ABCG2 Gene Expression Research Articles

Linifanib alone and in combination with metronomic chemotherapy is active on cutaneous T-cell lymphoma cells by targeting the AKT/mTOR signaling pathway.

Cutaneous T-cell lymphomas (CTCLs) are a rare and heterogeneous subset of skin-localized, non-Hodgkin lymphomas. Our aim was to evaluate the in vitro antitumor activity of the multi-kinase inhibitor linifanib, either alone or in combination with metronomic vinorelbine (mVNR) or etoposide (mETO), on CTCL cells. In vitro proliferation assay and Luminex analysis showed that long-term, daily exposure of linifanib significantly inhibited the proliferation of the human CTCL cell line HH, in a concentration-dependent manner (IC50 = 48.4 ± 20.4nM) and the phosphorylation of AKT/mTOR signaling pathway. The concomitant exposure of linifanib plus mVNR or mETO resulted in a strong synergism, with combination index values < 1. Linifanib significantly increased the VNR and ETO intracellular concentrations in HH cells, evaluated by UPLC-HRMS technology, and strongly reduced the ABCB1 and ABCG2 gene expression in HH. In conclusion, we reported a striking antitumor activity of daily, long-term linifanib and a clear synergistic effect when administered in combination with mCHEMO on CTCL cells, as a promising base for future clinical approaches in T-cell lymphomas.

ABCG2 Gene Expression in Non-Small Cell Lung Cancer.

Background/Objectives: ATP-binding cassette subfamily G member 2 [ABCG2/breast cancer resistance protein (BCRP)] contributes to mechanisms of multidrug resistance (MDR) and is a marker of side population (SP) cells in human cancers. The primary objective of this study was to investigate the impact of ABCG2 gene expression on the non-small cell lung cancer (NSCLC) development, course of cancer disease, and patient prognosis using publicly available data. Obtained results were supplemented with assessment of ABCG2 expression in blood of NSCLC patients. Methods: The dataset of lung cancer was analyzed utilizing the TIMER 2.0, UALCAN, TNMplot, MEXPRESS, cBioPortal, MethSurv, KM Plotter, STRING, and ShinyGO 0.80 databases. Blood samples from 50 patients were assessed using the real-time PCR method. Results: The ABCG2 gene was expressed at a low level in NSCLC, and did not correlate with clinical aggressiveness of lung cancer. Higher ABCG2 expression improved overall survival, but only in LUAD. In addition, CpG sites located on the CpG island affecting the NSCLC patient's prognosis were indicated. In the case of our own laboratory results, the study did not reveal any changes in the ABCG2 expression levels in blood collected from patients at different time points during the diagnostic-therapeutic procedure. In the in silico analysis, most ABCG2 protein interactors were associated with the development of drug resistance. Conclusions: ABCG2 appears to have a particularly significant impact on the survival of patients with lung cancer and on the effect of immunotherapy related to immune cell infiltration. Presented findings may support personalized medicine strategies based on bioinformatics findings.

Insights into multidrug resistance mechanisms: Exploring distinct miRNAs as prospective therapeutic agents in triple negative breast cancer

Triple Negative Breast Cancer (TNBC) constitutes 12–17 % of breast cancers and is distinguished by the absence of hormone receptor expression, deviating from other breast cancer types. Coupled with its elevated proliferation index, TNBC develops multidrug resistance, diminishing treatment efficacy, weakening disease prognosis, and leading to an aggressive clinical course. This study aimed to assess ABCB1, ABCC1, ABCG2, and ABCC9 gene expression, which play a primary role in the development of multidrug resistance, alongside miRNAs (miR-466, miR-4539, miR-659-3p, miR-3123, miR-3133, and miR-655-3p) targeting these genes. While ABCB1 (p = 0.433), ABCC1 (p < 0.05), and ABCG2 (p < 0.05) exhibited increased expression in tumor tissues, ABCC9 (p = 0.587) did not. miR-466 (p = 0.802), miR-4539 (p = 0.732), miR-659-3p (p = 0.807), and miR-3123 (p = 0.980) were upregulated, whereas miR-3133 (p < 0.05) and miR-655-3p (p = 0.190) were downregulated. Within the scope of our study, we also evaluated the clinical parameters like tumor size, stage, and neoadjuvant treatment that significantly impacted Progression Free Survival (PFS) and Overall Survival (OS). Considering these results, we found that the metastatic status significantly influenced PFS and OS. Chemotherapeutics were found to not affect survival times. Assessing the impact of miRNAs, which we view as potential therapeutic targets, on average survival revealed that elevated miR-3133 expression was correlated with shorter PFS and OS, whereas decreased miR-655-3p expression was associated with longer PFS and OS. In summary, the relevant miRNAs could serve as predictive biomarkers for drug response and aid in developing miRNA-targeted gene therapy strategies.

