Euphorbia factor L1 reverses ABCB1-mediated multidrug resistance involving interaction with ABCB1 independent of ABCB1 downregualtion

Euphorbia factor L1 (EFL1) belongs to diterpenoids of genus Euphorbia. In this article, its reversal activity against ABCB1-mediated MDR in KBv200 and MCF-7/adr cells was reported. However, EFL1 did not alter the sensitivity of KB and MCF-7 cells to chemotherapeutic agents. Meanwhile, EFL1 significantly increased accumulation of doxorubicin and rhodamine 123 in KBv200 and MCF-7/adr cells, showing no significant influence on that of KB and MCF-7 cells. Furthermore, EFL1 could enhance the ATP hydrolysis activity of ABCB1 stimulated by verapamil. At the same time, EFL1 inhibited the efflux of ABCB1 in KBv200 and MCF-7/adr cells. In addition, EFL1 did not downregulate expression of ABCB1 in KBv200 and MCF-7/adr cells either in mRNA or protein level.     

Two different docetaxel resistant MCF-7 sublines exhibited different gene expression pattern

The objective of the present study was to investigate gene expression pattern of two docetaxel resistant MCF-7 breast carcinoma sublines step wisely selected in 30 and 120 nM docetaxel. Cell proliferation assay was performed in order to demonstrate development of docetaxel resistance. cDNA microarray analysis was performed using Affymetrix^® Human Genome U133 Plus 2.0 Arrays in duplicate experiments. Quantitative and semi-quantitative gene expression analysis was also performed to confirm gene expression analysis for selected genes. XTT results demonstrated that 30 (MCF-7/30nM DOC) and 120 nM (MCF-7/120nM DOC) docetaxel selected cells were 13- and 47-fold resistant, respectively. cDNA microarray analysis demonstrated that expression profiles of MCF-7 and MCF-7/30nM DOC were more similar to each other where expression profile of MCF-7/120nM DOC was different as examined by line graphs and scatter plots. 2,837 and 4,036 genes were significantly altered in 30 and 120 nM docetaxel resistant sublines, respectively. Among these, 849 genes were altered in common in two docetaxel resistant sublines. Antiapoptotic gene expression (e.g., Bcl-2 and APRIL) were noticeably altered in MCF-7/30nM DOC. However, docetaxel resistance in MCF-7/120nM DOC were more complicated with the involvement of ECM related gene expression, cytokine and growth factor signaling, ROS metabolism and EMT related gene expression together with higher level of MDR1 expression. Expression profiles in 30 and 120 nM docetaxel resistant sublines changed gradually with increasing resistance index. Drug resistance development seems to be step wise event in MCF-7 cells.     

Androgen receptor transcriptional activity and chromatin modifications on the ABCB1/MDR gene are critical for taxol resistance in ovarian cancer cells

We report here that the androgen receptor (AR) and ABCB1 are upregulated in a model of acquired taxol resistance (txr) in ovarian carcinoma cells. AR silencing sensitizes txr cells to taxol threefold, whereas ectopic AR expression in AR-null HEK293 cells induces resistance to taxol by 1.7-fold. AR activation using the agonist dihydrotestosterone (DHT) or sublethal taxol treatment upregulates ABCB1 expression in both txr cells and AR-expressing HEK293 cells. In contrast, AR inactivation using the antagonist bicalutamide downregulates ABCB1 expression and enhances cytotoxicity to taxol. A functional ABCB1 promoter containing five predicted androgen-response elements (AREs) is cloned. Deletion assays reveal a taxol-responsive promoter segment which harbors ARE4. Notably, DHT- or taxol-activated AR potentiates binding of the AR to ARE4 as revealed by the chromatin immunoprecipitation. On the other hand, txr cells display an increase in chromatin remodeling. AR/H3K9ac and AR/H3K14ac complexes bind specifically to ARE4 in response to taxol. Furthermore, acetyltransferase protein levels (p300 and GCN5) are upregulated in txr cells. Silencing of p300 or GCN5 reduces chromatin modification and enhances cytotoxicity in both parental and txr SKOV3 cells. While the phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase (AKT) pathway is significantly activated by taxol, taxol-induced ABCB1 expression, histone posttranslational modifications, and p300 binding to ARE4 are suppressed following inhibition of the PI3K/AKT cellular pathway. These results demonstrate that the AKT/p300/AR axis can be activated to target ABCB1 gene expression in response to taxol, thus revealing a new treatment target to counter taxol resistance.     

