Cellular uptake and efflux and cytostatic activity of 4'-O-tetrahydropyranyladriamycin in adriamycin-sensitive and resistant tumor cell lines

Cellular uptake and cytostatic activity of 4'-O-tetrahydropyranyladriamycin (THP) in various sublines resistant to anthracycline antibiotics of mouse lymphoblastoma L5178Y, Chinese hamster ovary (CHO) and mouse leukemia P388 cells were studied. All the sublines resistant to adriamycin (ADR) showed slightly decreased uptake of THP as compared with each sensitive lines, but THP was still taken up much more quickly than ADR by each of the ADR-resistant cell lines. Efflux of both anthracycline glycosides from the ADR-resistant P388 cells was faster than that from the ADR-sensitive P388 cells. The percentage of THP retained at equilibrium was higher than that of ADR in both ADR-resistant and -sensitive P388 cells. Cytotoxicity of THP to ADR-resistant cell lines was considerably lower compared with that for each of the sensitive lines but THP inhibited growth of ADR-resistant tumor cells at a concentration about 10 times lower than that for ADR. Thus THP was taken up more quickly, effluxed more slowly than ADR from the ADR-resistant cells, and showed stronger cytostatic activity than ADR on the cells.     

Characterization of a subline of P388 leukemia resistant to amsacrine: evidence of altered topoisomerase II function

Sensitive (P388/S) and amsacrine-resistant (P388/amsacrine) sublines of P388 leukemia were cloned in vitro and tested for differential chemosensitivity against a panel of drugs. P388/amsacrine, resistant both in vivo and in vitro to amsacrine, was cross-resistant to other putative topoisomerase II inhibitors including teniposide, etoposide, bisantrene, and doxorubicin. P388/amsacrine, was however, as sensitive as cloned P388/S to camptothecin, an inhibitor of topoisomerase I. The pattern of cross-resistance suggested that an alteration in topoisomerase II may be involved in the resistance of P388/amsacrine to these drugs. No differences in the uptake of amsacrine were detected between the two sublines. Cross-resistance to vinblastine was evident in P388/amsacrine; however resistance to vinblastine was associated with alterations in uptake or efflux of the drug. The number of protein-concealed single-strand breaks induced in whole cells by amsacrine, teniposide, bisantrene, and camptothecin was measured. Diminished numbers of strand breaks in the resistant subline were consistent with decreases in DNA-protein crosslinks. In the absence of drug treatment, resistant cells sustained approximately one-half as many single-strand breaks and DNA-protein crosslinks as the sensitive cells during preparation of nuclei. As measured by the P4 phage DNA unknotting assay, 0.35 M NaCl nuclear extracts from P388/S contained approximately 2.3-fold more topoisomerase II catalytic activity than did extracts from P388/amsacrine. The amount of protein that immunoreacted with a specific antibody to calf thymus topoisomerase II was also decreased in the resistant cells. These data suggest that alterations in topoisomerase II which lead to differential drug sensitivities are partially responsible for the resistance of P388/amsacrine to a specific group of drugs.     

The effect of lysosomotropic agents and secretory inhibitors on anthracycline retention and activity in multiple drug-resistant cells

The effect of lysosomotropic agents and secretory inhibitors were compared with verapamil for their effect on the activity of doxorubicin (DOX) in multiple drug-resistant (MDR) P388 leukemia cells (P388R) and in blocking anthracycline efflux from these cells. Agents known to interact with the plasma membrane did not potentiate DOX activity in P388R cells unless these same agents were also capable of interacting with acidic compartments within the cell. The lysosomotropic detergent Triton WR-1339, for example, potentiated DOX activity in P388R cells and stimulated the net accumulation of daunorubicin (DAU) in P388R cells by inhibiting drug exodus. However, another detergent, deoxycholate, and two membrane active antibiotics, amphotericin B and filipin, had no effect on DOX activity and/or DAU efflux in P388R cells. Lysosomotropic agents such as chloroquine and secretory inhibitors such as monensin, cytochalasin B, and vinblastine all inhibited DAU efflux from P388R cells. In a MDR B16 melanoma cell line, the activity of DOX was potentiated by both verapamil and reserpine. These same two agents also inhibited melanin secretion from this same cell line. Based on these observations, we propose that secretory vesicles derived from the Golgi apparatus might be involved in the MDR phenomenon. We further suggest that drugs such as DOX might be concentrated in these acidic vesicles, where they would be released to the outside of the cell by exocytosis.     

