Multidrug-resistant Human Tumor Research Articles

Reversal of drug resistance by JS-K and nitric oxide in ABCB1- and ABCG2-expressing multi-drug resistant human tumor cells

Development of resistance to chemotherapy drugs is a significant problem in treating human malignancies in the clinic. Overexpression of ABC transporter proteins, including P-170 glycoprotein (P-gp), and breast cancer resistance protein (BCRP, ABCG2) have been implicated in this multi-drug resistance (MDR). These ABC transporters are ATP-dependent efflux proteins. We have recently shown that nitric oxide (NO) inhibits the ATPase activities of P-gp, resulting in a significant enhancement of drug accumulation and the reversal of multi-drug resistance in NCI/ADR-RES cells, a P-gp-overexpressing human MDR cell line. In this study, we used [O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)-piperazin-1 yl]-diazene-1-ium-1-2-diolate] (JS-K), a tumor-specific NO-donor to study the reversal of drug resistance in both P-gp- and BCRP-overexpressing human tumor cells. We report here that while JS-K was extremely effective in reversing adriamycin resistance in the P-gp-overexpressing tumor cells (NCI/ADR-RES); it was significantly resistant to BCRP-overexpressing (MCF-7/MX) tumor cells, suggesting that JS-K may be a substrate for BCRP. Using another NO-donor (DETNO), we show that NO directly inhibits the ATP activities of BCRP, inducing significant increases in the accumulations of both Hoechst 33342 dye and topotecan, substrates for BCRP. Furthermore, NO treatment significantly reversed topotecan and mitoxantrone resistance to MCF-7/MX tumor cells. Molecular docking studies indicated that while DETNO and JS-K bind to ATP binding site in both ABC proteins, binding score was significantly reduced, compared to the ATP binding. Our results indicate that appropriately designed NO donors may find success in reversing multidrug resistance in the clinic.

Ameliorative Potential of Tamoxifen/Thymoquinone Combination in Patients with Breast Cancer: A Biochemical and Immunohistochemical Study

Background: Breast cancer is one of the most common types of malignancies in females worldwide. Targeting the estrogen receptors alone with tamoxifen, reduces the incidence of estrogen receptor positive tumors but the possibility of development of tamoxifen resistance remains the most important adverse effect. Thymoquinone is an important constituent of the Nigella sativa plant (Black seed). It was found to be cytotoxic in several types of multidrug resistant human tumor cell lines. Objective: To detect the effects of the combination between tamoxifen and thymoquinone in patients with breast cancer. Methods: Eighty female patients with breast cancer were divided into four equal groups: control untreated group, tamoxifen treated group, thymoquinone treated group and tamoxifen + thymoquinone treated group. Tissue malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and transforming growth factor beta1 (TGF-β1) were determined in the tumor tissues. Parts of the tumor were subjected to immunohistochemical examination. The survival rate and the relapse rate were measured every year for five years. Results: Tamoxifen or thymoquinone alone or in combination induced significant increase in the 5-years survival rate, tumor CAT and SOD with significant decrease in the relapse rate, tumor tissue MDA, TNF-α, IL-6 and TGF-β1 and alleviated the immunohistochemical changes with significant increase in tissue caspase-3 expression and significant decrease in tissue bcl2 compared to the control untreated group. Conclusion: Tamoxifen/ thymoquinone combination had a better effect than each of these drugs alone on patients with breast cancer. So, the addition of thymoquinone to tamoxifen may represent a new therapeutic modality for management of breast cancer.

Structure–activity relationships of diverse xanthones against multidrug resistant human tumor cells

Thirteen xanthones were isolated naturally from the stem of Securidaca inappendiculata Hassk, and structure-activity relationships (SARs) of these compounds were comparatively predicted for their cytotoxic activity against three human multidrug resistant (MDR) cell lines MCF-7/ADR, SMMC-7721/Taxol, and A549/Taxol cells. The results showed that the selected xanthones exhibited different potent cytotoxic activity against the growth of different human tumor cell lines, and most of the xanthones exhibited selective cytotoxicity against SMMC-7721/Taxol cells. Furthermore, some tested xanthones showed stronger cytotoxicity than Cisplatin, which has been used in clinical application extensively. The SARs analysis revealed that the cytotoxic activities of diverse xanthones were affected mostly by the number and position of methoxyl and hydroxyl groups. Xanthones with more free hydroxyl and methoxyl groups increased the cytotoxic activity significantly, especially for those with the presence of C-3 hydroxyl and C-4 methoxyl groups.

