CC12 Induces Apoptotic Cell Death and Cell Cycle Arrest in Human Glioblastoma Cell Lines and Mouse Xenograft Model.

Among central nervous system tumors, glioblastoma (GBM) is the most common and the most malignant type. Even under current standard treatments, the overall survival rate is still low and the recurrence rate is high. Therefore, developing novel and effective therapy is urgently needed. CC12, a synthesized small molecule, was evaluated for the potential anti-GBM effects in two GBM cell lines, U87MG and U118MG. The observations of cell morphology, MTT assay, flow cytometry-based apoptosis after CC12 treatment, were conducted. Western blot was performed for the investigation of the apoptotic mechanism. Positron emission tomography scan analysis and bioluminescent imaging assay using a mouse xenograft model were performed for the effect of CC12 in vivo. After treated by 10 μM CC12 for 24 h, both U118MG and U87MG cells showed tumor cell death. MTT assay results showed that the survival rates decreased when the CC12 concentrations or the treatment periods increased. Ki-67 expression and flow cytometry results indicated that the proliferation was inhibited in GBM cells, and G1 phase arrest was shown. The results of 7-AAD, Br-dUTP, and JC-1 staining all showed the apoptosis of GBM cells after CC12 treatment. Increased γH2AX, caspase-3, and poly (ADP-ribose) polymerase (PARP) levels meant the DNA damage, and increased Bcl2 family proteins after CC12 treatment indicated the intrinsic apoptotic pathway was involved in CC12 induced apoptosis. Furthermore, CC12 can induce the decrease of tumor prognostic marker DcR3. In vivo experiment results showed the effect of CC12 on tumor size reduction of CC12. In addition, the ability to cross the brain–blood barrier of CC12 was also confirmed. CC12 may have anti-tumor ability through the regulation of cell cycle and apoptosis in vitro and in vivo.


Introduction
Primary central nervous system (CNS) tumors are a group of malignancies with diversely histological characteristics. In the World Health Organization (WHO) classification system, over one hundred distinct entities were identified [1]. Gliomas is one of the major types of CNS tumors and is classified to several subtypes, including oligodendroglioma, oligoastrocytoma, astrocytoma,

MTT Assay
The 4 × 10 4 cells were plated in 24-well plates and incubated at 37 °C for 24 h, and then CC12 was added into the medium with 2.5, 5, 10, 15, 20 μM for 24, 48, or 72 h. After treatment, 500 μL MTT solution (5 mg/mL in phosphate-buffered saline) was added into each well, the cells were continuously incubated for additional 4 h. The formazan crystals were solubilized with 500 μL DMSO, and the optical density values were detected at wavelength 570 nm by Synergy HT multidetection micro plate reader (Awareness Technology, Palm City, FL, USA). All experiments were performed in triplicate, and the relative cell growth was expressed as a percentage of the untreated control cells (set to 100%) to allow for unwanted source of variation.

Flow Cytometry-Based Apoptosisdetections
A total of 2×10 5 cells per well were seeded in 6-well plates and incubated for 24h, then CC12 was added. After the incubation with CC12 for 24 h or 48 h, cells were suspected with 0.05% trypsin and gathered, fixed with 75% ethanol at −20°C overnight, washed with phosphate buffered saline, and stained with 500 μL propidium iodide/RNase staining solution (BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at room temperature in the dark. The 10 4 stained cells were analyzed using FACSVerse™ laser flow cytometric analysis system (BD Biosciences) for each sample. Each experiment was done, at least, quantic. Annexin V-PE Dead Cell Apoptosis kit (BD Biosciences) was utilized for apoptotic cell death analysis, according to the manufacturer's protocol. Cells were trypsinized and washed twice with phosphate-buffer saline, then resuspended in 100 μL Binding

MTT Assay
The 4 × 10 4 cells were plated in 24-well plates and incubated at 37 • C for 24 h, and then CC12 was added into the medium with 2.5, 5, 10, 15, 20 µM for 24, 48, or 72 h. After treatment, 500 µL MTT solution (5 mg/mL in phosphate-buffered saline) was added into each well, the cells were continuously incubated for additional 4 h. The formazan crystals were solubilized with 500 µL DMSO, and the optical density values were detected at wavelength 570 nm by Synergy HT multi-detection micro plate reader (Awareness Technology, Palm City, FL, USA). All experiments were performed in triplicate, and the relative cell growth was expressed as a percentage of the untreated control cells (set to 100%) to allow for unwanted source of variation.

