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

Effect of mTOR inhibitors on the expression of pro-survival proteins

We investigated the effect of three generations of mTOR kinase inhibitors alone and in combination with the MEK1/2 kinase inhibitor AS-703026 on the expression of pro-survival proteins: p-Bcl-2 (T56), Bcl-2, Bcl-xL, and Mcl-1 in MEWO melanoma cell line. All the used mTOR kinase inhibitors, both of the first generation—everolimus and Torkinib, as well as the mTOR 1/2 -OSI-027 inhibitor, and dual PI3K and mTOR inhibitor, BEZ-235, Omipalisib, inhibited the expression of the phosphorylated form of phospho-mTOR (Ser2481) almost completely and significantly inhibited forms of phospho-mTOR (Ser2448) (Fig. 1). The AKT kinase inhibitor—MK-2206 showed similar results. The effect was even more pronounced for the combination of mTOR inhibitors with the MEK1/2 inhibitor—AS-703026 (Fig. 1).

Fig. 1figure 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

The used mTOR kinase inhibitors decreased the expression of pro-survival proteins significantly; the addition of the combination with the MEK1/2—AS-703026 inhibitor resulted in an additional synergistic effect in inhibiting the expression of Bcl-2 family proteins (Fig. 2).

Fig. 2figure 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

The expression of the p-Bcl-2 (T56) protein decreased by about 80% after the use of Omipalisib inhibitors; a slightly less pronounced effect was observed for the everolimus inhibitor and the dual inhibitor of the mTOR and AKT pathway, BEZ-235 (Fig. 2). Bcl-2 protein levels also decreased significantly (Fig. 2). The use of a combination of mTOR inhibitors with the MEK1/2 inhibitor AS-703026 almost stopped the expression of these proteins (Fig. 2).

The Bcl-xL protein level was also reduced to approximately 40–60%, in which case the AS-703026 inhibitor showed no clear enhancing effect (Fig. 2). On the other hand, for the Mcl-1 protein, a weaker decrease in expression was obtained after the use of mTOR kinase inhibitors at the level of 40%; the use of combination with an AS-703026 inhibitor did not result in an enhancement (Fig. 2).

Effect of mTOR and PI3K kinase inhibitors on caspase-3 activity and proliferation

We investigated the effect of protein kinase inhibitors involved in the AKT, MEK, and mTOR kinase signaling pathways on caspase-3 activation and proliferation in WM3211, Mel-1359, and MEWO melanoma cell lines. We used protein kinase inhibitors such as AKT—MK-2206, MEK1/2—AS-703026, mTOR—everolimus and Torkinib, mTOR1/2—OSI-027 inhibitor, as well as dual PI3K and mTOR inhibitor—BEZ-235, and Omipalisib in single mode, and their combinations with the MEK1/2 kinase inhibitor AS-703026.

For the primary melanoma cell line—WM3211 (VGP), with a wild type for BRAF, PTEN, N-RAS, and CDK4, and with a mutation at position 576 in the c-KIT gene, caspase 3 activation compared to the control was several times lower than for the metastatic lines Mel-1359 and MEWO (Figs. 3, 4 and 5a).

Fig. 3figure 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. 4figure 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. 5figure 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

In both metastasis-derived lines: Mel-1359 and MEWO, the use of single inhibitors and their combination with the MEK1/2-AS703026 inhibitor produced very similar trends of increased caspase 3 activity, with the increase slightly higher for the latter (Figs. 4, 5a).

The highest increases in caspase 3 activity were observed for the use of individual inhibitors compared to the control for the MEK1/2-AS-703026 inhibitor for the Mel-1359 line. The observed increase was approximately tenfold (p < 0.001) and 15-fold (p < 0.001) for 24 and 48 h (Fig. 4a), respectively. It was slightly lower for the AKT inhibitor—MK-2206: approximately 8 times (p < 0.001) for 24 h and 12 times (p < 0.001) for 48 h for the MEWO cell line (Fig. 5a). The remaining inhibitors showed increases in caspase activity of 3 to fivefold (p < 0.001) for 24 h and up to about sevenfold (p < 0.001) for 48 h (Figs. 4, 5a).

Treatment of melanoma cells with single inhibitors of PI3K and mTOR caused a decrease in proliferation compared to control cells, an effect that was the most pronounced for the Mel-1359 cell line (Figs. 3, 4 and 5b). Three inhibitors caused the highest decreases in proliferation during 24 h: BEZ-235, AS-703026, which saw a result of approximately 20% (p < 0.001) (Fig. 4b). The situation was similar for the 48 h period, during which time the decrease in proliferation reached 40% (p < 0.001) (Fig. 4b).

The use of the combination of PI3K and mTOR inhibitors with the MEK1/2 kinase inhibitor -AS-703026 resulted in the marked increase in caspase 3 activity compared to the use of single inhibitors (Figs. 3, 4 and 5a).

For both metastasis-derived lines: Mel-1359 and MEWO, a very similar trend of caspase 3 activation and a synergistic effect of the combination of inhibitors with the MEK1/2-AS703026 inhibitor (Figs. 4, 5a) were observed. For the primary line WM3211, an increase in caspase 3 activity was also observed after the use of the inhibitor combination, but these increases were up to 5 times lower than those of the metastatic lines and also showed a slightly different inhibitor preference (Figs. 3, 4 and 5a).

The largest and very similar activation of caspase 3 compared to the control occurred after the use of combinations of the MEK1/2-AS703026 inhibitor with dual PI3K kinase and mTOR kinase inhibitors: Omipalisib or BEZ-235.

