ANG1005 for breast cancer brain metastases: correlation between 18F-FLT-PET after first cycle and MRI in response assessment.

PURPOSE: Improved therapies and imaging modalities are needed for the treatment of breast cancer brain metastases (BCBM). ANG1005 is a drug conjugate consisting of paclitaxel covalently linked to Angiopep-2, designed to cross the blood-brain barrier. We conducted a biomarker substudy to evaluate 18F-FLT-PET for response assessment. METHODS: Ten patients with measurable BCBM received ANG1005 at a dose of 550 mg/m2 IV every 21 days. Before and after cycle 1, patients underwent PET imaging with 18F-FLT, a thymidine analog, retention of which reflects cellular proliferation, for comparison with gadolinium-contrast magnetic resonance imaging (Gd-MRI) in brain metastases detection and response assessment. A 20 % change in uptake after one cycle of ANG1005 was deemed significant. RESULTS: Thirty-two target and twenty non-target metastatic brain lesions were analyzed. The median tumor reduction by MRI after cycle 1 was -17.5 % (n = 10 patients, lower, upper quartiles: -25.5, -4.8 %) in target lesion size compared with baseline. Fifteen of twenty-nine target lesions (52 %) and 12/20 nontarget lesions (60 %) showed a ≥20 % decrease post-therapy in FLT-PET SUV change (odds ratio 0.71, 95 % CI: 0.19, 2.61). The median percentage change in SUVmax was -20.9 % (n = 29 lesions; lower, upper quartiles: -42.4, 2.0 %), and the median percentage change in SUV80 was also -20.9 % (n = 29; lower, upper quartiles: -49.0, 0.0 %). Two patients had confirmed partial responses by PET and MRI lasting 6 and 18 cycles, respectively. Seven patients had stable disease, receiving a median of six cycles. CONCLUSIONS: ANG1005 warrants further study in BCBM. Results demonstrated a moderately strong association between MRI and 18F-FLT-PET imaging.

The linker region of breast cancer resistance protein ABCG2 is critical for coupling of ATP-dependent drug transport.

The ATP-binding cassette (ABC) transporters of class G display a different domain organisation than P-glycoprotein/ABCB1 and bacterial homologues with a nucleotide-binding domain preceding the transmembrane domain. The linker region connecting these domains is unique and its function and structure cannot be predicted. Sequence analysis revealed that the human ABCG2 linker contains a LSGGE sequence, homologous to the canonical C-motif/ABC signature present in all ABC nucleotide-binding domains. Predictions of disorder and of secondary structures indicated that this C2-sequence was highly mobile and located between an α-helix and a loop similarly to the C-motif. Point mutations of the two first residues of the C2-sequence fully abolished the transport-coupled ATPase activity, and led to the complete loss of cell resistance to mitoxantrone. The interaction with potent, selective and non-competitive, ABCG2 inhibitors was also significantly altered upon mutation. These results suggest an important mechanistic role for the C2-sequence of the ABCG2 linker region in ATP binding and/or hydrolysis coupled to drug efflux.

Shared medical appointments for bariatric surgery follow-up: a patient satisfaction questionnaire.

BACKGROUND: Shared medical appointments (SMAs) are group clinics where practitioners see several patients, with common health needs, at once. There is a great financial strain on the National Health Service (NHS) to provide bariatric surgery. The aim of this study was to review patient satisfaction with the SMA that is the default means of following up patients after bariatric surgery at one particular NHS trust. METHODS: A patient-validated questionnaire was designed and handed out at the end of the SMAs. Patients who attended an SMA earlier in 2011 were also retrospectively sent questionnaires via post. RESULTS: A total of 47 patients completed the questionnaire from seven different SMAs covering the period from January to July 2011. All patients underwent laparoscopic adjustable gastric banding. After attending an SMA, patients gave an overall mean satisfaction rating of 4.13 ± 0.163 (on a scale of 1 to 5, 1 = very poor and 5 = excellent) which represented an increase (p < 0.01) compared to preconceptions before the clinic (3.59 ± 0.175). A cost analysis estimated a yearly saving of £4,617 or 65.1% made by the SMAs compared to 1:1 appointments. CONCLUSIONS: The bariatric surgery SMA demonstrates high levels of patient satisfaction and is cost-effective.

