TH588 effectively decreases cellular survival in heterogeneous neuroendocrine tumor cells.

<p>(A) Basal protein expression level of endogenous MTH1 in all four NET cell lines. The expression of MTH1 is evaluated by Western blot analysis. A representative blot out of three independently performed experiments is shown, together with densitometry quantification of 3 independent Western blots. (B) The effects of different concentrations of TH588 (100 nM to 10 μM) on cellular survival in neuroendocrine pancreatic BON1, pancreatic islet QGP1, bronchopulmonary H727 and ileal GOT1 cells are displayed after 144 h of incubation. The arithmetic means and standard deviation of at least three independent experiments are shown. Statistical significant different results in comparison to either single substance treatment are shown considering p<0,05 = *; p<0,01 = **; p<0,001 = ***. (C) 20% inhibitory concentration (IC₂₀) of TH588 (100 nM to 10 μM) in four different NET cell lines after 144 h of incubation.</p

TH588 treatment causes apoptotic cell death.

<p>(A) FACS analysis of BON1 and QGP1 cells after 72 h of incubation with TH588. The arithmetic means and standard deviation of at least three independent experiments are shown. Statistical significant different results in comparison to either sinlge substance treatment are shown considering p<0,05 = *; p<0,01 = **; p<0,001 = ***. (B) Western blot analysis of PARP and Caspase 3 cleavage in NETs. A representative blot out of three independently performed experiments is shown. (C) Caspase 3/7 activity in BON1 and QGP1 cells upon 72 h of incubation with TH588. The arithmetic means and standard deviation of at least three independent experiments are shown. Statistical significant different results in comparison to either sinlge substance treatment are shown considering p<0,05 = *; p<0,01 = **; p<0,001 = ***.</p

TH588 as a radio-sensitizing adjuvant in BON1 and QGP1 cells.

<p>The arithmetic means and standard deviation of at least three independent experiments are shown. Statistical significant different results in comparison to either single x-ray treatment are shown, considering p<0,05 = *; p<0,01 = **; p<0,001 = ***.</p

TH588 causes PI3K-Akt-mTOR pathway and pathway related growth factor receptor downregulation.

<p>Western blot analysis of components from the PI3K-Akt-mTOR pathway (Akt and 4EBP1) and 2 types of pathway related growth factor receptors (EGFR and IGFR) were analysed after 72 h of incubation with TH588 (5 μM and 10 μM). A representative blot out of three independently performed experiments is shown.</p

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

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

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