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Down-Regulation of SOX2 Underlies the Inhibitory Effects of the Triphenylmethane Gentian Violet on Melanoma Cell Self-Renewal and Survival

Open AccessPublished:June 29, 2016DOI:https://doi.org/10.1016/j.jid.2016.06.610
      Human melanomas contain a population of tumor-initiating cells that are able to maintain the growth of the tumor. We previously showed that the embryonic transcription factor SOX2 is essential for self-renewal and tumorigenicity of human melanoma-initiating cells. However, targeting a transcription factor is still challenging. Gentian violet (GV) is a cationic triphenylmethane dye with potent antifungal and antibacterial activity. Recently, a combination therapy of imiquimod and GV has shown an inhibitory effect against melanoma metastases. Whether and how GV affects melanoma cells remains unknown. Here we show that GV represses melanoma stem cell self-renewal through inhibition of SOX2. Mechanistically, GV hinders EGFR activation and inhibits the signal transducer and activator of transcription-3 [(STAT3)/SOX2] axis. Importantly, we show that GV treatment decreases STAT3 phosphorylation at residue tyrosine 705, thus preventing the translocation of STAT3 into the nucleus and its binding to SOX2 promoter. In addition, GV affects melanoma cell growth by promoting mitochondrial apoptosis and G2 cell cycle arrest. This study shows that in melanoma, GV affects both the stem cell and the tumor bulk compartments, suggesting the potential use of GV in treating human melanoma alone or in combination with targeted therapy and/or immunotherapy.

      Abbreviations:

      7-AAD (7-amino actinomycin D), ALDH (aldehyde dehydrogenase), GV (gentian violet), MIC (melanoma initiating cells), NOX (reduced nicotinamide adenine dinucleotide phosphate oxidase), ROS (reactive oxygen species), STAT (signal transducer and activator of transcription)

      Introduction

      Cutaneous melanoma is one of the most aggressive types of skin cancer. Early-stage melanoma can be cured in most patients by surgical excision. On the contrary, once disseminated, melanoma becomes a highly lethal condition. Targeted therapies and immunotherapies have improved progression-free and overall survival in this disease, extending median survival from 9 to over 25 months. However, resistance emerges in most patients treated with targeted therapy, and not all patients respond to immunotherapy; therefore, most patients still die from metastatic disease (
      • Lo J.A.
      • Fisher D.E.
      The melanoma revolution: from UV carcinogenesis to a new era in therapeutics.
      ).
      Accumulating evidence supports the existence of cancer stem cells in human melanomas that possess the ability to self-renew and differentiate into tumor bulk cells. These cells drive tumor initiation, progression, and metastasis, and they might contribute to relapse after therapy (
      • Murphy G.F.
      • Wilson B.J.
      • Girouard S.D.
      • Frank N.Y.
      • Frank M.H.
      Stem cells and targeted approaches to melanoma cure.
      ). Our group has previously shown that the embryonic transcription factor SOX2 is required for self-renewal and tumorigenicity of human melanoma-initiating cells (MICs) (
      • Santini R.
      • Pietrobono S.
      • Pandolfi S.
      • Montagnani V.
      • D’Amico M.
      • Penachioni J.Y.
      • et al.
      SOX2 regulates self-renewal and tumorigenicity of human melanoma-initiating cells.
      ). Although preclinical studies began to evaluate strategies to inhibit SOX2 (
      • Favaro R.
      • Appolloni I.
      • Pellegatta S.
      • Sanga A.B.
      • Pagella P.
      • Gambini E.
      • et al.
      Sox2 is required to maintain cancer stem cells in a mouse model of high-grade oligodendroglioma.
      ,
      • Narasimhan K.
      • Pillay S.
      • Bin Ahmad N.R.
      • Bikadi Z.
      • Hazai E.
      • Yan L.
      • et al.
      Identification of a polyoxometalate inhibitor of the DNA binding activity of Sox2.
      ,
      • Stolzenburg S.
      • Rots M.G.
      • Beltran A.S.
      • Rivenbark A.G.
      • Yuan X.
      • Qian H.
      • et al.
      Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer.
      ), the possibility to selectively target SOX2 remains a challenge.
      The cationic triphenylmethane pharmacophore gentian violet (GV) has been used for a long time as an antimycotic and antibacterial agent (
      • Docampo R.
      • Moreno S.N.
      The metabolism and mode of action of gentian violet.
      ). Recent studies suggest that GV has also a potent inhibitory activity against reduced nicotinamide adenine dinucleotide phosphate oxidases (NOX) Nox2 and Nox4 (
      • Bhandarkar S.S.
      • Jaconi M.
      • Fried L.E.
      • Bonner M.Y.
      • Lefkove B.
      • Govindarajan B.
      • et al.
      Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice.
      ), whose expression is linked to tumor progression, and that it is able to induce cell death by disrupting the mitochondrial system (
      • Zhang X.
      • Zheng Y.
      • Fried L.E.
      • Du Y.
      • Montano S.J.
      • Sohn A.
      • et al.
      Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes.
      ). A recent study reported a case of cutaneous melanoma metastasis successfully treated with a combination of GV and imiquimod. The patient was free of melanoma with no recurrence for 2 years, until death from congestive heart failure at 94 years of age (
      • Arbiser J.L.
      • Bips M.
      • Seidler A.
      • Bonner M.Y.
      • Kovach C.
      Combination therapy of imiquimod and gentian violet for cutaneous melanoma metastases.
      ,
      • Bonner M.Y.
      • Arbiser J.L.
      The antioxidant paradox: what are antioxidants and how should they be used in a therapeutic context for cancer.
      ). These findings prompted us to investigate the mechanism of action of GV in melanoma.
      Here, we show that GV impairs melanoma stem cell self-renewal and survival through the inhibition of SOX2. Mechanistically, GV negatively affects the signal transducer and activator of transcription (STAT) 3/SOX2 axis by reducing STAT3 phosphorylation, with consequent decreased STAT3 translocation into the nucleus and binding to SOX2 promoter. In addition, we show that GV exerts a potent antiproliferative effect on melanoma cells by promoting mitochondrial apoptosis and G2 cell cycle arrest.

