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Purpose: Pluronic block copolymers are potent sensitizers of multidrug resistant cancers. SP1049C, a Pluronic-based micellar formulation of doxorubicin (Dox) has completed Phase II clinical trial and demonstrated safety and efficacy in patients with advanced adenocarcinoma of the esophagus and gastroesophageal junction. This study elucidates the ability of SP1049C to deplete cancer stem cells (CSC) and decrease tumorigenicity of cancer cells in vivo., Experimental Design: P388 murine leukemia ascitic tumor was grown in BDF1 mice. The animals were treated with: (a) saline, (b) Pluronics alone, (c) Dox or (d) SP1049C. The ascitic cancer cells were isolated at different passages and examined for 1) in vitro colony formation potential, 2) in vivo tumorigenicity and aggressiveness, 3) development of drug resistance and Wnt signaling activation 4) global DNA methylation profiles, and 5) expression of CSC markers., Results: SP1049C treatment reduced tumor aggressiveness, in vivo tumor formation frequency and in vitro clonogenic potential of the ascitic cells compared to drug, saline and polymer controls. SP1049C also prevented overexpression of BCRP and activation of Wnt-β-catenin signaling observed with Dox alone. Moreover, SP1049C significantly altered the DNA methylation profiles of the cells. Finally, SP1049C decreased CD133(+) P388 cells populations, which displayed CSC-like properties and were more tumorigenic compared to CD133(-) cells., Conclusions: SP1049C therapy effectively suppresses the tumorigenicity and aggressiveness of P388 cells in a mouse model. This may be due to enhanced activity of SP1049C against CSC and/or altered epigenetic regulation restricting appearance of malignant cancer cell phenotype.

Cucurbitacin E increases the doxorubicin (DOX) level in M5076 ovarian sarcoma via suppressed DOX efflux in vitro. An increase in DOX induced antitumor activity by cucurbitacin E in vivo has been reported previously. This paper attempts to clarify the mechanism of cucurbitacin E induced increments in the antitumor activity of DOX. MK-571, a multidrug resistance associated protein (MRP) inhibitor, significantly suppressed DOX efflux from M5076 ovarian sarcoma cells. The combination of cucurbitacin E with MK-571 also inhibited DOX efflux, whereas the efficacy was the same in each treatment. Namely, the inhibition of DOX efflux by cucurbitacin E was expected to be related to MRP. In contrast, it appeared that the effect of cucurbitacin E on DOX permeability did not relate to P-gp. The cucurbitacin E co-treatment significantly increased DOX concentration in the tumor within a short time after DOX administration, whereas the same treatment decreased the DOX concentration in normal tissues. The different effects of cucurbitacin E between tumor and normal tissues was speculated to be related to differences in DOX transport system on cell membrane. In DOX therapy, cucurbitacin E co-treatment was expected to increase DOX induced antitumor activity without an increase in adverse reactions due to DOX.

1. The present study found that, compared with mouse heart and liver, P388 ascitic tumour had significantly lower superoxide dismutase (SOD) activity and that compared with the mouse liver, the heart had significantly lower SOD and catalase activities, as well as a lower glutathione content. 2. At 7.5 mg/kg, doxorubicin (DOX), a superoxide radical inducer, induced significant lipid peroxidation only in the tumour, whereas 15.0 mg/kg DOX induced lipid peroxidation in both the tumour and heart, but not in the liver. 3. Overall, the results of the present study suggest that the differential anti-oxidant activities in P388 ascitic tumour, heart and liver in mice may explain their differential responses and, hence, susceptibility to DOX-induced lipid peroxidation.

It was reported that xanthine derivatives (caffeine and 1-methyl-3-propyl-7-butylxanthine) enhanced the antitumor activity of doxorubicin (DOX) with increasing DOX concentrations in tumors in vivo in our previous papers. In addition, these actions were found to be related to the inhibitory activity toward DOX efflux from tumor cells in vitro. In this study, we searched for novel biochemical modulators of DOX among 3-n-propylxanthines with functional groups at the 1- or 7-position by using an assay system for their inhibitory effect on DOX efflux from P388 leukemia and DOX resistant P388 leukemia (P388/DOX) cells. 1-Substituted xanthines facilitated the DOX efflux from P388 cells. In contrast, among 7-substituted xanthines, XT-141 and XT-139 significantly inhibited the DOX efflux from P388 cells. In addition, XT-141 inhibited the DOX efflux from P388/DOX cells, and P-glycoprotein (P-gp) inhibitor facilitated DOX influx and inhibited DOX efflux from P388/DOX cells in a dose-dependent manner. These results indicated that the resistance of P388/DOX might depend on the over-expression of P-gp, and that XT-141 inhibited DOX efflux through its interaction with P-gp. We suspect that XT-141 is a useful biochemical modulator of DOX in DOX-resistant tumors with over-expression of P-gp in addition in DOX-sensitive tumors.

Although the ability of chemosensitizers to modulate P-glycoprotein (PGP)-based multidrug resistance (MDR) has been extensively studied, relatively little is known about the cellular pharmacology of the PGP inhibitors themselves in MDR cells. The studies described here have correlated the in vitro accumulation and retention properties of verapamil (VRP) in murine P388 (sensitive) and P388/ADR (MDR) cells with doxorubicin (DOX) uptake and cytotoxicity modulation characteristics in order to better understand VRP-tumor cell interactions that give rise to MDR modulation. VRP is rapidly taken up by DOX-sensitive and -resistant P388 cells where greater than 50% maximal VRP uptake occurs within 10 min of initial exposure at 37 degrees C. Whereas chemosensitization and DOX uptake in P388/ADR cells increase with increasing VRP concentration until a plateau is achieved at approximately 5 microM VRP, cellular modulator levels increase proportionally with increasing VPR concentrations beyond 20 microM. Subsequent to removal of noncell-associated modulator, VRP levels in both sensitive and resistant cells rapidly fall below 10% of those obtained at uptake equilibrium. However, a residual amount of VRP remains associated with the cells for extended time periods after the cells are washed. Pulse exposures of P388/ADR cells to high concentrations of VRP (50-100 microM) are capable of providing extended cell-associated VRP levels comparable to those obtained with continuous exposure at biologically active VRP concentrations (1-3 microM) and this leads to chemosensitization. These results are consistent with the existence of high- and low-affinity intracellular VRP pools in P388 MDR cells, both of which can contribute to the reversal of drug resistance. It is suggested that these properties should be taken into consideration during the design and evaluation of preclinical in vivo MDR models where pulsed exposure to high concentrations of resistance modulators often occurs. Special attention must be given to whether such high concentration pulses are desirable and/or achievable in relevant clinical settings.

