The MTH1 inhibitor TH588 demonstrates anti-tumoral effects alone and in combination with everolimus, 5-FU and gamma-irradiation in neuroendocrine tumor cells

Abstract

Modulation of the redox system in cancer cells has been considered a promising target for anti-cancer therapy. The novel MTH1 inhibitor TH588 proved tremendous potential in terms of cancer cell eradication, yet its specificity has been questioned by recent reports, indicating that TH588 may also induce cancer cell death by alternative mechanisms than MTH1 inhibition. Here we used a panel of heterogeneous neuroendocrine tumor cells in order to assess cellular mechanisms and molecular signaling pathways implicated in the effects of TH588 alone as well as dual-targeting approaches combining TH588 with everolimus, cytotoxic 5-fluorouracil or γ-irradiation. Our results reflect that TH588 alone efficiently decreased the survival of neuroendocrine cancer cells by PI3K-Akt-mTOR axis downregulation, increased apoptosis and oxidative stress. However, in the dual-targeting approaches cell survival was further decreased due to an even stronger downregulation of the PI3K-Akt-mTOR axis and augmentation of apoptosis but not oxidative stress. Furthermore, we could attribute TH588 chemo- and radio-sensitizing properties. Collectively our data not only provide insights into how TH588 exactly kills cancer cells but also depict novel perspectives for combinatorial treatment approaches encompassing TH588.

Fig 1. TH588 effectively decreases cellular survival in heterogeneous neuroendocrine tumor cells.

(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.

Fig 2. TH588 treatment causes apoptotic cell death.

(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 = ***.

Fig 3. TH588 causes PI3K-Akt-mTOR pathway and pathway related growth factor receptor downregulation.

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.

Fig 4. Dual-targeting approaches show agonistic effects in cell survival decrease due to either apoptotic cell death enhancement or cooperative PI3K-Akt-mTOR pathway downregulation.

(A) Effect of TH588 on cell survival. Human neuroendocrine pancreatic BON1 and pancreatic islet QGP1 cells were incubated with TH588 (5 μM and 10 μM) alone and in combination (TH588 (5 μM)) with 5-FU (5 μM) and everolimus (10 nM) for 96 h and 144 h. 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 components from PI3K-Akt-mTOR pathway and the apoptotic cell apparatus were analyzed with TH588 alone (5 μM and 10 μM) alone and in combination with 5-FU (5 μM) and everolimus (10 nM) after 96 h. A representative blot out of three independently performed experiments is shown, together with the densitometry quantification.

Fig 5. TH588 causes oxidative stress and serves as radio-sensitizing adjuvant.

(A) Displayed is the relative oxidative stress after 96 h of incubation with TH588 alone (5 μM) and in combination with 5-FU (5 μM) and everolimus (10 nM). 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 = ***. (B) Western blot analysis of components from DNA damage response and MTH1 are displayed after 96 h of incubation with TH588 alone and in combination with 5-FU and everolimus. A representative blot out of three independently performed experiments is shown.

Fig 6. TH588 as a radio-sensitizing adjuvant in BON1 and QGP1 cells.

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 = ***.