VALD-3, a Schiff base ligand synthesized from o-vanillin derivatives, induces cell cycle arrest and apoptosis in breast cancer cells by inhibiting the Wnt/β-catenin pathway

Schiff base compounds and their metal complexes have become important synthetic organic drugs due to their extensive biological activities, which include anticancer, antibacterial and antiviral effects. In this study, we investigated the cytotoxic and apoptotic effects of VALD-3, a Schiff base ligand synthesized from o-vanillin derivatives, on human breast cancer cells and the possible underlying mechanisms. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-test was used to observe the proliferation of human breast cancer MCF-7 and MDA-MB-231 cells induced by VALD-3. Flow cytometry analysis showed that VALD-3 triggered cell cycle arrest and induced apoptosis of breast cancer cells. Western blot analysis revealed that VALD-3 upregulated pro-apoptotic proteins (Bad and Bax), downregulated anti-apoptotic proteins (Bcl-2, Bcl-xl, survivin and XIAP) and increased the expression of cleaved caspase-3, cleaved caspase-8, Cyto-c and cleaved PARP. VALD-3 also regulated the Wnt/β-catenin signaling pathway in breast cancer cells, inhibiting the activation of downstream molecules. By xenografting human breast cancer cells into nude mice, we found that VALD-3 significantly suppressed tumor cell growth while showing low toxicity against major organs. In addition, survival analysis showed that VALD-3 can significantly prolong the survival time of mice (P = 0.036). This study is the first to show that VALD-3 induces apoptosis and cell cycle arrest in human breast cancer cells by suppressing Wnt/β-catenin signaling, indicating that it could be a potential drug for the treatment of breast cancer.

Cell culture. Human breast cancer MCF-7 and MDA-MB-231 cell lines were purchased from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (HyClone) containing 10% FBS and 1% penicillin streptomycin (HyClone) at 37℃ in a humidified 5% CO 2 atmosphere.

Xenograft of human breast cancer cells into nude mice.
Six-week-old female BALB/c athymic nude mice (specific pathogen free, SPF) (18 ± 4 g) were purchased from Beijing Weitonglihua Laboratory Animal Co., Ltd. (Beijing, China; animal quality license, SCXK (Jing) 2016-0011). Mice were maintained in sterile conditions at a constant temperature of 22-24 °C, 50-55% humidity, and under a 12 h light/dark cycle. All methods were approved by the Institutional Animal Care and Use Committee of Gansu Provincial Hospital (No. 2017-015).
The study was carried out in compliance with the ARRIVE guidelines and all experiments were approved and performed in accordance with the guidelines of Institutional Animal Care and Use Committee of Gansu Provincial Hospital. MCF-7 tumor cells were resuspended at a density of 1.2 × 10 7 cells/200 µL in saline solution were injected subcutaneously in the front armpit of the nude mice, followed by tumor growth measurements every 3 days. Negative controls were injected with 200 µL of phosphate buffered saline containing no cells. When the tumor size reached 100 mm 3 , 44 tumor-bearing mice were randomly assigned to the time course and survival experiments, which were conducted simultaneously. The time course experiment included five groups (negative control, control, 5 mg/kg/3d cisplatin, 20 mg/kg/d VALD-3 and 5 mg/kg/3d cisplatin + 20 mg/kg/d VALD-3, n = 8), whereas the survival experiment included two groups (control, 20 mg/kg/d VALD-3, n = 6). In the time course experiment, mice were sacrificed by cervical dislocation on day 13 and tumor tissues and blood samples were collected for further analysis. One mouse in the control group died on day 10 due to an oversized tumor. In the survival experiment, mice were treated until they died, the time of death of each mouse was recorded, and the survival analysis curve was plotted. During the course of the experiment, tumor size and body weight were measured every 3 days, and tumor volume was calculated based on the formula length × width 2 /2.  Histopathology and immunohistochemistry. Organs and tumors were fixed with 4% paraformaldehyde and embedded in paraffin. The paraffin-embedded specimens were cut into 4 µm thick slides and stained with hematoxylin and eosin (H&E) for pathological analysis. Tumor tissues were immunostained with antibodies specific for Bax and Bad. Images were captured using a light microscope (Nikon, Japan). In the case of MDA-MB-231, cell viability was significantly reduced to only 9.69 ± 1.31% of control. To visually analyze the inhibitory effect, the cell morphology was observed under an inverted microscope after treating with VALD-3 for 48 h. In the VALD-3-treated groups, the number of breast cancer cells was significantly less than in the control group. Particularly, when the concentration of VALD-3 reached 10 mg/l, the cells contracted, became deformed and detached (Fig. 1C).