The acridone derivative MBLI-87 sensitizes breast cancer resistance protein-expressing xenografts to irinotecan.

The breast cancer resistance protein ABCG2 confers cellular resistance to irinotecan (CPT-11) and its active metabolite SN-38. We utilised ABCG2-expressing xenografts as a model to evaluate the ability of a non-toxic ABCG2 inhibitor to increase intracellular drug accumulation. We assessed the activity of irinotecan in vivo in SCID mice: irinotecan completely inhibited the development of control pcDNA3.1 xenografts, whilst only delaying the growth of ABCG2-expressing xenografts. Addition of MBLI-87, an acridone derivative inhibitor, significantly increased the irinotecan effect against the growth of ABCG2-expressing xenografts. In vitro, MBLI-87 was as potent as GF120918 against ABCG2-mediated irinotecan efflux, and additionally was specific for ABCG2. A significant sensitisation to irinotecan was achieved despite the fact that doses remained well below the maximum tolerated dose (due to the rather limited solubility of MBLI-87). This suggested that MBLI-87 is an excellent candidate to prevent drug efflux by ABCG2, without altering plasma concentrations of irinotecan and SN-38 after IP (intra-peritoneal) injections. This could constitute a useful strategy to improve drug pharmacology, to facilitate drug penetration into normal tissue compartments protected by ABCG2, and potentially to reverse drug resistance in cancer cells.

An overview of the management of morbid obesity.

Obesity is a modern-day epidemic with serious physical, psychological and economic implications for the patients. Tackling obesity is now a priority for most healthcare providers. Managing such patients can be complex, emotional, time consuming and often frustrating. Obesity surgery, in its various forms, has revolutionised this struggle. With appropriate selection of patients, adequate resources and a multidisciplinary team involvement, obesity can now effectively be "cured". It is vital that those who deal with obese patients know how to access these services and understand the processes involved in the journey from initial assessment to postoperative follow-up. Obesity surgery has a major impact in reducing obesity-related comorbidities such as diabetes and hypertension and contributes to society by returning patients to work. Prevention must be at the heart of any strategy to manage obesity, but, for established cases, surgery is taking centre stage and will continue to flourish as new techniques and procedures are developed.

Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes.

Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.

Imatinib mesylate and nilotinib (AMN107) exhibit high-affinity interaction with ABCG2 on primitive hematopoietic stem cells.

The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dose-dependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [(125)I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.

ABC transporters in the balance: is there a role in multidrug resistance?

Drug resistance can occur at several levels and is the major cause of treatment failure in oncology. The ABC (ATP-binding cassette) transporters, beginning with the discovery of P-glycoprotein (Pgp) almost 30 years ago, have been intensively studied as potential mediators of drug resistance. Although we understand that drug resistance is almost certainly multifactorial, investigators have attempted to link anticancer drug resistance to overexpression of ABC transporters and the consequent reduction in drug accumulation. A body of evidence implicated Pgp as being important in clinical outcome; however, critical studies aimed at proving the hypothesis using Pgp inhibitors in clinical trials have to date failed. Identification of the MRP (multidrug resistance protein)/ABCC subfamily expanded the possible mechanisms of reduced drug accumulation, and the discovery of ABCG2 added a new chapter in these investigations. Correlative studies examining ABCG2 and the ABCC subfamily members in clinical drug resistance have been less avidly pursued, while basic molecular studies of structure and function have proceeded briskly. Recently, studies have focused on how single nucleotide polymorphism in multidrug transporters might affect the pharmacokinetics and pharmacodynamics of anticancer agents. These studies suggest an important role for ABC transporters in pharmacology, independent of the ultimate determination of their role in multidrug resistance.

Plasma pharmacokinetics and tissue distribution of the breast cancer resistance protein (BCRP/ABCG2) inhibitor fumitremorgin C in SCID mice bearing T8 tumors.