Acquired drug resistance is a major factor in the failure of doxorubicin-based cancer chemotherapy. We determined the ability of cinchonine to reverse doxorubicin drug resistance in a doxorubicin-resistant leukaemia cell line (mouse P388/DOX). A non-cytotoxic concentration of cinchonine (10 microM) increased the sensitivity to doxorubicin of multidrug-resistant P388/DOX cells and significantly enhanced the doxorubicin-induced apoptosis and DNA fragmentation in resistant cells, but had no effect in parent cells. Time-course studies demonstrated that DNA fragmentation was present 24 h after incubation with doxorubicin and cinchonine, indicating that DNA degradation was a preceding event. In cultured cells, cinchonine increased the intracellular accumulation of doxorubicin in the resistant cells in a dose-dependent manner. Using flow cytometry to measure the inhibition of the P-glycoprotein (P-gp) dependent efflux of rhodamine 123, cinchonine was found to be considerably more effective than quinine. The results with cinchonine suggest that there may be quinine derivatives with a similar capacity to inhibit drug transport by P-gp. Additionally, the G2/M phase cell population in resistant cells is increased by doxorubicin/cinchonine treatment. Exposure of resistant cells to 1 microM doxorubicin and 10 microM cinchonine resulted in the expression of Fas (APO-1/CD95) in cells after 6 h. These studies demonstrate that the cell killing effects of doxorubicin and cinchonine in resistant cells

It has been reported that several cis-unsaturated fatty acids (c-UFAs) could increase doxorubicin (DOX) accumulation in cancer cells and hence elevate its cytotoxicity. However, some researchers showed that c-UFA pretreatment did not affect its cytotoxicity in special cell lines. It is possible that the different results occurred due to different cellular characteristics. We hypothesized that c-UFA treatment might modulate the activities of some antioxidant enzymes to affect the resistance of cells to DOX. In the present study, we examined how c-UFA pretreatment affected DOX cytotoxicity on mouse leukemia cell line, P388, and its resistant subline, P388/DOX, which we found to have significantly higher glutathione peroxidase (GPx) activity as well as P-glycoprotein (p-gp) overexpression. We chose two c-UFAs, gamma-linolenic acid (GLA) (18:3n-6) and docosahexaenoic acid (DHA) (22:6n-3). Cytotoxicity was measured by MTT (3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue exclusion assays. DOX accumulation and p-gp expression were measured by flow cytometry. The activities of catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and GPx were determined for both cell lines with and without treatment with GLA or DHA. Significant DOX accumulation occurred in both cell lines with GLA or DHA pretreatment, but without any change in p-gp expression in either cell line. Sensitivity to DOX cytotoxicity was improved by GLA or DHA pretreatment in P388/DOX in which only SOD activity was significantly increased, but not in the parental cell line P388 in which both SOD and CAT were significantly increased by the pretreatment. However, combined pretreatment of GLA or DHA with antioxidants, pyrrolidinedithiocarbamate (PDTC) or Vitamin C, could sensitize not only P388/DOX but also P388 cells to DOX. We conclude that the effects of c-UFA pretreatment on the sensitivity of cancer cells to DOX not only depend on the change in drug accumulation but also the change in the levels of antioxidant enzyme activities, and suggest that combined administration of c-UFAs, antioxidants, and DOX may be more effective in treating leukemia.

The objective of this study was to develop a simulation system that optimizes the pharmacokinetic parameters of drug carriers for anticancer agents in order to maximize their anticancer effects. The pharmacokinetic/pharmacodynamic (PK/PD) model of doxorubicin (DOX) encapsulated into liposomes has been developed for mice and each parameter required for simulations was obtained in the peritoneally inoculated P388 leukemia model in mice. PK parameters, which describe the dispositions of free and liposomally encapsulated DOX, were obtained by kinetic analysis of experimental data in this study, as well as from literature. PD parameters, which describe the growth and death rate of cancer cells in vivo, were also determined. The PK/PD model developed in this study is capable of simulating the time course of the number of cancer cells quantitatively and evaluating the significance of each parameter on the carrier system for DOX. Simulations based on the PK/PD model predict the optimum rate of drug release from long circulating liposomes as 0.06 h(-1) for maximum anticancer effect. Thus, this simulation system provides useful information relative to the optimization of drug carriers for DOX.

The effects of 1-methyl-3-propyl-7-butylxanthine (MPBX), a xanthine derivative, on doxorubicin (DOX)-induced antitumor activity against DOX-sensitive P388 leukemia (P388) and DOX-resistant P388 leukemia (P388/DOX) have been examined. In P388-bearing mice, the combination of MPBX with DOX increased the antitumor activity of DOX 1.6-fold. In contrast, in P388/DOX-bearing mice, DOX alone did not decrease the tumor weight, whereas in combination with MPBX it significantly decreased the tumor weight in the control group by 50%. The increase in DOX-induced antitumor activity caused by MPBX was correlated with the DOX concentration in the tumors. The DOX concentration in the tumors of P388- and P388/DOX-bearing mice in the MPBX combination group increased by 1.3-fold and 2.2-fold, respectively, compared to the level in the DOX-alone group. On the other hand, there was no increase in the DOX concentration in the heart or liver in both types of tumor-bearing mice treated with MPBX. In vitro, the facilitated DOX influx and suppressed efflux by MPBX in both types of tumor cells were similar, suggesting that MPBX acts on the same site in both types of cells. P388/DOX overexpressed P-glycoprotein, i.e. the inhibitory order of DOX efflux caused by the inhibitor of P-glycoprotein was P388 < P388/DOX. However, the effect of MPBX was P388 > P388/DOX. Therefore, we expect that the site of attack by MPBX is not P-glycoprotein.

The effect of the anti-tuberculosis drug rifampicin on pirarubicin activity was investigated in multidrug-resistant cells overexpressing P-glycoprotein. Rifampicin increased the sensitivity of pirarubicin to anthracycline-resistant mouse leukemic P388 cells and significantly enhanced the cytotoxicity and intracellular accumulation of pirarubicin in resistant cells, but had no effect in parent cells. By contrast, two other rifamycins, rifamycin B and SV, had no effect on pirarubicin accumulation in resistant cells. Rifampicin also enhanced pirarubicin-induced apoptosis and G2/M blockade on the cell cycle in resistant cells. These results show that rifampicin enhances the cytotoxic action of pirarubicin in resistant cells, at least partly via the inhibition of cellular pirarubicin efflux.

