Inhibitory effect of valproic acid on bladder cancer in combination with chemotherapeutic agents in vitro and in vivo

Abstract

Histone deacetylase inhibitors (HDACIs) are a promising class of drugs that act as antiproliferative agents by promoting differentiation and inducing apoptosis. Valproic acid (VPA) is an HDACI that has been widely used as an anti-convulsant and shows promise as a chemotherapeutic drug for a number of tumor cells. The present study aimed to investigate the inhibitory effect of VPA on the viability of bladder cancer cells and its synergistic effect with chemotherapeutic agents in vitro and in vivo. The cell viability of human bladder cancer cell lines following treatment with VPA and/or VPA in combination with mitomycin C, cisplatin (DDP) and adriamycin were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining was used to observe the morphology of the apoptotic cells. Survivin protein and acetylated histone H3 levels were quantified using western blot analysis. The in vivo tumor growth inhibition of VPA was determined in rats with N-methyl-N-nitrosourea-induced bladder cancer. VPA significantly inhibited the growth of the bladder cancer cells in a concentration- and time-dependent manner. Furthermore, improved results were achieved for tumor inhibition when VPA was combined with chemotherapeutic agents in vitro and in vivo. Survivin expression decreased and acetylated histone H3 expression increased in the bladder cancer cells following the treatment with VPA. Intravesical injections of VPA were able to inhibit tumor progression when combined with DDP. In conclusion, VPA acts as an HDACI that has a direct anticancer effect and markedly enhances the action of several chemotherapy agents. VPA may sensitize bladder cancer to anticancer drugs by downregulating survivin expression.

Keywords: apoptosis; bladder cancer; combination; therapy; valproic acid.

Figure 1

Effect of valproic acid (VPA) on bladder cancer cell survival. Cells were seeded at 3×10³ cells/well in 96 multiwell plates and treated with medium alone or with medium containing various doses of VPA (0.5, 1, 1.5 or 3 mM) for up to 10 days. At days 1, 4, 7 and 10, the viable cells were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The VPA significantly reduced the number of surviving bladder cancer cells. The cell viability decreased in a dose-dependent manner in all the cell lines. VPA (1 mM) was able to inhibit the growth of all the bladder cancer cell lines significantly.

Figure 2

Cell morphology observations following Hoechst 33258 staining. The bladder cancer cells were separately incubated with 1 mM valproic acid (VPA) and/or cisplatin (DDP) for 72 h. The rats with bladder cancer were treated with VPA and/or DDP. The cells and sections were fixed with 4% paraformaldehyde and stained with Hoechst 33258. The group treated with VPA or VPA combined with DDP exhibited numerous apoptotic cells, while apoptosis of the control cells was not observed. Magnification, ×400.

Figure 3

Effect of valproic acid (VPA) in combination with cisplatin (DDP), mitomycin C (MMC) and adriamycin (ADM) on bladder cancer cell survival. Using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the synergistic effects were observed in the inhibition of bladder cancer survival using a combination of 1 mM VPA with 5 mg/l DDP, 5 mg/l MMC and 2 mg/l ADM.

Figure 4

Flow cytometry of apoptosis by Annexin V and propidium iodude (PI) double staining. T24 cells were treated with valproic acid (VPA) and/or cisplatin (DDP) for 72 h. Following the treatment, the cells were harvested and apoptosis was assesed using Annexin V/PI double staining. Following the treatment with VPA, DDP or VPA and DDP, the number of apoptotic and necrotic cells increased dramatically. The number of living cells decreased significantly.

Figure 5

Western blot analysis of acetylated histone H3 and survivin expression in T24 cells. The T24 cells were treated with 0, 1 and 1.5 mM valproic acid (VPA) and analyzed for acetylated histone H3 and survivin expression using western blot analysis. The relative fold increase was determined by scanning densitometry of the western blot analysis normalized to β-actin. VPA treatment resulted in an increase in acetylated H3 expression and a decrease in survivin expression in the T24 cells.

Figure 6

Histopathological findings in female Wistar rat bladders treated with five doses of intravesical N-methyl-N-nitrosourea (MNU). The rats commenced therapy at 11 weeks with valproic acid (VPA) and/or cisplatin (DDP) and were sacrificed at 25 weeks. The female Wistar rat bladders that were treated with five doses of MNU developed progressive neoplastic changes. These lesions progressed from hyperplasia or superficial transitional cell carcinoma (TCC) to large bulky muscle-invasive TCCs. Hematoxylin and eosin staining; magnification, ×400.

Figure 7

In vivo effects of valproic acid (VPA) and/or cisplatin (DDP) on N-methyl-N-nitrosourea (MNU)-induced bladder cancer. Histopathological findings in the female Wistar rat bladders that were treated with four doses of intravesical MNU. The rats commenced therapy at 11 weeks and were sacrificed at 25 weeks. TTC, transitional cell carcinoma.