Multidrug resistance (MDR) remains a major obstacle in the treatment of human cancers. The recently discovered breast cancer resistance protein (BCRP/ABCG2) has been found to be an important mediator of chemotherapeutic MDR. Fumitremorgin C (FTC) is a selective and potent inhibitor of BCRP that completely inhibits and reverses BCRP-mediated resistance at micromolar concentrations. We report a study of the pharmacokinetics and tissue distribution of FTC when administered intravenously (IV) at a dose of 25 mg/kg to female SCID mice bearing the BCRP-overexpressing human ovarian xenograft Igrov1/T8 tumors. Plasma pharmacokinetics and tissue distribution of FTC in various organs and tissues were studied. In addition, the effect of FTC administration on the expression of BCRP in T8 tumors was also assessed by RT-PCR. Administration of a single FTC IV dose did not appear to cause any major toxicities. The resulting pharmacokinetic data were fit to a two-compartment model using NONMEM and the FTC clearance was determined to be 0.55 ml/min (25.0 ml/min/kg) with a 56% inter-animal variability. Area under the plasma concentration time curve was determined by Bailer's method and was calculated to be 1128+/-111 microg min/ml. FTC was widely distributed in all tissues assayed with highest concentrations found in lungs, liver and kidney in decreasing order, respectively. FTC did not appear to have any effect on the expression of BCRP in T8 tumors. Less than 2% of the administered dose was recovered in the urine and feces after 24 h, suggesting hepatic metabolism as a primary mechanism of elimination. The current study can be used as a basis for future animal or in vivo studies with FTC designed to further understand the impact of BCRP on drug resistance.

Intractable cancers: the many faces of multidrug resistance and the many targets it presents for therapeutic attack.

Some types of cancer respond far less favorably to treatment than do others. A quantitative estimate of this intuition can be obtained from the SEER (Surveillance, Epidemiology and End-Results) Cancer Statistics Review. Of particular interest, from a drug resistance perspective, are the five-year survival data for patients presenting with tumors that were diagnosed as "distant". A good correlation can be found between those numbers and an estimate of treatment successes obtained from a survey of current literature on chemotherapy applied to cancers originating from these various tissues. These two measures, considered together, define "the axis of intractability", a parameter that characterizes the (possibly) inherent, physiological basis of the tissue-by-tissue intractability of cancers. Exploring the basis of this intractability, it appears that factors other than the classical ABC transporter-based, multidrug resistance systems probably play a major role. An ineffective DNA repair system, coupled to reduced apoptosis, is the basis for the inherent tractability of testicular cancer. For other tissues, important contributions to resistance arise from cell adhesion-mediated drug resistance, which is overcome when cells are released from tissues during anoikis. Making a direct comparison between gene expression in solid tumors and their corresponding cell lines, genes controlling the extracellular matrix and cell-cell communication appear among the genes that are over-expressed in the solid tumors, while genes coding for the protein biosynthesis system are over-expressed in the cell lines. The more tractable cancers are closer to the cell lines in their expression profiles of these sets of genes.

Repair rates of R-band, G-band and C-band DNA in murine and human cultured cells.

Repair of cyclobutane pyrimidine dimers (CPDs) in cultured neonatal human fibroblasts and in Mus spretus x M. castaneus F1 neonatal skin fibroblasts was analyzed after UVC-irradiation by cleavage with T4 endonuclease V cyclopyrimidine dimer glycosylase, alkaline-agarose gel electrophoresis, and Southern blotting. The blots were sequentially probed with 32P-labeled Alu, or B2, to preferentially illuminate R-band DNA, by L1 to preferentially illuminate G-band DNA, and by satellite DNA to illuminate C-band DNA. These three different DNA populations showed slightly different global nucleotide excision repair rates that are in the order of speed, R-band DNA > G-band DNA > C-band DNA. Fibroblasts from out-bred neonatal mice and humans showed similar band-specific repair rate ratios and the global repair rate of murine fibroblasts was almost as rapid as that of the human fibroblasts. The mass distribution of the human Alu-probed signal was further analyzed. Gel mobility data was fitted to a logistic equation to include all M(r) values. Hypothetical distributions of DNA randomly cleaved to a particular number-average molecular weight were fit to the logistic gel mobility function to determine how such a randomly cleaved distribution of a particular cleavage frequency would be displayed along the experimental gel. This revealed a rapidly repaired kinetic fraction that represented 17% of the Alu-probed signal (R-band DNA), almost none of the L1 probed signal (G-band DNA), and reflects transcription coupled repair of active genes. The remaining Alu-probed DNA showed a random distribution of UVC-induced CPDs throughout all stages of global nucleotide excision repair. The Alu-probed CPDs disappeared with an excellent fit to first order kinetics and with a half-life of seven hours.

Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity.

Recent studies have shown that mutations at amino-acid 482 in the ABCG2 gene affect the substrate specificity of the protein. To delineate the effects of these mutations clearly, human embryonic kidney cells (HEK-293) were stably transfected with wild-type 482R or mutant 482G and 482T ABCG2. By flow cytometry, mitoxantrone, BODIPY-prazosin, and Hoechst 33342 were found to be substrates of all ABCG2 proteins, while rhodamine 123, daunorubicin, and LysoTracker Green were transported only by mutant ABCG2. In cytotoxicity assays, all ABCG2 proteins conferred high levels of resistance to mitoxantrone, SN-38, and topotecan, while mutant ABCG2 also exhibited a gain of function for mitoxantrone as they conferred a four-fold greater resistance compared to wild type. Cells transfected with mutant ABCG2 were 13- to 71- fold resistant to the P-glycoprotein substrates doxorubicin, daunorubicin, epirubicin, bisantrene, and rhodamine 123 compared to cells transfected with wild-type ABCG2, which were only three- to four-fold resistant to these compounds. ABCG2 did not confer appreciable resistance to etoposide, taxol or the histone deacetylase inhibitor depsipeptide. None of the transfected cell lines demonstrated resistance to flavopiridol despite our previous observation that ABCG2-overexpressing cell lines are cross-resistant to the drug. Recently reported inhibitors of ABCG2 were evaluated and 50 microM novobiocin was found to reverse wild-type ABCG2 completely, but only reverse mutant ABCG2 partially. The studies presented here serve to underscore the importance of amino-acid 482 in defining the substrate specificity of the ABCG2 protein and raise the possibility that amino-acid 482 mutations in human cancers could affect the clinical application of antagonists for ABCG2.

Inhibitor of histone deacetylation, depsipeptide (FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a case report.

Depsipeptide, FR901228, has demonstrated potent in vitro and in vivo cytotoxic activity against murine and human tumor cell lines. In the laboratory, it has been shown to be a histone deacetylase (HDAC) inhibitor. In a phase I trial of depsipeptide conducted at the National Cancer Institute, 3 patients with cutaneous T-cell lymphoma had a partial response, and 1 patient with peripheral T-cell lymphoma, unspecified, had a complete response. Sézary cells isolated from patients after treatment had increased histone acetylation. These results suggest that inhibition of HDAC is a novel and potentially effective therapy for patients with T-cell lymphoma.

Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells.

A disparity was noted in the transport of rhodamine 123 among nine MXR/BCRP/ABCP-overexpressing cells studied; all demonstrated mitoxantrone transport, whereas only two effluxed rhodamine 123. When the MXR/BCRP/ABCP gene was sequenced in the cell lines studied, differences were noted at amino acid 482, predicted to be at the start of the third transmembrane domain. Sequencing genomic DNA revealed wild-type MXR/BCRP/ABCP to have an arginine at position 482. Cells having a threonine or glycine at position 482 were able to efflux rhodamine 123, whereas cells having an arginine were not. A vaccinia virus expression system confirmed that rhodamine as well as doxorubicin efflux is observed with R482T or R482G but not with the wild-type R482; all three MXR/BCRP/ABCP forms transported mitoxantrone. Cross-resistance studies suggest that, compared with wild-type MXR/BCRP/ABCP, cells having an R482T mutation have higher anthracycline resistance, whereas an R482G mutation seems to confer relatively less resistance to SN-38 and topotecan. These results suggest that amino acid 482 has a crucial role in MXR/BCRP/ABCP function and that mutation of a single amino acid residue significantly changes substrate specificity, thus altering the drug resistance phenotype.

From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance.

