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)     

Differential effect of the calmodulin inhibitor trifluoperazine in modulating cellular accumulation, retention and cytotoxicity of doxorubicin in progressively doxorubicin-resistant L1210 mouse leukemia cells. Lack of correlation between cellular doxorubicin levels and expression of resistance

Calmodulin inhibitors are effective in enhancing cytotoxic effects of doxorubicin (DOX) in DOX-resistant cells, possibly by enhancing cellular levels of drug. In the present study, L1210 mouse leukemia cells adapted to grow in vitro, in the presence of 0.025 to 0.25 microgram/ml DOX, and identified as L1210/DOX0.025, L1210/DOX0.05, L1210/DOX0.1, and L1210/DOX0.25 were approximately 5-, 10-, 20-, and 40-fold DOX resistant, respectively, compared to parent-sensitive cells (L1210/S). Using a soft agar colony assay and 3-hr drug exposure, the IC50 concentration of DOX in the progressively DOX-resistant (5- to 40-fold) L1210 cells ranged from 0.25 to 2.0 micrograms/ml and from 0.08 to 0.25 microgram/ml in the absence and presence of a non-cytotoxic concentration of 5 microM trifluoperazine (TFP) respectively. Further, based on the observed in vitro cytotoxic response, the IC50 concentration of DOX in the presence of 5 microM TFP was 2.5-, 4-, 6.7- and 8-fold lower than DOX without 5 microM TFP in the L1210/DOX0.025, L1210/DOX0.05, L1210/DOX0.1, and L1210/DOX0.25 resistant sublines respectively. In contrast, the IC50 of DOX in L1210/S cells was approximately 0.05 microgram/ml with or without 5 microM TFP. Cellular accumulation of DOX was 15-50% lower in the progressively resistant L1210 sublines compared to similarly treated L1210/S cells. However, in the presence of 5 microM TFP, cellular accumulation of DOX in the L1210/DOX0.05 and L1210/DOX0.1 but not L1210/DOX0.25 was comparable to the L1210/S cells. Cellular retention of DOX in the absence or presence of 5 microM TFP was comparable in similarly treated L1210/S, L1210/DOX0.05 and L1210/DOX0.1 cells, and a 2-fold reduction in the retention of DOX in the absence versus the presence of 5 microM TFP was apparent only in L1210/DOX0.25 cells. At the IC50 of DOX in the presence of 5 microM TFP, although cellular accumulation of DOX was concentration dependent over the range of 1-20 microM TFP, enhancement in cytotoxicity of DOX was dose dependent at 1-5 microM TFP but not 5-20 microM TFP. In cells treated for 3 hr at the IC50 concentration of DOX alone or DOX plus 5 microM TFP, cellular accumulation of DOX was 7- to 14-fold and 2.5- to 3.5-fold higher, respectively, in resistant than in sensitive cells. Additionally, following treatment for 3 hr at the IC50 dose of DOX in the absence or presence of 5 microM TFP, drug retention at 3 hr was 4- to 6-fold and 1.5-fold higher, respectively, in the resistant versus sensitive cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anthracycline-induced DNA breaks and resealing in doxorubicin-resistant murine leukemic P388 cells

