Topotecan Resistance Research Articles

The contribution of ABC transporters to drug resistance in a realistic mouse mammary tumor model

Although ABC transporters are brilliant in causing drug resistance in cultured cells, their contribution to drug resistance in patients has remained controversial. We have tackled this problem in a mammary tumor model in mice. These Brca1 / , p53 / tumors arise ‘spontaneously’ and closely resemble human breast cancer in BRCA1 carriers (1). These tumors respond to a range of standard chemotherapeutic agents (1), but eventually end up being resistant (1e3) to most drugs, but not to cisplatin (1,4). Resistance to doxorubicin in this tumor model is nearly always due to P-glycoprotein upregulation at the transcriptional level (1,3). A 5-fold increase in the low basal level of P-glycoprotein RNA in these tumors is sufficient for complete resistance. These levels of P-glycoprotein are not detectable with standard immunocytochemistry (3), showing that our current tools are inadequate to detect relevant amounts of P-gp in clinical samples. Crossing in null alleles for Mdr1a/b made the tumors hypersensitive to doxorubicin and docetaxel, showing the important contribution of P-gp to resistance in this tumor model. P-gp also causes resistance to the PARP inhibitor olaparib (2). In about half of the mice treated with topotecan, resistance is associated with upregulation of Abcg2 (Bcrp) (5). Tumors in Abcg2 / mice take longer to become resistant, showing the importance of Abcg2 as a defense system (5). We have not observed upregulation of any of the other ABC-transporters associated with topotecan resistance in cultured cells, such as Abcc2 and 4. Notwithstanding the complete remissions often obtained with some drugs, we are rarely able to eradicate the tumor (1e5). Our evidence indicates that this is not due to the hypothetical unique properties of tumor stem cells. We are also studying potential markers that determine initial drug response, but detect none at the RNA level before tumor treatment.

Sensitivity and Acquired Resistance of BRCA1;p53-Deficient Mouse Mammary Tumors to the Topoisomerase I Inhibitor Topotecan

There is no tailored therapy yet for human basal-like mammary carcinomas. However, BRCA1 dysfunction is frequently present in these malignancies, compromising homology-directed DNA repair. This defect may serve as the tumor's Achilles heel and make the tumor hypersensitive to DNA breaks. We have evaluated this putative synthetic lethality in a genetically engineered mouse model for BRCA1-associated breast cancer, using the topoisomerase I (Top1) poison topotecan as monotherapy and in combination with poly(ADP-ribose) polymerase inhibition by olaparib. All 20 tumors tested were topotecan sensitive, but response heterogeneity was substantial. Although topotecan increased mouse survival, all tumors eventually acquired resistance. As mechanisms of in vivo resistance, we identified overexpression of Abcg2/Bcrp and markedly reduced protein levels of the drug target Top1 (without altered mRNA levels). Tumor-specific genetic ablation of Abcg2 significantly increased overall survival of topotecan-treated animals (P < 0.001), confirming the in vivo relevance of ABCG2 for topotecan resistance in a novel approach. Despite the lack of ABCG2, a putative tumor-initiating cell marker, none of the 11 Abcg2(-/-);Brca1(-/-);p53(-/-) tumors were eradicated, not even by the combination topotecan-olaparib. We find that olaparib substantially increases topotecan toxicity in this model, and we suggest that this might also happen in humans.

Efficacy of ST1968 (namitecan) on a topotecan-resistant squamous cell carcinoma

ST1968 (namitecan), a novel 7-modified hydrophilic camptothecin, was found to be effective against tumor models relatively resistant to topotecan and irinotecan. Based on this observation, this study was designed to investigate the cellular and antitumor effects of ST1968 in a subline of A431, squamous cell carcinoma, selected for resistance to topotecan (A431/TPT). This model was characterized by a slow growth rate, associated with downregulation of EGFR and topoisomerase I. In contrast to other camptothecins (SN38 and gimatecan), ST1968 was able to overcome almost completely the resistance at cellular level. The cellular pharmacokinetics indicated a comparable accumulation and retention of ST1968 in sensitive and resistant cells, in spite of expression of the efflux transporter, P-glycoprotein, in resistant cells. The uptake and retention of topotecan were dramatically reduced in both tumor cell lines, but more evident in the resistant one. In contrast to topotecan, ST1968 retained an outstanding efficacy in vivo against the resistant tumor (A431/TPT). The results are consistent with the interpretation that ST1968 was able to overcome the most relevant mechanisms associated with the development of topotecan resistance (i.e., slow proliferation and target downregulation) owing to its peculiar pharmacokinetic behaviour.

