β-elemene Inhibits Proliferation Of Glioblastoma Cells Through Activation Of GMFβ-MKK3/6 Signaling Pathway

Elemene, a novel self-developed anticancer medicine in China with low cytotoxic-ity, is extracted from Curcuma wenyujin and exists as an essential oil mixture ofβ-,γ- andδ-elemenes. As the major active component,β-elemene has strong anti-proliferative effects on glioblastoma cells in vitro and in vivo. However, lack of a defined molecular mechanism for the antitumor action ofβ-elemene hinders its application in clinical treatment of glioblastoma. Mitogen-activated protein kinase kinase-3 (MKK3) and -6 (MKK6) are two upstream kinases of Mitogen-activated protein kinase p38 (p38 MAPK). Altered activity of MKK3/6 has also been seen in various malignant tumors. Glia maturation factorβ(GMFβ) is a 17 kDa intracellular protein and is necessary for the growth, development, proliferation, differentiation and apoptosis of glial cells and neurons. GMFβusually acts through changing activity of MAPK signaling pathway. Our previous research found thatβ-elemene inhibited glioblastoma cell proliferation by activating p38 MAPK and decreasing the expression of p-ERK1/2, BCL-2 and BCL-X/L. However, the mechanism underlying the change of aforementioned mole-cules activity remains unclear.Objective:1. To evaluate the anti-glioblastoma and cell-cycle arrest effect of ?-elemene on human U87 and rat C6 cells.2. To investigate the role of MKK3 and MKK6 in the anti-glioblastoma prolifera-tion effect of ?-elemene.3. To confirm the role of GMFβin the antitumor effect ofβ-elemene on glioblas-toma and the relationship between GMFβand MKK3/6. 4. To confirm whether treatment withβ-elemene sensitizes glioblastoma cells to cisplatin.Methods:1. The first section research: human U87 and rat C6 glioblastoma cells were treated withβ-elemene at different drug doses (0, 20, 40, 60 and 80μg/ml) or for dif-ferent time amounts (0, 12, 24, 36 and 48 h). Cell viability was determined using a methyl thiazolyl tetrazolium (MTT) assay. Cell cycle progression was evaluated by flow cytometry, and the percentages of cells in G0/G1 phase were calculated.2. The second section research: glioblastoma cells were treated with different concentrations of ?-elemene and extracted with protein lysis buffer. p-MKK3, MKK3, p-MKK6 and MKK6 were measured by Western blot analysis. Total RNA was ex-tracted and RT-PCR was performed to examine the mRNA expression of MKK3 and MKK6. Band results were further semi-quantitatively estimated using Gel-Pro Analyzer 4.0 software. Data were analyzed by statistics. We transfected U87 glioblastoma cells with Dominant Negative-MKK3 (DN-MKK3), Dominant Negative-MKK6 (DN-MKK6) to inhibit the activity of MKK3 and MKK6, and then examined transfection efficiency by fluorescence microscopy and cell growth by MTT assay. To confirm the relationship between glioblastoma and MKK3/6, immunohistochemistry on human glioblastoma and normal brain tissues was performed using antibodies against MKK3, p-MKK3, MKK6 and p-MKK6 to evaluate the expression levels of MKK3/6 and p-MKK3/6.3. The third section research: to investigate the role of GMFβin the anti-glioblastoma effect ofβ-elemene, we examined the levels of total GMFβand p-GMFβinβ-elemene-treated U87 cells by immunoprecipitation and Western blot analysis. Upon GMFβsilencing using RNA interference, the antitumor action ofβ-elemene was evaluated in a MTT assay, and MKK3/6 and p-MKK3/6 expressions were examined by semiquantitative Western blot analysis.4. The fourth section research: after single or combined treatment with cisplatin andβ-elemene, glioblastoma cell numbers were measured by cell counting. The cell growth inhibitory rate (GIR) was calculated to examine the chemosensitization to cis-platin byβ-elemene.Results:1. The first section research:β-elemene inhibited the proliferation activity of U87 and C6 glioblastoma cells and arrested cell cycle in the G0/G1 phase. The anti-proliferation effect ofβ-elemene is dose- and time-dependent. 2. The second section research:β-elemene increased phosphorylation of both MKK3 and MKK6 in human glioblastoma cells. In contrast, inhibition of MKK3 and MKK6 with dominant-negative plasmids reversed the antitumor effect of ?-elemene. Furthermore, when either MKK3 or MKK6 was inhibited, the other was compensatorily activated in the presence ofβ-elemene. The expression of both total and phosphorylated forms of MKK3/6 were up-regulated in human glioblastoma.3. The third section research:β-elemene increased phosphorylation of GMFβin human glioblastoma cells. Conversely, silencing the expression of GMFβreversed the anti-glioblastoma effect ofβ-elemene and impaired the phosphorylation of MKK3/6.4. The fourth section research: the GIR in the combination (β-elemene + cisplatin) group was higher than that in both the single cisplatin andβ-elemene groups.β-elemene increased the sensitivity of U87 glioblastoma cells to cisplatin-induced cytotoxicity.Conclusion:1.β-elemene inhibited the proliferation of U87 and C6 glioblastoma cells through arresting them in G0/G1 phase.2. The activation of the GMFβ-MKK3/6-p38 signaling pathway underlay the anti–proliferative effect ofβ-elemene in glioblastoma. MKK3 and MKK6 were in-volved in the development of human glioblastoma.3.β-elemene and cisplatin had synergistic inhibitory effects on glioblastoma cell proliferation.