Uptake of Compounds That Selectively Kill Multidrug-Resistant Cells: The Copper Transporter <i>SLC31A1</i> (CTR1) Increases Cellular Accumulation of the Thiosemicarbazone NSC73306

Acquired drug resistance in cancer continues to be a challenge in cancer therapy, in part due to overexpression of the drug efflux transporter P-glycoprotein (P-gp, MDR1, <i>ABCB1</i>). NSC73306 is a thiosemicarbazone compound that displays greater toxicity against cells expressing functional P-gp than against other cells. Here, we investigate the cellular uptake of NSC73306, and examine its interaction with P-gp and copper transporter 1 (CTR1, <i>SLC31A1</i>). Overexpression of P-gp sensitizes LLC-PK1 cells to NSC73306. Cisplatin (IC₅₀ = 77 μM), cyclosporin A (IC₅₀ = 500 μM), and verapamil (IC₅₀ = 700 μM) inhibited cellular accumulation of [³H]­NSC73306. Cellular hypertoxicity of NSC73306 to P-gp-expressing cells was inhibited by cisplatin in a dose-dependent manner. Cells transiently expressing the cisplatin uptake transporter CTR1 (<i>SLC31A1</i>) showed increased [³H]­NSC73306 accumulation. In contrast, CTR1 knockdown decreased [³H]­NSC73306 accumulation. The presence of NSC73306 reduced CTR1 levels, similar to the negative feedback of CTR1 levels by copper or cisplatin. Surprisingly, although cisplatin is a substrate of CTR1, we found that CTR1 protein was overexpressed in high-level cisplatin-resistant KB-CP20 and BEL7404-CP20 cell lines. We confirmed that the CTR1 protein was functional, as uptake of NSC73306 was increased in KB-CP20 cells compared to their drug-sensitive parental cells, and downregulation of CTR1 in KB-CP20 cells reduced [³H]­NSC73306 accumulation. These results suggest that NSC73306 is a transport substrate of CTR1

The Transcription Factor GCF2 Is an Upstream Repressor of the Small GTPAse RhoA, Regulating Membrane Protein Trafficking, Sensitivity to Doxorubicin, and Resistance to Cisplatin

Our aim was to explore the involvement of the transcriptional suppressor GCF2 in silencing RhoA, disorganization of the cytoskeleton, mislocalization of MRP1, and sensitivity to anticancer agents as an upstream gene target in cancer therapy. Increased expression of GCF2 was found in human cisplatin-resistant cells, and overexpression in GCF2-transfected cells results in loss of RhoA expression and disruption of the actin/filamin network. In consequence, the membrane transporter MRP1 was internalized from the cell surface into the cytoplasm, rendering cells sensitive to doxorubicin by more than 10-fold due to increased accumulation of doxorubicin in the cells. The GCF2 transfectants also showed reduced accumulation of cisplatin and increased resistance. siRNA targeted to GCF2 suppressed the expression of GCF2 in cisplatin-resistant cells, reactivated RhoA expression, and restored the fine structure of actin microfilaments. MRP1 was also relocated to the cell surface. siRNA targeted to RhoA increased resistance 3-fold in KB-3-1 and KB-CP.5 cells. These data for the first time demonstrate a novel complex regulatory pathway downstream from GCF2 involving the small GTPase RhoA, actin/filamin dynamics, and membrane protein trafficking. This pathway mediates diverse responses to cytotoxic compounds, and also provides a molecular basis for further investigation into the pleiotropic resistance mechanism at play in cisplatin-resistant cells

Clinical Relevance of Multidrug Resistance Gene Expression in Ovarian Serous Carcinoma Effusions

The presence of tumor cells in effusions within serosal cavities is a clinical manifestation of advanced-stage cancer and is generally associated with poor survival. Identifying molecular targets may help to design efficient treatments to eradicate these aggressive cancer cells and improve patient survival. Using a state-of-the-art TaqMan-based qRT-PCR assay, we investigated the multidrug resistance (MDR) transcriptome of 32 unpaired ovarian serous carcinoma effusion samples obtained at diagnosis or at disease recurrence following chemotherapy. MDR genes were selected a priori based on an extensive curation of the literature published during the last three decades. We found three gene signatures with a statistically significant correlation with overall survival (OS), response to treatment [complete response (CR) vs other], and progression free survival (PFS). The median log-rank <i>p</i>-values for the signatures were 0.023, 0.034, and 0.008, respectively. No correlation was found with residual tumor status after cytoreductive surgery, treatment (with or without chemotherapy) and stage defined according to the International Federation of Gynecology and Obstetrics. Further analyses demonstrated that gene expression alone can effectively predict the survival outcome of women with ovarian serous carcinoma (OS, log-rank <i>p</i> = 0.0000; and PFS, log-rank <i>p</i> = 0.002). Interestingly, the signature for overall survival is the same in patients at first presentation and those who had chemotherapy and relapsed. This pilot study highlights two new gene signatures that may help in optimizing the treatment for ovarian carcinoma patients with effusions

