WNT974 Inhibits Proliferation, Induces Apoptosis, and Enhances Chemosensitivity to Doxorubicin in Lymphoma Cells by Inhibiting Wnt/β-Catenin Signaling.

BACKGROUND Upregulation of the Wnt/ß-catenin pathway has been demonstrated to promote tumor proliferation and chemoresistance in lymphoma. Our objective was to evaluate the effect of the Wnt/ß-catenin pathway inhibitor WNT974 in lymphoma cells. MATERIAL AND METHODS Human lymphoma cell lines HUT-78 and BJAB were treated with or without 1 μM WNT974±0.15 μg/L doxorubicin (Dox). Cell viability and proliferation were evaluated by CCK-8 and colony formation assay. Expression of proliferating cell nuclear antigen (PCNA), KI67, and apoptotic-related proteins including Bcl-2, Bax, cleaved-caspase3, and cleaved-caspase9, together with Wnt pathway proteins Wnt, ß-catenin, Axin2, and c-Myc, were detected by Western blot analysis. Flow cytometry was used to calculate the ratio of apoptotic cells. RESULTS In HUT-78 and BJAB cells, 1 μM WNT974 significantly reduced viability and colony formation. The expression of 2 markers of tumor cell proliferation, protein PCNA and KI67, was also reduced by WNT974. Treatment with 1 μM WNT974 for 48 h increased the rate of cell apoptosis, inhibited the expression of anti-apoptotic protein Bcl-2, and enhanced pro-apoptotic proteins Bax, cleaved-caspase3, and cleaved-caspase9 expression in both cell lines. After treatment with WNT974 plus Dox, cell viability was markedly decreased compared with Dox treatment alone. Mechanistically, WNT974 prevented the expression of Wnt, Axin2, ß-catenin, and its target gene c-Myc. CONCLUSIONS WNT974 effectively treats lymphoma by inhibiting cell proliferation, inducing cell apoptosis, and enhancing chemosensitivity to Dox, and these effects are dependent on blocking Wnt/ß-catenin signaling.

Background

Lymphoma is a malignant tumor that originates from the lymphoid hematopoietic system and can affect any organ in the body, presenting with a wide range of symptoms. It is traditionally divided into Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) [1]. The incidence of lymphoma in China is about 5.94/100 000, and the rate is increasing steadily. HL and NHL are the main types of lymphoma [2,3]. NHL includes Burkitt lymphoma (BL), diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMLBCL), anaplastic large cell lymphoma (ALCL), and lymphoblastic lymphoma (LL), which are the most common malignancies in childhood and have higher prevalence in Central and South American and Asian populations [4-6]. Doxorubicin, a well-established cytotoxic drug, is one of the most common and effective anticancer drugs currently used for lymphoma and myeloma. Dox-based chemotherapy is the first-line treatment for HL and NHL, and has achieved curative effects since its introduction [7]. Cardiotoxicity and drug resistance are the main factors limiting the efficacy and long-term use of Dox [8-10]. Although efforts have been made to overcome Dox resistance in cancer therapy [11,12], further studies are still needed to explore effective strategies for restricting the drug resistance of Dox in lymphoma.

WNTs are a family of 19 secreted glycoproteins that have vital roles in the regulation of diverse processes, including cell proliferation, survival, migration and self-renewal in stem cells [13,14]. The Wnt/β-catenin pathway, also known as the classic Wnt pathway, is a crucial oncogenic pathway in various cancers and can regulate diverse biological processes necessary for cancer progression, including tumor initiation, tumor growth, cell senescence, cell death, differentiation, and metastasis [15]. In the absence of Wnt, β-catenin is degraded by the destruction complex containing glycogen synthase kinase 3β (GSK-3β), adenomatous polyposis coli (APC), and Axin. The binding of Wnt to frizzled (Fzd) and lipoprotein receptor-related protein 6 (LRP6) leads to phosphorylation of LRP6, which strengthens the interactions between LRP6 and Axin [16]. Consequently, the β-catenin destruction complex is destroyed, resulting in the stabilization and nuclear translocation of β-catenin, thereby activating the expression of nuclear target genes, including c-Myc, cyclin D1, matrix metalloprotein 9 (MMP-9), and cyclooxygenase 2 (cox-2) [17,18]. Moreover, Wnt/β-catenin was suggested to be involved in drug resistance in colorectal cancer, prostate cancer, acute myeloid leukemia, and other cancers. Aberrant activation of Wnt/β-catenin signaling not only regulates the development and progression of cancers, but also mediates resistance of chemotherapy, and Wnt inhibition can restore chemotherapy responsiveness [19-23].

