The effects of cucurbitacin E on GADD45β‐trigger G2/M arrest and JNK‐independent pathway in brain cancer cells

Abstract Cucurbitacin E (CuE), an active compound of the cucurbitacin family, possesses a variety of pharmacological functions and chemotherapy potential. Cucurbitacin E exhibits inhibitory effects in several types of cancer; however, its anticancer effects on brain cancer remain obscure and require further interpretation. In this study, efforts were initiated to inspect whether CuE can contribute to anti‐proliferation in human brain malignant glioma GBM 8401 cells and glioblastoma‐astrocytoma U‐87‐MG cells. An MTT assay measured CuE's inhibitory effect on the growth of glioblastomas (GBMs). A flow cytometry approach was used for the assessment of DNA content and cell cycle analysis. DNA damage 45β (GADD45β) gene expression and CDC2/cyclin‐B1 disassociation were investigated by quantitative real‐time PCR and Western blot analysis. Based on our results, CuE showed growth‐inhibiting effects on GBM 8401 and U‐87‐MG cells. Moreover, GADD45β caused the accumulation of CuE‐treated G2/M‐phase cells. The disassociation of the CDC2/cyclin‐B1 complex demonstrated the known effects of CuE against GBM 8401 and U‐87‐MG cancer cells. Additionally, CuE may also exert antitumour activities in established brain cancer cells. In conclusion, CuE inhibited cell proliferation and induced mitosis delay in cancer cells, suggesting its potential applicability as an antitumour agent.


| INTRODUC TI ON
A previous study reported that brain cancer is one of the most invasive and malignant cancers in developed countries. 1 However, the chemotherapy for brain cancer remains obscure. Two of the best strategies for tumour suppression are the induction of apoptosis (type I cell death) and cell cycle arrest in cancer cells. 2 Recent studies have demonstrated that phytochemicals synthesized from plants can suppress tumour growth by inducing apoptosis and cell cycle arrest, and down-regulating gap junctional intercellular communication. [3][4][5] Cucurbitacins, a class of tetracyclic triterpenoids with medicinal properties in Cucurbitaceae, are extracted from the climbing stems of Cucumis melo L. 6 Cucurbitacins have been widely used in inhibition of cancer cell progression as medicinal herbs throughout Asia. 7 In recent years, there is a growing interest in this herb because of its presumed beneficial pharmacological properties as anti-inflammatory 8 and antitumour agents. 9 Cucurbitacin E (CuE) is an active compound of the cucurbitacin family. 10 Recent reports have demonstrated that CuE possesses various pharmacological functions, such as antiviral, anti-inflammatory and anticancer effects. [11][12][13] Cucurbitacin E exhibits inhibitory effects in several types of cancer 10,14 ; however, its anticancer effect in brain cancer remains unclear. Therefore, the mechanism underlying the antitumour effect of CuE on brain cancer has yet to be identified.
Glioblastomas (GBMs) are highly invasive and recurrence brain tumours, 15 and have been shown to harbour therapy-resistant cancer stem cells (CSCs), and this is the main cause of death. 16,17 Recent studies indicated that GBMs contain a subpopulation of glioma-initiating tumour cells which exhibits stem cell characteristics and may be responsible for in vivo tumour growth. 18,19 Therefore, we chose the GBM 8401 and glioblastoma-astrocytoma U-87-MG cells as human brain cancer model to analyse the antitumour activity of CuE.
In the current study, efforts have been initiated to inspect whether CuE can contribute to the anti-proliferation of GBM 8401 and U-87-MG cells. Apoptosis 20 and cell cycle regulation 21 have been posited as possible targets for cancer therapy, and CuE was found to induce the regulation of cell cycle progression. 22 Therefore, we focused specifically on the effects of CuE on the induced delay of mitosis and gene expression in GBM 8401 and U-87-MG cells.
We expect that our study may provide a scientific foundation and technological support for brain GBM therapy.

| Materials
All reagents and chemicals were of analytical grade. Cell culture ma-

| Cell proliferation assay
Cells were seeded into a 96-well culture plate at 5000 cells/well followed by the addition of 0, 2.5, 5 or 10 μmol/L CuE for 1-3 days.

| Apoptosis measurement
The cells were plated in six-well culture plates (Orange Scientific, EU). The cells were harvested and centrifuged after the incubation with CuE for 4 hours, and the cell pellet was then resuspended in 1 × annexin-binding buffer containing 5 μL of annexin V-FITC and 1 μL of 100 μg/mL propidium iodide (PI) and incubated for 15 minutes at room temperature. The stained cells were detected by a FACSCalibur flow cytometer (BD Pharmingen) and analysed using WinMDI 2.9 free software (BD Pharmingen).

| Cell cycle analysis
A fluorescent nucleic acid dye PI was used to identify the proportion of cells in each interphase stage of the cell cycle. The cells were treated with CuE for 24 hours, and then harvested and fixed in 1 mL of cold 70% ethanol for at least 8 hours at −20°C. DNA was stained using a PI/RNaseA solution, and the DNA content was detected using a FACSCalibur flow cytometer. Data were analysed using WinMDI 2.9.  The antigens were then visualized using a near-infrared imaging system Li-COR, and the data were analysed using the software Odyssey 2.1 or a chemiluminescence detection kit (ECL; Amersham

| Coimmunoprecipitation
Briefly, 500 μg of cellular proteins was labelled by anti-CDC2 (p34; sc-747), and the protein-antibody immunoprecipitates were collected using protein A/G plus-agarose beads (SC-2003; Santa Cruz BioTechnology). DNA damage 45β protein levels was detected by Western blot analysis and visualized using a chemiluminescence detection kit (ECL; Amersham Corp.), and data were analysed using Odyssey 2.1 software.

