NSC23925 increases Pgp1 substrates accumulation in SKOV-3_TR.

<p>Images of SKOV-3_TR cells incubated with various fluorescent substrates of Pgp1 in the presence (bottom panel) and absence (top panel) of NSC23925. For visualization of effects of NSC23925 on the intracellular retention of rhodamine 123, mitoxantrone, Dioc2, doxorobucin, and calcein AM, 10,000 resistant cells were seeded on to Lab-Tek 8-well chamber slides on the day prior to the assay. SKOV-3_TR cells were then incubated with either 1 µM rhodamine 123, 10 µM mitoxantrone, 0.1 µM Dioc2, 10 µM doxorubicin or 0.25 µM calcein AM either alone or in the presence of NSC23925 in RPMI 1640 media for one hour at 37°C. Image were acquired a Nikon Eclipse Ti-U fluorescence microscope (Nikon Corp.) equipped with a SPOT RT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI).</p

Stimulation of Pgp1 ATPase activity by NSC23925 as compared with verapamil.

<p>Untreated, 100 µM Na₃VO₄, 100 µM verapamil and 10 µM NSC23925 -treated Pgp1 reactions were performed according to the manufacturer's protocol. Luminescence was read on a BMG LABTECH Polarstar Optima Luminometer. The decrease in luminescence of untreated samples compared to samples plus Na₃VO₄ (ARLU_basal) represents basal Pgp1 ATPase activity. The decrease in luminescence of verapamil or NSC23925 treated samples represent Pgp1 ATPase activity. <i>A</i>: Decrease in luminescence of NSC23925 treated sample as compared with Na₃VO₄ or Verapamil treated sample. <i>B</i>: Replotted the stimulation of Pgp1 ATPase activity by verapamil and NSC23925. <i>C</i> and <i>D</i>: Dose dependence of verapamil (<i>C</i>) and NSC23925 (<i>D</i>) stimulation of Pgp1 ATPase activity. The data were representative of one of three independent experiments. *, P<0.001. <i>E</i>: Effect of NSC23925 on expression of Pgp1 in paclitaxel resistant cells. The paclitaxel resistant cell line SKOV-3_TR were treated with different concentration of NSC23925 for 48 h. Equal amounts (20 µg protein) of total cell lysates were used for each sample. Pgp1 expression was determined by Western blot as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007415#s2" target="_blank">Materials and Methods</a>.</p

Reverse Drug Resistance by NSC23925

<p>Reverse Drug Resistance by NSC23925</p

Nonspecific toxicity of NSC23925 on SKOV3 vs SKOV-3_TR and OVCAR8 vs OVCAR8_TR.

<p>Cells were treated with the NSC23925 in RPMI1640 complete media at the indicated concentrations. The relative sensitivity of each line to NSC23925 was determined by MTT assay. <i>A</i>: Nonspecific toxicity of NSC23925 on SKOV3 vs SKOV-3_TR and OVCAR8 vs OVCAR8_TR. <i>B</i>: Nonspecific toxicity of NSC23925 on OVCAR8 vs OVCAR8_TR.</p

Intracellular retention of doxorubicin from multidrug resistant SKOV-3_TR and KHOSR2 cells in the presence of 5 µM doxorubicin with or without treatment of NSC23925 (0.5 µM).

<p>Cells were treated for 1 hour before the flow cytometry analysis.</p

Effect of NSC23925 Reversing Drug Resistance in Multidrug Resistant Cell Lines.

<p>IC₅₀ is the concentration of drug (µM) that produced 50% inhibition of cell growth.</p><p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007415#s3" target="_blank">Results</a> were calculated from one experiment with triplicate wells. ± reflect SDEV (SD).</p><p>Number in the parentheses represent fold-reversal of drug resistance.</p

Effect of NSC23925 to reverse drug resistance in MDR cell lines.

<p>Cells were treated with the paclitaxel and NSC23925 in RPMI1640 complete media at the indicated concentrations. The relative sensitivity of each line to paclitaxe was determined by MTT analysis 6 days posttreatment. <i>A</i>: Reversal of paclitaxel resistance by NSC23925 in SKOV-3_TR cells. <i>B</i>: Reversal of paclitaxel resistance by NSC23925 in OVCAR8_TR cells.</p

Chemical structure of NSC23925 and reverse paclitaxel resistance by NSC23925.

<p><i>A</i>: Structure of NSC23925. <i>B</i>: NSC23925 in combination with 0.1 µM paclitaxel induces significantly cell death in SKOV-3_TR.</p

Effect of verapamil on NSC23925 sensitivity in Pgp1-overexpressing cells and persistence of NSC23925 activity.

<p><i>A</i>: Effect of verapamil on NSC23925 sensitivity in SKOV-3_TR cells. SKOV-3_TR cells were treated with different concentration of NSC23925 alone or in combination with verapamil for 72 hours. Drug cytotoxicity was determined by MTT assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007415#s2" target="_blank">Materials and Methods</a>. <i>B</i>: Persistence of NSC23925 reverses paclitaxel resistance in SKOV-3_TR cells after incubation and washout of NSC23925 or verapamil.</p

Calcein AM efflux from treated SKOV-3 and SKOV-3_TR cells.

<p>The Calcein-AM assay was optimized and performed using the Vybrant Multidrug Resistance kit and SKOV-3_TR cells. Cells were seeded at 50,000 cells/well (100 µl of culture medium) in 96-well plate and incubated for 24 h. SKOV-3_TR cells in triplicate were treated with NSC23925, verapamil for one hour and then incubated in calcein AM for 30 min. The cell fluorescence images were acquired by a fluorescence microscope (<i>A</i>, <i>C</i>) and quantities of fluorescence were measured in a SPECTRAmax Microplate Spectrofluorometer (<i>B, D</i>). The data were representative of one of three independent experiments. *, P<0.001.</p