Aim: An in vitro cellular pharmacological study was undertaken to characterize the latency of modulating activity by the chemosensitizer, PSC 833., Methods: PSC 833 was evaluated for cytotoxicity in the MDR P388/ADR and MCF7/ADR cell lines. Cellular uptake levels and intracellular DOX distribution characteristics were assessed by flow cytometry and fluorescence microscopy, respectively. These parameters were correlated with the chemosensitivity of the MDR cells under varying conditions of exposure to PSC 833 and DOX., Results: PSC 833 (1 microM) provided near complete MDR reversal and exhibited significant latent modulating activity indicative of a sustained inhibition of the PGP pump in P388/ADR and MCF7/ADR cells after removal of the MDR modulator. Interestingly, complete latent chemosensitizing activity could be achieved by pulsing cells with PSC 833 prior to DOX exposure. Increases in cellular DOX uptake by individual P388/ADR and MCF7/ADR cells induced by MDR modulators correlated well with their ability to chemosensitize the resistant tumor cells where intracellular DOX levels approaching those obtained for sensitive wild type cells was observed for PSC 833., Conclusion: The MDR modulator PSC 833 exhibits latent MDR modulating activity that appears well suited for modulating PGP mediated MDR in vivo where chemosensitization is complicated by difficulties in synchronizing therapeutic levels of modulator and anticancer drug at the tumor site.

Polyalkylcyanoacrylate (PACA) nanoparticles loaded with doxorubicin allowed multidrug resistance to be overcome in vitro. However, increased cytotoxicity is not always correlated with an increased level of intracellular drug. Although we have previously shown that PACA nanoparticles are not endocytosed by tumour cells, we report here that a direct interaction between nanoparticles and cells is a necessary requirement for overcoming resistance. In addition, the results showed that the degradation products of PACA (mainly polycyanoacrylic acid) in the presence of doxorubicin are able to increase both accumulation and cytotoxicity, thus suggesting the formation of a doxorubicin-polycyanoacrylic acid ion pair. It is therefore concluded that resistance is overcome as a result of both the adsorption of nanoparticles to the cell surface and increased doxorubicin diffusion by the accumulation of an ion pair at the plasma membrane.

With few exceptions, the major limit to high-dose chemotherapeutic treatments is the severity and duration of drug-induced myelosuppression. We have recently developed a monoclonal antibody, MAD11, which reacts with the potent anti-tumour antibiotic doxorubicin and other anthracyclines. To protect directly pluripotent stem cells and cells of the haematopoietic microenvironment in the bone marrow against doxorubicin cytotoxicity, the monoclonal antibody MAD11 was injected into the tibial bone of mice before chemotherapeutic treatment. All mice pretreated intratibially with MAD11 and injected with 14 mg kg(-1) body weight of doxorubicin survived, whereas 41% of mice treated with doxorubicin alone died. At a higher dose of doxorubicin (18 mg kg(-1)), early mortality (first 6 days) was similar in the groups, but no deaths were observed thereafter in the intratibially MAD11-treated group, whereas most of the mice treated with doxorubicin alone died. Data obtained in mice injected with P388 leukaemia cells showed that the intratibial injection of MAD11 did not compromise the anti-tumoral activity of doxorubicin. Moreover, the administration of the anti-doxorubicin monoclonal antibody before chemotherapeutic treatment effectively reduced apoptosis induced by doxorubicin in the bone marrow cells. These data suggest the usefulness of monoclonal antibodies against chemotherapeutic drugs in the local protection of bone marrow without influencing the anti-tumour properties of the drug.

Antitumor activities of liposomes containing adriamycin (L-ADM) and their distribution process into tumour cells were analysed. The lipid composition of the liposomes was dimyristoylphosphatidylglycerol (DMPG)/egg phosphatidylcholine/cholesterol/adriamycin (ADM) in a molar ratio of 11.4:2:12:1.3. Liver-metastasizing murine tumour models, M5076 and L5178Y-ML, were used. In vivo antitumour effect against these tumour models was assessed from increase in life span (ILS). The survival prolongation effect of L-ADM in mice with liver failure caused by M5076 was significantly higher than that of F-ADM. In contrast, significant enhancement of the effects by encapsulation in liposomes was not observed in L5178Y-ML-bearing mice. In vitro cytostatic activities of L-ADM against M5076 cells as well as against other tumour cell lines were lower than those of F-ADM. The in vitro kinetic study on the distribution of L-ADM to the tumour cells revealed that ADM in L-ADM was taken up into the tumour cells mainly after it was released from the liposomes rather than taken up as the liposomal form. Among the cell lines tested, M5076 cells had the highest phagocytic activity and therefore the highest uptake activity of ADM during incubation with L-ADM. These findings suggest that the augmented antitumour activity of L-ADM in M5076-bearing mice was the result of phagocytosis of L-ADM by M5076 cells as well as the reduction of toxicity, prolonged retention of ADM in systemic circulation, and liver accumulation of ADM after administration of L-ADM.

SDZ PSC 833 (PSC 833) is a non-immunosuppressive analogue of cyclosporin A and is a potent modifier of P-glycoprotein (P-gp)-mediated multidrug resistance. The present study was undertaken to evaluate whether doxorubicin (DOX) pharmacokinetic and anti-tumour activity on P388- and P388/DOX-resistant leukaemia was modified by PSC 833 pretreatment. P388- or P388/DOX-bearing mice were given PSC 833 intraperitoneally 30 min before an intravenous injection of DOX. The levels of DOX were determined by a high-performance liquid chromatography method in leukaemic cells and in normal tissues (heart, lung, liver, small intestine, kidney and spleen). In all tissues, DOX concentrations were significantly increased in mice pretreated with PSC 833. The difference was greatest in P-gp-overexpressing P388/DOX cells, the DOX area under the curve being approximately seven times greater after PSC 833 and DOX than after DOX alone. In P388 cells the difference was approximately 2.5 times, as in the majority of normal tissues. As expected DOX levels in P388 cells were higher than in P388/DOX cells in mice treated with DOX alone, whereas after PSC 833 and DOX the levels of DOX were similar in the two leukaemic lines. In spite of this PSC 833 was unable to reverse the resistance to DOX of P388/DOX leukaemia in vivo, suggesting that mechanisms other than P-gp expression are responsible for resistance.