Effect of Melatonin on Chemoresistance Exhibited by Spheres Derived from Canine Mammary Carcinoma Cells.

Mammary cancer is a frequent disease in female dogs, where a high proportion of cases correspond to malignant tumors that may exhibit drug resistance. Within the mammary tumor microenvironment, there is a cell subpopulation called cancer stem cells (CSCs), which are capable of forming spheres in vitro and resisting anti-tumor treatments, partly explaining the recurrence of some tumors. Previously, it has been described that spheres derived from canine mammary carcinoma cells CF41.Mg and REM 134 exhibit stemness characteristics. Melatonin has shown anti-tumor effects on mammary tumor cells; however, its effects have been poorly evaluated in canine mammary CSCs. This study aimed to analyze the effect of melatonin on the chemoresistance exhibited by stem-like neoplastic cells derived from canine mammary carcinoma to cytotoxic drugs such as doxorubicin and mitoxantrone. CF41.Mg and REM 134 cells were cultured in high-glucose DMEM supplemented with fetal bovine serum and L-glutamine. The spheres were cultured in ultra-low attachment plates in DMEM/F12 medium without fetal bovine serum and with different growth factors. The CD44+/CD24-/low phenotype was analyzed by flow cytometry. The viability of sphere-derived cells (MTS reduction) was studied in the presence of melatonin (0.1 or 1 mM), doxorubicin, mitoxantrone, and luzindole. In addition, the gene (RT-qPCR) of the multidrug resistance bombs MDR1 and ABCG2 were analyzed in the presence of melatonin. Both cell types expressed the MT1 gene, which encodes the melatonin receptor MT1. Melatonin 1 mM does not modify the CD44+/CD24-/low phenotype; however, the hormone reduced viability (p < 0.0001) only in CF41.Mg spheres, without inducing an additive effect when co-incubated with cytotoxic drugs. These effects were independent of the binding of the hormone to its receptor MT1, since, by pharmacologically inhibiting them, the effect of melatonin was not blocked. In CF41.Mg spheres, the relative gene expression of ABCG2 and MDR1 was decreased in response to the hormone (p < 0.001). These results indicate that melatonin negatively modulates the cell survival of spheres derived from CF41.Mg cells, in a way that is independent of its MT1 receptor. These effects did not counteract the resistance to doxorubicin and mitoxantrone, even though the hormone negatively regulates the gene expression of MDR1 and ABCG2.

The contradictory role of febuxostat in ABCG2 expression and potentiating hypericin-mediated photodynamic therapy in colorectal cancers.

Photodynamic Therapy (PDT) is an emerging method to treat colorectal cancers (CRC). Hypericin (HYP) is an effective mediator of PDT and the ABCG2 inhibitor, Febuxostat (FBX) could augment PDT. HT29 and HEK293 cells showed light dependant cytotoxic response to PDT in both 2D and 3D cell models. FBX co-treatment was not found to improve PDT cytotoxicity. Next, ABCG2 protein expression was observed in HT29 but not in HEK293 cells. However, ABCG2 gene expression analysis did not support protein expression results as ABCG2 gene expression results were found to be higher in HEK293 cells. Although HYP treatment was found to significantly reduce ABCG2 gene expression levels in both cell lines, FBX treatment partially restored ABCG2 gene expression. Our findings indicate that FBX co-treatment may not be suitable for augmenting HYP-mediated PDT in CRC but could potentially be useful for other applications.

Paynantheine more effectively reverses multidrug resistance in malignant EPG85.257RDB and MCF7MX cells than morphine and speciociliatine.

