XAV939, a tankyrase 1 inhibitior, promotes cell apoptosis in neuroblastoma cell lines by inhibiting Wnt/β-catenin signaling pathway

Human NB SH-SY5Y, SK-N-SH and IMR-32 cells were obtained from the American Type Culture Collection (ATCC; Rockville, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone), with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Sigma Chemical Co., St Louis, Missouri) and were grown in a 5% CO₂ incubator at 37°C. The TNKS1 inhibitor XAV939 was purchased from Sigma Aldrich.

Cellular viability was assessed by MTT method. Briefly, equal numbers of NB SH-SY5Y, SK-N-SH and IMR-32 cells were plated at a density of 1 × 10⁴ per well in 96-well plates, and were treated with various concentrations of XAV939 for 24, 48, or 72 h. 20 μl MTT (5 mg/ml) were incubated with cells of each sample for 4 h, then were replaced with 150 μl DMSO and 96-well plates were rotated gently for 10 min. Cell viability was determined by measuring colorimetric absorbance at 490 nm, and was read with a microplate reader [25]. Experiments were done in triplicate and average activity rates relative to control and standard errors were calculated.

Colony formation assays were performed as described [26]. Briefly, SH-SY5Y cells were plated in triplicate at 100 cells per well in 6-well plates and cultured in DMEM medium supplemented with 10% FBS. After 4-5 h, cells were treated with DMSO or XAV939, as well as transfected with lentivirus-mediated scrambled-shRNA (SCR group) or TNKS1-shRNA (shRNA group). Colonies were allowed to form for 14 days and fixed in methanol for 15 minutes, and dyed with crystal violet for 15 minutes at room temperature. Afterward, the dye was washed off and colonies that contained more than 50 cells were counted. The colony formation efficiency was the ratio of the colony number to the planted cell number.

Apoptosis was measured using Annexin V/FITC Apoptosis Detection kit (KeyGEN Biotech, Nanjing, China) following the manufacturer’s protocol. Briefly, equal numbers of SH-SY5Y and SK-N-SH cells, treated with DMSO or XAV939 for 24, 48, or 72 h, were incubated with Annexin V-FITC, followed by staining of their DNA with propidium iodide (PI) in the dark. Then, each sample was analyzed by fluorescence-activated cell sorting (FACS) (BD, San Jose, CA, USA). The percentages of cells staining positive for Annexin V were calculated, and means as well as standard error were plotted. Alternatively, apoptosis was also determined using Hoechst 33342 staining. After treatment, cells were washed with PBS and stained with Hoechst 33342 (10 μg/mL, Sigma Aldrich). Then the cells were observed by fluorescent microscope (Olympus Inverted Fluorescence Microscope, I × 71) with excitation at 340 nm and approximately 100 cells from five random microscopic fields were counted. The percentage of apoptotic cells was calculated as the ratio of apoptotic cells to total cells. Mean and standard error were calculated for each time point and treatment group.

Equal numbers of SH-SY5Y, SK-N-SH and IMR-32 cells were plated in 10 cm dishes and treated with DMSO or XAV939 for 24, 48, or 72 h. 10⁶ cells were trypsinized, fixed with 70% ethanol, and incubated over night at 4°C, then were incubated in 100 μl RNase at 37°C for 30 min, followed by staining of their DNA with 400 μl PI for 30 min in the dark, and analyzed by FACS. The average percentages of cells in G0/G1, S or G2/M phases of the cell cycle were quantified and standard error was calculated for three experiments.

Equal numbers of SH-SY5Y and SK-N-SH cells were plated on 10 cm dishes and treated with DMSO or XAV939 for 24, 48 or 72 h. Then the cells were lysed with RIPA buffer and protein concentration was determined by the Bradford method. Equal amounts of protein (40 μg) were used for Western blot analysis with antibodies to anti-β-catenin (Santa Cruz, sc-7199), anti-Cyclin D1 (Santa Cruz, sc-718), anti-c-Myc (Santa Cruz: sc-789) and anti-Bcl-2 (Santa Cruz, sc-492). Specific antibody binding was detected by horseradish peroxidase-conjugated goat anti-rabbit antibodies and visualized with ECL reagent (Santa cruz) according to the manufacturer’s protocol. Antibody to actin was used to evaluate protein loading in each lane.