Flow Cytometry-Based Apoptosisdetections
A total of 2×10 5 cells per well were seeded in 6-well plates and incubated for 24h, then CC12 was added. After the incubation with CC12 for 24 h or 48 h, cells were suspected with 0.05% trypsin and gathered, fixed with 75% ethanol at −20 • C overnight, washed with phosphate buffered saline, and stained with 500 µL propidium iodide/RNase staining solution (BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at room temperature in the dark. The 10 4 stained cells were analyzed using FACSVerse™ laser flow cytometric analysis system (BD Biosciences) for each sample. Each experiment was done, at least, quantic. Annexin V-PE Dead Cell Apoptosis kit (BD Biosciences) was utilized for apoptotic cell death analysis, according to the manufacturer's protocol. Cells were trypsinized and washed twice with phosphate-buffer saline, then resuspended in 100 µL Binding Buffer. 5 µL Annexin V-PE and 10 µL 7-AAD then incubated for 15 min in the dark at room temperature. Next, 400 µL binding buffer was added to the cells and 10 4 events were acquired for each sample. The 7-AAD was analyzed at 488 nm and Annexin V-PE fluorescence was detected at 617 nm in a flow cytometer (FACSVerse™; BD Biosciences). An APO-DIRECTTM Kit (BD Biosciences) and a Flow Cytometry Mitochondrial Membrane Potential Detection Kit (BD Biosciences) were utilized for detecting DNA damage and the apoptotic cells. Each experiment was done, at least, in triplicate. Data were analyzed using FlowJo v7.6.5 software (Tree Star Inc., Ashland, OR, USA).

Data Analysis
All experiments were performed at least three times, and the values were reported as means ± SD (standard deviation). Differences between groups were evaluated using the Kruskal-Wallis test, followed by post hoc comparisons using the GraphPad Prime5.0 software (GraphPad Software, San Diego, CA). Statistical significance was set at p < 0.05.

CC12 Induced Tumor Cell Death and Inhibited Tumor Cell Proliferation
After treated with 10 µM CC12 for 24 h, both U118MG and U87MG cells were shrinking, meaning that CC12 may induce tumor cell death ( Figure 2A). This effect was time-and dose-dependent; results of MTT assay showed that the survival rates decreased when the CC12 concentrations or the treatment time increased. Both GBM cell lines showed this effect, in the U118MG cell line, when treated with CC12 for 48 h and 72 h; the IC50 were 41.87 and 5.791 µM, respectively. In the U87MG cell line, when treated with CC12 for 48 h and 72 h, the IC50 was 22.38 and 7.347 µM, respectively ( Figure 2B). CC12 also inhibited tumor cell proliferation, represented by both proliferation marker Ki-67 expression and the results of flow cytometric analysis. The expression of Ki-67 reduced after CC12 treatment, and the reduction levels increased with higher CC12 concentrations ( Figure 3A). The hypodiploid peaks were shown in the histograms of flow cytometry, meaning the increases in the proportion of apoptotic cells ( Figure 3B), and the proportions of sub-G1 phase cells also increased significantly after CC12 treatment, with 2.6% and 10.3% at 24 h, and 11.1% and 45.8% at 48 h after treatment, with 5 and 10 μM CC12, respectively ( Figure 3C). Western blot of the G1 phase drivers were performed to evaluate whether these proteins were involved in the regulation of cell cycle. The results showed that in both U118MG and U87MG cell lines, cyclinD1 decreased with the increased concentration of CC12. Further, the expressions of CDK2 and CDK4 decreased in U87MG cells; however, there was no change in U118MG cells ( Figure 3D). CC12 also inhibited tumor cell proliferation, represented by both proliferation marker Ki-67 expression and the results of flow cytometric analysis. The expression of Ki-67 reduced after CC12 treatment, and the reduction levels increased with higher CC12 concentrations ( Figure 3A). The hypodiploid peaks were shown in the histograms of flow cytometry, meaning the increases in the proportion of apoptotic cells ( Figure 3B), and the proportions of sub-G1 phase cells also increased significantly after CC12 treatment, with 2.6% and 10.3% at 24 h, and 11.1% and 45.8% at 48 h after treatment, with 5 and 10 µM CC12, respectively ( Figure 3C). Western blot of the G1 phase drivers were performed to evaluate whether these proteins were involved in the regulation of cell cycle. The results showed that in both U118MG and U87MG cell lines, cyclinD1 decreased with the increased concentration of CC12. Further, the expressions of CDK2 and CDK4 decreased in U87MG cells; however, there was no change in U118MG cells ( Figure 3D).