Two methods based on the reversion of adriamycin-resistance o the increase of Rhodamine 123 accumulation in a multidrug resistant (MDR) cell line have been simplified and adapted for the screening of MDR reversal agents. Both methods are carried out in microtiter plates, are highly sensitive and can be easily automated. In both assays verapamil and the cyclosporine derivative PSC 833 could be detected at concentrations lower than 1 and 0.05 microM, respectively. Depending on the MDR cell line used, drugs exhibiting the collateral sensitivity phenomenon can be selected in the cytotoxicity assay, while interferences due to sample toxicity are easily avoided in the dye accumulation assay.

Cancer chemotherapy frequently fails, because tumors develop multiple drug resistance (MDR). Pharmacological efforts to reverse this MDR phenotype and sensitize resistant tumor cells have utilized verapamil (VER) to inhibit p-glycoprotein function and buthionine sulfoximine (BSO) to inhibit glutathione synthesis. Our previous results indicate that restriction of two amino acids, tyrosine (Tyr) and phenylalanine (Phe), may potentially suppress the MDR phenotype. These results show that in vivo Tyr and Phe restriction improves the therapeutic response of a metastatic variant of B16-BL6 (BL6) murine melanoma to adriamycin (ADR) and B16 melanoma to levodopa methyl ester. We examine whether in vitro limitation of Tyr and Phe suppresses ADR resistance of BL6 cells and whether Tyr-Phe modulation of the MDR phenotype is applicable to other tumor types, particularly P388 murine leukemia. Mechanisms underlying Tyr-Phe modulation of ADR resistance are examined in the presence of VER and BSO, singly and in combination. Our results indicate that in vitro Tyr and Phe restriction has no effect on BL6 resistance to ADR. However, Tyr and Phe restriction does increase the sensitivity of ADR-resistant P388 cells to ADR without affecting drug efflux, ADR uptake, or glutathione levels. In addition, this enhanced ADR sensitivity of P388 cells is even more pronounced in the presence of BSO. Suppression of ADR resistance in P388-resistant cells by Tyr and Phe restriction indicates that Tyr- and Phe-mediated modulation of the MDR phenotype is possible and that Tyr and Phe restriction may be useful as a potential adjuvant to effective cancer chemotherapy.

We studied the effects of the protein synthesis inhibitor, cycloheximide (CH), on cell killing by doxorubicin (DOX) in vitro and in vivo. At the concentration of CH used (1 microgram/ml) the cytotoxicity of DOX was reduced in cultured P388 leukemia cells. An analysis of the DNA histogram obtained by flow cytometry showed that DOX exerts its growth-inhibitory effect by blocking the cell cycle at the G2/M phase in P388 cells. Treatment with CH diminished this blocking effect. When CH was added to the growth medium before DOX exposure, no change in intracellular DOX accumulation was observed. Treatment with CH (15 mg/kg) significantly diminished the lethality of DOX (20 mg/kg) in mice and it also reduced the antitumor activity of mice with P388 leukemia. Thus, CH inhibited cell death induced by DOX in vitro and in vivo. These results suggest that CH has an antagonistic effect on the pharmacological actions of DOX in cells and mice. The cytoprotective effect of CH may be due to protein synthesis inhibition.

We describe here investigations into the ability of a tiapamil derivative, Ro44-5912 to overcome multidrug resistance (MDR) in doxorubicin (ADR)-resistant murine leukemic P388 cells. This compound has the formula: C27H39NO4S2.1:2C6H8O6, a M. W. of 858 and is structurally similar to verapamil, an established inhibitor of P-glycoprotein (PGP). We have compared the MDR modulating properties of Ro44-5912 with verapamil in P388ADR cells. Doxorubicin concentration required to achieve 50% inhibition of growth (IC50) for P388ADR cells was found to be 24 microM. In contrast, treatment of P388ADR cells with Doxorubicin and 3 microM verapamil decreased the IC50 value to 2.5 microM. A further decrease was observed with 3 microM Ro44-5912 treatment, where an IC50 value of 1.1 microM was obtained. Doxorubicin accumulation was also determined by flow cytometry in order to determine whether the increased levels of chemosensitivity observed for Ro44-5912 were reflected by increased cellular drug uptake. The results revealed that Ro44-5912, at equivalent concentration, increased doxorubicin accumulation in P388ADR cells beyond that obtained with verapamil whereas no effects were seen with the parental P388 cells. The effect of Ro44-5912 on the binding of C219 monoclonal antibody to PGP in MDR cells was also studied and found not to decrease C219 expression on P388ADR cells.

We studied the potentiation of doxorubicin (DOX) activity in multidrug-resistant (MDR) cells by buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, and by cepharanthine (CE), which interacts with P-glycoprotein. The glutathione (GSH) of MDR cells was approximately 1.5-fold greater than that of the parental cell line. BSO reduced GSH content of MDR cells compared to that of the sensitive ones. The BSO treatment (50 microM) enhanced the effect of DOX by 1.8-fold, while CE caused a greater reversal of drug resistance. The combination of BSO with CE produced further potentiation of DOX activity in an antiproliferative effect. Pretreatment of cells with BSO did not alter the cellular accumulation of DOX in the absence or presence of CE. The addition of BSO (30 mM) to the drinking water of mice reduced the tissue levels of GSH in tumor cells, suggesting that the marked decrease in GSH might diminish the ability of that tumor to resist DOX. Combined administration of CE and DOX resulted in enhancement of DOX antitumor activity and prolongation of survival time. The survival of mice treated with BSO and CE as a supplement to DOX treatment was superior that of mice receiving DOX alone. These studies demonstrated that the combinations of BSO with CE may be useful for killing drug-resistant tumor cells.

