ABCB1 Expression Research Articles

Long non-coding RNA LUCAT1 regulates the RAS pathway to promote the proliferation and invasion of malignant glioma cells through ABCB1

Long non-coding RNAs (lncRNAs) are closely related to the occurrence and development of tumors and have gradually become a hot topic in the field of glioma research in recent years. In this study, the role of lung cancer associated transcript 1 (lncRNA LUCAT 1) in glioma occurrence and development, as well as its possible regulatory mechanism, was explored. We utilized the gene chip technology in the preliminary experiment, and based on the experiment results, selected LUCAT1（NONHSAT102745）, which was significantly upregulated in glioma, and ATP-binding cassette Subfamily B member l (ABCB1), which was significantly down-regulated in co-expression analysis, for study. Next, the expression of LUCAT1 and ABCB1 in cells and tissues was immediately evaluated. Subsequently, the cells were transfected with scrambled siRNA, LUCAT1-siRNA/ABCB 1-siRNA, or overexpressed LUCAT1/ABCB1 plasmid + RAS signaling pathway inhibitor-farnesylthiosalicylic acid (FTS). By comparing with the normal combination negative control group, the cell proliferation and invasion ability were evaluated. Finally, subcutaneous tumor formation experiments in the nude mice confirmed the association between LUCAT1 and ABCB1 and RAS signaling pathways. The expression of LUCAT 1 was up-regulated with an increase in WHO grade, and the lncRNA-mRNA co-expression analysis showed that the expression of ABCB1 was low. LUCAT 1 gene knockout reduced the mRNA and protein levels of Ras signaling pathway related factors (Ras, Raf-1, p-AKT, and p-ERK) as regulating ABCB1 expression and inhibiting the ability of tumor in proliferation and invasion no matter in vitro or in vivo. For overexpressing of LUCAT 1, the opposite was true. After we knocked out ABCB1, the LUCAT1 expression was reversely regulated while the level of RAS signaling pathway related factors increased, and the ability of tumors in proliferation and invasion was enhanced. The abnormal LUCAT1 expression affected the biological behaviors of glioma cells, such as proliferation, invasion, etc. by regulating ABCB1 and promoting the activation of the RAS signaling pathway. This provided a new drug target and therapeutic approach for gene therapy of glioma, which is expected to significantly improve the prognosis of relevant patients.

Consecutive dosing of UVB irradiation induces loss of ABCB5 expression and activation of EMT and fibrosis proteins in limbal epithelial cells similar to pterygium epithelium

Pterygium pathogenesis is often attributed to a population of altered limbal stem cells, which initiate corneal invasion and drive the hyperproliferation and fibrosis associated with the disease. These cells are thought to undergo epithelial to mesenchymal transition (EMT) and to contribute to subepithelial stromal fibrosis. In this study, the presence of the novel limbal stem cell marker ABCB5 in clusters of basal epithelial pterygium cells co-expressing with P63α and P40 is reported. ABCB5-positive pterygium cells also express EMT-associated fibrosis markers including vimentin and α-SMA while their β-catenin expression is reduced. By using a novel in vitro model of two-dose UV-induced EMT activation on limbal epithelial cells, we could observe the dysregulation of EMT-related proteins including an increase of vimentin and α-SMA as well as downregulation of β-catenin in epithelial cells correlating to downregulation of ABCB5. The sequential irradiation of limbal fibroblasts also induced an increase in vimentin and α-SMA. Taken together, these data demonstrate for the first time the expression of ABCB5 in pterygium stem cell activity and EMT-related events while the involvement of limbal stem cells in pterygium pathogenesis is exhibited via sequential irradiation of limbal epithelial cells. The later in vitro approach can be used to further study the involvement of limbal epithelium UV-induced EMT in pterygium pathogenesis and help identify novel treatments against pterygium growth and recurrence.

ABCB1 Is Frequently Methylated in Higher-Grade Gliomas and May Serve as a Diagnostic Biomarker of More Aggressive Tumors.

ABCB1 belongs to a superfamily of membrane transporters that use ATP hydrolysis to efflux various endogenous compounds and drugs outside the cell. Cancer cells upregulate ABCB1 expression as an adaptive response to evade chemotherapy-mediated cell death. On the other hand, several reports highlight the role of the epigenetic regulation of ABCB1 expression. In fact, the promoter methylation of ABCB1 was found to be methylated in several tumor types, including gliomas, but its role as a biomarker is not fully established yet. Thus, the aim of this study was to analyze the methylation of the ABCB1 promoter in tumor tissues from 50 glioma patients to verify its incidence and to semi-quantitively detect ABCB1 methylation levels in order to establish its utility as a potential biomarker. The results of this study show a high interindividual variability in the ABCB1 methylation level of the samples derived from gliomas of different grades. Additionally, a positive correlation between ABCB1 methylation, the WHO tumor grade, and an IDH1 wild-type status has been observed. Thus, ABCB1 methylation can be regarded as a potential diagnostic or prognostic biomarker for glioma patients, indicating more aggressive tumors.