Energy-dependent drug efflux is believed to be a major factor in cellular resistance to doxorubicin (DOX). However, recent studies have shown that decreased retention alone cannot account for anthracycline resistance, and possibly other factors, such as drug metabolism, free radical scavengers, and altered DNA damage/repair, may be involved. We have measured DOX-induced DNA damage and its repair in P388 cells sensitive (P388/S) and resistant (P388/R) to DOX. Our studies show 2- to 5-fold less DNA damage, measured as protein-associated single-strand DNA breaks, in P388/R cells when compared to similarly treated P388/S cells. The repair of DNA in whole cells, expressed as percent DNA rejoined, was complete in 4 hr in P388/R, whereas no repair was seen in P388/S cells until 20 hr. No difference in repair of DNA lesions was observed when nuclei were used in repair experiments. The absence of repair in sensitive whole cells may be due to high retention or slow drug efflux. Increase of cellular DOX retention by exposure of cells to trifluoperazine (TFP) or verapamil (VPL) did not result in the increase of DNA damage in P388/R cells. DOX analogs, N-trifluoroacetyladriamycin-14-valerate (AD 32), 4'-O-tetrahydropyranyladriamycin (THP-adriamycin), and N-benzyladriamycin-14-valerate (AD 198), induced 2- to 4-fold more DNA damage than DOX in resistant cells. There was no difference in the poly(ADP-ribose) synthesis of P388/S and P388/R cells exposed to DOX or AD 32. Since ADP-ribose polymer synthesis is associated with free radical-induced DNA damage and is indicative of DNA repair by an excision-repair mechanism, data from these studies suggest that DNA breaks in anthracycline-exposed cells may not be due to free radical production but rather to other mechanisms, such as inhibition of DNA topoisomerase II activity. The present studies, in addition to emphasizing the role of DNA damage in resistance, also underscore the relative importance of DNA topoisomerase II function in anthracycline cytotoxicity.     

Overexpression of P-glycoprotein and alterations in topoisomerase II in P388 mouse leukemia cells selected in vivo for resistance to mitoxantrone.

The overexpression of P-glycoprotein (PGP) and alterations in DNA topoisomerase II (TOPO II) were evaluated in mouse leukemia P388 cells selected in vivo for mitoxantrone (MTT) resistance (P388/MTT) and compared to doxorubicin (DOX) resistant (P388/DOX) or vincristine (VCR) resistant (P388/VCR) models. Among a panel of TOPO II inhibitors which included etoposide (VP-16), DOX, MTT and 4'-[(9-acridinyl)-amino]methanesulfon-m-anisidide (m-AMSA), the relative resistance compared to parental sensitive P388/S cells was: P388/DOX greater than P388/MTT greater than P388/VCR. All the resistant sublines exhibited minimal cell kill (less than 20%) at vincristine concentrations greater than 100-fold the IC50 for P388/S cells. In a soft-agar colony-forming assay, the modulation of cytotoxicity in P388/MTT cells by the calmodulin inhibitor trifluoperazine following a 3-hr drug treatment demonstrated a marked potentiation in cell kill with MTT, VP-16, DOX and m-AMSA but not VCR. Immunoblotting data revealed that while PGP was not detectable in P388/S cells, the overexpression of PGP was apparent in P388/MTT cells and the relative expression between the resistant sublines was: P388/DOX greater than P388/MTT greater than P388/VCR. Although the amount and DNA cleavage activity of TOPO II in nuclear extracts from P388/VCR cells were comparable to those in P388/S cells, they were markedly lower in both P388/DOX and P388/MTT cells. However, decatenation activity of TOPO II in nuclear extracts was comparable between the sensitive (P388/S) and resistant sublines (P388/MTT, P388/DOX, and P388/VCR). Results from the present study demonstrated that P388 cells selected for resistance to mitoxantrone exhibit changes in TOPO II and overexpression of PGP similar to P388/DOX cells, while vincristine resistant cells only overexpress PGP. Since therapeutic strategies are primarily designed to interfere with PGP-mediated drug efflux, the choice of agents for modulating resistance in tumors which overexpress PGP versus tumors which overexpress PGP with altered TOPO II could be different.     

Glutathione S-transferases and glutathione peroxidases in doxorubicin-resistant murine leukemic P388 cells

Energy-dependent rapid drug efflux is believed to be a major factor in cellular resistance to doxorubicin (DOX). However, several recent studies have demonstrated that cellular DOX retention alone does not always correlate with its cytotoxicity and suggest that mechanisms other than rapid drug efflux may also be important. In the present study, we have compared glutathione (GSH) S-transferase (GST), selenium-dependent GSH peroxidase and selenium-independent GSH peroxidase II activities in DOX-sensitive (P388/S) and resistant (P388/R) mouse leukemic cells. The GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) was markedly higher in P388/R cells compared to P388/S cells. Purification of GST by GSH-affinity chromatography from an equal number of P388/S and P388/R cells revealed an increased amount of GST protein in P388/R cells. Immunological studies indicated that alpha and pi type GST isoenzymes were 1.27- and 2.2-fold higher, respectively, in P388/R cells compared to P388/S cells. Selenium-dependent GSH peroxidase activity was similar in both the cell lines, whereas selenium-independent GSH peroxidase II activity was approximately 1.36-fold higher in P388/R cells compared to P388/S cells. These results suggest that increased GSH peroxidase II activity in P388/R cells may contribute to cellular DOX resistance by enhancing free radical detoxification in this cell line.     

