Inhibition of IGF-IR increases chemosensitivity in human colorectal cancer cells through MRP-2 promoter suppression

The emergence of multidrug resistance (MDR) in cancer cells has made many of the currently available chemotherapeutic agents ineffective. However, the mechanism involved in mediating this effect is not yet fully understood. Here, we found the overexpression of type I insulin-like growth factor receptor (IGF-IR) in established colorectal MDR cells. Specific siRNA of IGF-IR decreases cell proliferation, exert synergistic effect with anticancer drugs. The downstream signaling of IGF-IR, PI3K/AKT pathway, was altered upon IGF-IR silencing. The expression of multidrug-resistance-associated protein 2 (MRP-2) was suppressed due to the nuclear translocation of nuclear factor-like 2 (Nrf2). Then the intracellular drug concentration was increased and the drug-resistant phenotype was reversed. Our findings improve current understanding of the biology of IGF-IR and MDR and have significant therapeutic implications on colorectal MDR cancer.     

Genes of multidrug resistance in haematological malignancies

Since the early 1970s, multiple drug resistance has been known to exist in cancer cells and is thought to be attributable to a membrane-bound, energy-dependent pump protein (P-glycoprotein [P-gp]) capable of extruding various related and unrelated chemotherapeutic drugs. The development of refractory disease in haematological malignancies is frequently associated with the expression of one or several multidrug resistance (MDR) genes. MDR1, multidrug resistance-associated protein (MRP) and lung-resistance protein (LRP) have been identified as important adverse prognostic factors. Recently it has become possible to reverse clinical MDR by blocking P-gp-mediated drug efflux. The potential relevance of these reversal agents of MDR as well as the potential new approaches to treat the refractory disease are discussed in this article. In addition, an array of different molecules and mechanisms by which resistant cells can escape the cytotoxic effect of anticancer drugs has now been identified. These molecules and mechanisms include apoptosis-related proteins and drug inactivation enzymes. Resistance to chemotherapy is believed to cause treatment failure in more than 50% patients. Clearly, if drug resistance could be overcome, the impact on survival would be highly significant. This review focuses on molecular mechanism of drug resistance in haematological malignancies with emphasis on molecules involved in MDR. In addition, it brings the survey of methods involved in determination of MDR, in particular P-gp/MDR1, MRP and LRP.     

The drug resistance proteins, multidrug resistance-associated protein and P-glycoprotein, do not confer resistance to Fas-induced cell death

BACKGROUND: Multidrug resistance (MDR) is mediated by the drug resistance proteins, the multidrug resistance-associated protein (MRP) and P-glycoprotein, both of which confer resistance by the active efflux of chemotherapeutic drugs from the cell. Reduced Fas (CD95/APO-1) expression and resistance to Fas-mediated apoptosis have also been correlated with P-glycoprotein-mediated MDR. METHODS: We investigated cell surface Fas expression (using anti-Fas monoclonal antibody DX2.1) in a series of MRP-expressing drug-resistant leukemia sublines, and P-glycoprotein-expressing leukemia sublines, and their susceptibility to apoptosis induced by anti-Fas treatment (CH-11 monoclonal antibody). Caspase-3 activation was detected by Western blot and apoptosis was determined by flow cytometry with 7-aminoactinomycin D (7-AAD) staining of cells. RESULTS: Fas expression was not reduced in either the MRP- or P-glycoprotein-expressing drug-resistant cell lines, although expression was reduced by 15% in one low-level drug-resistant subline. Expression of MRP or P-glycoprotein did not confer resistance to caspase-3 activation or to anti-Fas-induced cell death. CONCLUSIONS: MDR mediated by the drug transport proteins MRP and P-glycoprotein does not correlate with resistance to Fas-mediated cell death or resistance to caspase-3 activation.     