Given the critical role of Wnt/β-catenin signaling in cancer and drug resistance, targeting this pathway may be an attractive therapeutic approach. However, due to the lack of effective target drugs in the Wnt pathway, success has been limited. Liu et al. discovered that a valid and specific porcine (PORCN) inhibitor, WNT974 (LGK974), can effectively inhibit Wnt signaling in vivo and in vitro [24]. The specific PORCN is dedicated to Wnt posttranslational acylation, which is essential for subsequent Wnt secretion [25]. Loss of PORCN results in inhibition of Wnt ligand-driven signaling activities in mouse models [26,27]. Since then, studies have reported the inhibitory effect of LGK974 on Wnt signaling and its protective effect on various diseases, including inflammation [28] and lung cancer [29], and its effect on pancreatic ductal adenocarcinoma [30] has been confirmed [31]. Furthermore, the Wnt signaling pathway was shown to be crucial in development of lymphomas, including HL, DLBCL, and mantle cell lymphoma (MCL) [32], but whether the inhibition of Wnt signaling could produce a curative effect and overcome Dox resistance in lymphoma remains to be elucidated. Here, we explored the effects of the Wnt pathway inhibitor WNT974 on lymphoma cell proliferation and its role in Dox resistance.

Material and Methods

Chemicals and reagents

WNT974 (Novartis China, Shanghai, China) was diluted in DMSO to 5 mM concentration and stored at 4°C. Dox (Novartis China) was dissolved to a concentration of 2 g/l in dH2O and divided into 25 aliquots (1.5 ml).

Cell culture

Human lymphoma cell lines HUT-78 and BJAB were provided by the American Type Culture Collection (ATCC, USA). All cells were cultured in RIPM (Roswell Park Memorial Institute) 1640 medium (Gibco, USA) containing 10% FBS and 1% antibiotics at 37°C in 5% CO2.

Cell proliferation assay

To determine the cell viability, cells in the 96-well plate treated with indicated conditions were assessed. Cell Counting Kit-8 (CCK-8) was used following the manufacturer’s instructions. We added 10 μl CCK-8 working solution to each well, followed by incubation for 2 h under normal cell culture conditions, and the optical density (OD) was evaluated at 450 nm wavelength using a microplate reader.

Colony formation assay

For colony formation assays, the cell suspension was resuspended in 1 ml medium with 20% FBS and 0.9% methylcellulose medium (Sigma, USA). Samples were plated in 24-well plates and incubated for 2 weeks. A colony with >50 cells was counted as a positive colony. The colony forming ability was expressed by the percentage of the number of colonies in the test group to the control group.

Western blot analysis

Total proteins were extracted from cells using RIPA lysis buffer (Beyotime, China) including proteasome inhibitors. All protein samples were quantified using a BCA protein assay kit (Beyotime, China), and equal amounts of each sample were subjected to 8–12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). Subsequently, the membranes were blotted with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) at room temperature for 1 h, and then incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: anti-PCAN, anti-KI67, anti-Bcl2, anti-Bax, anti-caspase3, anti-cleaved caspase3, anti-caspase9, anti-cleaved caspase9, anti-Axin2, anti-c-Myc, anti-β-catenin, anti-Wnt, and anti-GAPDH (Santa Cruz Biotechnology, USA). Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using an ECL (electrochemiluminescence) system (Amersham, USA).

Flow cytometry

Cell apoptosis was assessed by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, China). Cells were collected and washed with PBS, gently resuspended in Annexin V binding buffer, and incubated with Annexin V-FITC/PI. Flow cytometer (Becton Dickinson, USA) and flow cytometry software was used for analysis.

Statistical analysis

All statistical analyses were performed using GraphPrism 6.0 software (GraphPad Prism, USA). The data are shown as mean±SD. The t test or one-way ANOVA was used to analyze the differences between 2 or more groups. All results were considered statistically significant when P was <0.05.

Results

WNT974 inhibited proliferation of lymphoma cells

The ability of WNT974 to inhibit lymphoma cell proliferation was evaluated. HUT-78 and BJAB cells were exposed to 1 μM WNT974 for 24, 48, and 72 h, then cell viability was analyzed by CCK-8. As shown in Figure 1, HUT-78 and BJAB cells were sensitive to WNT974, and WNT974 reduced cell viability in a time-dependent manner.

