Yiqun Yao

β-elemene inhibits proliferation of human glioblastoma cells through the activation of glia maturation factor β and induces sensitization to cisplatin

β-elemene, a natural drug extracted from Curcuma wenyujin, strongly inhibits glioblastoma growth. However, the mechanism of β-elemene antitumor action remains unclear. Glia maturation factor β (GMFβ) regulates cellular growth, fission, differentiation and apoptosis. It has been reported that overexpression of GMFβ inhibits the growth of glioblastoma cells and decreases tumor volume. To illustrate the role of GMFβ in the anti-proliferative effect of β-elemene in glioblastoma, U87 cells were treated with β-elemene at various doses and for different periods of time, and levels of phospho-GMFβ (p-GMFβ) and total GMFβ were determined by immunoprecipitation and western blot analysis. Upon GMFβ silencing using RNA interference, the antitumor action of β-elemene was evaluated in a methyl thiazolyl tetrazolium assay and by semi-quantitative western blot analysis of MKK3/6 and p-MKK3/6 expression. Finally, chemosensitization to cisplatin by β-elemene was examined using a cell counting array, and the cell growth inhibitory rate was calculated. The results showed that β-elemene inhibits U87 cell viability through the activation of the GMFβ signaling pathway. Conversely, silencing the expression of GMFβ reversed the antitumor effect of β-elemene and impaired the phosphorylation of MKK3/6. Furthermore, β-elemene increased the sensitivity of U87 glioblastoma cells to the chemotherapeutic agent cisplatin. Taken together, these results suggest that activation of the GMFβ pathway mediates the antitumor effect of β-elemene in glioblastoma. GMFβ is a putative molecular target for glioblastoma therapy.

ß-Elemene inhibits proliferation of human glioblastoma cells and causes cell-cycle G0/G1 arrest via mutually compensatory activation of MKK3 and MKK6

ß-elemene, a natural plant drug extracted from Curcuma wenyujin, has shown a strong anti-glioblastoma effect. However, the antitumor mechanism of ß-elemene remains unclear. Mitogen-activated protein kinase kinase-3 (MKK3) and -6 (MKK6) can regulate cellular growth, fission, differentiation and apoptosis. To illustrate the role of MKK3 and MKK6 in the anti-glioblastoma proliferation effect of ß-elemene, U87 cells were treated with ß-elemene at various doses or for different times, and then phosphorylated MKK3 (p-MKK3), phosphorylated MKK6 (p-MKK6), MKK3 and MKK6 were detected by Western blot assay. After transient transfection with dominant-negative mutant plasmids of MKK3 and MKK6, cell viability and cell cycle stage were determined by methyl thiazolyl tetrazolium assay and flow cytometry, respectively. Results showed that ß-elemene inhibited the proliferation of U87 glioblastoma cells and arrested them in G0/G1 phase through up-regulating p-MKK3 and p-MKK6 levels. In contrast, inhibition of MKK3 and MKK6 reversed the antitumor effect of ß-elemene. Furthermore, when either MKK3 or MKK6 was inhibited by a dominant-negative plasmid, the other was compensatorily activated in the presence of ß-elemene. Taken together, our findings indicate that mutually compensatory activation of MKK3 and MKK6 mediates the anti-glioblastoma effect of ß-elemene. MKK3 and MKK6 might be two putative targets for molecular therapy against glioblastoma.

β-Elemene inhibits Hsp90/Raf-1 molecular complex inducing apoptosis of glioblastoma cells

Abstractβ-Elemene, an active component of herb medicine Curcuma wenyujin, has been shown to antagonize glioblastoma cells by inducing apoptosis. However, how β-elemene induces apoptosis of these cells remains unclear. In this study, we report that β-elemene disrupted the formation of the Hsp90/Raf-1 complex, a key step in maintaining the conformation stability of Raf-1, and caused deactivation of Raf-1 and inhibition of the ERK pathway, thereby leading to apoptosis of glioblastoma cells. Specifically, treatment of glioblastoma cell lines with β-elemene attenuated phosphorylation of multiple members of the kinase families in the Ras/Raf/MEK/ERK cascade, including Raf-1 and ERK as well as downstream signaling targets such as Bcl-2. These results suggest that the Hsp90/Raf-1 complex could be a promising molecular target for new drug development for the treatment of glioblastoma.