Potent and Selective Inhibitors of Breast Cancer Resistance Protein (ABCG2) Derived from the p-Glycoprotein (ABCB1) Modulator Tariquidar

The efflux pumps ABCB1 (p-gp, MDR1) and ABCG2 (BCRP) are expressed to a high extent by endothelial cells at the blood-brain barrier (BBB) and other barrier tissues and are involved in drug resistance of tumor (stem) cells. Whereas numerous ABCB1 inhibitors are known, only a few ABCG2 modulators with submicromolar activity have been published. Starting from tariquidar (4) analogues as ABCB1 modulators, minimal structural modifications resulted in a drastic shift in favor of ABCG2 inhibition. Highest potency was found when the 3,4-dimethoxy-2-(quinoline-3-carbonylamino)benzoyl moiety in 4 was replaced with a 4-methoxycarbonylbenzoyl moiety bearing a hetarylcarboxamido group in 3-position, e.g., quinoline-3-carboxamido (5, IC(50): 119 nM) or quinoline-2-carboxamido (6, IC(50): 60 nM, flow cytometric mitoxantrone efflux assay, topotecan-resistant MCF-7 breast cancer cells); the selectivity for ABCG2 over ABCB1 was about 100-500 fold and the compounds were inactive at ABCC2 (MRP2). Chemosensitivity assays against MCF-7/Topo cells revealed that the nontoxic inhibitor 6 completely reverted ABCG2-mediated topotecan resistance at concentrations >100 nM, whereas 5 showed ABCG2 independent cytotoxicity. ABCG2 inhibitors might be useful for cancer treatment with respect to reversal of multidrug resistance, overcoming the BBB and targeting of tumor stem cells.

Evaluation of Lapatinib and Topotecan Combination Therapy: Tissue Culture, Murine Xenograft, and Phase I Clinical Trial Data

Topotecan resistance can result from drug efflux by P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) as well as survival signals initiated by epidermal growth factor receptor family members. The present studies were done to determine the effect of combining topotecan and the dual epidermal growth factor receptor/HER2 inhibitor lapatinib in tissue culture, a murine xenograft model, and a phase I clinical trial. The effects of lapatinib on topotecan accumulation and cytotoxicity in vitro were examined in paired cell lines lacking or expressing Pgp or BCRP. Antiproliferative effects of the combination were assessed in mice bearing HER2+ BT474 breast cancer xenografts. Based on tolerability in this preclinical model, 37 patients with advanced-stage cancers received escalating doses of lapatinib and topotecan in a phase I trial. Lapatinib increased topotecan accumulation in BCRP- or Pgp-expressing cells in vitro, and the combination showed enhanced efficacy in HER2+ BT474 xenografts. In the phase I study, nausea, vomiting, diarrhea, and fatigue were dose limiting. The maximum tolerated doses were 1,250 mg/d lapatinib by mouth for 21 or 28 days with 3.2 mg/m2 topotecan i.v. on days 1, 8, and 15 of 28-day cycles. Pharmacokinetic analyses showed that combined drug administration resulted in decreased topotecan clearance consistent with transporter-mediated interactions. Seventeen (46%) patients had disease stabilization. The lapatinib/topotecan combination is well tolerated and warrants further study.

