ABCG2 Protein Expression Research Articles

Scutellaria Extract Decreases the Proportion of Side Population Cells in a Myeloma Cell Line by Down-regulating the Expression of ABCG2 Protein

Scutellaria is one of the most popular traditional Chinese herbal remedies against various human diseases, including cancer. In this study, we examined the active effects of Scutellaria extract and its main flavonoid constituents on the proportion of side population cells within human multiple myeloma cell line RPMI8226 in vitro and explored the potential molecular mechanisms involved. The contents of flavonoids in ethanolic extract of Scutellaria baicalensis Georgi were determined using high performance liquid chromatography. The antiproliferative effect of the ethanolic extract on RPMI-8226 was determined by CCK assay. Apoptosis was measured by annexin combining with propidium iodide in a flow cytometer. Cell cycle analysis was performed by propidium iodide staining in combination with flow cytometry analysis. Hoechst 33342 exclusion assay was used for the identification of side population within RPMI8226 cells. The expression of ABCG2 protein was assessed by Western blotting assay. The content of major flavonoids constitutents of Scutellaria extract was baicalin (10.2%), wogonoside (2.50%), baicalein (2.29%), and wogonin (0.99%), respectively. The crude Scutellaria extract did not show significant anti-proliferative effect, apoptosis induction and cell cycle arrest in RPMI-8226 within the concentrations of 1-75μg/mL. However, the ethanolic extract, baicalein, wogonin and baicalin reduced the side population cells in RPMI-8226, and data showed that baicalein and wogonin had stronger inhibitory effects. Correspondingly, they also exhibited significant effects on decreasing the expression level of ABCG2 protein in RPMI-8226 in vitro. Our results for the first time demonstrated a novel mechanism of action for Scutellaria extract and its main active flavonoids, namely targeting SP cells by modulating the expression of ABCG2 protein. This study provides an insight for new therapeutic strategies targeting cancer stem cells of multiple myeloma.

Inflammation Induces Hematopoietic Stem Cell/Progenitor Migration and Recruitment To Lymph Nodes In Mice and Humans

Under steady-state conditions a small fraction of hematopoietic stem and progenitor cells (HSPC) circulate in the bloodstream. Circulation of HSPC follows a circadian rhythm of migration from the bone marrow (BM) to blood and possibly, lymph. The functional purpose of circulating HSPC is unclear but it has been associated with homeostatic regulation of tissue-specific reservoir of HSPC, vasculogenesis and immunosurveillance. During infectious stress, HSPC are activated by inflammation mediators and chemokines and mobilized to circulation and extramedullary tissues. Side Population (SP) cells are stem cells with pluripotent differentiation ability found in a number of adult tissues. Here we have studied whether the lymph nodes (LN) are an obligatory site of transit/homing of HSPC in inflammation. To analyze whether there is accumulation of HSPC in inflammatory lymph nodes, we first analyzed the presence of SP cells in human LN. The patient median age was 34 (range 3-89). Patient-derived LN biopsies were diagnosed by the pathologist as having reactive lymphadenitis (RL, n=35, 50.7%) or cancer (n=34, 49.3.%). Cancer diagnoses were Hodgkin's Lymphoma (HL, n=12,17.4%), non-Hodgkin's lymphoma (NHL, n=20, 28.9%) or adenocarcinoma (AC, n=2, 2.9%) which associate with decreasing levels of inflammation. The biopsy-derived LN cells were stained with Hoechst 33342/propidium iodide and monoclonal antibodies against CD45, CD34, CD20, CD19, CD133 and ABCG2 surface markers. Flow cytometry analysis of LN specimens identified SP cells in 82.3% of RL LN samples (1.9±1.2%; range 0-39%), 50% of HL LN biopsies (0.7±0.6%; range 0-7.7%) and 20% of NHL LN biopsies (0,03±0.01%; range 0-0.23). Interestingly, SP cells were absent from AC LN specimens. The presence of LN SP cells was significantly associated with RL versus any of the analyzed cancer groups (P<0.001). The phenotype of human SP cells in all SP-positive LN specimens was CD45+/CD34-/CD19-/CD20-/CD133Low/neg. ABCG2 protein expression had a strong correlation with the presence of SP cell positive samples (c2=11.8, p=0.001). To identify whether inflammation was associated to HSPC migration to LN, we analyzed the SP cells and functional myeloid progenitor cell (CFU) content of different organs in adult mice treated with lipopolysaccharide (LPS, 3 mg/Kg) or vehicle control at 6 and 12 hours post-administration. LPS treatment resulted in migration of HSPC to LN (∼13-fold), liver (∼7-fold) and kidney (∼4 fold). Circulation of HSPC in peripheral blood increased (∼8 fold) but no HSPC was found in the thoracic duct or other organs after up to 12 hours post-infusion of LPS. These data suggest the co-existence of direct migration of HSPC to peripheral blood and lymphatic vessels. Migration of HSPC to the lymph is followed by efficient LN homing/retention of HSPC. In summary, we have found that inflammation associates with HSPC mobilization and recruitment to LN in mice and humans, strongly suggesting that local inflammatory signals are crucial in the regulation of the traffic and homing of HSPC. Disclosures:No relevant conflicts of interest to declare.