Doxorubicin and mitomycin C co-loaded polymer-lipid hybrid nanoparticles inhibit growth of sensitive and multidrug resistant human mammary tumor xenografts

Multidrug resistance (MDR) and drug toxicity are two major factors responsible for the failure of cancer chemotherapy. Herein the efficacy and safety of combination therapy using doxorubicin (Dox, D)–mitomycin C (MMC, M) co-loaded stealth polymer-lipid hybrid nanoparticles (DMsPLNs) were evaluated in sensitive and MDR human mammary tumor xenografts. DMsPLN demonstrated enhanced efficacy compared to liposomal Dox (PLD) with up to a 3-fold increase in animal life span, a 10–20% tumor cure rate, undetectable normal tissue toxicity and decreased tumor angiogenesis. These results suggest DMsPLN have potential as an effective treatment of breast cancer.

Delivering Drugs Into the Brain: Barriers and Possibilities

Delivering Drugs Into the Brain: Barriers and Possibilities

Abstract B42: Molecular and cytogenetic characterization of novel multidrug-resistant human tumor cell lines

Abstract Multidrug resistance (MDR) is the major cause of failure to a successful cancer treatment. Cancer cells manifesting MDR phenotype are cross-resistant to many structurally and functionally unrelated drugs. The purpose of this study was to establish in vitro models, i.e., resistant cell lines, to further investigate the mechanisms of MDR. We used the human colon cancer cell line DLD1 and the glioma cell line U87 to establish paclitaxel-resistant cell lines, DLD1 -TxR and U87-TxR, by continuous exposure to stepwise increasing concentrations of the drug. The newly established, DLD1 -TxR and U87-TxR cell lines showed extensive resistance to paclitaxel (301-fold and 103-fold respectively). They also demonstrated cross-resistance to other anticancer agents such as doxorubicin, epirubicin, vinblastin, and etoposide. Cytogenetic data based on inverted-DAPI Banding, demonstrated that both resistant cell lines retained several karyotypic characteristics of their sensitive counterparts and also acquired novel structural or numerical chromosomal aberrations that may be related to resistance to chemotherapy. Interestingly, while the dividing parental U87 cells were mainly near-tetraploid, chemoresistance in the U87-TxR cell line was accompanied by one level ploidy reduction as verified by the increased prevalence of near-diploid cell populations. In addition, the DLD1 -TxR cells showed an increase tendency to loose the Y chromosome. Gene expression analyses revealed a 4-fold increase in the level of mdr1 mRNA in DLD1 -TxR cells. U87-TxR cells demonstrated prominent mdr1 activity since the expression in parental cells was undetectable. In contrast, the level of mrp1 mRNA was 40% decreased in both resistant cell lines. The expression of other MDR-related genes, topo IIα, and lrp, was unaffected by paclitaxel resistance. Flow cytometry analyses showed that the accumulation of rhodamine in resistant cells DLD1 -TxR and U87-TxR was significantly lower than in their sensitive counterparts, 17-fold and 8-fold, respectively. These results indicate that P-gp, encoded by mdr1 gene, is significantly overexpressed in both resistant cell lines and may account for paclitaxel resistance. In conclusion, the two novel resistant cell lines, DLD1 -TxR and U87-TxR may serve as appropriate models for the study of the mechanisms of paclitaxel resistance offering novel insights into potential targets for overcoming MDR that would improve the response to chemotherapy and patient outcome. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B42.

Improvement of tumor targeting and antitumor activity by a disulphide bond stabilized diabody expressed in Escherichia coli.