BCRP/ABCG2 levels account for the resistance to topoisomerase I inhibitors and reversal effects by gefitinib in non-small cell lung cancer

Breast cancer resistance protein (BCRP) confers resistance against topoisomerase I inhibitors in cancer cells. Very recently, we reported that gefitinib reverses BCRP-mediated drug resistance by direct inhibition. However, it remains undetermined how much BCRP contributes to the resistance to topoisomerase I inhibitors in non-small cell lung cancer (NSCLC). The present study was designed to examine whether BCRP levels in NSCLC cells are correlated with the resistance to topoisomerase I inhibitors and the reversal effect by gefitinib. BCRP levels and its function were evaluated by Western blotting and flowcytometry, respectively. Gefitinib-insensitive NSCLC cells expressed various levels of BCRP, which were closely correlated not only with the IC50 values of SN-38 (r=0.874, P<0.05) and those of topotecan (r=0.968, P<0.001), but also with the reversal effects of 1 microM gefitinib on SN-38 resistance (r=0.956, P<0.001) and topotecan resistance (r=0.977, P=0.0001). BCRP levels accounted for between 80 and 90% of the variation in the resistance to topoisomerase I inhibitors and the reversal effects by gefitinib. Also, gefitinib increased intracellular topotecan accumulation in proportion to the BCRP levels. These findings suggest that BCRP is the most important molecule responsible for topoisomerase I inhibitor resistance, and that the development of BCRP inhibitors is an effective approach for overcoming this resistance. In addition, the examination of BCRP levels in NSCLC tissues may identify an optimal patient population for treatment with topoisomerase I inhibitors alone or in combination with BCRP inhibitors.

Breast cancer resistance protein-mediated topotecan resistance in ovarian cancer cells

Overexpression of breast cancer resistance protein (BCRP) and mitoxantrone (MX) resistance protein can confer resistance to a variety of cytostatic drugs, such as MX, topotecan (TPT), doxorubicin, and daunorubicin. This study investigates the role of BCRP in resistance of ovarian cancer to TPT treatment. We have developed TPT-resistant human ovarian cancer cell line. Intracellular concentration of fluorescent dye rhodamine 123 (Rh123) was measured by flow cytometry. The expression of several membrane transporter proteins including P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), and BCRP were determined by reverse transcription-polymerase chain reaction and Western blotting. The Rh123 concentration in parental cells was approximately three times of those in TPT-resistant cells. In contrast to undetectable level of P-gp messenger RNA (mRNA) and minimal level of MRP1 expression in TPT-resistant cells, overexpression of both the BCRP mRNA and the protein was detected in these cells. Introduction of antisense-phosphorothioate oligonucleotide derived from BCRP mRNA into TPT-resistant cells resulted in a significant increase in the concentration of intracellular Rh123. These results suggested a novel mechanism in which a reduced intracellular drug concentration may be mediated by BCRP gene products in human ovarian cancer cells.

Breast cancer resistance protein–mediated topotecan resistance in ovarian cancer cells

Breast cancer resistance protein–mediated topotecan resistance in ovarian cancer cells

1151 POSTER Accelerated high-dose radiotherapy by target splitting for inoperable non-small cell lung cancer: a phase I trial

Resistance to chemotherapy is a major challenge in the treatment of ovarian/peritoneal cancer. One purported mechanism of topotecan resistance is the breast cancer resistance protein (BCRP) and P-glycoprotein (Pgp). We designed a phase II clinical trial evaluating the efficacy and adverse event profile of concomitant topotecan and lapatinib, a small molecule pan-erbB inhibitor that can block BCRP/Pgp efflux of topotecan.Patients with platinum-refractory or resistant epithelial ovarian/peritoneal cancer were treated with topotecan 3.2 mg/m2 IV on Day 1, 8 and 15 and lapatinib 1250 mg PO daily, continuously in 28 day cycles. The primary endpoint was response rate. For correlative studies, archived tissue was assessed for expression of EGFR, HER2, HIF-1α, CD31, and BCRP.Eighteen patients were enrolled and treated. Four experienced evidence of clinical benefit: one partial response and three with stable disease. Using a two-stage Simon design, the trial was stopped after the first stage due to insufficient activity. Grades 3+ and 4+ adverse events (AE) were experienced in 14 and 4 patients, respectively. The most common grade 3/4 AE were neutropenia (56%), thrombocytopenia (28%), and diarrhea (22%).The combination of lapatinib plus topotecan for the treatment of platinum refractory/resistant epithelial ovarian cancer lacks sufficient activity to warrant further investigation. In particular, hematologic adverse events were substantial. Expression of correlative study markers did not reveal patterns of predicted benefit or toxicity. Disruption of erbB signaling and BCRP/Pgp efflux with lapatinib was insufficient for overcoming topotecan resistance, suggesting alternative mechanisms of resistance are involved.