Adagrasib, a KRAS G12C inhibitor, reverses the multidrug resistance mediated by ABCB1 in vitro and in vivo

BackgroundMultidrug resistance (MDR) is a complex phenomenon that frequently leads to chemotherapy failure during cancer treatment. The overexpression of ATP-binding cassette (ABC) transporters represents the major mechanism contributing to MDR. To date, no effective MDR modulator has been applied in clinic. Adagrasib (MRTX849), a specific inhibitor targeting KRAS G12C mutant, is currently under investigation in clinical trials for the treatment of non-small cell lung cancer (NSCLC). This study focused on investigating the circumvention of MDR by MRTX849.MethodsThe cytotoxicity and MDR reversal effect of MRTX849 were assessed by MTT assay. Drug accumulation and drug efflux were evaluated by flow cytometry. The MDR reversal by MRTX849 in vivo was investigated in two ABCB1-overexpressing tumor xenograft models in nude mice. The interaction between MRTX849 and ABCB1 substrate binding sites was studied by the [125I]-IAAP-photoaffinity labeling assay. The vanadate-sensitive ATPase assay was performed to identify whether MRTX849 would change ABCB1 ATPase activity. The effect of MRTX849 on expression of ABCB1 and PI3K/AKT signaling molecules was examined by flow cytometry, Western blot and Quantitative Real-time PCR analyses.ResultsMRTX849 was shown to enhance the anticancer efficacy of ABCB1 substrate drugs in the transporter-overexpressing cells both in vitro and in vivo. The MDR reversal effect was specific against ABCB1 because no similar effect was observed in the parental sensitive cells or in ABCG2-mediated MDR cells. Mechanistically, MRTX849 increased the cellular accumulation of ABCB1 substrates including doxorubicin (Dox) and rhodamine 123 (Rho123) in ABCB1-overexpressing MDR cells by suppressing ABCB1 efflux activity. Additionally, MRTX849 stimulated ABCB1 ATPase activity and competed with [125I]-IAAP for photolabeling of ABCB1 in a concentration-dependent manner. However, MRTX849 did not alter ABCB1 expression or phosphorylation of AKT/ERK at the effective MDR reversal drug concentrations.ConclusionsIn summary, MRTX849 was found to overcome ABCB1-mediated MDR both in vitro and in vivo by specifically attenuating ABCB1 efflux activity in drug-resistant cancer cells. Further studies are warranted to translate the combination of MRTX849 and conventional chemotherapy to clinical application for circumvention of MDR.3A9j1Q87GL5CKdhmy4PpapVideo

Selective Fluorescent Probes for High-Throughput Functional Diagnostics of the Human Multidrug Transporter P-Glycoprotein (ABCB1).

The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dyes to examine the functional expression and properties of the ABCB1 multidrug transporter, especially in microplate-based high-throughput flow cytometry assays. In addition, we demonstrate the applicability of the TP dyes to efficiently select and separate even a very low number of Pgp-expressing intact cells.

The WD repeat-containing protein 5 (WDR5) antagonist WDR5-0103 restores the efficacy of cytotoxic drugs in multidrug-resistant cancer cells overexpressing ABCB1 or ABCG2

The development of multidrug resistance (MDR) is one of the major challenges in the treatment of cancer which is caused by the overexpression of the ATP-binding cassette (ABC) transporters ABCB1 (P-glycoprotein) and/or ABCG2 (BCRP/MXR/ABCP) in cancer cells. These transporters are capable of reducing the efficacy of cytotoxic drugs by actively effluxing them out of cancer cells. Since there is currently no approved treatment for patients with multidrug-resistant tumors, the drug repurposing approach provides an alternative route to identify agents to reverse MDR mediated by ABCB1 and/or ABCG2 in multidrug-resistant cancer cells. WDR5–0103 is a histone H3 lysine 4 (H3K4) methyltransferase inhibitor that disrupts the interaction between the WD repeat-containing protein 5 (WDR5) and mixed-lineage leukemia (MLL) protein. In this study, the effect of WDR5–0103 on MDR mediated by ABCB1 and ABCG2 was determined. We found that in a concentration-dependent manner, WDR5–0103 could sensitize ABCB1- and ABCG2-overexpressing multidrug-resistant cancer cells to conventional cytotoxic drugs. Our results showed that WDR5–0103 reverses MDR and improves drug-induced apoptosis in multidrug-resistant cancer cells by inhibiting the drug-efflux function of ABCB1 and ABCG2, without altering the protein expression of ABCB1 or ABCG2. The potential sites of interactions of WDR5–0103 with the drug-binding pockets of ABCB1 and ABCG2 were predicted by molecular docking. In conclusion, the MDR reversal activity of WDR5–0103 demonstrated here indicates that it could be used in combination therapy to provide benefits to a subset of patients with tumor expressing high levels of ABCB1 or ABCG2.

