Spontaneous reversal of p-glycoprotein expression in multidrug resistant cell lines

Increased expression of P-glycoprotein encoded by the mdr-1 gene is a well-characterised mechanism for resistance to cancer chemotherapeutic drugs in cell lines. However, the P-glycoprotein expression after removal of the selection pressure has not fully been elucidated. The stability of P-glycoprotein expression in the presence (+) and absence (-) of vincristine (30 or 150 nM) was studied in multidrug resistant K562 cell lines (VCR30+, VCR150+, VCR30- and VCR150-) for 11 months. The P-glycoprotein protein and mdr-1 mRNA levels were determined at regular intervals using flow cytometry and real-time PCR, respectively. Chemosensitivity to a panel of antineoplastic drugs was measured using an MTT assay. The presence of vincristine (VCR30+ and VCR150+) resulted in high and stable levels of P-glycoprotein and mdr-1 mRNA during the whole period compared to wild type. As for the VCR30- and VCR150- subcultures, the expressions of P-glycoprotein and mdr-1 mRNA were stable for five months, and then the levels decreased rapidly. Concomitantly, the sensitivity to drugs known as P-glycoprotein substrates was restored. In conclusion, resistant cells growing in the presence of the inducing drug have a stable P-glycoprotein expression and resistance level, but removing the inducing drug may result in a sudden and rapid lowering of P-glycoprotein and mdr-1 mRNA levels as long as five months after drug withdrawal.     

KB细胞耐药株的建立及其耐药机制的探讨

用对长春新碱(VCR)敏感的KB细胞为亲本,通过诱变剂甲基磺酸乙酯刺激,然后在培养液中加入浓度递增的CVR,得到耐药细胞株KB_V200。此细胞株对VCR的耐受程度约为KB细胞的175倍。对其它抗肿瘤药物如紫杉醇、秋水仙碱和阿霉素等也有不同程度的交叉耐药性。进一步研究表明,KB_V200对³H-VCR的蓄积明显减少,且耐药基因(mdr1)表达增加。钙通道阻滞剂维拉帕米(Ver)可增加KB_V200对³H-VCR的蓄积和对VCR的敏感性。这些结果提示,KB_V200耐药的机制可能是由于mdr1基因表达增加,产生过量的p-糖蛋白,使药物外排增多所致。     

Relationship between expression of P-glycoprotein and efficacy of trifluoperazine in multidrug-resistant cells.

Tumor cell resistance due to enhanced efflux of drugs with diverse structures and/or mechanisms of action is termed multidrug resistance (MDR), and modulation of the MDR phenotype by calcium blockers or calmodulin inhibitors is suggested to involve P-glycoprotein. In drug-sensitive (S) and 5-fold doxorubicin (DOX)-resistant (R0) L1210 mouse leukemia cells, no obvious differences in mdr mRNA or P-glycoprotein expression or alterations in cellular uptake, retention, or cytotoxicity of vincristine (VCR) were observed. However, in the 10-fold (R1) and 40-fold (R2) DOX-resistant sublines, expression of P-glycoprotein was correlated with the level of resistance (R2 greater than R1). An RNase protection assay revealed that elevated levels of mdr1 and mdr2 mRNA were detected in R1 and R2 cells, with an additional increase in mdr3 mRNA in the R2 subline. Further, in the R1 and R2 sublines, no VCR dose-dependent cytotoxicity was apparent, and cell kill of greater than 40% was not achievable following a 3-hr drug exposure. Cellular uptake and retention of VCR were 2- to 4-fold lower in the R1 and R2 sublines, compared with similarly treated S or R0 cells. Potentiation of VCR cytotoxicity by a noncytotoxic concentration of 5 microM trifluoperazine (TFP) was greater than 2-fold in S and R0 cells and less than 1.3-fold in the R1 and R2 sublines. Modulation of VCR uptake by 5 microM TFP in the S and R0 cells was 2-fold and it was 4- to 7-fold in the R1 and R2 sublines. The presence of 5 microM TFP, by competing for efflux, enhanced VCR retention 1.5-fold in S and R0 cells and 2- to 4-fold in the R1 and R2 sublines. In contrast to these results with VCR, dose-dependent cytotoxicity of DOX was apparent in all the resistant sublines, and modulation of DOX cytotoxicity by 5 microM TFP was dependent on the level of resistance. Cellular accumulation of DOX was 20 and 50% lower in the R1 and R2 sublines, respectively, compared with similarly treated S or R0 cells. Marked increases (greater than 1.5-fold) in cellular accumulation of DOX by TFP were apparent only in the R2 subline. Results suggest that a relationship between overexpression of P-glycoprotein isoforms and their role in affecting cellular drug levels and consequent cytotoxicity in MDR L1210 cells determines resistance to VCR but not DOX.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin therapy restores impaired function and expression of P-glycoprotein in blood-brain barrier of experimental diabetes

