Anthracycline Resistance Research Articles

MAPK overexpression is associated with anthracycline resistance and increased risk for recurrence in patients with triple-negative breast cancer

BackgroundTriple-negative breast cancer is estimated to account for 15%–20% of all patients with breast cancer and is considered as a prognostically unfavorable subset. The aim of this study is to evaluate the prognostic impact of various molecular factors in patients with triple-negative breast cancer. Patients and methodsTumor specimens from 109 patients with receptor-negative (estrogen receptor and progesterone receptor) breast cancer were analyzed for mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR) and phosphoinositol-3-kinase (PI3K) expression by immunohistochemistry. The prognostic significance of these molecular factors, in addition to various prognostic variables, was investigated. ResultsFifteen (13.8%), 38 (34.9%) and 33 patients (30.3%) had positive staining for EGFR, MAPK and PI3K, respectively. MAPK was associated with anthracycline resistance (P=0.008) and lower MAPK score was significantly associated with shorter disease-free survival (P=0.029). Survival following relapse was significantly worse for those with a higher MAPK score (P=0.03). ConclusionMAPK is a significant prognostic and predictive factor in patients with triple-negative breast cancer. Furthermore, the level of staining among those with a positive MAPK expression may play a prognostic role at different stages of relapse. Further translational research is required to elucidate molecular mechanisms of tumor proliferation in this subset of patients.

Cellular Mechanisms of Resistance to Anthracyclines and Taxanes in Cancer: Intrinsic and Acquired

Taxanes and anthracyclines are two of the most potent and broadly effective classes of chemotherapeutic agents. However, resistance to these agents is common and significantly limits their potential. As such, there is a great need to understand the mechanisms underlying de novo and acquired resistance to these agents. Beyond the resistance barrier lies even greater potential to significantly alter the natural course of human cancer. This review discusses what we currently understand about the mechanisms of resistance to taxanes and anthracyclines. Preclinical models suggest a role for ATP-binding cassette transporters, tubulin isoforms, microtubule-associated proteins, tubulin gene mutations, and mitotic checkpoint signaling proteins in resistance to taxanes. Preclinical models also suggest that drug transport proteins, antioxidant defenses, apoptotic signaling, and topoisomerase modulation may mediate anthracycline resistance. Many of these hypotheses remain untested in appropriately designed clinical studies, but limited clinical evidence will be reviewed. Epothilones represent a novel class of non-taxane microtubule stabilizing agents with distinct drug-resistance profiles. Potential mechanisms behind these differences and their potential role in the treatment of both taxane- and anthracycline-refractory patients are discussed.

Lower Level of MAPK Expression Is Associated with Anthracycline Resistance and Decreased Survival in Patients with Hormone Receptor Negative Breast Cancer

Introduction: Hormone receptor negative breast cancer is encountered in about 30% of all patients with breast cancer and is considered as a prognostically unfavorable subset. The aim of this study is to evaluate the prognostic impact of various molecular markers in patients with receptor negative breast cancer. Methods: Tumor specimens from 140 patients with receptor negative (ER, PR) breast cancer were analyzed for MAPK, Her-2/neu, EGFR and PI3K expression by immunohistochemistry. The prognostic significance of these molecular factors, in addition to various prognostic variables were determined with respect to disease-free and overall survival.Results: Nineteen (13.6%), 45 (32.1%), 16 (11.4%) and 47 (33.5%) patients had positive staining for EGFR, PI3K, Her-2/neu and MAPK, respectively. Twenty-three patients with positive MAPK (16.4%) had a high level of expression (score 4–7) and 24 (17.1%) had a low score (1–3). A lower percentage of MAPK expression was significantly associated with a poorer OS (p = 0.03) and a tendency for shorter DFS (p = 0.08) among those who were positive for MAPK. Anthracycline resistance remained the only independent significant variable for OS by Cox regression analysis (p = 0.001, HR:26.1). In patients with recurrent disease, median survival after initial relapse was 16.8 months. MAPK was determined as the only prognostic factor for this endpoint. Patients with higher level of MAPK staining showed significantly shorter survival following initial recurrence (p = 0.04). Conclusion: MAPK expression is a significant prognostic factor for non-metastatic patients with hormone receptor breast cancer. A lower level of staining is shown to be associated with with antracycline resistance and oveall survival, whereas a higher expression level is correlated with shorter survival following initial relapse, suggesting possible role of different molecular mechanisms pertaining to tumor progression once recurrence occurs. Further translational research is required to elucidate molecular mechanisms of the cross-talk between intracellular signaling and molecular pathways leading to drug resistance in patients with receptor negative breast cancer.