Pharmacogenetic determinants associated with sunitinib-induced toxicity and ethnic difference in Korean metastatic renal cell carcinoma patients

The aim of this study was to investigate the pharmacogenetic determinants of sunitinib-related toxicity and ethnic difference in metastatic renal cell carcinoma (mRCC) among Korean patients. A pharmacogenetic study was performed in 65 patients with mRCC treated with the standard schedule of sunitinib (50mg orally once daily for 4weeks-on/2weeks-off). Detailed data regarding the toxicity of sunitinib, including thrombocytopenia, neutropenia, anemia, and hand-foot syndrome (HFS), were prospectively collected in a clinical trial program (n=38) or standard oncology practice (n=27). Total of 12 genetic polymorphisms in 8 candidate genes (CYP1A1, CYP3A5, ABCB1, ABCG2, PDGFRα, VEGFR2, RET, and FLT3) were analyzed for an association with treatment-related toxicity from sunitinib using Pearson χ (2) test. Common grade 3 or grade 4 treatment-related toxicities were thrombocytopenia (36.9%, 24/65), neutropenia (18.4%, 12/65), anemia (7.7%, 5/65), and HFS (12.3%, 8/65). Patients carrying an ABCG2 421 AA genotype developed significantly more grade 3 or grade 4 thrombocytopenia, neutropenia, and HFS adjusted for age, sex, and Eastern Cooperative Oncology Group performance status, and body surface area (odds ratio compared with AC/CC genotypes [OR] 9.90, P=0.04, thrombocytopenia; OR 18.20, P=0.02, neutropenia; and OR 28.46, P=0.01, HFS). In addition, total and surface protein ABCG2 protein expression was decreased in ABCG2 421 AA mutant cells compared to wild type. Among 12 genetic polymorphisms, polymorphism in the ABCG2 421C>A gene may be mostly associated with the risk of sunitinib-related toxicity in mRCC patients. Considering the high frequency of 421C>A SNP in Asian, this may be related to differential toxicities among ethnic groups.

Transcription factors Sp1 and Sp3 regulate expression of human ABCG2 gene and chemoresistance phenotype.

ABCG2 is a member of the ATP binding cassette (ABC) transmembrane proteins that plays an important role in stem cell biology and drug resistance of cancer cells. In this study, we investigated how expression of human ABCG2 gene is regulated in lung cancer A549 cells. Binding of Sp1 and Sp3 transcription factors to the ABCG2 promoter in vitro and in vivo was elucidated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ABCG2 promoter activity was impaired when Sp1 sites were mutated but was enhanced by overexpression of Sp1 or Sp3 proteins. Knockdown of Sp1 or Sp3 expression by short interfering RNA significantly decreased the expression of ABCG2 mRNA and protein, resulting in attenuated formation of the side population in A549 cells. In addition, Sp1 inhibition in vivo by mithramycin A suppressed the percentage of the side population fraction and sphere forming activities of A549 cells. Moreover, inhibiting Sp1- or Sp3-dependent ABCG2 expression caused chemosensitization to the anticancer drug cisplatin. Collectively, our results demonstrate that Sp1 and Sp3 transcription factors are the primary determinants for activating basal transcription of the ABCG2 gene and play an important role in maintaining the side population phenotype of lung cancer cells.