Tepoxalin increases chemotherapy efficacy in drug-resistant breast cancer cells overexpressing the multidrug transporter gene ABCB1

Effective cancer chemotherapy treatment requires both therapy delivery and retention by malignant cells. Cancer cell overexpression of the multidrug transmembrane transporter gene ABCB1 (MDR1, multi-drug resistance protein 1) thwarts therapy retention, leading to a drug-resistant phenotype. We explored the phenotypic impact of ABCB1 overexpression in normal human mammary epithelial cells (HMECs) via acute adenoviral delivery and in breast cancer cell lines with stable integration of inducible ABCB1 expression. One hundred sixty-two genes were differentially expressed between ABCB1-expressing and GFP-expressing HMECs, including the gene encoding the cyclooxygenase-2 protein, PTGS2. Several breast cancer cell lines with inducible ABCB1 expression demonstrated sensitivity to the 5-lipoxygenase, cyclooxygenase-1/2 inhibitor tepoxalin in two-dimensional drug response assays, and combination treatment of tepoxalin either with chemotherapies or with histone deacetylase (HDAC) inhibitors improved therapeutic efficacy in these lines. Moreover, selection for the ABCB1-expressing cell population was reduced in three-dimensional co-cultures of ABCB1-expressing and GFP-expressing cells when chemotherapy was given in combination with tepoxalin. Further study is warranted to ascertain the clinical potential of tepoxalin, an FDA-approved therapeutic for use in domesticated mammals, to restore chemosensitivity and improve drug response in patients with ABCB1-overexpressing drug-resistant breast cancers.     

The c‐MYC‐ABCB5 axis plays a pivotal role in 5‐fluorouracil resistance in human colon cancer cells

c‐MYC overexpression is frequently observed in various cancers including colon cancer and regulates many biological activities such as aberrant cell proliferation, apoptosis, genomic instability, immortalization and drug resistance. However, the mechanism by which c‐MYC confers drug resistance remains to be fully elucidated. In this study, we found that the c‐MYC expression level in primary colorectal cancer tissues correlated with the recurrence rate following 5‐fluorouracil (5‐FU)‐based adjuvant chemotherapy. Supporting this finding, overexpression of exogenous c‐MYC increased the survival rate following 5‐FU treatment in human colon cancer cells, and knockdown of endogenous c‐MYC decreased it. Furthermore, c‐MYC knockdown decreased the expression level of ABCB5, which is involved in 5‐FU resistance. Using a chromatin immunoprecipitation assay, we found that c‐MYC bound to the ABCB5 promoter region. c‐MYC inhibitor (10058‐F4) treatment inhibited c‐MYC binding to the ABCB5 promoter, leading to a decrease in ABCB5 expression level. ABCB5 knockdown decreased the survival rate following 5‐FU treatment as expected, and the ABCB5 expression level was increased in 5‐FU‐resistant human colon cancer cells. Finally, using a human colon cancer xenograft murine model, we found that the combined 5‐FU and 10058‐F4 treatment significantly decreased tumorigenicity in nude mice compared with 5‐FU or 10058‐F4 treatment alone. 10058‐F4 treatment decreased the ABCB5 expression level in the presence or absence of 5‐FU. In contrast, 5‐FU treatment alone increased the ABCB5 expression level. Taken together, these results suggest that c‐MYC confers resistance to 5‐FU through regulating ABCB5 expression in human colon cancer cells.     

Immediate early gene X-1 (IEX-1), a hydroxytamoxifen regulated gene with increased stimulation in MCF-7 derived resistant breast cancer cells

