Antioxidants impair anti-tumoral effects of Vorinostat, but not anti-neoplastic effects of Vorinostat and caspase-8 downregulation

Abstract

We have recently demonstrated that histone deacetylase inhibitor, Vorinostat, applied as a single therapy or in combination with caspase-8 downregulation exhibits high anti-tumoral activity on endometrial carcinoma cell lines. In the present study, we have assessed the signalling processes underlying anti-tumoral effects of Vorinostat. Increasing evidence suggests that reactive oxygen species are responsible for histone deacetylase inhibitor-induced cell killing. We have found that Vorinostat induces formation of reactive oxygen species and DNA damage. To investigate the role of oxidative stress as anti-neoplastic mechanism, we have evaluated the effects of different antioxidants (Bha, Nac and Tiron) on endometrial carcinoma cell line Ishikawa treated with Vorinostat. We show that Bha, Nac and Tiron markedly inhibited the cytotoxic effects of Vorinostat, increasing cell viability in vitro. We found that all three antioxidants did not inhibited accumulation of acetyl Histone H4, so that antioxidants did not inhibit Vorinostat activity. Finally, we have evaluated the effects of antioxidants on anti-tumoral activity of Vorinostat as monotherapy or in combination with caspase-8 downregulation in vivo. Interestingly, antioxidants blocked the reduction of tumour growth caused by Vorinostat, but they were unable to inhibit anti-tumoral activity of Vorinostat plus caspase-8 inhibition.

Figure 1. Vorinostat induces DNA damage and production of ROS in IK cell line.

A) Representative images (left) and quantification (right) of γH2AX immunofluorescence of IK cells showing foci formation after Vorinostat treatment. **p<0.001 compared with the control group. B) Ik cells were treated with the indicated doses of Vorinostat and cell lysates were subjected to western blot using antibodies anti-acetylated H4, anti-H2AX (Ser-139) and antitubulin. C) Quantification of ROS production determined by staining with DCFH-DA and quantified by flow cytometer (Left graph). D) Loss of mitochondrial membrane potential (MMP) was determined by staining with Rhodamine 123 and quantified by flow cytometer (Right graph). An absence of Rho123 from cells indicated the loss of MMP (ΔΨ_m). **p<0.001 compared with the control group. E) Quantification of ROS production on cells treated with Vorinostat or Vorinostat plus the anti-oxidant NaC. **p<0.001 compared with the control group.

Figure 2. Effects of Nac, Tiron and Bha on reduction of cell viability caused by Vorinostat.

A) IK cells were treated with 3 μM Vorinostat, alone or in combination with increasing doses of Nac, Tiron and Bha. Cell viability was assessed by MTT assay 24 h after treatment. Results are expressed as percent of viability. B) Quantification of Hoechst-stained apoptotic nuclei of IK cells was realized 24 h after being treated with Vorinostat, Vorinostat plus antioxidants or left untreated. Results are expressed as percent of the control values.

Figure 3. Nac treatment reduces DNA damage and ROS production in IK endometrial cell line.

A) Representative γH2AXimmunofluorescence corresponding to IK cells after the combination of Vorinostat and Nac treatment at indicated time. Cells were stained with Hoechst to evidence nuclei. B). Quantifification of the number of cells exhibiting γH2AX positive focis. **p<0.001 compared with the control group C) IK cells were treated with Vorinostat plus Nac, at indicate doses, or left untreated for 24 h. Cell lysates were subjected to western blot with, anti-H2AX (Ser-139) and anti-Tubulin.

Figure 4. Effect of co-treatment with Vorinostat and antioxidants on histone acetylation.

Representative western blot showing acetylated histone H4 and tubulin western blot analysis of IK cells treated or left untreated with 3 μM Vorinostat, IK cells treated with indicated doses of Nac, Tiron and Bha antioxidants and finally, IK cells treated with Vorinostat together with antioxidants at indicated doses for 24 hours. Mean and standard deviations of the ratios of intensities of Ac-histone H4 band-Tubulin bands from the corresponding lanes.

Figure 5. Nac treatment increases colony formation and reduces caspase-3 cleavage in IK endometrial cancer cells treated with Vorinostat.

A) IK cells were seeded as a single-cell suspension with a specified number of cells. After allowing cells time to attach (6 h), Vorinostat plus Nac or the vehicle control was added and treated for indicated times. Ten to fourteen days after seeding, survival curves (from counting the number of colonies) were generated after normalizing. *p<0.05, **p<0.001 compared with the control group. B) IK cells were treated with Vorinostat and Nac, at indicate doses, or left untreated (UN) for 24 h. Cell lysates were subjected to western blot with Acetylate H4, anti-caspase-3, 8 and anti-Tubulin.

Figure 6. Nac treatment do not reduce the effects of blocking caspase-8 and Vorinostat treatment in IK cancer cell line.

A) IK cells were infected with lentiviruses carrying Caspase-8 shRNA (FSV-Casp-8 shRNA) or vehicle control (FSV) and treated with 3 μM Vorinostat plus 5 mM Nac at indicated times. The graph represents a quantification of nuclei displaying nuclear apoptotic morphology. B) Western blot analysis of whole lysates from IK cells infected with lentiviruses carrying Caspase-8 shRNA (FSV-Casp-8 shRNA) or vehicle control (FSV). The membranes were reproved with Acetyl H4, Caspase-8, Caspase-3 and Tubulin to ensure equal protein loading.

Figure 7. Nac treatment impairs Vorinostat but not Vorinostat plus caspase-8 inhibition effects on endometrial cancer.

Tumour weight (TW) measuraments and representative images corresponding to tumours developed by IK cells infected with lentiviruses carrying control vector (FSV) or caspase-8 shRNA (FSV-Casp8 shRNA) and subcutaneously grafted in SCID mice. At day 42, mice were divided into four groups, and were untreated (with vehicle) or injected intraperitoneally with Nac alone (250 mg/kg/day), Vorinostat alone (50 mg/kg/day) or Vorinostat plus Nac. Graphs (A) In the presence of caspase-88, Nac blocks anti-tumor effect of Vorinostat (B). In the absence of caspase-8, Nac does not block anti-tumor effect of Vorinostat.