To identify shRNA sequences could knockdown TNKS1 in SH-SY5Y and SK-N-SH cells, we screened three MISSION shRNA clones NM_003747 (GENECHEM CO., LTD., Shanghai, China) targeted against the human TNKS1 sequence. MISSION shRNA clones together with packaging and envelope plasmids GV118 (GENECHEM CO., LTD., Shanghai, China), were transfected into HEK 293 T packaging cells using Lipofectamine 2000 (Invitrogen). At 48 h post-transfection, virus-containing media was used to infect NB cell lines. GFP was used to monitor the efficiency of HEK 293 T transfection and infection. After selection with puromycin (5 μg/ml) for 48 h, cells were tested for TNKS1 expression by qRT-PCR and then used for clonogenic survival assays and Western blot analyses.

The results were presented as Mean ± Standard deviation (S.D.) of triplicate determinations. The data were processed using the Statistical Package for the Social Sciences, version 16.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA was performed for comparison between different groups. Dunnett’s t (when homogeneity of variances existed) or Dunnett T3 (when heterogeneity of variances existed) was calculated. A P-value of < 0.05 was regarded as statistically significant difference.

XAV939 has been described as a potent, small molecule inhibitor of TNKS1 and 2 and could inhibit the growth of DLD-1 cancer cells [14]. To elucidate the role of XAV939 in NB, we investigated how XAV939 affects cell proliferation in NB cell lines with different concentrations. After that, both SH-SY5Y cells and IMR-32 cells showed reduction in cell proliferation after 24 h of treatment with 1 μM XAV939, with a maximum reduction at 72 h (Figure 1A, B). However, SK-N-SH cells showed the same effect only with 0.5 μM XAV939 treatment (Figure 1C). This anti-proliferative effect was dose and time dependent at 1, 5, 10 and 50 μM at 24, 48 and 72 h. These results indicate that inhibition of TNKS1 by small molecule inhibitor attenuates NB cell proliferation. Thus 1 or 0.5 μM XAV939 were used depending on the cell lines for further assays.

To determine whether TNKS1 inhibition reduces cell viability and survival of SH-SY5Y cells, we performed a colony formation assay in vitro. The number of colonies in the control and various treatment groups were counted and are summarized in Figure 2. From these results it is evident that the XAV939 caused 62.7% inhibition of colony formation in SH-SY5Y cells. In addition, we also observed the effect of shRNA for TNKS1 on cell colony formation. As shown in Figure 2, specific knockdown of TNKS1 by shRNA in SH-SY5Y cells resulted in a significant decrease (55.3%) in the number of colonies, as compared to SCR group (P < 0.01, Figure 2B). These results indicate that the growth inhibitory effects of XAV939 on SH-SY5Y cells are due to TNKS1-dependent inhibition.