DNA Damage-Induced NF-κB Activation in Human Glioblastoma Cells Promotes miR-181b Expression and Cell Proliferation

Background: Glioblastoma (GBM) is the most common and most aggressive form of brain cancer. After surgery, radiotherapy is the mainstay of treatment for GBM patients. Unfortunately, the vast majority of GBM patients fail responding to radiotherapy because GBM cells remain highly resistant to radiation. Radiotherapy-induced DNA damage response may correlate with therapeutic resistance. Methods: Ionizing radiation (IR) was used to induce DNA damage. Cell proliferation and migration were detected by wound-healing, MTT and apoptosis assays. Dual-luciferase assays and Western blot analysis were performed to evaluate NF-κB activation and validate microRNA targets. Real-time PCR was used to study mRNA and microRNA levels. Results: IR-induced DNA damage activated NF-κB in GBM cells which promoted expression of IL-6, IL-8 and Bcl-xL, thereby contributing to cell survival and invasion. Knockdown SENP2 expression enhanced NF-κB essential modulator (NEMO) SUMOylation and NF-κB activity following IR exposure. miR-181b targets SENP2 and positively regulated NF-κB activity. Conclusion: NF-κB activation by DNA damage in GBM cells confers resistance to radiation-induced death.

RNF135, RING finger protein, promotes the proliferation of human glioblastoma cells in vivo and in vitro via the ERK pathway

Ring finger protein 135 (RNF135), located on chromosome 17q11.2, is a RING finger domain-containing E3 ubiquitin ligase that was identified as a bio-marker and therapy target of glioblastoma. In our study, we confirmed that RNF135 was up-regulated in glioblastoma tissues compared with normal brain (NB) tissues, and that RNF135 knockdown inhibited proliferation and migration and led to cell cycle arrest in the G0/G1 phase in vivo. By lowering RNF135 expression, phosphorylated Erk and cell cycle protein CDK4 were down-regulated, while p27Kip1 and p21Waf1/Cip1 were up-regulated in U87 and U251 cells in vitro. In addition, using the immunofluorescence double labelling method, we found that RNF135 and P-Erk were co-localized in the cytoplasm and were highly expressed in glioblastoma samples compared with NB tissues. Moreover, the growth of U87 cell-transplanted tumours in nude mice was inhibited while transduced with Lv-shRNF135. Taken together, our findings demonstrate the biological effects of RNF135 in glioblastoma cell proliferation, migration and cell cycle, and its role in the progression of glioblastoma may be associated with the ERK signal transduction pathway.

Down-regulation of ribosomal protein S15A inhibits proliferation of human glioblastoma cells in vivo and in vitro via AKT pathway.

Ribosomal protein s15a (RPS15A), a highly conserved cytoplasmic protein, promotes mRNA/ribosome interaction in translation. Recent evidence showed that RPS15A is essential for tumor growth. RPS15A expression level was measured in glioblastoma tissue samples and normal brain (NB) tissue samples. RPS15A RNAi stable cell line U87 and U251 was generated by the pLVTHM-GFP lentiviral RNAi expression system. The knockdown efficiency was confirmed by quantitative real-time PCR and western blot. Molecular mechanisms and the effect of RPS15A on cell growth and migration were investigated by using western blot, MTT assay, wound healing assay, transwell migration assay, and tumorigenesis in nude mice. Here, we report that RPS15A is overexpressed in human glioblastoma tumor tissues. RPS15A knockdown inhibits proliferation and migration of glioblastoma cells in vitro. Knocking down RPS15A leads to the level of p-Akt decrease and cell cycle arrested in G0/G1 phase in U87 and U251 cells. Furthermore, the growth of glioblastoma cell-transplanted tumors in nude mice is inhibited by transduction with Lv-shRPS15A. Our findings indicate that RPS15A promotes cell proliferation and migration in glioblastoma for the first time. RPS15A might play a distinct role in glioblastoma and serve as a potential target for therapy.