Characterization of breast cancer resistance protein (BCRP) in epithelial ovarian cancer (EOC)

2020 Purpose: While the majority of patients with advanced EOC achieve an initial response with current chemotherapy regimens, most eventually relapse with resistant disease. One of the cellular mechanisms of drug resistance is over-expression of BCRP, a drug efflux pump that selectively pumps chemotherapeutic drugs out of cells. The objectives of this study are to characterize BCRP in EOC cell lines and investigate the relationship between BCRP expression and clinicopathological parameters in advanced EOC. Methodology: BCRP expression and cellular localization were evaluated in 10 EOC cell lines using QRT-PCR and fluorescence microscopy. Functional analysis was performed measuring topotecan efflux by flow cytometry in the presence and absence of Trypostatin-A, a specific BCRP inhibitor. A topotecan-resistant cell line was treated with DES (a known inhibitor of BCRP) and efflux of topotecan was measured by flow cytometry. BCRP expression was measured in 101 untreated primary EOC tissues using QRT-PCR. Statistical correlations between BCRP expression and clinicopathological parameters were analyzed using chi square test, log-rank test, and Cox proportional hazards models. Results: EOC cell lines exhibited varying degrees of BCRP expression. mRNA expression was correlated with protein levels and function. DES was able to reverse BCRP-mediated topotecan resistance in EOC cell lines. BCRP mRNA expression in EOC patient tissues was widely dispersed. No correlation was found between BCRP expression and age, stage, grade or debulking status. A significant correlation was demonstrated between BCRP expression and chemotherapeutic response (CR/PR vs. SD/PD, p=2.75x10–14). Comparison of survival (<24 vs. >24 months) showed a trend of decreased survival with high BCRP expression (p=0.076). Conclusions: BCRP is both present and functionally active in EOC cell lines. Further, preliminary studies with DES have shown a dose-dependent reversal of topotecan uptake. Additionally, we have demonstrated a highly significant association between elevated BCRP expression and poor chemotherapeutic response in patient samples. BCRP may serve as a molecular target for reducing drug resistance to chemotherapy in advanced EOC. No significant financial relationships to disclose.

FUNCTIONAL ANALYSIS OF THE HUMAN VARIANTS OF BREAST CANCER RESISTANCE PROTEIN: I206L, N590Y, AND D620N

Previous studies have shown that the V12M and Q141K variants of breast cancer resistance protein (BCRP) can affect expression and function of the transporter. In this study, the effects of the I206L, N590Y, and D620N variants on protein expression, plasma membrane localization, and transport activity of BCRP were investigated. Wild-type BCRP and the three variants were stably expressed in human embryonic kidney (HEK) cells. Confocal microscopy analysis showed that the three variants were predominantly routed to the plasma membrane of HEK cells. The expression level of I206L in the plasma membrane was approximately 45% of that of wild-type protein, whereas the N590Y and D620N levels were increased approximately 3.6-fold and 2.4-fold, respectively, as determined by immunoblotting. All three variants transported mitoxantrone, pheophorbide a, and BODIPY FL-prazosin. After normalization for differences in BCRP expression, I206L, N590Y, and D620N exhibited approximately 2-fold, 0.3-fold, and 0.5-fold wild-type efflux activities, respectively. The variants also conferred resistance to mitoxantrone and topotecan. Mitoxantrone and topotecan resistance by I206L and N590Y was approximately 2-fold and 0.3-fold of the wild-type BCRP resistance levels, respectively. Although D620N conferred a topotecan resistance similar to that of the wild-type protein, its level of mitoxantrone resistance was decreased by 50%. After normalization to BCRP expression levels, ATPase activities of I206L were not significantly different from those of wild-type protein, whereas N590Y and D620N exhibited approximately 30% and 50% of wild-type ATPase activities, respectively. These results suggest that I206L has the lowest protein expression and the highest activity, whereas N590Y and D620N display higher expression and lower activity, relative to wild-type BCRP.

