Effects of iRF on B16F10 cell viability.

<p>(A) B16F10 and HaCaT cells were treated with different concentrations of RF and (B) iRF for 24 h, and cell viability was assessed by MTT reduction assay. (C) B16F10 were treated with iRF for 12, 24 and 48 h. (D) Cell proliferation evaluation by BrdU assay of B16F10 treated with iRF and (E) representative pictures of colony assay, and the number of colonies. B16F10 cells were treated for 2 and 10 days. The extent of MTT reduction and colony assay content only medium with SFB was considered as 100%. The results represent the means ± SD (n = 9). p<0.01, p<0.001 versus control.</p

Schematic representation of the molecular mechanism by which iRF decreases the aggressiveness of B16F10 cells.

<p>iRF decreases the capacity of cell migration through inhibition of MMP-9 and MMP-2 activities as well as expression. Furthermore, the expression of a physiological inhibitor of MMPs, TIMP-1, was augmented by iRF. In addition, cell survival mediator, mTOR, was less active as well as Src kinase, an important key player of cell invasion. As we have shown previously in other cancer cell lines, iRF also induced B16F10 cells death by apoptosis since the expression of anti-apoptotic protein Bcl-2 is reduced. Bcl-2 acts by inhibiting Bax, a pro-apoptotic protein responsible to cytochrome-C release, and activation of caspases, leading cells to apoptosis. Furthermore, the expression of cIAP, a caspase 3, was diminished. HH is another pathway that seems to be important for iRF antimelanoma action. iRF inhibits HH pathway by downregulating GLI-1 and PATCH expression and overexpression of SUFU. GLI-1 can be sequestered by SUFU forming a complex with kif7 (Cos homologue in mice), thus keeping GLI-1 in the cytoplasm and preventing translocation to the nucleus, making it unable to activate the transcriptional program of the HH pathway.</p

The effect of iRF on melanoma cell migration.

<p>(A) Scratch assay of B16F10 cells exposed to 1 and 10 µM of iRF. The images were acquired at 0 and 16 hours. The ratio of wound area at 16 h and at 0 h represent the inhibition of migration. (B) Representative pictures of scratch assay of each group after 0 and 16 h. (C) B16F10 cells were incubated with CellTracker green and then treated with different concentrations of iRF in serum free DMEM. Fluorescence was measured every 2 min for 4 h. (D) Migration capacity of B16F10 cells through the 2H11 endothelial cells monolayer was analyzed by transmigration assay using Fluoroblock™ inserts at 3 and 18 h. The results represent the means ± SD (n = 9). p<0.05, p<0.01, p<0.001 versus control.</p

Effect of flavins on the number of B16F10 pulmonary tumor foci.

<p>After administration of 3×10⁵ B16F10 melanoma cells into the lateral tail vein,C57Bl/6 mice were treated intravenously with riboflavin or irradiated riboflavin 0.1 and 0.2 mg/kg, 6 times within 2 weeks. Mice were sacrificed 14 days after cancer cell injection and the number of tumor foci at the surface of the lungs was determined. The results are expressed as the mean ± SD (n = 8); * p<0.05, ** p<0.01 versus control.</p

Effect of irradiated riboflavin on mTOR and Src activities and expression of antiapoptotic proteins Bcl-2 and cIAP.

<p>Analysis of the expression of p-mTOR (Ser 2448), mTOR, p70S6K and p-p70S6K (Thr 389) (A) and Bcl-2 and cIAP (B) in B16F10 treated with different concentrations of iRF for 24 h. The results are shown as mean ± SD of three independent experiments. p<0.001 versus control.</p

The effect of irradiated riboflavin on activity and expression of MMPs e TIMP-1.

<p>(A) B16F10 cells were treated with different concentrations of iRF for 24 h. Equal amounts of protein from each sample were applied to gel zymography. The activity of MMP-9 and MMP-2 was measured by densitometry of the gelatin zymography. (B) Western blotting analysis of the expression of MMP-9, MMP-2 and TIMP-1 in B16F10 treated with different concentrations of iRF for 24 h. The results are shown as mean ± SD of three independent experiments. p<0.01, p<0.001 versus control.</p

Evaluation of P-glycoprotein activity.

<p>To measure P-gp activity the substrate Rhodamine 123 was used as substrate. <i>LMW-PTP</i> was silenced and after 24 h cells were further incubated for 1 h in the presence of 200 ng/mL Rhodamine 123 with or without a P-gp inhibitor (5 µM verapamil). Afterwards, cells were washed with PBS and Rhodamine 123 mean fluorescence intensity was assessed by flow cytometry analysis within the live-gate, using a FACScalibur (Becton and Dickinson, USA). Decreasing of mean fluorescence intensity indicates higher P-gp activity.</p

Knocking down <i>LMW-PTP</i> decreased phosphorylation of Src and ABL.

<p>(<b>A</b>) Phosphorylation status of Src kinase and ABL in leukemia cells with silenced <i>LMW-PTP</i>. (<b>B</b>) The ratio between p-Brc-Abl(210 kDa)/GAPDH and p-c-Abl (135 kDa)/GAPDH, from K562 and Lucena-1 cells with silenced <i>LMW-PTP</i> (<i>LMW-PTP</i> siRNA) or not (scramble siRNA) were quantified by densitometry and plot in the graph. *** p<0.001- significant differences relative to the respective scramble siRNA.</p

Expression of LMW-PTP and modulation of c-Src and FAK in leukemia cells treated with vincristine.

<p>Cells were treated with VCR for 24 h and LMW-PTP expression, phosphorylation status of c-Src and FAK were analyzed. (A) K562 and Lucena-1 cells were treated with VCR for 24 h and the protein levels were analyzed by Western blotting. (B) The ratio between LMW-PTP/GAPDH, Src/GAPDH, FAK/GAPDH, p-Src/Src, p-FAK/FAK from VCR treatment samples were quantified by densitometry and plot in the graph.</p

<i>LMW-PTP</i> silencing increases the cytotoxicity effect of chemotherapeutics on Lucena-1 cells.

<p>(<b>A</b>) LWM-PTP was knocked down in Lucena-1 cells and its expression was checked by Western (<b>B</b>) After 48 h of <i>LMW-PTP</i> silencing Lucena-1 cells (100.000 cells/ml) were treated with VCR 500 nM or imatinib 500 nM for 24 h, subsequently, the viable cells were assessed by trypan blue exclusion and (<b>C</b>) apoptosis induction by caspase 3 activity. Each value represents the mean ± S.E.M. of three independent experiments (n = 3) and has been normalized to scramble siRNA sample. *** p<0.001 - significant differences relative to its respective scramble siRNA sample.</p