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. 2015 Mar 6;6(11):8722–8735. doi: 10.18632/oncotarget.2726

Figure 7. GANT61 and PI103 cooperate to suppress clonogenic survival, 3D sphere formation and tumor growth in vivo.

Figure 7

(A and B) RD cells were treated for 18 hours with 1 μM PI103 and/or 6 μM GANT61 and clonogenic survival was assessed by colony formation assay at day 10. Representative images are shown (A); the number of colonies was counted after crystal violet staining and is expressed as percentage of untreated controls (B) (C) RD cells were treated for 24 hours with 1 μM PI103 and/or 6 μM and spheres were counted after 15 days. (D and E), RD cells were seeded on the CAM of fertilized chicken eggs, treated with 2 μM PI103 and/or 20 μM GANT61 for three days and tumor growth was analyzed using H/E-stained paraffin sections of the CAM. Representative pictures of at least 15 tumors (D) and quantification of tumor area (E) are shown. Mean + S.D. of three independent experiments performed at least in triplicate are shown (B, C, E); *p < 0.05; **p < 0.01. (F) Scheme of the proposed mechanism of GANT61/PI103 induced mitochondrial apoptosis. GANT61 and PI103 treatment results in NOXA and BMF upregulation and subsequently BAX and BAK activation. BAX/BAK activation leads to cleavage of caspase-9 and activation of caspase-3 as execution pathway of apoptosis. GANT61/PI103-induced apoptosis is inhibited by overexpression of BCL-2 or phospho-mutant MCL-1, by knockdown of NOXA, BMF or BAK or by caspase inhibitor zVAD.fmk.