Figures & data
Figure 1. PDE5 inhibitors interact with established cytotoxic chemotherapy agents to kill multiple medulloblastoma cell lines. (A–D) D283, DAOY, HOSS1, and VC312 cells were treated with chemotherapy drugs and sildenafil, as indicated. Cells were isolated after 24 h and viability determined by trypan blue exclusion (n = 3, ± SEM) *P < 0.05 greater than corresponding value in vehicle control. (E) DAOY cells were treated with chemotherapy drugs and tadalafil, as indicated. Cells were isolated after 24 h and viability determined by trypan blue exclusion (n = 3, ± SEM) *P < 0.05 greater than corresponding value in vehicle control. (F) DAOY cells, plated as single cells were treated with etoposide and/or sildenafil for 24 h. Colonies were permitted to form (n = 3, ± SEM) #P < 0.05 less than ETO alone value. (G) DAOY cells were transfected with scrambled siRNA or to knock down expression of PDE5. Thirty-six hours after transfection cells were treated with increasing doses of etoposide. Cells were isolated after 24 h and viability determined by trypan blue exclusion (n = 3, ± SEM) *P < 0.05 greater than corresponding value in siSCR control. (H) HOSS1 cells were pre-treated with PBS vehicle (VEH) or the iNOS inhibitor L-NAME (10 μM). Thirty minutes later cells were treated with vehicle, etoposide, sildenafil, or the drugs in combination. Cells were isolated after 24 h and viability determined by trypan blue exclusion (n = 3, ± SEM) #P < 0.05 less than corresponding value in VEH control.
Figure 2. The toxic interaction between PDE5 inhibitors and chemotherapy is blocked by overexpression of BCL-XL or c-FLIP-s. (A) DAOY cells were treated with zVAD, LEHD, or IETD (all 50 μM) followed by drugs and viability determined by trypan blue exclusion (n = 3, ± SEM). (B–E) HOSS1, D283, and DAOY cells were infected with empty vector adenovirus (CMV) or three other viruses to express dominant negative caspase 9 (dn casp 9), BCL-XL, and c-FLIP-s respectively. Thirty-six hours after infection cells were treated with drugs and viability determined by trypan blue exclusion (n = 3, ± SEM) #P < 0.05 less than corresponding value in CMV control. (F) DAOY cells were transfected with scrambled siRNA (siSCR) or siRNA molecules to knock down expression of CD95 or FADD (siCD95, siFADD). Thirty-six hours after infection cells were treated with drugs and viability determined by trypan blue exclusion (n = 3, ± SEM) #P < 0.05 less than corresponding value in siSCR control.
Figure 3. Sildenafil and chemotherapy-induced lethality is mediated through increased autophagy. (A and B) DAOY cells were transfected to express LC3-GFP-RFP. Twenty-four hours after transfection cells were treated with drugs and cells were examined 6 h and 12 h after treatment using a fluorescent microscope and the mean number of LC3-GFP+ and LC3-RFP+ vesicles determined in >40 cells (n = 3, ± SEM). *P < 0.05 greater than vehicle control. (C–F) DAOY, D283, and HOSS1 cells were transfected with scrambled siRNA (siSCR) or siRNA molecules to knock down expression of ATG5 or Beclin1 (siATG5, siBeclin1). Thirty-six hours after transfection cells were treated with drugs and viability determined by trypan blue exclusion (n = 3, ± SEM) #P < 0.05 less than corresponding value in siSCR control. (C upper) DAOY cells were treated with drugs as indicated. Cells were isolated 12 h after treatment and immunoblotting performed to examine the expression of the indicated proteins (n = 3).
Figure 4. Modulation of cell signaling pathways controls etoposide and sildenafil lethality. (A) DAOY cells were treated with drugs for 24 h before isolation, SDS PAGE, and determination of the phosphorylation and total expression of the indicated proteins. (B and C) DAOY cells were transfected with empty vector plasmid (CMV) or plasmids to express: an activated form of mTOR (ca-mTOR), an activated form of p70S6K (ca-p70), an activated form of MEK1 (caMEK1), and an activated form of AKT (caAKT). Thirty-six hours after transfection cells were treated with drugs and viability determined by trypan blue exclusion (n = 3, ± SEM) #P < 0.05 less than corresponding value in CMV control. (D) DAOY cells were treated with vehicle or the JNK inhibitory peptide (JNK-IP, 10 μM). Thirty minutes later cells were treated with drugs and viability determined by trypan blue exclusion (n = 3, ± SEM) #P < 0.05 less than corresponding value in vehicle control.
Figure 5. Sildenafil increases and prolongs chemotherapy-induced DNA damage; knockdown of ATM enhances drug combination toxicity. (A) DAOY cells were grown in soft agar, and treated as indicated with drugs for 6 h and 12 h. Cells were subjected to electrophoresis and stained. Images are a representative (n = 4). Blot: DAOY cells were treated with drugs and were isolated 6 h and 12 h after exposure and the phosphorylation of γH2AX determined, with the fold increase in phosphorylation shown (n = 3, ± SEM). (B) Graph: The tail moments of cells imaged in (A) were measured and plotted (n = 4, ± SEM) #P < 0.05 greater than corresponding value in vehicle-treated cells. (C) HOSS1 cells were grown in soft agar, in the presence or absence of L-NAME (10 mM), and treated as indicated with drugs, as indicated, for 6 h and 12 h. Cells were subjected to electrophoresis and stained. The tail moments of cells were measured and presented below each image (n = 4, ± SEM) *P < 0.05 less than corresponding value in vehicle-treated cells. (D and E) DAOY cells and HOSS1 cells were transfected with scrambled siRNA (siSCR) or to knock down expression of ATM (siATM). Thirty-six hours after transfection cells were treated with drugs and viability determined by trypan blue exclusion (n = 3, ± SEM) *P < 0.05 greater than corresponding value in siSCR control.
