Pentoxifylline Enhances the Apoptotic Effect of Carboplatin in Y79 Retinoblastoma Cells

Abstract

Background/aim: Retinoblastoma (RB) is the most common primary intraocular malignancy. Carboplatin (CPt) is a DNA damage-inducing agent that is widely used for the treatment of RB. Unfortunately, this drug also activates the transcription factor nuclear factor-kappa B (NF-ĸB), leading to promotion of tumor survival. Pentoxifylline (PTX) is a drug that inhibits the phosphorylation of I kappa B-alpha (IĸBα) in serines 32 and 36, and this disrupts NF-ĸB activity that promotes tumor survival. The goal of this study was to evaluate the effect of the PTX on the antitumor activity of CPt.

Materials and methods: Y79 RB cells were treated with CPt, PTX, or both. Cell viability, apoptosis, loss of mitochondrial membrane potential, the activity of caspase-9, -8, and -3, cytochrome c release, cell-cycle progression, p53, and phosphorylation of IĸBα, and pro- and anti-apoptotic genes were evaluated.

Results: Both drugs significantly affected the viability of the Y79 RB cells in a time- and dose-dependent manner. The PTX+CPt combination exhibited the highest rate of apoptosis, a decrease in cell viability and significant caspase activation, as well as loss of mitochondrial membrane potential, release of cytochrome c, and increased p53 protein levels. Cells treated with PTX alone displayed decreased I kappa B-alpha phosphorylation, compared to the CPt treated group. In addition, the PTX+CPt combination treatment induced up-regulation of the proapoptotic genes Bax, Bad, Bak, and caspases- 3, -8, and -9, compared to the CPt and PTX individual treated groups.

Conclusion: PTX induces apoptosis per se and increases the CPt-induced apoptosis, augmenting its antitumor effectiveness.

Keywords: NF-ĸB; Pentoxifylline; Y79 cells; apoptosis; carboplatin.

Figure 1. Cell viability of Y79 RB cells treated with carboplatin (CPt)(A) and pentoxifylline (PTX) (B) at different doses and for different amounts of time. The effects of CPt, PTX, and PTX and CPt combined on cell viability (C) are expressed as a percentage of non-viable cells in comparison with the untreated control group (UCG) considered as 100%. The results are presented as means±standard deviation. **p<0.001 (PTX+CPt compared to the UCG), *p<0.05 (CPt and PTX alone groups compared to the UCG, and PTX+CPt treated group compared to the CPt and PTX groups).

Figure 2. Analysis of caspase activity. Pancaspase activity (A) and caspases 9, 8 and 3 activity (B) in Y79 cells treated with pentoxifylline (PTX; 4 mM), carboplatin (CPt; 30 μg/ml) or their combination for 24 h. The results are presented as means±standard deviation. **p<0.007 [CPt, PTX or PTX+CPt groups compared to untreated control group (UCG)] *p<0.001(CPt, PTX or PTX+CPt compared to the UCG).

Figure 3. Pentoxifylline (PTX) alone and combined with carboplatin (CPt) decrease mitochondrial and increase cytosolic levels of cytochrome C (CytC) in Y79 cells. Mitochondrial (A) and cytosolic (B) CytC levels in Y79 cells treated with PTX, CPt or PTX and CPt combined. The results are presented as means±standard deviation. *p<0.05, [PTX and PTX+CPt groups compared to the untreated control group (UCG) and CPt treated group] in the mitochondrial CytC analysis. **p<0.001, (PTX and PTX+CPt groups compared to the UCG and CPt alone) in the cytosolic CytC analysis.

Figure 4. Pentoxifylline (PTX) and carboplatin (CPt) modulate the cell cycle checkpoints of Y79 cells. Data are representative of 3 independent experiments, which yielded a similar result *p<0.05; **p<0.01 compared to the untreated control group (UCG).

Figure 5. Determination of Total-IĸBa and phosphorylated IĸBα (pS32/36) and in Y79 cells treated with carboplatin (CPt), pentoxifylline (PTX), and PTX+CPt. The results are presented as means±standard deviation. **p<0.001, [PTX and PTX+CPt treated groups compared to the untreated control group (UCG) and CPt alone group]. *p<0.05 (PTX and PTX+CPt treated groups compared to UCG and CPt group, and CPt alone group compared to the UCG).

Figure 6. Protein levels of p53 in Y79 cells treated with pentoxifylline (PTX), carboplatin (CPt), or PTX+CPt for 18, 24 or 48 h were analyzed by western blot. The results in the charts are presented as means±standard deviation. *p<0.05 [CPt and PTX+CPt groups, compared to the untreated control group (UCG), and CPt alone group compared to PTX and PTX+CPt treated groups].

Figure 7. Expression of pro- and anti-apoptotic genes in Y79 cells treated with pentoxifylline (PTX), carboplatin (CPt), and PTX combined with CPt. The data are expressed as fold change of mRNA expression versus untreated control group (UCG). *p<0.05 (PTX and PTX+CPt treated groups compared to CPt group and UCG).