Gene/environment interaction in the susceptibility of Crohn's disease patients to aluminum

Background & aimThe key role of environmental factors in the pathogenesis of Inflammatory Bowel Diseases (IBD) is recognized. Aluminum is suspected to be a risk factor for IBD. However, mechanisms linking aluminum exposure to disease development are unknown. We examined the role of aluminum transport and subcellular localisation on human colon susceptibility to aluminum-induced inflammation. MethodsHuman colon biopsies isolated from Crohn's disease (CD) or control patients and Caco-2 cells were incubated with aluminum. The effects of aluminum were evaluated on cytokine secretion and transporter expression. The role of aluminum kinetics parameters was studied in Caco-2 using transport inhibitors and in human colon biopsies by assessing genetic polymorphisms of transporters. ResultsAluminum exposure was shown to induce cytokine secretion in colon of CD but not healthy patients. In Caco-2 cells, aluminum internalisation was correlated with inflammatory status. In human colon, analysis of genetic polymorphisms and expression of ABCB1 and SLC26A3 transporters showed that their decreased activity was involved in aluminum-induced inflammation. ConclusionsWe hypothesize that alteration in detoxifying response would lead to a deregulation of intestinal homeostasis and to the expression of IBD. Our study emphasizes the complexity of gene/environment interaction for aluminum adverse health effect, highlighting at risk populations or subtypes of patients. A better understanding of correlations between gene expression or SNP and xenobiotic kinetics parameters would shift the medical paradigm to more personalized disease management and treatment.

ISG15 as a prognostic biomarker in solitary fibrous tumour

BackgroundSolitary fibrous tumour (SFT) is a rare mesenchymal malignancy that lacks robust prognostic and predictive biomarkers. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier, associated with tumour progression, and with poor survival of SFT patients, as previous published by our group. Here, we describe the role of ISG15 in the biology of this rare tumour.MethodsISG15 expression was assessed by immunohistochemistry in tissue microarrays from SFT patients and tested for correlation with progression-free survival and overall survival (OS). The effects of ISG15 knockdown or induction were investigated for cancer stem cell (CSC) characteristics and for drug sensitivity in unique in vitro models of SFT.ResultsThe prognostic value of ISG15 for OS was validated at protein level in malignant SFT patients, prospectively treated with pazopanib and enrolled in GEIS-32 trial. In SFT in vitro models, ISG15 knockdown lead to a decrease in the expression of CSC-related genes, including SOX2, NANOG, ALDH1A1, ABCB1 and ABCC1. Likewise, ISG15 downregulation decreased the clonogenic/ tumoursphere-forming ability of SFT cells, while enhancing apoptotic cell death after doxorubicin, pazopanib or trabectedin treatment in 3D cell cultures. The regulation of CSC-related genes by ISG15 was confirmed after inducing its expression with interferon-β1; ISG15 induction upregulated 1.28- to 451-fold the expression of CSC-associated genes.ConclusionsISG15 is a prognostic factor in malignant SFT, regulating the expression of CSC-related genes and CSCs maintenance. Our results suggest that ISG15 could be a novel therapeutic target in SFT, which could improve the efficacy of the currently available treatments.

Muramyl Dipeptide Administration Delays Alzheimer's Disease Physiopathology via NOD2 Receptors.