Anthracycline resistance in murine leukemic P388 cells. Role of drug efflux and glutathione related enzymes

Energy-dependent drug efflux is a major factor in cellular resistance of P388/R84 mouse leukemic cells to anthracyclines such as doxorubicin (DOX), and blocking of efflux increases sensitivity. However, efflux does not play a significant role in resistance to N-trifluoroacetyladriamycin-14-valerate (AD 32), a DOX analog. Since drug efflux alone cannot account for resistance to anthracyclines, we have, in the present study, measured cellular glutathione (GSH) content and activity of GSH cycle related enzymes to determine their role in resistance. Cellular GSH content was similar in DOX-sensitive and -resistant mouse leukemic cells (P388 and P388/R84). GSH peroxidase, glucose-6-phosphate dehydrogenase and glutathione reductase activities were 1.36-, 1.58- and 1.14-fold higher in P388/R84 cells. Incubation of P388/R84 cells with 100 microM buthionine-S,R-sulfoximine (BSO) for 24 hr reduced cellular GSH content to 6% of control and reduced their resistance to DOX [dose modification factor (DMF) 3.9]. GSH depletion had no significant effect on the cytotoxicity of AD 32 (DMF 1.5). Exposure of P388/R84 cells to BSO (for GSH depletion) and trifluoperazine (for efflux blocking) further reduced their resistance to DOX (DMF 14). These results indicate that DOX resistance in P388/R84 cells is multifactorial and that changes in GSH cycle related enzymes such as GSH peroxidase may also contribute to their resistance.     

Studies with 4'-deoxyepivincristine (vinepidine), a semisynthetic vinca alkaloid

The antitumor activity of 4'-deoxyepivincristine (vinepidine, VNP) was examined against a human rhabdomyosarcoma line, HxRh12, grown as a xenograft in immune-deprived mice. The efficacy of VNP was lower than that of vincristine (VCR) but far superior to that of vinblastine (VLB) in this model. After i.p. administration, accumulation of [G-3H]VNP in tumors was biphasic and progressive for at least 72 hr. In contrast, VCR and VLB achieved maximal tumor levels within 4 hr, after which the level of VCR was maintained but VLB levels decreased 3-fold by 72 hr. Analysis of tumor extracts by high performance liquid chromatography showed that at 72 hr after VNP injection 98% of the radiolabel chromatographed with parent compound. In normal tissues, VNP was cleared less rapidly than VCR or VLB, and analyses of tissue extracts suggested that VNP was less rapidly metabolized than VCR or VLB. This may account for why the potency of VNP is greater than that of VCR in mice.     

Effects of a new triazinoaminopiperidine derivative on adriamycin accumulation and retention in cells displaying P-glycoprotein-mediated multidrug resistance.

A new triazinoaminopiperidine derivative, Servier 9788 (S9788), was investigated for its ability to increase Adriamycin (ADR) accumulation and retention in two rodent (P388/ADR and DC-3F/AD) and three human (KB-A1, K562/R and COLO 320DM) cell lines displaying the P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) phenotype. Depending on the cell line S9788 was shown to be two to five times more active and five to 15 times more potent than Verapamil (VRP) in increasing ADR accumulation in resistant cells. ADR retention in KB-A1 cells maintained in a concentration of 10 microM S9788 was twice that in VRP-treated cells, and similar to that measured in the untreated sensitive KB-3-1 cells. Although 5 microM S9788 and 50 microM VRP gave the same values of ADR uptake in KB-A1 cells, S9788 was shown to induce a greater ADR retention following cell wash and post-incubation in resistance modifier- and ADR-free medium. Taking into account that S9788 had no effects on ADR accumulation and retention in sensitive KB-3-1 cells, it can be suggested that S9788 inhibits specifically the P-gp dependent ADR efflux, and in a manner less reversible than that observed with VRP. Moreover, [3H]azidopine photolabeling of P-gp, in P388/ADR plasma membranes, was completely inhibited by 100 microM S9788. Although S9788, as VRP, had no effect on the cell cycle of P388 cells, 5 microM S9788 increased 700-fold the efficacy of ADR to block P388/ADR cells in the G2+M phase of the cell cycle. Together, these results show that the sensitization, by S9788, of cell lines resistant to ADR is mainly due to an increase in ADR accumulation and retention, leading to an increase in the number of resistant cells blocked in the G2+M phase.     