We have generated an anti-Pgp/anti-CD3 diabody which can effectively inhibit the growth of multidrug-resistant human tumors. However, the two chains of the diabody are associated non-covalently and are therefore capable of dissociation. Cysteine residues were introduced into the V-domains to promote disulphide cross-linking of the dimer as secreted by Escherichia coli. Compared with the parent diabody, the ds-Diabody obtained was more stable in human serum at 37 degrees C, without loss of affinity or cytotoxicity activity in vitro. Furthermore, the ds-Diabody showed improved tumor localization and a twofold improved antitumor activity over the parent diabody in nude mice bearing Pgp-overexpressing K562/A02 xenografts. Our data demonstrate that ds-Diabody may be more useful in therapeutic applications than the parent diabody.

ARRY-649, a member of a novel class of Eg5 kinesin inhibitors

13045 Background: Inhibitors of the Eg5 motor kinesin selectively disrupt mitotic spindles in dividing cells. This selective targeting of microtubule dynamics in dividing cells is expected to translate to broad-spectrum anti-tumor activity while avoiding neuropathic side effects caused by taxanes. To date, quinazolinone Eg5 inhibitors, exemplified by ispinesib, represent the only Eg5 inhibitors reported in clinical trials. Methods: We used X-ray crystallography, biochemical and cell-based assays, pharmacokinetic profiling and in vivo efficacy studies to identify and optimize a potent series of Eg5 inhibitors. Results: We have discovered a distinct series of potent Eg5 inhibitors, exemplified by ARRY-649. ARRY-649 inhibited Eg5 with an IC50 of 0.7 nM, blocked phosphorylation of histone H3 in HeLa cells with an IC50 of 0.3 nM and showed sub-nanomolar activity against a broad panel of human tumor cells lines in in vitro viability assays. Further evaluation of mechanism-of-action in tumor cell lines revealed that ARRY-649 induced a monopolar spindle phenotype and subsequent apoptosis characteristic of Eg5 inhibition. In contrast to paclitaxel, ARRY-649 retained potent activity against multi-drug resistant human tumor cells lines selected to overexpress P-glycoprotein. ARRY-649 demonstrated robust efficacy in the HT-29 human colon tumor xenograft model and was well-tolerated at efficacious doses and schedules. Durable complete- and partial-regressions were observed upon intraperitoneal administration on a 5 mg/kg q4dx3 schedule in the HT-29 model. Similar results were obtained using the HL-60 myelomonocytic xenograft model. Conclusions: ARRY-649 represents a distinct class of potent Eg5 inhibitors with robust activity in preclinical models of human cancer. Based on these observations, we have progressed novel Eg5 inhibitors toward clinical development. [Table: see text]

Therapy of multidrug resistant human prostate tumors in the prostate of nude mice by simultaneous targeting of the epidermal growth factor receptor and vascular endothelial growth factor receptor on tumor-associated endothelial cells.

Inhibiting epidermal growth factor receptor (EGF-R) and vascular endothelial growth factor receptor (VEGF-R) activation with AEE788 can decrease prostate cancer (CaP) growth/progression. We determined whether tumor cells or tumor-associated endothelial cells were the primary target by treating multidrug-resistant (MDR) CaP growing in the prostate of nude mice. MDR human CaP cells with 30-fold increased taxane-resistance were implanted into nude mouse prostates. After 2 weeks, mice were randomized to control, paclitaxel, AEE788, and AEE788/paclitaxel for 10 weeks. Mice were necropsied and tumors stained. AEE788 or AEE788 plus paclitaxel significantly reduced tumor incidence and tumor weight, and eradicated lymph node metastasis. Inhibiting VEGF-R and EGF-R phosphorylation induced apoptosis of tumor-associated endothelial cells causing a second apoptotic wave of surrounding tumor cells. Inhibiting VEGF-R and EGF-R activation on tumor-associated endothelial cells with AEE788 combined with paclitaxel can bypass CaP cell resistance and prevent lymph node metastasis.

Efficient inhibition of multidrug-resistant human tumors with a recombinant bispecific anti-P-glycoprotein x anti-CD3 diabody.