The ATP binding cassette (ABC) superfamily of membrane transporters is one of the largest protein classes known, and counts numerous proteins involved in the trafficking of biological molecules across cell membranes. The first known human ABC transporter was P-glycoprotein (P-gp), which confers multidrug resistance (MDR) to anticancer drugs. In recent years, we have obtained an increased understanding of the mechanism of action of P-gp as its ATPase activity, substrate specificity and pharmacokinetic interactions have been investigated. This review focuses on the functional characterization of P-gp, as well as other ABC transporters involved in MDR: the family of multidrug-resistance-associated proteins (MRP1-7), and the recently discovered ABC half-transporter MXR (also known as BCRP, ABCP and ABCG2). We describe recent progress in the analysis of protein structure-function relationships, and consider the conceptual problem of defining and identifying substrates and inhibitors of MDR. An in-depth discussion follows of how coupling of nucleotide hydrolysis to substrate transport takes place, and we propose a scheme for the mechanism of P-gp function. Finally, the clinical correlations, both for reversal of MDR in cancer and for drug delivery, are discussed.

A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2).

The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compound in a series of MXR-overexpressing drug-selected cell lines and in ten unselected cell lines which were used to determine if the four fluorescent compounds were sensitive enough to detect the low MXR levels found in drug-sensitive cell lines. FTC-inhibitable efflux of mitoxantrone and prazosin was found in four of the ten cell lines, SF295, KM12, NCI-H460, and A549, and low but detectable levels of MXR mRNA were also observed by Northern analysis in these cells. FTC-inhibitable mitoxantrone and prazosin efflux in both selected and unselected cell lines was found to correlate well with MXR levels as determined by Northern blotting, r(2)=0.89 and r(2)=0.70 respectively. In contrast, rhodamine and LysoTracker were not able to reliably detect MXR. Cytotoxicity assays performed on two of the four unselected cell lines confirmed increased sensitivity to mitoxantrone in the presence of FTC. FTC was found to be a specific inhibitor of MXR, with half-maximal inhibition of MXR-associated ATPase activity at 1 microM FTC. Short term selections of the SF295, KM12, NCI-H460 and A549 cell lines in mitoxantrone resulted in a small but measurable increase in MXR by both Northern blot and functional assay. These studies show that flow cytometric measurement of FTC-inhibitable mitoxantrone or prazosin efflux is a sensitive and specific method for measuring the function of the MXR half-transporter in both selected and unselected cell lines.

The P-glycoprotein antagonist PSC 833 increases the plasma concentrations of 6alpha-hydroxypaclitaxel, a major metabolite of paclitaxel.

PURPOSE: Overexpression of P-glycoprotein (Pgp) is one mechanism of drug resistance in cancer chemotherapy. A Phase I trial was conducted using PSC 833, a Pgp antagonist, in combination with paclitaxel in patients with refractory cancer. The objective of this study was to assess the effect of PSC 833 on the metabolism of paclitaxel and characterize the differences in 6alpha-hydroxypaclitaxel pharmacokinetics. In addition, we examined the possibility of enhanced cytotoxicity of paclitaxel by the coexistence of 6alpha-hydroxypaclitaxel. EXPERIMENTAL DESIGN: Patients received paclitaxel 35 mg/m(2)/day by continuous intravenous infusion (CIVI) x 4 days without PSC 833 in cycle 1 and escalating doses of paclitaxel (13.1, 17.5, or 21.3 mg/m(2)/day CIVI x 4 days) with 5 mg/kg PSC 833 by mouth every 6 h x 7 days in cycle 2. Plasma samples were analyzed for both paclitaxel and its major metabolite with high-performance liquid chromatography methods. Using human liver microsomes, we studied the effect of PSC 833 on the metabolism of paclitaxel. In addition, the in vitro cytotoxicity of 6alpha-hydroxypaclitaxel alone and in combination with paclitaxel was evaluated. RESULTS: Twenty-one of 22 patients had a metabolite peak (6alpha-hydroxypaclitaxel) observed in the chromatogram of plasma samples from cycle 2 when they received paclitaxel in combination with PSC 833. This metabolite was not detectable in plasma obtained during the first cycle when they received paclitaxel without PSC 833. During cycle 2, the mean concentrations of 6alpha-hydroxypaclitaxel and paclitaxel were 0.10 +/- 0.074 and 0.079 +/- 0.041 microg/ml, respectively. A moderate association was observed between total bilirubin and 6alpha-hydroxypaclitaxel concentrations (P = 0.015, r = 0.52; n = 21). Human liver microsome experiments showed that a PSC 833 concentration as high as 10 microM did not affect the production of 6alpha-hydroxypaclitaxel. Paclitaxel cytotoxicity in HL60 and K562 human leukemia cells was increased in the presence of noncytotoxic concentrations of 6alpha-hydroxypaclitaxel. CONCLUSIONS: PSC 833 increases the plasma concentration of 6alpha-hydroxypaclitaxel during paclitaxel therapy. Inhibition of cytochrome P-450 3A4 by PSC 833 may explain this in part, although other mechanisms cannot be excluded.

