Sensitivity Of Human Glioma Cells Research Articles

Oxidative Stress Activated by Sorafenib Alters the Temozolomide Sensitivity of Human Glioma Cells Through Autophagy and JAK2/STAT3-AIF Axis.

The development of temozolomide (TMZ) resistance in glioma leads to poor patient prognosis. Sorafenib, a novel diaryl urea compound and multikinase inhibitor, has the ability to effectively cross the blood-brain barrier. However, the effect of sorafenib on glioma cells and the molecular mechanism underlying the ability of sorafenib to enhance the antitumor effects of TMZ remain elusive. Here, we found that sorafenib could enhance the cytotoxic effects of TMZ in glioma cells in vitro and in vivo. Mechanistically, the combination of sorafenib and TMZ induced mitochondrial depolarization and apoptosis inducing factor (AIF) translocation from mitochondria to nuclei, and this process was dependent on STAT3 inhibition. Moreover, the combination of sorafenib and TMZ inhibited JAK2/STAT3 phosphorylation and STAT3 translocation to mitochondria. Inhibition of STAT3 activation promoted the autophagy-associated apoptosis induced by the combination of sorafenib and TMZ. Furthermore, the combined sorafenib and TMZ treatment induced oxidative stress while reactive oxygen species (ROS) clearance reversed the treatment-induced inhibition of JAK2/STAT3. The results indicate that sorafenib enhanced the temozolomide sensitivity of human glioma cells by inducing oxidative stress-mediated autophagy and JAK2/STAT3-AIF axis.

Elevated GIGYF2 expression suppresses tumor migration and enhances sensitivity to temozolomide in malignant glioma.

Glioma is a common type of malignant and aggressive tumor in the brain. Despite progress on mechanistic studies, current understanding of the initiation and progression of glioma remains incomplete. GIGYF2 is a critical regulator in neural development and degeneration, however, its contribution in glioma is not yet elucidated. In this study, using an integrative approach spanning bioinformatic analysis and functional approaches, we explored the potential contribution of GIGYF2 in glioma. Bioinformatic data from public database and our cohort showed that GIGYF2 expression was closely associated with low glioma malignancy and better patient survival. Elevation of GIGYF2 expression impaired cell migration and enhanced temozolomide sensitivity of human glioma cells. We further establish its molecular mechanism by demonstrating that GIGYF2 inhibits MMP-9 mediated cell migration pathway and pro-survival AKT/Bax/Caspase-3 signaling. Our work identifies the suppressive role of GIGYF2 in gliomas, and clarifies the relationship between GIGYF2 expression and glioma malignancy, which may provide a potential target for future interventions.

Inhibition of mitochondrial carrier homolog 2 (MTCH2) suppresses tumor invasion and enhances sensitivity to temozolomide in malignant glioma

BackgroundMalignant glioma exerts a metabolic shift from oxidative phosphorylation (OXPHOs) to aerobic glycolysis, with suppressed mitochondrial functions. This phenomenon offers a proliferation advantage to tumor cells and decrease mitochondria-dependent cell death. However, the underlying mechanism for mitochondrial dysfunction in glioma is not well elucidated. MTCH2 is a mitochondrial outer membrane protein that regulates mitochondrial metabolism and related cell death. This study aims to clarify the role of MTCH2 in glioma.MethodsBioinformatic analysis from TCGA and CGGA databases were used to investigate the association of MTCH2 with glioma malignancy and clinical significance. The expression of MTCH2 was verified from clinical specimens using real-time PCR and western blots in our cohorts. siRNA-mediated MTCH2 knockdown were used to assess the biological functions of MTCH2 in glioma progression, including cell invasion and temozolomide-induced cell death. Biochemical investigations of mitochondrial and cellular signaling alternations were performed to detect the mechanism by which MTCH2 regulates glioma malignancy.ResultsBioinformatic data from public database and our cohort showed that MTCH2 expression was closely associated with glioma malignancy and poor patient survival. Silencing of MTCH2 expression impaired cell migration/invasion and enhanced temozolomide sensitivity of human glioma cells. Mechanistically, MTCH2 knockdown may increase mitochondrial OXPHOs and thus oxidative damage, decreased migration/invasion pathways, and repressed pro-survival AKT signaling.ConclusionOur work establishes the relationship between MTCH2 expression and glioma malignancy, and provides a potential target for future interventions.

