Inducement of mitosis delay by cucurbitacin E, a novel tetracyclic triterpene from climbing stem of Cucumis melo L., through GADD45γ in human brain malignant glioma (GBM) 8401 cells

Abstract

Cucurbitacin E (CuE) is a natural compound previously shown to have anti-feedant, antioxidant and antitumor activities as well as a potent chemo-preventive action against cancer. The present study investigates its anti-proliferative property using MTT assay; CuE demonstrated cytotoxic activity against malignant glioma GBM 8401 cells and induced cell cycle G2/M arrest in these cells. CuE-treated cells accumulated in metaphase (CuE 2.5-10 μM) as determined using MPM-2 by flow cytometry. We attempted to characterize the molecular pathways responsible for cytotoxic effects of CuE in GBM 8401 cells. We studied the genome-wide gene expression profile on microarrays and molecular networks by using pathway analysis tools of bioinformatics. The CuE reduced the expression of 558 genes and elevated the levels of 1354 genes, suggesting an existence of the common pathways involved in induction of G2/M arrest. We identified the RB (GADD45β and GADD45γ) and the p53 (GADD45α) signaling pathways as the common pathways, serving as key molecules that regulate cell cycle. Results indicate that CuE produced G2/M arrest as well as the upregulation of GADD45 γ and binding with CDC2. Both effects increased proportionally with the dose of CuE, suggesting that the CuE-induced mitosis delay is regulated by GADD45γ overexpression. Our findings suggest that, in addition to the known effects on cancer prevention, CuE may have antitumor activity in glioma therapy.

Figure 1

(a) CuE mediates the survival of GBM8401 cells (n=6 per group) and thus inhibits their proliferation. In vitro study was initiated by treating each of the cell lines to the increasing doses of CuE (0, 2.5, 5 and 10 μM) for 24 h. Reversibility of the growth-inhibitory effect of CuE in Figure 1A. The GBM8401 cells were each first treated with CuE for 24 h. After the treatment was terminated by washing off CuE, the cultures were reincubated for 24–48 h to check the extent of recovery of cancer cells. The survival of these CuE-treated GBM8401 cells was then measured by MTT assay. (b) Influence of CuE on apoptosis in GBM8401 cells. (c) Total apoptosis in GBM8401 cells after 4 h of incubation with CuE. Results were expressed as a percentage of total apoptotic cells (early and late apoptosis). Results were expressed as a percentage of control, which was considered as 100%. All data were reported as the means (±S.E.M.) of at least three separate experiments. Statistical analysis used the t-test, with the significant differences determined at the level of *P<0.05 versus control group, ^#P<0.05 versus 24 h-treated group

Figure 2

(a) Arrest of cell cycle progression at G₂/M in response to CuE treatment. The distribution of the cell cycle of GBM8401 cells was assessed by flow cytometry after staining with PI. (b) Results were expressed as a percentage of G₂/M. (c) MPM-2 (anti-phospho-Ser/Thr-Pro) expression in untreated and treated cancer cells. MPM-2 is an antibody that recognizes proteins that are only phosphorylated in mitosis. Cells were dually stained using propidium iodide to analyze DNA content and protein expression was quantified by flow cytometry. As a positive control, separate groups of cells were treated for 24 h with nocodazole (15 μg/ml), an antifungal agent known to induce metaphase arrest. Cell cycle analysis and quantification of MPM-2 expression were performed by flow cytometry following treatment with CuE for 24 h. (d) CuE enhanced the level of MPM-2 in CRC cells. Symbol (*) in each group of bars indicates that the difference resulting from treatment with CuE 0 μM is statistically significant at P<0.05

Figure 3

The genes downregulated or upregulated in GBM8401 cells following exposure to CuE. (a) The JunD, cyclinB1, CDC2 and GADD45-α, −β, −γ gene expression profile was studied in GBM8401 cells exposed for 4 h to the vehicle (DMSO) or to the CuE 5 μM. (b) The RT-PCR of JunD, cyclin B1, CDC2 and GADD45-α, −β, −γ mRNAs in GBM8401 cells following exposure to the CuE. The panels (c) indicate quantitative RT-PCR (qPCR) analysis of JunD, cyclin B1, CDC2 and GADD45-α, −β, −γ mRNA expression standardized against the levels of GAPDH in GBM8401 cells exposed for 4 h to DMSO (CuE 0 μM control) or CuE. Statistical analysis used the t-test, with the significant differences determined at the level of *P<0.05 versus the control group

Figure 4

Cell cycle arrest by CuE in GBM8401 cells via GADD45γ/CDC2 complex formation. Cells were treated with CuE followed by (a) western blot analysis (upper) and Co-IP (lower) (b) quantification of intensities by Li-COR near infrared imaging system. (c) Mitosis delay by CuE in GBM8401 cells via inhibition of Cdc2-cyclin B1 complex dissociation by GADD45γ binding with CDC2. Significant differences were determined at a level of *P<0.05 versus the 0 μM control group