C-Jun N-Terminal Kinase (JNK) Activation Failure Confers a New Mechanism of Anthracycline Resistance in Acute Myeloid Leukemia (AML).

Chemotherapy resistance remains a major challenge in the treatment of acute myeloid leukemia (AML). Besides the P-glycoprotein efflux of chemotherapeutics, additional cellular factors may contribute to drug resistance in AML. c- Jun N-terminal Kinase (JNK) is a protein kinase activated after exposure of cells to chemotherapeutic agents. Recently, studies in solid tumours have associated chemoresistance with failure of cancer cells to activate JNK. We asked whether drug resistance in AML is also attributed to intrinsic failure of the AML blasts to activate JNK. In vitro treatment of U937 AML cell line with anthracyclines induced a rapid and robust JNK phosphorylation and apoptosis. In contrast, the anthracyline-resistant derivative U937R cells showed no JNK activation after exposure to anthracyclines, also at doses that resulted in high accumulation of the drug within the cells. Inhibition of JNK in drug-sensitive U937 cells made them resistant to anthracyclines. First, JNK1-siRNA transfected U937 cells exhibited a 50.4% and 61.3% reduced daunorubicin- (DNR, 1μM 24hr) and doxorubicin- (DOX, 1.5 μM 24hr) induced apoptosis respectively; as compared to empty vector or untransfected U937 cells (P< 0.001). Second, pretreatment of U937 cells with the 420116 cell-permeable JNK inhibitor (1 μM) reduced to a less but yet significant extent DNR-induced apoptosis as compared to cells treated with a negative control peptide (P = 0.013<0.05). On the other hand, selective restoration of the inactive JNK pathway in resistant U937R cells by transfection with a mutant form of the SEK1/MKK4 upstream activator of JNK sensitized U937R cells to anthracyclines, compared to empty vector transfected cells (3.3-fold increase in DNR-induced apoptosis, 1μM DNR, 24hr and 3.1-fold increase in DOX-induced apoptosis, 1.5μM DOX, 24hr). Furthermore, we assessed the activation of JNK pathway in 29 primary AML bone marrow samples after short term (30-60 min) in vitro exposure to DNR (1 μM) and correlated it with clinical data. We found a strong correlation between the in vitro pharmacodymanic changes of phospho-JNK levels in AML primary blasts and the response of the AML patients to standard induction chemotherapy (P = 0.012). In addition, the drug-induced JNK activation pattern correlated with AML evolving from antecedent MDS (P = 0.017) and patient age (P = 0.046). In summary, our in vitro and in vivo results suggest that JNK activation failure is another mechanism of anthracycline resistance in AML. Elucidating the ultimate mechanisms leading to JNK suppression in chemoresistant AML may be of major therapeutic value.

Lymphocyte expression of CD95 at rest and in response to acute exercise in healthy children and adolescents

CD95 is essential in regulating the immune system, and its reduced expression has been associated with anthracycline resistance in some childhood leukemias. We determined whether an acute bout of exercise would influence the mixed lymphocyte expression of CD95 in healthy children and adolescents. Fifty healthy pediatric subjects (female n = 25) cycled for 60 min @ 70% V ˙ O 2 max . Blood was collected before, during, and after exercise to determine CD95 expression on T (CD3 +CD19 −) and B (CD3 −CD19 +) cells using flow cytometry. At rest, the percentage of CD95 hi T cells was 29% higher in girls versus boys ( p < 0.001). The percentage of CD95 hi T cells and the median fluorescence intensity (MFI) of CD95 hi were higher during recovery versus rest ( p < 0.0001). The percentage of CD95 lo T cells was lower during recovery versus rest, whereas the MFI of CD95 lo was higher during exercise versus rest. The CD95 hi T cell count increased during exercise, but only in 14-yr-old girls, whereas CD95 lo T cell counts increased similarly in all groups. CD95 + B cells were relatively unresponsive to exercise. We conclude that lymphocyte expression of CD95 is influenced by acute exercise in healthy children and adolescents. Exercise-induced alterations in CD95 expression may have clinical implications for some pediatric conditions.