VALD-3 induces apoptosis of MCF-7 and MDA-MB-231 cells. Hoechst 33258 staining was per-
formed to determine if VALD-3 induced apoptosis of MCF-7 and MDA-MB-231 cells. The results showed that both cell lines exhibited evident apoptotic features after being treated with VALD-3, including nuclear fragmentation, irregular chromatin condensation and apoptotic body formation ( Fig. 2A, B). Annexin V/PI staining was performed to quantify apoptosis. Flow cytometry analysis showed that the percentage of apoptotic MCF-7 cells increased significantly after treatment with VALD-3 for 24 h, reaching 24.09% at 40 mg/l (Fig. 3A). Treating for 48 h, 72 h with the same concentration of VALD-3 resulted in a significantly higher cell apoptosis rate than when the treatment was for 24 h (Fig. 3C). Similarly, the percentage of apoptotic MDA-MB-231 cells increased significantly with increasing VALD-3 concentrations when compared with control ( Fig. 3B, D). These results indicate that VALD-3 induces apoptosis of breast cancer cells in a dose-and time-dependent manner.  Compared with the control group, the levels of cleaved Caspase-3, cleaved Caspase-8 and cleaved PARP were significantly increased in VALD-3-treated breast cancer cells, indicating that VALD-3 could simultaneously activate these proteins (Fig. 5A, C, D). Furthermore, the levels of Cytochrome C, a molecule upstream of Caspase-9, were examined by Western blot. The results showed that the levels of Cytochrome C were markedly higher than in the control group (Fig. 5A). These results indicate that VALD-3 triggers MCF-7 and MDA-MB-231 apoptosis by a mitochondrial-mediated apoptotic signaling pathway.

VALD-3 promotes the expression of pro-apoptotic proteins and inhibits the expression of anti-apoptotic proteins in MCF-7 and MDA-MB-231 cells. To verify that apoptosis of MCF-7 and
MDA-MB-231 cells induced by VALD-3 was related to activation of the mitochondrial apoptotic pathway, we measured the levels of Bad, Bax, Bcl-2, Bcl-xl, Survivin and XIAP by Western blot. We found that the levels of pro-apoptotic Bad and Bax proteins were significantly increased. In contrast, the levels of anti-apoptotic proteins like Bcl-2, Bcl-xl, Survivin and XIAP, were markedly reduced in VALD-3-treated MCF-7 and MDA-MB-231 cells when compared with control cells (Fig. 5E). As shown in Fig. 5G, H, analyses of the relative levels of proapoptotic and anti-apoptotic proteins were consistent with the above results. In addition, Bcl-2 and Bad mRNA expression levels in MCF-7 cells and MDA-MB-231 cells were analyzed by qRT-PCR. The results showed that VALD-3 downregulated Bcl-2 and upregulated Bax expression at the mRNA level (Fig. 5B, F). Finally, immunohistochemistry tests were performed to detect the expression of Bad and Bax in tumor tissues. The results showed that treatment with VALD-3 significantly increased the expression of Bad and Bax in tumor tissues (Fig. 7I). These results demonstrate that VALD-3 triggers apoptosis via a caspase-dependent intrinsic pathway.  (Fig. 6A). Analysis of the  www.nature.com/scientificreports/ relative protein levels showed that VALD-3 inhibited the Wnt/β-catenin pathway and its downstream molecules in a concentration dependent manner (Fig. 6B, D). In addition, qRT-PCR analysis showed that VALD-3 reduced β-catenin, C-myc and CyclinD1 mRNA levels in MCF-7 and MDA-MB-231 cells (Fig. 6C, E).  The percent inhibitions were 57.4% for cisplatin (5 mg/kg/3d), 51.0% for VALD-3 (20 mg/kg/d) and 58.2% for VALD-3 + cisplatin ( Fig. 7D; Table1). In addition, the weight curve showed important differences between the control and experimental groups. As shown in Fig. 7B, the body weights of mice belonging to the cisplatin (5 mg/ kg/3d) and VALD-3 + cisplatin groups were significantly lower than the control. Importantly, whereas the body www.nature.com/scientificreports/ weights of mice in the cisplatin group were lower than in the control group, we observed no significant differences in body weights between the VALD-3 (20 mg/kg/d) and control groups. On the day of sacrifice, the body weights for the cisplatin, VALD-3, VALD-3 + cisplatin and control groups were 14.90 ± 0.87 g, 17.83 ± 0.73 g, 14.69 ± 0.97 g and 17.66 ± 0.48 g, respectively (Table 1). Western blots were also performed to examine the levels of β-catenin and downstream molecules. Compared with the control group, the expression levels of β-catenin, c-myc, cyclinD, LEF-1, CD44 and Mmmp7 in VALD-3 (20 mg/kg/d) group were significantly reduced (Fig. 7F).