Cross resistance between actinomycin-D,adriamycin and vincristine in a murine solid tumour in vivo

In experimental tumour systems, patterns of cross-resistance to the cytotoxic drugs of the anthracycline group, the vinca alkaloids and actinomycin-D have been well documented, (1 – 3). Early work suggested that resistance to these agents resulted from failure of drug uptake (4 – 6), and in addition changes in membrane glycoprotein content have been identified as important factors underlying this decrease in membrane permeability in resistant tumour cells (7). However, recent evidence from $\text{in vitro}$ experiments suggests that a common mechanism of enhanced active efflux of these drugs, rather than of impaired influx, may underly the development of cellular drug resistance (8, 9).Previous $\text{in vivo}$ studies of cross-resistance have been performed using murine tumours in the ascites form, either sublines of P 388 leukaemia (3, 10) or of the Ehrlich ascites tumour (2). In this study, resistance to actinomycin-D was developed $\text{in vivo}$ in a solid tumour, the Ridgway osteogenic sarcoma (ROS) in AKH mice, and patterns of cross resistance to adriamycin, vincristine and cyclophosphamide were then examined. In addition a preliminary investigation of $\text{in vivo}$ drug uptake and retention in sensitive and resistant ROS tumour sublines has been performed, by estimating concentrations of radiolabelled actinomycin-D and vincristine in tumour digests at intervals following injection.     

Saturable Function of P-Glycoprotein as a Drug-efflux Pump in Multidrug-resistant Tumour Cells

P-glycoprotein acts as an active drug-efflux pump in multidrug-resistant tumour cells. We studied the capacity of P-glycoprotein to extrude drugs from the cells. For nanomolar concentrations of vinblastine P388/ADR cells, which overexpress P-glycoprotein in the plasma membrane, accumulated vinblastine, at 37°C for 30 min, to a much lower extent than the sensitive cells (P388/S), while in the micromolar range the cellular concentration was similar for both types of cells. When cells were incubated with a low (10 nM) or high concentration (1 μM) of vinblastine while energy deprived, the vinblastine concentration increased only in the resistant cells incubated with the low concentration of vinblastine, and this increased level was lowered to the level under the normal conditions by addition of glucose. In contrast, the cellular concentrations in other cases were increased to the normal level by glucose. After cells were loaded with the low concentration of vinblastine, the cellular vinblastine was extruded more rapidly from the resistant cells than from the sensitive cells. The courses of vinblastine efflux from the cells loaded with the high concentration of vinblastine were similar in both types of cells. NA-382, a reported P-glycoprotein inhibitor, effectively increased the intracellular vinblastine and inhibited the drug efflux only from multidrug-resistant cells, P388/ADR and AH66 cells, which were incubated with the low concentration of vinblastine. Cellular uptake of NA-382 was also less in P388/ADR cells than in P388/S cells in culture with 10 nM but not 1 μM of the agent, and this low level was reversed to the level in the sensitive cells by 10 μM vinblastine. These results indicate that P-glycoprotein as a drug-efflux pump works effectively under low extracellular concentrations of substrates, but does not under the high concentrations.     

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.     

Role of differential drug uptake, efflux, and binding of etoposide in sensitive and resistant human tumor cell lines: implications for the mechanisms of drug resistance