Alcohol Cirrhosis Alters Nuclear Receptor and Drug Transporter Expression in Human Liver

Unsafe use of alcohol results in approximately 2.5 million deaths worldwide, with cirrhosis contributing to 16.6% of reported deaths. Serum insulin levels are often elevated in alcoholism and may result in diabetes, which is why alcoholic liver disease and diabetes often are present together. Because there is a sizable population with these diseases alone or in combination, the purpose of this study was to determine whether transporter expression in human liver is affected by alcoholic cirrhosis, diabetes, and alcoholic cirrhosis coexisting with diabetes. Transporters aid in hepatobiliary excretion of many drugs and toxic chemicals and can be determinants of drug-induced liver injury. Drug transporter expression and transcription factor-relative mRNA and protein expression in normal, diabetic, cirrhotic, and cirrhosis with diabetes human livers were quantified. Cirrhosis significantly increased ABCC4, 5, ABCG2, and solute carrier organic anion (SLCO) 2B1 mRNA expression and decreased SLCO1B3 mRNA expression in the liver. ABCC1, 3-5, and ABCG2 protein expression was also upregulated by alcoholic cirrhosis. ABCC3-5 and ABCG2 protein expression was also upregulated in diabetic cirrhosis. Cirrhosis increased nuclear factor E2-related factor 2 mRNA expression, whereas it decreased pregnane-X-receptor and farnesoid-X-receptor mRNA expression in comparison with normal livers. Hierarchical cluster analysis indicated that expressions of ABCC2, 3, and 6; SLCO1B1 and 1B3; and ABCC4 and 5 were more closely related in the livers from this cohort. Overall, alcoholic cirrhosis altered transporter expression in human liver.

The effects of aflatoxin B1 on transporters and steroid metabolizing enzymes in JEG-3 cells

Effects of 96h aflatoxin B1 (AFB1) exposure at concentrations from 0.2μM to 6μM on the mRNA and protein expression levels of the following transporters ABCB1/B4, ABCC1, ABCC2, ABCG2, OAT4 and the mRNA expression of steroid-metabolizing enzymes CYP1A1, CYP19A1, HSD3B1 and HSD17B1, and conjugating enzyme family UGT1A were evaluated in trophoblastic JEG-3 cells. Statistically significant dose-dependent five-fold increases in the expression levels with ABCC2 and OAT4 were recorded at 2 and 6μM AFB1. Protein expression of ABCG2 was decreased dose-dependently with 0.2–6μM AFB1. With the other transporters, only a trend of increased expression was observed. Analogously, a three-fold increase in the expressions of CYP19A1, HSD3B1, HSD17B1 and UGT1A-family were observed at 0.3μM AFB1. When an inhibitor of CYP19A1, finrozole, was dosed simultaneously with AFB1, no increases in the transcripts of transporters or steroid hydroxylases or CYP19A1 were observed. This delayed increase in the expression levels – only after 96h incubations – may indicate that the response is due to a secondary metabolite of AFB1 or other secondary controlling cascades rather than the parent compound itself. In conclusion, AFB1 affected the placental steroid synthesizing, metabolizing and conjugating enzymes as well as the expression levels of several transporter proteins in JEG-3 cells. These alterations may lead to anomalies in the foetoplacental hormonal homeostasis.

Reversal of ABCG2-mediated multidrug resistance by human cathelicidin and its analogs in cancer cells