Lung resistance protein analysis in testicular cancer

Germ cell testicular cancers are well-curable neoplasms, because total remission can be achieved in about 80% of the cases. However, 15-20% of the patients die due to drug resistance (DR). A number of mechanisms of the multidrug resistance phenotype are known, including MDR/P-glycoprotein (P-gp) and the so-called multidrug resistance associated protein (MRP). Lung Resistance Protein (LRP) is an ATP dependent membrane transporter protein associated with MDR. In our present work we studied the expression of LRP in testicular cancers. LRP expression was determined by immunohistochemistry (IH), Western blot (WB) and RT-PCR techniques. Clinical resistance was defined in accordance with the clinical oncologic rules. In 29 (41%) of 70 primary testicular tumours and in 22 (63%) of 35 cases, elevated LRP levels were established by IH and WB, respectively. In the latter 63%, the LRP mRNA levels were elevated as well. Six cases of the 15 seminomas and 23 cases of the nonseminomatous germ cell tumours (NSGCT) proved to be positive. No relationship was demonstrated between LRP expression and the stage of the disease. Despite the LRP positivity of 6 tumour samples, all of the seminomas proved sensitive. Of the 39 sensitive NSGCT, 27 cases were LRP-negative, whereas 11 tumour samples of 16 patients belonging to the resistant group proved LRP-positive (p=0.04). The authors concluded that the expression of LRP is responsible for clinical drug resistance in non-seminomatous testicular cancer patients.     

Fractionated irradiation can induce functionally relevant multidrug resistance gene and protein expression in human tumor cell lines

The molecular basis of radiotherapy-related multidrug resistance (MDR) is still unclear. Here we report on a study investigating the effect of fractionated irradiation on expression of the MDR-associated proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and lung resistance-related protein (LRP), the respective mRNAs, and the functional consequences. Cells of six colon and five breast cancer cell lines were irradiated with a total dose of 27 Gy, five fractions of 1.8 Gy per week. The mRNA expression was measured by quantitative RT-PCR, protein levels and drug sensitivity to cisplatin, doxorubicin and bendamustine were assessed by flow cytometry. Breast cancer cell lines showed enhancement of the mRNAs encoding for P-gp, MRP1 and LRP in comparison to nonirradiated cells. No up-regulation of the three mRNA species was observed in the colon cancer cell lines. After irradiation, three breast cancer cell lines showed an up-regulation of LRP, one line an up-regulation of MRP1, and four lines a small up-regulation of P-gp. In the colon cancer cell lines, radiation induced significant enhancement of all three proteins. In comparison to controls, the irradiated cells lines showed a significant resistance to cisplatin, doxorubicin and bendamustine. This study confirms the prior reports of enhancement of P-gp and MRP1 after irradiation, which is accompanied by a multidrug resistance phenomenon, but in addition proposes a novel mechanism in the appearance of MDR after radiation-induced enhancement of LRP.     

Mechanisms for resistance to anticancer agents and the reversal of the resistance

MDR results from overexpression of P-glycoprotein (Pgp) and multidrug resistance protein (MRP or MRP1) that function as ATP-dependent efflux pumps. Lung resistance related protein (LRP) is also supposed to be involved in MDR. The human canalicular multispecific organic anion transporter (cMOAT) gene that is responsible for the defects in Dubin-Johnson syndrome was isolated. cMOAT is homologous to MRP1 and supposed to be involved in drug resistance. Human cMOAT cDNA transfected LLC-PK1 cells, LLC/cMOAT-1, have increased resistance to vincristine (VCR), 7-ethyl-10-hydroxycamptothecin (SN-38), and cisplatin. The multidrug resistance (MDR)-reversing agents, cyclosporin A (CsA) and PAK-104P, almost completely reversed the resistance to VCR, SN-38 and cisplatin of LLC/cMOAT-1 cells by interacting with the substrate binding site of cMOAT. Treatment of human colorectal carcinoma SW-620 cells with sodium butyrate(NaB) induced LRP in the cells and conferred resistance to Adrianycin(ADM), VCR, VP-16, gramicidin D and taxol. Two LRP-specific ribozymes inhibited the NaB-induced expression of LRP in SW-620 cells and almost completely abolished their acquisition of the MDR phenotype. The accumulation of ADM, VCR and taxol was not decreased in NaB-treated cells, suggesting that ATP-binding cassette transporters are not involved in the MDR of NaB-treated cells. ADM was mainly located in the nuclei of untreated and the cytoplasm of NaB-treated cells. The accumulation level of ADM in the nuclei isolated from untreated cells or those from treated cells in the presence of anti-LRP polyclonal antibody was higher than that from treated cells in the absence of the antibody. Efflux of ADM from nuclei isolated from NaB-treated cells was enhanced compared with those from untreated cells and NaB-treated cells transfected with a LRP-specific ribozyme. The polyclonal antibody against LRP inhibited the enhanced efflux of ADM from nuclei isolated from NaB-treated cells. These findings indicate that LRP is involved in resistance to ADM, VCR, VP-16, taxol and gramicidin D, and has an important role in the transport of ADM from the nucleus to the cytoplasm.     