The possible interactions of morphine, paynantheine and speciociliatine alkaloids with ATP-binding cassette (ABC) transporters was investigated. The compounds were docked against ABCG2 and ABCB1 to predict the binding mode of alkaloids in active binding sites. The cytotoxicity of morphine, paynantheine and speciociliatine for EPG85.257RDB and MCF7MX cells was determined and ABCB1 and ABCG2 gene and protein expression were determined. The binding score of paynantheine to ABCB1 was higher in the docking studies. Paynantheine and speciociliatine had similar binding scores to ABCB1, but higher binding scores to ABCG2 than did morphine. Paynantheine and speciociliatine were more effective against MCF7MX and EPG85.257RDB cells and showed greater cyctotoxicity in the MTT assay. The effect of morphine and paynantheine on the ABCB1 gene and protein expression suggests these compounds can reduce resistance in cancer patients, but that speciociliatine may not be a suitable candidate because of its increased ABCB1 expression while speciociliatine decreased the expression of ABCG2 in MCF7MX cells. This indicates that speciociliatine is a better candidate for reducing drug resistance in this cell line. Structural modification, drug-metabolizing enzymes and differences in the binding sites could cause functional differences between these compounds.

Promotion of ABCG2 gene expression by neolignans from Piper longum L.

We focused on Piper longum L., a herbal drug produced in Myanmar, which has a renoprotective effect. Thus, we attempted to isolate and identify compounds that enhance the expression of the ABCG2 gene from the aerial parts of the plant except for the fruit. Among the various P. longum extracts, we isolated and identified the components. Using Caco-2 cells, the hABCG2 mRNA expression-enhancing effects of the isolated compounds were compared with the positive reference compound (3-methylcholanthrene [3MC]) using real-time polymerase chain reaction. Six compounds were isolated and identified from the methanol extract of P. longum. Among the isolated compounds, licarin A and neopomatene had lower toxicity and higher hABCG2 mRNA expression-enhancing effects in Caco-2 cells. Suppression of hAhR expression by siRNA reduced the activity of licarin A and neopomatene, as well as the hAhR agonist 3MC, suggesting that these 2 compounds may act as hAhR agonists to promote hABCG2 expression.

Targeting liver cancer stem cell through EpCAM therapy targeted with chemotherapy endorse enhanced progression in hepatocellular carcinoma

BackgroundTwo chief hurdles in most cancer treatments are chemoresistance and tumor recurrence, especially counting hepatocellular carcinoma (HCC). Most conformist chemotherapy fails to completely cure HCC patients because of its susceptibility to develop multidrug resistance (MDR) through factors such as hypoxia, cancer stem cells, and drug efflux mechanism cancer stem cells (CSC) which are significant factors involved in chemoresistance. It has been exposed that targeting liver cancer stem cells and chemotherapeutic drugs have a better selected, overall survival rate for hepatocellular carcinoma patients.AimThis study aims to investigate the effectiveness of targeting stem cells for liver cancer using a therapy that targets EpCAM in combination with chemotherapy and how this approach can enhance the treatment outcomes in hepatocellular carcinoma, the most prevalent kind of liver cancer.ResultsThe outcome was studied by flow cytometry, Western blot, RT-PCR, and cytotoxicity assays. EpCAM gene silenced and XAV939-treated cells showed decreased expression of CD133, a liver cancer stem cell (LCSC) marker in flow cytometry analysis, and reduced expression of ABCG2 gene, which is a reliable marker for chemoresistance in RT-PCR and western blot analysis; it was also unable to form colonies in colony forming assay. Similarly, in the spheroid formation assay, EpCAM gene silenced cells and XAV939-treated cells in combinations with cisplatin treatment were powerless to appear spheroid, whereas cisplatin alone-treated cells showed spheroids. In the cytotoxicity assay, cisplatin alone and combined with EpCAM silenced and XAV939-treated cells showed more lactate dehydrogenase (LDH) release than EpCAM silenced arm XAV939 treated components.ConclusionThese findings confirm our hypothesis that conventional chemotherapy kills cancer cells but not cancer stem cells. We believe EpCAM-targeted therapy enhances chemosensitivity and decreases relapsed chances. This approach might be the best option for a better prognosis for hepatocellular carcinoma patients.