Multidrug resistance (MDR) of cancer cells to a wide spectrum of anticancer drugs is a major obstacle to successful chemotherapy. It is usually mediated by the overexpression of one of the three major ABC transporters actively pumping cytotoxic drugs out of the cells. There has been great interest in the search for inhibitors toward these transporters with an aim to circumvent resistance. This is usually achieved by screening from natural product library and the subsequent structural modifications. This study reported the reversal of ABCG2-mediated MDR in drug-selected resistant cancer cell lines by a class of host defense antimicrobial peptides, the human cathelicidin LL37 and its fragments. The effective human cathelicidin peptides (LL17-32 and LL13-37) were found to increase the accumulation of mitoxantrone in cancer cell lines with ABCG2 overexpression, thereby circumventing resistance to mitoxantrone. At the effective concentrations of the cathelicidin peptides, cell proliferation of the parental cells without elevated ABCG2 expression was not affected. Result from drug efflux and ATPase assays suggested that both LL17-32 and LL13-37 interact with ABCG2 and inhibit its transport activity in an uncompetitive manner. The peptides were also found to downregulate ABCG2 protein expression in the resistant cells, probably through a lysosomal degradation pathway. Our data suggest that the human cathelicidin may be further developed for sensitizing resistant cancer cells to chemotherapy.

Expression Levels of the ABCG2 Multidrug Transporter in Human Erythrocytes Correspond to Pharmacologically Relevant Genetic Variations

We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.

Cadmium inhibits ABCG2 transporter function in BeWo choriocarcinoma cells and MCF-7 cells overexpressing ABCG2

Heavy metals such as cadmium, lead and methylmercury are known to be neurotoxic to developing fetus. ABCG2 which is an efflux transporter located in the maternal facing membranes of human placenta protects fetus from xenobiotics by transferring compounds from syncytiotrophoblast to maternal circulation. The aim of this study was to clarify whether heavy metal compounds (CdCl2, PbCl2 and MeHgCl) affect the expression and function of ABCG2 transporter in human placental BeWo choriocarcinoma cells. The expression of ABCG2 was determined by immunoblotting and RT-PCR. The functional activity of ABCG2 was evaluated by measuring the efflux of two known ABCG2 substrates: fluorescent mitoxantrone and 14C-labeled food carcinogen PhIP. According to MTT assay all compounds were cytotoxic as expected (MeHgCl > CdCl2 > PbCl2). CdCl2 inhibited the efflux of mitoxantrone and 14C-PhIP suggesting inhibition of ABCG2 transporter function. PbCl2 had no effect on mitoxantrone efflux. Because of high toxicity, the inhibitory potency of MeHgCl was not tested. According to protein data these heavy metals did not affect ABCG2 transporter protein expression. Also, the expression of ABCC1, ABCC2 or ABCG2 mRNA were not affected by heavy metals. In conclusion, although the studied metal salts did not affect mRNA or protein expression of ABCG2, CdCl2 inhibited its function. Further studies to evaluate whether this leads to elevated placental transfer of ABCG2 substrates are needed.

Increased JNK1 Signaling Pathway Is Responsible for ABCG2-Mediated Multidrug Resistance in Human Colon Cancer

Multidrug resistance remains a major obstacle to effective chemotherapy of colon cancer. ABCG2, as a half-transporter of the G subfamily of ATP-binding cassette transporter genes (ABC transporters), is known to play a crucial role in multidrug resistance. However, the molecular mechanism of controlling ABCG2 expression in drug resistance of colon cancer is unclear and scarcely reported. In the present study, we systematically investigate the potential role of the c-Jun NH2-terminal kinase (JNK) signal pathway in ABCG2-induced multidrug resistance in colon cancer. In the hydroxycamptothecin (HCPT) resistant cell line SW1116/HCPT from human colon cancer cell line SW1116, ABCG2 is the major factor for multidrug resistance, other than well-studied ABCB1 or ABCC1. Our findings indicate that blocking the JNK pathway by pathway inhibitor SP600125 reduces the expression level and transport function of ABCG2 in drug-resistant cells SW116/HCPT. Notably, the experiments of small interfering RNA directed against JNK1 and JNK2 show that only silence of JNK1 gene has the equal effect as SP600125 on dephosphorylation of transcription factor c-Jun and the expression of ABCG2 protein, while the corresponding phenomena were not observed after silence of JNK2 gene. Meanwhile, SP600125 induces the apoptosis of SW116/HCPT cells by promoting the cleavage of PARP and suppressing the anti-apoptotic protein survivin and bcl-2, and increases the sensitivity of SW1116/HCPT to HCPT. Taken together, our work demonstrated that JNK1/c-jun signaling pathway was involved in ABCG2-mediated multidrug resistance in colon cancer cells. Definitely, inhibition of the JNK1/c-jun pathway is useful for reversing ABCG2-mediated drug resistance in HCPT-resistant colon cancer cells.

