Irradiated riboflavin diminishes the aggressiveness of melanoma in vitro and in vivo

Abstract

Melanoma is one of the most aggressive skin cancers due to its high capacity to metastasize. Treatment of metastatic melanomas is challenging for clinicians, as most therapeutic agents have failed to demonstrate improved survival. Thus, new candidates with antimetastatic activity are much needed. Riboavin (RF) is a component of the vitamin B complex and a potent photosensitizer. Previously, our group showed that the RF photoproducts (iRF) have potential as an antitumoral agent. Hence, we investigated the capacity of iRF on modulating melanoma B16F10 cells aggressiveness in vitro and in vivo. iRF decreases B16F10 cells survival by inhibiting mTOR as well as Src kinase. Moreover, melanoma cell migration was disrupted after treatment with iRF, mainly by inhibition of metalloproteinase (MMP) activity and expression, and by increasing TIMP expression. Interestingly, we observed that the Hedgehog (HH) pathway was inhibited by iRF. Two mediators of HH signaling, GLI1 and PTCH, were downregulated, while SUFU expression (an inhibitor of this cascade) was enhanced. Furthermore, inhibition of HH pathway signaling by cyclopamine and Gant 61 potentiated the antiproliferative action of RF. Accordingly, when a HH ligand was applied, the effect of iRF was almost completely abrogated. Our findings indicate that Hedgehog pathway is involved on the modulation of melanoma cell aggressiveness by iRF. Moreover, iRF treatment decreased pulmonary tumor formation in a murine experimental metastasis model. Research to clarify the molecular action of flavins, in vivo, is currently in progress. Taken together, the present data provides evidence that riboflavin photoproducts may provide potential candidates for improving the efficiency of melanoma treatment.

Figure 1. Effects of iRF on B16F10 cell viability.

(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.

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

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.

Figure 3. The effect of iRF on melanoma cell migration.

(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.

Figure 4. The effect of irradiated riboflavin on activity and expression of MMPs e TIMP-1.

(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.

Figure 5. The effect of iRF on Hedgehog pathway.

(A) Western blotting analysis of the expression of GLI1, PTCH and SUFU in B16F10 treated with different concentrations of iRF for 24 h. (B) Cell viability (MTT assay) of B16F10 cells with or without pretreatment with 5 µM cyclopamine, 5 µM Gant61 and 0.5 µg/mL SHH for 6 h and following iRF treatment for 24 h. (C) Cell proliferation BrdU assay with or without HH modulator pretreatment followed by iRF treatment and (D) representative pictures of colony assay and the number of colonies of B16F10 cells treated for 10 days. The results are expressed as the mean ± SD and are representative of three independent experiments to western blotting and n = 9 for others experiments. p<0.05, p<0.01, p<0.001 versus control.

Figure 6. Effect of flavins on the number of B16F10 pulmonary tumor foci.

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.

Figure 7. Schematic representation of the molecular mechanism by which iRF decreases the aggressiveness of B16F10 cells.

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.