MiR-548 K regulatory effect on the ABCG2 gene expression in MDR breast cancer cells.

multidrug resistance (MDR) is One of the foremost challenges in overcoming breast cancer. Various molecular processes are involved in the development of MDR in breast cancer cells, including over expression of ABC transporters such as ABCG2 (BCRP), increase breast cancer stem cells drug resistance, and epithelial mesenchymal transition. In the present study, we used bioinformatics and experimental analysis to investigate the role of miR-548 K, in the modulating of ABCG2, in MDR breast cancer cells. In silico inspections introduce 14 microRNAs targeting 3'-UTR region of ABCG2 transcripts, which are probably involved in breast cancer drug resistance. An association was highlighted between miR-548 k with ABC transporter family. The expression level of ABCG2 gene in MCF7-MX cell lines was significantly more than MCF7 cell lines. On the other hand, we increased the expression of miR-548 K in MCF7-MX and MCF7 cell lines through its transfection, which dramatically coincided with decreasion in the ABCG2 transcripts level. Additional studies on patient samples revealed that the expression of ABCG2 showed an increase in ABCG2 level in neoadjuvant chemotherapy drugs resistance (NCDR) patients compared to primary pre-operative chemotherapy drugs response (PCDR) patients. Also, a reduction in the expression of miR-548 K in NCDR patients was revealed. The results of our study suggest that miR-548 K may be involved in modulating the expression of ABCG2 in MDR breast cancer cells.

Molecular aspects of ABCB1 and ABCG2 in Gallbladder cancer and its clinical relevance.

The function of ABC transporters in the body is manifold; such as maintenance of homeostasis, effect on multi-drug resistance and their role in tumor initiation & progression. Evidence pointing towards the direct or indirect role of ABC transporter genes in particular; ABCB1 and ABCG2 in cancer genesis is increasing. However, their role in gallbladder cancer is unexplored. Therefore, we investigated the methylation status and expression pattern of ABCB1 and ABCG2in gallbladder carcinogenesis. The methylation and expression study of ABCB1/MDR1 and ABCG2/BCRP was performed in tumour and normal fresh tissue samples collected from 61 histopathologically diagnosed gallbladder cancer patients. The methylation status was analysed by Methylation-Specific PCR and expression was determined by Real-Time PCR and Immunohistochemistry. Hypomethylation of ABCB1 and ABCG2 was found in 44 (72.13%) and 48 (78.6%) cases, respectively. ABCB1 hypomethylation pattern showed association with female patients (p = 0.040) and GradeII tumors (p = 0.036) while, ABCG2hypomethylation was more frequent in early tumors (T1-T2). The mRNA expression ofABCB1 and ABCG2 was up-regulated in 33 (54.10%) and 41 (67.21%) patients with fold change of 4.7 and 5.5, respectively. The mRNA expression of both genes showed association with Grade II tumours and the increased fold change of ABCG2 was higher in (T1-T2) depth of invasion (p = 0.02) and Stage I-II disease (p = 0.08). The protein expression on IHC was strongly positive for ABCB1/MDR1and ABCG2/BCRP in 32 (52.46%) and 45 (73.77%) patients, respectively. The protein expression in ABCG2 showed association with patients age > 50years (p = 0.04) and GradeII differentiation (p = 0.07). Interestingly, the hypomethylation of both the genes showed significant correlation with increased expression. ABCB1/MDR1 and ABCG2/BCRP hypomethylation and overexpression could have a potential role in gallbladder cancer tumorigenesis especially in early stages. The epigenetic change might be a plausible factor for altered gene expression of ABCB1 and ABCG2 in gallbladder cancer.

A Study to Explore the Role of IDH1 (R132) Mutation on Imatinib Toxicity and Effect of ABCG2/OCT1 Expression on N-Desmethyl Imatinib Plasma Level in Egyptian Chronic Myeloid Leukemia Patients.