In order to study the mechanism of etoposide (VP-16) resistance in human tumor cells and to assess the role of P-170 glycoprotein in VP-16 accumulation, we have examined the uptake and efflux of VP-16 in both sensitive and multidrug-resistant MCF-7 human breast and HL60 human promyelocytic leukemia cells. The drug-resistant cells, MCF-7/ADR and HL60/ADR, were selected for resistance to adriamycin and were 200- to 250-fold resistant to VP-16. Whereas MCF-7/ADR cells overexpress the P-170 glycoprotein and show the multidrug-resistant phenotype, HL60/ADR cells do not overexpress the P-170 glycoprotein. Although there was a 2-fold decrease in accumulation of VP-16 in MCF-7/ADR cells, this decrease did not correlate with a 250-fold resistance to the drug. VP-16 efflux was rapid and almost complete from MCF-7 cell lines and it was decreased at 4 degrees. Further, there was a significant increase in VP-16 accumulation in the MCF-7/ADR cells in the presence of glucose-free medium supplemented with sodium azide. However, no change in the pattern of VP-16 efflux was observed. Under these conditions, addition of glucose caused release of VP-16 from MCF-7/ADR cells, suggesting energy-dependent modifications in the drug binding. Coincubation of vincristine with VP-16 also increased the drug accumulation and decreased the rate of efflux of VP-16 in both sensitive and resistant MCF-7 cells, suggesting that vincristine and VP-16 may compete for similar binding and efflux mechanisms in these cell lines. In contrast, daunorubicin increased VP-16 accumulation only in the sensitive MCF-7 cell line, whereas the efflux rate of VP-16 was not significantly changed in either cell line. HL60 sensitive cells accumulated 4- to 5-fold more VP-16 than the resistant subline. Both sensitive and resistant cells showed an important noneffluxable pool of the drug, 3-fold larger for sensitive cells (79 +/- 12 versus 25 +/- 2 pmol of VP-16/mg of protein, for sensitive and resistant cells, respectively). The efflux of VP-16 was temperature dependent only in sensitive cells. VP-16 accumulation in HL60/ADR cells was increased in glucose-free medium supplemented with sodium azide; however, the noneffluxable pool of VP-16 was not significantly changed. In contrast, although these conditions had no effect on the drug accumulation in the parental line, they caused a decrease in the noneffluxable pool of VP-16, suggesting an energy-dependent binding and retention of VP-16.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Transport and binding of daunorubicin, adriamycin, and rubidazone in Ehrlich ascites tumour cells.

The determinants of cellular net uptake of three anthracyclines—daunorubicin, adriamycin, and rubidazone—were studied in Ehrlich ascites tumour cells in vitro. A striking finding was the marked effect of the pH on net uptake in the way that lowering of the pH decreased the rate of uptake and steady-state level. As pK_A of the drugs proved to be 8.15–8.45, this finding suggests a penetration of the drugs by passive transport of the unionized form of the molecules. Several findings indicated an uphill efflux of the drugs from the cells. (1) The estimate of the cytoplasm/medium concentration ratio at steady-state for daunorubicin and rubidazone was considerably below that predicted when assuming passive distribution. (2) The cellular accumulation of the drugs was significantly enhanced by sodium azide. (3) The cellular release of the drugs was inhibited by sodium azide. Together these findings suggest a membrane transport of the drugs as a “leak-and-pump” system. Studies of the intracellular binding showed that the affinity of adriamycin for isolated nuclei and for total cellular homogenate was about 50 per cent higher than that of daunorubicin; rubidazone possessed the lowest affinity for cellular binding sites. The affinity of the drugs for intracellular binding sites was not influenced by the pH.The results indicate that the net uptake of anthracycline derivatives in these tumour cells is determined by: (1) the permeability of the membrane to the unionized form of the molecule ; (2) the specificity of the drug for the mechanism of active efflux; (3) the relationship between intracellular and extracellular pH; (4) the pK_A, value of the drug; (5) the affinity of the drug for the intracellular binding sites.     

Membrane-associated proteins of adriamycin sensitive and resistant murine leukemic P388 cells

We have isolated an 84-fold adriamycin resistant subline, P388/R84, from mouse leukemia P388 cells by serial cultivation in methylcellulose in the presence of increasing drug concentrations. Electrophoresis of detergent soluble fractions of radiolabeled sensitive and resistant cells suggested marked alterations in the protein fractions of 160, 100, 60, 45, and 30 kd. In resistant clones labeled with ¹²⁵1 an increase in 160 and 100 kd proteins was accompanied by concomitant reduction in the 60, 45, and 30 kd proteins. In ³⁵S methionine-labeled resistant cells, similar increases in the 160 and 100 kd components were observed but in contrast to ¹²⁵I-labeled cells the 30 kd component was also higher. Alterations in surface proteins were confirmed in experiments where the cell extracts were adsorbed to concanavalin A polymers and extracted with 0.26 M methyl-α-D-mannopyranoside. Our data confirm earlier reported observations on cell-surface protein changes in cells resistant to anthracyclines and alkaloids.     

Nuclear targeting and nuclear retention of anthracycline-formaldehyde conjugates implicates DNA covalent bonding in the cytotoxic mechanism of anthracyclines.