Mrp4 confers resistance to topotecan and protects the brain from chemotherapy.

The role of the multidrug resistance protein MRP4/ABCC4 in vivo remains undefined. To explore this role, we generated Mrp4-deficient mice. Unexpectedly, these mice showed enhanced accumulation of the anticancer agent topotecan in brain tissue and cerebrospinal fluid (CSF). Further studies demonstrated that topotecan was an Mrp4 substrate and that cells overexpressing Mrp4 were resistant to its cytotoxic effects. We then used new antibodies to discover that Mrp4 is unique among the anionic ATP-dependent transporters in its dual localization at the basolateral membrane of the choroid plexus epithelium and in the apical membrane of the endothelial cells of the brain capillaries. Microdialysis sampling of ventricular CSF demonstrated that localization of Mrp4 at the choroid epithelium is integral to its function in limiting drug penetration into the CSF. The topotecan resistance of cells overexpressing Mrp4 and the polarized expression of Mrp4 in the choroid plexus and brain capillary endothelial cells indicate that Mrp4 has a dual role in protecting the brain from cytotoxins and suggest that the therapeutic efficacy of central nervous system-directed drugs that are Mrp4 substrates may be improved by developing Mrp4 inhibitors.

Overexpression of IL-6 but not IL-8 increases paclitaxel resistance of U-2OS human osteosarcoma cells.

The cytokines IL-6, initially recognized as a regulator of immune and inflammatory response and IL-8, a potential regulator of angiogenesis, also regulate the growth of many tumor cells. Human cancer cells selected for multidrug resistance to common chemotherapeutic agents demonstrate increased expression of IL-6 and IL-8. To determine whether IL-6 or IL-8 overexpression contributes directly to the drug resistant phenotype, IL-6 or IL-8 cDNA were introduced into the paclitaxel sensitive human osteosarcoma cell line U-2OS using the pIRESneo bicistronic expression vector. Interleukin-6 and IL-8 transfectants were selected for either high IL-6 or IL-8 secretion and evaluated in drug resistance assays. Two IL-6 and two IL-8 secreting clones express IL-6 or IL-8 levels of 10 ng/ml and 1 ng/ml in culture, while parental U-2OS and pIRESneo vector transfected control cells express IL-6 and IL-8 levels of 0.005 ng/ml and 0.1 ng/ml, respectively. MTT cytotoxicity with IL-6 transfected cells demonstrates a five-fold increase in resistance to paclitaxel and a four-fold increase in resistance to doxorubicin as compared to U-2OS. There are no changes in mitoxantrone or topotecan resistance in the IL-6 transfectants as compared to parental U-2OS. Northern analysis of IL-6 transfectants demonstrates that the resistant phenotype is not related to increased levels of MDR-1, MRP-1, or LRP. Western analysis also confirms that P-glycoprotein levels are not altered in IL-6 transfectants. Further supporting an MDR-1 independent mechanism of drug resistance, verapamil cannot reverse paclitaxel resistance in transfected cells, findings further supported by rhodamine 123 exclusion data. Treatment of IL-6 transfected cells with paclitaxel, compared with drug-sensitive parental U-2OS, shows U-2OS(IL-6) are significantly more resistant to apoptosis induced by paclitaxel and exhibit decreased proteolytic activation of caspase-3. In contrast U-2OS(IL-8) transfectants demonstrate no appreciable increase in paclitaxel resistance when compared with parental cells. In summary, while both IL-6 and IL-8 are overexpressed in paclitaxel resistant cell lines, only IL-6 has the potential to contribute directly to paclitaxel and doxorubicin resistance in U-2OS. This resistance is through a non-MDR-1 pathway.

