Anticancer Activity of γ-Bisabolene in Human Neuroblastoma Cells via Induction of p53-Mediated Mitochondrial Apoptosis

γ-Bisabolene has demonstrated antiproliferative activities against several human cancer cell lines. This study first discloses the antiproliferative and apoptosis induction activities of γ-bisabolene to human neuroblastoma TE671 cells. A CC50 value of γ-bisabolene was 8.2 μM to TE671 cells. Cell cycle analysis with PI staining showed γ-bisabolene elevating the sub-G1 fractions in a time-dependent manner. In addition, annexin V-FITC/PI staining showed γ-bisabolene significantly triggering early (annexin-V positive/PI negative) and late (annexin-V positive/PI positive) apoptosis in dose-dependent manners. γ-Bisabolene induced caspase 3/8/9 activation, intracellular ROS increase, and mitochondrial membrane potential decrease in apoptosis of human neuro-blastoma cells. Moreover, γ-bisabolene increased p53 phosphorylation and up-regulated p53-mediated apoptotic genes Bim and PUMA, as well as decreased the mRNA and protein levels of CK2α. Notably, the results indicated the involvement of CK2α-p53 pathways in mitochondria-mediated apoptosis of human neuroblastoma cells treated with γ-bisabolene. This study elucidated the apoptosis induction pathways of γ-bisabolene-treated neuroblastoma cells, in which could be useful for developing anti-neuroblastoma drugs.


Introduction
Neuroblastoma is a tumor of neural progenitor cells, which widely metastasizes to skin, bone marrow, liver, and non-contiguous lymph nodes, and then becomes the most common extracranial solid tumor in children [1]. Neuroblastoma is found in a greater than 7% of malignancies and near 15% of oncology deaths in children [2]. The infant patients with metastasis only to skin, bone marrow, and liver have the good prognosis post treatment with the current anticancer drugs. The 5-year event-free survival rate of children with disseminated neuroblastoma (Stage 4) is still less than 40% post-treatment [3,4]. In addition, neuroblastoma has resistance to chemotherapeutic drugs, and even

Growth Inhibition and Apoptosis Induction of γ-Bisabolene to Human Neuroblastoma
To examine the growth inhibitory ability of γ-bisabolene, the survival rates of human neuroblastoma TE671 cells were examined using MTT assays 2 days post-treatment ( Figure 1B). γ-Bisabolene inhibited the growth of TE671 cells in a concentration-dependent manner, exhibiting a CC 50 value of 8.2 µM ( Figure 1B). recurrence [2]. Therefore, the development of novel anti-neuroblastoma agents is still recommended to improve the patient outcomes. Apoptotic inducers have been widely regarded as potential anti-cancer therapeutics and evaluated in pre-clinical or clinical trials [5]. Apoptosis results from initiating Fas receptor-mediated or mitochondria-dependent pathways, subsequently converting DNA fragmentation, cytoskeletal protein degradation, and apoptotic body formation [6]. Anti-and pro-apoptotic molecules included Bcl-2, BclXL, Bax, Bak, BAD, BIM, and BclXS, regulate in mitochondria-dependent apoptotic pathways [6]. Tumor suppressor protein p53 with proapoptotic activity is the key transcription factor for up-regulation of pro-apoptotic genes like PUMA and Noxa.
γ-Bisabolene shows in vitro and in vivo antiproliferative and apoptotic activities against human oral squamous cell carcinoma [7]. γ-Bisabolene induces the apoptosis of oral squamous cell carcinoma via p53-medaited signaling pathways. This study further investigates the antiproliferative and apoptotic mechanisms of γ-bisabolene against human neuroblastoma.
Meanwhile, cell cycle analysis of flow cytometry with PI staining showed the increase in the sub-G1 fractions and the decrease in the G1 fractions of γ-bisabolene-treated cells compared to mock controls in a time-dependent manner ( Figure 1C,D). The results indicated anti-proliferative activity of γ-bisabolene to human neuroblastoma cells.