      Results

      GV inhibits self-renewal and survival of melanoma spheres

      Melanomas contain a subpopulation of MICs, which drives melanoma initiation and progression and is responsible for tumor relapse after therapy (
      • Murphy G.F.
      • Wilson B.J.
      • Girouard S.D.
      • Frank N.Y.
      • Frank M.H.
      Stem cells and targeted approaches to melanoma cure.
      ). Hence, we tested whether GV administration might affect the behavior of melanoma spheres, which display self-renewal ability and mimic the original tumor after transplantation into athymic nude mice (
      • Santini R.
      • Vinci M.C.
      • Pandolfi S.
      • Penachioni J.Y.
      • Montagnani V.
      • Olivito B.
      • et al.
      Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.
      ). Treatment of M33x, SSM2c, and A375 cells with increasing doses (0.1, 0.25 and 0.5 μmol/L) of GV led to a dose-dependent reduction in the number of primary spheres and a progressive loss of their ability to self-renew as secondary spheres (Figure 1a and b). GV-treated primary and secondary melanoma spheres were reduced in size (Figure 1c), thus suggesting an effect on proliferation and/or death of progenitors or more differentiated cells composing the sphere. Indeed, GV decreased proliferation, as determined by analysis of BrdU incorporation, and increased both early and late apoptosis and the number of cells testing positive for cleaved caspase-3 (Figure 1d–f).
      Figure 1
      Figure 1GV inhibits melanoma sphere self-renewal and survival. (a) Primary and secondary melanoma spheres. Melanoma cells were treated for 72 hours during primary sphere formation and left untreated to form secondary spheres. (b) Representative phase-contrast images of secondary SSM2c spheres as indicated in a. Scale bar = 100 μm. (c) Size of primary and secondary spheres treated with increasing doses of GV. (d) Quantification of BrdU incorporation and (e) of early (annexin V+/7-AAD) and late (annexin V+/7-AAD+) apoptotic cells in SSM2c primary spheres after GV treatment. (f) Percentage of caspase 3-positive melanoma cells after GV treatment. (g) Western blot of SOX2 in adherent melanoma cells and spheres after GV treatment. HSP90 was used as a loading control. Data shown are mean ± standard error of the mean. n = 3. P ≤ 0.05 versus control. 7-AAD, 7-amino actinomycin D; GV, gentian violet; N, number.
      We next assessed whether GV might affect the expression of stemness genes. Quantitative real-time PCR analysis showed that GV treatment consistently reduced SOX2 mRNA levels in M33x, SSM2c, and A375 melanoma spheres (see Supplementary Figure S1a online). KLF4 expression was not significantly affected by GV treatment, whereas OCT4 was slightly decreased in SSM2c and increased in M33x and A375 spheres (see Supplementary Figure S1a), likely because of a compensatory effect. Western blot analysis confirmed the GV-mediated decrease of SOX2 expression also at the protein level in both melanoma cells and their derived melanoma spheres (Figure 1g). Interestingly, SOX2 protein levels correlated with sphere-formation and self-renewal abilities of M33x, SSM2c, and A375 cells treated with increasing doses of GV (Figure 1g vs. Figure 1a). Collectively, these data indicate that GV impairs self-renewal and survival of melanoma spheres by reducing the expression of the embryonic transcription factor SOX2.

      GV affects maintenance of putative melanoma stem cells

      As an alternative approach to test the effect of GV on melanoma stem cells, we sorted melanoma cells with high aldehyde dehydrogenase (ALDHhigh) activity (
      • Luo Y.
      • Dallaglio K.
      • Chen Y.
      • Robinson W.A.
      • Robinson S.E.
      • McCarter M.D.
      • et al.
      ALDH1A isozymes are markers of human melanoma stem cells and potential therapeutic targets.
      ,
      • Santini R.
      • Vinci M.C.
      • Pandolfi S.
      • Penachioni J.Y.
      • Montagnani V.
      • Olivito B.
      • et al.
      Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.
      ). Cytometric analysis showed a significant reduction in the number of ALDHhigh M33x, SSM2c, and A375 cells treated with increasing doses of GV (Figure 2a and b). Treatment with GV reduced the expression of SOX2 in ALDHhigh putative MICs and, to a lesser extent, in ALDHlow tumor bulk cells (Figure 2c). Moreover, GV drastically reduced the self-renewal ability of ALDHhigh, and only marginally that of ALDHlow spheres (Figure 2d), consistent with lower SOX2 levels in the ALDHlow subpopulation (
      • Santini R.
      • Pietrobono S.
      • Pandolfi S.
      • Montagnani V.
      • D’Amico M.
      • Penachioni J.Y.
      • et al.
      SOX2 regulates self-renewal and tumorigenicity of human melanoma-initiating cells.
      ). AnnexinV/7-amino actinomycin D (7-AAD) staining indicated that GV increased apoptosis in both ALDHhigh and ALDHlow subpopulations (Figure 2e), as also shown by the drastic reduction of the anti-apoptotic factor BCL2 (Figure 2c). To determine whether GV-induced cell death in ALDHhigh cells could be attributable to the ability of GV to lead to a more differentiated status characterized by lower ALDH activity, we sorted both ALDHhigh and ALDHlow fractions after GV treatment (Figure 2f). Western blot analysis showed activation of caspase-3 only in the GV-enriched ALDHlow fraction. Notably, no induction of apoptosis was observed in the residual ALDHhigh subpopulation (Figure 2g). In an effort to provide additional evidence that GV affects melanoma stem cells, we used CD271/p75NTR, an established melanoma stem cell marker (
      • Boiko A.D.
      • Razorenova O.V.
      • van de Rijn M.
      • Swetter S.M.
      • Johnson D.L.
      • Ly D.P.
      • et al.
      Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271.
      ,
      • Civenni G.
      • Walter A.
      • Kobert N.
      • Mihic-Probst D.
      • Zipser M.
      • Belloni B.
      • et al.
      Human CD271-positive melanoma stem cells associated with metastasis establish tumor heterogeneity and long-term growth.
      ). FACS analysis showed that GV treatment reduced the number of CD271+ putative melanoma stem cells in a dose-dependent manner in all three cell types (see Supplementary Figure S1b and c), confirming results obtained with Aldefluor (StemCell Technologies, Vancouver, Canada) and sphere assays. Altogether, these results indicate that GV decreases MIC survival and self-renewal by affecting cancer stem cell features.
      Figure 2
      Figure 2GV reduces the number and self-renewal of ALDHhigh melanoma stem cells. (a, b) Quantification and representative images of ALDHhigh cells after GV treatment for 24 hours. ALDH inhibitor diethylaminobenzaldehyde was used as negative control. (c) Western blot of BCL2 and SOX2 in ALDHhigh and ALDHlow subpopulations after GV treatment. (d) Number of secondary spheres in ALDHhigh and ALDHlow SSM2c cells treated with GV. (e) Cytometric analysis of apoptotic cells in ALDHhigh and ALDHlow fractions 48 hours after GV treatment. (f) Representative images of sorted ALDHhigh and ALDHlow subpopulations. Sorting gates were set at least one logarithm apart from the relative baseline fluorescence (drawn after the incubation with DEAB). (g) Western blot of cleaved caspase-3 in ALDHlow- and ALDHhigh-sorted cells, as shown in f. HSP90 was used as loading control. Data shown are mean ± standard error of the mean. n = 3. P ≤ 0.05 versus control. ALDH, aldehyde dehydrogenase; Cleav, cleaved; DEAB, diethylaminobenzaldehyde; GV, gentian violet; N, number.

      Enhanced expression of SOX2 partially rescues the inhibitory effect of GV on melanoma stem cell self-renewal

      We have previously shown that SOX2 is critical for the maintenance of melanoma stem cells and for the survival and proliferation of melanoma cells that constitute the tumor bulk (
      • Santini R.
      • Pietrobono S.
      • Pandolfi S.
      • Montagnani V.
      • D’Amico M.
      • Penachioni J.Y.
      • et al.
      SOX2 regulates self-renewal and tumorigenicity of human melanoma-initiating cells.
      ). Our data indicate that GV significantly decreases SOX2 expression in melanoma cells and MICs; therefore, we assessed whether SOX2 is involved in mediating the effects of GV on MIC self-renewal and survival. To test this hypothesis, we overexpressed SOX2 in patient-derived melanoma cells SSM2c and M33x, using a replication-incompetent lentivirus expressing SOX2 (LV-SOX2) (Figure 3a). SOX2 overexpression reduced (in M33x spheres) and nearly abolished (in SSM2c spheres) the inhibitory effect of GV on sphere formation (Figure 3b) and self-renewal as secondary spheres (Figure 3c). On the other hand, GV was able to overcome the effect of SOX2 on survival of more differentiated progenitors composing the sphere. Indeed, ectopic SOX2 expression failed to prevent the decrease in sphere size observed after GV treatment (Figure 3d). Consistently, AnnexinV/7-AAD staining confirmed the induction of apoptosis in both control and SOX2-overexpressing spheres (Figure 3e). These results suggest that enhanced SOX2 expression is able to counteract the apoptotic effect of GV on sphere-initiating cells but does not prevent the apoptotic effect of GV on the tumor bulk. Altogether these data indicate that down-regulation of SOX2 mediates, at least in part, the inhibitory effect of GV on melanoma stem cell self-renewal.
      Figure 3
      Figure 3SOX2 mediates the inhibitory effect of GV on melanoma stem cell self-renewal. (a) Western blot of SOX2 in M33x and SSM2c spheres transduced with LV-c or LV-SOX2. HSP90 was used as loading control. (b) Primary and (c) secondary sphere assay shows that ectopic SOX2 counteracts the decrease in the number of spheres induced by GV treatment. (d) Size of primary SSM2c spheres transduced with LV-c and LV-SOX2. (e) Quantification of the percentage of early (annexin V+/7-AAD) and late (annexin V+/7-AAD+) apoptotic cells in SSM2c spheres transduced with LV-c and LV-SOX2. Data shown are mean ± standard error of the mean. n = 3. P ≤ 0.05 versus control. GV, gentian violet.