Partial circumvention of P-glycoprotein-mediated multidrug resistance by doxorubicin-14-O-hemiadipate

Previously, we have reported partial circumvention ofP-glycoprotein (Pgp)-associated resistance to doxorubicin(Dox) in MCF7/R human breast carcinoma and P388/R murineleukemia cell lines by doxorubicin-14-O-hemiadipate (H-Dox)[Povarov L.S. et al. (1995) Russian J. Bioorganic Chemistry21: 797–803]. We felt that these changes were due toalterations in the cellular pharmacokinetics of the analog inmultidrug (MDR) resistant cells, as compared to that of Dox.To address this hypothesis, we performed comparative studiesof the accumulation, retention and intracellular localizationof H-Dox and Dox in Dox-sensitive murine leukemia cell lineP388/S and its Dox-selected, Pgp-positive drug resistantP388/R subline. These studies were performed in the presenceor absence of cyclosporin A (CsA), a competitive inhibitor ofPgp. Flow cytometric analysis revealed significant differencesin Dox and H-Dox accumulation in P388/R cells when compared toP388/S cells. In P388/R versus P388/S cells, there was a 38-fold decrease in Dox accumulation, but only a 5-fold decreasein H-Dox accumulation, indicating over a 7-fold increase in H-Dox buildup in resistant cells. CsA did not affect uptake orretention of either drug by sensitive cells. However,coincubation with CsA resulted in a 54-fold increase in Doxaccumulation and only a 5-fold increase in H-Dox uptake inP388/R cells, restoring anthracycline levels in P388/R to100% of that found in P388/S cells. Once internalized by theresistant cells, H-Dox was retained better than Dox regardlessof presence or absence of CsA. Confocal microscopic analysisrevealed the presence of H-Dox but no Dox in cellular nucleiof P388/R cells. Thus, increased activity of H-Dox towardP388/R cells was correlated with its enhanced ability to enterand be retained in these cells, and also with redistributionof H-Dox into the nuclei of the resistant cells as compared toDox. Overall, our findings support our initial hypothesis andprovide evidence that H-Dox, a 14-O-hemiadipate ofdoxorubicin, is affected by Pgp-mediated MDR to a lesserextent than parental Dox due to changes in the cellularpharmacokinetics of the analog.     

CQ11逆转多药耐药人胃癌细胞株对多柔比星的耐药

目的:探讨氯喹衍生物CQ11对耐长春新碱(vincristine,VCR)人胃癌多药耐药(multidrug resistance,MDR)细胞株SGC7901/VCR的耐药逆转作用。方法:将SGC7901和SGC7901/VCR细胞分别与各种浓度的多柔比星(doxorubicin,DOX)和/或CQ11在体外共同培养,采用MTT法检测其细胞毒作用;采用荧光分光光度计测定细胞内DOX蓄积量。结果:SGC7901/VCR细胞对DOX的耐药程度是SGC7901细胞的37.5倍。1.0、2.5和5.0 mol/L的CQ11分别使DOX对SGC7901/VCR细胞的敏感性分别增加到2.2倍(P<0.01)、5.5倍(P<0.01)和14倍(P<0.01)。DOX蓄积实验表明,CQ11能显著增加SGC7901/VCR细胞内DOX蓄积,而对SGC7901细胞内DOX蓄积无明显影响。结论:通过增加细胞内DOX蓄积量,CQ11在体外能有效逆转SGC7901/VCR细胞对DOX的耐药性。     