Apoptosis of Neuroblastoma Cells Induced by γ-Bisabolene
To test whether γ-bisabolene induces apoptosis of human neuroblastoma cells, the fractions of early (annexin-V positive/PI negative) and late (annexin-V positive/PI positive) apoptosis in treated were determined by flow cytometer with annexin V-FITC and PI staining (Figure 2A-C). γ-Bisabolene triggered the significant increase of early and late apoptosis on TE671 cells in dose-dependent manners. To further examine the mRNA and protein levels of caspases 3, 8 and 9 in treated cells, TE671 cells treated were harvested 48 h post treatment for total RNA extraction and the lysate preparation. Quantitative RT-PCR revealed that γ-bisabolene significantly induced the mRNA expression of caspases 3, 8, and 9 in dose-dependent manners ( Figure 3A). Caspases 3, 8 and 9 were activated to greater than 5 folds in response to 10 µM γ-bisabolene. Subsequently, western blots indicated the dose-dependent increase in pro-and active forms of caspases 3, 8, and 9 in γ-bisabolene-treated cells 48 h post treatment ( Figure 3B-E). Active forms of caspases 3, 8, and 9 exhibited 3.5-, 1.6-, and 2.3-fold increases post-treatment with 10 µM γ-bisabolene, respectively. The results demonstrated γ-bisabolene induces extrinsic and intrinsic apoptosis of human neuroblastoma. Meanwhile, cell cycle analysis of flow cytometry with PI staining showed the increase in the sub-G1 fractions and the decrease in the G1 fractions of γ-bisabolene-treated cells compared to mock controls in a time-dependent manner ( Figure 1C,D). The results indicated anti-proliferative activity of γ-bisabolene to human neuroblastoma cells.

Apoptosis of Neuroblastoma Cells Induced by γ-Bisabolene
To test whether γ-bisabolene induces apoptosis of human neuroblastoma cells, the fractions of early (annexin-V positive/PI negative) and late (annexin-V positive/PI positive) apoptosis in treated were determined by flow cytometer with annexin V-FITC and PI staining (

ROS Production Increase and Mitochondrial Membrane Potential Decrease in Treated Cells
To examine the apoptotic pathways of γ-bisabolene-induced apoptosis, the changes in the intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP) in γ-bisabolene-treated cancer cells was subsequently surveyed using flow cytometry analysis with DCFH-DA and DiOC6(3) staining, respectively (Figures 4 and 5). γ-Bisabolene treatment induced ROS production in human neuroblastom cells in a dose-dependent manner ( Figure 4).
Quantitative analysis of MMP changes using DiOC6(3) staining demonstrated that γ-bisabolene significantly decreased the MMP of human neuroblastom cells ( Figure 5A,B); a 97% decrease in MMP was found in TE671 cells treated with 5 μM of γ-bisabolene ( Figure 5B). Therefore, the results indicated γ-bisabolene induces ROS production and injures the mitochondria in apoptosis of human neuroblastom cells.  in TE671 were characterized using western blotting (B); Relative band intensity of indicated caspase or active caspase was normalized by β actin, compared to the mock cells, and quantified using image J based on triplicate replicates of each experiment (C-E). **, p value < 0.01; ***, p value < 0.001 compared with untreated cells.

ROS Production Increase and Mitochondrial Membrane Potential Decrease in Treated Cells
To examine the apoptotic pathways of γ-bisabolene-induced apoptosis, the changes in the intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP) in γ-bisabolene-treated cancer cells was subsequently surveyed using flow cytometry analysis with DCFH-DA and DiOC6 (3)

