Chalcone 9X Contributed to Repressing Glioma Cell Growth and Migration and Inducing Cell Apoptosis by Reducing FOXM1 Expression In Vitro and Repressing Tumor Growth In Vivo

Objective Natural and synthetic chalcones played roles in inflammation and cancers. Chalcone 9X was an aromatic ketone that was found to inhibit cell growth of hepatic cancer and lung cancer cells. In this study, we wanted to investigate the functions of Chalcone 9X in glioma. Materials and Methods Chemical Chalcone 9X was added in human glioma cell lines (U87 and T98G cells) and normal astrocyte cell lines (HA1800) with various concentrations (0 μmol/L, 20 μmol/L, 50 μmol/L, and 100 μmol/L). CCK-8 assay was used to measure cell viability. Flow cytometric assay was used to measure cell apoptotic rates. Wound healing assay and transwell assay were used to measure cell invasion. RT-PCR was used to detect relative mRNA expressions, and the protein expressions were detected by western blot (WB) and immunohistochemical staining (IHC). Finally, nude mouse xenograft assay was performed to prove the effects of Chalcone 9X in vivo. Results Results revealed that Chalcone 9X treatment suppressed cell viability and cell migration capacity; it could also induce cell apoptosis in U87 and T98G cells with dose dependence. However, it had little cytotoxicity to normal astrocyte HA1800 cells. Moreover, Chalcone 9X treatment could repress the mRNA and protein expressions of FOXM1 in human glioma cell lines, which was an oncogene that could promote the progression and malignancy of glioma. In addition, FOXM1 overexpression dismissed the Chalcone 9X effects on cell proliferation, apoptosis, and migration in human glioma cell lines. Finally, in vivo assay showed that Chalcone 9X treatment repressed the expression of FOXM1, which inhibited the tumor growth of a xenograft model injected with U87 in nude mice. Conclusions In all, we found that Chalcone 9X could suppress cell proliferation and migration and induce cell apoptosis in human glioma cells, while it has little cytotoxicity to normal astrocyte cells. Therefore, we uncovered a novel way that Chalcone 9X could inhibit FOXM1 expression and repress the progression and biofunctions of glioma cells, which might be a potential therapeutic drug for treating human glioma.


Introduction
Glioma is a king of prevalent cancer on central nervous system worldwide [1][2][3][4], and it is divided into astrocytoma, oligodendroglioma, and ependymal tumor [1]. Researchers reported that its incidence has been obviously increased for the past 20 years [1,3]. Because of its variability, no specific treatments and strategies have been developed and applied [5,6]. Therefore, it is urgent to find some new effective therapies and drugs with significant effects and low cytotoxicity to hinder the development of glioma.
Natural and chemical synthetic chalcones have been revealed to play critical roles in some biological functions, such as inflammation [7][8][9] and antibacterial effects [10,11]. Furthermore, more studies demonstrated that chalcones could inhibit tumor progression in various cancers [12][13][14][15][16][17][18][19], including glioma [15,18]. For example, Mendanha et al. found that Chalcone 1 could induce cell apoptosis of glioblastoma (GBM), which was triggered by the cell cycle arrest in the G2/M checkpoint, and might be a potential to provide a new treatment alternative for GBM 15 . Moreover, Wang et al. also discovered that flavokawain B (FKB), a natural kava chalcone, induced protective autophagy signaling pathway in GBM cells, which indicated that the combination treatment of FKB with autophagy inhibitors might be an effective strategy for GBM 18 .
The chemical synthetic chalcone 4 ′ -hydroxyl-3,4dimethoxyl-3 ′ -methoxylchalcone (Chalcone 9X) was an aromatic ketone that had been demonstrated to have some antitumor activities in human lung cancer cells and hepatic cancer cells [19]. Researches revealed that Chalcone 9X could induce cell apoptosis and inhibit invasion and migration capacities through activating Caspase-3 and Caspase-8 signaling pathways in lung cancer cells and hepatic cancer cells in vitro and in vivo [19]. However, the functions of Chalcone 9X in other tumors remained unknown, and whether Chalcone 9X could affect glioma cells remained unclear.
In this study, we wanted to investigate whether Chalcone 9X played some roles in glioma. Our preliminary results hinted that it showed some antitumor activities in human glioma cells. As a result, we explored the potential molecular mechanism of Chalcone 9X in glioma in vitro and in vivo.