The anthracycline, antitumor drugs doxorubicin (DOX), daunorubicin (DAU), and epidoxorubicin (EPI) catalyze production of formaldehyde through induction of oxidative stress. The formaldehyde then mediates covalent bonding of the drugs to DNA. Synthetic formaldehyde conjugates of DOX, DAU, and EPI, denoted Doxoform (DOXF), Daunoform (DAUF), and Epidoxoform (EPIF), exhibit enhanced toxicity to anthracycline-sensitive and -resistant tumor cells. Uptake and retention of parent anthracycline antitumor drugs (DOX, DAU, and EPI) relative to those of their formaldehyde conjugates (DOXF, DAUF, and EPIF) were assessed by flow cytometry in both drug-sensitive MCF-7 cells and drug-resistant MCF-7/ADR cells. The MCF-7 cells took up more than twice as much drug as the MCF-7/ADR cells, and both cell lines took up substantially more of the formaldehyde conjugates than the parent drugs. Both MCF-7 and MCF-7/ADR cells retained fluorophore from DOXF, DAUF, and EPIF hours after drug removal, while both cell lines almost completely expelled DOX, DAU, and EPI within 1 h. Longer treatment with DOX, DAU, and EPI resulted in modest drug retention in MCF-7 cells following drug removal but poor retention of DOX, DAU, and EPI in MCF-7/ADR cells. Fluorescence microscopy showed that the formaldehyde conjugates targeted the nuclei of both sensitive and resistant cells, and remained in the nucleus hours after drug removal. Experiments in which [(3)H]Doxoform was used, synthesized from doxorubicin and [(3)H]formaldehyde, also indicated that Doxoform targeted the nucleus. Elevated levels of (3)H were observed in DNA isolated from [(3)H]Doxoform-treated MCF-7 and MCF-7/ADR cells relative to controls. The results implicate drug-DNA covalent bonding in the tumor cell toxicity mechanism of these anthracyclines.     

Reversal of multidrug resistance by 7-O-benzoylpyripyropene A in multidrug-resistant tumor cells

7-O-Benzoylpyripyropene A (7-O-BzP), a semi-synthetic analog of pyripyropene, was investigated for its reversing effect on multidrug-resistant (MDR) tumor cells. 7-O-BzP (6.25 microg/ml) completely reversed resistance against vincristine and adriamycin in vincristine-resistant KB cells (VJ-300) and adriamycin-resistant P388 cells (P388/ADR), respectively. 7-O-BzP alone had no effect on the growth of drug sensitive and drug-resistant cells. 7-O-BzP (6.25 microg/ml) significantly enhanced accumulation of [3H]vincristine in VJ-300 cells and completely inhibited the binding of [3H]azidopine to the P-glycoprotein in VJ-300 cells and P388/ADR cells. The result suggests that 7-O-BzP effectively reverses P-glycoprotein-related MDR by interacting directly with P-glycoprotein in drug resistant VJ-300 and P388/ADR cells.     

Bioactive Lipids-Based pH Sensitive Micelles for Co-Delivery of Doxorubicin and Ceramide to Overcome Multidrug Resistance in Leukemia

Purpose

Construction of a novel PEGylated bioactive lipids-based micelle system for co-delivery of doxorubicin (DOX) and short chain ceramide (C6-ceramide) to overcome multidrug resistance in leukemia.

Methods

The PEGylated bioactive lipids-based micelle system was constructed via electrostatic and hydrophobic interactions among DOX, bioactive lipids PazPC and C6-ceramide. The micellar formulation was characterized in terms of size, zeta potential, stability and release behavior, etc., and in vitro cytotoxicity, in vivo antitumor efficacy and the underlying mechanism were further evaluated.

Results

This novel micellar system showed small size (~15 nm), high drug encapsulation efficiency (>90%), good stability and endosomal acid-triggered release of DOX. Synergistic cytotoxic effects between DOX and bioactive lipid C6-ceramide in P-gp overexpressing drug resistant leukemia P388/ADR cells were observed. The mechanistic studies demonstrated that modulation of drug efflux system and induction of apoptotic effects by lipids were responsible for the synergistic effects between DOX and C6-ceramide in drug resistant leukemia P388/ADR cells. Using an in-vivo P388/ADR leukemia mouse model, the median survival time of the DOX-loaded PEGylated micelles with PazPC and C6-ceramide as major components was significantly greater than that of free DOX and control group.