      GV inhibits melanoma cell viability by promoting mitochondrial apoptosis and G2 cell cycle arrest

      Our data indicate that GV acts through two parallel mechanisms: the impairment of self-renewal ability of MICs in a SOX2-dependent manner and the decrease of survival and proliferation of melanoma tumor bulk cells independently of SOX2 (Figure 3). To better define the pro-apoptotic and antiproliferative effects of GV we used adherent M33x, SSM2c, A375, M51, and SK-MEL-28 melanoma cells. GV treatment resulted in a dose-dependent decrease of melanoma cell viability in all cell types (Figure 4a and see Supplementary Figure S2a online). Cell cycle analysis showed that GV induced a dose-dependent increase in the percentage of cells in the G2 phase to the detriment of G0/G1 in SSM2c and M33x cells and, to a lesser extent, in A375 (Figure 4b). Consistently, the expression level of the G2/M checkpoint regulator cyclin B1 was decreased in a dose-dependent manner in all three cell lines (Figure 4c). Conversely, GV failed to induce G2 cell cycle arrest in M51 and SK-MEL-28 melanoma cells (see Supplementary Figure S2b). AnnexinV/7-AAD staining showed dose-dependent induction of both early (annexin V+/7-AAD) and late (annexin V+/7-AAD+) apoptosis in all melanoma cells treated with increasing doses of GV (Figure 4d and see Supplementary Figure S2c).
      Figure 4
      Figure 4GV impairs melanoma cell viability by promoting mitochondrial apoptosis and G2 cell cycle arrest. (a) Cell viability assay 48 hours after GV treatment. Controls (DMSO) were set to 100%. (b) Cell cycle analysis after GV treatment (48 hours). (c) Western blot of cyclin B1, BCL2, BAX, total and cleaved caspase-3, and p53 in melanoma cells treated with GV for 48 hours. HSP90 was used as loading control. Densitometric quantification of BAX/BCL2 ratio is shown. (d) Cytometric analysis of apoptotic cells after GV treatment for 48 hours. (e) Western blot of cytochrome C in cytosolic extracts 48 hours after GV treatment. Fibrillarin (nuclear) and GAPDH (cytosolic) were used as loading control. (f) Quantification of mitochondrial ROS in cells treated with GV, stained with MitoSOX (Thermo Fisher Scientific, Waltham, MA) and analyzed by fluorescence-activated cell sorting. Data shown are mean ± standard error of the mean. n = 3. P ≤ 0.05 versus control. Cleav, cleaved; Cytc, cytochrome; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GV, gentian violet; ROS, reactive oxygen species; WCE, whole cell extract.
      A previous study showed that cationic triphenylmethanes can induce cell death by disrupting the mitochondrial system, followed by release of cytochrome C and apoptosis-inducing factors (
      • Zhang X.
      • Zheng Y.
      • Fried L.E.
      • Du Y.
      • Montano S.J.
      • Sohn A.
      • et al.
      Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes.
      ). Indeed, GV treatment increased the BAX/BCL2 ratio, an indicator of susceptibility to mitochondrial apoptosis (
      • Oltvai Z.N.
      • Milliman C.L.
      • Korsmeyer S.J.
      Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death.
      ), and induced cleaved caspase-3 in all cell lines at nanomolar doses (Figure 4c and see Supplementary Figure S2d). In addition, treatment with a high dose of GV (≥ 0.5 μmol/L in SSM2c and ≥0.25 μmol/L in A375) induced an increase of the cytoplasmic fraction of cytochrome C (Figure 4e) and an enhancement of mitochondrial reactive oxygen species (ROS) production (Figure 4f), suggesting that high doses of GV activate the mitochondrial apoptotic pathway in melanoma cells. Inhibition of mitochondrial ROS with 2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (i.e., mitoTEMPO), a specific scavenger of mitochondrial superoxide (
      • Dikalova A.E.
      • Bikineyeva A.T.
      • Budzyn K.
      • Nazarewicz R.R.
      • McCann L.
      • Lewis W.
      • et al.
      Therapeutic targeting of mitochondrial superoxide in hypertension.
      ), partially prevented late apoptosis without affecting early apoptosis in all melanoma cell types (see Supplementary Figure S3 online).
      We next tested whether GV treatment might affect p53 activity (
      • Garufi A.
      • D’Orazi V.
      • Arbiser J.L.
      • D’Orazi G.
      Gentian violet induces wtp53 transactivation in cancer cells.
      ). Although GV increased total p53 levels in A375 but not in SSM2c and M33x cells (Figure 4c), quantitative real-time PCR analysis showed that GV treatment increased the expression of the p53-target genes PUMA, PIG3, and, partially, NOXA in all cell types (see Supplementary Figure S4a online). In addition, silencing of p53 in A375 and SSM2c cells decreased basal apoptosis compared with in control cells and reduced sensitivity to GV in terms of early apoptosis (see Supplementary Figure S4b–d), suggesting that GV-induced cell death is partially dependent on p53 function.
      Altogether these data indicate that GV inhibits melanoma cell growth with different dose-dependent effects: at low doses it induces G2 cell cycle arrest, whereas at high doses it mainly promotes mitochondrial apoptosis.