Inhibition of cathepsin L sensitizes human glioma cells to ionizing radiation in vitro through NF-κB signaling pathway.

Cathepsin L, a lysosomal cysteine proteinase, is exclusively elevated in a variety of malignancies, including gliomas. In this study we investigated the relationship between cathepsin L and NF-κB, two radiation-responsive elements, in regulating the sensitivity of human glioma cells ionizing radiation (IR) in vitro. Human glioma U251 cells were exposed to IR (10 Gy), and the expression of cathepsin L and NF-κB was measured using Western blotting. The nuclear translocation of NF-κB p65 and p50 was analyzed with immunofluorescence assays. Cell apoptosis was examined with clonogenic assays. NF-κB transcription and NF-κB-dependent cyclin D1 and ATM transactivation were monitored using luciferase reporter and ChIP assays, respectively. DNA damage repair was investigated using the comet assay. IR significantly increased expression of cathepsin L and NF-κB p65 and p50 in the cells. Furthermore, IR significantly increased the nuclear translocation of NF-κB, and NF-κB-dependent cyclin D1 and ATM transactivation in the cells. Knockdown of p65 did not change the expression of cathepsin L in IR-treated cells. Pretreatment with Z-FY-CHO (a selective cathepsin L inhibitor), or knockdown of cathepsin L significantly attenuated IR-induced nuclear translocation of NF-κB and cyclin D1 and ATM transactivation, and sensitized the cells to IR. Pretreatment with Z-FY-CHO, or knockdown of p65 also decreased IR-induced DNA damage repair and clonogenic cell survival, and sensitized the cells to IR. Cathepsin L acts as an upstream regulator of NF-κB activation in human glioma cells and contributes to their sensitivity to IR in vitro. Inhibition of cathepsin L can sensitize the cells to IR.

4E-BP1 regulates the sensitivity of human glioma cells to chemotherapy through PI3K/Akt/mTOR-independent pathway

Drug resistance is one of the most formidable obstacles for treatment of glioma. Eukaryotic initiation factor 4E-binding protein (4E-BP1), a key component in the rate-limiting step of protein translation initiation, is closely associated with poor prognosis in multiple tumor types. However, it is unclear whether 4E-BP1 is involved in the drug resistance of human glioma. Herein we show that the expression of 4E-BP1 in human SWOZ2-BCNU drug-resistant glioma cells is significantly lower than that of the parent SWOZ2 cell line. Moreover, down-regulation of 4E-BP1 by short interfering RNA significantly impaired the sensitivity of SWOZ2 and U251 cells to carmustine (BCNU). Furthermore, overexpression of 4E-BP1 with plasmid transfection regained this sensitivity. Clinical studies showed that the expression levels of 4E-BP1 in primary glioma tissues were markedly higher than those of recrudescent glioma tissues. Taken together, our results suggest that 4E-BP1 is a novel protein that contributes to acquired drug resistance and it may be a potential target for reversing drug resistance in human glioma.

Interaction of Beclin 1 with survivin regulates sensitivity of human glioma cells to TRAIL-induced apoptosis

We reported a novel interaction between Beclin 1, a key regulator of autophagy, and survivin, a member of the inhibitor of apoptosis protein family. We found that knock-down of Beclin 1 down-regulated survivin protein, and the turnover rate of survivin was increased when Beclin 1 expression was silenced. Knock-down of Beclin 1 sensitized glioma cells to TRAIL-induced apoptosis, and introduction of survivin antagonized the sensitizing effect, suggesting that down-regulation of survivin mediates the enhanced sensitivity to TRAIL-induced apoptosis. These results demonstrate a novel interaction between Beclin 1 and survivin, and may provide a potential mechanism underlying the cross-talk between autophagy and apoptosis. Structured summary MINT- 7969366: Beclin-1 (uniprotkb: Q14457) physically interacts (MI: 0915) with survivin (uniprotkb: O15392) by anti tag coimmunoprecipitation (MI: 0007) MINT- 7968986, MINT- 7969161: survivin (uniprotkb: O15392) physically interacts (MI: 0915) with Beclin-1 (uniprotkb: Q14457) by anti bait coimmunoprecipitation (MI: 0006)