Modulation of doxorubicin-induced chromosomal damage by calmodulin inhibitors and its relationship to cytotoxicity in progressively doxorubicin-resistant tumor cells

Modulation of doxorubicin (DOX) cytotoxicity by the calmodulin inhibitor trifluoperazine (TFP) in progressively doxorubicin-resistant L1210 mouse leukemia cells is unrelated to effects on drug accumulation. Based on the clastogenic activity of DOX, the effects of TFP and the selective calmodulin inhibitor 1,3-dihydro-1-[1-[4-methyl-4H,6H-pyrrolo[1,2-a][4,1]- benzoxazepin-4-yl-methyl]-4-piperidinyl]-2H-benzimidazol-2-o ne(1:1) maleate (CGS9343B) on DOX-induced chromosomal damage and its relationship to cytotoxicity were evaluated in sensitive and progressively DOX-resistant L1210 cells. Potentiation of DOX cytotoxicity by CGS9343B (a potent inhibitor of calmodulin which does not inhibit protein kinase C) was related to the level of resistance. Further, for equivalent cytotoxicity, cellular DOX levels in the absence versus the presence of TFP or CGS9343B were markedly higher. Exposure to calmodulin inhibitors following DOX treatment enhanced chromosomal aberrations and cytotoxicity. Maximal effects of calmodulin inhibitors were apparent when used during and after DOX treatment, and potentiation of cytotoxicity was related to modulation of DOX-induced chromosomal aberrations. Results suggest that inhibition of calmodulin-regulated processes is a potential target in the modulation of DNA damage/repair, and could play a pivotal role in the expression of "acquired resistance" to DOX.     

Modulation by verapamil of vincristine pharmacokinetics and toxicity in mice bearing human tumor xenografts

The effect of the calcium channel blocker verapamil (VRP) on the accumulation and retention of vincristine (VCR) has been examined in mice bearing xenografts of human rhabdomyosarcomas. The tumors were Rh18, moderately sensitive to VCR, and its subline, Rh18/VCR3, selected in vivo for primary resistance to VCR. Administration of VRP by i.p. bolus at dose levels above 75 mg/kg was limited by acute lethality. At this dose, the maximal concentration in plasma was 24 microM, with rapid elimination such that plasma concentrations reported to modulate resistance in vitro (approximately 5-10 microM) were maintained for less than 60 min. To sustain a 10 microM plasma concentration, mice were infused with VRP at 6.25 mg/kg/hr (150 mg/kg/day) for up to 7 days using osmotic pumps implanted in the peritoneal cavity. Steady-state plasma levels were greater than or equal to 10 microM for at least 96 hr, and this schedule demonstrated minimal toxicity. Administration of VCR 20 hr after the start of VRP infusion produced significant lethality, requiring an 8-fold reduction in the VCR dose. Pharmacokinetic studies showed that VRP markedly increased the uptake and retention of VCR in small intestine, liver and kidney of mice. In small intestine, 8-fold greater levels of VCR were determined 24 hr after VCR administration, and this was associated with in increase in T1/2 for elimination from 350 to 913 min. HPLC analysis of extracts from small intestine showed that greater than 90% of the radiolabel eluted with VCR or 4-desacetyl-VCR. Modulation of VCR retention was also related to the dose of VCR administered. The VRP-sensitive efflux pathway appeared more effective in certain tissues only at higher concentrations of VCR. In contrast, VRP did not alter significantly the uptake and retention of VCR in either the parent or VCR-resistant human xenografts. The data demonstrated that, in the mouse, VRP modulates the uptake and retention of VCR in several tissues, and this may indicate that drug efflux mediated by a VRP-sensitive mechanism (e.g. GP-170, associated with the multiple drug resistance phenotype) has a protective function against xenobiotics in these tissues.     