New Pinostilbene Analogues Overcome Anthracycline Resistance in Acute Lymphoplastic Leukemia Ex Vivo.

Anthracylines play a very important role in the treatment of acute lymphoblastic leukemia (ALL) and relapsed ALL in childhood, however resistance to anthracyclines leads to a poor prognosis. In the present study, we have synthesized two new pinostilbene analogues, i.e.trans-3,4′-dihydroxy-5-methoxystilbene and(E)-resveratrol 4′-O-ß-D-glucopyranoside, which are able to overcome anthracycline resistance in childhood acute lymphoblastic leukemia (ALL) ex vivo and induce apoptosis in established leukemia cell lines via mitochondrial pathway.Apoptosis induction has been investigated by flowcytometric measurement of DNA-fragmentation [LC 50: 10 μM for (1) and (2)], mitochondrial membrane potential reduction and phosphatedylserin-staining on cell membrane surface. Cell death by necrosis could be excluded by a lactatdehydrogenase-release assay. For the first time we analysed the antileukemic and chemopreventive potentials of the pinostilbene analogues (1) and (2) ex vivo in a significant number of primary lymphoblasts of patients suffering from childhood ALL.Patients: Primary lymphoblasts isolated from 22 children with de novo ALL (median: 6.8 years; range 0,6–16.9 years) and relapsed ALL (median:7.2 years) were tested for ex vivo drug response with the anthracyclines daunorubicin (10 μmol/l) and doxorubicin (10 μmol/l) and two new pinostilbene analogues (1) and (2) (10μmol/l), according to their LC50 values in established cell lines. We could demonstrate in these primary cells that the pinostilbenes (1) and (2) were even more effective as compared with the anthracyclines. Out of 22 patients 14 were female (14 de novo ALL) and 8 were male (6 de novo ALL, 2 relapsed ALL). Within these cell populations following immunologic subgroups were found: c-ALL, pre-B-ALL, pro-B-ALL, T-ALL and pre-T-ALL.Results: Daunorubicin induced apoptosis in 6 out of 22 lymphoblast populations (response rate 27,3 %). A similar response rate was observed after treatment with doxorubicin: only 5 of 22 lymphoblast populations responded (22,7%). Nevertheless, far higher response rates were observed for (1) with 11/15 (73,3 %; p<0.005) and for (2) with 15/17 (88,2%; p<0.0002, all p-values by t-test). Interestingly, treatment of daunorubicin-resistant lymphoblasts resulted in significant apoptosis induction in 6 out of 10 cell populations after treatment with compound (1) (response rate 60 %) and in 6/6 after treatment with compound (2) (response rate 100%). Furthermore, pinostilbene (2) showed significant synergistic activity with daunorubicin in 2 of 3 lymphoblast populations. We clearly demonstrated that the ex vivo treatment of lymphoblasts from children with de novo and relapsed ALL with the new pinostilbenes (1) and (2) induced significantly higher response rates than daunorubicin or doxorubicin treatment. In conclusion, the high ex vivo sensitivity of anthracycline resistant leukemia cells to pinostilbene treatment reveals the great proapoptotic and chemopreventive potential of this new class of antileukemic agents.

