Figure 1.
Chemical structure of purine nucleoside analog – sulfinosine (SF).
Figure 2.
SF inhibits the cell growth and suppresses the cell proliferation.
The growth inhibitory effects of SF on NCI-H460 and NCI-H460/R (A), U87, U87-TxR and HaCaT (C) cells grown on plastic after 72 h treatment were assessed by SRB assay. Average ± S.D. values were calculated from five independent experiments (n = 5). NCI-H460 and NCI-H460/R (B), U87 and U87-TxR (D) cells were stained with CFSE and incubated for 72 h with 10 µM SF. The rate of proliferation (CFSE declination) was determined by flow-cytometry on channel FL1. Light microscopy of NCI-H460 and NCI-H460/R (E), U87 and U87-TxR (F) cell growth on plastic (2-D culture) and matrigel growth (3-D culture) after 72 h of 10 µM SF treatment.
Figure 3.
SF induces caspase activation, apoptosis and autophagy in MDR cancer cell lines.
Cell death analysis of NCI-H460/R (A) and U87-TxR (C) cells untreated and treated with 5 µM SF (B, D) for 72 h. The samples were analyzed for green fuorescence (Annexin-V-FITC) and red fluorescence (Propidium Iodide) by flow-cytometry. The assay distinguishes viable cells (AV− PI−), apoptotic cells (AV+ PI−), late apoptotic and necrotic cells (AV+ PI+) and secondary necrotic or dead cells (AV− PI+). Pancaspase activation in NCI-H460/R (E) and U87-TxR (F) cells untreated and treated with 10 µM SF was assessed after 72 h by flow-cytometry. The cells were labelled with a cell-permeable, FITC-conjugated pan-caspases inhibitor (ApoStat). Western blot analysis of procaspase-3, p17 cleaved caspase-3 and β-actin in NCI-H460/R (G) and U87-TxR (H) cells. (I) The autophagy in NCI-H460/R and U87-TxR cell lines assessed after 144 h incubation period (72 h of SF treatment followed by next 72 h of cell recovery in fresh medium). Fold of increase in red vs. green (FL3/FL1) fluorescence ratio after acridine orange-staining was determined using a FacsCalibur flow cytometer and Cell Quest Pro software. The statistical significance between the controls and treatments is presented as p<0.01 (**) and p<0.001 (***).
Figure 4.
SF increases ROS concentration and inhibits GSH detoxification in MDR cancer cell lines.
Flow-cytometric measurements of dihydroethidium (DHE)-fluorescence correspond to cellular ROS levels (A). Mean fluorescence intensity (MFI) calculated after correction for autofluorescence is presented. The data indicate the average ± S.D. of three experiments carried out in triplicate. Statistical significance of SF treatment compared to untreated control: p<0.01 (**) and p<0.001 (***). Detection of GSH concentration (B) for each sample was performed at least six times. The data indicate the average ± S.D. Statistical significance of SF treatment compared to untreated control: p<0.01 (**). The expression of gst-π mRNA in NCI-H460/R (C) and U87-TxR cells (D) was evaluated relative to the internal control - β-actin. The PCR products were separated on agarose gels beside a 100 bp DNA ladder. Statistical significance between treated and untreated cells is presented as p<0.001 (***).The decreases in γGCS expression in NCI-H460/R (E) and U87-TxR cells (F) after SF treatments were calculated relative to β-actin expression and untreated samples. Statistical significance between treated and untreated cells is presented as p<0.01 (**).
Figure 5.
SF decreases the expression of mRNAs involved in tumor progression.
The expression of gst-π, hif-1α, mdr1 and vegf mRNAs in NCI-H460/R (A) and U87-TxR cells (B) after 10 µM SF treatment was evaluated relative to the internal control – gapdh and β-actin, and calculated in relation to the untreated control. Statistical significance between treated and untreated cells is presented as p<0.05 (*) and p<0.01 (**). Relative hif-1α (C), mdr1 (D) and vegf (E) mRNAs expression after 10 µM SF treatment in normoxic (absence of CoCl2) and hypoxic (presence of 50 µM CoCl2) conditions studied in NCI-H460/R cells. Statistical significance between SF treated and SF untreated cells is presented as p<0.05 (*) and p<0.01 (**).Statistical significance between CoCl2 treated and CoCl2 untreated cells is presented as p<0.01 (##) and p<0.001 (###).
Figure 6.
SF inhibits P-gp expression and increases DOX accumulation.
P-gp expression was detected after 24 h, 48 h and 72 h in NCI-H460/R (A) and U87-TxR (B) cells untreated and treated with 5 and 10 µM SF. The percentage of P-gp positive cells (stained with FITC-labeled direct antibody) was determined after elimination of the cells that were stained with FITC-labeled isotypic antibody. The corresponding flow-cytometric histograms illustrate the effect of 10 µM SF after 72 h in NCI-H460/R (C) and U87-TxR (D) cells. DOX accumulation in NCI-H460/R (E) and U87-TxR (F) cells untreated and pretreated with, 5 and 10 µM SF. DOX accumulation was assessed at each time point (24 h, 48 h and 72 h) after 120 min of 20 µM DOX treatment. Flow cytometric profiles after 72 h pretratment compare the effects of 10 µM SF, 50 nM TQ and 10 µM Dex-VER in NCI-H460/R (G) and U87-TxR (H) cells Three independent experiments were performed (a minimum of 10,000 events were collected for each experimental sample). Statistical significance between treated and untreated cells is presented as p<0.05 (*), p<0.01 (**) and p<0.001 (***).
Table 1.
Relative reversion of resistance to DOX in simultaneous and subsequent treatments with SF.
Figure 7.
SF modulates VEGF165 expression and secretion in MDR cancer cell lines.
The amplified NCI-H460/R (A) and U87-TxR samples (B) of the vegf gene (four splicing products of vegf mRNA) were visualized with ethidium-bromide in agarose gel next to a DNA ladder (100 bp). The PCR product of β-actin was co-amplified with vegf. The relative expression of vegf165 was calculated in relation to β-actin expression. Statistical significance between treated and untreated cells is presented as p<0.05 (*) and p<0.001 (***). VEGF expression was detected after 24 h, 48 h and 72 h in NCI-H460/R (C) and U87-TxR (D) cells untreated and treated with 10 µM SF. The mean fluorescence of VEGF (PE-labeled direct antibody) was determined after elimination of the signal obtained with PE-labeled isotypic antibody. The corresponding flow-cytometric histograms illustrate the effect of 10 µM SF after 24 h in NCI-H460/R (E) and 48 h in U87-TxR (F) cells. VEGF secretion levels in culture medium were evaluated after 24 h, 48 h, and 72 h using Human VEGF Immunoassay Kit. NCI-H460/R (G) and U87-TxR cells (H) were treated with 10 µM SF. The data indicate the average ± S.D. of four experiments carried out in triplicate. Statistical significance of SF treatment compared to untreated control when the secretion levels were normalized based on the same amount of cells analyzed in untreated and treated samples, grey bars: p<0.05 (*) and p<0.01 (**). Statistical significance of SF treatment compared to untreated control when the secretion levels of treated cells were not normalized, black bars: p<0.05 (*) and p<0.01 (**).