Alzheimer’s disease (AD) is the most common form of dementia in the world. The prevalence is steadily increasing due to an aging population and the lack of effective treatments. However, modulation of innate immune cells is a new therapeutic avenue, which is quite effective at delaying disease onset and improving cognitive decline. Methods: We studied the effect of the NOD2 receptor ligand muramyl dipeptide (MDP) on the modulation of the innate immune cells, namely patrolling monocytes and microglia. We administrated MDP once a week for 3 months in an APPswe/PS1 mouse model in both sexes. We started the treatment at 3 months before plaque formation and evaluated its effects at 6 months. Results: We showed that the MDP injections delay cognitive decline in both sexes via different mechanisms and protect the blood brain barrier (BBB). In males, MDP triggers the sink effect from the BBB, leading to a diminution in the amyloid load in the brain. This phenomenon is underlined by the increased expression of phagocytosis markers such as TREM2, CD68, and LAMP2 and a higher expression of ABCB1 and LRP1 at the BBB level. The beneficial effect seems more restricted to the brain in females treated with MDP, where microglia surround amyloid plaques and prevent the spreading of amyloid peptides. This phenomenon is also associated with an increase in TREM2 expression. Interestingly, both treated groups showed an increase in Arg-1 expression compared to controls, suggesting that MDP modulates the inflammatory response. Conclusion: These results indicate that stimulation of the NOD2 receptor in innate immune cells is a promising therapeutic avenue with potential different mechanisms between males and females.

The role of ABCB1 gene polymorphisms in steroid-resistant nephrotic syndrome: Evidence from a meta-analysis of steroid-receiving patients.

Previous studies in nephrotic syndrome have shown that three common variants in the ABCB1 gene, including rs1128503, rs2032582, and rs1045642, change the expression and activity of ABCB1, which may be responsible for the drug resistance. However, as a result of the inconclusive outcomes of these studies, we performed a meta-analysis to validate the association between ABCB1 polymorphisms and the susceptibility of steroid-resistant nephrotic syndrome (SRNS). The association was evaluated by calculating the odds ratio (OR) and 95% confidence interval. A total of 12 studies containing 1,463 subjects (514 steroid-resistant and 949 steroid-sensitive) were included. Single nucleotide polymorphism rs1128503 showed a significant association with SRNS (p< 0.05) only in the allele model (OR =1.40) in Africans. A statistically significant association was found for rs2032582 in codominant 2, dominant, recessive, and allele models (OR =1.85, 1.52, 1.38, and 1.34, respectively). Subgroup analysis revealed that rs2032582 showed a significant correlation with SRNS in codominant 1, 2, dominant, over-dominant, and allele models in Africans (OR =3.22, 3.52, 3.29, 1.74, and 1.83, respectively). In the case of rs1045642, codominant 1 (OR =0.72) and recessive models (OR =1.34) revealed a significant correlation with SRNS. Again, codominant 1 (OR =0.58), dominant (OR =0.69), and over-dominant models (OR =0.62) showed a protective effect in Asians. Haplotype analysis showed that the TGC haplotype is associated with a 1.83, 1.77, and 2.17 times significant correlation in overall, Asian, and African populations, respectively. By contrast, the CGC haplotype showed a 0.69 and 0.57 times lower association in the overall and African populations, respectively. The CTC haplotype also showed a 1.79 times enhanced susceptibility for SRNS in the overall population. Our study suggests that ABCB1 polymorphisms are associated with SRNS development, especially in Africans and Asians.

PB1790: ABCB1 AS A POTENTIAL BENEFICIAL TARGET OF MIDOSTAURIN IN ACUTE MYELOID LEUKEMIA

Background: In AML, therapeutic outcome of anthracycline-based induction therapy often can be compromised by a resistant phenotype associated with overexpression of ABCB1 transporters. Novel anti-AML agent, midostaurin, was designed to aim at multiple targets including FLT3 mutation. Besides, it was also identified as ABCB1 inhibitor. Aims: To evaluate ABCB1 efflux activity and expression in primary AML samples in relation to multiple clinical parameters and investigate ABCB1 regulation by microRNA. Methods: ABCB1 gene expression was absolutely quantified in 28 primary AML samples collected prior to any treatment. Efflux activity was examined using combination of mitoxantrone (ABCB1 substrate) and midostaurin (ABCB1/FLT3 inhibitor). Results were compared in molecular classes defined by CD34 marker, FLT3-ITD and NPM1 mutations, ELN risk classification and (un)achieved complete remission (CR). Enhancement of daunorubicin proapoptotic effect mediated by midostaurin was determined in HL-60 wt and HL-60 overexpressing ABCB1. ABCB1 regulation by microRNAs (miR-9, miR-27a, miR-331) was evaluated by ddPCR and correlated with ABCB1 efflux activity. Results: High ABCB1 expression was associated with unachieved CR, adverse ELN risk and CD34 positivity. ABCB1 efflux activity was observed in a functional study when mitoxantrone accumulation increased as a direct consequence of midostaurin in CD34+ AML and patients with unachieved CR. ABCB1 efflux activity and expression were found to be linked to miR-9 downregulation Summary/Conclusion: We propose therapeutic value of midostaurin for drug-resistant patients beyond the FLT3 inhibition based on ABCB1 expression and CD34 positivity. We also propose miR-9 as a predictive ABCB1-related biomarker that could be helpful in identifying ABCB1-resistant AML phenotype.