We aimed to investigate effects of insulin on function and expression of P-glycoprotein (P-GP) in the blood-brain barrier of streptozotocin (STZ)-induced diabetic rats. Brain-to-plasma concentration ratio of vincristine (VCR) in rats was used as an indicator of in vivo function of P-GP. Western blot and quantitative real time-polymerase chain reaction were used to determine protein levels of P-GP and its mdr1a/mdr1b mRNA levels, respectively, in cerebral cortex of rats. In vitro effects of insulin on function and expression of P-GP in primarily cultured rat brain microvessel endothelial cells (rBMECs) were evaluated using rhodamine 123 (Rho123) uptakes and Western blot, respectively. The results showed that 3- and 5-week insulin treatment alleviated the impaired efflux function, expression and mdr1a/mdr1b mRNA levels of P-GP in cerebral cortex of diabetic rats. The 3- and 5-week insulin treatments also significantly enhanced P-GP levels and mdr1a/mdr1b mRNA levels in the cerebral cortex of normal rats. Addition of insulin to the insulin-deficient diabetic rat serum normalized the impaired function and expression of P-GP in rBMECs cultured in diabetic rat serum. When incubated with normal culture medium containing different levels of insulin, the rBMECs exhibited the enhanced P-GP levels and the reduced Rho123 uptake in a concentration-dependent manner. So we may conclude that appropriate level of insulin plays an important role in maintaining the normal function of BBB through regulating the function and expression of P-GP in the diabetic and normal rats.     

Overcoming vincristine resistance in cancer: Computational design and discovery of piperine‐inspired P‐glycoprotein inhibitors

P‐glycoprotein (P‐gp)/MDR‐1 plays a major role in the development of multidrug resistance (MDR) by pumping the chemotherapeutic drugs out of the cancer cells and reducing their efficacy. A number of P‐gp inhibitors were reported to reverse the MDR when co‐administered with chemotherapeutic drugs. Unfortunately, none has approved for clinical use due to toxicity issues. Some of the P‐gp inhibitors tested in the clinics are reported to have cross‐reactivity with CYP450 drug‐metabolizing enzymes, resulting in unpredictable pharmacokinetics and toxicity of co‐administered chemotherapeutic drugs. In this study, two piperine analogs ( 3 and 4 ) having lower cross‐reactivity with CYP3A4 drug‐metabolizing enzyme are identified as P‐glycoprotein (P‐gp) inhibitors through computational design, followed by synthesis and testing in MDR cancer cell lines over‐expressing P‐gp (KB Ch^R 8–5, SW480‐VCR, and HCT‐15). Both the analogs significantly increased the vincristine efficacy in MDR cancer cell lines at low micromole concentrations. Specifically, 3 caused complete reversal of vincristine resistance in KB Ch^R 8–5 cells and found to act as competitive inhibitor of P‐gp as well as potentiated the vincristine‐induced NF‐KB‐mediated apoptosis. Therefore, 3 ((2E,4E)‐1‐(6,7‐dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)‐5‐(4‐hydroxy‐3‐methoxyphenyl)penta‐2,4‐dien‐1‐one) can serve as a potential P‐gp inhibitor for in vivo investigations, to reverse multidrug resistance in cancer.     

