Three generations of mTOR kinase inhibitors in the activation of the apoptosis process in melanoma cells

Abstract

Many signaling pathways are involved in the mammalian target of rapamycin (mTOR), and this serine/threonine kinase regulates the most important cellular processes such as cell proliferation, autophagy, and apoptosis. The subject of this research was the effect of protein kinase inhibitors involved in the AKT, MEK, and mTOR kinase signaling pathways on the expression of pro-survival proteins, activity of caspase-3, proliferation, and induction of apoptosis in melanoma cells. The following inhibitors were used: protein kinase inhibitors such as AKT-MK-2206, MEK-AS-703026, mTOR-everolimus and Torkinib, as well as dual PI3K and mTOR inhibitor-BEZ-235 and Omipalisib, and mTOR1/2-OSI-027 inhibitor in single-mode and their combinations with MEK1/2 kinase inhibitor AS-703026. The obtained results confirm the synergistic effect of nanomolar concentrations of mTOR inhibitors, especially the dual PI3K and mTOR inhibitors (Omipalisib, BEZ-235) in combination with the MAP kinase inhibitor (AS-703026) in the activation of caspase 3, induction of apoptosis, and inhibition of proliferation in melanoma cell lines. Our previous and current studies confirm the importance of the mTOR signal transduction pathway in the neoplastic transformation process. Melanoma is a case of a very heterogeneous neoplasm, which causes great difficulties in treating this neoplasm in an advanced stage, and the standard approach to this topic does not bring the expected results. There is a need for research on the search for new therapeutic strategies aimed at particular groups of patients. Effect of three generations of mTOR kinase inhibitors on caspase-3 activity, apoptosis and proliferation in melanoma cell lines.

Keywords: Apoptosis; Caspase-3 activity; Immunosuppressive treatment; Melanoma; Proliferation; mTOR inhibitors.

Fig. 1

Effect of mTOR inhibitors on the expression of phospho-mTOR (Ser2441) and Phospho-mTOR (Ser2448). Actin was used as a loading control. The densitometric analysis of phospho-mTOR (Ser2441) and Phospho-mTOR (Ser2448) was normalized against its corresponding β-actin data point. The data obtained from three separate analyses are expressed as mean ± SD. Statistical analyses were performed using one-way ANOVA with a post hoc Dunett test (Statistica 12.0 StatSoft); significant differences from control values are indicated as (*) p < 0.05, (**) p < 0.01, (***) p < 0.001

Fig. 2

Effect of the combination of mTOR inhibitors with the MEK1/2 inhibitor AS-703026 on the expression of pro-survival proteins in MEWO melanoma cell lines. Expression of pro-survival proteins. Actin was used as a loading control. The densitometric analysis of pro-survival protein was normalized against its corresponding β-actin data point. The data obtained from three separate analyses are expressed as mean ± SD. Statistical analyses were performed using one-way ANOVA with a post hoc Dunett test (Statistica 12.0 StatSoft); significant differences from control values are indicated as (*) p < 0.05, (**) p < 0.01, (***) p < 0.001

Fig. 3

Effect of mTOR kinase inhibitors on caspase-3 activity (A) and cell proliferation in WM 3211 melanoma cell lines (B). Caspase-3 activity (A) and cell proliferation—crystal violet assay (B) were calculated from the mean values of three independent experiments. Each value was expressed as a ratio of caspase-3 activity or cell proliferation level to the control level; the control value was set to 1 for caspase-3 activity and 100% for cell proliferation. The data are presented as mean ± standard deviation; Statistical analyses were performed using one-way ANOVA with a post hoc Dunett test (Statistica 12.0 StatSoft); significant differences from control values are indicated as (*) p < 0.05, (**) p < 0.01, (***) p < 0.001

Fig. 4

Effect of mTOR kinase inhibitors on caspase-3 activity (A) and cell proliferation in Mel-1359 melanoma cell lines (B). Caspase-3 activity (A) and cell proliferation—crystal violet assay (B) were calculated from the mean values of three independent experiments. Each value was expressed as the ratio of caspase-3 activity or cell proliferation level to the control level; the control value was set at 1 for caspase-3 activity and 100% for cell proliferation. The data are presented as mean ± standard deviation; Statistical analyses were performed using one-way ANOVA with a post hoc Dunett test (Statistica 12.0 StatSoft); significant differences from control values are indicated as (*) p < 0.05, (**) p < 0.01, (***) p < 0.001

Fig. 5

The effect of mTOR kinase inhibitors on caspase-3 activity (A) and cell proliferation in MEWO melanoma cell lines (B). Caspase-3 activity (A) and cell proliferation—crystal violet assay (B) were calculated from the mean values of three independent experiments. Each value was expressed as the ratio of caspase-3 activity or cell proliferation level to the control level; the control value was set at 1 for caspase-3 activity and 100% for cell proliferation. The data are presented as mean ± standard deviation; Statistical analyses were performed using one-way ANOVA with a post hoc Dunett test (Statistica 12.0 StatSoft); significant differences from control values are indicated as (*) p < 0.05, (**) p < 0.01, (***) p < 0.001

Fig. 6

The effect of mTOR kinase inhibitors on melanoma cell apoptosis—Mel-1359 (A) and MEWO (B). The data are presented as mean ± standard deviation; Statistical analyses were performed using one-way ANOVA with a post hoc Dunett test (Statistica 12.0 StatSoft); significant differences from control values are indicated as (*) p < 0.05, (***) p < 0.001. EF, enrichment factor (calculated to estimate the fold increase in DNA fragmentation in treated samples with reference to the control)

Fig. 7

Morphological changes of MEWO cells after treatment with mTOR inhibitors for 24 h followed by DAPI staining. Apoptosis was confirmed by DAPI staining, which showed apparent changes in the nuclear morphology (chromatin condensation and nuclear fragmentation) of the MEWO cells. The concentrations of the inhibitors used are described in the Materials and Methods. The experiments were performed in triplicate