Volasertib suppresses the growth of human hepatocellular carcinoma in vitro and in vivo

Abstract

Hepatocellular carcinoma (HCC) is the sixth most frequent malignant tumor with poor prognosis, and its clinical therapeutic outcome is poor. Volasertib, a potent small molecular inhibitor of polo-like kinase 1 (PLK1), is currently tested for treatment of multiple cancers in the clinical trials. However, the antitumor effect of volasertib on HCC is still unknown. In this study, our data show that volasertib is able to induce cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with the spindle abnormalities in human HCC cells. Furthermore, volasertib also increases the intracellular reactive oxidative species (ROS) levels, and pretreated with ROS scavenger N-acety-L-cysteine partly reverses volasertib-induced apoptosis. Moreover, volasertib markedly inhibits the subcutaneous xenograft growth of HCC in nude mice. Overall, our study provides new therapeutic potential of volasertib on hepatocellular carcinoma.

Keywords: Hepatocellular carcinoma; ROS; apoptosis; volasertib.

Figure 1

Volasertib inhibits the growth of HCC cells in vitro. A: Chemical structure of volasertib. B: The representative growth curves of cells treated with volasertib are shown. C: Summary of IC₅₀ of volasertib in the indicated HCC cells is shown. Cells were treated with various concentrations of volasertib for 72 h and cell survival was determined by MTT assay. Datas were mean ± S.D. of three independent experiments.

Figure 2

Volasertib induces cell cycle arrest at G2/M Phase in HCC cells. BEL7402 (A) and HepG2 (B) cells were treated with volasertib at the indicated concentrations. The distribution of cell cycle was detected by FCM with PI staining. The percentages of sub G1, G1/G0, S, G2/M phase were calculated using ModFit LT 3.0 software. The representative charts, quantified results (C, D) of three independent experiments were shown. *P<0.05 and **P<0.01 vs. corresponding control.

Figure 3

Volasertib induces apoptosis in HCC cells. BEL7402 (A) and HepG2 (B) cells were treated with volasertib at the indicated time and concentrations. The apoptosis was detected by FCM with Annexin V/PI staining. The proportions of Annexin V+/PI- and Annexin V+/PI+ cells indicated the early and late stage of apoptosis. The protein expression was examined by Western blot, and GAPDH was used as loading control. The representative charts, quantified results (C, D) and Western blot results (E, F) of three independent experiments were shown. *P<0.05 and **P<0.01 vs. corresponding control.

Figure 4

Volasertib promotes spindle abnormalities in HCC cells. BEL7402 (A) and HepG2 (B) cells were treated with 10 nM and 100 nM of volasertib for 24 h respectively. Cells were fixed and stained with Hoechst 33342 (to stain DNA, blue) as well as with β-tubulin antibody (to visualize tubulin spindles, red). Cells were analyzed by immunofluorescence microscopy. Arrows indicate the spindle abnormalities of cells.

Figure 5

Volasertib enhances ROS accumulation in HCC cells. BEL7402 and HepG2 cells were treated with volasertib at the indicated times and concentrations, stained with DHE and photographed under florescent microscope. The representative micrographs (bright field - A, B and fluorescence - C, D) and quantified results (E, F) of three independent experiments were shown. *P<0.05; **P<0.01 vs. corresponding control.

Figure 6

Inhibition of ROS partially rescues volasertib-induced apoptosis in HCC cells. BEL7402 (A) and HepG2 (B) cells were treated with volasertib at 30 nM and 10 μM respectively for 48 h in the presence or absence of 5 mM NAC pretreatment for 1 h, stained with DHE and photographed under florescent microscope. The representative micrographs and quantified results (C, D) of three independent experiments were shown. The apoptosis was detected by FCM with Annexin V/PI staining. The representative charts (E, F) and quantified results (G, H) of three independent experiments were shown. Vol: Volasertib. *P<0.05 and **P<0.01 versus corresponding control.

Figure 7

Volasertib suppresses the subcutaneous xenograft growth of HCC in nude mice. Each mouse was injected subcutaneously with BEL7402 and HepG2 cells (2×10⁶ in 100 μl of medium) under the shoulder. When the tumors developed into 0.3×0.3 cm² (two perpendicular diameters) in size, mice were randomized into two groups, and were intraperitoneally injection with vehicle (0.5% carboxymethyl cellulose) and volasertib (15 mg/kg) every two days, respectively. The body weights of mice and the two perpendicular diameters of the tumor were recorded. The mice were anaesthetized after experiment, and tumor tissue was excised from the mice and weighed. The original tumors (A, B), tumor volume (C, D), tumor weight (E, F), body weight (G) and summary data (H) were shown. The values presented are the means ± SD for each group. *P<0.05 and **P<0.01 vs. corresponding control.