      GV suppresses STAT3 activation through an EGFR-dependent mechanism

      Triphenylmethane dyes have been show to inhibit ROS production by targeting Nox4 and Nox2 in different biological contexts (
      • Bhandarkar S.S.
      • Jaconi M.
      • Fried L.E.
      • Bonner M.Y.
      • Lefkove B.
      • Govindarajan B.
      • et al.
      Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice.
      ,
      • Mukawera E.
      • Chartier S.
      • Williams V.
      • Pagano P.J.
      • Lapointe R.
      • Grandvaux N.
      Redox-modulating agents target NOX2-dependent IKKε oncogenic kinase expression and proliferation in human breast cancer cell lines.
      ,
      • Perry B.N.
      • Govindarajan B.
      • Bhandarkar S.S.
      • Knaus U.G.
      • Valo M.
      • Sturk C.
      • et al.
      Pharmacologic blockade of angiopoietin-2 is efficacious against model hemangiomas in mice.
      ). Expression of NOX genes was investigated in six patient-derived primary melanoma cells, in four melanoma cell lines and in normal human epidermal melanocytes. Quantitative real-time PCR showed that NOX1 and NOX4 were variably expressed among melanoma cells, whereas NOX2 was expressed in only two cell lines (see Supplementary Figure S5a online). One possible explanation for lack of G2 cell cycle arrest in M51 and SK-MEL-28 melanoma cells upon GV treatment (see Supplementary Figure S2b) might be the absence of NOX4 expression in these two cell lines (
      • Yamaura M.
      • Mitsushita J.
      • Furuta S.
      • Kiniwa Y.
      • Ashida A.
      • Goto Y.
      • et al.
      NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression.
      ). To determine whether GV inhibited ROS production in melanoma cells, we used different assays. Using the oxidant-sensing fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (i.e., DCFH-DA), we failed to observe a decrease of intracellular ROS upon GV treatment in M33x and SSM2c cells. Unexpectedly, we documented an increase in ROS level in A375 cells, which was also confirmed by confocal microscopy using CellRox dye (CellRox, Tel Aviv, Israel) (see Supplementary Figure S5b and c). Consistently, GV treatment decreased the reduced form of Src in A375 cells without affecting phosphorylated Src (see Supplementary Figure S5d and e).
      A previous study showed that triphenylmethane analogs exert a strong inhibitory activity against tyrosine-kinase receptors (
      • Antipova A.A.
      • Stockwell B.R.
      • Golub T.R.
      Gene expression-based screening for inhibitors of PDGFR signaling.
      ). Therefore, we hypothesized that GV might act on EGFR, which is expressed in almost all melanoma cell lines (see Supplementary Figure S6a online). To test whether GV affects EGFR activation, we immunoprecipitated endogenous EGFR and performed Western blot with an anti-phospho-Tyr antibody. The experiment showed that GV treatment reduced EGFR phosphorylation but did not change total levels of EGFR (Figure 5a). Consistently, GV reduced phospho-AKT (Ser473) and phospho–extracellular signal-regulated kinase–1/2 levels (see Supplementary Figure S6b), two downstream mediators of EGFR activation. GV strongly decreased STAT3 phosphorylation at Tyr705 and hence its activation both in adherent and melanoma spheres (Figure 5b), thus suggesting the existence of an inhibitory mechanism on STAT3.
      Figure 5
      Figure 5GV inhibits STAT3/SOX2 axis. (a) Immunoprecipitation of endogenous EGFR in A375 cells. (b) Western blot of pSTAT3Y705 and STAT3 in adherent cells and spheres treated with GV, for 24 and 72 hours, respectively. HSP90 was used as loading control. (c) STAT3 cellular localization 24 hours after GV treatment. GAPDH (cytoplasm) and fibrillarin (nuclear) were used as loading control. (d) Chromatin immunoprecipitation on SOX2 proximal promoter. IgG was used as negative control and set to 1. Data represent mean ± standard error of the mean. n = 3. (e) Quantification of dual-luciferase reporter assay. Relative luciferase activities were firefly/Renilla ratios, with the level induced by control equated to 1. Data represent mean ± standard error of the mean. n = 4. (f) Western blot of SOX2 and BCL2 in SSM2c transfected with Myc-STAT3wt, Myc-STAT3Y705E, or Myc after 250 nmol/L of GV for 24 hours. (g, h) Primary spheres formation and representative images in cells transfected with Myc-STAT3wt, Myc-STAT3Y705E, or Myc. Scale bar = 100 μm. P < 0.05 versus control. (i) Proposed mechanism of action of GV. P < 0.05 versus control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GV, gentian violet; Rel, relative; STAT3, signal transducer and activator of transcription-3.
      To further confirm the involvement of EGFR on STAT3 activation, melanoma cells were treated with GV alone or in the presence of the EGFR inhibitor gefitinib (
      • Patel S.P.
      • Kim K.B.
      • Papadopoulos N.E.
      • Hwu W.J.
      • Hwu P.
      • Prieto V.G.
      • et al.
      A phase II study of gefitinib in patients with metastatic melanoma.
      ,
      • Wakeling A.E.
      • Guy S.P.
      • Woodburn J.R.
      • Ashton S.E.
      • Curry B.J.
      • Barker A.J.
      • et al.
      ZD1839 (Iressa): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy.
      ). Western blot analysis showed that gefitinib down-regulated STAT3 by itself, and that combination of gefitinib and GV prevented the effect of GV on STAT3 activation (see Supplementary Figure S6c). Furthermore, treatment of melanoma cells with ruxolitinib, a specific JAK1/2 inhibitor (
      • Ostojic A.
      • Vrhovac R.
      • Verstovsek S.
      Ruxolitinib: a new JAK1/2 inhibitor that offers promising options for treatment of myelofibrosis.
      ), completely abrogated STAT3 phosphorylation (see Supplementary Figure S6d). Altogether, these data suggest that GV reduces phospho-STAT3 by inhibiting EGFR activation, likely through a JAK-dependent and ROS/Src-independent mechanism.
      The inhibitory effect of GV on EGFR activation might also explain the induction of apoptosis on adherent melanoma cells. PI3K/AKT signaling has been shown to inhibit programmed cell death through a shift from pro- to anti-apoptotic signals (
      • Fulda S.
      Shifting the balance of mitochondrial apoptosis: therapeutic perspectives.
      ) or by inducing Mdm2-mediated p53 degradation (
      • Ogawara Y.
      • Kishishita S.
      • Obata T.
      • Isazawa Y.
      • Suzuki T.
      • Tanaka K.
      • et al.
      Akt enhances Mdm2-mediated ubiquitination and degradation of p53.
      ). Consistently, our data showed that inhibition of PI3K signaling by LY294002 (
      • Aziz S.A.
      • Davies M.
      • Pick E.
      • Zito C.
      • Jilaveanu L.
      • Camp R.L.
      • et al.
      Phosphatidylinositol-3-kinase as a therapeutic target in melanoma.
      ) primed melanoma cells to mitochondrial apoptosis and minimized the ability of GV to further enhance both early and late apoptosis (see Supplementary Figure S7 online).

      GV down-regulates SOX2 expression by inhibiting STAT3 in melanoma cells

      STAT3 phosphorylation at tyrosine 705 has been shown to mediate STAT3 translocation into the nucleus as a dimer, thus allowing dimeric STAT3 to bind target genes and to promote gene transcription (
      • Darnell J.E.
      STATs and gene regulation.
      ). Western blot analysis of STAT3 confirmed that treatment with GV reduced nuclear STAT3 levels and increased its cytoplasmic fraction in both SSM2c and A375 melanoma cells (Figure 5c).
      Because STAT3 positively correlates with SOX2 in several contexts (
      • Foshay K.M.
      • Gallicano G.I.
      Regulation of Sox2 by STAT3 initiates commitment to the neural precursor cell fate.
      ,
      • Zhao D.
      • Pan C.
      • Sun J.
      • Gilbert C.
      • Drews-Elger K.
      • Azzam D.J.
      • et al.
      VEGF drives cancer-initiating stem cells through VEGFR-2/Stat3 signaling to upregulate Myc and Sox2.
      ), we investigated whether STAT3 directly regulates SOX2 expression in melanoma cells. Bioinformatic analysis identified a putative site possessing the STAT3 canonical binding motif TTC(N)2-4GAA (
      • Darnell J.E.
      STATs and gene regulation.
      ,
      • Ehret G.B.
      • Reichenbach P.
      • Schindler U.
      • Horvath C.M.
      • Fritz S.
      • Nabholz M.
      • et al.
      DNA binding specificity of different STAT proteins. Comparison of in vitro specificity with natural target sites.
      ) within the proximal region of the SOX2 promoter (–447 base pairs [bp]/–426 bp) (Figure 5d). Chromatin immunoprecipitation in melanoma cells showed STAT3 bound to SOX2 promoter at approximately 400 bp upstream to the SOX2 transcription start site (Figure 5d). Interestingly, treatment with GV prevented STAT3 binding to SOX2 promoter, consistent with its cytoplasmic accumulation (Figure 5c). To confirm that SOX2 is a direct transcriptional target of STAT3 in melanoma, we co-transfected STAT3 expression construct with the SOX2 promoter driven by a luciferase reporter (–601bp/+293bp), as previously described (
      • Eberl M.
      • Klingler S.
      • Mangelberger D.
      • Loipetzberger A.
      • Damhofer H.
      • Zoidl K.
      • et al.
      Hedgehog-EGFR cooperation response genes determine the oncogenic phenotype of basal cell carcinoma and tumour-initiating pancreatic cancer cells.
      ). Luciferase assay showed that STAT3 increased SOX2 promoter activity by 3-fold and that treatment with GV reverted the effect of STAT3 on the endogenous SOX2 promoter (Figure 5e), indicating that GV reduces SOX2 expression through inhibition of STAT3 transcriptional activity. To further validate these results, we performed site-directed mutagenesis to mutate the tyrosine in position 705 into a glutamic acid to mimic STAT3 phosphorylation and activation, and we overexpressed both wild-type and STAT3 mutant Tyr705Glu (Y705E). Western blot analysis in SSM2c cells showed that ectopic expression of the constitutive active STAT3 mutant, but not of the wild-type, counteracted the decrease of SOX2 protein levels induced by GV treatment (Figure 5f). Consistently, overexpression of both wild-type STAT3 and STAT3-Y705E increased the number of primary spheres compared with the empty vector, but only STAT3-Y705E prevented the inhibitory effect of GV on sphere formation (Figure 5g and h). Collectively, our results indicate that GV inhibits melanoma stem cell survival and self-renewal by affecting the EGFR/STAT3/SOX2 axis (Figure 5i).