Development of a cancer cells self‑activating and miR‑125a‑5p expressing poly‑pharmacological nanodrug for cancer treatment.

Cancer cells can acquire resistance to targeted therapeutic agents when the designated targets or their downstream signaling molecules develop protein conformational or activity changes. There is an increasing interest in developing poly-pharmacologic anticancer agents to target multiple oncoproteins or signaling pathways in cancer cells. The microRNA 125a-5p (miR-125a-5p) is a tumor suppressor, and its expression has frequently been downregulated in tumors. By contrast, the anti-apoptotic molecule BIRC5/SURVIVIN is highly expressed in tumors but not in the differentiated normal tissues. In the present study, the development of a BIRC5 gene promoter-driven, miR-125a-5p expressing, poly-L-lysine-conjugated magnetite iron poly-pharmacologic nanodrug (pL-MNP-pSur-125a) was reported. The cancer cells self-activating property and the anticancer effects of this nanodrug were examined in both the multidrug efflux protein ABCB1/MDR1-expressing/-non-expressing cancer cells in vitro and in vivo. It was demonstrated that pL-MNP-pSur-125a decreased the expression of ERBB2/HER2, HDAC5, BIRC5, and SP1, which are hot therapeutic targets for cancer in vitro. Notably, pL-MNP-pSur-125a also downregulated the expression of TDO2 in the human KB cervical carcinoma cells. PL-MNP-pSur-125a decreased the viability of various BIRC5-expressing cancer cells, regardless of the tissue origin or the expression of ABCB1, but not of the human BIRC5-non-expressing HMEC-1 endothelial cells. In vivo, pL-MNP-pSur-125a exhibited potent antitumor growth effects, but without inducing liver toxicity, in various zebrafish human-ABCB1-expressing and ABCB1-non-expressing tumor xenograft models. In conclusion, pL-MNP-pSur-125a is an easy-to-prepare and a promising poly-pharmacological anticancer nanodrug that has the potential to manage numerous malignancies, particularly for patients with BIRC5/ABCB1-related drug resistance after prolonged chemotherapeutic treatments.

Screening Biomarkers for Systemic Lupus Erythematosus Based on Machine Learning and Exploring Their Expression Correlations With the Ratios of Various Immune Cells.

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune illness caused by a malfunctioning immunomodulatory system. China has the second highest prevalence of SLE in the world, from 0.03% to 0.07%. SLE is diagnosed using a combination of immunological markers, clinical symptoms, and even invasive biopsy. As a result, genetic diagnostic biomarkers for SLE diagnosis are desperately needed.MethodFrom the Gene Expression Omnibus (GEO) database, we downloaded three array data sets of SLE patients’ and healthy people’s peripheral blood mononuclear cells (PBMC) (GSE65391, GSE121239 and GSE61635) as the discovery metadata (nSLE = 1315, nnormal = 122), and pooled four data sets (GSE4588, GSE50772, GSE99967, and GSE24706) as the validate data set (nSLE = 146, nnormal = 76). We screened the differentially expressed genes (DEGs) between the SLE and control samples, and employed the least absolute shrinkage and selection operator (LASSO) regression, and support vector machine recursive feature elimination (SVM-RFE) analyze to discover possible diagnostic biomarkers. The candidate markers’ diagnostic efficacy was assessed using the receiver operating characteristic (ROC) curve. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was utilized to confirm the expression of the putative biomarkers using our own Chinese cohort (nSLE = 13, nnormal = 10). Finally, the proportion of 22 immune cells in SLE patients was determined using the CIBERSORT algorithm, and the correlations between the biomarkers’ expression and immune cell ratios were also investigated.ResultsWe obtained a total of 284 DEGs and uncovered that they were largely involved in several immune relevant pathways, such as type І interferon signaling pathway, defense response to virus, and inflammatory response. Following that, six candidate diagnostic biomarkers for SLE were selected, namely ABCB1, EIF2AK2, HERC6, ID3, IFI27, and PLSCR1, whose expression levels were validated by the discovery and validation cohort data sets. As a signature, the area under curve (AUC) values of these six genes reached to 0.96 and 0.913, respectively, in the discovery and validation data sets. After that, we checked to see if the expression of ABCB1, IFI27, and PLSCR1 in our own Chinese cohort matched that of the discovery and validation sets. Subsequently, we revealed the potentially disturbed immune cell types in SLE patients using the CIBERSORT analysis, and uncovered the most relevant immune cells with the expression of ABCB1, IFI27, and PLSCR1.ConclusionOur study identified ABCB1, IFI27, and PLSCR1 as potential diagnostic genes for Chinese SLE patients, and uncovered their most relevant immune cells. The findings in this paper provide possible biomarkers for diagnosing Chinese SLE patients.