The clinical benefit of pegylated liposomal doxorubicin in patients with metastatic breast cancer previously treated with conventional anthracyclines: a multicentre phase II trial

This study evaluates the clinical benefit of pegylated liposomal doxorubicin (PLD) in patients with metastatic breast cancer (MBC), previously treated with conventional anthracyclines. Seventy-nine women with MBC previously treated with anthracyclines received PLD 50 mg m−2 every 4 weeks. All patients were previously treated with chemotherapy and 30% of patients had ⩾3 prior chemotherapies for metastatic disease. Patients were considered anthracycline resistant when they had disease progression on anthracycline therapy for MBC or within 6 months of adjuvant therapy. The overall clinical benefit rate (objective response+stable disease ⩾24 weeks) was 24% (16.1% in patients with documented anthracycline resistance vs 29% in patients classified as having non-anthracycline-resistant disease). There was no difference with respect to the clinical benefit between patients who received PLD >12 months and those who received PLD ⩽12 months since last anthracycline treatment for metastatic disease (clinical benefit 25 vs 24.1%, respectively). Median time to progression and overall survival were 3.6 and 12.3 months, respectively. The median duration of response was 12 months, and the median time to progression in patients with stable disease (any) was 9.5 months. Fourteen patients (17.7%) had a prolonged clinical benefit lasting ⩾12 months. In conclusion, PLD was associated with an evident clinical benefit in anthracycline-pretreated patients with MBC.

The nitroxide Tempol modulates anthracycline resistance in breast cancer cells

The occurrence of multidrug resistance (MDR) is the major obstacle to successful anthracycline-based cancer chemotherapy. In the present study, we assessed the effects of Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine- N-oxyl, TPL), a piperidine nitroxide with growth-inhibitory properties in tumor cell lines, on a number of molecular mechanisms involved in the resistance of human breast adenocarcinoma cell lines to doxorubicin (DOX). Cytotoxicity studies in MCF-7 wildtype and their MDR variant MCF-7 Adr R cells showed a synergistic effect between TPL and DOX when exposure to TPL preceded or was simultaneous with DOX treatment in MCF-7 Adr R cells. This effect of TPL seems to be due in part to its ability to increase peroxide levels and to deplete cellular glutathione pools. In addition, TPL increased DOX accumulation in MCF-7 Adr R cells by interfering with P-glycoprotein-mediated DOX efflux, as evidenced using a specific antibody that recognizes the active form of the protein. TPL was also found to affect the expression levels of proteins involved in response to drug treatment (e.g., p53, bcl2, bax, p21). Taken together, our results indicate that TPL is a potential new agent that may improve the clinical effect of DOX in tumors exhibiting a MDR phenotype.

Effects of the plant alkaloid thaliblastine on non-cross-resistant and sensitive human leukemia cells in relation with reversal of acquired anthracycline resistance

Thaliblastine exhibits dose dependent cytotoxic effect on HL-60, HL-60/DOX, RHE and HD-MY-2 leukemia cells. Additionally, typical for apoptosis oligonucleosomal DNA fragmentation could be detected in leukemia cells treated with thaliblastine. Moreover, an MDR-phenotype reversing effect of thaliblastine was also identified.

Breast cancer resistance protein (BCRP/MXR/ABCG2) in adult acute lymphoblastic leukaemia: frequent expression and possible correlation with shorter disease‐free survival

Drugs used in treatment of adult acute lymphoblastic leukaemia (ALL) are substrates for breast cancer resistance protein (BCRP, MXR, ABCG2), which may thus play a role in resistance in this disease. Pretreatment blasts from 30 adult ALL patients were studied for BCRP mRNA by quantitative reverse transcription polymerase chain reaction analysis, BCRP protein by immunophenotyping with three antibodies and BCRP function by fumitremorgin C modulation of intracellular mitoxantrone retention, measured by flow cytometry. BCRP mRNA in all cases encoded wild type protein (BCRP(R482)), which mediates mitoxantrone and methotrexate resistance, but only low-level anthracycline resistance. The BXP-21, BXP-34 and anti-ABCG2 antibodies stained blasts in 13, 11 and 14 cases (43%, 37% and 47%); BXP-21 correlated well with BXP-34 and anti-ABCG2, but BXP-34 and anti-ABCG2 did not correlate, and antibody staining did not correlate with mRNA levels. BCRP function was seen in 21 cases (70%), but correlated poorly with antibody staining. An exploratory statistical analysis indicated that BXP-21 staining was predictive of shorter disease-free survival (DFS) (P = 0.0374) in this small patient population. Poor correlations between mRNA, protein and function indicate the complex biology of BCRP in adult ALL, and the possible correlation of BCRP expression with DFS should be studied in larger series.