Figure 1

WNT974 inhibited HUT-78 and BJAB cells proliferation. (A, B) The cell viability of HUT-78 (A) and BJAB (B) before and after WNT974 treatment was measured by CCK-8 at different times (n=3). (C, D) Colony formation of HUT-78 (C) and BJAB (D) cells was examined in the presence or absence of WNT974. ** P<0.01 and *** P<0.001 vs. control.

In addition, colony formation ability of HUT-78 and BJAB cells was assessed, showing that WNT974 significantly decreased the numbers of colonies in both cell lines.

To further investigate the effect of WNT974 on lymphoma cell proliferation, the protein expression of tumor cell proliferation factors proliferating cell nuclear antigen (PCNA) and KI67 was detected by Western blotting. Consistent with the results of cell viability and colony formation assay, the expression of PCNA and KI67 was also reduced by WNT974 (Figure 2). These results revealed the inhibitory effect of WNT974 on human lymphoma cell proliferation.

Figure 2

WNT974 downregulated PCAN and KI67 expression of HUT-78 and BJAB cells. (A) Representative immunoblot analysis together with relative protein expression for PCAN and KI67 in HUT-78 cells with or without WNT974 treatment (n=3). (B) Representative immunoblot analysis together with relative protein expression for PCAN and KI67 in BJAB cells before and after WNT974 treatment (n=3). *** P<0.001 vs. control.

WNT974 promoted apoptosis of lymphoma cells

To evaluate the effect of WNT974 on lymphoma cell apoptosis, HUT-78 and BJAB cells were treated with or without 1 μM WNT974 for 48 h, and then detected by flow cytometry and Western blot. Results showed that WNT974 increased the apoptotic rate of HUT-78 and BJAB cells (Figure 3). The expression of anti-apoptotic protein Bcl-2 was prevented, while the expression of pro-apoptotic proteins Bax, cleaved-caspase3 and cleaved-caspase9 was enhanced by WNT974 (Figures 4, 5). These results confirmed that WNT974 induced apoptosis of lymphoma cells.

Figure 3

WNT974 increased apoptotic rate of HUT-78 and BJAB cells. (A) Cell apoptosis of HUT-78 was assessed by flow cytometry and the ratio of apoptotic cells with or without WNT974 treatment was calculated (n=3). (B) Cell apoptosis of BJAB was assessed by flow cytometry, and the ratio of apoptotic cells with or without WNT974 treatment was calculated (n=3). *** P<0.001 vs. control.

Figure 4

Effect of WNT974 on expression of apoptosis-related proteins in HUT-78 cells. (A) Representative immunoblot analysis for apoptosis-related proteins in HUT-78 cells before and after WNT974 treatment. (B–E) Relative protein expression of (B) anti-apoptosis protein Bcl-2, pro-apoptosis proteins (C) Bax, (D) cleaved- and pro-caspase 3 and (E) cleaved- and pro-caspase 9 after normalization to GAPDH (n=3). ** P<0.01 and *** P<0.001 vs. control.

Figure 5

Effect of WNT974 on expression of apoptosis-related proteins in BJAB cells. (A) Representative immunoblot analysis for apoptosis-related proteins in BJAB cells before and after WNT974 treatment. (B–E) Relative protein expression of (B) anti-apoptosis protein Bcl-2, pro-apoptosis proteins (C) Bax, (D) cleaved- and pro-caspase 3, (E) cleaved- and pro-caspase9 after normalization to GAPDH (n=3). ** P<0.01 and *** P<0.001 vs. control.

WNT974 enhanced the sensitivity of lymphoma cells to doxorubicin

To assess whether WNT974 can restore responsiveness of lymphoma cells to Dox, HUT-78 and BJAB cells were incubated with 0.15 μg/l Dox for 24 h, and then exposed to 1 μM WNT974 for 48 h. The inhibition of cell proliferation was determined by CCK-8 assay. As demonstrated in Figure 6, Dox significantly inhibited cell proliferation, and WNT974 enhanced the Dox-induced inhibitory rate of cell proliferation compared with Dox treatment in the absence of WNT974, which indicated that WNT974 enhanced the sensitivity of lymphoma to Dox.

Figure 6

WNT974 enhanced the sensitivity of HUT-78 and BJAB lymphoma cells to doxorubicin. Cell growth inhibition (proportion of dead cells) of (A) HUT-78 cells and (B) BJAB cells treated with indicated reagents (n=3). Dox, doxorubicin. *** P<0.001 vs. control; ### P<0.001 vs. Dox; @@@ P<0.001 vs. WNT974.