CC12 Induced Apoptosis of Tumor Cells
We used 7-AAD, Br-dUTP, and JC-1 staining to evaluate whether the cell death included by CC12 was through apoptosis. The increased annexin V-PE positive and 7-AAD negative cells, or increased cells in the A4 area, indicated the occurrence of apoptosis were shown in both U118MG and U87MG cell lines after treatment with different concentrations of CC12 (data not shown). The apoptosis proportion, after treatment, with 10 μM CC12 for 24 h in U118MG cells was 9.5%, as well as 13.2% in U87MG cells. In Br-dUTP staining, the apoptotic cells of the U118MG cell line significantly increased to 4.5% and 13.4% after treatment of 5 and 10 μM CC12 for 24 h, respectively. Moreover, the proportion of U87MG cells with DNA fragmentation content increased from 11.5% to 34.2%. JC-1 staining also showed similar results, the apoptotic cells increased in both bU118MG and U87MG cell lines (Figure 4). The quantification analysis of the western blot results. # and ## indicates significant difference compared with the control group, p < 0.05 and < 0.01, respectively; * and *** indicates the significant difference between 24 h and 48 h treatments within the same concentration groups, p < 0.05 and < 0.01, respectively.

CC12 Induced Apoptosis of Tumor Cells
We used 7-AAD, Br-dUTP, and JC-1 staining to evaluate whether the cell death included by CC12 was through apoptosis. The increased annexin V-PE positive and 7-AAD negative cells, or increased cells in the A4 area, indicated the occurrence of apoptosis were shown in both U118MG and U87MG cell lines after treatment with different concentrations of CC12 (data not shown). The apoptosis proportion, after treatment, with 10 µM CC12 for 24 h in U118MG cells was 9.5%, as well as 13.2% in U87MG cells. In Br-dUTP staining, the apoptotic cells of the U118MG cell line significantly increased to 4.5% and 13.4% after treatment of 5 and 10 µM CC12 for 24 h, respectively. Moreover, the proportion of U87MG cells with DNA fragmentation content increased from 11.5% to 34.2%. JC-1 staining also showed similar results, the apoptotic cells increased in both bU118MG and U87MG cell lines (Figure 4). Both intrinsic and extrinsic apoptotic pathway were evaluated for the mechanism of CC12associated tumor cell apoptosis by Western blot ( Figure 5A). γH2AX is a marker of DNA damage, which is one of the major changes when apoptosis. The expression of γH2AX was induced by CC12 treatment. ATR, P53, and phosphor-P53, Bid, Bax, caspase-3, phosphorylated caspase-9, caspase-8, and PARP increased after CC12 treatment and were dose-dependent in both U118MG and U87MG cell lines; Bcl2 expression reduced after CC12 treatment. No change was found in FasL levels. As a tumor prognostic marker, we found that DcR3 expressed in GBM, like other malignancies, and the amount decreased after CC12 treatment and with dose-dependent manner in both GBM cell lines. A proposed mechanism base on the results is shown in Figure 5B. Both intrinsic and extrinsic apoptotic pathway were evaluated for the mechanism of CC12-associated tumor cell apoptosis by Western blot ( Figure 5A). γH2AX is a marker of DNA damage, which is one of the major changes when apoptosis. The expression of γH2AX was induced by CC12 treatment. ATR, P53, and phosphor-P53, Bid, Bax, caspase-3, phosphorylated caspase-9, caspase-8, and PARP increased after CC12 treatment and were dose-dependent in both U118MG and U87MG cell lines; Bcl2 expression reduced after CC12 treatment. No change was found in FasL levels. As a tumor prognostic marker, we found that DcR3 expressed in GBM, like other malignancies, and the amount decreased after CC12 treatment and with dose-dependent manner in both GBM cell lines. A proposed mechanism base on the results is shown in Figure 5B.

CC12 Showed Tumor Elimination Effects in Vivo
Further, we used a xenograft mouse model to evaluate the potential therapeutic effects of CC12 in vivo. On Day 0, the mean uptake value of [ 18 F]-FDG in CC12 group was 0.139 ± 0.02, consistent with those in the DMSO group, as 0.136 ± 0.0, (p > 0.05), while those in the CC12 group were significantly lower than the DMSO group on Day 28, with the mean values as 0.097 ± 0.02 vs. 0.138 ± 0.01 (p < 0.01). Furthermore, the tumor volumes reduced in mice of the CC12 group; in contrast, the tumor volume enlarged in mice of the DMSO group. On Day 0, mean tumor volume of the CC12 group was 61.15 ± 6.89 mm 3 , similar to that in the DMSO group, as 64.01 ± 14.08 mm 3 (p > 0.05). On Day 28, the tumor volumes were significantly different between the two treatment groups, as 44 ± 12 mm 3 in the CC12 group and 496 ± 480 mm 3 in the DMSO group (p < 0.05). At the end of the experiment (Day 29), the tumors were isolated after the mice were sacrificed. The tumors were significantly shrunken in the mice of the CC12 group, compared to that in the DMSO group, with the tumor weight 43.6 ± 12. mg and 548 ± 554 mg in CC12 and DMSO groups, respectively (p < 0.01) ( Figure 6).