Imatinib mesylate (IM) is the gold standard for treatment of Chronic Myeloid Leukemia (CML). This study aimed to gain more knowledge of the altered PK, pharmacogenetic factors, and gene expression leading to variable IM levels. Fifty patients with chronic phase-CML were enrolled in this study and divided as 25 responders and 25 non-responders (patients are directly recruited after response assessment). HPLC/MS/MS was used to determine trough and peak concentration of imatinib and N-desmethyl imatinib in the blood. PCR-RFLP technique was used to detect IDH1 gene mutation (R132). The median value of IM trough level was significantly higher, the P/T ratio was significantly lower and the α-1-acid glycoprotein (AGP) was significantly higher among responders compared to non-responders (P=0.007, 0.009 and 0.048, respectively). Higher N-desmethyl imatinib peak plasma concentration was observed with low mRNA expression of ABCG2 and OCT1 (P=0.01 and 0.037, respectively). IDH1 R132 gene mutation was associated with a significant increase in toxicities (P=0.028). In conclusion, IM trough level, P/T ratio and AGP was significantly higher in responders. In addition, ABCG2 and OCT1 gene expression may affect the interindividual PK variation. Although a prospective study with a larger patient population is necessary to validate these findings. IDH1 mutation is a predictor of increased toxicity with IM treatment.

Differential gene expression of ABCG2, SLC22A12, IL-1\u03b2, and ALPK1 in peripheral blood leukocytes of primary gout patients with hyperuricemia and their comorbidities: a case\u2013control study

BackgroundThe ABCG2, SLC22A12, and ALPK1 genes have been strongly associated with dysfunction of urate metabolism in patients with gout, but it is unknown how these transporters are expressed in patients with acute or chronic gout. Our objectives were to: (a) analyze the gene expression of urate transporters and of inflammation genes in peripheral blood from gout patients and controls; (b) determine whether the metabolic profile of gout patients can influence the gene expression profile and the expression of urate transporters, ABCG2 and SLC22A12, and inflammation molecules, ALPK1 and IL-1β, in peripheral blood leukocytes from gout patients; (c) compare them with their metabolic profile and the gene expression of people without gout and without hyperuricemia.MethodsA total of 36 chronic and acute patients and 52 controls were recruited, and ABCG2, SLC22A12, IL-1β, and ALPK1 gene expression was evaluated by quantitative real-time PCR. Correlations of gene expression with clinical and laboratory parameters of patients were also analyzed.ResultsIL-1β was significantly increased in peripheral blood mononuclear cells (PBMCs) of patients compared with their polymorphonuclear leukocytes white blood cells (PMNLs, p < 0.05). A significant increase in ABCG2 and IL-1β was found in PMNLs from patients compared to controls (p < 0.05). Correlations of gene expression in patients were found with levels of serum uric acid (sUA), serum creatinine, C-reactive protein (CRP), triglycerides, body mass index (BMI), kidney disease, hypertension, and metabolic syndrome.ConclusionsOur data suggest that leukocytes of patients respond to the presence of hyperuricemia and comorbidities, expressing ABCG2 and IL-1β genes differentially compared to normouricemic and nondisease states. Hyperuricemia, dyslipidemia, and obesity probably stimulate the differential gene expression of peripheral blood leukocytes (neutrophils and monocytes), even in an asymptomatic state.

KRAS and EGFR Mutations Differentially Alter ABC Drug Transporter Expression in Cisplatin-Resistant Non-Small Cell Lung Cancer.

Lung carcinoma is still the most common malignancy worldwide. One of the major subtypes of non-small cell lung cancer (NSCLC) is adenocarcinoma (AC). As driver mutations and hence therapies differ in AC subtypes, we theorized that the expression and function of ABC drug transporters important in multidrug resistance (MDR) would correlate with characteristic driver mutations KRAS or EGFR. Cisplatin resistance (CR) was generated in A549 (KRAS) and PC9 (EGFR) cell lines and gene expression was tested. In three-dimensional (3D) multicellular aggregate cultures, both ABCB1 and ABCG2 transporters, as well as the WNT microenvironment, were investigated. ABCB1 and ABCG2 gene expression levels were different in primary AC samples and correlated with specific driver mutations. The drug transporter expression pattern of parental A549 and PC9, as well as A549-CR and PC9-CR, cell lines differed. Increased mRNA levels of ABCB1 and ABCG2 were detected in A549-CR cells, compared to parental A549, while the trend observed in the case of PC9 cells was different. Dominant alterations were observed in LEF1, RHOU and DACT1 genes of the WNT signalling pathway in a mutation-dependent manner. The study confirmed that, in lung AC-s, KRAS and EGFR driver mutations differentially affect both drug transporter expression and the cisplatin-induced WNT signalling microenvironment.