Human–Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P‑Glycoprotein (ABCB1)

The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter’s broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human–mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [¹²⁵I]­iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes

Human–Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P‑Glycoprotein (ABCB1)

The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter’s broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human–mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [¹²⁵I]­iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes

Identification of a Cryptic Bacterial Promoter in Mouse (<i>mdr1a</i>) P-Glycoprotein cDNA

<div><p>The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse <i>mdr1a</i> cDNA display significant genetic instability during cloning in bacteria, indicating that <i>mdr1a</i> cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse <i>mdr1a</i> cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of <i>mdr1a</i> DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of <i>mdr1a</i> cDNA capable of initiating bacterial protein expression. An <i>mdr1a</i> M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of <i>mdr1a</i> in <i>E</i>. <i>coli</i> while retaining transport properties similar to wild-type P-gp. This mutant <i>mdr1a</i> cDNA may prove useful for efficient cloning of <i>mdr1a</i> in <i>E</i>. <i>coli</i>.</p></div

Identification of putative transcriptional and translational start sites in <i>mdr1a</i> cDNA.

<p>An <i>in silico</i> sigma 70 binding site analysis was used to screen for a putative cryptic bacterial promoter in <i>mdr1a</i> cDNA. (A) Sequence of <i>mdr1a</i> cDNA shown with predicted -35 and extended -10 elements (red). The location in the cDNA for each element is noted above the sequence. (B) Predicted Shine-Dalgarno and ATG of a methionine that is in-frame with regards to the <i>mdr1a</i> ORF.</p

Mutations and their position in <i>mdr1a</i> cDNA after transformation into <i>E</i>. <i>coli</i>.

<p>The pCR-Blunt-II-TOPO vector containing <i>mdr1a</i> cDNA was used to transform <i>E</i>.<i>coli</i> and 20 colonies were selected for small-scale bacterial growth, plasmid purification, and analysis of plasmid DNA by agarose gel electrophoresis. Sixteen of the colonies selected for analysis contained large insertion or deletion mutations (not shown), and the remaining four colonies containing potentially correct <i>mdr1a</i> cDNA were sequenced. Each of the four sequenced plasmids contained mutated <i>mdr1a</i> cDNA, indicating a 100% mutational rate. Mutations observed were classified as point, insertion, or deletion mutations. Wild-type <i>mdr1a</i> cDNA (top) is compared to sequenced, “mutant” <i>mdr1a</i> cDNA (bottom). Insertion and point mutations are indicated in red in the mutant cDNA. Deleted base pairs are highlighted in red in wild-type cDNA, and the resulting mutated cDNA sequence is shown below. A) A point mutation (C→T) at location 1027 bp resulting in an introduced stop codon into <i>mdr1a</i> cDNA. B) A single adenine base pair insertion at location 1033 bp. C) Two examples of deletion mutations. All mutations resulted either directly (point mutations) or indirectly (insertion or deletion mutations) in the introduction of a stop codon into <i>mdr1a</i> cDNA. Locations of the introduced stop codons are indicated.</p

Mouse P-gp model in apo (open) conformation with methionine residue at position 107 highlighted.

<p>(A) Mouse P-gp is shown with the methionine to leucine mutation highlighted in TM1-ECL1 by the presence of a space filled leucine residue. The region of the drug-binding pocket (DBP) is indicated. (B) Enlarged view of M107L mutation. Figures were generated using PyMOL from PDB deposit 4M1M [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136396#pone.0136396.ref016" target="_blank">16</a>].</p

Fluorescence of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids.

<p>(A) Confocal images of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids. From left to right, columns represent fluorescence, bright field, and a merged image. Yellow bars indicate 20 μm. (B) Fluorescence of <i>E</i>. <i>coli</i> transformed with GFP fusion plasmids. Fluorescence spectroscopy was used to determine levels of GFP fluorescence in bacteria transformed with GFP plasmids. Confocal images and fluorescence spectroscopy values for bacteria transformed with λpR-GFP plasmids were omitted due to the signal saturation resulting from high GFP fluorescence. Data represent the mean and standard deviation of five individual colonies from one transformation event. Multiple groups were analyzed using one-way ANOVA where significance was defined as p < 0.05 in GraphPad Prism.</p