CC12 Showed Tumor Elimination Effects In Vivo
Further, we used a xenograft mouse model to evaluate the potential therapeutic effects of CC12 in vivo. On Day 0, the mean uptake value of [ 18 F]-FDG in CC12 group was 0.139 ± 0.02, consistent with those in the DMSO group, as 0.136 ± 0.0, (p > 0.05), while those in the CC12 group were significantly lower than the DMSO group on Day 28, with the mean values as 0.097 ± 0.02 vs. 0.138 ± 0.01 (p < 0.01). Furthermore, the tumor volumes reduced in mice of the CC12 group; in contrast, the tumor volume enlarged in mice of the DMSO group. On Day 0, mean tumor volume of the CC12 group was 61.15 ± 6.89 mm 3 , similar to that in the DMSO group, as 64.01 ± 14.08 mm 3 (p > 0.05). On Day 28, the tumor volumes were significantly different between the two treatment groups, as 44 ± 12 mm 3 in the CC12 group and 496 ± 480 mm 3 in the DMSO group (p < 0.05). At the end of the experiment (Day 29), the tumors were isolated after the mice were sacrificed. The tumors were significantly shrunken in the mice of the CC12 group, compared to that in the DMSO group, with the tumor weight 43.6 ± 12. mg and 548 ± 554 mg in CC12 and DMSO groups, respectively (p < 0.01) ( Figure 6).  The tumor volumes continuously increased in mice of the DMSO group; however, no significant change was found in those of CC12 treatment group. On Day 28, the difference between the CC12 and DMSO groups were significant. (E) The tumor weight at the end of the in vivo study showed the significant difference between the CC12 treatment group and the DMSO control group. Data are presented as mean ± SD, statistically signification values of ** p < 0.01, *** p < 0.001 were appraised compared with the control, # p < 0.05, ## p < 0.01, ### p < 0.001 comparing days 0 and 28. Significantly different from control; ANOVA with Tukey's test.
Since GBM is the major type of brain malignancies, the effective treatment should have ability to cross BBB. We measured the concentration of CC12 in different sources of the samples from mice received 5 mg/kg CC12 for 28 days. CC12 can be detected in the CNS by the HPLC/MS method, including cortex, striatum, and CSF, indicating that CC12 can diffuse into the brain, then induce the apoptosis of the brain tumor (Figure 7). The tumor volumes continuously increased in mice of the DMSO group; however, no significant change was found in those of CC12 treatment group. On Day 28, the difference between the CC12 and DMSO groups were significant. (E) The tumor weight at the end of the in vivo study showed the significant difference between the CC12 treatment group and the DMSO control group. Data are presented as mean ± SD, statistically signification values of ** p < 0.01, *** p < 0.001 were appraised compared with the control, # p < 0.05, ## p < 0.01, ### p < 0.001 comparing days 0 and 28. Significantly different from control; ANOVA with Tukey's test.
Since GBM is the major type of brain malignancies, the effective treatment should have ability to cross BBB. We measured the concentration of CC12 in different sources of the samples from mice received 5 mg/kg CC12 for 28 days. CC12 can be detected in the CNS by the HPLC/MS method, including cortex, striatum, and CSF, indicating that CC12 can diffuse into the brain, then induce the apoptosis of the brain tumor (Figure 7).

Figure 7.
CC12 can cross the brain-blood barrier. HPLC/MS analysis results indicated that CC12 can be detected in plasma, cortex, striatum, and CSF from mice that received 5 mg/kg CC12 for 28 days.

Discussion
In both U118MG and U87MG GBM cell lines, CC12 incubation reduced the cell survival rate, and the level of reduction increased by treatment time and CC12 dose. The tumor cell proliferation was inhibited, represented by decreased Ki-67 expression and increased sub-G1 phase cells, significantly. CC12 also increased the apoptosis rate observed by flow cytometry-base analysis. For the mechanism of cell cycle, apoptosis, and tumor prognosis, we found that G1 phase drivers decreased after CC12 treatment in U87MG cells, including cyclinD1, CDK2, and CDK4; apoptotic proteins γ-H2AX, Bid, Bax, caspase 3,8 Figure 7. CC12 can cross the brain-blood barrier. HPLC/MS analysis results indicated that CC12 can be detected in plasma, cortex, striatum, and CSF from mice that received 5 mg/kg CC12 for 28 days.

