/FU cells., Conclusion: Sorafenib can inhibit the expression of the mdr1 gene product P-gp and increase the intracellular concentration of chemotherapeutic drugs, so as to reverse the multidrug resistance of HCC cells.

Objective: To investigate the effect of Tween-80 in modulating rhodamine123 (R123) permeability across the intestinal membranes., Methods: The permeability of R123 or fluorescein sodium (CF) across isolated rat intestinal membranes at the concentration of 5 microg/ml was evaluated using an in vitro diffusion chamber system, in the presence or absence of Tween-80 at different low concentrations. The concentration of R123 or CF in the receptor chamber was determined using fluorospectrophotometry., Results: The intestinal membrane permeability of R123 gradually decreased from the jejunum to the ileum and then to the colon. The serosal-to-mucosal transport of R123 was much greater than its mucosal-to-serosal transport. In the presence of low concentrations of Tween-80, the absorptive transport of R123 was significantly increased while its secretory transport decreased depending on the concentration of Tween-80. However, Tween-80 at the experimental concentrations was found to obviously affect the CF transport across the intestinal membranes., Conclusion: Low concentrations of Tween-80 may promote the absorption of P-gp-mediated drugs and therefore improve the oral bioavailability of these drugs.

Cell lines of Bcap37 and Bcap37/MDR1 (the high P-glycoprotein (P-gp) expressing cell line) were used as model to investigate the different accumulations of (E)-2-(4-(diethylamino methyl) benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one (BYZX) in the two kinds of cells. It was authenticated that whether BYZX was the substrate of P-gp. Meanwhile, the inhibitive effects of BYZX on the P-gp were investigated by determining the fluorescence intensity of rhodamine 123 in the model cells, with and without BYZX. A reversed-phase high-performance liquid chromatography (RP-HPLC) method was used to determine the accumulations of BYZX in the two cells. The results showed that the amount of BYZX accumulation in Bcap37/MDR1 cells were as many as those in Bcap37 cells (P > 0.05), and the concentrations of BYZX accumulated in the Bcap37/MDR1 cells did not increase when co-incubated with P-gp inhibitor verapamil. Furthermore, different concentrations of BYZX also had no effects on the efflux of rhodamine 123 (P > 0.05). These results indicated that there were no interactions between BYZX and P-gp. BYZX will not be pumped out of the cells, and it also not inhibited the P-gp. It was the useful advantage for its absorption.

Objective: To study the toxicological effects of copper on human intestinal Caco-2 cells., Methods: The effects of copper on human intestinal Caco-2 cells were evaluated by MTT conversion, intra-cellular production of reactive oxygen species (ROS), cloning efficiency and changes of P-glycoprotein (P-gp) activities in Caco-2 cells. In addition, Caco-2 cells and Salmonella enteritidis served as the models to examine the effect of copper on the host-parasite interaction., Results: Copper induced a dose-dependent decrease of cell viability measured by MTT assay; by using the fluorescent probe 2, 7-dichlorofluorescin diacetate, a significant increase of ROS production in Cu-treated cells was detected; the significant time- and dose-dependent decrease of P-gp activities in Cu-treated cells was demonstrated by increased accumulation of rhodamine 123 in cells. Copper treatment also increased the efficiency of the bacterial invasion, but decreased the number of bacteria surviving in the intracellular environment., Conclusion: Copper induced a variety of toxicological endpoints which might be caused by oxidative damage, but the effect of copper on cell-bacteria interaction needs to be further investigated.

Aim: To study the effect of lomerizine (Lom) on the reversal of multidrug resistance (MDR) in K562/ADM cells and its mechanism., Methods: MTT assay was used to determine the influence of Lom on the cytotoxicity of adriamycin (ADM). The effect of Lom on the apoptosis induced by ADM and vincristine (VCR) in K562/ADM cells was detected using flow cytometry. Intracellular accumulation of ADM was measured by fluorescence spectrophotometry. Flow cytometry was used to investigate the efflux of rhodamine 123 (Rh123) and the expression of P-glycoprotein (P-gp) in K562/ADM cells., Results: Lom increased the cytotoxicity of ADM and the apoptosis induced by ADM or VCR in K562/ADM cells. At the concentration of 3, 10 and 30 micromol x L(-1), Lom reduced the IC50 value of ADM from 79.03 micromol x L(-1) to 28.14, 8.16 and 3.16 micromol x L(-1), respectively. Lom increased the intracellular accumulation of ADM and inhibited the efflux of Rh123 in K562/ ADM cells. No change in P-gp expression was observed after the treatment of Lom for 72 h., Conclusion: Lom had strong reversal effect on MDR in K562/ADM cells by inhibiting P-gp function.

Objectives: To investigate the reversal effect of gene MDR1 and MRP with combinational antisense phosphorothioate oligonucleotide on Drug-resistant human hepatocellular carcinoma cells SMMC-7721/ADM., Methods: SMMC-7721/ADM was transfected with synthetic antisense phosphorothioate oligonucleotides complementary to gene MDR1 and MRP mediated by Lipofectamine. Drug sensitivity was measured by MTT assay, Fluorescence intensity of cells was determined by flow cytometric analysis, RH123 and DNR retention was assayed by confocal scanning laser microscopy., Results: ASODN of MDR1+MRP increased the sensitivity of SMMC-7721/ADM to chemotherapeutic drug more significantly than that any of MDR1 and MRP did separately. But they did not enhance the inhibition expression of protein of p190 or p170., Conclusion: Drug-resistance could be reversed significantly when antisense phosphorothioate oligonucleotide of MDR1+MRP were transfected into drug-resistant human hepatocellular carcinoma cells SMMC-7721/ADM together.

To explore Rh123 ion concentration effect of "Bushen huayu jiedu fang" (BSHYJD) on drug-resistance lung cancer cells, tumor-bearing animal drug-serum pharmacology and flow cytometry method were utilitied. In contrast with normal animal drug serum, BSHYJD drug-serum of tumor-bearing animal would markedly increase Rh123 concentration in drug-resistance lung cancer cells(P < 0.05), but which hasn't marked discrepancy in contrast with normal animal drug-serum(P > 0.05), that indicated BSHYJD drug-serum has increased drug concentration in drug-resistance lung cancer cells.

The cyproheptadine (CYP) reversal of multidrug resistance (MDR) and its mechanism in KBV200 cell line were studies. MTT assay showed that CYP 15.0 mumol.L-1 could reverse vincristine, adriamycin (ADR) and etoposide resistance in KBV200 cells by a factor of 5.5, 2.0 and 1.9, respectively. CYP appeared to have no influence on the cytotoxicity of 5-fluorouracil (5-FU) and melphalan (MEL) in the cells. These results indicate that CYP is a MDR reversing agent. CYP 15.0 mumol.L-1 increased the ADR accumulation in KBV200 cells from 0.68 +/- 0.03 microgram/10(6) cells to 1.36 +/- 0.08 micrograms/10(6) cells (P < 0.01). CYP 15.0 mumol.L-1 was shown to obviously increase rhodamine 123 (R123) accumulation in and decrease its efflux from the cells. There was no change in PGP dying intensity under immunocytochemical assay and in mdr1 RNA level through slot blot analysis in the KBV200 cells exposed continuously to CYP 15.0 mumol.L-1 for 72 h. These results suggest that CYP acts by inhibiting the pumping function of PGP.