Modulation of anthracycline resistance by reserpine in P388 leukemia cells

The activity of reserpine and a possible mechanism by which it reverses the resistance to both doxorubicin and pirarubicin in doxorubicin-resistant P388 leukemia (P388/DOX) cells were examined in vitro. During 48 hr drug-exposure, the sensitivity of doxorubicin and pirarubicin were potentiated markedly when reserpine was present at the concentration of 1 microgram/ml, which is not toxic to P388 leukemia (P388/S) cells. However, reserpine had little effect on the cytotoxicity of doxorubicin and pirarubicin in the sensitive parent cell. Reserpine at 0.5-20 micrograms/ml increased intracellular accumulation of doxorubicin and pirarubicin in the drug-resistant cells. The potentiating action of reserpine was stronger when the cells were preincubated with reserpine within 30 min. Efflux of doxorubicin and pirarubicin was greater in drug-resistant cells compared to sensitive cells. This enhanced efflux of drug resulted in a decrease in the intracellular accumulation of doxorubicin in the drug-resistant cells. When the resistant cells were exposed to 2 micrograms/ml of reserpine, this enhanced efflux was blocked. A similar effect of reserpine on doxorubicin was seen with the efflux pattern of pirarubicin. From the measurements of drug uptake and efflux, it seems that like other multiple drug resistance modifiers, reserpine modulates anthracycline resistance by increasing intracellular accumulation of drug.     

Modulation by verapamil of vincristine pharmacokinetics and sensitivity to metaphase arrest of the normal rat colon in organ culture

The in-vitro pharmacokinetics of vincristine (VCR) in normal rat colonic mucosa were studied. Two complementary approaches were adopted using an explant organ-culture system. Firstly [G-3H]vincristine (3HVCR) accumulation, retention and efflux were characterized under basal conditions and compared with measurements made either under energy-depleted conditions, or in the presence of VRP. Secondly, a histological method--the postmetaphase index (PMI)--was used to compare the sensitivity of explants to VCR in the presence or absence of verapamil (VRP). This latter technique involves the measurement, by counting, of the proportion of mitotic figures escaping from metaphase arrest. The studies yielded the following results: 3HVCR accumulation in colonic mucosa showed no evidence of saturability up to the maximum dose studied (130 nM), at a dose of 52 nM accumulation was enhanced in energy-depleted conditions by a factor of 1.8, and in the presence of VRP (6.6 microM) by a factor of 1.4. In the presence of VRP (6.6 microM) retention of 3HVCR was increased by a factor of 1.3 and efflux was reduced by a factor of 0.8 after 2 hr. VRP (6.6 microM) reduced the PMI of colonic mucosal epithelial cells exposed to 11 nM VCR from 18.8% to 11.4% (i.e. 40% reduction) indicating sensitization of the cells to this property of VCR. These results provide evidence that the sensitivity of normal colonic mucosa to vincristine is, at least in part, regulated by drug transport. Qualitatively our observations resemble those described in multidrug resistance. Given that P-glycoprotein has been demonstrated by several groups in colonic mucosal cells, the results support a normal role for this membrane transport molecule in the protection of intestinal cells from plant alkaloids and other xenobiotic agents ingested in the diet.     

Relationship between binding affinity, retention and sensitivity of human rhabdomyosarcoma xenografts to Vinca alkaloids