Effects of ABCB1 DNA methylation in donors on tacrolimus blood concentrations in recipients following liver transplantation.

AimsTo investigate the effects of ABCB1 DNA methylation in donors on individual differences in tacrolimus blood concentrations following liver transplantation.MethodsTwenty‐three donor liver samples carrying the CYP3A5*3/*3 genotype were classified into 2 groups based on their initial tacrolimus blood concentrations (C0 >10 μg/L or <5 μg/L) following liver transplantation. ABCB1 mRNA levels in liver tissues and HepG2 cells were determined by quantitative reverse transcriptase polymerase chain reaction. DNA methylation status in liver tissues and HepG2 cells was determined using Illumina 850 methylation chip sequencing technology and pyrosequencing. 5‐Aza‐2dC was used to reverse methylation in HepG2 cells. Intracellular tacrolimus concentrations were determined by liquid mass spectrometry.ResultsGenome‐wide methylation sequencing and pyrosequencing analyses showed that the methylation levels of 3 ABCB1 CpG sites (cg12501229, cg00634941 and cg05496710) were significantly different between groups with different tacrolimus concentration/dose (C0/D) ratios. ABCB1 mRNA expression in donor livers was found to be positively correlated with tacrolimus C0/D ratio (R = .458, P < .05). After treatment with 5‐Aza‐2‐Dc, the methylation levels of the ABCB1 CpG sites in HepG2 cells significantly decreased, and this was confirmed by pyrosequencing; there was also a significant increase in ABCB1 transcription, which induced a decrease in intracellular tacrolimus concentrations.Conclusion ABCB1 CpG site methylation affects tacrolimus metabolism in humans by regulating ABCB1 expression. Therefore, ABCB1 DNA methylation in donor livers might be an important epigenetic factor that affects tacrolimus blood concentrations following liver transplantation.

Overcoming Resistance to Anti–Nectin-4 Antibody-Drug Conjugate

Antibody-drug conjugates (ADC) represent a fast-growing drug class in oncology. However, ADCs are associated with resistance, and therapies able to overcome it are of utmost importance. Recently, enfortumab vedotin-ejfv (EV) was approved in nectin-4+ metastatic urothelial cancer. We previously described PVRL4/nectin-4 as a new therapeutic target in breast cancer and produced an efficient EV-like ADC comprising a human anti-nectin-4 mAb conjugated to monomethyl auristatin-E (MMAE) named N41mab-vcMMAE. To study the consequence of the long-term treatment with this ADC, we developed a preclinical breast cancer model in mice, and report a mechanism of resistance to N41mab-vcMMAE after 9-month treatment and a way to reverse it. RNA-sequencing pointed to an upregulation in resistant tumors of ABCB1 expression, encoding the multidrug resistance protein MDR-1/P-glycoprotein (P-gp), associated with focal gene amplification and high protein expression. Sensitivity to N41mab-vcMMAE of the resistant model was restored in vitro by P-gp pharmacologic inhibitors, like tariquidar. P-gp is expressed in a variety of normal tissues. By delivering the drug to the tumor more specifically than classical chemotherapy, we hypothesized that the combined use of ADC with P-gp inhibitors might reverse resistance in vivo without toxicity. Indeed, we showed that the tariquidar/N41mab-vcMMAE combination was well tolerated and induced a rapid regression of ADC-resistant tumors in mice. In contrast, the tariquidar/docetaxel combination was toxic and poorly efficient. These results show that ABC transporter inhibitors can be safely used with ADC to reverse ADC-induced resistance and open new opportunities in the fight against multidrug resistance.