BCRP/MXR/ABCP expression in topotecan-resistant human breast carcinoma cells

We have previously described a mitoxantrone-resistant MCF7 cell line that is cross-resistant to topotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin (CPT-11), and 9-aminocamptothecin, but not to camptothecin. A novel mechanism that resulted in decreased topotecan accumulation in MCF7/MX cells was proposed (Yang et al. Cancer Res 55: 4004–4009, 1995). We now have developed a topotecan-resistant cancer cell line from wild-type MCF7 cells. MCF7/TPT300 cells were 68.9-fold resistant to topotecan, 68.3-fold to 10-hydroxy-7-ethylcamptothecin (SN-38), and 116-fold to mitoxantrone, but only 4.1-fold to camptothecin. Topotecan efflux was increased in MCF7/TPT300 cells compared with MCF7/WT cells, and this increase was reversed upon ATP depletion by sodium azide, suggesting an energy-dependent drug efflux mechanism. However, MCF7/TPT300 cells did not overexpress P-glycoprotein or the multidrug resistance-associated protein (MRP1). In contrast, overexpression of the breast cancer resistance protein (BCRP/MXR/ABCP) was observed in MCF7/TPT300 cells as well as DNA topoisomerase I down-regulation. Our data suggest that enhanced topotecan efflux contributes partly to topotecan resistance in MCF7/TPT300 cells, possibly mediated by BCRP/MXR/ABCP.

The Product of the ABC Half-Transporter Gene ABCG2 (BCRP/MXR/ABCP) Is Expressed in the Plasma Membrane

The products of the ABC gene family can be generally classified as either full-transporters of half-transporters. Full-transporters are expressed in the plasma membrane, whereas half-transporters are usually found in intracellular membranes. Recently, an ABC half-transporter, the ABCG2 gene product Breast Cancer/Mitoxantrone Resistance Protein (BCRP/MXR), has been shown to cause mitoxantrone and topotecan resistance. The purpose of this study was to determine the expression and the intracellular localization of this protein in various drug-resistant cell lines. BCRP/MXR expression was determined by Western blot and immunohistochemistry. This protein is highly overexpressed in several drug-resistant cell lines and localizes predominantly to the plasma membrane, instead of to intracellular membranes as seen with all other known half-transporters. Therefore, BCRP/MXR is unique among the ABC half-transporters by being localized to the plasma membrane.

High-dose topotecan with granulocyte-colony stimulating factor in fluoropyrimidine-refractory colorectal cancer: A phase II and pharmacodynamic study

The premise for this study was that topotecan (TPT) resistance in preclinical studies is associated with low level expression of the p-glycoprotein (Pgp) multi-drug transporter conferred by the multi-drug resistant (MDR) phenotype, which might be overcome in clinical practice by administering moderately (2.3-fold) higher doses of TPT that have shown to be feasible with granulocyte colony-stimulating factor (G-CSF) support. This phase II study evaluated the anti-tumor activity of TPT administered at its highest possible solid tumor dose with G-SCF in patients with fluoropyrimidine-refractory advanced colorectal carcinoma. The study also sought to identify pharmacodynamic (PD) determinants of both activity and toxicity. TPT was administered as a 30-minute infusion daily for five days every three weeks at a dose of 3.5 mg/m2/day to patients with advance colorectal carcinoma who developed progressive disease either during treatment with fluoropyrimidine-based chemotherapy for advanced disease or within six months after receiving fluoropyrimidine-based adjuvant chemotherapy. This dose of TPT was previously determined to be the maximal tolerated dose (MTD) with G-CSF support in a phase I study involving solid tumor patients with similar risk factors for myelosuppression. Plasma sampling with performed during course 1 to characterize the pharmacokinetic (PK) and PD behavior of TPT. Seventeen patients who received 89 courses of TPT and G-CSF were evaluable for toxicity; 16 patients were evaluable for anti-tumor response. Toxicity, particularly myelosuppression, was substantial. At the 3.5 mg/m2/day dose level, absolute neutrophil counts (ANC) were less than 500/microliters for longer than 5 days in 17% of courses involving seven of seventeen (41%) patients. Severe neutropenia associated with fever occurred in 12.3% of courses; and platelet counts below 25,000/microliters were noted in 26.9% of courses. These toxicities resulted in dose reductions in seven of 17 (41%) patients. Nevertheless, 90% of the planned total dose of TPT was administered. No major responses were observed, though minor activity was noted in several patients. Both the median time to progression and the median survival time were short--2.5 and 4 months respectively. Although interindividual variability in the disposition of total TPT was observed, the lack of objective responses precluded PD assessments related to disease activity. Total TPT exposure was significantly higher than drug exposure achieved in similar patients at an identical dose in a previous phase I study of TPT and G-CSF, which may explain why more severe myelosuppressive effects occurred in the present study. There were no PD relationships evident between relevant PK parameters and the percent decrements in platelets and ANC's during course 1, although patients with severe toxic effects (ANC below 500/microliters for more than five days and/or platelets < 25,000/microliters) had higher drug exposure than patients with less severe toxicity (P < 0.018 and P = 0.09, respectively). Based on these results, the true response rate of TPT at its solid tumor MTD with G-CSF support is unlikely to approach 20%. Although a response rate of less than 20% might be viewed as significant in this disease setting and might be confirmed with sufficient statistical certainty by treating additional patients, the substantial toxicity, inconvenience, and cost associated with this high dose TPT/G-CSF regimen does not warrant the acceptance of a lower level of anti-tumor activity as a criterion for further development.