Glutathione depletion upregulates P‐glycoprotein expression at the blood‐brain barrier in rats

Objectives Glutathione (GSH) depletion has been implicated in the pathogenesis of neurological diseases. During GSH depletion, cells of the blood‐brain barrier are subjected to chronic oxidative stress. Using an in‐vivo system, we have investigated whether glutathione depletion changed expression of P‐glycoprotein at the blood‐brain barrier in rats. Methods Diethyl maleate was intraperitoneally injected to induce GSH depletion in rats. P‐glycoprotein expression at the blood‐brain barrier was examined by Western blotting and RT‐PCR, and its function was assessed by measuring the brain‐to‐plasma concentration ratios (Kp values) of rhodamine 123 (Rh123). Evans Blue dye was used as a blood‐brain barrier indicator for examining the extravasation from the blood to the brain. Key findings Four hours after treatment of rats with diethyl maleate, the brain GSH content significantly reduced. The mdr1a mRNA expression at the blood‐brain barrier was upregulated, whereas no significant change in mdr1b mRNA expression was found. The P‐glycoprotein level was significantly increased compared with control rats. At the same time, the Kp values of Rh123 suggested that function of P‐glycoprotein was significantly enhanced at the blood‐brain barrier in rats with GSH depletion induced by diethyl maleate. No significant difference of the Evans Blue dye concentration in the brain cortex was found between GSH depletion rats and control rats. Treatment of rats with N‐acetylcysteine decreased P‐glycoprotein upregulation induced by diethyl maleate. Conclusions The oxidative stress induced by GSH depletion played a positive role in the regulation of function and expression of P‐glycoprotein at the blood‐brain barrier in rats.     

Modulation of P-glycoprotein expression in hyperthyroid rat tissues.

P-glycoprotein (Pgp) is expressed in various normal tissues and plays an important role in drug absorption and disposition. In addition, it is supposed that alterations in the expression levels of Pgp are involved in the inter- and intraindividual variability of pharmacokinetics of many drugs. Since pharmacokinetic properties of various drugs are altered in patients with thyroid disorders, we examined the expression of Pgp and mdr1a/1b mRNA in the kidney, liver, jejunum, and ileum from euthyroid and hyperthyroid rats. Western blot analysis revealed that Pgp expression was markedly increased in the kidney and liver of hyperthyroid rats. In contrast, it was slightly increased in the jejunum and ileum. mdr1a/1b mRNA levels were significantly increased in the kidney of hyperthyroid rats. However, they were not increased in the liver as well as in the jejunum and ileum of hyperthyroid rats. Expression levels of bile salt export pump and mdr2 mRNA were also unchanged in hyperthyroid rat liver. Taken together, these findings suggest that thyroid hormone induces Pgp expression in a tissue-selective manner, and that the modulation of mdr1a/1b mRNA expression in the hyperthyroid state varies among tissues.     

Combined treatment of P-gp-positive L1210/VCR cells by verapamil and all-trans retinoic acid induces down-regulation of P-glycoprotein expression and transport activity.