The efficacy of tamoxifen in breast cancer treatment only lasts a few years and the tumor eventually recurs. We performed selective subtractive hybridization to isolate mRNAs that were differentially expressed in MCF-7 derived cells, in which resistance had been induced through long-term culture in the presence of hydroxytamoxifen (OHT). Among the 15 mRNAs found to be overexpressed, we focused on Immediate early gene X-1 (IEX-1) mRNA because of the recognized contribution of its expression to apoptosis or cell cycle progression, depending on the cell type and culture conditions. We observed that IEX-1 expression was stimulated by OHT, that the degree of increase was greater in resistant cells (four-fold versus 1.5-fold) and that this OHT regulation was estrogen receptor dependent. A detailed study of the IEX-1 promoter indicated that it involved NF-kappaB. Our cells were not cross-resistant to faslodex, a pure antiestrogen, which moreover was inefficient in regulating IEX-1 expression. Altogether, our data suggest that the greater IEX-1 expression in OHT resistant cells is related to their ability to grow in the presence of OHT. Knowledge on the capacity of OHT to stimulate gene expression and its NF-kappaB dependence should contribute to a better understanding of tamoxifen pharmacology and allow new drug strategies to be designed that would delay antiestrogen resistance acquisition.     

Modulation of function of three ABC drug transporters, P-glycoprotein (ABCB1), mitoxantrone resistance protein (ABCG2) and multidrug resistance protein 1 (ABCC1) by tetrahydrocurcumin, a major metabolite of curcumin

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [³H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [³H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [¹²⁵I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [¹²⁵I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-gp, MRP1, and MXR were determined by MTT assay. THC significantly increased the sensitivity of vinblastine, mitoxantrone and etoposide in drug resistance KB-V-1, MCF7AdrVp3000 and MRP1-HEK 293 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Taken together, this study clearly showed that THC inhibits the efflux function of P-gp, MXR and MRP1 and it is able to extend the MDR reversing activity of curcuminoids in vivo.     

Long Noncoding RNA MRUL Promotes ABCB1 Expression in Multidrug-Resistant Gastric Cancer Cell Sublines

Multidrug resistance (MDR) is the most common cause of chemotherapy failure in gastric cancer (GC) treatment; however, the underlying molecular mechanisms remain elusive. Long noncoding RNAs (lncRNAs) can be involved in carcinogenesis, but the effects of lncRNAs on MDR are poorly understood. We show here that the lncRNA MRUL (MDR-related and upregulated lncRNA), located 400 kb downstream of ABCB1 (ATP-binding cassette, subfamily B, member 1), was significantly upregulated in two multidrug-resistant GC cell sublines, SGC7901/ADR and SGC7901/VCR. Furthermore, the relative expression levels of MRUL in GC tissues were negatively correlated with in vitro growth inhibition rates of GC specimens treated with chemotherapeutic drugs and indicated a poor prognosis for GC patients. MRUL knockdown in SGC7901/ADR and SGC7901/VCR cells led to increased rates of apoptosis, increased accumulation, and reduced doxorubicin (Adriamycin [ADR]) release in the presence of ADR or vincristine. Moreover, MRUL depletion reduced ABCB1 mRNA levels in a dose- and time-dependent manner. Heterologous luciferase reporter assays demonstrated that MRUL might positively affect ABCB1 expression in an orientation- and position-independent manner. Our findings indicate that MRUL promotes ABCB1 expression and is a potential target to reverse the MDR phenotype of GC MDR cell sublines.     

Estimation of ABCB1 concentration in plasma membrane

The interaction between ABCB1 transporter and its substrates takes place in cell membranes but the available data precludes quantitative analysis of the interaction between transporter and substrate molecules. Further, the amount of transporter is usually expressed as a number of ABCB1 molecules per cell. In contrast, the substrate concentration in cell membranes is estimated by determination of substrate-lipid partition coefficient, as examples. In this study, we demonstrate an approach, which enables us to estimate the concentration of ABCB1 molecules within plasma membranes. For this purpose, human leukemia K562 cells with varying expression levels of ABCB1 were used: drug selected K562/Dox and K562/HHT cells with very high transporter expression, and K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with gradually decreased expression of ABCB1 derived from K562/Dox cells using RNA interference technology. First, we determined the absolute amount of ABCB1 in cell lysates using immunoblotting and recombinant ABCB1 as a standard. We then determined the relative portion of transporter residing in the plasma membrane using immunohistochemistry in nonpermeabilized and permeabilized cells. These results enabled us to estimate the concentration of ABCB1 in the plasma membrane in resistant cells. The ABCB1 concentrations in the plasma membrane of drug selected K562/Dox and K562/HHT cells containing the highest amount of transporter reached millimolar levels. Concentrations of ABCB1 in the plasma membrane of resistant K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with lower transporter expression were proportionally decreased.