Gefitinib enhances the efficacy of photodynamic therapy using 5-aminolevulinic acid in malignant brain tumor cells

Since the ATP-binding cassette transporter ABCG2 plays a physiologically significant role of porphyrin efflux from living cells, the expression of ABCG2 may influences the efficacy of photodynamic therapy (PDT). In this study, we evaluated the effect of gefitinib, a potent ABCG2 inhibitor, on 5-aminolevulinic acid (5-ALA)-PDT in brain tumor cell lines in vitro. Four human glioma cell lines (U87MG, U118MG, A172, and T98G) and a malignant meningioma cell line (IOMM-Lee) were incubated with gefitinib (0.01-1.0μM) before incubation with 5-ALA (1mM). The effects gefitinib on intracellular protoporphyrin IX (PpIX), mRNA and protein expression of ABCG2, and PDT were evaluated, in vitro. At concentrations of 0.1μM or higher, gefitinib enhanced intracellular levels of PpIX in a dose-dependent manner in all those cell lines. Gefitinib decreased mRNA and plasma membrane protein expression of ABCG2, and efficiently enhanced the effect of 5-ALA-PDT in malignant brain tumor cells. Gefitinib can inhibit ABCG2-mediated PpIX efflux from malignant brain tumor cells to increase the intracellular PpIX and thereby enhance the PDT effect.

Breast cancer, side population cells and ABCG2 expression

Recurrent metastatic breast cancer may arise in part due to the presence of drug resistant adult stem cells such as Side Population (SP) cells, whose phenotype has been demonstrated to be due to the expression of ABCG2. We hypothesised that SP may be identified in Fine Needle Aspirates (FNAs) and their presence may be determined by expression of ABCG2 in breast tumours. SP and non-side population cells (NSP) were isolated using dual wavelength flow cytometry combined with Hoechst 33342 dye efflux and analysed for expression of ABCG2 and chemoresistance. FNA samples used in SP analysis were matched with paraffin-embedded tissue which was used in immunohistochemical analysis to assess ABCG2 expression. Results were correlated to the pathobiology of the tumour. MCF7 and MDA-MB-231 cell lines contain SP cells. MCF7 SP have increased expression of ABCG2 and increased resistance to mitoxantrone compared to NSP cells. The presence of SP in FNAs were significantly associated with ER-negative (p=0.008) and with triple negative breast cancers (p=0.011) which were also found to have a significant increase in ABCG2 protein expression. ABCG2 transcript was detected in some but not all SP cell populations isolated from FNA samples.

MicroRNA profiling in K-562 cells under imatinib treatment

Despite the enormous success of imatinib in chronic myeloid leukemia (CML), therapy resistance has emerged in a significant proportion of patients, partly because of the overexpression of ABC efflux transporters. Using an array comprising 667 miRNAs, we investigated whether the expression of microRNAs (miRNAs) is altered in CML K-562 cells becoming resistant to increasing concentrations of imatinib. ABCB1 and ABCG2 mRNA (quantitative real-time PCR) and protein expression (western blot) were quantified under short-term and 4 months' imatinib treatment. Interaction of miR-212 and miR-328 with ABCG2 was investigated by transfection experiments and reporter gene assays using respective miRNA precursors or miRNA inhibitors. Although ABCB1 protein was not expressed, ABCG2 protein was 7.2-fold elevated after long-term treatment with 0.3 µmol/l imatinib and decreased gradually at higher concentrations. miRNAs miR-212 and miR-328 were identified to correlate inversely with ABCG2 expression under these conditions. Short-term treatment also induced ABCG2 protein concentration dependently and caused a downregulation of miR-212, but not of miR-328 at all tested concentrations (P=0.050). Reporter gene assays confirmed miR-212 to target the 3'-UTR region of ABCG2. In contrast, transfection of anti-miR-212 revealed an upregulation of ABCG2 protein expression, whereas the effect of anti-miR-328 was weak. Our study suggests an association of imatinib treatment, miRNA downregulation and ABCG2 overexpression, possibly contributing to the mechanisms involved in imatinib distribution and response in CML therapy.