Apoptosis plays an important role in both the cause and treatment of tumor [27]. The early apoptotic cells could be stainned by Annexin V, which located in the right lower quadrant (Figure 3A, E). Both the SH-SY5Y and SK-N-SH cell lines showed that no significant difference in the percentage of apoptotic cells between the control and XAV939 group for 24 h (Figure 3B, G). However, after 48 h or 72 h treatment, the apoptotic rates in XAV939 group were 3.31 ± 0.17% and 5.41 ± 0.63% respectively in SH-SY5Y cells (Figure 3B), which were significantly higher than those in control group (P < 0.05, Figure 3B). Similarly, the apoptotic rates in XAV939 group were 3.69 ± 0.31% and 5.44 ± 0.24% respectively in SK-N-SH cells (Figure 3G, P < 0.05). To further confirm that TNKS1 inhibition induced apoptosis in NB cell lines, we studied the nuclear morphology of SH-SY5Y and SK-N-SH cells following Hoechst 33342 staining (Figure 3C, F). As depicted in Figure 3C and F, control cells without XAV939 treatment were uniformly stained with and displayed equally disseminated chromatin, normal and intact cell membrane. In contrast, cells treated with XAV939 for 24, 48, or 72 h illustrated varying degrees of archetypal characteristics of apoptotic cells, including the condensation of chromatin, shrinkage of nuclei, and presence of apoptotic bodies with intense blue fluorescence (Figure 3C, F). The major findings were showed by arrows in Figure 3C, F. In SH-SY5Y cells, the percentages of cells with apoptotic nuclei in XAV939 group were 9.2%, 25.0% and 52.3% respectively at 24 h, 48 h and 72 h, which in control group were 8.8%, 13.8% and 15.0% respectively (Figure 3D). In SK-N-SH cells, The percentages of cells with apoptotic nuclei in XAV939 group were 5.7%, 35.5% and 53.5% respectively at 24 h, 48 h and 72 h, which in control group were 4.5%, 13.2% and 13.5% respectively (Figure 3H). The statistical analysis showed that there was no significant difference of apoptotic cells between the control and XAV939 groups at 24 h, but the percentages of apoptotic cells in XAV939 group were significant higher than those in control group at 48 h and 72 h respectively (P < 0.05, Figure 3D, H), confirming the induction of apoptosis following treatment. Together, these results suggest that apoptosis is promoted by TNKS1 inhibition in NB cell lines.

TNKS1 was proposed to be required for the resolution of sister telomere association or assembly of bipolar spindles, and TNKS1 knockdown was reported to cause strong mitotic arrest [28, 29]. We detected the effect of XAV939 on cell cycle in SH-SY5Y, SK-N-SH and IMR-32 cells. As shown in Figure 4A, the number of SH-SY5Y cells in the G0/G1 phase decreased, while those in the S and G2/M phase both increased after XAV939 treatment (Figure 4A). At 24 h after treatment, 58.25% of the DMSO-treated SH-SY5Y cells were at G0/G1, 28.02% at S and 13.73% at G2/M (Figure 4B, P < 0.05). In comparison, 48.38% of the XAV939-treated SH-SY5Y cells were at G0/G1, 33.68% at S and 17.94% at G2/M (Figure 4B, P < 0.05). This trend was continued at later time points (Figure 4B). Figure 4C, E indicated the cell cycle of SK-N-SH and IMR-32 cells respectively, and showed the same tendency with that of SH-SY5Y cells (Figure 4D, F, P < 0.05). This suggested that TNKS1 plays a role in cell cycle regulation and that TNKS1 inhibition induces an accumulation of NB cell lines at G2/M and S phase of the cell cycle.

It has been reported that XAV939 could inhibit the proliferation of DLD-1 cells growth by attenuating the expression of Wnt signaling [14]. Western blot was performed to determine if TNKS1 inhibition induces apoptosis in SH-SY5Y and SK-N-SH cells and if so whether Wnt/β-catenin signalling and Bcl-2 plays a role. TNKS1 in SH-SY5Y and SK-N-SH cells was inhibited by XAV939 of 1 and 0.5 μM, or by specific shRNA to TNKS1. Following inhibition of TNKS1 in SH-SY5Y and SK-N-SH cells, protein levels of Bcl-2 were both reduced (P < 0.05, Figure 5A, B; P < 0.01, Figure 5C, D). This suggests that TNKS1 inhibition might induce apoptosis in NB cell lines in part by reducing expression of the anti-apoptotic protein Bcl-2. After treating with XAV939 or specific shRNA to TNKS1, we noted that the accumulation of β-catenin reduced as well as Cyclin D1 and c-Myc in both NB cell lines (Figure 5A, C). Quantification analysis revealed these results (P < 0.05, Figure 5B, D). As a result, the anti-apoptotic protein Bcl-2 also decreased.