Suppression of Esophageal Cancer Stem-like Cells by SNX-2112 Is Enhanced by STAT3 Silencing.

Many studies have demonstrated that cancer stem cells (CSCs) or tumor-initiating cells (TICs) are responsible for tumor cell proliferation, chemotherapy resistance, metastasis, and relapse in various cancers. We, and others, have previously shown that the signal transducer and activator of transcription 3 (STAT3) signaling pathway is responsible for CSCs and TICs growth. Recent reports have indicated that the heat shock protein 90 (Hsp90) is also essential for the survival of CSCs and TICs. SNX-2112 is an Hsp90 inhibitor. However, it remains unclear whether proliferation of esophageal cancer stem-like cells (ECSLCs) is suppressed by SNX-2112 with knockdown of STAT3 (shSTAT3). Here, we explored the association between SNX-2112 with shSTAT3 and the suppression of ECSLCs growth. We found that the expression level of both STAT3 and p-STAT3 was higher in clinical esophageal cancer tissue than in the adjacent normal tissue, using western blot and qPCR analysis. Furthermore, differential expression analysis demonstrated that STAT3 was overexpressed in clinical specimens. We demonstrated that SNX-2112 inhibited cancer cell proliferation, decreased ABCB1 and ABCG2 gene expression levels and reduced the colony formation capacity of ECSLCs, which was enhanced by STAT3 silencing. Flow cytometry analysis revealed that the combination of SNX-2112 and shSTAT3 significantly induced apoptosis and cell cycle arrest at G2/M phase in ECSLCs. Levels of proliferation pathway proteins, including p38, c-Jun N-terminal kinase (JNK), and extracellular signal–regulated kinase (ERK) which were also client proteins of Hsp90, were also reduced. In addition, SNX-2112 with shSTAT3 inhibited the proliferation of ECSLCs in vivo. Finally, STAT3 overexpression eliminated the apoptotic and antiproliferative effects of SNX-2112 on ECSLCs. Hence, these results provide a rationale for the therapeutic potential of the combination of SNX-2112 with shSTAT3 in esophageal cancer, and may indicate new targets for clinical intervention in human cancer.

Cloning and tissue distribution of the ATP-binding cassette subfamily G member 2 gene in the marine pufferfish Takifugu rubripes

In this study, we cloned the ATP-binding cassette (ABC) subfamily G member 2 gene (Abcg2 gene; breast cancer resistance protein) from the liver of the marine pufferfish Takifugu rubripes, and examined its tissue distribution and gene structure. The full-length complementary DNA (cDNA) was 1977 base pairs (bp), comprising 15 exons in chromosome 17, and was assigned to the teleost Abcg2b paralogue. The open reading frame was 1839 bp in length, and encodes 612 amino acids constituting Walker A, Q-loop, C motif, Walker B, D-loop, and H-loop sequences in the first half and six transmembrane domains in the second half. Gene expression of Abcg2 was predominantly found in the intestines, followed by the liver and kidneys. The Kozak motif and the polyadenylation signal sequences were at respective ends of the transcript. The putative core promoter sequences including the transcription factor II B recognition elements, the TATA box, the initiator element, and the downstream core promoter element were found in the transcript and its upstream genomic DNA sequence. The hypoxia response element and the estrogen response element were located in the upstream genomic DNA sequence, suggesting that the pufferfish Abcg2 gene possesses the same regulation mechanism of transcription as the human ABCG2 gene.

AhR-activating pesticides increase the bovine ABCG2 efflux activity in MDCKII-bABCG2 cells.