Impact of Genetic Variation in Breast Cancer Resistance Protein (BCRP/ABCG2) on Sunitinib Pharmacokinetics

To elucidate the impact of genetic variations in breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (MDR1/ABCB1) on the pharmacokinetics of sunitinib, we carried out a pharmacogenetic study in a clinical setting and pharmacokinetic analysis using Abcg2(-/-), Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice. Nineteen renal cell carcinoma patients were enrolled in this study. The plasma concentrations of sunitinib and its active metabolite were determined and the area under the concentration-time curve (AUC) was calculated. Genetic polymorphisms in ABCG2 (421C>A) and ABCB1 (1236C>T, 2677G>T/A and 3435C>T) were examined. The dose-adjusted AUC(0-24) of sunitinib was significantly higher in patients with a heterozygous variant for ABCG2 421C>A than in wild-type patients (p = 0.02), and one homozygous patient showed the highest dose-adjusted AUC(0-24). The ABCB1 polymorphisms were not associated with the dose-adjusted AUC(0-24). The maximum concentration and AUC(0-4) of sunitinib were significantly higher in Abcg2(-/-), Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice than wild-type mice when sunitinib was given orally but not intraperitoneally. Incidence of thrombocytopenia and hypertension and poor compliance were associated with the systemic exposure to sunitinib and its active metabolite. These results suggest that the loss of protein expression of ABCG2 by genetic polymorphism is associated with an increase in the systemic exposure to sunitinib and sunitinib-induced toxicity.

In vitro and in vivo modulation of ABCG2 by functionalized aurones and structurally related analogs

Over-expression of ABCG2 is linked to multidrug resistance in cancer chemotherapy. We have previously shown that functionalized aurones effectively reduced the efflux of pheophorbide A (an ABCG2 substrate) from ABCG2 over-expressing MDA-MB-231/R (“R”) cells. In the present report, we investigated the functional relevance of this observation and the mechanisms by which it occurs. Aurones and related analogs were investigated for re-sensitization of R cells to mitoxantrone (MX, a chemotherapeutic substrate of ABCG2) in cell-based assays, accumulation of intracellular MX by cell cytometry, interaction with ABCG2 by biochemical assays and in vivo efficacy in MX resistant nude mice xenografts. We found that methoxylated aurones interacted directly with ABCG2 to inhibit efflux activity, possibly by competing for occupancy of one of the substrate binding sites on ABCG2. The present evidence suggests that they are not transported by ABCG2 although they stimulate ABCG2-ATPase activity. Alteration of ABCG2 protein expression was also discounted. One member was found to re-sensitize R cells to MX in both in vitro and in vivo settings. Our study identified methoxylated aurones as promising compounds associated with low toxicities and potent modulatory effects on the ABCG2 efflux protein. Thus, they warrant further scrutiny as lead templates for development as reversal agents of multidrug resistance.

Regulation of drug transporter expression by oncostatin M in human hepatocytes

The cytokine oncostatin M (OSM) is a member of the interleukin (IL)-6 family, known to down-regulate expression of drug metabolizing cytochromes P-450 in human hepatocytes. The present study was designed to determine whether OSM may also impair expression of sinusoidal and canalicular drug transporters, which constitute important determinants of drug hepatic clearance. Exposure of primary human hepatocytes to OSM down-regulated mRNA levels of major sinusoidal solute carrier (SLC) influx transporters, including sodium-taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, organic cation transporter 1 and organic anion transporter 2. OSM also repressed mRNA expressions of ATP binding cassette (ABC) efflux transporters such as multidrug resistance protein (MRP) 2/ABCC2 and breast cancer resistance protein/ABCG2, without however impairing those of multidrug resistance gene 1/P-glycoprotein/ABCB1, MRP3/ABCC3, MRP4/ABCC4 and bile salt export pump/ABCB11. The cytokine concomitantly reduced NTCP, OATP1B1, OATP2B1 and ABCG2 protein expression and NTCP and OATP transport activities. OSM effects towards transporters were found to be dose-dependent and highly correlated with those of IL-6, but not with those of other inflammatory cytokines such as tumor necrosis factor-α or interferon-γ. In addition, OSM-mediated repression of some transporters such as NTCP, OATP1B1 and OATP2B1, was counteracted by knocking-down expression of the type II OSM receptor subunits through siRNA transfection. This OSM-mediated down-regulation of drug SLC transporters and ABCG2 in human hepatocytes may contribute to alterations of pharmacokinetics in patients suffering from diseases associated with increased production of OSM.

