Cell viability in HepG2 cells: HepG2 cells were pre-treated with lovastatin for 24h before everolimus was added, and the combination of both drugs was incubated for 48h (A) or 120h (B).

<p>The single drugs were incubated for the same time as in combination. The mean percentage of cell viability, relative to the untreated control, ± SEM (error bars) is shown. (A) Treatment with 5–20 μM lovastatin or with 10 nM everolimus alone significantly decreased HepG2 cell viability. Combination treatment with 10 μM lovastatin and 10 nM everolimus significantly more strongly reduced cell viability than each drug separately. (B) Treatment with 5–20 μM lovastatin or with 10 nM everolimus separately significantly decreased HepG2 cell viability. Combination treatment with 10–20 μM lovastatin and 10 nM everolimus significantly more strongly reduced cell viability compared to each drug given alone. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (compared to the control); @ P ≤ 0.05, @@ P ≤ 0.01, @@@ P ≤ 0.001 (combination compared to each drug separately).</p

Cell viability in MPC (A) and MTT (B) cells: MPC and MTT cells were pre-treated with lovastatin for 24h before everolimus was added, and the combination of both drugs was incubated for 48h.

<p>The single drugs were incubated for the same time as in combination. The mean percentage of cell viability, relative to the untreated control, ± SEM (error bars) is shown. Treatment with 10 μM lovastatin or with 10 nM everolimus separately significantly reduced MPC and MTT cell viability, compared to the control. 48h combination treatment with 10 μM lovastatin and 10 nM everolimus significantly more potently decreased MPC and MTT cell viability compared to each drug given separately. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (compared to the control); @ P ≤ 0.05, @@ P ≤ 0.01, @@@ P ≤ 0.001 (combination compared to each drug separately).</p

Cell viability in H727 cells: H727 cells were pre-treated with lovastatin for 24h before everolimus was added, and the combination of both drugs was incubated for 48h (A) or 120h (B).

<p>The single drugs were incubated for the same time as in combination. The mean percentage of cell viability, relative to the untreated control, ± SEM (error bars) is shown. (A) Treatment with 5–20 μM lovastatin or with 10 nM everolimus alone significantly decreased H727 cell viability. Combination treatment with 20 μM lovastatin and 10 nM everolimus significantly more strongly reduced cell viability than each drug separately. (B) Treatment with 5–20 μM lovastatin or with 10 nM everolimus separately significantly reduced H727 cell viability. The combination of 5–20 μM lovastatin with 10 nM everolimus significantly more strongly reduced cell viability compared to each drug given separately. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (compared to the control); @ P ≤ 0.05, @@ P ≤ 0.01, @@@ P ≤ 0.001 (combination compared to each drug separately).</p

Cell viability in BON1 cells: BON1 cells were pre-treated with lovastatin for 24h before everolimus was added, and the combination of both drugs was incubated for 48h (A) or 120h (B).

<p>The single drugs were incubated for the same time as in combination. The mean percentage of cell viability, relative to the untreated control, ± SEM (error bars) is shown. (A) Treatment with 5–10 μM lovastatin had no significant effect on BON1 cell viability. Treatment with 20 μM lovastatin or with 10 nM everolimus alone significantly decreased BON1 cell viability. There was no additive effect of both drugs. (B) Treatment with 5–10 μM lovastatin had no significant effect on BON1 cell viability. Treatment with 20 μM lovastatin or with 10 nM everolimus separately significantly reduced BON1 cell viability; combination treatment showed no additive effect. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (compared to the control); @ P ≤ 0.05, @@ P ≤ 0.01, @@@ P ≤ 0.001 (combination compared to each drug separately).</p

The effects of lovastatin and everolimus on the signaling pathways found in the present study: The dashed arrows show inconsistent effects on the signaling pathways in different cell lines.

<p>In all cell lines investigated, lovastatin led to suppression of EGFR and AKT signaling (green lines). Moreover, lovastatin was associated with ERK inhibition in BON1, HepG2 and Huh7 cell lines, but not in H727 cells (no effect on ERK signaling) (dashed green line). While leading to a massive increase of pp70S6K in HepG2 cells, lovastatin was accompanied by a mild to moderate decrease of pp70S6K in BON1, H727 and Huh7 cells (green dashed arrow). Everolimus, in contrast, always led to mTORC1/p70S6K inhibition (red line). However, everolimus was inconsistently associated with an increase of EGFR and AKT signaling: everolimus was accompanied by a strong increase of pEGFR in BON1 cells. In H727, HepG2 and Huh7 cells EGFR signaling was not markedly affected by everolimus (red dashed arrow). In H727 and Huh7 cells, everolimus led to a strong increase of pAKT, but in BON1 and HepG2 cells to a decrease of pAKT (red dashed arrows).</p

Signaling pathways in BON1, H727, HepG2 and Huh7 cells.