Discussion
In both U118MG and U87MG GBM cell lines, CC12 incubation reduced the cell survival rate, and the level of reduction increased by treatment time and CC12 dose. The tumor cell proliferation was inhibited, represented by decreased Ki-67 expression and increased sub-G1 phase cells, significantly. CC12 also increased the apoptosis rate observed by flow cytometry-base analysis. For the mechanism of cell cycle, apoptosis, and tumor prognosis, we found that G1 phase drivers decreased after CC12 treatment in U87MG cells, including cyclinD1, CDK2, and CDK4; apoptotic proteins γ-H2AX, Bid, Bax, caspase 3,8 and 9, PARP, P53, and ATR increased, and the anti-apoptotic protein Bcl2 decreased in U118MG and U87MG cells after CC12 treatment. Furthermore, CC12 can induced the decrease of tumor prognostic marker DcR3. From the results of the in vivo experiments, we observed the effect of tumor size reduction of CC12; in addition, we confirmed that CC12 could cross the BBB by HPLC analysis.
Cell death pathways are frequently inactivated in human cancers, including GBM. For development of therapeutic strategy, re-activate the normal cell death pathway is a promising target. As well as oncogenesis, inactivated cell death signaling is the major source of treatment resistance; thus, we tested the apoptosis-regulation capability of the synthesized compound CC12 on GBM cell lines, and both intrinsic and extrinsic apoptotic pathways were evaluated. In GBM, high expression levels of Bcl2 proteins expression in several GBM cell lines have been reported, indicating that Bcl2 with a partial caspase-dependent mechanism is important in the immortalization process of GBM [13]. Many GBM drugs have been developed to target the Bcl2 pathway, for example, ABT-737 inhibits both Bcl2 and Bcl-XL, and can induce apoptosis of GBM in vitro and in vivo [14]. The downstream proteins Bid was accumulated, Bax was activated, and then led to caspase-dependent apoptosis [14,15]. CC12 may-through this pathway-induce GBM cell apoptosis.
However, we did not find any change in the expression level of FasL after CC12 treatment, neither in U118MG nor in U87MG cells. Because FasL is the starter of the extrinsic apoptotic pathways, CC12 s anti-tumor effects probably via another mechanism. The downstream molecules of FasL we evaluated in the present study included caspase 3 and PARP. Caspase 3 can also be regulated by intrinsic apoptotic proteins, including Bid [15], so the changes of caspase 3 after CC12 treatment were caused by the alteration of the intrinsic apoptotic signal pathway. Studies of the mechanisms of treatment and resistance of temozolomide, the most frequently used GBM drug, indicated that PARP can improve the sensitivity to temozolomide in malignances, including GBM [16]. PARP is a DNA damage sensor of single strand breaks and participates in single strand break repair. The increase of PARP after CC12 treatment was consistent with our results of Br-dUTP staining, indicating the DNA fragmentation, and the higher levels of γH2AX expression induced by CC12, a symbol of DNA damage. However, not only DNA damage, the association of γH2AX and cell cycle arrest at the G1 phase was reported in GBM cells [17], which were seen in our results, too.
DcR3 is a soluble receptor belonging to the tumor necrosis factor receptor superfamily that binds competitively to FasL [18]. High level expression of DcR3 is associated with poor prognosis of many different malignancies, including GBM [19]. CC12 can reduce the expression of DcR3; however, the mechanism may be FasL independent because of the 'no changed' level after CC12 treatment, which we mentioned above. DcR3 can bind to other members of the tumor necrosis factor receptor superfamily, including LIGHT and TNF-like molecule 1A [20,21]; the actual binding molecules in GBM after CC12 treatment need further investigation. The oncogenesis mechanism of DcR3 have been evaluated in a mouse tumor model, indicating that DcR3 regulated macrophage differentiation and contributed to tumor progression [22]. The role of immune moderation and inflammation of tumor initiation and progression via DcR3 in GBM can be evaluated for developing new treatment targets.
In conclusion, a newly synthesized compound CC12 was investigated for the potential therapy for GBM; the results indicated that CC12 may have anti-tumor ability through the regulation of cell cycle and apoptosis, in vitro and in vivo, and has the ability to cross BBB.

Conflicts of Interest:
The authors declare that they have no competing interests.