Abstract 596: ABCG2 is upregulated in SN-38 resistant colorectal cancer cells

Abstract Irinotecan, a member of the camptothecin chemotherapeutic drug family, is a first and second line therapy for patients with colorectal cancer. However, as with most chemotherapeutic therapies, patients’ tumors eventually develop drug resistance. Therefore, identifying mechanisms of chemotherapeutic resistance will allow for the development of additional molecular therapeutic targets. In this study SN-38, the highly insoluble active metabolite of irinotecan, was used to investigate corollary resistance to irinotecan. SN-38 resistant HT-29 cells were generated and compared to parental untreated HT-29 cells. Cells were characterized following treatment with either DMSO soulubilized SN-38 or IVECT encapsulated SN-38, a triblock copolymer micelle, in aqueous solution. The ATP-binding cassette transmembrane transporter protein, ABCG2, was found to have more than 40-fold expression in the SN-38 resistant HT-29 cells compared to the parental line. The increased expression of ABCG2 protein correlated with increased resistance to SN-38 in HT-29 cells and also with increased cell efflux. Cytotoxicity studies demonstrated that SN-38 resistant HT-29 cells were affected in a similar manner by IVECT polymer encapsulated SN-38 micelles and DMSO dissolved free SN-38. Cell cycle analysis showed that SN-38 resistant cells had an abrogated S phase arrest compared to parental cells. Cell treatment with free SN-38 was limited by its solubility in addition to DMSO toxicity. Further, IVECT polymer encapsulation improved SN-38 aqueous solubility allowing for increased concentration administration. Efflux proteins such as ABCG2 have been found to be upregulated in numerous types of cancers by several classes of chemotherapeutic drugs. Since transporter proteins have an important role in cell efflux, ABCG2 overexpression could serve as an additional target for pharmacological modulation. Finally, the increased solubility of SN-38 by IVECT polymer encapsulation allows for potential in vivo clinical applications of SN-38, a molecule that is 1000 times more active than its prodrug irinotecan. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 596. doi:10.1158/1538-7445.AM2011-596

Abstract 1740: Analogs of OSI-930 as potent ABCG2-mediated multidrug resistance reversal agents

Abstract OSI-930 {N-(4-trifluoromethoxyphenyl) 3-((quinolin-4-ylmethyl) amino) thiophene-2-carboxamide}, a dual c-Kit and KDR tyrosine kinase inhibitor, has been reported to be undergoing Phase I dose escalation study in cancer patients. A series of sixteen pyridyl and phenyl analogs (JP compounds) of OSI-930 was designed and synthesized. Extensive screening of these compounds led to the discovery that nitropyridyl and nitrophenyl analogs of OSI-930, JP1 and JP3, were effective in reversing ABC subfamily G member 2 (ABCG2/BCRP/MXR) transporter-mediated multidrug resistance (MDR). In the present study, JP1 and JP3 significantly sensitized ABCG2-expressing cells to established substrates of ABCG2 including mitoxantrone, SN-38 and doxorubicin in a concentration-dependent manner, but not to the non-ABCG2 substrate cisplatin. Furthermore, JP1 and JP3 were unable to reverse ABCB1- or ABCC1-mediated MDR indicating their selectivity for ABCG2. Western blot analysis was used to evaluate ABCG2 expression and it was found that neither JP1 nor JP3 significantly altered ABCG2 protein expression during the course of our experiment. Accumulation study with an ABCG2 substrate [3H]-mitoxantrone demonstrated that JP1 and JP3 increased the intracellular accumulation of [3H]-mitoxantrone. JP1 and JP3 also significantly inhibited the transport of [3H]-methotrexate by ABCG2 in the in vitro transport assays using membrane vesicles. Importantly, both JP1 and JP3 efficaciously stimulated the activity of ATPase of ABCG2 and inhibited the photoaffinity labeling of this transporter with its substrate [125I]-iodoarylazidoprazosin. Results from the present study suggest that analogs of OSI-930, JP1 and JP3, specifically inhibit the function of ABCG2 through direct interaction with its substrate binding site(s). Thus the OSI-930 analogs represent a new class of potential reversal compounds for the treatment of MDR in ABCG2 over-expressing tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1740. doi:10.1158/1538-7445.AM2011-1740