Regulation of PITX2 and ABCB1 in oncogenic multidrug resistance by tumorigenic HIF2 in VHL‐deficient renal cancer cells

The tumour suppressor von Hippel‐Lindau (VHL) is involved in ubiquitination and subsequent proteasomal degradation of hypoxia‐inducible factors (HIFs). Under hypoxic conditions, HIFα‐subunits are stabilized, leading to dimerization with HIFβ and transactivation of genes for metabolism, survival and angiogenesis. Human renal cancer cells A498 are VHL‐deficient and harbor stabilized HIF‐2, whereas Caki‐1 cells are VHL‐intact and HIF‐1‐dominant. Previous studies demonstrated increased paired‐like homeodomain transcription factor 2 (PITX2) and its target gene, the ABCB1 drug transporter, in A498 cells. The relationship between VHL, HIF, PITX2 and ABCB1 remains unclear.Transient transfection of A498 cells with HIF‐1α or HIF‐2α siRNA (25 nM, 72 h, &gt;85% protein knockdown) attenuated PITX2 protein by up to 75% by immunoblotting and PITX2 promoter activity by ~40% in a luciferase reporter gene assay. Immunoprecipitation studies did not propose direct HIF‐PITX2 protein‐protein interaction. HIF‐regulated PITX2 expression was proportional to ABCB1 expression, which was decreased at both mRNA (~20%) and protein (~70%) levels after HIF‐1α or HIF‐2α knockdown. Furthermore, HIF/PITX2 target gene CCND1 was reduced by &gt;60%, but PITX2 targets LCN2 and WNT4 were increased &gt;3‐fold following transient HIF downregulation. HIF‐2α siRNA was generally more effective than HIF‐1α siRNA in altering PITX2 and its target genes. Conversely, in Caki‐1 cells, HIF siRNA augmented PITX2 and ABCB1 mRNA and protein by ~1.5‐fold and ~2‐fold, respectively, with no significant changes in CCND1, LCN2 and WNT4. Polysome profiling show HIF‐2α siRNA negatively impacts active translation of PITX2 and ABCB1 mRNA. Finally, HIF2 knockdown sensitizes A498 cells to cell death by the chemotherapeutic drug and ABCB1 substrate, doxorubicin (1 µM, 24 h), as determined by trypan blue staining.In summary, these findings suggest PITX2 and ABCB1 are positively regulated by tumorigenic HIF2 in drug‐resistant A498 cells whereas they are suppressed by HIF‐1 in drug‐sensitive Caki‐1 cells. These data may provide better understanding of membrane transporters and possible upstream causes of multidrug resistance in different renal cancers.

Down-regulation of ABCB1 by collateral sensitivity drugs reverses multidrug resistance and up-regulates enolase I.

The emergence of drug resistance remains an obstacle in the clinical treatment of cancer. Recent developments in the studies of drug resistance have identified compounds such as verapamil and tamoxifen that specifically target ABCB1-expressing multidrug-resistant (MDR) cells, through an ATP-dependent ROS-generating mechanism. In this report, we demonstrate that treatment of ABCB1-expressing MDR cells (CHORC5 or MDA-Doxo400) or individual clones of the latter with sub-lethal concentrations of tamoxifen or verapamil down-regulates ABCB1 protein and mRNA expression in surviving clones. Consequently, tamoxifen- and verapamil-treated cells show increased sensitivity to chemotherapeutic drugs (e.g., colchicine and doxorubicin) and decreased sensitivity to collateral sensitivity drugs (e.g., verapamil and tamoxifen). Importantly, we show for the first time that down-regulation of ABCB1 expression resulting from tamoxifen treatment and CRISPR-knockout of ABCB1 expression up-regulate α-enolase (enolase I) protein levels and activity. These findings demonstrate a possible effect of ABCB1 expression on the metabolic homeostasis of MDR cells. Moreover, given the use of tamoxifen to prevent the recurrence of oestrogen receptor-positive breast cancer, the findings of this study may be clinically important in modulating activity of other drugs.

CDK6-PI3K signaling axis is an efficient target for attenuating ABCB1/P-gp mediated multi-drug resistance (MDR) in cancer cells

BackgroundMultidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1/P-gp) is a major cause of cancer chemotherapy failure, but the regulation mechanisms are largely unknown.MethodsBased on single gene knockout, we studied the regulation of CDK6-PI3K axis on ABCB1-mediated MDR in human cancer cells. CRISPR/Cas9 technique was performed in KB-C2 cells to knockout cdk6 or cdk4 gene. Western blot, RT-PCR and transcriptome analysis were performed to investigate target gene deletion and expression of critical signaling factors. The effect of cdk4 or cdk6 deficiency on cell apoptosis and the cell cycle was analyzed using flow cytometry. In vivo studies were performed to study the sensitivity of KB-C2 tumors to doxorubicin, tumor growth and metastasis.ResultsDeficiency of cdk6 led to remarkable downregulation of ABCB1 expression and reversal of ABCB1-mediated MDR. Transcriptomic analysis revealed that CDK6 knockout regulated a series of signaling factors, among them, PI3K 110α and 110β, KRAS and MAPK10 were downregulated, and FOS-promoting cell autophagy and CXCL1-regulating multiple factors were upregulated. Notably, PI3K 110α/110β deficiency in-return downregulated CDK6 and the CDK6-PI3K axis synergizes in regulating ABCB1 expression, which strengthened the regulation of ABCB1 over single regulation by either CDK6 or PI3K 110α/110β. High frequency of alternative splicing (AS) of premature ABCB1 mRNA induced by CDK6, CDK4 or PI3K 110α/110β level change was confirmed to alter the ABCB1 level, among them 10 common skipped exon (SE) events were found. In vivo experiments demonstrated that loss of cdk6 remarkably increased the sensitivity of KB-C2 tumors to doxorubicin by increasing drug accumulation of the tumors, resulting in remarkable inhibition of tumor growth and metastasis, as well as KB-C2 survival in the nude mice.ConclusionsCDK6-PI3K as a new target signaling axis to reverse ABCB1-mediated MDR is reported for the first time in cancers. Pathways leading to inhibition of cancer cell proliferation were revealed to be accompanied by CDK6 deficiency.

Clinical significance and correlation of miR-200c and P-gp expression in gastric cancer and the effects on multidrug resistance.

Poor prognosis is common in gastric cancer patients due to multidrug resistance (MDR)-induced recurrence and metastasis. In the present study, we investigated the expression of microRNA (miR)-200c in gastric cancer tissues and cell lines and its relationship with the expression of the drug resistant gene ABCB1, which encodes P-glycoprotein (P-gp). The basic characteristics of 102 patients with gastric cancer were reviewed. Real time-polymerase chain reaction (PCR), immunohistochemistry, and Western blot were employed to detect the expression levels of miR-200c and P-gp in gastric carcinoma tissues and cell lines. The correlation of miR-200c messenger RNA (mRNA) level with clinicopathological characteristics and P-gp protein expression were analyzed. SGC7901/vincristine (VCR) cells were transfected with miR-200c mimics or a specific small interfering RNA (siRNA) targeting the ABCB1 gene. The methyl thiazolyl tetrazolium (MTT) assay and flow cytometry were used to determine the role of miR-200c and ABCB1 on the viability and apoptosis of gastric carcinoma cell lines. The level of miR-200c in carcinoma tissues was significantly lower than that in adjacent tissues, and the expression level of P-gp in carcinoma tissues was obviously higher than that in adjacent tissues (P<0.01, P=0.029). The expression levels of miR-200c and P-gp were associated with the malignant characteristics of gastric cancer, and patients with high expression of miR-200c or negative expression of P-gp had a better prognosis (P=0.006, P=0.022). MiR-200c negatively regulated the ABCB1 gene in gastric cancer cell lines. MiR-200c overexpression and ABCB1 down-regulation increased the sensitivity of SGC7901/VCR cells to VCR and reversed MDR by promoting cell apoptosis. The expression level of miR-200c decreases in gastric carcinoma tissues and drug-resistant gastric cancer SGC7901/VCR cells. Overexpression of miR-200c may enhance the sensitivity of SGC7901/VCR cells to VCR by regulating the expression of P-gp.

Swietephragmin D, a limonoid from Swietenia mahagoni, reverses multidrug resistance by inhibiting P-glycoprotein dependent efflux

Background: P-glycoprotein mediated efflux leads to multidrug resistance of tumor cells, which is an important reason for chemotherapy failure. Thus inhibiting P-gp is an effective approach to reverse multidrug resistance. Purpose: Swietephragmin D is a limonoid compound isolated from the fruits and leaves of Swietenia mahagoni (L.) Jacq.. The reversal efficiency of swietephragmin D was examined in this study. Methods: Rhodamine 123 efflux assays were used to measure inhibition efficiency of swietephragmin D. Cytotoxicity assays were used to measure the reversal effect of swietephragmin D. Real-time PCR and ATPase assays were performed to explore the mechanisms of reversion. Results: Swietephragmin D inhibited P-glycoprotein-induced efflux in a dose and time dependent manner. This compound inhibited P-glycoprotein more effectively than verapamil. The IC50 values of the multidrug resistant cells were reduces by swietephragmin D significantly. The transcription levels of ABCB1 (encoded P-glycoprotein) were not affected by swietephragmin D. However, the ATPase activity of P-glycoprotein was stimulated by swietephragmin D. Conclusion: Swietephragmin D was a substrate of P-glycoprotein and inhibited P-gp mediated efflux. It did not interfere with expression of ABCB1. Thus swietephragmin D reversed multidrug resistance as a competitive inhibitor of P-glycoprotein.