Sodium valproate inhibits the growth of human cholangiocarcinoma in vitro and in vivo - PubMed (original) (raw)
Sodium valproate inhibits the growth of human cholangiocarcinoma in vitro and in vivo
Bing Wang et al. Gastroenterol Res Pract. 2013.
Abstract
Background. None of treatment options for Cholangiocarcinoma (CCA), including surgery, adjuvant radiotherapy and chemotherapy, and ultimately liver transplantation, have been shown to substantially improve the survival rate in patients with CCA. Valproic acid (VPA), a histone deacetylase inhibitor, has been shown to display potent antitumor effects. In this study, sodium valproate, the clinically available form of VPA, was tested for its ability to inhibit the growth of cholangiocarcinoma cells, both in vitro and in vivo. Materials and Methods. Cholangiocarcinoma cells (TFK-1, QBC939, and CCLP1) of different origins were treated with sodium valproate to determine their effects on cell proliferation and differentiation, cell cycle regulation, apoptosis, and autophagy. The in vivo effects of sodium valproate on cholangiocarcinoma growth were assessed using a xenograft mouse model injected with TFK-1 cells. Results. Sodium valproate inhibited cholangiocarcinoma cell growth by inducing cell cycle arrest, cell differentiation, and apoptosis; sodium valproate effects were independent of autophagy. Tumor growth inhibition was also observed in vivo using TFK-1 xenografts. Conclusion. The in vitro and in vivo outcomes provide preclinical rationale for clinical evaluation of sodium valproate, alone or in combination with other drugs, to improve patient outcome in cholangiocarcinoma.
Figures
Figure 1
Chemical structure of valproate (VPA, 2-propylpentanoic acid).
Figure 2
Effects of sodium valproate on cell growth of cholangiocarcinoma cell lines. After exposure to sodium valproate for 24 h–120 h, cell inhibition of TFK-1 (a), QBC939 (b), and CCLP1 (c) was measured by CCK-8 assay. All assays were conducted at least in triplicate. The inhibition rate was in comparison with untreated cells. *P < 0.05 and # P < 0.01.
Figure 3
Inductive effect of sodium valproate on TFK-1 cell differentiation. Dendrite-like structure formation in TFK-1 cells treated for 120 h with (a) 0 mM (control), (b) 0.5 mM, and (c) 2 mM sodium valproate. With the increase of sodium valproate concentration, the dendrite-like structures became progressively longer. The morphology of cells was analyzed using a Nikon FX-35A camera (200x magnification). Each experiment was performed in triplicate.
Figure 4
Induction of apoptosis in TFK-1 cells by sodium valproate. TFK-1 cells were incubated with 0–8 mM sodium valproate for 120 h. Apoptosis was measured by Annexin-V and PI double staining. The values represent the mean ± S.D. (n = 3). *P < 0.05 versus control.
Figure 5
Apoptotic morphological changes in TFK-1 cells after treatment with sodium valproate. After 120 h, untreated control group (a) and 2 mM sodium valproate-treated TFK-1 cells (b) were loaded with Hoechst 33342/PI and then observed via fluorescence microscopy (100x magnification). Mixture of low blue with low pink fluorescence indicates live cells ((a), short arrow), while high blue fluorescence indicates apoptotic cells ((b), long arrow), and pink represents dead cells ((b), short arrow).
Figure 6
Effects of sodium valproate on cell cycle distribution in cholangiocarcinoma TFK-1 cells and QBC939 cells. (a) TFK-1 cells were incubated with 0–8 mM sodium valproate, and (b) QBC939 cells were incubated with 0–10 mM sodium valproate. On 24 h, 72 h, and 120 h, cells were harvested, and the fractions of cells in G0/G1-phase, S-phase, and G2/M-phase were determined by flow cytometry.
Figure 7
Sodium Valproate induces autophagy in TFK-1 cells and QBC939 cells. TFK-1 cells and QBC939 cells were transfected with green fluorescence protein (GFP) and tagged with MAP-LC3 (GFP-LC3) plasmid and treated with sodium valproate at 2 mM and 8 mM, respectively. The formation of punctate GFP-LC3 spots is indicative of autophagy (short arrow).
Figure 8
The effect of sodium valproate treatment on cholangiocarcinoma xenografts. (a) Tumor xenografts were established in mice by implanting TFK-1 cells on the upper-right flank. Animals were randomly divided into 2 groups and injected through i.p. with vehicle (control group) or sodium valproate (300 mg/kg, every day) for 2 weeks. Values were presented as mean ± SD. *P < 0.05 compared with the control. (b) Representative tumor tissue excised from control (left) and treated groups (right).
Figure 9
Percentage survival of animals in control and sodium valproate-treated groups. Animals in both sodium valproate-treated and control groups were monitored every day from the first day of treatment until death. By using the Kaplan-Meier method, survival times were determined. The mean percent survival of sodium valproate treated animals was significantly higher, 60% compared to the untreated control, which was 20% over 22 days.
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