Overexpressing of P-glycoprotein (Pgp) has been shown to be responsible for cancer resistance to multiple chemotherapeutic agents. Immunotherapy with biological agents, such as bispecific antibodies (BsAbs), may represent a promising approach to overcome the emergence of drug resistance. Here we constructed a recombinant BsAb, a diabody, with specificities to both CD3 on human T-lymphocyte and Pgp on cancer cells. The diabody was produced in Escherichia coli in a soluble functional form and purified by an affinity chromatography with a yield of >4 mg/l culture medium in shaker flask. The diabody binds to both CD3 on T-lymphocytes and Pgp on multidrug-resistant (MDR) tumor cells with affinities that are comparable to its respective parental single chain Fv molecules. In the presence of activated human peripheral blood lymphocytes (PBLs), the diabody mediates effectively the lysis of the Pgp-overexpressing human leukemia K562/A02 and epidermoid carcinoma KBv(200) cells, but is much less potent in mediating the lysis of the parent K562 and KB cells. Further, the diabody localized selectively within the K562/A02 xenografts in mice. When combined with activated PBL, the diabody significantly inhibited the growth of K562/A02 and KBv(200), but had no effect on K562 and KB xenografts. In contrast, treatment with doxorubicin, a standard chemotherapeutic agent, only inhibited the growth of K562 and KB, but had no effect on K562/A02 and KBv(200) xenografts. Taken together, our results suggest that the anti-Pgp x anti-CD3 diabody may have a great potential in the treatment of various MDR cancers.

L-Canavanine modulates cellular growth, chemosensitivity and P-glycoprotein substrate accumulation in cultured human tumor cell lines

l-Canavanine (L-CAV) is a naturally occurring l-arginine analog that induces the formation of non-functional proteins in a variety of organisms. Previous studies have shown that L-CAV is cytotoxic for several human tumor cell lines. In this study, we have evaluated the cytotoxicity of L-CAV for both parental and multi-drug resistant (MDR) human tumor cells. We have also determined the effect of L-CAV exposure on cellular expression and activity of the MDR P-glycoprotein (P-gp) membrane efflux pump, and the effect of L-CAV on cellular accumulation of P-gp substrates. The effect of pre-treatment with non-cytotoxic doses of L-CAV on cellular sensitivity to ten standard antineoplastic agents was also evaluated, in order to assess the chemosensitization potential of L-CAV. 3-(4,5-Dimethylthiazol-)2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assays revealed that the MDR variants of human uterine sarcoma and leukemic cells were equally sensitive to L-CAV as compared with their respective parental controls. Although the presence of free L-CAV in the uptake media did not influence cellular accumulation of P-gp substrates, cells cultured for 72 h in 250 μM L-CAV accumulated from 16 to 23% less P-gp substrate than untreated controls. Although L-CAV-cultured sarcoma cells accumulated 17% less doxorubicin (DOX) than untreated controls, they were three times more sensitive to its cytotoxic effects. L-CAV-treated cells were also significantly more sensitive to cisplatin, 5-fluorouracil, mitoxantrone and bleomycin than were untreated controls. Indirect immunofluorescence revealed that 72-h exposure to as much as 1000 μM L-CAV did not alter cellular expression of P-gp. These studies suggest that L-CAV may be equally cytotoxic for both parental and MDR tumor cells, and that L-CAV neither induces the expression of, nor is a substrate for, P-gp. The observation that L-CAV pre-treatment reduces cellular accumulation of DOX, yet sensitizes tumor cells to DOX and other DNA-targeting antineoplastic drugs, suggests a role for L-CAV as a chemosensitizer for the chemotherapy of cancer.

The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis.

Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents. However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive. In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas. The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors. Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death. Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells. By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression. These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.