Xenografts of human rhabdomyosarcoma (RMS) have been derived that differ in their degree of sensitivity to Vinca alkaloids. Lines Rh12 and Rh18 demonstrated, respectively, high and moderate sensitivity to vincristine (VCR), but showed little responsiveness to vinblastine (VLB) in vivo. Rh18/VCR-3, a subline of Rh18 selected for resistance to VCR under in situ conditions, was insensitive to further challenge with VCR. Resistance was associated with elimination of the agent in a biphasic manner, whereas sensitivity to VCR corresponded to very prolonged drug retention in sensitive neoplastic tissues. The initial half-times for drug retention in tumors in vivo (t1/2 alpha) correlated with the degree of sensitivity of tumors to Vinca alkaloids, decreasing t1/2 alpha being associated with decreased sensitivity. A single binding species was observed when membrane-free supernatant fractions were incubated at 37 degrees for 15 min with 10.4 nM [3H]VCR and analyzed by gel filtration HPLC. The protein eluted with a retention time of 57 min and corresponded to a molecular weight (Mr) of approximately 113,000 daltons, agreeing very closely with the Mr of dimeric tubulin (approximately equal to 110,000 daltons). Two fractions were collected and eluted on a one-dimensional denaturing gel. Proteins were transferred subsequently to nitrocellulose and probed with an 125I-labeled monoclonal antibody specific for beta-tubulins. Only the fraction containing bound [3H]VCR contained tubulin. Estimates for the dissociation constants (Kd) for the binding affinity of VCR and VLB in crude, membrane-free supernatant fractions from RMS xenografts were obtained by computer curve fitting using a mathematical binding model. Data fitted a two-site binding model, with Kd values for the high-affinity site ranging from 61 to 160 nM, and for the low-affinity site, from 42 to 94 microM. At physiologically achievable drug concentrations, the relationship between binding affinity, drug retention and tumor sensitivity was examined further. A close relationship was apparent between the Kd values for VCR in Rh12, Rh18 and Rh18/VCR-3 tumor supernatant fractions and VLB in Rh12 preparations, and t1/2 alpha values for drug retention. Prolonged drug retention correlated with a low binding constant. As t1/2 alpha decreased, binding affinity also decreased, as demonstrated by an increase in the Kd value. Consequently, the tightness of drug binding in tumors also correlated with the degree of sensitivity of the xenografts to Vinca alkaloids.(ABSTRACT TRUNCATED AT 400 WORDS)     

GP与抗癌药物合用对肿瘤多药耐药的逆转作用

目的研究GP与抗癌药物合用对肿瘤多药耐药的逆转。方法MTT法测定GP与抗癌药物合用对K562/DOX和A549/DOX细胞的细胞毒作用;流式细胞术测定GP对K562/DOX细胞内阿霉素累积的影响。结果GP与阿霉素合用,能够明显逆转K562/DOX细胞对阿霉素的耐药性,并且能够浓度依赖性增加K562/DOX细胞内阿霉素的剂量;GP与阿霉素、顺铂、长春新碱合用时,对A549/DOX耐药性逆转作用相对较弱。结论GP能促进抗癌药物在MDR细胞内的累积,增强抗癌药物对MDR细胞的细胞毒作用,逆转肿瘤多药耐药。     

Potentiation of etoposide and vincristine by two synthetic 1,4-dihydropyridine derivatives in multidrug-resistant and atypical multidrug-resistant human cancer cells

Newly synthesized 1,4-dihydropyridine derivatives had been screened to determine whether they could overcome vincristine (VCR)-resistance in VCR-resistant (P388/VCR) leukemia-bearing mice, and six compounds had strong reversing ability among the screened compounds. We further determined whether NK-250 and NK-252 among the six compounds could potentiate cytocidal activities of etoposide (VP16) as well as VCR against both multidrug-resistant (MDR) cell line (VJ-300) and atypical MDR cell line (KB/VM-4). Both VJ-300 and KB/VM-4 were derived from the same parental human cancer KB cell line: VJ-300 cells showed enhanced expression of a MDR-specific glycoprotein of molecular weight of 170,000 Da (gp170) while KB/VM-4 cells were selected as teniposide (VM26)-resistant cell line with no expression of gp170. NK-250 and NK-252 potentiated the cytotoxic action of VCR about 2- to 10-fold against KB and KB/VM-4 cells, and they almost completely reversed VCR-resistance in VJ-300 cells. By contrast, NK-250 and NK-252 potentiated the cytotoxic action of VP16 about 2-fold against KB cells while they reversed 5- to 10-fold VP16-resistance in both VJ-300 and KB/VM-4 cells. The reversal effect by NK-250 and NK-252 of VCR-resistance in VJ-300 cells appeared to be due to enhanced cellular accumulation of radioactive VCR through interaction to 170-kDa P-glycoprotein. The potentiation effects by these dihydropyridines of VCR and VP16 on KB or KB/VM-4 cells also appeared to be due to enhanced accumulation of radioactive VP16 or VCR, but the effects might be mediated through other mechanisms, plausibly enhanced cellular uptake of the drugs.     