ROS Production Increase and Mitochondrial Membrane Potential Decrease in Treated Cells
To examine the apoptotic pathways of γ-bisabolene-induced apoptosis, the changes in the intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP) in γ-bisabolene-treated cancer cells was subsequently surveyed using flow cytometry analysis with DCFH-DA and DiOC6(3) staining, respectively (Figures 4 and 5). γ-Bisabolene treatment induced ROS production in human neuroblastom cells in a dose-dependent manner ( Figure 4).
Quantitative analysis of MMP changes using DiOC6(3) staining demonstrated that γ-bisabolene significantly decreased the MMP of human neuroblastom cells ( Figure 5A,B); a 97% decrease in MMP was found in TE671 cells treated with 5 μM of γ-bisabolene ( Figure 5B). Therefore, the results indicated γ-bisabolene induces ROS production and injures the mitochondria in apoptosis of human neuroblastom cells.      Quantitative analysis of MMP changes using DiOC6(3) staining demonstrated that γ-bisabolene significantly decreased the MMP of human neuroblastom cells ( Figure 5A,B); a 97% decrease in MMP was found in TE671 cells treated with 5 µM of γ-bisabolene ( Figure 5B). Therefore, the results indicated γ-bisabolene induces ROS production and injures the mitochondria in apoptosis of human neuroblastom cells.

The Increase of p53-Mediated Transcription in γ-Bisabolene-Treated Cells
To examine the γ-bisabolene-induced apoptotic pathways of human neuroblastom cells, p53-mediated transcriptional activities for apoptosis induction were subsequently analyzed ( Figure 6). Western blotting showed γ-bisabolene elevating the phosphorylation of p53 and p21 in TE671 cells ( Figure 6A,B), linked with activation of PUMA and Bim expression, but not Noxa, in dose-dependent manners quantified using real-time RT-PCR ( Figure 6C). Since down-regulation of casein kinase 2α (CK2α) was associated with activation of p53 transcriptional activities in apoptosis of γ-bisabolene-treated oral cancer cells [7], the CK2α and CK2β expression in γ-bisabolene-treated neuroblastom cells was further characterized ( Figure 7A-C). Real-time RT-PCR and western blotting demonstrated that γ-bisabolene significantly reduced the CK2α and CK2β expression in treated human neuroblastom cells. Therefore, CK2 down-regulation was suggested to be associated with p53-mediated apoptosis of γ-bisabolene-treated neuroblastom cells.

The Increase of p53-Mediated Transcription in γ-Bisabolene-Treated Cells
To examine the γ-bisabolene-induced apoptotic pathways of human neuroblastom cells, p53mediated transcriptional activities for apoptosis induction were subsequently analyzed ( Figure 6). Western blotting showed γ-bisabolene elevating the phosphorylation of p53 and p21 in TE671 cells ( Figure 6A,B), linked with activation of PUMA and Bim expression, but not Noxa, in dose-dependent manners quantified using real-time RT-PCR ( Figure 6C). Since down-regulation of casein kinase 2α (CK2α) was associated with activation of p53 transcriptional activities in apoptosis of γ-bisabolenetreated oral cancer cells [7], the CK2α and CK2β expression in γ-bisabolene-treated neuroblastom cells was further characterized ( Figure 7A-C). Real-time RT-PCR and western blotting demonstrated that γ-bisabolene significantly reduced the CK2α and CK2β expression in treated human neuroblastom cells. Therefore, CK2 down-regulation was suggested to be associated with p53-mediated apoptosis of γ-bisabolene-treated neuroblastom cells.
Tumor suppressor p53 has demonstrated its involvement in the mitochondrial apoptotic pathway, exerting transcriptional activities on regulating the expression of pro-apoptotic genes: e.g., PUMA, Bim, and NOXA [6,11,12]. γ-Bisabolene concentration-dependently increased p53 phosphorylation that linked with up-regulation of p53 transcriptional activities on apoptotic genes PUMA and Bim in human neuroblastom cells ( Figure 6). Since PUMA and Bim have been known as the activator for Bax involved in mitochondria-mediated apoptosis [13], γ-Bisabolene-induced p53mediated PUMA and Bim up-regulation correlated with the MMP decreases in treated neuroblastom Figure 7. Decrease of CK2α/β expression in γ-bisabolene-treated cells. Cells were treated with γ-bisabolene for 48 h, and then harvested for real time PCR (A) and western blot assays (B,C); Relative mRNA levels of CK2α and CK-2β were normalized by GAPDH, and then compared to un-treated cells, respectively. Relative band intensity of CK2α was normalized by β actin, compared to the mock cells, and quantified using image J based on triplicate replicates of each experiment (C); GeneGO Meta-Core pathway analysis of the results predicts p53-mediated apoptosis pathway in treated cells (D). *, p value < 0.05; **, p value < 0.01 compared with untreated cells.
Tumor suppressor p53 has demonstrated its involvement in the mitochondrial apoptotic pathway, exerting transcriptional activities on regulating the expression of pro-apoptotic genes: e.g., PUMA, Bim, and NOXA [6,11,12]. γ-Bisabolene concentration-dependently increased p53 phosphorylation that linked with up-regulation of p53 transcriptional activities on apoptotic genes PUMA and Bim in human neuroblastom cells ( Figure 6). Since PUMA and Bim have been known as the activator for Bax involved in mitochondria-mediated apoptosis [13], γ-Bisabolene-induced p53-mediated PUMA and Bim up-regulation correlated with the MMP decreases in treated neuroblastom cells ( Figure 5). In addition, p53 could induce the Fas up-regulation that activates the extrinsic apoptotic pathway [14]. Thus, p53 activation induced by γ-bisabolene might be responsible for increasing the expression and activity of caspase 8 (Figure 3). Results demonstrated γ-bisabolene triggering p53-mediated apoptosis of human neuroblastom cells.
Protein kinase CK2α participates in heart and neural tube development, maintains cell viability, and regulates cell cycle stages [15][16][17][18]. CK2α has been identified to overexpress in human colorectal cancer [19]; gene silence of CK2α small interfering RNA was associated with elevating the expression of p53/p21 and decreasing the expression of C-myc [20]. Therefore, suppression of CK2α was suggested to a novel therapeutic approach for human colorectal cancer. This study demonstrated γ-bisabolene down-regulating the CK2α and CK2β expression in human neuroblastom cells ( Figure 7A-C). Thus, down-regulation of CK2 was proposed to as one of promising mechanisms for activating p53-mediated apoptosis of human neuroblastom cells treated with γ-bisabolene ( Figure 7D).