      Discussion

      Current therapies to treat melanoma induce only temporary benefits because they are predominantly directed toward bulk tumor cells and fail to eradicate MICs, which drive tumor regrowth and originate recurrent tumors characterized by high aggressiveness, fast spreading, and resistance to therapy (
      • Frank N.Y.
      • Schatton T.
      • Frank M.H.
      The therapeutic promise of the cancer stem cell concept.
      ,
      • Murphy G.F.
      • Wilson B.J.
      • Girouard S.D.
      • Frank N.Y.
      • Frank M.H.
      Stem cells and targeted approaches to melanoma cure.
      ). Hence, strategies that kill or induce differentiation of MICs may improve prognosis and contribute to curing patients (
      • Schlaak M.
      • Schmidt P.
      • Bangard C.
      • Kurschat P.
      • Mauch C.
      • Abken H.
      Regression of metastatic melanoma in a patient by antibody targeting of cancer stem cells.
      ,
      • Mukherjee N.
      • Schwan J.V.
      • Fujita M.
      • Norris D.A.
      • Shellman Y.G.
      Alternative treatments for melanoma: targeting BCL-2 family members to de-bulk and kill cancer stem cells.
      ). Treatment of cutaneous melanoma metastases with a combination of GV and imiquimod has been shown to prevent melanoma recurrence (
      • Arbiser J.L.
      • Bips M.
      • Seidler A.
      • Bonner M.Y.
      • Kovach C.
      Combination therapy of imiquimod and gentian violet for cutaneous melanoma metastases.
      ,
      • Bonner M.Y.
      • Arbiser J.L.
      The antioxidant paradox: what are antioxidants and how should they be used in a therapeutic context for cancer.
      ). However, it is not known whether and how GV affects melanoma cell growth and stemness.
      In this study, we elucidated two antitumor mechanisms of GV. First, we show that GV inhibits melanoma stem cell self-renewal by down-regulating the transcription factor SOX2 through STAT3 (Figure 5). Second, we show that GV harbors a potent antiproliferative effect on melanoma bulk by promoting mitochondrial apoptosis and G2 cell cycle arrest. Altogether these findings suggest that GV might become an effective compound in the treatment of human melanoma, alone or in combinatory regimen.
      The major finding of this study is that GV acts on melanoma stem cells by inhibiting SOX2 through the EGFR/STAT3 signaling axis. Indeed, ectopic SOX2 expression protects MICs from the effects of GV. This result is in agreement with the critical role of SOX2 in MIC maintenance (
      • Santini R.
      • Pietrobono S.
      • Pandolfi S.
      • Montagnani V.
      • D’Amico M.
      • Penachioni J.Y.
      • et al.
      SOX2 regulates self-renewal and tumorigenicity of human melanoma-initiating cells.
      ) and with studies showing the importance of SOX2 in mediating the effects of EGFR activation in other cancer types (
      • Chou Y.T.
      • Lee C.C.
      • Hsiao S.H.
      • Lin S.E.
      • Lin S.C.
      • Chung C.H.
      • et al.
      The emerging role of SOX2 in cell proliferation and survival and its crosstalk with oncogenic signaling in lung cancer.
      ,
      • Eberl M.
      • Klingler S.
      • Mangelberger D.
      • Loipetzberger A.
      • Damhofer H.
      • Zoidl K.
      • et al.
      Hedgehog-EGFR cooperation response genes determine the oncogenic phenotype of basal cell carcinoma and tumour-initiating pancreatic cancer cells.
      ,
      • Rybak A.P.
      • Tang D.
      SOX2 plays a critical role in EGFR-mediated self-renewal of human prostate cancer stem-like cells.
      ). Because SOX2 has been shown to induce de-differentiation and to impart stem-like signatures in several tumors (
      • Weina K.
      • Utikal J.
      SOX2 and cancer: current research and its implications in the clinic.
      ), inhibition of SOX2 by GV may increase the number of differentiated cells, thus sensitizing them to GV-mediated apoptosis. This hypothesis is consistent with the observed decrease of melanoma cells expressing high ALDH activity and CD271, two melanoma stem cell markers (
      • Boiko A.D.
      • Razorenova O.V.
      • van de Rijn M.
      • Swetter S.M.
      • Johnson D.L.
      • Ly D.P.
      • et al.
      Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271.
      ,
      • Civenni G.
      • Walter A.
      • Kobert N.
      • Mihic-Probst D.
      • Zipser M.
      • Belloni B.
      • et al.
      Human CD271-positive melanoma stem cells associated with metastasis establish tumor heterogeneity and long-term growth.
      ,
      • Luo Y.
      • Dallaglio K.
      • Chen Y.
      • Robinson W.A.
      • Robinson S.E.
      • McCarter M.D.
      • et al.
      ALDH1A isozymes are markers of human melanoma stem cells and potential therapeutic targets.
      ,
      • Santini R.
      • Vinci M.C.
      • Pandolfi S.
      • Penachioni J.Y.
      • Montagnani V.
      • Olivito B.
      • et al.
      Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.
      ), and with the observation that ALDHhigh putative MICs become sensitive to apoptotic stimuli induced by GV when they are differentiated.
      Although several studies have highlighted potential strategies to inhibit SOX2, to date the ability to selectively target SOX2 remains a challenge. In this respect, a molecule such as GV that is well tolerated in humans and can be applied topically on cutaneous melanoma lesions might be an interesting agent to block SOX2 activity and can be regarded as a therapeutic option in combination with targeted therapy and immunotherapy.
      Previous studies indicated that GV affects ROS production by targeting NOX enzymes (
      • Bhandarkar S.S.
      • Jaconi M.
      • Fried L.E.
      • Bonner M.Y.
      • Lefkove B.
      • Govindarajan B.
      • et al.
      Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice.
      ,
      • Mukawera E.
      • Chartier S.
      • Williams V.
      • Pagano P.J.
      • Lapointe R.
      • Grandvaux N.
      Redox-modulating agents target NOX2-dependent IKKε oncogenic kinase expression and proliferation in human breast cancer cell lines.
      ,
      • Perry B.N.
      • Govindarajan B.
      • Bhandarkar S.S.
      • Knaus U.G.
      • Valo M.
      • Sturk C.
      • et al.
      Pharmacologic blockade of angiopoietin-2 is efficacious against model hemangiomas in mice.
      ). Nevertheless, here we show that GV blocks melanoma cell growth in vitro and negatively affects the melanoma stem cell compartment without reducing ROS production. This may depend on the fact that melanoma cells undergo mitochondrial apoptosis that may mask the effect of GV. On the other hand, our data indicate that both NOX4-positive (A375, SSM2c, and M33x) and NOX4-negative (SK-MEL-28 and M51) melanoma cells are equally sensitive to GV-induced apoptotic stimuli, whereas only NOX4-positive cells display G2 cell cycle arrest (Figure 4 and see Supplementary Figure S2), consistent with a previous report (
      • Yamaura M.
      • Mitsushita J.
      • Furuta S.
      • Kiniwa Y.
      • Ashida A.
      • Goto Y.
      • et al.
      NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression.
      ).
      Our study shows that GV inhibits activation of STAT3 by EGFR in a Src-independent and JAK-dependent manner. This is supported by studies showing that STAT3 activation in melanoma cells relies on JAK or protein-tyrosine phosphatase rather than on Src (
      • Kreis S.
      • Munz G.A.
      • Haan S.
      • Heinrich P.C.
      • Behrmann I.
      Cell density dependent increase of constitutive signal transducers and activators of transcription 3 activity in melanoma cells is mediated by Janus kinases.
      ,
      • Flashner-Abramson E.
      • Klein S.
      • Mullin G.
      • Shoshan E.
      • Song R.
      • Shir A.
      • et al.
      Targeting melanoma with NT157 by blocking Stat3 and IGF1R signaling.
      ;). STAT3 has been reported to enhance invasion and metastasis (
      • Fofaria N.M.
      • Srivastava S.K.
      Critical role of STAT3 in melanoma metastasis through anoikis resistance.
      ) and to mediate the acquisition of a stemness phenotype in melanoma cells (
      • Ohanna M.
      • Cheli Y.
      • Bonet C.
      • Bonazzi V.F.
      • Allegra M.
      • Giuliano S.
      • et al.
      Secretome from senescent melanoma engages the STAT3 pathway to favor reprogramming of naïve melanoma towards a tumor-initiating cell phenotype.
      ). In addition, inhibition of STAT3 has been shown to induce a significant regression of melanoma growth in vivo (
      • Yin D.
      • Li Y.
      • Lin H.
      • Guo B.
      • Du Y.
      • Li X.
      • et al.
      Functional graphene oxide as a plasmid-based Stat3 siRNA carrier inhibits mouse malignant melanoma growth in vivo.
      ). Hence, targeting the EGFR-JAK-STAT3 signaling by GV could represent a valid strategy to treat melanoma.
      In conclusion, we uncovered an EGFR/STAT3/SOX2 signaling cascade that regulates melanoma stem cell behavior and identified GV as an inhibitor of this axis. GV has been used as an antibacterial, antimycotic, and antiangiogenic agent. More recently, GV has shown a strong anticancer activity both in vitro and in vivo (
      • Bhandarkar S.S.
      • Jaconi M.
      • Fried L.E.
      • Bonner M.Y.
      • Lefkove B.
      • Govindarajan B.
      • et al.
      Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice.
      ,
      • Cunniff B.
      • Newick K.
      • Nelson K.J.
      • Wozniak A.N.
      • Beuschel S.
      • Leavitt B.
      • et al.
      Disabling mitochondrial peroxide metabolism via combinatorial targeting of peroxiredoxin 3 as an effective therapeutic approach for malignant mesothelioma.
      ,
      • Mukawera E.
      • Chartier S.
      • Williams V.
      • Pagano P.J.
      • Lapointe R.
      • Grandvaux N.
      Redox-modulating agents target NOX2-dependent IKKε oncogenic kinase expression and proliferation in human breast cancer cell lines.
      ,
      • Perry B.N.
      • Govindarajan B.
      • Bhandarkar S.S.
      • Knaus U.G.
      • Valo M.
      • Sturk C.
      • et al.
      Pharmacologic blockade of angiopoietin-2 is efficacious against model hemangiomas in mice.
      ,
      • Yamaguchi M.
      • Vikulina T.
      • Weitzmann M.N.
      Gentian violet inhibits MDA-MB-231 human breast cancer cell proliferation, and reverses the stimulation of osteoclastogenesis and suppression of osteoblast activity induced by cancer cells.
      ) with minimal adverse effects, thus making it safe for use in humans (
      • Arbiser J.L.
      Gentian violet is safe.
      ). Taken together, our data suggest that GV could be a good candidate for both chemoprevention and therapeutic treatment of human melanoma. Further studies will help give insights into the potential use of GV in combination with other drugs targeting signaling pathways involved in melanoma progression.