The observed increase in caspase 3 activity was 30 and 27 times (p < 0.001) for the Mel-1359 line (Fig. 4a) for each of the combinations, respectively. A slightly lower increase in caspase 3 activity was observed (by approximately 20–25 times) for the use of the combination of inhibitors PI3K-MK-2206 and mTOR-everolimus together with the inhibitor MEK1/2—AS703026 (Fig. 4a).

On the other hand, the highest caspase 3 activation was achieved after cell treatment with the combination of mTOR inhibitors (everolimus or OSI-027 or Torkinib) with the MEK1/2 kinase inhibitor AS703026 (Fig. 4a). It was approximately 23 times (p < 0.001) after 48 h compared to the control. For the primary line WM3211, the highest, although small compared to other cell lines, were increases in caspase 3 activity after the combination of the MEK1/2 inhibitor-AS703026 with the following inhibitors: PI3K—MK2206 approximately 9 times (p < 0.001); the double inhibitors PI3K and mTOR BEZ-235 approximately 7 times (p < 0.001); and the inhibitor Omipalisib approximately 6 times (p < 0.001) (Fig. 3a).

The use of the combination of PI3K inhibitors, mTOR, with the MEK1/2 inhibitor AS703026 resulted in a marked decrease in melanoma cell proliferation (Figs. 3, 4 and 5b). These decreases reached even 50% for the combination with the inhibitor BEZ-235, Omipalisib, or MK-2206 (Figs. 3, 4 and 5b).

Effect of mTOR kinase inhibitors on DNA fragmentation ELISA assay: detection of apoptosis

For metastatic melanoma cell lines: Mel 1359 and MEWO, which showed the highest caspase 3 activity, the cell death detection ELISA assay that reflects DNA fragmentation in apoptotic cells was performed to verify the induction of apoptosis (Fig. 6).

Fig. 6figure 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)

As shown in Fig. 6, the activation profile of the apoptosis process for the Mel-1359 and MEWO melanoma cell lines, depending on the mTOR kinase inhibitor used, was quite similar and only slightly higher for the Mel-1359 cell line.

Except for the enrichment of the MEK1/2 inhibitor AS-703026 in the fragmented DNA content, none of the inhibitors used alone was considerably effective in triggering apoptosis.

The highest level of DNA apoptotic degradation was observed in response to concurrent application of the MEK 1/2 inhibitor—AS-703026 with dual inhibitors of PI3K kinase and mTOR kinase (Omipalisib or BEZ-235). A slightly lower effect was observed for the combination with the AKT inhibitor—MK-2206 or mTOR—everolimus (Fig. 6).

The apoptosis process was manifested by a significant increase in absorbance value compared to the untreated sample, with the enrichment factor EF (calculated to estimate the fold in-crease in DNA fragmentation in treated samples with reference to control one) for the Mel-1359 cell line reaching 276.98, and 285.74 for the MEK 1/2 inhibitor—AS-703026 combined with the dual PI3K kinase and mTOR kinase inhibitors Omipalisib or BEZ-235 (p < 0.001). A slightly lower effect was observed for the combination with the AKT inhibitor—MK-2206 or mTOR—everolimus, in which cases it reached 197.09 and 157.38, respectively (p < 0.001) (Fig. 6a). The result seems to suggest a synergistic effect of the applied agents.

Similarly, in the case of MEWO melanoma cells (Fig. 6b), each of the inhibitors used alone was hardly effective in induction of apoptosis, except the MEK1/2 inhibitor, AS-703026, manifested by a high EF value (~ 56). The use of the combination with the MEK1/2 AS-703036 inhibitor resulted in a slightly higher absorbance value, but the enrichment factor—EF values were slightly lower and were 204.79 and 219.12 (p < 0.001) for the combination with the dual inhibitors of PI3K kinase and mTOR kinase Omipalisib or BEZ-235, respectively. For the combination with mTOR—everolimus or the AKT inhibitor MK-2206, they reached 110.10 and 135.35 (p < 0.001), respectively (Fig. 6b).

To confirm the morphological changes at the level of the cell nucleus and lysosomes, we stained the cells with DAPI (Fig. 7) and acridine orange (supplementary materials).

Fig. 7figure 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

Cytotoxicity assay

Cytotoxicity of selected kinase inhibitors: AKT—MK-2206 (2 μM), MEK1/2—AS-703026 (10 μM), mTOR—everolimus (20 nM), dual PI3K and mTOR inhibitor—BEZ-235 (20 nM), dual PI3K and mTOR inhibitor—Omipalisib (20 nM), mTOR1/2—OSI-027 (20 nM), mTOR—Torkinib (20 nM) was determined using Cytotoxicity Detection Kit LDH, Roche, Germany.

The inhibitors MK-2206, AS-703026, everolimus, BEZ-235, Omipalisib, OSI-027 and Torkinib showed no cytotoxicity effect at 24 h of treatment in any of the examined melanoma cell lines. LDH activity in culture medium in no case exceeded 3.9%; however, after 72 h of treatment, the effect of cytotoxicity was observed for the combination of inhibitors: BEZ-235 with AS-703026 (10.5%) and Omipalisib with AS-703026 (11%). We also observed an increase in lactate dehydrogenase activity for the Mel-1359 cell line for a combination of inhibitors: AS-703026 and MK-2206 (5.9%). No cytotoxic effect was observed for the primary melanoma cell line WM3211 (VGP), with a wild type for BRAF, PTEN, N-RAS, and CDK4, and mutation at position 576 in the c-KIT gene, even for long incubation times—72 h.

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