Cell Cultures
Human neuroblastoma TE671 cells were cultured in MEM medium (HyClone Laboratories, South Logan, UT, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM glutamine and 1 mM sodium pyruvate at 37˝C in a humidified atmosphere of 5% CO 2 .

Apoptosis and Cell Cycle Assay by Flow Cytometry
Cells were treated with γ-bisabolene at 10 µM for 24 and 48 h, and harvested for cell cycle analysis by propidium iodide (PI) staining, and apoptosis assays with γ-bisabolene at 1, 5, 10, and 20 µM for 48 h by Annexin V-FITC apoptosis Detection Kit with PI (BioVision, Milpitas, CA, USA). Cell cycle and apoptosis assays were performed as described in our prior report [7].

Mitochondrial Membrane Potential (MMP) Detection Assays
Cells were treated with γ-bisabolene at 1, 5, 10, and 20 µM for 48 h; MMP changes were quantitated using flow cytometry with DiOC6(3) staining (Calbiochem, San Diego, CA, USA). Cells were stained with the DiOC6(3) solution at 37˝C in the dark for 1 h, and then measured using a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Detecting Intracellular Reactive Oxygen Species (ROS) by Flow Cytometry
Cells were treated with γ-bisabolene at 1, 5, 10, and 20 µM for 48 h, harvested, and then stained with 10 µM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma, Saint Louis, MO, USA) at 37˝C for 30 min in darkroom. DCF fluorescence converted from DCFH-DA by ROS was detected by flow cytometry with excitation wavelength of 485 nm and emission wavelength of 530 nm, as described in our prior report [21].

Statistical Analysis
All data from three independent experiments were used for measuring mean˘standard error (mean˘S.E.) that was compared using Student's t-test. p < 0.05 was considered as statistically significant.

Conclusions
This study demonstrated that γ-bisabolene has potential to inhibit the growth and inducing apoptosis of human neuroblastom cells. γ-Bisabolene reduced the CK2α expression and activated p53-mediated mitochondrial apoptosis pathway in human neuroblastom cell. The results suggested γ-bisabolene as a potential agent of anti-proliferative and apoptosis-inducing activities for treating human neuroblastom cells.