The multidrug-resistant human lung tumour cell line, DLKP-A10, expresses novel drug accumulation and sequestration systems

Drug accumulation studies with the anticancer agents adriamycin and vincristine were carried out on the MDR variant of the human lung cell lines DLKP, DLKP-A10 which overexpresses the MDR associated P-glycoprotein efflux pump. Reduced cellular accumulation of both agents was observed in the resistant variant. The subsequent addition of verapamil and cyclosporin A resulted in partial restoration of cellular accumulation of both drugs in the DLKP-A10 resistant variant while complete restoration of cellular drug levels was observed in the SKMES-1/ADR cell line. These results suggested that the accumulation defect observed in the SKMES-1/ADR cell line was P-glycoprotein mediated and that accordingly, the cells exhibited characteristics consistent with the classical MDR phenotype. In contrast, while P-glycoprotein also appears to mediate a reduction in cellular drug accumulation in the DLKP-A10 cells, an alternative transport mechanism may also be present. No significant increase in the expression of either the MRP or LRP transport proteins was observed in the resistant cells. Metabolic inhibition by antimycin A (but not sodium azide or 2-deoxy- d-glucose) resulted in complete restoration of drug accumulation suggesting the presence of an alternative energy dependent transport mechanism. Fluorescent microscopy studies indicated different cellular localisation of the drug within the parental and resistant cells despite equivalent intracellular concentrations. These studies also revealed the presence of an ATP-dependent, vesicular sequestration mechanism which may be involved in the reduction of nuclear adriamycin accumulation in the DLKP-A10 cell line. This was indicated by observation of the disruption of cytoplasmic vesicles by antimycin A and also inhibition of cytoplasmic drug sequestration by the carboxylic ionophores, monensin and nigericin, accompanied by increased adriamycin accumulation and redistribution of the drug from the cytoplasm to the nucleus.

Regulation by glutathione of drug transport in multidrug-resistant human lung tumour cell lines overexpressing multidrug resistance-associated protein.

Previous studies have shown that multidrug resistance (MDR) in the doxorubicin-selected lung tumour cell lines COR-L23/R, GLC4/ADR and MOR/R is associated with overexpression of the MRP gene. In this study we report that resistance to daunorubicin, vincristine and rhodamine 123 can be partially reversed in these cell lines by exposing the cells to buthionine sulphoximine (BSO), an inhibitor of glutathione (GSH) synthesis. This effect of BSO on drug resistance was associated with an increased intracellular accumulation of daunorubicin and rhodamine 123, owing to inhibition of the enhanced drug efflux. In contrast, the accumulation of daunorubicin was not increased by BSO treatment in a P-glycoprotein (P-gp)-mediated MDR cell line. BSO treatment (25 microM, 20 h) of the cell lines resulted in 60-80% depletion of cellular GSH levels. The effects of BSO on daunorubicin accumulation in the COR-L23/R and GLC4/ADR cells were associated with cellular GSH depletion. In addition, increase of cellular GSH levels in BSO-treated COR-L23/R and GLC4/ADR cells as a result of incubation with 5 mM GSH ethyl ester restored the accumulation deficit of daunorubicin. However, the transport of daunorubicin did not increase the GSH release in any of the cell lines. These results demonstrate that drug transport in MRP- but not in P-gp-overexpressing MDR tumour cell lines can be regulated by intracellular GSH levels.

Enhancement by Recombinant Human Interferon Alfa of the Reversal of Multidrug Resistance by MRK-16 Monoclonal Antibody