VALD-3 reduced tumor growth in an MCF
The results of Western blots also showed the levels of apoptosis related protein (XIAP and Survivin) significantly down-regulated in VALD-3 (20 mg/kg/d) tumor tissues (Fig. 7E) and the increased cellular apoptosis were confirmed by TUNEL staining (Fig. 7H). In addition, survival analysis results showed that VALD-3 can significantly prolong the survival time of mice (P = 0.036) (Fig. 7G). Furthermore, H&E staining showed that VALD-3 at a concentration of 20 mg/kg did not induce significant heart, hepatic or kidney damage when compared with control (Fig. 7J). These results suggest that VALD-3 has potent anti-breast cancer effects but with low toxicity.

Discussion
Schiff bases are mainly organic compounds containing imine or methylamine specific groups (-RC=N-), usually formed by condensation of ammonia and reactive carbonyl compounds. Over the past few decades, Schiff bases and their complexes have been the focus of increasing attention due to their pharmacological activities, which include anti-inflammatory, anti-microbial, anti-viral and anti-tumor effects 8,[20][21][22] . Previous studies have confirmed significant anti-tumor effects of Valdien, which belongs to the family of Schiff bases, against colon cancer and human non-Hodgkin's lymphoma. However, Valdien has poor water solubility, limiting its clinical application, and its effects against breast cancer as well as the underlying mechanisms have not been elucidated. Therefore, we synthesized Schiff base ligands from o-vanillin derivatives, identified one with high water solubility (VALD-3), and investigated its anti-tumor effects and potential mechanisms of action in vivo and in vitro.
To measure the cytotoxic effects of VALD-3 against MCF-7 and MDA-MB-231 cells, we employed the MTT assay. The results showed that VALD-3 exerted significant time-and dose-dependent anti-proliferative effects against MCF-7 and MDA-MB-231 cells, indicating that VALD-3 had the ability to inhibit the growth of breast cancer cells in vitro. At the concentration 40 mg/l, VALD-3 reduced the viability of MCF-7 and MDA-MB-231 cells to only 24.01 ± 5.87% and 9.69 ± 1.31% of the control values, respectively. These results indicate that VALD-3 inhibits cell proliferation and induces cytotoxicity.
Apoptosis is a form of induced cell death triggered by multiple signaling pathways. It involves regulation at the gene level and results in the orderly and efficient removal of damaged cells 23,24 . Studies have demonstrated that apoptosis can be initiated by engagement of a death receptor (the so-called extrinsic pathway), or by the mitochondrial (or intrinsic) pathway 25 . The activation of caspases ultimately results in morphological and biochemical changes common to both the death receptor and mitochondrial pathways 26,27 . The best characterized death receptors of the extrinsic pathway are Fas and TNFR1, in which caspase 8, caspase 3 and other downstream caspases are activated, triggering the apoptotic cascade 28 . An intracellular protein-protein interaction domain, called the death domain (DD), structurally defines the death receptors and is closely related to the induction of apoptosis signals 29 . In the mitochondrial-mediated pathway, different stress conditions cause cytochrome c, an apoptosis-inducing factor, to be released from mitochondria into the cytosol. Cytochrome c can then form the apoptosome by binding to cytosolic Apaf-1 (apoptosis protease activating factor-1), activating caspase 9 and downstream effectors (caspase-8, caspase-3 and PARP), and ultimately triggering auto-activation and apoptosis 30,31 . In the current study, Hoechst 33258 staining showed that VALD-3 induced apoptotic body formation in MCF-7 and MDA-MB-231 cells. In addition, we quantified early and late apoptosis by flow cytometry to determine the effects of VALD-3 on breast cancer cells. Consistent with the Hoechst 33258 staining results, we found that VALD-3 induced apoptosis of breast cancer cells. VALD-3 seems to have a stronger apoptosisinducing effect against triple-negative breast cancer MDA-MB-231 cells than against MCF-7 cells. Western blotting results showed that treatment of breast cancer cells with VALD-3 resulted in cleavage/activation of cytochrome c, caspase-8, caspase-3 and PARP.