The prognostic role of serum telomerase activity in advanced breast cancer receiving anthracyclines

861 Background: Breast cancer is the second leading cause of cancer deaths throughout the world. Cytotoxic chemotherapy including anthracyclines is the main stay of the treatment of the advanced breast cancer. However, the resistance is an important problem for the most of the patients. Various prognostic factors have been studied to determine the anthracycline resistance and prognosis. In this preliminary study, we aimed to assess the relation of serum telomerase activity with the response to anthracyclines and role of the serum enzyme activity as a prognostic factor in patients with advanced breast cancer. Methods: Nineteen chemo naive patients with advanced breast cancer were included. The first line chemotherapy regimens included the standard doses of doxorubicine plus cyclophosphamide in 14 patients, cyclophosphamide, fluorouracil and doxorubicine in 4 and cyclophosphamide, fluorouracil and epirubicine in 1 patient. Serum telomerase activity was measured before chemotherapy and every 2 cycles thereafter by using a PCR-ELISA trap assay. ER/PR, c-erbB2 expression of tumor tissues were determined by immunohistochemistry. Results: The median age was 47 years. The overall response rate was 57.9%. Half of the patients have ER/PR and 44.4% have erb-B2 positive tumors. The rate of serum telomerase positivity was 39.5%. Though not significant the patients without erb-B2 expression or non-detectable serum telomerase activity has tended to have better response rates when compared to positive ones (83.3%, P=0.177 and 83.3%, P=0.206, respectively). Though not significant, the patients without serum telomerase have better 1-year survival rate when compared to positive ones (62.5% vs. 47.6%, P=0.350). Conclusions: In conclusion, the results of the c current preliminary study shows that serum telomerase activity could be detected in the sera of the cancer patients and detectable serum levels could be a poor prognostic factor for response to the anthracycline containing regimens and prognosis in patients with advanced breast cancer. No significant financial relationships to disclose.

Neoadjuvant Chemotherapy of Breast Cancer

The use of neoadjuvant chemotherapy in the treatment of breast cancer has been evolving during the past two decades. Fisher's group accomplished the scientific rationale in mouse models in the 1970s. The Milan group was the first to use neoadjuvant therapy in locally advanced breast cancer and also determined that adjuvant sequential doxorubicin with cyclophosphamide/methotrexate/fluorouracil (CMF) offered improved survival when compared to alternating CMF with doxorubicin. Other groups (M. D. Anderson and NSABP) have evaluated similar doxorubicin-based neoadjuvant therapies in locally advanced breast cancer (LABC) and in early disease. These studies have shown an increase in breast-conserving therapy (BCT) and an increase in pathologic complete response (pCR) when neoadjuvant therapy was used. Due to anthracycline resistance, taxanes were added to the doxorubicin-based neoadjuvant chemotherapy regimen with an increase in BCT and pCR than when an anthracycline regimen was used alone. Overall survival has yet to be determined when comparing an anthracycline-based regimen to the same regimen with the addition of a sequential taxane. Therefore, a combined treatment of an anthracycline/cyclophosphamide/taxane regimen is the recommended neoadjuvant chemotherapy for patients with breast cancer.

Treatment of Anthracycline-Resistant Breast Cancer

The anthracyclines are commonly used for the treatment of early stage and advanced stage breast cancer, but many patients develop resistance to therapy. The definition of anthracycline resistance varies considerably in the literature, but in most cases includes disease progression during or within 6–12 months after completion of anthracycline therapy. Some authors have distinguished true anthracycline resistance (defined as progression during anthracycline therapy) from anthracycline pretreatment (defined as progression after completion of therapy).