      Materials and Methods

      Human melanoma samples and cell cultures

      Commercial melanoma cell lines used in this study were A375 and SK-MEL-28 (a gift from Laura Poliseno, Istituto Toscano Tumori) (
      • Marranci A.
      • Tuccoli A.
      • Vitiello M.
      • Mercoledi E.
      • Sarti S.
      • Lubrano S.
      • et al.
      Identification of BRAF 3'UTR isoforms in melanoma.
      ). Human melanoma samples were obtained after approved protocol by the institutional review board of the hospital, and written informed consent of patients was obtained following the Helsinki protocol. Cells were periodically screened for contamination with Mycoplasma species by PCR. For cell viability assay, 15,000 cells/well were plated in 12-well plates and treated with GV at indicated concentrations for 48 hours in 1% fetal bovine serum.

      Primary sphere formation and self-renewal assay

      For melanoma sphere cultures, cells were seeded in human embryonic stem cell medium supplemented with 4ng/ml basic fibroblast growth factor, as reported (
      • Santini R.
      • Vinci M.C.
      • Pandolfi S.
      • Penachioni J.Y.
      • Montagnani V.
      • Olivito B.
      • et al.
      Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.
      ). For primary sphere formation assay, melanoma cells were seeded in 12-well plates at 1cell/μl dilution, and spheres were counted after 72 hours. For self-renewal assay, primary melanoma spheres were dissociated into single cells and plated at 1cell/μl dilution in 12-well plates. After 1 week secondary spheres were counted (
      • Santini R.
      • Pietrobono S.
      • Pandolfi S.
      • Montagnani V.
      • D’Amico M.
      • Penachioni J.Y.
      • et al.
      SOX2 regulates self-renewal and tumorigenicity of human melanoma-initiating cells.
      ).

      Aldefluor assay and flow cytometry analysis

      Aldefluor assay was performed using the Aldefluor kit (Stemcell Technologies, Vancouver, Canada) as previously reported (
      • Santini R.
      • Vinci M.C.
      • Pandolfi S.
      • Penachioni J.Y.
      • Montagnani V.
      • Olivito B.
      • et al.
      Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.
      ). For cell cycle analysis, melanoma cells were resuspended in 50μg/ml propidium iodide, 0.1% Triton X-100, and 0.1% sodium citrate 48 hours after GV treatment. Data were collected on BD Accuri C6 software (BD Biosciences, Franklin Lakes, NJ) and analyzed using ModFit LT software (Verity Software House, Topsham, ME). Apoptosis was measured after 48 (adherent cells) or 72 (spheres) hours of GV treatment using annexin V-phycoerythrin/7-AAD apoptosis kit (BD Biosciences) according to the manufacturer’s instructions. The number of apoptotic cells was detected and analyzed using BD Accuri C6 software.

      Plasmids, cloning, mutagenesis, and lentiviral vectors

      See Supplementary Materials online for details.