The development of the most common multidrug resistance (MDR) phenotype associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells may result in more than one hundred fold higher resistance of these cells to several drugs. L1210/VCR is a P-gp-positive drug resistant cell line in which P-gp overexpression was achieved by repeated cultivation of parental cells with a stepwise increasing concentration of vincristine. Relatively little is known about regulation of P-gp expression. Therefore, serious efforts have been made to recognize all aspects involved in regulation of P-gp expression. Retinoic acid nuclear receptors are involved in regulating expression of a large number of different proteins. Several authors have described that all-trans retinoic acid (ATRA, ligand of retinoic acid receptors, RARs) may induce alterations in P-gp expression and/or activity in drug resistant malignant cell lines. There are also other nuclear receptors for retinoids--retinoid X receptors (RXRs)--that may be involved in the development of the P-gp-mediated MDR phenotype. The topic of the present paper is a study of the relationship, if any, between the regulatory pathways of nuclear receptors for retinoids and P-glycoprotein expression. Increased levels of mRNAs encoding the retinoic acid nuclear receptors RARalpha and gamma, as well as decreased levels of the mRNAs encoding RARbeta and the retinoid X receptor RXRgamma or slightly decreased levels of RXRbeta mRNA, were observed in L1210/VCR cells in comparison with parental L1210 cells. Neither L1210 cells nor L1210/VCR cells contained measurable amounts of mRNA encoding the RXRalpha receptor. ATRA did not influence the viability of L1210/VCR cells differently from L1210 cells. A combined treatment of L1210/VCR cells with vincristine (1.08 micromol/l) and ATRA induced slightly higher cell death than that observed with ATRA alone. When applied alone, ATRA did not influence P-gp expression (monitored by anti P-gp antibody c219 using western blot analysis) or transport activity (monitored by use of calcein/AM as a P-gp substrate by FACS) in L1210/VCR cells. In contrast, when ATRA was applied together with verapamil (an often used P-gp inhibitor), a significant decrease in P-gp expression and transport activity were observed. However, no significant differences in [11, 12-(3)H]-ATRA uptake were observed in either sensitive or resistant cells, in the latter case in the absence or presence of vincristine. Moreover, verapamil did not influence ATRA uptake under any conditions. Thus, we can conclude that the combined treatment of L1210/VCR cells with ATRA and verapamil is able to depress P-gp expression, and consequently its activity. ATRA is not a P-gp-transportable substance, and thus this effect could not be attributed to verapamil-induced inhibition of P-gp that would allow ATRA to reach retinoic acid nuclear receptors and activate them.     

Using real-time quantitative TaqMan RT-PCR to evaluate the role of dexamethasone in gene regulation of rat P-glycoproteins mdr1a/1b and cytochrome P450 3A1/2

Cytochromes P450 (CYPs) and p-glycoproteins (Pgps) are believed to play important roles in drug absorption, metabolism, and elimination. Numerous drugs and environmental chemicals can modulate expression of these two classes of genes in different species. The present study investigated the effect of dexamethasone (Dex) on gene expression on both message and protein levels of mdr1a, mdr1b, CYP3A1, and CYP3A2 in small intestine, colon, liver, kidney, and brain microvessels of the rats treated orally with Dex at 1 or 20 mg/kg/day for 3 days. The basal expression of mdr1a mRNA was highest in the brain microvessels followed by colon, small intestine, liver, and kidney, and mdr1b mRNA was highest in the brain microvessels followed by kidney, liver, colon, and small intestine. After Dex treatment, mdr1a mRNA was increased by 5.5- and 10.7-fold in the small intestine, decreased extensively by 85-90% in the liver, and showed little or no change in the colon, kidney, and brain microvessels compared to the control rats. A similar pattern was observed for mdr1b mRNA. CYP3A1 mRNA was increased in all tissues examined. CYP3A2 mRNA was not significantly changed with the exception that at 20 mg/kg CYP3A2 mRNA was increased 5- and 30-fold in the colon and kidney. In general, Western blot analyses were consistent with mRNA changes. CYP3A protein expression was increased in all tissues examined. The disparity of the impact of Dex on the CYP 3A and Pgp expression in these studies suggest that the regulation of Pgp expression is very complex and is difficult to predict solely based on the PXR response to xenobiotics.     

Effect of endogenous multidrug resistance 1 and P‐glycoprotein expression on anticancer drug resistance in colon cancer cell lines