Materials and Methods
2.1. Chalcone 9X Synthesis. The chemical Chalcone 9X was synthesized with the standard Claisen-Schmidt aldol condensation protocols from El-Mahdy et al. [20] and Konoike et al. [21] The 3,4-dimethoxyl benzaldehyde was added into a stirred solution of 4-hydroxyl-3-methoxylacetophenone, and the solution was added with NaOH and ethanol. The synthetic reaction was finished at room temperature for 48 h, which was then quenched in ice-cold water and acidized with 18% HCl. The separated product was filtered. Finally, the crude product was recrystallized from ethanol and stored until usage. The chemical formula of Chalcone 9X is C 18 H 18 O 5 , and the molecular weight is 314 g/mol. Chalcone 9X was diluted into concentrations of 20 μmol/L, 50 μmol/L, and 100 μmol/L to treat cells.

Construction of Plasmid and Cell
Transfection. The full length of human FOXM1 cDNA was synthesized and constructed into a pCDNA3.1 (Invitrogen, Carlsbad, CA, USA) vector, resulting in FOXM1-pcDNA for its overexpression, which was named p-FOXM1. And scrambled oligonucleotides were synthesized to be used as the negative control, which was named p-NC. U87 and T98G cells were preincubated on six-well plates to about 50% confluence, which was then transfected by incubation with p-FOXM1 or p-NC with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. U87 and T98G cells with FOXM1 stable overexpression were obtained. Then, Chalcone 9X was used to treat these prepared cells, and cells were harvested after 48 h for further analysis.
2.4. CCK-8 Assay. Cell viability of human normal astrocyte cell line HA1800 and glioma cells (U87 and T98G) were detected by a cell counting kit-8 (CCK-8) assay (Dojindo, Rockville, Japan). Cells were seeded on 96-well plates (2 × 10 3 cells/mL) and cultivated in an incubator. Then, 10 μL CCK-8 agent was added to the prepared cells at 24 h, 48 h, and 72 h. Finally, the absorbance of each well was measured at 450 nm using a microplate reader (Thermo Fisher, USA). Three independent experiments were repeated to get the mean value.
2.5. Flow Cytometric Analysis. Cell apoptotic rates were measured by flow cytometric analysis. After Chalcone 9X treatment for the indicated time, prepared glioma cells were washed with PBS three times and digested with 0.25% trypsin. Then cells were stained with FITC-Annexin V and Propidium iodide (PI) solution with 50 μg/mL at room temperature for 30 min. At last, stained cells were subjected to a FACScan Calibur flow cytometer (BD Biosciences Becton-Dickinson, Franklin Lakes, USA) to calculate apoptotic cell rates.
2.6. Transwell Assay. Cell migration ability was measured by Transwell assay. U87 and T98G cells (5 × 10 4 cells/well) were plated to the top of upper chambers (Corning, NY, USA) on the noncoated membranes with a 200 μL serumfree medium. The lower chambers were added with 500 μL 10% FBS and DMEM (Hyclone, Massachusetts, USA). After 24 h, the cells that remained in the upper chambers were removed; however, migrated cells through the membranes were stained with 95% methanol for 15 min and then stained with 0.1% crystal violet for 20 min. Finally, the migrated cells were counted and examined under a light microscope (Olympus, Tokyo, Japan).

Wound
Healing Assay. Wound healing assay was used to measure glioma cell migration ability. U87 and T98G cells were cultured in 60-mm tissue culture plates for 24 h. Then a pipette tip was used to create an artificial wound, and the wells were washed with PBS three times to remove the detached cells. Furthermore, U87 and T98G cells were treated with Chalcone 9X (0 μmol/L, 20 μmol/L, 50 μmol/L, and 100 μmol/L) in new media for 48 h. The wounds were photographed and measured using an inverted microscope (Leica, Germany).