      Luciferase reporter assays

      Luciferase reporters were used in combination with Renilla luciferase pRL-TK reporter vector (Promega, Madison, WI) to normalize luciferase activities; pGL3Basic vector (Promega) was used to equal DNA amounts. Luminescence was measured using the Dual-Glo Luciferase Assay System (Promega) and the GloMax 20/20 Luminometer (Promega).

      Western blot analysis

      Western blot was performed as already described (
      • Pandolfi S.
      • Montagnani V.
      • Lapucci A.
      • Stecca B.
      HEDGEHOG/GLI-E2F1 axis modulates iASPP expression and function and regulates melanoma cell growth.
      ). See Supplementary Materials for antibodies.

      Chromatin immunoprecipitation

      Chromatin immunoprecipitation was performed as already described (
      • Pandolfi S.
      • Montagnani V.
      • Lapucci A.
      • Stecca B.
      HEDGEHOG/GLI-E2F1 axis modulates iASPP expression and function and regulates melanoma cell growth.
      ). Sonicated cell lysates were incubated overnight at 4 °C with 20 μl of protein G magnetic dynabeads (Invitrogen, Carlsbad, CA) coupled to 3 μg of anti-STAT3 antibody (H-190) (Santa Cruz Biotechnology, Santa Cruz, CA) or IgG control. DNA was purified, and quantitative real-time PCR was carried out at 60 °C using FastStart SYBR Green Master (Roche Diagnostics, Basel, Switzerland). Primers are listed in Supplementary Table S1 online.

      Statistical analysis

      Data represent mean ± standard error of the mean values calculated on at least three independent experiments. P-values were calculated using one-way analysis of variance and Bonferroni correction when more than two samples were analyzed and Student t test when two samples were compared. A two-tailed value of P < 0.05 was considered statistically significant.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We thank Fritz Aberger (University of Salzburg, Austria) for SOX2 promoter reporter plasmid, Michela Sica (Istituto Toscano Tumori, Florence, Italy) for assistance with cell sorting, Matteo Lulli (University of Florence, Italy) for confocal images acquisition, and Valentina Montagnani and Maria Riverso (Istituto Toscano Tumori, Florence, Italy) for helpful comments on the paper. This work was supported by grants from the Italian Association for Cancer Research (AIRC) to BS (IG-14184) and to PC (IG-8797); from Istituto Toscano Tumori (0203607) to PC; and from NIH AR47901, VA Merit Award, the Jamie Rabinowitch Davis Foundation, and the Robert Margolis Foundation to JLA. AM was supported by a Fondazione Umberto Veronesi postdoctoral fellowship.