In bovine mammary glands, the ABCG2 transporter actively secretes xenobiotics into dairy milk. This can have significant implications when cattle are exposed to pesticide residues in feed. Recent studies indicate that the fungicide prochloraz activates the aryl hydrocarbon receptor (AhR) pathway, increasing bovine ABCG2 (bABCG2) gene expression and efflux activity. This could enhance the accumulation of bABCG2 substrates in dairy milk, impacting pesticide risk assessment. We therefore investigated whether 13 commonly used pesticides in Europe are inducers of AhR and bABCG2 activity. MDCKII cells expressing mammary bABCG2 were incubated with pesticides for up to 72 h. To reflect an in vivo situation, applied pesticide concentrations corresponded to the maximum residue levels (MRLs) permitted in bovine fat or muscle. AhR activation was ascertained through CYP1A mRNA expression and enzyme activity, measured by qPCR and 7-ethoxyresorufin-Ο-deethylase (EROD) assay, respectively. Pesticide-mediated increase of bABCG2 efflux activity was assessed using the Hoechst 33342 accumulation assay. For all assays, the known AhR-activating pesticide prochloraz served as a positive control, while the non-activating tolclofos-methyl provided the negative control. At 10-fold MRL concentrations, chlorpyrifos-methyl, diflufenican, ioxynil, rimsulfuron, and tebuconazole significantly increased CYP1A1 mRNA levels, CYP1A activity, and bABCG2 efflux activity compared to the vehicle control. In contrast, dimethoate, dimethomorph, glyphosate, iprodione, methiocarb and thiacloprid had no impact on AhR-mediated CYP1A1 mRNA levels, CYP1A activity or bABCG2 efflux. In conclusion, the MDCKII-bABCG2 cell model proved an appropriate tool for identifying AhR- and bABCG2-inducing pesticides. This provides an in vitro approach that could reduce the number of animals required in pesticide approval studies.

Entrectinib reverses cytostatic resistance through the inhibition of ABCB1 efflux transporter, but not the CYP3A4 drug-metabolizing enzyme

Entrectinib is a new tyrosine kinase inhibitor that was recently approved for the treatment of ROS1-positive metastatic non-small cell lung cancer (NSCLC). In this study, we aimed to characterize its potential to act as a modulator of pharmacokinetic cytostatic resistance and perpetrator of drug interactions. In accumulation studies, entrectinib exhibited potent inhibition of ABCB1, while only moderate interaction was recorded for ABCG2 and ABCC1 efflux transporters. Furthermore, incubation assays revealed the potential of this drug to inhibit various recombinant cytochrome P450 enzymes, which can be ranked according to inhibitory affinities as follows: CYP2C8 ≈ CYP3A4 > CYP2C9 > CYP2C19 ≈ CYP3A5 > CYP2D6 > CYP2B6 > CYP1A2. Additionally, in silico docking analysis confirmed entrectinib’s interactions with ABCB1 and CYP3A4 and resolved their possible molecular background. In subsequent drug combination experiments, we demonstrated the ability of entrectinib to synergize with daunorubicin in various ABCB1-expressing cellular models. Moreover, the comparative proliferation study results suggested that the anticancer efficacy of entrectinib is not affected by the functional presence of tested ABC transporters. In contrast to ABCB1-related data, no resistance reversal effect was recorded for the combination with docetaxel in HepG2-CYP3A4 cells. In the final experimental set, we observed no significant changes in ABCB1, ABCG2, ABCC1 or CYP3A4 gene expression in NSCLC cells exposed to entrectinib. In summary, our work indicates that entrectinib may be a perpetrator of clinically relevant pharmacokinetic drug interactions and modulator of ABCB1-mediated resistance. Our in vitro results might provide a valuable foundation for future clinical investigations.

An Explorative Analysis of ABCG2/TOP-1 mRNA Expression as a Biomarker Test for FOLFIRI Treatment in Stage III Colon Cancer Patients: Results from Retrospective Analyses of the PETACC-3 Trial.