New triazine derivatives as potent modulators of multidrug resistance.

A series of 70 triazine derivatives have been synthesized and tested for their capacity to modulate multidrug resistance (MDR) in DC-3F/AD and KB-A1 tumor cells in vitro, in comparison with verapamil (VRP), a calcium channel antagonist currently used in therapy as an antihypertensive drug, which also shows MDR modulating activity. Among the 12 selected compounds, 16 (S9788) showed high MDR reversing properties in vitro (300- and 6-fold VRP at 5 microM in DC-3F/AD and KB-A1 cells, respectively) and induced a strong accumulation of adriamycin. The relationship between the increase of ADR accumulation and the fold reversal induced by these compounds and their lack of effects on the sensitive DC-3F cells suggest that they act mainly by inhibiting the P-glycoprotein (Pgp) catalyzed efflux of cytotoxic agents, as already described for a majority of MDR modulators. In vivo, in association with the antitumor drug vincristine (0.25 mg/kg), 16 (100 mg/kg) increased the T/C by 39% in mice bearing the resistant tumor cell line P388/VCR. According to these interesting properties, 16 was selected for a clinical development because it was more bioavailable than 34, even though it was less active.     

Mechanism of resistance to oxidative stress in doxorubicin resistant cells

Doxorubicin (DOX) is an anthracycline drug widely used in chemotherapy for cancer patients, but it often gives rise to multidrug resistance in cancer cells. The purpose of this work was to study the effect of hydrogen peroxide in DOX-sensitive mouse P388/S leukemia cells and in the DOX-resistant cell line. Hydrogen peroxide induced a significant increase in dose- and time-response cell death in cultured P388/S cells. The degree of cell death in P388/DOX cells induced by hydrogen peroxide was less than that in P388/S cells treated with hydrogen peroxide. Parent cells exposed to 3 mM of hydrogen peroxide showed a loss of mitochondrial membrane potential correlated with cell death. Hydrogen peroxide at a concentration greater than 0.3 mM increased the intracellular Ca2+ of P388/S cells dose-dependently; however, no change following addition of hydrogen peroxide (0.3-1 mM) was observed in the resistant cells. Hydrogen peroxide (0.1 and 1 mM) treatment also induced the production of intracellular ROS in P388/S cells, while no such increase was produced by this substance in P388/DOX cells. Resistant cells also showed a significant level of glutathione (GSH) compared with the parent cells. In addition, N-acetyl-L-cysteine and reduced GSH antioxidants abolished death of P388/S cells caused by hydrogen peroxide. Therefore, it is believed that the reduced effect of oxidative stress towards the resistant cells may be related to an increase in intracellular GSH level.     

Increase of daunorubicin and vincristine accumulation in multidrug resistant human ovarian carcinoma cells by a monoclonal antibody reacting with P-glycoprotein

An overexpression of plasma membrane 170-180 kDa P-glycoproteins is consistently found in multidrug-resistant (MDR) cell lines. In this study MRK-16, a monoclonal antibody (mAb) reacting with P-glycoprotein is used to study the putative functional role of this protein in vincristine (VCR) and daunorubicin (DNR) cellular accumulation in the MDR human ovarian carcinoma cell line 2780AD. We established that this cell line is highly cross-resistant to vincristine and daunomycin, related to a greatly reduced drug accumulation. Verapamil (Vp) (8 microM) caused a 3.6-fold increase in DNR as well as VCR accumulation. Exposition of 2780AD cells to MRK-16 led to an increase of 30% in cellular accumulation of VCR, both in normal growth medium as well as in medium without added glucose and with sodium azide, which largely depleted cellular ATP levels. No increase in DNR accumulation was found under these conditions. However, in the presence of 8 microM Vp, MRK-16 increased not only VCR but also DNR accumulation with about 30%. The relative increase of DNR accumulation was constant in a concentration range of 0.2-4 microM DNR. These data indicate that mAbs against P-glycoprotein might potentiate the action of calcium antagonists like Vp to increase cellular anthracycline accumulation.