P‐glycoprotein (P‐gp, multidrug resistance 1 (MDR1)) overexpression confers multidrug resistance to cancer cells, and P‐gp in cell lines transfected with MDR1 or selected with chemotherapeutics significantly affect the anticancer drug efficacy. Although human cancer cell line panels consisting of defined tumor cell lines expressing endogenous P‐gp have been used to screen drugs in pharmaceutical industries, endogenous P‐gp affecting in vitro anticancer drug efficacy is unclear. The impact of P‐gp expression on anticancer drug efficacy was assessed by using five colon cancer cell lines expressing varying endogenous P‐gp levels and by selecting from the Cancer Cell Line Encyclopedia (CCLE). mRNA expression of MDR1 was considered as a surrogate of the protein expression of its gene product, P‐gp, in CL‐11, C2BBe1 and RKO cells, whereas P‐gp protein expression in plasma membranes or crude membrane fractions was lower than expected from mRNA expression in CW‐2 and CL‐40 cells. The EC₅₀ of paclitaxel and vinorelbine decreased in the presence of a P‐gp inhibitor in CW‐2 and CL‐11 cells that highly express P‐gp. No significant alterations in EC₅₀ were observed in the CL‐40, C2BBe1 and RKO cells, which show lower P‐gp expression. Accordingly, the apparent in vitro efficacy of anticancer drugs could be underestimated if the endogenous P‐gp expression is higher than in CL‐11 cells. The effect of P‐gp needs to be carefully evaluated in cell lines that highly express P‐gp, which account for 1.5% of cancer cell lines, including all cancer types, and 14.5% of colon cancer cell lines in CCLE, considering the protein expression levels in plasma membranes.     

High cell density-dependent resistance and P-glycoprotein-mediated multidrug resistance in mitoxantrone-selected Chinese hamster cells.

Mitoxantrone (MIT) resistance has been studied in a colony selected from the CHO AA8 parental line in one step under a low degree of selective pressure (9 nM). The cells of the clonal isolate AA8/MIT C1(0) were sensitive to 9 nM MIT at low cell density but able to grow at high density. Parental AA8 cells were not able to grow under the latter condition. Decreased MIT accumulation (-20%) was observed at this step (step 0) in the absence of overexpression of mdr RNA coding for the drug efflux pump P-glycoprotein. Furthermore, AA8/MIT C1(0) did not exhibit cross resistance to vincristine, Adriamycin and etoposide at low cell density. During subsequent controlled growth for 2 months at high cell density in the presence of 9 nM drug, an additional selection occurred leading to a 4-fold MIT-resistant subline AA8/MIT C1(+). This subline was characterized at this step (step I) and after an additional 4 months of culture in the presence of 9 nM MIT (step II). Analysis of mdr gene expression and gene copy number showed an increase in mdr RNA and a pattern of mdr gene amplification which changed between step I and II. AA8/MIT C1(+)II exhibited a classical multidrug resistance phenotype with decreased accumulation of [14C]MIT and cross-resistance to vincristine, Adriamycin and etoposide. The ability to form the cleavable complex in the presence of etoposide in DNA topoisomerase II-containing nuclear extracts was identical in AA8/MIT C1(+)II and AA8 cell lines. These results demonstrate a new sequence of events in MIT resistance: low level of drug resistance at high cell density followed by mdr gene amplification.     

盐酸小檗碱与环孢素A合用对大鼠肝P450同工酶和mdr1的影响

目的　阐明盐酸小檗碱 (berberinechloride ,Ber)及其与环孢素A (cyclosporin ,CsA)合用对大鼠肝脏P4 5 0同工酶和多药耐药基因的影响。方法　采用分光光度法测定大鼠肝微粒体红霉素N 脱甲基酶 (ERD)、氨基比林N 脱甲基酶(ADM )的活性 ,采用RT PCR法测定大鼠肝脏CYP3A1、CYP1A1、CYP2E1、mdr1a和mdr1b基因的水平。结果　灌胃给药 6d后 ,除外 10 0mg·kg-1Ber组 ,其余各组对ERD活性有明显的抑制作用 ;给药 12d时 ,所有用药组对ERD活性均有抑制作用。给药 6d后 ,CsA单用组、Ber与CsA合用组对ADM均有抑制作用 ;给药 12d后 ,除了 15 0mg·kg-1酮康唑组和 10 0mg·kg-1Ber组外 ,其余各用药组对ADM均有抑制作用。给药 12d时 ,除了 10 0mg·kg-1Ber组外 ,其余各用药组对大鼠肝脏CYP3A1、CYP2E1、mdr1a、mdr1b基因均有抑制作用。各组的CYP1A1基因均未能检出。结论　抑制CYP3A基因的表达及酶的活性 ,抑制mdr1a、mdr1b基因的表达 ,从而减少CsA在肝脏的代谢及消除 ,可能是Ber增加CsA血浓度的重要机制     