Expression of multidrug resistance-related protein (MRP-1), lung resistance-related protein (LRP) and topoisomerase-II (TOPO-II) in Wilms' tumor: immunohistochemical study using TMA methodology

Aims: MRP-1, LRP and TOPO-II are all associated with protection of the cells from the adverse effects of various chemotherapeutics. The aim of this study was to measure the expression of these proteins in Wilms' tumor (WT). Materials and Methods: TMA block was constructed from 14 samples of WT's and from xenografts derived from them. Sections of the TMA were used for immunostaining against MRP-1, LRP and TOPO-IIa. Results: All normal kidneys expressed MRP-1 but were either weakly or negatively stained for LRP and TOPO-IIa. In WT samples, MRP-1 was universally expressed, exclusively in the tubular component, while there was no expression of LRP and TOPO-IIa showed heterogeneous distribution. The xenografts varied in their MRP-1 and TOPO-IIa expression and exhibited weak/negative staining of LRP. Conclusions: This study shows that although all the proteins evaluated, had different expression patterns in the tumor samples, the most prominent changes in expression were found for MRP-1. The exact clinical implications of these changes in expression and their relevance to the resistance of these tumors to chemotherapy requires further investigation. The finding of different expression profiles for the multidrug resistance proteins in the original WT's and their xenografts suggests that the results of animal cancer models may be difficult to interpret.     

Synthesis of curcumin mimics with multidrug resistance reversal activities

In order to discover novel multidrug resistance (MDR) reversal agents for efficient cancer chemotherapy, the unsymmetrical curcumin mimics with various amide moieties (6-19) were synthesized and evaluated their MDR reversal activities in MDR cell line KBV20C. Among the tested compounds, 13, 16, and 17 showed potent MDR reversal activities by inhibiting drug efflux function of P-glycoprotein in KB20C cells, and almost recovered the cytotoxicity of vincristine and paclitaxel against KBV20C cell to the degree of potency against drug sensitive KB cells.     

Relationship of LRP-human major vault protein toin vitro and clinical resistance to anticancer drugs

Multidrug resistance (MDR) has been related to two members of the ABC-superfamily of transporters, P-glycoprotein (Pgp) and Multidrug Resistance-associated Protein (MRP). We have described a 110 kD protein termed the Lung Resistance-related Protein (LRP) that is overexpressed in several non-Pgp MDR cell lines of different histogenetic origin. Reversal of MDR parallels a decrease in LRP expression. In a panel of 61 cancer cell lines which have not been subjected to laboratory drug selection, LRP was a superior predictor forin vitro resistance to MDR-related drugs when compared to Pgp and MRP, and LRP's predictive value extended to MDR unrelated drugs, such as platinum compounds. LRP is widely distributed in clinical cancer specimens, but the frequency of LRP expression inversely correlates with the known chemosensitivity of different tumour types. Furthermore, LRP expression at diagnosis has been shown to be a strong and independent prognostic factor for response to chemotherapy and outcome in acute myeloid leukemia and ovarian carcinoma (platinum-based treatment) patients. Recently, LRP has been identified as the human major protein. Vaults are novel cellular organelles broadly distributed and highly conserved among diverse eukaryotic cells, suggesting that they play a role in fundamental cell processes. Vaults localise to nuclear pore complexes and may be the central plug of the nuclear pore complexes. Vaults structure and localisation support a transport function for this particle which could involve a variety of substrates. Vaults may therefore play a role in drug resistance by regulating the nucleocytoplasmic transport of drugs.Abbreviations LRP Lung Resistance-related Protein - MVP Major Vault Protein - MDR Multidrug resistance - MRP Multidrug resistance-associated Protein - NPC Nuclear Pore Complex - Pgp P-glycoprotein     