Bcl-2 family proteins are pivotal regulators of apoptosis, and their anti-apoptotic and pro-apoptotic members play vital roles in the mitochondrial pathway 32 . Whereas anti-apoptotic proteins (Bcl-2, Bcl-xL) can block apoptosis by inhibiting their pro-apoptotic counterparts through protein-protein interactions, pro-apoptotic proteins (Bax, Bad) facilitate this process by promoting the mitochondrial release of cytochrome-c, ultimately resulting in the cleavage of critical cellular proteins 30,[33][34][35] . Our results showed that VALD-3 upregulated the levels of pro-apoptotic Bax and Bad proteins while simultaneously downregulating the levels of anti-apoptotic Table 1. Tumor growth parameters of VALD-3 treatment on the breast cancer xenografts model. *P < 0.05 compared with the control group with significant differences. www.nature.com/scientificreports/ Bcl-2, Bcl-xl proteins. On the other hand, X-linked inhibitor of apoptosis protein (XIAP) and survivin are two factors showing the strongest apoptosis-inhibitory effects of the known inhibitor of apoptosis proteins (IAPs). IAPs are endogenous proteins that can strongly inhibit apoptosis triggered by various factors. Studies have shown that XIAP is a crucial therapeutic target in cancer 36,37 . We found that the expression of Survivin and XIAP protein was markedly reduced in VALD-3-treated MCF-7 and MDA-MB-231 cells. These results indicate that VALD-3 induces apoptosis of MCF-7 and MDA-MB-231 cells partly through the mitochondrial apoptotic pathway. It is widely accepted that cell cycle arrest can induce apoptosis. Many anticancer agents alter the regulation of the cell-cycle machinery, leading to cell cycle arrest at any phase and inhibition of tumor cell growth 38,39 . Cell cycle regulation is mediated by a combination of cyclins, CDKs and CDK inhibitors (CDKI). Cyclin D specifically regulates the G1/S phase, and Cyclin D overexpression accelerates progression from G0/G1 to S. Cyclin B and CDK1, two specific regulators of the G2/M phase, interact with each other and form the maturation promoting factor (MPF) [40][41][42] . The S phase arrest induced by VALD-3 in MCF-7 and MDA-MB-231 cells may be linked to downregulation of Cyclin D. On the other hand, based on flow cytometric analysis, the G2/M arrest of MCF-7 cells may be related to Cyclin B and CDK1 downregulation. However, it is surprising that although VALD-3 downregulated cyclin B1 and CDK1 in MDA-MB-231 cells, the induction of G2/M phase arrest was not observed. Collectively, these results indicate that inhibition of cell cycle progression may underlie the anti-cancer effects of VALD-3 against breast cancer cells.
The Wnt pathway is one of the most important pathways regulating the development of breast cancer 43 , andβ-catenin is a key regulator of the Wnt pathway. This pathway is not active in normal mature cells. When the Wnt pathway is activated, β-catenin is dephosphorylated and enters the nucleus to bind to the transcription factor TCF/LEF, thereby turning on the transcription of downstream c-myc and other target genes 44 . For this reason, we explored the effects of VALD-3 on the Wnt/β-catenin pathway in breast cancer cells by Western blotting. We found evidence that β-catenin was significantly downregulated, and transcription factor TCF/LEF and downstream proteins were also reduced, indicating inactivation of the Wnt/β-catenin signaling pathway.
However, anti-tumor effects of VALD-3 in vitro does not necessarily mean that it will also have anti-tumor effects in vivo. Therefore, we established an MCF-7 tumor xenograft model to evaluate the anticancer effects of VALD-3 in vivo. We found that VALD-3 at 20 mg/kg inhibited tumor growth with a potency similar to cisplatin. We also evaluated the expression of β-catenin and its downstream proteins, XIAP and Survivin, in tumor tissues. The results showed that VALD-3 lowered the expression of XIAP, Survivin and the Wnt/β-catenin signaling pathway, in agreement with the in vitro results. In addition, the Tunel assay showed important differences between the control and experimental groups. VALD-3 (20 mg/kg/d) obviously induced apoptosis of breast cancer cells. The survival analysis showed that VALD-3 prolonged the survival of tumor-bearing mice when compared with the control group. Moreover, H&E staining showed that VALD-3 markedly suppressed tumor growth with no clear signs of damage to the major organs. Therefore, VALD-3 showed marked tumor inhibitory effects and low toxicity in vivo. These results indicate that it may be a good anticancer drug candidate.
In conclusion, this study provides evidence that VALD-3 inhibits the proliferation of breast cancer cells in vitro and in vivo. Our study demonstrated that VALD-3 reduced cell viability and colony formation, and induced S phase arrest and cellular apoptosis. In addition, modulation of the Wnt/β-catenin signaling pathway was involved in the VALD-3-induced apoptosis of MCF-7 and MDA-MB-231 cells. Collectively, these results demonstrate the anti-tumor effects of VALD-3 and its probable mechanisms of action. Therefore, VALD-3 may be an alternative strategy for the treatment of breast cancer. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.