The lipophilic anthracycline antibiotic annamycin (Ann) was entrapped in liposomes of different size [median diameter: 1.64 microns, multilamellar liposomal Ann (L-Ann); 0.030 micron, small unilamellar Ann (S-Ann)] with > 90% entrapment efficiency and tested in vitro against four pairs of sensitive and multidrug-resistant (MDR) tumor cell lines and in vivo by the i.v. route in five tumor models: advanced s.c. B16 melanoma; s.c. M5076 reticulosarcoma; lung metastases of Lewis lung carcinoma; and s.c. KB and KB-V1 xenografts in nude mice. Predetermined optimal doses of the different formulations were used and the results were compared with doxorubicin (Dox). In vitro, Ann, either in suspension in 10% dimethyl sulfoxide (F-Ann) (1 mg/ml) or entrapped in liposomes, was able to partially overcome resistance in all four pairs of sensitive and MDR KB, 8226, P388, and CEM cell lines (resistance indexes 63, 269, 333, and 356 for Dox versus 4, 5, 19, and 8.7 for L-Ann, respectively). In vivo, both F-Ann and liposome-entrapped Ann were slightly more effective than Dox in inhibiting the growth of advanced s.c. B16 melanoma tumors. L-Ann was markedly more effective than Dox and moderately more effective than F-Ann in prolonging the life span of animals bearing s.c. M5076 and lung metastases of Lewis lung carcinoma tumors. All drugs were equally effective at optimal doses in delaying the growth of s.c. KB xenografts, whereas all Ann formulations were markedly more effective than Dox in delaying the growth of s.c. KB-V1 (MDR) xenografts. In all in vivo experiments, S-Ann was consistently more effective than L-Ann and L-Ann was more effective than F-Ann. These results indicate that (a) Ann is more effective than Dox by the i.v. route against several tumor models and that MDR tumors are partially not cross-resistant to Ann both in vitro and in vivo, (b) liposomes enhance the in vivo antitumor properties of Ann, and (c) small liposomes are more effective than large liposomes in enhancing Ann antitumor activity.

This paper reports studies on P388 leukemic cells sensitive and resistant to ADR and VCR. The P450 dependent enzyme system and the cytosolic calcium were estimated and are discussed in relation to MDR. It could be shown, in comparison to sensitive P388 cells, that the plasma membrane permeability for fluorescent dyes like rhodamine 123 and fura-2 and the biotransformation of xenobiotics are changed in resistant cells. Whereas the transport behaviour for the dyes is similarly induced in resistant cells independent of the drug which made the MDR, the P450 dependent enzyme activities are strongly increased in P388/ADR in comparison to the P388 and P388/VCR. The cell cycle analysis shows the same effect. Many more cells of P388/ADR are in the S-phase in comparison to P388 or P388/VCR. The LI is also increased in this direction. Therefore, it can be concluded that, depending on the kind of drug which made the MDR, different biochemical mechanisms are activated.

Previous studies have clearly demonstrated that polyisobutylcyanoacrylate (PIBCA) doxorubicin-loaded nanoparticles (NS-Dox PIBCA) can overcome the resistance of P388/ADR cells. In the present paper, we found that overcoming multidrug resistance with the aid of doxorubicin (Dox) loaded onto these nanoparticles was associated with an increased intracellular drug content. Indeed, after a 6-h incubation period, the amount of cell-associated drug was 5 times higher when the cells were incubated with NS-Dox PIBCA as compared with free Dox. Further experiments, such as uptake studies in the presence of cytochalasin B or efflux studies, indicated a possible mechanism of nanoparticle/cell interaction. These results suggested that nanoparticles did not enter the cells by an endocytic process, in contrast to a previous hypothesis.

The effect of thaliblastine (TBL, NSC-68075), a plant alkaloid, in over-coming multidrug resistance was investigated in doxorubicin (ADR)-resistant murine leukemic P388/R-84 cells. In the soft agar clonogenic assay, a nontoxic concentration of TBL (2 microM) reduced the 50% inhibitory dose of ADR (1-h exposure) from 10.8 to 1.4 microM with a dose modification factor of 7.7. Continuous treatment of P388/R-84 cells with ADR and TBL for 24 h further lowered the 50% inhibitory dose from 3.5 to 0.07 microM, the resistance level being decreased from 233-fold in the absence of TBL to 4.7-fold in the presence of TBL as compared to the parental P388 cells. Although ADR or TBL individually had no detectable effects on cell cycle traverse, the combination of the two drugs caused a significant G2 block. Flow cytometric analysis showed that TBL enhanced ADR retention in P388/R-84 cells in a dose- and time-dependent manner. TBL partially blocked the photolabeling of P-glycoprotein with [3H]azidopine, and this blocking effect was further enhanced in combination with ADR. Our results indicate that TBL can reverse multidrug resistance by direct interaction with P-glycoprotein, thereby increasing cellular ADR retention.

Annamycin (AN) is an anthracycline antibiotic with high affinity for lipid membranes which is being developed for clinical studies formulated in liposomes. We studied the in vitro cytotoxicity, cellular pharmacology, and DNA damage induced by AN in P388 cells sensitive and resistant to doxorubicin (DOX). AN was as cytotoxic as DOX against P388-sensitive cells and about 50 times more cytotoxic than DOX against P388-resistant cells (resistance index 5 for AN versus 250 for DOX). Cellular uptake of AN by sensitive cells was 2-3-fold higher than that of DOX. In resistant cells, cellular uptake of AN and DOX was approximately 65% and 30%, respectively, of the cellular uptake in sensitive cells. As a result, cellular uptake of AN by resistant cells was higher than uptake of DOX by sensitive cells. DOX was fully retained in sensitive cells while it was effluxed rapidly from resistant cells. In contrast, efflux of AN was similar in sensitive and resistant cells, thus suggesting that it is not mediated by P-glycoprotein. AN was more effective than DOX in inducing single DNA breaks, double DNA breaks, and DNA-protein cross-links, both in sensitive and resistant cells, although DNA damage was lower in resistant cells than in sensitive cells. DNA lesions induced by AN in resistant cells were similar to or greater than those induced by DOX in sensitive cells. These studies indicate that the lack of cross-resistance between DOX and AN appears to be related, at least in part, to the relatively higher cellular uptake of AN compared with DOX and is associated with the ability of AN to induce significant DNA damage in resistant cells.