Isolation and identification of side population cells in human lung adenocarcinoma cell line A549

To isolate and characterize the side population cells (SP cells) in the lung adenocarcinomas cell line A549. The protein expression of ABCG2 in human lung adenocarcinoma cell line A549 was detected by immunohistochemistry. SP and NSP cells in the cell line A549 were isolated by FACS, and their differentiation was analysed. ABCG2 expression in the two cell subsets was detected by RT-PCR. The cell growth curves, cell division indexes, cell cycles, plate clone formation tests, migration and invasion assays, chemotherapeutic susceptibility tests, tests of the intracellular drug levels, and the tumor cell implantation experiments on nude mice were applied to study the biological properties of the two cell subsets. The expression of ABCG2 in the transplanted tumor in nude mice was detected by immunohistochemistry and RT-PCR. The positive rate of ABCG2 expression in the A549 cells by immunohistochemistry was 2.13%. SP and NSP cells were isolated by FACS. The SP cells could produce both SP and NSP cells, while NSP cells only produced NSP cells. SP cells expressed ABCG2, but NSP cells did not. The proliferation and migration abilities of the two cell subsets were similar, but the invasion and tumorigenic ability of SP cells was significantly higher than that of NSP cells. The susceptibilities to DDP and its intracellular levels of the two cell subsets were similar, but the susceptibilities to 5-FU, VP16, NVB and GEM and their intracellular levels of NSP cells were significantly higher than those of the SP cells. SP cells in the human lung adenocarcinomas cell line A549 is enriched with tumor stem cells. An effective way to get lung adenocarcinomas stem cells is to isolate SP cells by FACS.

Breast cancer resistance protein BCRP/ABCG2 regulatory microRNAs (hsa-miR-328, -519c and -520h) and their differential expression in stem-like ABCG2+ cancer cells

Recent studies have shown that a number of microRNAs (miRNA or miR) may regulate human breast cancer resistance protein (BCRP/ABCG2), an important efflux transporter responsible for cellular drug disposition, whereas their effects on ABCG2 protein expression are not compared. In this study, we first identified a new proximal miRNA response element (MRE) for hsa-miR-519c within ABCG2 3′-untranslated region (3′UTR) through computational analyses. This miR-519c MRE site was confirmed using dual luciferase reporter assay and site-directed mutagenesis. Immunoblot analyses indicated that ABCG2 protein expression was significantly down-regulated in MCF-7/MX100 cells after transfection with hsa-miR-328- or -519c expression plasmids, and was markedly up-regulated in MCF-7 cells after transfection with miR-328 or -519c antagomir. However, ABCG2 protein expression was unchanged in MCF-7/MX100 cells after transfection with hsa-miR-520h expression plasmids, which was associated with undetectable miR-520h expression. Furthermore, ABCG2 mRNA degradation was accelerated dramatically in cells transfected with miR-519c expression plasmid, suggesting the involvement of mRNA degradation mechanism. Intervention of miR-328 or -519c signaling led to significant change in intracellular mitoxantrone accumulation, as determined by flow cytometry analyses. In addition, we separated RB143 human retinoblastoma cells into stem-like (ABCG2+) and non-stem-like (ABCG2−) populations through immunomagnetic selection, and found that miR-328, -519c and -520h levels were 9-, 15- and 3-fold lower in the ABCG2+ cells, respectively. Our data suggest that miR-519c and -328 have greater impact on ABCG2 expression than miR-520h in MCF-7 human breast cancer cells, and the presence of proximal miR-519c MRE explains the action of miR-519c on shortened ABCG2 3′UTR.