Insulin induces drug resistance in melanoma through activation of the PI3K/Akt pathway

Abstract

Introduction: There is currently no curative treatment for melanoma once the disease spreads beyond the original site. Although activation of the PI3K/Akt pathway resulting from genetic mutations and epigenetic deregulation of its major regulators is known to cause resistance of melanoma to therapeutic agents, including the conventional chemotherapeutic drug dacarbazine and the Food and Drug Administration-approved mutant BRAF inhibitors vemurafenib and dabrafenib, the role of extracellular stimuli of the pathway, such as insulin, in drug resistance of melanoma remains less understood.

Objective: To investigate the effect of insulin on the response of melanoma cells to dacarbazine, and in particular, the effect of insulin on the response of melanoma cells carrying the BRAF(V600E) mutation to mutant BRAF inhibitors. An additional aim was to define the role of the PI3K/Akt pathway in the insulin-triggered drug resistance.

Methods: The effect of insulin on cytotoxicity induced by dacarbazine or the mutant BRAF inhibitor PLX4720 was tested by pre-incubation of melanoma cells with insulin. Cytotoxicity was determined by the MTS assay. The role of the PI3K/Akt pathway in the insulin-triggered drug resistance was examined using the PI3K inhibitor LY294002 and the PI3K and mammalian target of rapamycin dual inhibitor BEZ-235. Activation of the PI3K/Akt pathway was monitored by Western blot analysis of phosphorylated levels of Akt.

Results: Recombinant insulin attenuated dacarbazine-induced cytotoxicity in both wild-type BRAF and BRAF(V600E) melanoma cells, whereas it also reduced killing of BRAF(V600E) melanoma cells by PLX4720. Nevertheless, the protective effect of insulin was abolished by the PI3K and mTOR dual inhibitor BEZ-235 or the PI3K inhibitor LY294002.

Conclusion: Insulin attenuates the therapeutic efficacy of dacarbazine and PLX4720 in melanoma cells, which is mediated by activation of the PI3K/Akt pathway and can be overcome by PI3K inhibitors.

Keywords: BRAF inhibitors; DTIC; PI3K/Akt; drug resistance; insulin; melanoma.

Figure 1

Cytotoxicity of DTIC towards melanoma cells. Notes: B16 mouse melanoma cells and Mel-RMu human melanoma cells were treated with DTIC at indicated concentrations for 24 hours. Cell viability was measured by MTS assays and expressed as a relative value of control. The data shown are the mean ± standard error of three individual experiments. Abbreviations: DTIC, dacarbazine; MTS, CellTiter 96® aqueous one solution cell proliferation.

Figure 2

Insulin protects melanoma cells against DTIC. Notes: B16 (upper panel) and Mel-RMu (lower panel) cells with or without pretreatment with insulin at indicated concentrations for 15 minutes were treated with DTIC (25 μg/mL) for a further 24 hours. Cell viability was measured by MTS assays. The data shown are the mean ± standard error of three individual experiments (*P<0.01, Student’s t-test). Abbreviations: DTIC, dacarbazine; MTS, CellTiter 96® aqueous one solution cell proliferation.

Figure 3

Insulin protects BRAF^V600E melanoma cells against the BRAF inhibitor PLX4720. Notes: Mel-RMu cells with or without pretreatment with insulin (250 nM) for 15 minutes were treated with PLX4720 (5 μM) for a further 24 hours. Cell viability was measured by MTS assays. The data shown are the mean ± standard error of three individual experiments (*P<0.01, Student’s t-test). Abbreviations: MTS, CellTiter 96® aqueous one solution cell proliferation; BRAF, v-Raf murine sarcoma viral oncogene homolog B1.

Figure 4

Insulin activates the PI3K/Akt in melanoma cells. Notes: Whole cell lysates from B16 and Mel-RMu cells with or without treatment with insulin (250 nM) for 15 minutes were subjected to Western blot analysis of phosphorylated Akt, Akt, and GAPDH (as a loading control). The data shown are representative of three individual experiments. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Akt, protein kinase B.

Figure 5

The PI3K and mTOR dual inhibitor BEZ-235 reverses protection of melanoma cells against DTIC and/or PLX4720 by insulin. Notes: (A) Whole cell lysates from B16 and Mel-RMu cells with or without pretreatment with BEZ-235 (50 nM) for 1 hour followed by exposure to insulin (250 nM) for a further 15 minutes were subjected to Western blot analysis of phosphorylated Akt, Akt, and GAPDH (as a loading control). The data shown are representative of three individual experiments. (B) B16 (upper panel) and Mel-RMu (lower panel) cells were pretreated with BEZ-235 (50 nM) for 1 hour before the addition of insulin (250 nM) for 15 minutes followed by exposure to DTIC (25 μg/mL) for a further 24 hours. Cell viability was measured by MTS assays. The data shown are the mean ± standard error of three individual experiments (*P,0.01, Student’s t-test). (C) Mel-RMu cells were pretreated with BEZ-235 (50 nM) for 1 hour before the addition of insulin (250 nM) for 15 minutes followed by exposure to PLX4720 (5 μM) for a further 24 hours. Cell viability was measured by MTS assays. The data shown are the mean ± standard error of three individual experiments (*P<0.01, Student’s t-test). Abbreviations: DTIC, dacarbazine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Akt, protein kinase B; MTS, CellTiter 96® aqueous one solution cell proliferation.

Figure 6

The PI3K inhibitor LY294002 reverses protection of melanoma cells against DTIC and/or PLX4720 by insulin. Notes: (A) Whole cell lysates from B16 and Mel-RMu cells with or without pretreatment with LY294002 (20 μM) for 1 hour followed by exposure to insulin (250 nM) for a further 15 minutes were subjected to Western blot analysis of phosphorylated Akt, Akt, and GAPDH (as a loading control). The data shown are representative of three individual experiments. (B) B16 (upper panel) and Mel-RMu (lower panel) cells were pretreated with LY294002 (20 μM) for 1 hour before the addition of insulin (250 nM) for 15 minutes followed by exposure to DTIC (25 μg/mL) for a further 24 hours. Cell viability was measured by MTS assays. The data shown are the mean ± standard error of three individual experiments (*P<0.01, Student’s t-test). (C) Mel-RMu cells were pretreated with LY294002 (20 μM) for 1 hour before the addition of insulin (250 nM) for 15 minutes followed by exposure to PLX4720 (5 μM) for a further 24 hours. Cell viability was measured by MTS assays. The data shown are the mean ± standard error of three individual experiments (*P<0.01, Student’s t-test). Abbreviations: DTIC, dacarbazine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Akt, protein kinase B; MTS, CellTiter 96® aqueous one solution cell proliferation.