Tumor Xenograft in Nude
Mice. Eighteen nude male BALB/c mice were included (SLAC Laboratory Animal, Shanghai, China), which were 4 weeks old and weigh 15-20 g. Mice were raised in standard laboratory conditions and fed food and water. Then, the nude mice were injected with U87 cells (5 × 10 6 cells in 0.1 mL of PBS) on the right flanks. After two weeks, these mice were divided into three groups (n = 6), including the model group, Chalcone 9X 20 mg/kg group, and Chalcone 9X 40 mg/kg group. Chalcone 9X was provided by tail vein injection once a day for four weeks, and tumor volumes of nude mice in three groups were observed every week. The tumor volumes were calculated with V = π/6 × length × width × height. Mice were sacrificed; tumor tissues were obtained for further research. The extracted tumors were frozen with optimal cutting temperature (OCT) compound (Sigma-Aldrich; Merck KgaA, USA) at −20°C, which were then stored at −80°C until use. All procedures were carried out according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. This study was approved by the Animal Care Ethics Committee of our hospital.
2.11. Immunohistochemical Staining. The extracted tumor tissues were sliced at a thickness of 10 μm and were placed on gelatin-coated glass slides. Furthermore, the sliced tumor tissues were fixed with 4% paraformaldehyde (PFA) for 15 min, which were incubated with methanol containing 3% hydrogen peroxide for another 20 min. The sliced tumor tissues were placed with 0.3% Triton X-100 in PBS for 20 min. Besides, the tissues were blocked with the blocking solution for 1 h, which was fixed with 0.1% BSA (Sigma-Aldrich; Merck KgaA, USA), 0.3% Triton X-100, and 1.5% FBS (Hyclone; Cytiva, USA). Then, the tissues were incubated with the primary antibodies overnight at 4°C, including FOXM1 (ab207298, Abcam, Cambridge, USA) and Caspase-3 (ab13847, Abcam, Cambridge, USA). Finally, the incubated tissues were subsequently washed with PBS two times and then incubated with biotinylated anti-rabbit (cat. no. BA-1000), and antirat (cat. no. BA-4000) IgG (H+L) antibodies (1 : 400, Abcam, Cambridge, MA, USA) were labeled secondary antibodies. The tumor tissues were sealed and observed under an inverted microscope (IX73, Olympus, Tokyo, Japan). To determine cell proliferation of tumor tissues, four high-powered fields of vision were obtained from each group. The average number of FOXM1+ cells or Caspase-3+ cells was used for statistical analysis, which was quantified as the protein expression.
2.12. Statistical Analysis. The data were analyzed by SPSS 18.0 (Chicago, IL, USA) and GraphPad Prism 5.0 (La Jolla, CA, USA). Data were analyzed according to three independent experiments. Statistical significance between groups were analyzed by the SNK method followed by one-way ANOVA. p < 0:05 was considered to be a statistically significant difference.    In addition, we detected some gene expressions of migration, such as Vimentin [22,23], N-cadherin [24][25][26], and Ecadherin [27,28]. Results showed that Chalcone 9X treatment repressed protein levels of Vimentin and N-cadherin and promoted E-cadherin expression with dose dependency in U87 and T98G cells after 48 h (Figures 3(i)-3(k)) (p < 0:05), which suggested that Chalcone 9X repressed cell migration in glioma cells. The above data indicated that Chalcone 9X could suppress cell proliferation and migration in glioma cells. However, the mechanism was not precise. Therefore, we intended to explore the potential mechanism of Chalcone 9X in treating glioma.
Results indicated that Chalcone 9X repressed the protein levels of FOXM1 in a dose-dependent manner in U87 and T98G cells after 48 h ( Figure 4) (p < 0:05), which might suggest that Chalcone 9X might play some roles in glioma cell proliferation via the oncogenic factor FOXM1.

FOXM1
Overexpression Removed the Chalcone 9X Effects on Cell Proliferation, Apoptosis, and Migration in U87 and T98G Cells. To verify that Chalcone 9X repressed cell proliferation, migration, and induced cell apoptosis through repressing FOXM1 expression in glioma, the total length of FOXM1 was constructed into the plasmid, which was named p-FOXM1 and transfected into U87 and T98G cells. Then Chalcone 9X with 100 μmol/L was added into the glioma cells with p-FOXM1 or p-NC for 48 h. The cell proliferation and migration capacities were observed. Results showed that the protein expressions of FOXM1 were upregulated after p-FOXM1 transfection into U87 and T98G cells, which suggested FOXM1 was successfully transfected into glioma cells, while FOXM1 protein levels were reduced after treating with Chalcone 9X (Figure 5(a)) (p < 0:001). Furthermore, the CCK-8 assay showed that the promoted cell viability in the p-FOXM1 group was repressed after treatment with Chalcone 9X (Figure 5(b)) (p < 0:001). Moreover, FACS assay showed that the reduced cell apoptotic rates in the p-FOXM1 group was increased after treating with Chalcone 9X (Figure 5(c)) (p < 0:001). Besides, the transwell assay illustrated that the promoted cell migration capacity in p-FOXM1 group was repressed after treating with Chalcone 9X (Figures 5(d) and 5(e)) (p < 0:001). In addition, the increased protein levels of Vimentin and N-cadherin and repressed protein levels of E-cadherin were reversed after treatment with Chalcone 9X in glioma cells (Figures 5(f)-5(h)) (p < 0:001). Above all, these data suggested that FOXM1 overexpression removed the Chalcone 9X effects on cell proliferation, apoptosis, and migration in U87 and T98G cells.

BioMed Research International
In addition, as previously introduced, the FOXM1-positive index is an oncogene for cancer development and a marker for cell proliferation [36][37][38], and the Caspase-3 is a marker for cell apoptosis [19]. As shown in   BioMed Research International (p < 0:05), which indicated that the migrated capacity was also repressed in vivo. Collectively, these data suggested that Chalcone 9X could inhibit U87 tumor growth of a xenograft model in nude mice.