Evaluation of a novel bis-naphthalimide anticancer agent, DMP 840, against human xenografts derived from adult, juvenile, and pediatric cancers.

The new bis-naphthalimide antitumor agent (R,R)2,2'-[1,2-ethanediylbis[imino(1-methyl-2.1-ethanediyl)]-bis(5 -nitro 1H-benz[de]-isoquinoline-1,3-2H) dione] dimethanesulfonate (DMP 840) was evaluated against parental and multidrug-resistant human KB cell lines in vitro and against these lines growing as xenografts in immune-deprived mice. In vitro, KB8-5 cells were 50-fold resistant to vincristine but only 16-fold resistant to DMP 840 as measured by clonogenic survival. For in vivo evaluation, DMP 840 was given by i.v. injection daily for 9 days or for 5 days/week for 2 consecutive weeks [(dx5)2]. In contrast to the cross-resistance of KB cell lines in vitro, both KB3-1 and KB8-5 tumors were highly and equally sensitive to DMP 840; only KB3-1 xenografts demonstrated sensitivity to vincristine, which was consistent with the in vitro results. DMP 840 was also evaluated against a panel of human tumors comprising colon adenocarcinoma and rhabdomyosarcoma xenografts. Against eight lines of colon adenocarcinoma, DMP 840 caused a high frequency of partial and complete regressions in two lines and significant inhibition of growth in two lines. DMP 840 caused complete regressions in five of six lines of advanced rhabdomyosarcomas, demonstrating a broad range of effective dose levels. The pattern of activity against this tumor panel was similar but not identical to that of two inhibitors of topoisomerase I. There was no cross-resistance to DMP 840 in xenografts selected for resistance to vincristine or in a rhabdomyosarcoma selected for resistance to the topoisomerase I inhibitor topotecan. In contrast, a colon tumor selected for topotecan resistance was completely resistant to DMP 840. Slight cross-resistance to DMP 840 was demonstrated in a rhabdomyosarcoma xenograft that was selected for primary resistance to melphalan and was cross-resistant to topoisomerase I inhibitors. The pattern of activity and cross-resistance in these tumors was compared with that shown by two agents that inhibit topoisomerase I: topotecan and CPT-11.

Author Index

The products of the ABC gene family can be generally classified as either full-transporters of half-transporters. Full-transporters are expressed in the plasma membrane, whereas half-transporters are usually found in intracellular membranes. Recently, an ABC half-transporter, the ABCG2 gene product Breast Cancer/Mitoxantrone Resistance Protein (BCRP/MXR), has been shown to cause mitoxantrone and topotecan resistance. The purpose of this study was to determine the expression and the intracellular localization of this protein in various drug-resistant cell lines. BCRP/MXR expression was determined by Western blot and immunohistochemistry. This protein is highly overexpressed in several drug-resistant cell lines and localizes predominantly to the plasma membrane, instead of to intracellular membranes as seen with all other known half-transporters. Therefore, BCRP/MXR is unique among the ABC half-transporters by being localized to the plasma membrane.