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.2013 Jul 30;8(7):e69064.
doi: 10.1371/journal.pone.0069064. Print 2013.

NVP-LDE225, a potent and selective SMOOTHENED antagonist reduces melanoma growth in vitro and in vivo

Affiliations

NVP-LDE225, a potent and selective SMOOTHENED antagonist reduces melanoma growth in vitro and in vivo

Ahmad Jalili et al. PLoS One..

Erratum in

  • PLoS One. 2013;8(9). doi:10.1371/annotation/ddd22094-5d8d-43ef-ad81-b95afe392ec7

Abstract

Melanoma is one of the most aggressive cancers and its incidence is increasing worldwide. So far there are no curable therapies especially after metastasis. Due to frequent mutations in members of the mitogen-activated protein kinase (MAPK) signaling pathway, this pathway is constitutively active in melanoma. It has been shown that the SONIC HEDGEHOG (SHH)-GLI and MAPK signaling pathway regulate cell growth in many tumors including melanoma and interact with each other in the regulation of cell proliferation and survival. Here we show that the SHH-GLI pathway is active in human melanoma cell lines as they express downstream target of this pathway GLI1. Expression of GLI1 was significantly higher in human primary melanoma tissues harboring BRAF(V600E) mutation than those with wild type BRAF. Pharmacologic inhibition of BRAF(V600E) in human melanoma cell lines resulted in decreased expression of GLI1 thus demonstrating interaction of SHH-GLI and MAPK pathways. Inhibition of SHH-GLI pathway by the novel small molecule inhibitor of smoothened NVP-LDE225 was followed by inhibition of cell growth and induction of apoptosis in human melanoma cell lines, interestingly with both BRAF(V600E) and BRAF(Wild Type) status. NVP-LDE225 was potent in reducing cell proliferation and inducing tumor growth arrest in vitro and in vivo, respectively and these effects were superior to the natural compound cyclopamine. Finally, we conclude that inhibition of SHH-GLI signaling pathway in human melanoma by the specific smoothened inhibitor NVP-LDE225 could have potential therapeutic application in human melanoma even in the absence of BRAF(V600E) mutation and warrants further investigations.

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Conflict of interest statement

Competing Interests:Frank Kalthoff and Anton Stuetz are employed by Novartis Institutes for Biomedical Research in Vienna, Austria. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Evaluation of theGLI1 expression in human melanoma cell lines and human primary melanoma tissues.
A) RelativeGLI1 RNA expression in human melanoma cell lines and normal human melanocytes as measured by quantitative RT-PCR.B)GLI1 RNA expression in human primary melanoma tissues harboring wild type (n = 7) or V600E activating mutation in BRAF (n = 15) evaluated by Affymetrix gene profiling. P<0.05 is considered significant. Mean values with SD are shown. ANOVA test with Tukey's post-test.
Figure 2
Figure 2. Inhibition of BRAFV600E, expression of GLI1 and SHH-GLI pathway inhibition by NVP-LDE225 in human melanoma cellsin vitro.
A) LOX IMVI and UACC 257 with PLX-4032 at the dose of 1 µM for 24 hr. subsequently protein lystaes prepared and subjected to WB analysis for the expression of GLI1 and phospho-ERK1/2. B) Effect of NVP-LDE225 on PTCH1 promoter. In total, 1 µg ofPTCH1 pGL3b-hPTCH1-prom-wt or pGL3b-hPTCH1-prom-mut luciferase construct and reporter were cotransfected into LOX IMVI cells. Cells were subsequently treated with 10 µM of NVP-LDE225 or cyclopamine for 4 hours (time point selected base on kinetic experiments). Fold activation was calculated relative to cells transfected with 3 µg of pB-actin-RL. One representative experiment of 2 is shown.
Figure 3
Figure 3. NVP-LDE225 and human melanoma cell cycle.
Human melanoma cell lines were synchronized by thymidine and then treated with vehicle (DMSO), NVP-LDE225 or cyclopamine (each at 10 µM concentration). Cell cycle analysis performed 8 hr later. One representative experiment of 3 is shown.
Figure 4
Figure 4. The effect of NVP-LDE225 on human melanoma cell viability.
Human melanoma cell lines LOX IMVI, MEWO, SK-MEL-2, UACC 257, WM 115 and MEL FH were treated with different concentrations of NVP-LDE225, cyclopamine or the vehicle (DMSO). Cell viability at specific time points was measured by MTT assay. Experiments were performed in triplicates. One representative experiment is shown. Mean values with SD are shown.
Figure 5
Figure 5. LDE225 induces apoptosis in human melanoma cell lines.
Annexin V/PI staining of human melanoma cell lines after 48 hr of treatment with NVP-LDE225, cyclopamine (each at 10 µM concentration) or DMSO. Annexin V+/PI− are apoptotic cells. Experiments were performed 3 times with similar results. One representative experiment is shown.
Figure 6
Figure 6. LDE225 antitumor activity in xenotransplantation model of human melanoma.
A & B) 1×106 LOX OMVI human melanoma cells suspended in PBS containing 10% FCS were injected s.c into both flanks. As tumors reach the mean volume of 48 mm3, NVP-LDE225 or cyclopamine were injected on daily basis at doses of 2, 20 or 200 µg/shot. (* p<0.05, ** p<0.01).C) 7.5×105 of LOX IMVI cells were inoculated as above. Tumors with the mean volume of 18 mm3 were treated intratumorally on daily basis with NVP-LDE225 or vehicle. D) Photographs of mice after treatment with NVP-LDE225, cyclopamine or vehicle showing tumor volumes.
Figure 7
Figure 7.In vivo GLI1 expression after intratumoral administration of NVP-LDE225.
LOX OMVI human melanoma cells were injected s.c into both flanks as mentioned above. Tumors were treated intratumorally on daily basis with vehicle (A & B) or NVP-LDE225 (C & D). Immunofluorescent microscopy of GLI1was performed on isolated tumor tissues. GLI1 staining was performed by overnight incubation of sections at 4°C with rabbit anti-human polyclonal Ab (B & D, NBP1-78259, Novus Biologicals, Littleton, CO) or isotype control (A & C) followed by an 1 hr-incubation with Alexa Fluor® 488 Donkey IgG, anti-rabbit (A21206, Invitrogen, Carlsbad, CA) at RT (green). Counterstaining of nuclei was performed with propidium iodide (red). Pictures were taken on a confocal laser-scanning microscope system (LSM 410; Zeiss). Yellow color corresponds to double positive (anti-GLI1 and propidium iodide) nuclear staining.
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