Screening for and identification of novel agents directed at renal cell carcinoma.

We were interested in identifying novel agents for renal cell carcinoma (RCC) by screening for activities that model renal tumor biology. Searching for relative renal cell sensitivity and leukemia insensitivity among cytotoxicity profiles in the NCI Drug Screen database, we identified 16 potential agents with renal selectivity. We evaluated the agents in 10 RCC cell lines (of primary and metastatic origin) isolated from 5 patients. The 50% inhibitory concentrations (IC50) in these cell lines ranged from 0.019 +/- 0.013 to 11.4 +/- 0.55 microM and were comparable with values obtained with renal cell lines in the NCI Drug Screen panel. Because RCC are slowly growing tumors, we evaluated the compounds on rapidly (27% S phase) or slowly (6% S phase) growing cells. In contrast to doxorubicin, where cytotoxicity was restricted to rapidly proliferating cells, three compounds (NSC 280074, 281613, and 281817) were more cytotoxic in slowly proliferating cells. NSC 72151 and 268965 were equitoxic for both populations. NSC 94889, 638850, and 630938 were more cytotoxic in rapidly growing cells. In in vitro time exposure studies, four compounds, NSC 268965, 280074, 281613, and 281817, were maximally cytotoxic with as little as 3 h exposure time. From an analysis comparing the p53 genotype of the 60 cell lines of the National Cancer Institute (NCI) Drug Screen with the cytotoxicity profiles for the 16 putative renal compounds, 13 compounds were classified as likely to be indifferent to p53 status. We also developed a panel specificity detection method for the NCI Drug Screen database to evaluate the prevalence of renal sensitive compounds. Of the 16 studied compounds, 14 were among those identified as renal sensitive by the statistical analysis. Lastly, we found reduced tumor growth in mice with established renal human tumor xenografts after treatment with two of the renal active compounds. These studies describe compounds with potential renal activity that are candidates for preclinical development for renal cell carcinoma.

Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells.

We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100 microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated in the presence of 5 microM of the ABCG2 inhibitor fumitremorgin C (FTC), and sensitivity of MCF-7 FLV1000 cells to flavopiridol, mitoxantrone, SN-38, and topotecan was restored. Mitoxantrone efflux studies were performed, and high levels of FTC-reversible mitoxantrone efflux were found. Northern blot and PCR analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol.

The role of half-transporters in multidrug resistance.

ATP-binding cassette proteins comprise a superfamily of transporter proteins, a subset of which have been implicated in multidrug resistance. Although P-glycoprotein was described over 15 years ago, the recent expansion in the number of transporters identified has prompted renewed interest in the role of drug transporters in clinical drug resistance. These newly identified transporters include additional members of the MRP family, ABC2, and a new half-transporter, MXR/BCRP/ABCP1. This half-transporter confers high levels of resistance to mitoxantrone, anthracyclines, and the camptothecins SN-38 and topotecan. At 72 kDa, MXR localizes to the plasma membrane in cells which highly overexpress the protein either through gene amplification or though gene rearrangement. Future studies will be aimed at identifying an inhibitor, and attempting to translate recognition of this new transporter into a target for anticancer treatment.