The impact of the novel chemosensitizer ((2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxy-beta- phenethyl)amino]propyloxy)benzenesulfonyl))indolizine (SR33557) on the intracellular distribution of doxorubicin (DOX) within the multidrug-resistant murine P388/ADR leukemia cell line was studied by fluorescence microscopy. We found that under conditions which modulated multidrug-resistant (30 microM SR33557 for 1 h), P388/ADR cells presented an original sequestration of DOX in large intracellular vesicles, where SR33557 is itself sequestered, as seen by colocalization studies. Colocalization experiments with lysosomal and mitochondrial probes suggest that these vesicles are neither mitochondrial in nature nor functional lysosomes. To investigate the biochemical basis for this effect, we studied the impact of SR33557 on the sphingolipid metabolism of P388/ADR cells. We observed that although P388/ADR cells normally catabolized exogenous [3H]sphingomyelin, when pretreated with SR33557 they showed almost complete inhibition of sphingomyelin breakdown. Finally, in order to demonstrate that the inability of P388/ADR cells to degrade sphingomyelin in the presence of SR33557 (which is a potent inhibitor of acid lysosomal sphingomyelinase) leads to phospholipid accumulation, we performed electron microscopy where we observed laminated inclusions. These morphological modifications are similar to those observed in Niemann-Pick disease lymphoblastoid cell lines which are inherently deficient in acid sphingomyelinase activity. The observation that, in the absence of SR33557, these Niemann-Pick disease cell lines presented similar DOX sequestration to that of SR33557-treated P388/ADR cells strongly suggests that DOX accumulates in SR33557-induced myeloid bodies. The redistribution of DOX within these vesicles, perhaps by preventing its expulsion by P-glycoprotein, may be a key in discovering the mechanism of action of SR33557.

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.

Taking into account the possible regulatory influences of the beta-adrenergic system on lymphocyte proliferation as well as the proposed role of cyclic 3'-5'-adenosine monophosphate (cAMP) in the modulation of multidrug resistance (MDR) in tumour cells, we have tried to assess the status of the interactions between the beta-adrenergic system and a mouse lymphocytic leukemia, the P388, both as a doxorubicin-sensitive (P388) and -resistant (MDR) variant (P388/DXR). P388 showed a low total number of high affinity 125I-pindolol binding sites (340 +/- 33/cell, Kd 108 pM) when compared with normal splenocytes (1221 +/- 67 sites/cell, Kd 97 pM). The number of beta-adrenergic receptors was even lower in P388/DXR cells (230 +/- 41 sites/cell, Kd 101 pM). In addition, these receptors were subnormally expressed on the cell surface: only 26% and 52% of the total receptors were surface receptors in P388 and P388/DXR, respectively, whereas it was 87% in normal splenocytes. Isoproterenol slightly (less than 1-fold) stimulated cAMP accumulation in P388 and P388/DXR; the stimulation observed in splenocytes was 2.5-fold. In addition, the basal levels of cAMP appeared to be low (0.48 +/- 0.05 pmoles/10(6) cells in P388 and 0.71 +/- 0.08 pmoles/10(6) cells in P388/DXR; 3.47 +/- 0.28 pmoles/10(6) cells in splenocytes) in the two leukemias and they were only slightly (less than 2-fold) increased by forskolin, which otherwise stimulated about 15-fold cAMP accumulation in splenocytes; thus, P388 and P388/DXR were probably also defective in their adenylate cyclase activity. It can be concluded that, owing to multifactorial mechanisms, the lymphocytic leukemia P388, also as an MDR variant, is minimally sensitive to the direct influences of the beta-adrenergic system, probably including any effect of this system on drug-sensitivity.

The biochemical activity of cepharanthine and the possible mechanism by which it reverses the resistance to doxorubicin in P388 leukemia cells were examined in vitro. The microfluorometric analysis of the cellular level of doxorubicin in drug-resistant cells showed that cepharanthine markedly enhanced the sensitivity of doxorubicin against resistant cells in the cellular level. Cepharanthine also enhanced the inhibitory effect of doxorubicin on the incorporation of thymidine into DNA in resistant cells. The analysis of DNA histogram obtained by flow cytometry showed that doxorubicin exerted its growth-inhibitory effect by blocking the cell cycle at the G2 phase in P388 cells. At higher concentrations, doxorubicin prolonged the S phase and inhibited cell cycle progression to the G2/M phase in cells. The treatment with cepharanthine potentiated these blocking effects induced by doxorubicin in cells. It seems that the modifications of the biological effect of doxorubicin by cepharanthine are due to the change of their ability to induce DNA damage in cells.

We investigated the effects of seven isoquinoline derivatives in overcoming resistance to vinblastine in Adriamycin-resistant mouse leukemia P388/ADR cells and human myelogeneous leukemia K562/ADR cells. N-(2-Methylpiperazyl)-5-isoquinoline-sulfonamide (H-7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) did not reverse resistance to vinblastine in these resistant cells. N-[2-[N-[3-(4-Chlorophenyl)-2-propenyl]amino]ethyl]-5- isoquinolinesulfonamide (H-86) and N-[2-[N-[3-(4-chlorophenyl)-1-methyl-2-propenyl]- amino]ethyl]-5-isoquinolinesulfonamide (H-87) caused significant accumulation of intracellular vinblastine and marked reversal of the resistance to vinblastine in both resistant cell lines. Addition of a formyl group at the terminal amino group of H-86 (H-85) or addition of an aminoethyl group to the nitrogen atom at the sulfonamide group of H-86 (W-66) reduced those activities. The activity on vinblastine accumulation seems to correlated with the hydrophobicity of the compounds. The compounds that effectively reversed resistance to vinblastine inhibited [3H]vinblastine efflux and photoaffinity labeling of P-glycoprotein with a photosensitive analogue of vinblastine, N-(p-azido-(3-[125I]iodo)-salicyl)-N'-beta-aminoethylvindesine. Although these isoquinoline derivatives inhibited protein kinase A and protein kinase C with various potencies, these inhibitory activities did not correlate with the reversal of drug resistance. These results indicate that hydrophobic isoquinoline derivatives reverse multidrug resistance due to the suppression of drug binding to P-glycoprotein, without involvement of their activities on protein kinase A and protein kinase C.

The effect of sodium chloride on the cytotoxicity of 4'-O-tetrahydropyranyldoxorubicin (pirarubicin), a novel anthracycline derivative, was investigated on P388 mouse leukemia cells in vitro. The modifications in efficiency of uptake and killing action of pirarubicin were found to be dependent on the ionic strength of the medium. The relationship between intracellular drug accumulation into cells and cell killing was pointed out.

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.