Induction of cytochrome P450 (CYP)1A1, CYP1A2, and CYP3A4 but not of CYP2C9, CYP2C19, multidrug resistance (MDR-1) and multidrug resistance associated protein (MRP-1) by prototypical inducers in human hepatocytes

Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism. Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6beta-hydroxylase(T6H)-activity. Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistance-associated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses. Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and beta-naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days. CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days. Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly.     

Detection of the Mr 110,000 lung resistance-related protein LRP/MVP with monoclonal antibodies.

The Mr 110,000 lung resistance-related protein (LRP), also termed the major vault protein (MVP), constitutes >70% of subcellular ribonucleoprotein particles called vaults. Overexpression of LRP/MVP and vaults has been linked directly to MDR in cancer cells. Clinically, LRP/MVP expression can be of value to predict response to chemotherapy and prognosis. Monoclonal antibodies (MAbs) against LRP/MVP have played a critical role in determining the relevance of this protein in clinical drug resistance. We compared the applicability of the previously described MAbs LRP-56, LMR-5, LRP, 1027, 1032, and newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37 for the immunodetection of LRP/MVP by immunoblotting analysis and by immunocyto- and histochemistry. The availability of a broader panel of reagents for the specific and sensitive immunodetection of LRP/MVP should greatly facilitate biological and clinical studies of vault-related MDR.     

Identification of residues in the first cytoplasmic loop of P-glycoprotein involved in the function of chimeric human MDR1-MDR2 transporters.

The human MDR1 gene encodes the multidrug transporter (P-glycoprotein), a multidrug efflux pump. The highly homologous MDR2 gene product does not appear to be a functional multidrug pump. We have constructed a chimeric protein in which the first intracytoplasmic loop and the third and fourth transmembrane domains of the MDR1 protein were replaced by the analogous region of MDR2. Substitution of the MDR2 sequences encompassing amino acid residues 140 to 229 resulted in 17 amino acid changes, 10 in the intracytoplasmic loop (amino acids 141-188) and 7 in the transmembrane regions. This chimeric protein was expressed on the surface of NIH 3T3 cells where it bound [3H]azidopine but did not confer drug resistance. When only 4 residues, 165, 166, 168, and 169, were changed back to MDR1 amino acids, a functional drug transporter was recovered. When residues 165, 166, 168, and 169 from MDR2 were substituted into a functional MDR1 cDNA, the resulting construction was not able to confer drug resistance. These results indicate that the major functional differences between MDR1 and MDR2 in this region of P-glycoprotein reside in a small segment of the first intracytoplasmic loop. We also independently analyzed the effect of replacing Asn183 of MDR1 with Ser which occurs in MDR2. Substitution of Ser at position 183 in combination with Val at position 185 in P-glycoprotein resulted in a relative increase in resistance to actinomycin D, vinblastine, and doxorubicin in transfected NIH 3T3 cells. These results emphasize the importance of the first intracytoplasmic loop in P-glycoprotein in determining function and relative drug specificity of the transporter.     

Immunolocalization and cell expression of lung resistance-related protein (LRP) in normal and tumoral human respiratory cells.

Lung resistance-related protein (LRP) is an integral part of the multidrug resistance (MDR) phenotype involved in cell resistance toward xenobiotics or chemotherapy. The aim of this study was to compare the intracellular localization and cell expression of LRP in normal bronchial cells and their tumoral counterparts from non-small cell lung cancer (NSCLC). LRP expression was also investigated concurrently with DNA ploidy and chromosome 16 (lrp gene locus) aberrations. Confocal microscopy showed that LRP localization was exclusively intracytoplasmic regardless of the cell type and was never observed in the nuclear pore complex. Flow cytometry demonstrated a similar level of LRP expression in normal bronchial cells and in cancer cells from NSCLC samples. FISH analysis, performed to evaluate the number of chromosome 16 and lrp loci, demonstrated a significant gain of chromosome 16 in DNA aneuploid tumors. Furthermore, we did not find any link between LRP expression and DNA ploidy status or chromosome 16 number. These results suggest that LRP expression observed in NSCLC, maintained through the carcinogenesis process of respiratory cells, is not altered by the increased number of copies of chromosome 16 and probably controlled by mechanisms different from those of MRP1 expression, whereas both proteins are associated with the MDR phenotype.     