The anti-P-glycoprotein monoclonal antibody MRK-16 mediates the reversal of multidrug resistance. Recombinant human interferon alfa (rHuIFN alpha) enhances the cytotoxic activity of diverse chemotherapeutics and may modulate multidrug resistance. Our purpose was to determine the outcome of combination treatment with MRK-16, rHuIFN alpha-2a, and cytotoxic agents on tumor cells that express P-glycoprotein (Pgp). Three Pgp-expressing, multidrug-resistant human tumor cell lines were used: the MDR1 retrovirus-infected HT-29 colon adenocarcinoma (HT-29mdr1), the doxorubicin (Adriamycin)-resistant MCF-7 (AdrR MCF-7) breast carcinoma, and the de novo Pgp-acquired, HCT-15 colon carcinoma. The parental cell lines HT-29par and MCF-7 were used as controls. The in vitro effects of MRK-16 and rHuIFN alpha-2a were studied on: (a) chemosensitivity of parental and multidrug-resistant cell lines to vincristine, doxorubicin, or paclitaxel (Taxol); (b) intracellular drug concentrations; and (c) Pgp expression. The efficacy of vincristine alone or in combination with MRK-16 and/or rHuIFN alpha-2a was assessed against HT-29mdr1 cells in female, athymic NCr-nu/nu mice. For vincristine, the IC50 (i.e., the concentration that causes 50% inhibition of cell growth) was 7.0 ng/mL in HT-29mdr1 cells. Pretreatment of HT-29mdr1 cells with MRK-16 partially restored vincristine sensitivity (IC50 = 4.8 ng/mL), which was enhanced by noncytotoxic concentrations of rHuIFN alpha-2a (IC50 = 2.9 ng/mL) via a mechanism independent of Pgp modulation or [3H]vincristine efflux. rHuIFN alpha-2a potentiated MRK-16 reversal of multidrug resistance with both doxorubicin and paclitaxel on HT-29mdr1 cells and with vincristine on AdrR MCF-7 and HCT-15 tumor cells. Treatment of mice with 1 mg/kg vincristine weekly for 3 weeks, beginning 10 days after tumor injection, significantly increased the median survival times of the HT-29par tumor-bearing mice (60 days versus 35 days; P < .0001) but was only marginally therapeutic for HT-29mdr1 tumor-bearing mice (52 days versus 46 days). Pretreatment with MRK-16 (500 micrograms) and rHuIFN alpha-2a (5 x 10(4) U), alone or in combination, 24 hours before vincristine therapy did not affect the survival of HT-29par tumor-bearing mice. In contrast, the survival of mice bearing HT-29mdr1 tumors was significantly increased following treatment with MRK-16 before vincristine (80 days; P < .0001). Administration of a nontherapeutic dose of rHuIFN alpha-2a (5 x 10(4) U) with MRK-16 before vincristine treatment further increased the median survival times of HT-29mdr1 tumor-bearing mice (116 days; P < .0001). MRK-16 used in combination with rHuIFN alpha-2a was significantly more effective than MRK-16 in overcoming multidrug resistance.

Free radical formation by ansamycin benzoquinone in human breast tumor cells: Implications for cytotoxicity and resistance

The benzoquinonoid ansamycin antibiotics, geldanamycin and herbimycin A, are potent cytotoxins against tumor cells in vitro. We have examined the mechanism of their in vitro cytotoxicity against human breast adenocarcinoma (MCF-7) cells and we have found that multidrug-resistant MCF-7/ADR R cells that exhibit the MDR phenotype and the overexpression of P-170-glycoprotein, were cross-resistant to geldanamycin and herbimycin A. Verapamil, which binds competitively with P-170-glycoprotein, enhanced geldanamycin cytotoxicity 12-fold only in resistant cells, suggesting that geldanamycin may interact with the drug efflux protein. Geldanamycin and herbimycin A, like adriamycin, were reductively activated by the NADPH-cytochrome cytochrome P450-reductase and formed reactive ·OH. The formation of ·OH was signifacantly lower in resistant cells. In contrast to adriamycin, the formation of ·OH was unaffected by the addition of DNA, indicating that a DNA-complexed drug was redoxactive and may, therefore, may be more effective in killing tumor cells at the DNA level. These observations indicate that both the decreased free radical formation and interactions with P170 glycoprotein may be important in geldanamycin and herbimycin A resistance in multidrug resistant human breast tumor cells.

Cooperative P-glycoprotein mediated daunorubicin transport into DNA-loaded plasma membrane vesicles

Most of the multidrug resistant human tumor cell lines overexpress the MDR1 gene product P-glycoprotein (P-gp) which is believed to function as an energy-dependent drug efflux pump. Here we describe a novel method that allows the kinetic characterization of P-gp-mediated active drug transport. This method is based on the fluorescence quenching of anthracyclines transported into DNA-loaded plasma membrane vesicles. The uptake of daunorubicin (DNR) into the plasma membrane vesicles was saturable in terms of the extravesicular DNR concentration with a K m of 1.5 ± 0.1 μM. This transport occured by a cooperative process with a Hill coefficient close to 2 for DNR. A model is discussed in which P-gp pumps two molecules of drug per turnover.

