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Vemurafenib synergizes with nutlin-3 to deplete survivin and suppresses melanoma viability and tumor growth.

ABSTRACT: For patients with advanced melanoma, primary and secondary resistance to selective BRAF inhibition remains one of the most critically compelling challenges. One rationale argues that novel biologically informed strategies are needed to maximally cripple melanoma cells up front before compensatory mechanisms emerge. As p53 is uncommonly mutated in melanoma, restoration of its function represents an attractive adjunct to selective BRAF inhibition.Thirty-seven BRAF(V600E)-mutated melanoma lines were subjected to synergy studies in vitro using a combination of vemurafenib and nutlin-3 (Nt-3). In addition, cellular responses and in vivo efficacy were also determined. We also analyzed changes in the levels of canonical apoptotic/survival factors in response to vemurafenib.Dual targeting of BRAF(V600E) and Hdm2 with vemurafenib and Nt-3, respectively, synergistically induced apoptosis and suppressed melanoma viability in vitro and tumor growth in vivo. Suppression of p53 in melanoma cells abrogated Nt-3's effects fully and vemurafenib's effects partially. A survey of canonical survival factors revealed that both vemurafenib and Nt-3 independently attenuated levels of the antiapoptotic protein, survivin. Genetic depletion of survivin reproduces the cytotoxic effects of the combination strategy.These results show preclinical feasibility for overcoming primary vemurafenib resistance by restoring p53 function. Moreover, it identifies survivin as one downstream mediator of the observed synergism and a potential secondary target.

SUBMITTER: Ji Z

PROVIDER: S-EPMC3777641 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Vemurafenib synergizes with nutlin-3 to deplete survivin and suppresses melanoma viability and tumor growth.

Ji Zhenyu Z Kumar Raj R Taylor Michael M Rajadurai Anpuchchelvi A Marzuka-Alcalá Alexander A Chen Y Erin YE Njauw Ching-Ni Jenny CN Flaherty Keith K Jönsson Göran G Tsao Hensin H

Clinical cancer research : an official journal of the American Association for Cancer Research 20130627 16

<h4>Purpose</h4>For patients with advanced melanoma, primary and secondary resistance to selective BRAF inhibition remains one of the most critically compelling challenges. One rationale argues that novel biologically informed strategies are needed to maximally cripple melanoma cells up front before compensatory mechanisms emerge. As p53 is uncommonly mutated in melanoma, restoration of its function represents an attractive adjunct to selective BRAF inhibition.<h4>Experimental design</h4>Thirty- ...[more]

PMID: 23812671

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Project description:BACKGROUND:As the selective inhibitor of BRAF kinase, vemurafenib exhibits effective antitumor activities in patients with V600 BRAF mutant melanomas. However, acquired drug resistance invariably develops after its initial treatment. METHODS:Immunohistochemical staining was performed to detect the expression of iNOS and hTERT, p-p65, Epcam, CD44, PCNA in mice with melanoma xenografts. The proliferation and migration of melanoma cells were detected by MTT, tumorsphere culture, cell cycle, cell apoptosis, AO/EB assay and colony formation, transwell assay and scratch assay in vitro, and tumor growth differences were observed in xenograft nude mice. Changes in the expression of key molecules in the iNOS/hTERT signaling pathways were detected by western blot. Nucleus-cytoplasm separation, and immunofluorescence analyses were conducted to explore the location of p50/p65 in melanoma cell lines. Flow cytometry assay were performed to determine the expression of CD44. Pull down assay and ChIP assay were performed to detect the binding ability of p65 at iNOS and hTERT promoters. Additionally, hTERT promoter-driven luciferase plasmids were transfected in to melanoma cells with indicated treatment to determine luciferase activity of hTERT. RESULTS:Melatonin significantly and synergistically enhanced vemurafenib-mediated inhibitions of proliferation, colony formation, migration and invasion and promoted vemurafenib-induced apoptosis, cell cycle arresting and stemness weakening in melanoma cells. Further mechanism study revealed that melatonin enhanced the antitumor effect of vemurafenib by abrogating nucleus translocation of NF-?B p50/p65 and their binding at iNOS and hTERT promoters, thereby suppressing the expression of iNOS and hTERT. The elevated anti-tumor capacity of vemurafenib upon co-treatment with melatonin was also evaluated and confirmed in mice with melanoma xenografts. CONCLUSIONS:Collectively, our results demonstrate melatonin synergizes the antitumor effect of vemurafenib in human melanoma by inhibiting cell proliferation and cancer-stem cell traits via targeting NF-?B/iNOS/hTERT signaling pathway, and suggest the potential of melatonin in antagonizing the toxicity of vemurafenib and augmenting its sensitivities in melanoma treatment.

Project description:Melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppresses the growth of melanoma cells and induces reactive oxygen species generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here, we determined the potential mechanism of CAPE against melanoma in vivo and in vitro. Administration of 10 mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE-treated mice showed reduced phosphorylation of phosphoinositide 3-kinase, AKT, mammalian target of rapamycin and protein level of X-linked inhibitor of apoptosis protein (XIAP) and enhanced the cleavage of caspase-3 and poly (ADP ribose) polymerase. In order to confirm the in vivo observations, melanoma cells were treated with CAPE. CAPE treatment suppressed the activating phosphorylation of phosphoinositide 3-kinase at Tyr 458, phosphoinositide-dependent kinase-1 at Ser 241, mammalian target of rapamycin at Ser 2448 and AKT at Ser 473 in B16F0 and SK-MEL-28 cells in a concentration and time-dependent study. Furthermore, the expression of XIAP, survivin and BCL-2 was downregulated by CAPE treatment in both cell lines. Significant apoptosis was observed by CAPE treatment as indicated by cleavage of caspase-3 and poly (ADP ribose) polymerase. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. CAPE treatment increased the nuclear translocation of XIAP, indicating increased apoptosis in melanoma cells. To confirm the involvement of reactive oxygen species in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant N-acetyl-cysteine (NAC) prior to CAPE treatment. Our results indicate that NAC blocked CAPE-mediated AKT/XIAP inhibition and protected the cells from apoptosis. Because AKT regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE treatment decreased the interaction of AKT with XIAP. To establish the involvement of AKT in the apoptosis-inducing effects of CAPE, cells were transfected with AKT. Our results revealed that AKT overexpression attenuated the decrease in XIAP and significantly blocked CAPE-mediated apoptosis. Similarly, overexpression of XIAP further decreased CAPE-induced apoptosis. Taken together, our results suggest that CAPE suppresses phosphoinositide 3-kinase/AKT/XIAP pathway leading to apoptosis in melanoma tumor cells in vitro and in vivo.

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Vemurafenib synergizes with nutlin-3 to deplete survivin and suppresses melanoma viability and tumor growth.

Publications

Vemurafenib synergizes with nutlin-3 to deplete survivin and suppresses melanoma viability and tumor growth.

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