Evaluation of the binding of the tricyclic isoxazole photoaffinity label LY475776 to multidrug resistance associated protein 1 (MRP1) orthologs and several ATP-binding cassette (ABC) drug transporters

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB 100 cells to both vincristine and doxorubicin. LY475776 photolabeled 170 kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190 kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C 4, vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.

Functional Importance of Polar and Charged Amino Acid Residues in Transmembrane Helix 14 of Multidrug Resistance Protein 1 (MRP1/ABCC1): IDENTIFICATION OF AN ASPARTATE RESIDUE CRITICAL FOR CONVERSION FROM A HIGH TO LOW AFFINITY SUBSTRATE BINDING STATE

Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions. We previously demonstrated that Glu1089 in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance. We have now assessed the functional importance of all polar and charged amino acids in this TM helix. Asn1100, Ser1097, and Lys1092, which are all predicted to be on the same face of the helix as to Glu1089, are involved in determining the substrate specificity of the protein. Notably, elimination of the positively charged side chain of Lys1092, increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16). In addition, mutations S1097A and N1100A selectively decreased transport of 17beta-estradiol 17-(beta-d-glucuronide) (E217betaG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity. In contrast, mutations of Asp1084 that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E217betaG and LTC4, despite the fact that LTC4 binding was unaffected. We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2. Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp1084 may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs.

Inhibitors of vacuolar H+-ATPase impair the preferential accumulation of daunomycin in lysosomes and reverse the resistance to anthracyclines in drug-resistant renal epithelial cells.

It has been suggested that the inappropriate sequestration of weak-base chemotherapeutic drugs in acidic vesicles by multidrug-resistance (MDR) cells contributes to the mechanisms of drug resistance. The function of the acidic lysosomes can be altered in MDR cells, and so we investigated the effects of lysosomotropic agents on the secretion of lysosomal enzymes and on the intracellular distribution of the weak-base anthracycline daunomycin in drug-resistant renal proximal tubule PKSV-PR(col50) cells and their drug-sensitive PKSV-PR cell counterparts. Imaging studies using pH-dependent lysosomotropic dyes revealed that drug-sensitive and drug-resistant cells exhibited a similar acidic lysosomal pH (around 5.6-5.7), but that PKSV-PR(col50) cells contained more acidic lysosomes and secreted more of the lysosomal enzymes N -acetyl-beta-hexosaminidase and beta-glucuronidase than their parent PKSV-PR cells. Concanamycin A (CCM A), a potent inhibitor of the vacuolar H(+)-ATPase, but not the P-glycoprotein modulator verapamil, stimulated the secretion of N -acetyl-beta-hexosaminidase in both drug-sensitive and drug-resistant cells. Fluorescent studies and Percoll density gradient fractionation studies revealed that daunomycin accumulated predominantly in the lysosomes of PKSV-PR(col50) cells, whereas in PKSV-PR cells the drug was distributed evenly throughout the nucleo-cytoplasmic compartments. CCM A did not impair the cellular efflux of daunomycin, but induced the rapid nucleo-cytoplasmic redistribution of the drug in PKSV-PR(col50) cells. In addition, CCM A and bafilomycin A1 almost completely restored the sensitivity of these drug-resistant cells to daunomycin, doxorubicin and epirubicin. These findings indicate that lysosomotropic agents that impair the acidic-pH-dependent accumulation of weak-base chemotherapeutic drugs may reverse anthracycline resistance in MDR cells with an expanded acidic lysosomal compartment.