Biomarker-guided treatment for patients with colon cancer is needed. We tested ABCG2 and topoisomerase 1 (TOP1) mRNA expression as predictive biomarkers for irinotecan benefit in the PETACC-3 patient cohort. The present study included 580 patients with mRNA expression data from Stage III colon cancer samples from the PETACC-3 study, which randomized the patients to Fluorouracil/leucovorin (5FUL) +/− irinotecan. The primary end-points were recurrence free survival (RFS) and overall survival (OS). Patients were divided into one group with high ABCG2 expression (above median) and low TOP-1 expression (below 75 percentile) (“resistant”) (n = 216) and another group including all other combinations of these two genes (“sensitive”) (n = 364). The rationale for the cut-offs were based on the distribution of expression levels in the PETACC-3 Stage II set of patients, where ABCG2 was unimodal and TOP1 was bimodal with a high expression level mode in the top quarter of the patients. Cox proportional hazards regression was used to estimate the hazard ratios and the association between variables and end-points and log-rank tests to assess the statistical significance of differences in survival between groups. Kaplan-Meier estimates of the survival functions were used for visualization and estimation of survival rates at specific time points. Significant differences were found for both RFS (Hazard ratio (HR): 0.63 (0.44–0.92); p = 0.016) and OS (HR: 0.60 (0.39–0.93); p = 0.02) between the two biomarker groups when the patients received FOLFIRI (5FUL+irinotecan). Considering only the Microsatellite Stable (MSS) and Microsatellite Instability-Low (MSI-L) patients (n = 470), the differences were even more pronounced. In contrast, no significant differences were observed between the groups when patients received 5FUL alone. This study shows that the combination of ABCG2 and TOP1 gene expression significantly divided the Stage III colon cancer patients into two groups regarding benefit from adjuvant treatment with FOLFIRI but not 5FUL.

Identification of Functional Transcriptional Binding Sites within Chicken Abcg2 Gene Promoter and Screening Its Regulators.

Breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) half transporter encoded by the Abcg2 gene, is reported to influence the pharmacokinetics of substrate drugs during clinical therapy. The aim of this study was to clarify the mechanisms that regulate the transcription of the chicken Abcg2 gene through cloning and characterization of its promoter region. Results showed that the Abcg2 gene is transcribed by a TATA-less promoter with several putative Sp1 sites upstream from two putative CpG islands. A luciferase reporter assay conducted both in chicken leghorn male hepatoma (LMH) cells and chicken primary hepatocytes mapped a basal promoter to nucleotides −110 to +30, which is responsible for the constitutive expression of Abcg2. The 5′-region upstream of the basal promoter was characterized by both positive and negative regulatory domains. Further, using the cell-based reporter gene assay combined with RT-PCR and drug accumulation analysis, we found that four xenobiotics, daidzein, clotrimazole, ivermectin, and lipopolysaccharide (LPS), influence the expression and function of BCRP through significant regulation of the Abcg2 gene promoter. Interaction sites with the Abcg2 gene promoter of these four selected regulators were clarified by progressive deletions and mutation assays. This study shed some light on the regulatory mechanisms involved in chicken Abcg2 gene expression and the results may have far-reaching significance regarding the usage and development of veterinary drugs.

Depressive Disorder promotes Hepatocellular Carcinoma metastasis via upregulation of ABCG2 gene expression and maintenance of self-renewal.

Depressive disorder (DD) is the leading cause of disability worldwide and is the most prevalent mood disorder. Accumulative evidence from epidemiological studies has shown that DD is a risk factor for cancer. However, the role and molecular mechanism of DD in hepatocellular carcinoma (HCC) are still unknown. In this study, 30 mice were randomly divided into two groups: the HCC group and the HCC-DD group. The DD mouse model of HCC was established by induction with reserpine every other day and with monthly doses of diethylnitrosamine (DEN). All of the molecular studies were based on primary cell culture, and the effects of DD on HCC cell proliferation and migration and cancer stem cell (CSC) self-renewal were determined by colony formation, wound healing, and sphere culture assays. We found that the CSC markers ABCG2 and CD133 were upregulated in HCC-DD primary cells compared with HCC primary cells. Moreover, HCC-DD primary cells were more aggressive in terms of metastasis and self-renewal than HCC primary cells. Further study revealed that DD promoted tumor growth and metastasis by activating the AKT signaling pathway followed by an increased ABCG2 expression. Taken together, our novel findings indicate that DD promotes proliferation, self-renewal, and metastasis by upregulating ABCG2 in the AKT pathway.