Conclusions

We developed a novel pH sensitive bioactive lipids-based micellar formulation which could potentially be useful in delivering chemotherapeutic drug DOX and provide a novel strategy to increase the therapeutic index for drug resistant leukemia treatment.     

m-AMSA as a probe for transport phenomena associated with anthracycline resistance

Kinetics of transport of the acridine derivative 4′-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) were examined in P388 murine leukemia cells and in P388/ADR, a subline selected for adriamycin resistance and cross-resistant to variety of drugs including m-AMSA. Compared with the drug-responsive parent cell line, P388/ADR cells showed impaired accumulation of m-AMSA and an enhanced rate of drug exodus. Competition studies demonstrated structural specificity of the outward transport process. There was a low degree of intracellular m-AMSA binding, and steady-state drug levels were reached in <1in. These results suggest that m-AMSA will be a useful probe for studying transport systems associated with anthracycline resistance.     

Vinflunine (20′,20′-difluoro- 3′,4′-dihydrovinorelbine), a novel Vinca alkaloid, which participates in P-glycoprotein (Pgp)-mediated multidrug resistance in vivo and in vitro

Vinflunine (VFL) is a novel derivative of vinorelbine (NVB, Navelbine®), which has shown markedly superior antitumor activity to NVB, in various experimental animal models. To establish whether this new Vinca alkaloid participates in P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), VFL-resistant murine P388 cells (P388/VFL) were established in vivo and used in conjunction with the well established MDR P388/ADR subline, to define the in vivo resistance profile for VFL. P388/VFL cells proved cross-resistant to drugs implicated in MDR (other Vinca alkaloids, doxorubicin, etoposide), but not to campothecin or cisplatin and showed an increased expression of Pgp, without any detectable alterations in topoisomerase II or in glutathione metabolism. The P388/ADR cells proved cross-resistant to VFL both in vivo and in vitro, and this VFL resistance was efficiently modulated by verapamil in vitro. Cellular transport experiments with tritiated-VFL revealed differential uptake by P388 sensitive and P388/ADR resistant cells, comparable with data obtained using tritiated-NVB. In various in vitro models of human MDR tumor cells, whilst full sensitivity was retained in cells expressing alternative non-Pgp-mediated MDR mechanisms, cross resistance was identified in Pgp-overexpressing cells. Differences were, however, noted in terms of the drug resistance profiles relative to the other Vincas, with tumor cell lines proving generally least cross-resistant to VFL. Overall, these results suggest that VFL, like other Vinca alkaloids, participates in Pgp-mediated MDR, with tumor cells selected for resistance to VFL overexpressing Pgp, yet MDR tumor cell lines proved generally less cross resistant to VFL relative to the other Vinca alkaloids.     

Vinflunine (20′,20′-difluoro- 3′,4′-dihydrovinorelbine), a novel Vinca alkaloid, which participates in P-glycoprotein (Pgp)-mediated multidrug resistance in vivo and in vitro

Vinflunine (VFL) is a novel derivative of vinorelbine (NVB, Navelbine®), which has shown markedly superior antitumor activity to NVB, in various experimental animal models. To establish whether this new Vinca alkaloid participates in P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), VFL-resistant murine P388 cells (P388/VFL) were established in vivo and used in conjunction with the well established MDR P388/ADR subline, to define the in vivo resistance profile for VFL. P388/VFL cells proved cross-resistant to drugs implicated in MDR (other Vinca alkaloids, doxorubicin, etoposide), but not to campothecin or cisplatin and showed an increased expression of Pgp, without any detectable alterations in topoisomerase II or in glutathione metabolism. The P388/ADR cells proved cross-resistant to VFL both in vivo and in vitro, and this VFL resistance was efficiently modulated by verapamil in vitro. Cellular transport experiments with tritiated-VFL revealed differential uptake by P388 sensitive and P388/ADR resistant cells, comparable with data obtained using tritiated-NVB. In various in vitro models of human MDR tumor cells, whilst full sensitivity was retained in cells expressing alternative non-Pgp-mediated MDR mechanisms, cross resistance was identified in Pgp-overexpressing cells. Differences were, however, noted in terms of the drug resistance profiles relative to the other Vincas, with tumor cell lines proving generally least cross-resistant to VFL. Overall, these results suggest that VFL, like other Vinca alkaloids, participates in Pgp-mediated MDR, with tumor cells selected for resistance to VFL overexpressing Pgp, yet MDR tumor cell lines proved generally less cross resistant to VFL relative to the other Vinca alkaloids.     