<p>Effects of 48h combination treatment with 20 μM lovastatin and 10 nM everolimus after 24h pre-treatment with lovastatin on signaling pathways in BON1, H727, HepG2 and Huh7 cells compared to treatment with each drug separately. Data are shown as mean percentage of the absolute phospho-protein expression, relative to the untreated control, ± SEM.</p

Representative Western blots: Effects of lovastatin and everolimus alone and in combination on EGFR, AKT, ERK and p70S6K signaling in BON1, H727, HepG2 and Huh7 cells: pEGFR was decreased in all cell lines after lovastatin treatment compared to the control—this was least in BON1 cells and increasingly stronger in Huh7, H727 and HepG2 cells.

<p>Apart from a clear up-regulation in BON1 cells after treatment with everolimus, everolimus hardly changed pEGFR in H727, HepG2 or Huh7 cells, compared to the control. Except for a very slight increase of pEGFR in BON1 cells, combination treatment decreased pEGFR in H727, HepG2 and Huh7 cells, compared to the control. pAKT was consistently suppressed in all cell lines after lovastatin treatment. After treatment with everolimus, there was a decrease of pAKT in BON1 and HepG2 cells, but a strong increase in H727 and Huh7 cells. Combination treatment led to a stronger suppression of pAKT than everolimus alone in all cell lines, and reduced pAKT compared to the control in BON1, H727 and HepG2. In Huh7 cells, combination treatment clearly attenuated the everolimus-induced increase of pAKT. Lovastatin treatment was accompanied by a strong decrease of pERK in BON1 cells, a clearly milder decrease in HepG2 and Huh7 cells, and no change of pERK in H727 cells. Everolimus led to a mild to moderate decrease of pERK in all cell lines. Apart from a slight increase of pERK in H727 cells, combination treatment was associated with suppression of pERK in BON1, HepG2 and Huh7 cells. Lovastatin treatment was accompanied by a very strong increase of pp70S6K in HepG2 cells. In BON1, H727 and Huh7 cells lovastatin led to a mild to moderate decrease of pp70S6K. Everolimus and combination treatment suppressed pp70S6K in all cell lines much more strongly than lovastatin did. Combination treatment completely inhibited p70S6K signaling in BON1, H727 and Huh7 cells, and clearly attenuated the lovastatin-induced pp70S6K up-regulation in HepG2 cells.</p

Neuroendocrine marker expression was increased by BYL719 treatment.

<p>A: BON-1, H727 and QGP-1 cells were treated with 10 μM BYL719 for 96 h and mRNA expression of Chromogranin A <i>(CHGA)</i>, <i>SSTR1</i>, <i>SSTR2</i> and <i>SSTR5</i> was analyzed by qPCR. B: BON-1, H727 and QGP-1 cells were treated with 1 μM BYL719 for 96 h and mRNA expression of <i>SSTR2</i> was analyzed by qPCR; Values are summarized from three independent experiments of three replicates per data point. * p<0.05; ** p≤0.01; *** p≤0.001.</p

Representative western blots of the investigated signaling pathways, apoptosis and cell cycle markers in BON-1, QGP-1 and H727 cells: The means, standard deviations and p-values of the western blot quantification and statistical analysis from at least three independent replicates are given in S1 Table.

<p>Representative western blots of the investigated signaling pathways, apoptosis and cell cycle markers in BON-1, QGP-1 and H727 cells: The means, standard deviations and p-values of the western blot quantification and statistical analysis from at least three independent replicates are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182852#pone.0182852.s031" target="_blank">S1 Table</a>.</p

CgA secretion of NET cells was not altered by BYL719 treatment.

<p>BON-1, H727 and QGP-1 cells were treated with vehicle, PDBu (positive control) or BYL719 (1 μM, 10 μM) for 24 h; the supernatant was collected for CgA measurements (two independent experiments with three replicates per data point).</p