Fig. 8.

Interfering Notch and MAP4K4 signaling pathways in OC cells. Panel A: combination treatment of cisplatin and GSIs. SKOV3^CP cells were treated under the following conditions: (A) medium control, (B) 2 µg/ml cisplatin, (C) 2 µg/ml cisplatin + 5 µM DAPT, and (D) 2 µg/ml cisplatin + 1 µM RG-4733 for 120 h. The simultaneous administration of cisplatin and GSIs did not reverse the neuronal morphology of SKOV3^CP cells (A-D). Additionally, this combination did not lead to significant changes in Notch3 expression (E). However, the incubation with both GSIs induced an upregulation of PAX6 and ChgA, with RG-4733 exerting the most pronounced effect. Panel B: combination treatment of MAP4K4i and GSIs in OC Cells. The exposure of OC cells to MAP4K4i did not induce general changes as expected in HES1 protein levels across various OC cell lines. However, both GSIs significantly downregulated HES1 protein expression (A-D). Notch3 expression was stimulated by all inhibitors in both SKOV3^WT and SKOV3^CP cells (E). In OVCAR-3 and primary OC cells OC236, only the combination of MAP4K4i with RG-4733 synergistically enhanced Notch3 expression (G-H). Furthermore, βIII-tubulin expression was strongly stimulated by the MAP4K4i in SKOV3^WT cells, an effect not observed in the resistant SKOV3^CP subline. Nevertheless, the combination of MAP4K4i with GSIs led to an enhanced expression of this neuronal-related structural protein (I-J). ChgA expression was slightly repressed in SKOV3^WT cells but enhanced in SKOV3^CP cells after 24 h of MAP4K4 inhibitor treatment. Both GSIs increased ChgA expression, and this effect was slightly potentiated in the presence of the MAP4K4 inhibitor (K-L). Abbreviations: M: MAP4K4i; D: DAPT; RG: RG-4733; AU: arbitrary units. Bar graphs represent densitometric analyses of western blots performed to measure protein expression following drug exposure. Results are representative of n = 3 independent experiments.