      Supplementary Material

      References

        • Antipova A.A.
        • Stockwell B.R.
        • Golub T.R.
        Gene expression-based screening for inhibitors of PDGFR signaling.
        Genome Biol. 2008; 9: R47
        • Arbiser J.L.
        Gentian violet is safe.
        J Am Acad Dermatol. 2009; 61: 359
        • Arbiser J.L.
        • Bips M.
        • Seidler A.
        • Bonner M.Y.
        • Kovach C.
        Combination therapy of imiquimod and gentian violet for cutaneous melanoma metastases.
        J Am Acad Dermatol. 2012; 67: e81-e83
        • Aziz S.A.
        • Davies M.
        • Pick E.
        • Zito C.
        • Jilaveanu L.
        • Camp R.L.
        • et al.
        Phosphatidylinositol-3-kinase as a therapeutic target in melanoma.
        Clin Cancer Res. 2009; 15: 3029-3036
        • Bhandarkar S.S.
        • Jaconi M.
        • Fried L.E.
        • Bonner M.Y.
        • Lefkove B.
        • Govindarajan B.
        • et al.
        Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice.
        J Clin Invest. 2009; 119: 2359-2365
        • Boiko A.D.
        • Razorenova O.V.
        • van de Rijn M.
        • Swetter S.M.
        • Johnson D.L.
        • Ly D.P.
        • et al.
        Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271.
        Nature. 2010; 466: 133-137
        • Bonner M.Y.
        • Arbiser J.L.
        The antioxidant paradox: what are antioxidants and how should they be used in a therapeutic context for cancer.
        Future Med Chem. 2014; 6: 1413-1422
        • Chou Y.T.
        • Lee C.C.
        • Hsiao S.H.
        • Lin S.E.
        • Lin S.C.
        • Chung C.H.
        • et al.
        The emerging role of SOX2 in cell proliferation and survival and its crosstalk with oncogenic signaling in lung cancer.
        Stem Cells. 2013; 31: 2607-2619
        • Civenni G.
        • Walter A.
        • Kobert N.
        • Mihic-Probst D.
        • Zipser M.
        • Belloni B.
        • et al.
        Human CD271-positive melanoma stem cells associated with metastasis establish tumor heterogeneity and long-term growth.
        Cancer Res. 2011; 71: 3098-3109
        • Cunniff B.
        • Newick K.
        • Nelson K.J.
        • Wozniak A.N.
        • Beuschel S.
        • Leavitt B.
        • et al.
        Disabling mitochondrial peroxide metabolism via combinatorial targeting of peroxiredoxin 3 as an effective therapeutic approach for malignant mesothelioma.
        PLoS One. 2015; 10: e0127310
        • Darnell J.E.
        STATs and gene regulation.
        Science. 1997; 277: 1630-1635
        • Dikalova A.E.
        • Bikineyeva A.T.
        • Budzyn K.
        • Nazarewicz R.R.
        • McCann L.
        • Lewis W.
        • et al.
        Therapeutic targeting of mitochondrial superoxide in hypertension.
        Circ Res. 2010; 107: 106-116
        • Docampo R.
        • Moreno S.N.
        The metabolism and mode of action of gentian violet.
        Drug Metab Rev. 1990; 22: 161-178
        • Eberl M.
        • Klingler S.
        • Mangelberger D.
        • Loipetzberger A.
        • Damhofer H.
        • Zoidl K.
        • et al.
        Hedgehog-EGFR cooperation response genes determine the oncogenic phenotype of basal cell carcinoma and tumour-initiating pancreatic cancer cells.
        EMBO Mol Med. 2012; 4: 218-233
        • Ehret G.B.
        • Reichenbach P.
        • Schindler U.
        • Horvath C.M.
        • Fritz S.
        • Nabholz M.
        • et al.
        DNA binding specificity of different STAT proteins. Comparison of in vitro specificity with natural target sites.
        J Biol Chem. 2001; 276: 6675-6688
        • Favaro R.
        • Appolloni I.
        • Pellegatta S.
        • Sanga A.B.
        • Pagella P.
        • Gambini E.
        • et al.
        Sox2 is required to maintain cancer stem cells in a mouse model of high-grade oligodendroglioma.
        Cancer Res. 2014; 74: 1833-1844
        • Flashner-Abramson E.
        • Klein S.
        • Mullin G.
        • Shoshan E.
        • Song R.
        • Shir A.
        • et al.
        Targeting melanoma with NT157 by blocking Stat3 and IGF1R signaling.
        Oncogene. 2016; 35: 2675-2680
        • Fofaria N.M.
        • Srivastava S.K.
        Critical role of STAT3 in melanoma metastasis through anoikis resistance.
        Oncotarget. 2014; 5: 7051-7064
        • Foshay K.M.
        • Gallicano G.I.
        Regulation of Sox2 by STAT3 initiates commitment to the neural precursor cell fate.
        Stem Cells Dev. 2008; 17: 269-278
        • Frank N.Y.
        • Schatton T.
        • Frank M.H.
        The therapeutic promise of the cancer stem cell concept.
        J Clin Invest. 2010; 120: 41-50
        • Fulda S.
        Shifting the balance of mitochondrial apoptosis: therapeutic perspectives.
        Front Oncol. 2012; 2: 121
        • Garufi A.
        • D’Orazi V.
        • Arbiser J.L.
        • D’Orazi G.
        Gentian violet induces wtp53 transactivation in cancer cells.
        Int J Oncol. 2014; 44: 1084-1090
        • Kreis S.
        • Munz G.A.
        • Haan S.
        • Heinrich P.C.
        • Behrmann I.
        Cell density dependent increase of constitutive signal transducers and activators of transcription 3 activity in melanoma cells is mediated by Janus kinases.
        Mol Cancer Res. 2007; 5: 1331-1341
        • Lo J.A.
        • Fisher D.E.
        The melanoma revolution: from UV carcinogenesis to a new era in therapeutics.
        Science. 2014; 346: 945-949
        • Luo Y.
        • Dallaglio K.
        • Chen Y.
        • Robinson W.A.
        • Robinson S.E.
        • McCarter M.D.
        • et al.
        ALDH1A isozymes are markers of human melanoma stem cells and potential therapeutic targets.
        Stem Cells. 2012; 30: 2100-2113
        • Marranci A.
        • Tuccoli A.
        • Vitiello M.
        • Mercoledi E.
        • Sarti S.
        • Lubrano S.
        • et al.
        Identification of BRAF 3'UTR isoforms in melanoma.
        J Invest Dermatol. 2015; 135: 1694-1697
        • Mukawera E.
        • Chartier S.
        • Williams V.
        • Pagano P.J.
        • Lapointe R.
        • Grandvaux N.
        Redox-modulating agents target NOX2-dependent IKKε oncogenic kinase expression and proliferation in human breast cancer cell lines.
        Redox Biol. 2015; 6: 9-18
        • Mukherjee N.
        • Schwan J.V.
        • Fujita M.
        • Norris D.A.
        • Shellman Y.G.
        Alternative treatments for melanoma: targeting BCL-2 family members to de-bulk and kill cancer stem cells.
        J Invest Dermatol. 2015; 135: 2155-2161
        • Murphy G.F.
        • Wilson B.J.
        • Girouard S.D.
        • Frank N.Y.
        • Frank M.H.
        Stem cells and targeted approaches to melanoma cure.
        Mol Aspects Med. 2014; 39: 33-49
        • Narasimhan K.
        • Pillay S.
        • Bin Ahmad N.R.
        • Bikadi Z.
        • Hazai E.
        • Yan L.
        • et al.
        Identification of a polyoxometalate inhibitor of the DNA binding activity of Sox2.
        ACS Chem Biol. 2011; 6: 573-581
        • Ogawara Y.
        • Kishishita S.
        • Obata T.
        • Isazawa Y.
        • Suzuki T.
        • Tanaka K.
        • et al.
        Akt enhances Mdm2-mediated ubiquitination and degradation of p53.
        J Biol Chem. 2002; 277: 21843-21850
        • Ohanna M.
        • Cheli Y.
        • Bonet C.
        • Bonazzi V.F.
        • Allegra M.
        • Giuliano S.
        • et al.
        Secretome from senescent melanoma engages the STAT3 pathway to favor reprogramming of naïve melanoma towards a tumor-initiating cell phenotype.
        Oncotarget. 2013; 4: 2212-2224
        • Oltvai Z.N.
        • Milliman C.L.
        • Korsmeyer S.J.
        Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death.
        Cell. 1993; 74: 609-619
        • Ostojic A.
        • Vrhovac R.
        • Verstovsek S.
        Ruxolitinib: a new JAK1/2 inhibitor that offers promising options for treatment of myelofibrosis.
        Future Oncol. 2011; 7: 1035-1043
        • Pandolfi S.
        • Montagnani V.
        • Lapucci A.
        • Stecca B.
        HEDGEHOG/GLI-E2F1 axis modulates iASPP expression and function and regulates melanoma cell growth.
        Cell Death Differ. 2015; 22: 2006-2019
        • Patel S.P.
        • Kim K.B.
        • Papadopoulos N.E.
        • Hwu W.J.
        • Hwu P.
        • Prieto V.G.
        • et al.
        A phase II study of gefitinib in patients with metastatic melanoma.
        Melanoma Res. 2011; 21: 357-363
        • Perry B.N.
        • Govindarajan B.
        • Bhandarkar S.S.
        • Knaus U.G.
        • Valo M.
        • Sturk C.
        • et al.
        Pharmacologic blockade of angiopoietin-2 is efficacious against model hemangiomas in mice.
        J Invest Dermatol. 2006; 126: 2316-2322
        • Rybak A.P.
        • Tang D.
        SOX2 plays a critical role in EGFR-mediated self-renewal of human prostate cancer stem-like cells.
        Cell Signal. 2013; 25: 2734-2742
        • Santini R.
        • Pietrobono S.
        • Pandolfi S.
        • Montagnani V.
        • D’Amico M.
        • Penachioni J.Y.
        • et al.
        SOX2 regulates self-renewal and tumorigenicity of human melanoma-initiating cells.
        Oncogene. 2014; 33: 4697-4708
        • Santini R.
        • Vinci M.C.
        • Pandolfi S.
        • Penachioni J.Y.
        • Montagnani V.
        • Olivito B.
        • et al.
        Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.
        Stem Cells. 2012; 30: 1808-1818
        • Schlaak M.
        • Schmidt P.
        • Bangard C.
        • Kurschat P.
        • Mauch C.
        • Abken H.
        Regression of metastatic melanoma in a patient by antibody targeting of cancer stem cells.
        Oncotarget. 2012; 3: 22-30
        • Stolzenburg S.
        • Rots M.G.
        • Beltran A.S.
        • Rivenbark A.G.
        • Yuan X.
        • Qian H.
        • et al.
        Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer.
        Nucleic Acids Res. 2012; 40: 6725-6740
        • Wakeling A.E.
        • Guy S.P.
        • Woodburn J.R.
        • Ashton S.E.
        • Curry B.J.
        • Barker A.J.
        • et al.
        ZD1839 (Iressa): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy.
        Cancer Res. 2002; 62: 5749-5754
        • Weina K.
        • Utikal J.
        SOX2 and cancer: current research and its implications in the clinic.
        Clin Transl Med. 2014; 3: 19
        • Yamaguchi M.
        • Vikulina T.
        • Weitzmann M.N.
        Gentian violet inhibits MDA-MB-231 human breast cancer cell proliferation, and reverses the stimulation of osteoclastogenesis and suppression of osteoblast activity induced by cancer cells.
        Oncol Rep. 2015; 34: 2156-2162
        • Yamaura M.
        • Mitsushita J.
        • Furuta S.
        • Kiniwa Y.
        • Ashida A.
        • Goto Y.
        • et al.
        NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression.
        Cancer Res. 2009; 69: 2647-2654
        • Yin D.
        • Li Y.
        • Lin H.
        • Guo B.
        • Du Y.
        • Li X.
        • et al.
        Functional graphene oxide as a plasmid-based Stat3 siRNA carrier inhibits mouse malignant melanoma growth in vivo.
        Nanotechnology. 2013; 24: 105102
        • Zhang X.
        • Zheng Y.
        • Fried L.E.
        • Du Y.
        • Montano S.J.
        • Sohn A.
        • et al.
        Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes.
        Free Radic Biol Med. 2011; 50: 811-820
        • Zhao D.
        • Pan C.
        • Sun J.
        • Gilbert C.
        • Drews-Elger K.
        • Azzam D.J.
        • et al.
        VEGF drives cancer-initiating stem cells through VEGFR-2/Stat3 signaling to upregulate Myc and Sox2.
        Oncogene. 2015; 34: 3107-3119