LY294002对多药耐药急性白血病细胞株HL60/VCR的凋亡及细胞周期的影响研究

目的:研究磷脂酰肌醇-3-激酶(PI3K)抑制剂LY294002对多药耐药急性白血病细胞株HL60/VCR凋亡与细胞周期的影响及可能机制。方法:取耐长春新碱(VCR)的HL60/VCR多药耐药急性白血病细胞株分为未加药的对照组及加入不同浓度的LY294002(其终浓度为1.0、2.5、5.0、10μmol.L-1)组,MTT法检测2种细胞的半数抑制浓度(IC50),流式细胞仪检测细胞凋亡及细胞周期分布,半定量逆转录-聚合酶链反应(RT-PCR)检测抗凋亡蛋白Bcl-2、细胞周期调控分子CyclinD1mRNA表达。结果:与对照组比较,LY294002可明显降低HL60/VCR对VCR的IC50(P<0.05),且与剂量相关;LY294002可显著增加HL60/VCR细胞凋亡率,阻滞细胞于G0/G1期,降低HL60/VCR细胞的Bcl-2、CyclinD1mRNA的表达(P均<0.05)。结论:LY294002可能是通过影响Bcl-2及CyclinD1基因的表达,从而促进HL60/VCR细胞凋亡及抑制细胞增殖。     

抑制PI3K/PKB信号通路提高胃癌细胞化疗敏感性的研究

目的探讨磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)抑制剂LY294002提高胃癌耐药细胞SGC7901/VCR和亲代细胞SGC7901对化疗药物敏感性的作用及其机制。方法MTT法分别检测胃癌细胞SGC7901和SGC7901/VCR对化疗药长春新碱(VCR)的敏感性;RT-PCR法和免疫细胞化学法检测LY294002处理前后多药耐药蛋白1(multidrug resistance protein-1,MDR1)和X染色体连锁的凋亡抑制蛋白(X-linked inhibitor of apoptosis,XIAP)基因和蛋白水平;Westernblot法检测LY294002处理前后细胞PKB总蛋白水平和磷酸化水平;并用流式细胞仪检测细胞的凋亡率。结果2×10-5mol·L-1 LY294002能明显增加SGC7901和SGC7901/VCR细胞对VCR的敏感性(P<0.01),其IC50分别由(0.20±0.03)和(8.09±0.60)mg·L-1降至(0.05±0.006)和(1.70±0.20)mg·L-1;降低细胞MDR1和XIAP的基因和蛋白水平;降低磷酸化PKB水平,而对其总蛋白水平无影响;LY294002联合VCR用药后细胞凋亡率明显高于单独VCR处理(P<0.01)。结论LY294002通过降低耐药基因MDR1和抗凋亡基因XIAP的表达,提高耐药和非耐药胃癌细胞对化疗药物的敏感性,此过程与抑制PI3K/PKB通路密切相关。     