A mutant TP53 gene status is associated with a poor prognosis and anthracycline-resistance in breast cancer patients

This study evaluates the prognostic and predictive relevance of a mutated p53 in a series of 254 samples from primary breast cancer patients. C-erbB-2 analysis was defined in a limited subpopulation of 79 patients. p53 and c-erbB-2 status was analysed by immunohistochemical staining of the tumour samples. Positive p53 immunostaining was present in 86 cases (34%) and correlated with a high malignant grade, negative progesterone receptor status and ductal histology of tumour. C-erbB-2 positivity was seen in 38 samples (48%). Within an average follow-up time of 74 months, 121 patients developed recurrent or metastatic disease. Patients with mutated p53 showed a statistically significant shorter overall survival and disease-free survival in both univariate and multivariate analyses. The worst clinical outcome was seen in patients who were both p53- and c-erbB-2-positive. The response rate to anthracycline-based chemotherapy in metastatic disease was low in the p53-positive cases. Our results help to clarify the independent prognostic role of a mutated p53 status in breast cancer patients, indicating that this gene might be predictive of anthracycline resistance. Patients with a mutant p53 status and overexpressing c-erbB-2 should be regarded as high-risk cases.

Role of the lung resistance-related protein (LRP) in the drug sensitivity of cultured tumor cells

Drug resistance, one of the major obstacle in the successful anticancer therapy, can be observed at the outset of therapy (intrinsic resistance) or after exposure to the antitumor agent (acquired resistance). To gain a better insight into the mechanisms of intrinsic resistance we have analyzed two human cell types derived from untreated tumors: MCF-7 breast cancer and A549 non small cell lung cancer (NSCLC). We have examined: the cytotoxic effect induced by doxorubicin (DOX); the time course of drug accumulation by flow cytometry and intracellular drug distribution by confocal microscopy; the expression and distribution of proteins related to anthracycline resistance, such as P-gp (P-glycoprotein), MRP1 (multidrug resistance-associated protein) and LRP (lung resistance-related protein). The cytotoxicity assays showed that A549 cells were less sensitive than MCF-7 cells to the DOX treatment in agreement with the different DOX uptake. Moreover, while in A549 cells DOX was mostly located in well defined intracytoplasmic vesicles, in MCF-7 cells it was mainly revealed inside the nuclei. The analysis of P-gp and MRP expression did not show significant differences between the two cell lines while a high expression of LRP was detected at the nuclear envelope and cytoplasmic levels in A549 cells. These findings suggest that the lower sensitivity to DOX treatment showed by lung carcinoma cells could be ascribed to drug sequestration by LRP inside the cytoplasmic compartments.

Identification of a Nonconserved Amino Acid Residue in Multidrug Resistance Protein 1 Important for Determining Substrate Specificity: EVIDENCE FOR FUNCTIONAL INTERACTION BETWEEN TRANSMEMBRANE HELICES 14 AND 17

Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), very poorly. By mutating variant residues in mrp1 to those present in MRP1, we identified Glu(1089) of MRP1 as being critical for anthracycline resistance. However, Glu(1089) mutations had no effect on E(2)17betaG transport. We have now identified a nonconserved amino acid within the highly conserved COOH-proximal transmembrane helix of MRP1/mrp1 that is important for transport of the conjugated estrogen. Converting Ala(1239) in mrp1 to Thr, as in the corresponding position (1242) in MRP1, increased E(2)17betaG transport 3-fold. Any mutation of mrp1 Ala(1239), including substitution with Thr, decreased resistance to vincristine and VP-16 without altering anthracycline resistance. However, introduction of a second murine to human mutation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16. To confirm the importance of MRP1 Thr(1242) for E(2)17betaG transport and drug resistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys. These mutations decreased E(2)17betaG transport 2-fold. Conversion to Asp eliminated transport of the estrogen conjugate and also decreased leukotriene C(4) transport approximately 2-fold. The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested. As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C(4) and E(2)17betaG. These results demonstrate the important role of Thr(1242) for E(2)17betaG transport. They also reveal a highly specific functional relationship between nonconserved amino acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both proteins to confer similar levels of resistance to vincristine and VP-16.

Glutathione-dependent Binding of a Photoaffinity Analog of Agosterol A to the C-terminal Half of Human Multidrug Resistance Protein

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD(0)), which is connected to a P-glycoprotein-like core region (DeltaMRP) by a cytoplasmic linker domain zero (L(0)). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD(0) and L(0) are not known. We synthesized [(125)I]11-azidophenyl agosterol A ([(125)I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C(932-1531)) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C(4). Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L(0) is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L(0) of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.