The activity of dipyridamole and its possible mechanisms which reverse the resistance of pirarubicin were studied in a P388 mouse leukemia cell lines. Dipyridamole alone was minimally cytotoxic in both of the doxorubicin-resistant cell line (P388/DOX) and the sensitive parent cell line (P388/S), but reversed pirarubicin-resistance in a dose-related manner in P388/DOX cells. A similar dose-response relationship was observed for dipyridamole by increasing net intracellular pirarubicin accumulation. The increase was a result of secondary blocking of enhanced pirarubicin efflux from P388/DOX cells. In contrast, dipyridamole did not affect cytotoxicity and transport in the drug-sensitive cell line. It is suggested that dipyridamole is a useful drug for modulation of the multidrug-resistance of cells.

beta-D-galactose-containing glycoproteins were prepared from cells P-388 leukemia and from P-388 leukemia cells with induced resistance to doxorubicin. It was shown by HPLC method that plasma membranes from resistant cells contain 4-4.5% P-glycoproteins and plasma membranes from sensitive cells contain P-glycoproteins about 10 times lower.

Lipids extracted from doxorubicin-resistant murine leukemia cells (P388/ADR) contained greater relative amounts of myristic and palmitoleic acids than lipids from sensitive cells (P388). This was seen in both the phospholipid and neutral lipid fractions under two nutritional conditions. Correspondingly in P388/ADR cells, myristic acid comprised a greater proportion of the products of the fatty acid synthetase system, and acyl-CoA 9-desaturase activity was transiently greater than in P388. Similar alterations in myristic acid synthesis were exhibited by DC3F/AD X, N417/VP-16, and P388/AZQ30U cells but not by CHRC5 or HL60/AR cells. This alterations was independent of alterations in the P180 glycoprotein and might be linked via the myristoylation of proteins to a different mechanism of drug resistance. Doxorubicin-resistant P388/ADR cells also exhibited a much higher rate of oxidative energy production.

The intracellular uptake, retention and cytotoxicity of pirarubicin, an anthracycline derivative, combined with chlorpromazine were investigated in doxorubicin-resistant mouse P388 leukemia (P388/DXR) cells. The number of viable cells was determined by the dye exclusion method. Chlorpromazine increased the cytotoxicity of pirarubicin in a dose-related manner in P388/DXR cells. A similar dose-response relationship was observed for chlorpromazine in increasing net intracellular pirarubicin accumulation. The accumulation was based on block of enhanced pirarubicin efflux from resistant cells by chlorpromazine. However, chlorpromazine did not affect cytotoxicity or transport of pirarubicin in the drug-sensitive cell line. The possible mechanisms of the restoration of pirarubicin sensitivity in P388/DXR cells by chlorpromazine are discussed.

Lymphoid cell lines resistant to doxorubicin, P388/R, were characterized by: 1) decreased intracellular acumulatin and retention of DOX; 2) decreased amount of DOX-induced DNA lesions; and 3) rapid repair of DOX-induced DNA lesions. Using the highest noncytotoxic concentrations of three modulators; 2 calcium channel blockers, Verapamil (VEP) and DMDP and a nucleoside transport inhibitor, Dipyridamole (DIP), restoration of Doxorubicin (DOX) sensitivity in vitro against P388/R cells was partial; resistance was reduced from approximately 200 to 10 fold, although DOX accumulation in the resistant cells in the presence of the modulators was completely restored. The DNA single-strand break (SSB) level induced by DOX in P388/S cells (1371 +/- 144 rad equivalents) was significantly higher than in P388/R cells (74 +/- 17 rad equivalents). The effects of VEP, DMDP and DIP on the induction of DNA SSBs by DOX in P388/R were different. DMDP and DIP potentiated the DOX-induced DNA SSBs by 30% each and VEP by 15%. Furthermore, while VEP and DIP had no significant effects on the rapid repair of DOX-induced SSBs, no significant repair of DNA lesion was observed in P388/R treated with DMDP at 1.2 microM, a non-cytotoxic concentration. These data indicate that although these modulators can effectively restore the intracellular accumulation and retention of DOX, these conditions although essential are not sufficient for the complete restoration of DOX sensitivity in this highly resistant cll line. The ability of a calcium antagonist, DMDP, to circumvent DOX resistance might be related not only to the modulation of drug retention, but also to the ability to retard the repair of DOX-induced DNA SSBs.

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.

Electron microscopy of cultured P388 leukemia cells revealed morphological differences between the Adriamycin-resistant (P388/ADR) and -sensitive (P388/0) cell lines. P388/ADR cells showed longer villus-like protrusions and large foldings of the plasma cell membrane, whereas P388/0 cells had only short membrane protrusions. Western blot analysis of cells revealed the increased expression of a glycoprotein of 170-180 kDa (P-glycoprotein) in P388/ADR cells as compared with P388/0 cells, which is consistent with the findings of other studies. A modulation of the membrane morphology of in vitro P388/ADR cells was evident from one i.p. in vivo passage for 10 days, by a reversion to the non-ruffled sensitive-cell morphology. Long-term P388/ADR culture cells showed a reduction in membrane folding with as little as a 4-h exposure to the peritoneal ascitic fluid obtained when the tumor was harvested. However, there was no alteration in the expression of P-glycoprotein or in the sensitivity to Adriamycin in either of the P388 cell lines when grown in vivo or when exposed to ascites fluid from an i.p. tumor. Thus, the modulation of membrane morphology was related to the tumor cell environment rather than the abundance of P-glycoprotein in the plasma membrane.

Using mice BDF1 it has been shown that the period of retention of Doxorubicin (Dx) is shorter in the leukemia P388 cells with induced antibiotic resistance (P388/Dx) as compared to P388 cells sensitive to Dx. Administration of Verapamil (Vp) to animals leads to an increase of Dx concentration in the leukemia P388/Dx cells during a 240 min observation period. Vp promotes the therapeutic effect of Dx on P388/Dx bearing mice. It can be suggested that the mechanism of Vp action consists in the damaged Dx elimination from cells with induced resistance, since Vp doesn't change the period of circulation of the antibiotic in the blood plasma of mice.

The effects of certain compounds on the in vitro growth rate and the sensitivity to doxorubicin of P388 murine leukaemia cell line and of a doxorubicin-resistant subline (P388/ADR) were studied. The calcium channel blocking activity of these compounds was evaluated by measuring their effects on the sodium-dependent and membrane potential-dependent calcium uptake in synaptic plasma membrane vesicles. At non-inhibitory concentrations, verapamil, dipyridamole, meclizine and nicardipine were highly active in restoring the sensitivity to doxorubicin of P388/ADR cells. Moderately active were propranolol, N-(beta-diethylaminoethyl)-N-(beta-hydroxy-beta-phenylethyl)-2,5-dich loranaline (MDL-6792), thioridazine and chlorocyclizine, while nifedipine, guanethidine, phentolamine, chloroquine and papaverine had zero or only minimal synergistic activity to doxorubicin in this cell line. Doxorubicin synergistic activity could not be demonstrated in the parent drug-sensitive cell line. No sodium-dependent or membrane potential-dependent calcium uptake could be demonstrated in vesicles prepared from plasma membranes of either cell line. There is no correlation between the ability of these compounds to inhibit calcium uptake in synaptic vesicles and their potency in restoring the sensitivity of P388/ADR cells to doxorubicin.