维拉帕米及艾灸对长春新碱耐药胃癌细胞株SGC7901逆转增效作用

目的 观察维拉帕米(VPM)及艾灸对小鼠长春新碱(VCR)耐药SGC7901胃癌移植后的瘤体重量、抑瘤率及P-糖蛋白(P-gp)表达的影响,以了解两者对长春新碱耐药逆转增效作用.方法 分别进行不同剂量维拉帕米及艾灸联合不同剂量维拉帕米抗肿瘤耐药的实验.第一组实验将SD小鼠分成等渗盐水组、VCR对照组、VPM对照组、0.5 mg/kg VPM+VCR组、1.0 mg/kg VPM+VCR组、1.5 mg/kg VPM+VCR组、2.0 mg/kg VPM+VCR组,,每组16只,雌雄各半,所有模型组小鼠在左前肢腋下接种瘤组织,按组给予等渗盐水和不同剂量药物,15 d后,取出瘤体称重,计算抑瘤率,同时留取瘤体标本,检测多药耐药(MDR)相关蛋白P-gp的变化.第二组实验将SD小鼠分成等渗盐水组、VCR对照组、VPM对照组、艾灸对照组、0.5 mg/kg VPM+VCR+艾灸组、1.0 mg/kg VPM+VCR+艾灸组、1.5 mg/kg VPM+VCR+艾灸组、2.0 mg/kg VPM+VCR+艾灸组,每组16只,雌雄各半,所有模型组小鼠在左前肢腋下接种瘤组织,按组给予等渗盐水和不同剂量药物及艾灸,15 d后,取出瘤体称重,计算抑瘤率,同时留取瘤体标本,检测MDR相关蛋白P-gp的变化.结果 第一组实验中给予维拉帕米时,等渗盐水组与不同剂量药物组间差异有统计学意义(P<0.05),随剂量增加瘤体重量下降、抑瘤率增加和蛋白P-gp表达下降,但1.5 mg/kg和2.0 mg/kg时,蛋白P-gp值差异无统计学意义.第二组实验中给予维拉帕米和艾灸时,等渗盐水组与不同剂量药物组间差异也有统计学意义(P<0.05),随剂量增加瘤体重量下降、抑瘤率增加和蛋白P-gp表达下降,其中剂量组:1.5 mg/kg和2.0 mg/kg与仅给予维拉帕米组相比差异有统计学意义(P<0.05).结论 维拉帕米和艾灸对小鼠长春新碱耐药SGC7901胃癌耐药具有逆转增效作用,在2.0 mg/kg维拉帕米、0.5 mg/kg长春新碱、艾灸联合用药时可达到单用1.0 mg/kg长春新碱用药效果.     

Evaluation of a vincristine resistant Caco-2 cell line for use in a calcein AM extrusion screening assay for P-glycoprotein interaction.

AIM: To develop a fast fluorometric screening assay based on vincristine resistant Caco-2 cells (Caco-2VCR) in order to elucidate potential P-glycoprotein (Pgp) interactions of compounds, and to characterise Caco-2VCR cells with regard to their expression of the efflux transporters Pgp, MRP1 and MRP2. METHODS: We applied the Caco-2VCR cells to a 96-well plate-based calcein AM extrusion assay. The Caco-2VCR cells were cultured as monolayers and incubated with calcein AM with/without addition of Pgp modulators. Fourteen known Pgp modulators were tested in the assay (chloropromazine, cyclosporin A, domperidone, digoxin, ivermectin, ketoconazole, loperamide, metoprolol, propranolol, progesterone, quinidine, quinine, verapamil and vincristine). For each compound an EC50 value was calculated. Protein and mRNA levels of the efflux transporters were analysed by Western blot and polymerase chain reaction techniques. RESULTS: All compounds with the exception of digoxin displayed increased calcein levels. Protein and mRNA analysis showed increased levels of Pgp after vincristine exposure, while expression of the efflux transporters MRP1 and MRP2 remained unchanged. CONCLUSIONS: The calcein AM extrusion assay applied to Caco-2VCR cells can be a valuable tool as a screening assay for new compounds and their potential interaction with P-glycoprotein.     

mdr1反义寡核苷酸增强多药耐药肝癌细胞HepG2/ADM化疗敏感性的实验研究

目的：探讨在体外实验条件下mdr1反义寡核苷酸对多药耐药肝癌细胞株化疗敏感性的影响。方法以肝癌细胞HepG2/ADM为研究对象,设立mdr1反义寡核苷酸组和空白试剂组作对照,利用脂质体包载肿瘤耐药基因mdr1的反义寡核苷酸进行细胞转染,通过反转录聚合酶链反应（RT-PCR）、免疫印迹实验（Western blotting）分别检测mdr1基因mRNA和P-gp蛋白表达,通过MTT实验检测细胞转染前后对阿霉素（ADM）、顺铂（DDP）和5-氟尿嘧啶（5-FU）的化疗敏感性。结果 HepG2/AMD肝癌细胞经反义寡核苷酸处理后,mdr1 mRNA、P-gp蛋白表达水平均明显降低,对ADM、DDP 和5-FU的化疗敏感性明显增强。结论反义寡核苷酸能在体外有效增加肝癌细胞HepG2/ADM对化疗药物的敏感性。