Discussion
Chalcones have been demonstrated to inhibit tumor progression and development in cancers [12][13][14][15][16][17][18], including glioma [15,18]. The synthetic Chalcone 9X is an aromatic ketone, which has been proved to own antitumor activities in lung cancer cells and hepatic cancer cells through activating Caspase-3 and Caspase-8 signaling pathway [19]. The functions of Chalcone 9X in glioma cells remained unclear. In this study, we found that Chalcone 9X could suppress cell growth and induce cell apoptosis in glioma cells, and it showed little cytotoxicity to normal astrocyte cell line HA1800. As a result, we intended to explore the molecular mechanisms of Chalcone 9X in glioma cells.
In addition to its apoptotic and growth inhibitory functions, Chalcone 9X was also found to suppress the migration of glioma cells. N-cadherin and E-cadherin belong to the cadherin family, which have been found to regulate migration and metastasis in glioma [24][25][26][27][28]. Vimentin is a protein in the intermediate filament, which forms the cytoskeleton and is associated with cancer migration [22,23]. We found 9 BioMed Research International that Chalcone 9X treatment inhibited cell migration and protein expressions of N-cadherin and Vimentin were repressed, while E-cadherin was increased, which confirmed that Chalcone 9X could repress migration in glioma cells, but the potential mechanisms remained unclear.
In addition, we found that FOXM1 overexpression blocked the effects of Chalcone 9X on cell proliferation, apoptosis, and migration in glioma cells. Finally, a xenograft tumor in vivo model of U87 cells in nude mice was used. Results proved that Chalcone 9X treatment could inhibit tumor growth and the tumor migrated protein expressions of N-cadherin and Vimentin in vivo. In this study, we found that Chalcone 9X showed a lower cytotoxic effect than Chalcones 1 for human normal cell lines, and the security is much better than Chalcones 1. 100 μmol/L Chalcone 9X had little cytotoxicity to normal astrocyte HA1800 cells, while 100 μmol/L Chalcone 1 showed very high cytotoxicity to nontumor cell line [15].  Figure 5: FOXM1 overexpression removed the Chalcone 9X effects on cell proliferation, apoptosis, and migration in U87 and T98G cells. P-NC and p-FOXM1 were, respectively, glioma cells, and then 100 μmol/L Chalcone 9X was treated in these cells for 48 h. (a) The mRNA levels of FOXM1 were detected by RT-PCR. (b) CCK8 assay was used to measure cell viability. (c) FACS assay was used to measure cell apoptosis. (d, e) Transwell assay was used to detect cell migration capacities. (f-h) The protein levels of Vimentin, N-cadherin, and Ecadherin in glioma cells were detected by WB (magnifications: ×0.8). * * p < 0:01 and * * * p < 0:001.
A report found that chalcone 9X could induce conformation changes of Caspase-3 protein into mature Caspase-3 or cleaved Caspase-3, which could induce cell apoptosis [19]. In this study, our data uncovered that chalcone 9X could repress cell proliferation and the migration and induce cell apoptosis, which is likely conferred by the ability of this compound to promote the activation of FOXM1, thereby downregulating N-cadherin and Vimentin expression, upregulating Caspase-3 and N-cadherin expression. Moreover, that might be a potential treatment for glioma. Vimentin N-cadherin E-cadherin (g) Figure 6: Chalcone 9X inhibited U87 tumor growth of a xenograft model in nude mice. U87 cells were injected into nude mice for two weeks; then Chalcone 9X (20 mg/kg, 40 mg/kg) was provided by gavage to these mice for another four weeks. (a) Images of 9 primary tumors at day 28 from the model group, Chalcone 9X 20 mg/kg group, and Chalcone 9X 40 mg/kg group after mouse scarification. (b) Tumor volumes were measured every seven days. (c) Tumor weights on day 28 after injection was calculated. (d, e) Immunohistochemical staining of FOXM1 and Caspase-3 in tumor tissue sections. (f, g) WB assay was performed to detect the protein expressions of FOXM1, Vimentin, N-cadherin, and E-cadherin. # p < 0:05 and ### p < 0:001, model group versus Chalcone 9X 20 mg/kg; * * * p < 0:001, model group versus Chalcone 9X 40 mg/kg.

Conclusion
In conclusion, this study showed that Chalcone 9X, a synthetic chemical chalcone, suppressed cell proliferation, the migration, and induced cell apoptosis in human glioma cells, and it has little cytotoxicity to normal astrocyte cells. Therefore, we uncovered a novel way that Chalcone 9X could inhibit FOXM1 expression and repress the progression and bio-functions of glioma cells, which might be a potential therapeutic drug for treating human glioma.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
The authors declare that they have no conflict of interest.