Calmodulin inhibitors enhance the cytotoxic effects of doxorubicin (DOX) in DOX-resistant (P388/DOX) P388 mouse leukemia cells by augmenting cellular accumulation and retention of drug. In P388/DOX cells which are cross-resistant to vinblastine (VLB) and vincristine (VCR), cell kill following treatment with VLB and VCR alone was evident only after 12 hr of treatment. Additionally, the 2- to 10-fold increase in cytotoxicity of the vinca alkaloids in the presence of 2 and 4 microM trifluoperazine (TFP) was observed only in P388/DOX cells treated for 12 hr, but not for 3 or 6 hr. However, in DOX-sensitive (P388/S) P388 mouse leukemia cells, cytotoxic effects of VCR but not VLB were apparent after treatment for 3 hr, and cell kill with VLB and VCR was enhanced 2- to 20-fold in the presence of 2 and 4 microM TFP following treatment for 12 hr. Cellular accumulation of [3H]VLB in P388/DOX cells was 12-fold lower than in similarly treated P388/S cells and, in the presence of 2 and 4 microM TFP, cellular VLB levels were enhanced 1.3- to 2.0-fold in P388/S cells and 2- to 8-fold in P388/DOX cells. The effect of TFP in increasing cellular retention of [3H]VLB was more apparent with P388/DOX cells, and retention of [3H]VLB in the presence of 4 microM TFP was enhanced less than 1.5-fold and greater than 4-fold in P388/S and P388/DOX cells respectively. Results from this study and our earlier observations with DOX and TFP in P388/DOX cells demonstrate that: (1) TFP potentiates the cytotoxicity of VLB and VCR in P388/S and P388/DOX cells by augmenting drug accumulation and retention; (2) enhanced cell kill in the presence of TFP with P388/DOX cells is apparent at 1 hr for DOX vs 12 hr for VLB and VCR; and (3) in P388/S cells, TFP has a more striking effect on the cellular accumulation, retention and cytotoxicity of VLB and VCR rather than DOX.

An anthracycline-resistant subline of P388 murine leukemia cells, P388/ADR, has been shown to have an altered total lipid content and composition when compared to the parent line. When specific lipids of the cell lines are compared, it is found that P388/ADR cells contain approximately 3.6 times the amount of triglycerides as P388 cells. Although no differences are noted in the amounts of unesterified cholesterol or total phospholipids in the two cell lines, they do differ in specific phospholipid patterns. P388/ADR cells contain relatively less phosphatidylcholine and more sphingomyelin than drug-sensitive cells. No differences are observed in the content of phosphatidylethanolamine and cardiolipin. The difference in phosphatidylcholine/sphingomyelin ratio (5.57 for ADR-sensitive cells and 3.28 for ADR-resistant cells) may account for the previously observed difference in plasma membrane lipid structural order between these cell lines. Measurements of the relative activity of phosphocholine transferase in the two lines, using the rate of incorporation of 3H-choline into phosphatidylcholine, indicate that lower phosphocholine transferase activity in P388/ADR cells may account for the observed changes in cellular lipid and phospholipid composition.

Experiments were carried out in vitro on adriamycin (ADR) accumulation and cytotoxicity alone and in combination with calcium channel antagonist, verapamil (VRP), in ascites murine tumour models of Ehrlich carcinoma (EAC), sarcoma 180 (S180) and P388 lymphocytic leukaemia (P388). The cytotoxicity was assayed as the inhibition of [3H]-thymidine incorporation into the cellular DNA. ADR, a broad-spectrum anticancer drug, at a concentration of 10 micrograms/ml showed a cytotoxic effect in the order S180 greater than P388 greater than EAC. VRP alone exhibited the DNA synthesis inhibiting activity. The enhanced DNA biosynthesis inhibition of ADR along with VRP was maximal in S180 and marginal in P388 and EAC. The ADR retention after 3 hr of incubation in these tumour models correlated with the cytotoxicity. VRP enhanced the accumulation of ADR in all these cell lines. The property of potentiation in the activity and accumulation of ADR in these tumours exposed to a non-toxic concentration of VRP can best be utilized in cancer chemotherapy where the massive cytotoxic therapy, with a single large dose of ADR, can be substituted with a low dose, along with this drug-response modulator, for better therapeutic results.

A new analog of doxorubicin, 4'-O-methyldoxorubicin, was previously reported to have a pronounced activity against L1210 leukemia, which shows a natural partial resistance to doxorubicin itself. In the present study, lines of P388 leukemia and Ehrlich ascites tumor with acquired resistance to doxorubicin were found to be cross-resistant to 4'-O-methyldoxorubicin, indicating that the natural and the acquired resistance to doxorubicin involve different mechanisms. In vitro studies on the uptake of 4'-O-methyldoxorubicin in the Ehrlich ascites tumor cells indicated that the observed cross-resistance was partly due to a decreased drug uptake in the resistant cells because of an increased extrusion of the drug, in accordance with previous findings on the mechanism of acquired resistance to doxorubicin.

Anticalmodulin and calcium channel blockers have been shown to reverse Adriamycin resistance by reducing the drug efflux from resistant cells. Since cellular calcium and calmodulin levels are probably related to these effects, we have measured the total, membrane-bound, and intracellular calcium levels in Adriamycin-resistant (P388/R) and -sensitive (P388/S) leukemia cells. In P388/R cells, total calcium was approximately 1.4-fold higher than that of P388/S cells. Membrane-bound and intracellular calcium levels were also higher in P388/R cells. No major difference was observed in the calmodulin content of these cells. The P388/R cells had a higher (approximately 1.4-fold) protein content. When calculated on the basis of per unit protein, P388/S and P388/R cells had similar total calcium but a higher intracellular free calcium and calmodulin content in P388/S cells. Thus our studies suggested that the lower drug efflux and increased drug retention in P388/R cells may not be related to calcium and calmodulin levels but may be due to some other membrane-related factors.