Analysis of MRP mRNA in mitoxantrone-selected, multidrug-resistant human tumor cells

MRP, a gene recently isolated from a non-P-glycoprotein-mediated multidrug-resistant small cell lung cancer line, is a candidate multidrug-resistance gene. Mitoxantrone, an anthracenedione antitumor agent, frequently selects for non-P-glycoprotein-mediated multidrug resistance in in vitro models. To determine whether mitoxantrone-selected multidrug resistance wasdue to overexpression of MRP, we examined the expression of MRP in four mitoxantrone-selected, multidrug-resistant human tumor cell lines, using a reverse transcriptase/polymerase chain reaction assay. Results from these experiments suggest that overexpression of MRP does not appear to play a primary role in mitoxantrone-selected multidrug resistance in these cell lines, and that other novel drug-resistance mechanisms are likely.

Identification of a multidrug resistance modulator with clinical potential by analysis of synergistic activity in vitro, toxicity in vivo and growth delay in a solid human tumour xenograft

Circumvention of multidrug resistance in vitro by resistance modulators is well documented but their clinical use may be limited by effects on normal tissues. We have compared four resistance modifiers, both in terms of modulation of doxorubicin sensitivity in vitro and toxicity in vivo, in order to determine whether it is possible to select agents with clinical potential. Verapamil, d-verapamil and quinidine are all maximally active in the multidrug resistant cell line at about 7 μM and are not cytotoxic at this concentration. The tiapamil analogue Roll-2933 is a highly potent resistance modulator such that at only 2 μM sensitization is greater than is seen with the other modulators at 7 μM. Since the id 50 concentration for Ro11-2933 is 17.7 μM (5–12-fold less than the other modifiers) we have used isobologram analysis to demonstrate that the interaction with doxorubucin is supra-additive and cannot be explained by additive toxicity. This method of analysis also revealed that when resistance modulation is related to the cytotoxicity of the modulator itself, all four modulators show comparable activity. On the other hand, measurement of the acute toxicity in mice of the modulators did reveal differences. The ld 10 for verapamil (51 mg kg ) was about one third of that for quinidine (185 mg kg ) and this is consistent with the known maximum tolerated plasma levels in patients. Furthermore, whilst epirubicin alone was unable to reduce the growth rate of a multidrug resistant human tumour xenograft, the addition of quinidine, but not verapamil, at the maximum tolerated dose did do so. d-Verapamil was only about half as toxic as racemic verapamil and this too is consistent with clinical observations. The ld 10 for Ro11-2933 (152 mg kg ) was comparable with that for quinidine. In the human tumour xenograft model maximal growth inhibition was observed with the combination of epirubicin and Ro11-2933 (45 mg kg ) and this degree of growth inhibition was comparable to that obtained with epirubicin alone in the drug sensitive xerografts. Ro11-2933 had no measurable effects on the plasma or tumour pharmacokinetics of epirubicin. These results suggest that it is possible to predict the clinical potential of a resistance modulator. Furthermore, Ro11-2933 is a promising agent for use in the clinic since maximal resistance modulation in vivo is observed at about one third of the ld 10 dose.

5-N-acetylardeemin, a novel heterocyclic compound which reverses multiple drug resistance in tumor cells. I. Taxonomy and fermentation of the producing organism and biological activity.

The ardeemins are a new family of secondary metabolites produced by submerged fermentation of a fungus which was isolated from a soil sample collected in Brazil. Based on taxonomic studies, the producing culture was identified as Aspergillus fischeri var. brasiliensis strain AB 1826M-35. 5-N-Acetylardeemin potentiated the cytotoxicity of the anticancer agent vinblastine in multidrug resistant human tumor cells.