WNT974 downregulated the Wnt/β-catenin pathway

To determine whether the inhibitory effect of WNT974 on lymphoma is dependent on the Wnt/β-catenin pathway, the protein expressions of Wnt, β-catenin, Axin2, and c-Myc were assessed. Results from Western blot analysis showed that WNT974 obviously prevented the expression of these proteins (Figure 7). These results further confirmed that WNT974 blocked Wnt/β-catenin signaling.

Figure 7

WNT974 blocked the Wnt/β-catenin pathway. (A, B) Representative immunoblot analysis together with relative protein expression for Axin2, c-Myc, β-catenin, and Wnt in HUT-78 cells with or without WNT974 treatment (n=3). (C, D) Representative immunoblot analysis together with relative protein expression for Axin2, c-Myc, β-catenin, and Wnt in BJAB cells with or without WNT974 treatment (n=3). *** P<0.001 vs. control.

Discussion

The role of dysregulated Wnt signaling in a variety of cancers, including lymphoma, has been well established. It is reported that Wnt signaling is involved in the microenvironmental interactions between endothelial and lymphoma cells [33], thereby accelerating the pathogenesis of lymphoma [34,35]. Considering the crucial role of Wnt signaling in cancer, the potential of targeting this pathway therapeutically has attracted much research attention. In fact, multiple efforts have been made to exploit antibodies against key Wnt receptors, including Fzd and LRP6 [36-38]. The PORCN inhibitors IWP2, C59, and WNT974 used in this study have been found to possess good potency and specificity in inhibiting Wnt signaling in vitro and in animal models [24,39,40]. Here, we observed the effect of the Wnt inhibitor WNT974 on lymphoma cell proliferation and found that WNT974 reduced cell viability, colony formation ability, and the protein expression of tumor cell proliferation factors PCNA and KI67 in HUT-78 and BJAB cells. Therefore, the results of our research demonstrated that WNT974 treatment inhibited lymphoma cells proliferation in vitro.

Apoptosis is a form of programmed cell death and plays an important role in cell homeostasis and cancer treatment [41]. In the present study, WNT974 induced apoptosis of lymphoma cells as determined by flow cytometry (Figure 3). Apoptosis is triggered by the receptor-mediated extrinsic pathway and mitochondrial-dependent intrinsic pathway. Anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax are the most characteristic apoptosis regulators in mitochondrial-dependent apoptosis. Upon apoptotic stimulation, Bax translocates to the mitochondria and facilitates the release of cytochrome c, resulting in activation of caspase9, which can in turn activate caspase3, leading ultimately to apoptotic cell death. Bcl-2 can inhibit cell apoptosis by maintaining mitochondrial membrane integrity and preventing the release of cytochrome c [42,43]. It has been demonstrated that the reduced level of Bax and increased level of Bcl-2 exacerbate the development of lymphoma [44]. In the present study, results from Western blot analysis revealed that WNT974 downregulated the protein expression of Bcl-2 and enhanced the levels of Bax, cleaved-caspase3, and cleaved-caspase9, which further confirmed the occurrence of apoptosis induced by WNT974 in lymphoma cells.

Doxorubicin is an effective anticancer drug used for lymphoma and myeloma, but chemoresistance is an urgent problem in treating cancers. Because the inhibitory effect of WNT974 on HUT-78 and BJAB cells and Wnt pathway was indicated to participate in chemoresistance [45], we next assessed the effect of WNT974 on lymphoma cells resistant to Dox. Consistent with our hypothesis, the presence of WNT974 significantly increased the inhibitory effect of Dox on lymphoma cells viability. These results suggest the potential of WNT974 as a therapeutic strategy for lymphoma.

Finally, to determine whether the effect of WNT974 on lymphoma is dependent on blocking of the Wnt/β-catenin pathway, we performed Western blot analysis to assess the protein expression of key proteins involved in the Wnt/β-catenin pathway. Results showed that WNT974 inhibited the expression of these proteins. The specific functions of WNT974 in lymphoma and chemoresistance in vivo and in vitro, together with the in-depth mechanisms, need to be clarified.

Conclusions

In conclusion, our study revealed that WNT974 possesses antitumor activity in lymphoma cells, and this effect may be associated with blocking of the Wnt/β-catenin pathway, leading to inhibition of proliferation and induction of apoptosis. Moreover, WNT974 increased the chemosensitivity of lymphoma cells to Dox. Taken together, our findings may provide a novel approach for the development of lymphoma therapy using WNT974.