Identification and characterization of a plasma membrane phosphoprotein which is present in chinese hamster lung cells resistant to adriamycin

Studies have been carried out to analyze the phosphoproiein composition of plasma membranes from Chinese hamster lung cells resistant to the action of adriamycin. Gel electrophoretic analysis of [³²P_i]-labeled proteins revealed that plasma membranes from resistant cells contain a phosphoprotein of 180,000 molecular weight (P180) which is not detected in drug sensitive cells. Protein P180 can also be identified after phosphorylation of resistant plasma membranes in an in vitro protein kinase system. Pulse-chase experiments indicated that the P180 was metabolically active and underwent cycles of phosporylation and dephosphorylation in the cell. Additional studies showed that, in the presence of N-ethylmaleimide (NEM), there was a major increase in the uptake of adriamycin in resistant cells. A similar effect was observed with KCN but not with sodium azide. When resistant cells were grown in the presence of [³²P_i] and then incubated in the presence of NEM, there was a considerable increase in the phosphorylation of P180. In contrast, many other plasma membrane proteins were dephosphorylated under these incubation conditions. The results suggest the possibility that, as P180 was hyperphosphorylated, the protein was inactivated and this contributed to the ability of resistant cells to accumulate adriamycin.     

In vitro cytotoxicity of S16020-2, a new olivacine derivative

Summary S16020-2 is a new olivacine derivative which has recently shown a marked antitumor activity in various experimental models. This study was undertaken in order to measure the inhibition of the proliferation of various sensitive and resistant tumor cell lines, by S16020-2, and to obtain information concerning its mechanism of action.For a continuous exposure, S16020-2 was as cytotoxic as adriamycin (ADR) (mean IC₅₀ of about 28 nM) and on average, 46 fold more potent than elliptinium acetate (ELP), against a panel of 20 non-multidrug resistant cell lines. With a short exposure (1 hour) followed by a post-incubation of 95 hours in drug-free medium, S16020-2 was 5 and 6 fold more cytotoxic than ADR for human lung A549 and murine melanoma B16 cells, respectively. Furthermore, S16020-2 inhibited more actively the formation of colonies issued from proliferating cells, compared to colonies issued from quiescent A549 cells. Because quiescent cells demonstrated a 3 fold lower level of topoisomerase II (topo II) than proliferating cells, these results suggest that this enzyme could be a potential target for S16020-2. In addition, as demonstrated by flow cytometric studies, S16020-2 intercalated into DNA and induced a cell cycle arrest in G2.Cell lines displaying the multidrug resistance (MDR) phenotype, P388/ADR-1, P388/ADR, P388/VCR-20, KB-A1, DC-3F/AD, S1/tMDR, and Colo320DM, were more sensitive to S16020-2 than to ADR or ELP, as shown by the mean resistance factors, 8, 201, and 23 respectively. In addition, the two cell lines displaying the pure classical MDR phenotype, linked exclusively to the P-glycoprotein (P-gp) overexpression (P388/VCR-20 and S1/tMDR), were as sensitive to S16020-2 as their sensitive parental counterparts, although they were resistant to ADR.S16020-2 is thus one of the most potent olivacine and ellipticine derivative yet characterized. The good cytotoxicity of S16020-2 against cells displaying a P-gp-mediated multidrug resistance, and its antitumor activity in vivo delineate an important chemotherapeutic potential for this drug.     

Ability of Doxorubicin-Loaded Nanoparticles to Overcome Multidrug Resistance of Tumor Cells After Their Capture by Macrophages

Purpose. Investigation of the ability of doxorubicin-loaded nanoparticles (NP/Dox) to overcome multidrug resistance (MDR) when they have first been taken up by macrophages. Methods. The growth inhibition of P388 sensitive (P388) and resistant (P388/ADR) tumor cells was evaluated in a coculture system consisting of wells with two compartments. The tumor cells were seeded into the lower compartment, the macrophages were introduced into the upper part in which the drug preparations were also added. Results. Doxorubicin exerted lower cytotoxicity on tumor cells in coculture compared with direct contact. In P388/ADR, NP/Dox cytotoxicity was far higher than that of free doxorubicin (Dox). Three different formulations of cyclosporin A (either free (CyA), loaded to nanoparticles (NP/CyA) or in a combined formulation with doxorubicin (NP/Dox-CyA)), were added to modulate doxorubicin efficacy. The addition of cyclosporin A to Dox increased drug cytotoxicity. Both CyA added to NP/Dox and NP/Dox-CyA were able to bypass drug resistance. Conclusions. Despite the barrier role of macrophages, NP/Dox remained far more cytotoxic than Dox against P388/ADR. Both NP/ Dox + CyA and NP/Dox-CyA allowed to overcome MDR, but the last one should present greater advantagein vivo by confining both drugs in the same compartment, hence reducing the adverse effects.