Knockdown of NUPR1 inhibits the proliferation of glioblastoma cells via ERK1/2, p38 MAPK and caspase-3

Nuclear protein-1 (NUPR1), located on chromosome 16p11.2, is a stress response factor that plays an important role in the growth and migration of human malignant tumor cells. However, the role of NUPR1 in glioblastoma remains poorly understood. The expression level of NUPR1 was detected by quantitative real-time PCR and immunohistochemistry (IHC). Wound healing, MTT, cell counting and BrdU assays were used to analyze the migration and proliferation of glioblastoma cells after down-regulating NUPR1 expression using a lentiviral vector. FACS analysis and a signaling antibody array kit were used to detect the mechanism by which NUPR1 modulates cell cycle and apoptosis activities in glioblastoma cells. We confirmed that NUPR1 was up-regulated in glioblastoma tissues compared to NB tissues. Down-regulation of NUPR1 suppressed cell migration and proliferation, arrested the cell cycle in the G0/G1 phase and promoted apoptosis in U251 and U87 cells in vitro. Furthermore, the expression levels of phosphorylated ERK1/2, p38 MAPK and cleaved caspase-3 were decreased upon silencing NUPR1 expression in U251 and U87 cells. In summary, NUPR1 plays an important role in the growth and migration of human glioblastoma cells. Knockdown of NUPR1 suppressed glioblastoma cell growth by arresting the cell cycle and inducing cell apoptosis via decreases in the expression of ERK1/2, p38 MAPK and caspase-3.

MUC4 modulates human glioblastoma cell proliferation and invasion by upregulating EGFR expression

Glioblastoma (GBM), the most common primary brain tumor, is the leading cause of deaths related to tumors in the central nervous system. The prognosis of GBM patients is currently poor, and the mechanisms underlying GBM genesis remain unclear. The expression of MUC4, a high-molecular-weight and highly glycosylated protein, has been studied in many cancers. However, information on MUC4 expression in GBM is limited. In this study, we found that MUC4 was overexpressed in GBM cell lines and tissues. The proliferation and invasive potential of GBM cells were significantly increased by the ectopic expression of MUC4. By contrast, RNA interference targeting MUC4 in GBM cells significantly decreased the proliferation and invasive potential of GBM cells. We also found that the expression of epidermal growth factor receptor (EGFR) was modulated by MUC4. EGFR inhibition by siRNA reversed the MUC4-induced proliferation and invasion. These results indicated that MUC4 expression in GBM was important in GBM cell proliferation and invasion, which may be partly associated with EGFR overexpression.

Trefoil factor 3 contributes to the malignancy of glioma via regulating HIF-1α

Trefoil factor 3 (TFF3) plays significant roles in several solid tumors. However, the expression pattern and function of TFF3 in glioblastoma (GBM) have not been reported. Here, we report that expression level of TFF3 significantly elevated in glioma and correlated with the prognosis of glioma patients. Then we found TFF3 promotes proliferation, invasion, and migration and inhibits apoptosis of glioma cells in vitro, and delayed tumor progression in subcutaneous xenograft nude mice, and prolonged the median survival time in orthotopic xenograft mice. Moreover, knockdown of TFF3 reduced the expression of HIF-1α through a hypoxia-independent manner. These findings suggest that targeting TFF3 may offer a novel strategy for therapeutic intervention of malignant gliomas.

Somatic mutations in myeloid cell leukemia‑1 contribute to the pathogenesis of glioma by prolonging its half‑life

The identification of mutated genes in glioblastoma multiforme (GBM) is an essential step towards improving current understanding of the molecular mechanism underlying the disease and establishing novel targets for diagnostic and therapeutic purposes. The present study used direct sequencing to screen 20 malignancy-associated genes, which have either been well described in the literature or observed multiple times in human cancer sequencing, in cancerous and normal control tissue samples from 20 patients with histologically confirmed GBM. The investigation identified five somatic non-synonymous coding mutations in four candidate genes, with two located in the proline, glutamic acid, serine, threonine-rich region of myeloid cell leukemia sequence 1 (Mcl)-1, (D155G and L174S). The sample pool was then expanded by sequencing Mcl-1 in a further 43 patients with GBM and another somatic mutation in the same region, D155H, was identified. The subsequent functional investigation confirmed that these somatic mutations affected the degradation of Mcl-1, and the growth of glioma cells transfected with mutant plasmids was significantly accelerated compared with cells overexpressing wild-type Mcl-1. The mutational profiling of GBM in the present study revealed for the first time, to the best of our knowledge, several mutations in Mcl-1, and identified this gene as a novel therapeutic target for the treatment of GBM.