A key structural domain of the Candida albicans Mdr1 protein

A major multidrug transporter, MDR1 (multidrug resistance 1), a member of the MFS (major facilitator superfamily), invariably contributes to an increased efflux of commonly used azoles and thus corroborates their direct involvement in MDR in Candida albicans. The Mdr1 protein has two transmembrane domains, each comprising six transmembrane helices, interconnected with extracellular loops and ICLs (intracellular loops). The introduction of deletions and insertions through mutagenesis was used to address the role of the largest interdomain ICL3 of the MDR1 protein. Most of the progressive deletants, when overexpressed, eliminated the drug resistance. Notably, restoration of the length of the ICL3 by insertional mutagenesis did not restore the functionality of the protein. Interestingly, most of the insertion and deletion variants of ICL3 became amenable to trypsinization, yielding peptide fragments. The homology model of the Mdr1 protein showed that the molecular surface-charge distribution was perturbed in most of the ICL3 mutant variants. Taken together, these results provide the first evidence that the CCL (central cytoplasmic loop) of the fungal MFS transporter of the DHA1 (drug/proton antiporter) family is critical for the function of MDR. Unlike other homologous proteins, ICL3 has no apparent role in imparting substrate specificity or in the recruitment of the transporter protein.     

Functional analysis of ABCA8, a new drug transporter

We examined the transport capacity in Xenopus laevis oocytes of human EST KIAA0822/ABCA8, a member of the ABC superfamily. Substrates of ABCC2/MRP-2 such as [14C]estradiol-beta-glucuronide, taurocholate, and LTC4, and of organic anion transporter (OAT), such as para-aminohippuric acid, ochratoxin-A, were significantly accumulated while tetraethylammonium and doxorubicin were not. The transport of [14C]estradiol-beta-glucuronide was ATP-dependent and K(m) and V(max) values of 30.4microM and 66.9pmol/h/egg, respectively, were estimated. The transport of [14C]estradiol-beta-glucuronide was inhibited by substrates/inhibitors of ABCC2/MRP-2, but not by those of the organic cation transporter and multidrug resistance protein (MDR)-1. KIAA0822/ABCA8 possesses two ATP-binding sites and fourteen transmembrane domains. Northern blot analysis revealed expression in most organs, especially in heart, skeletal muscle, and liver. Thus, ABCA8 is a new member of the xenobiotic transporter ABC-subfamily.     

Axitinib targeted cancer stemlike cells to enhance efficacy of chemotherapeutic drugs via inhibiting the drug transport function of ABCG2

Stemlike cells have been isolated by their ability to efflux Hoechst 33342 dye and are called the side population (SP). We evaluated the effect of axitinib on targeting cancer stemlike cells and enhancing the efficacy of chemotherapeutical agents. We found that axitinib enhanced the cytotoxicity of topotecan and mitoxantrone in SP cells sorted from human lung cancer A549 cells and increased cell apoptosis induced by chemotherapeutical agents. Moreover, axitinib particularly inhibited the function of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) and reversed ABCG2-mediated multidrug resistance (MDR) in vitro. However, no significant reversal effect was observed in ABCB1-, ABCC1- or lung resistance-related protein (LRP)-mediated MDR. Furthermore, in both sensitive and MDR cancer cells axitinib neither altered the expression of ABCG2 at the mRNA or protein levels nor blocked the phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2. In nude mice bearing ABCG2-overexpressing S1-M1-80 xenografts, axitinib significantly enhanced the antitumor activity of topotecan without causing additional toxicity. Taken together, these data suggest that axitinib particularly targets cancer stemlike cells and reverses ABCG2-mediated drug resistance by inhibiting the transporter activity of ABCG2.