| Small-interfering RNA
The specific small-interfering RNA (siRNA) of GADD45β (Level com, Taiwan) and Lipofectamine RNAiMAX gene transfection system were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). The transfection protocol was in accordance with manufacturer's instructions.

| Statistical analysis
All data are reported as the mean (±SEM) of at least three separate experiments. The t test or one-way ANOVA with a Scheffe's post hoc test was used for statistical analysis, with significant differences determined at P < 0.05.

| CuE inhibits the cell viabilities of GBM8401and U-87-MG cells
To investigate the antitumour effects of CuE on cell survival and proliferation, an in vitro study was applied to treat GBM8401

| Non-significant changes in apoptosis/necrosis in CuE-treated brain cancer cells
To clarify the apoptosis/necrosis effects of CuE on the brain cancer cells, the cells were treated with CuE for 4 hours followed by detecting the generation of sub-G1 cells by Annexin V-FITC and PI staining, and the apoptotic ratios were quantified with flow cytometry.
Flow cytometry dot-plots of Annexin V-FITC/PI staining illustrated non-significant changes in apoptosis or necrosis ratios in CuEtreated cells compared with that of untreated (control) cells ( Figure   S1A,B). Moreover, no significant increase in the ratio of caspase-3 activity (data not shown) was observed in CuE-treated cancer cells.

| CuE enhances the numbers cell populations in the G2/M phase
To further investigate the effect of CuE on GBM8401 and U-87-MG cell growth, the cell cycle distribution among CuE-treated cells was analysed and quantified using flow cytometry. As shown in Figure 1B, treatment with CuE resulted in accumulation of cells in the G 2 /M phase, implying that the brain cancer cell lines underwent mitosis delay. Our observed delay in mitosis implied that exposure to CuE enhanced the cell number in the G 2 /M phase while synchronously decrease the cell populations in other phases ( Figure 1C).

| Effects of CuE on the mitotic index
MPM-2 is a commonly used marker of mitotic disturbance that can bind to phosphorylated amino acid epitopes during mitosis, especially with maximum staining at the G2/M phase.

| G 2 /M-phase cell cycle arrest by CuE in GBM8401 and U-87-MG cells through CDC2 and cyclin B1disassociation with GADD45β up-regulation
As shown in Figure 3, cellular proteins were extracted from the brain cancer cell lines treated with CuE, which was followed by qRT-PCR, co-immunoprecipitation (Co-IP) and Western blot analysis. CDC2, cyclin B1 and GADD45β gene expression levels were quantified based on measurements of relative intensities.
There was no significant change in mRNA levels following treatment with CuE ( Figure S2A); in contrast, the protein expressions of CDC2 and cyclin B1 were significantly down-regulated in CuE-treated GBM8401 and U-87-MG cells ( Figure S2B,C).
Furthermore, Co-IP test was performed to quantify the activities of GADD45β/CDC2 and cyclin B1/CDC 2, the critical roles for G 2 -M transition during the cell cycle ( Figure 3D). The results indicated that the increasing number of the brain cancer  Non-specific rabbit Immunoglobulin G (IgGs) was used as negative control; and CDC2 was used as an internal control. E, Quantification of band intensities. All data are reported as the mean (±SEM) of three separate experiments. Statistical analysis was performed using the t test, with differences considered significant at a level of *P < 0.05 vs the 0 μmol/L CuE control group In summary, in this study, efforts were made to determine that CuE is an effective inhibitor of brain cancer cell lines medicated by

| Down-regulation of GADD45β in GBM8401 and U-87-MG cells by silencing GADD45β was reversed by CuE-induced G 2 /M arrest
GADD45β. The mechanism of CuE involvement in the inhibition of tumour growth was highlighted by the delay of mitosis via the upregulation of GADD45β expression. The present results speculate the appropriateness of CuE as a potential anticancer agent for the chemoprevention of brain cancer.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. F I G U R E 5 Inhibition of DNA damage 45β (GADD45β) could alleviate cell cycle arrest upon cucurbitacin E (CuE) treatment in GBM8401 and U-87-MG cells. A, Expression of GADD45β in brain cancer cells treated CuE and/or GADD45β siRNA. B, Representative blots and quantification of band intensities from three independent experiments. C, Cucurbitacin E delayed the progression of mitosis, whereas the addition of GADD45β siRNA could resume cell cycle. D, Cell cycle distribution (%) in GBM8401 cells. Cells underwent staining with propidium iodide to analyse DNA content, followed by quantification through flow cytometry. The control group was used as 100% (target gene/beta actin × 100%) to calculate the ratio of each group. All data are reported as the mean (±SEM) of three separate experiments. *P < 0.05 compared with control; @ P < 0.05 compared with CuE 5 μmol/L