C-Met antisense oligodeoxynucleotides increase sensitivity of human glioma cells to paclitaxel

Cell culture, tissue chemistry and flow cytometry were used to determine whether antisense c-Met oligodeoxynucleotides enhanced the sensitivity of human glioma cells to paclitaxel. A combination of paclitaxel with antisense c-Met oligodeoxynucleotides inhibited cell growth, induced apoptosis and induced c-Met protein expression in U251 and SHG44 human glioma cells more significantly than either paclitaxel or the oligodeoxynucleotides on their own (P<0.01). Thus, c-Met antisense oligodeoxynucleotides increase the sensitivity of human glioma cells to paclitaxel. Combined use of the two agents could be a novel and attractive strategy in human glioma treatment.

Inhibition of invasion-associated thromboxane synthase sensitizes experimental gliomas to γ-radiation

The invasion- and apoptosis-associated thromboxane synthase gene encoding an enzyme of the arachidonic acid pathway has been implicated in glioma progression. Furegrelate, a specific inhibitor of thromboxane synthase, blocks cell motility, induces apoptosis and increases sensitivity to drug induced apoptosis in human glioma cells in vitro. The impact of furegrelate on the sensitivity of human glioma cells to gamma-irradiation was analyzed using colony formation assay in vitro and an orthotopic mouse model in vivo. Pre-treatment of glioma cells with furegrelate increases radiation sensitivity of cultured glioma cells. Treatment of experimental gliomas with suboptimal doses of radiation and furegrelate results in a significant decrease in tumor volumes compared to untreated controls. Thus, the specific thromboxane synthase inhibitor furegrelate increases death response induced by gamma-radiation in glioma cells in vitro and sensitizes experimental gliomas to radiation treatment in vivo.

Human Glioma Cell Sensitivity to the Sequence-Specific Alkylating Agent Methyl-Lexitropsin

Defining the cytotoxicity of individual adducts in DNA is necessary for mechanistic understanding of human brain tumor resistance to therapeutic alkylating agents and for design of DNA repair-related antiresistance strategies. Our purpose is to characterize the sensitivity of human glioma cells to methyl-lexitropsin (Me-lex), a sequence-specific alkylator that produces 3-methyladenine (3-meA) as the predominant (>90%) DNA lesion. We quantitated the Me-lex cytotoxicity of 10 human glioma cell lines that differ in O(6)-methylguanine (O(6)-meG)-DNA methyltransferase (MGMT) and mismatch repair activity. We used antisense suppression of alkyladenine DNA glycosylase (AAG) and Ape1 to assess the contribution of 3-meA and abasic sites to lethality and measured abasic sites. (a) The LD(10) for Me-lex varied widely among the cell lines. (b) MGMT-proficient lines were more resistant than MGMT-deficient lines, an unexpected finding because Me-lex produces very little O(6)-meG. (c) Suppression of AAG increased Me-lex killing and reduced abasic site content. (d) Suppression of Ape1 increased Me-lex killing and increased abasic site content. (e) Ablation of MGMT had no effect on Me-lex cytotoxicity. (a) Me-lex is cytotoxic in human glioma cells and AAG promotes resistance, indicating that 3-meA is a lethal lesion in these cells. (b) Abasic sites resulting from 3-meA repair are cytotoxic and Ape1 promotes resistance to these derivative lesions. (c) A factor(s) associated with MGMT expression, other than repair of O(6)-meG, contributes to Me-lex resistance. (d) Me-lex may have clinical utility in the adjuvant therapy of gliomas. (e) AAG and Ape1 inhibitors may be useful in targeting alkylating agent resistance.

Effect of wild-type p53 gene transfection on the growth and radiotherapeutic sensitivity of human glioma cells

To evaluate the effect of wild-type p53 gene on the growth and radiotherapeutic sensitivity of human glioma cells, plasmid PC53-SN3 carrying wild-type p53 gene was transfected into U251 cells. p53 gene expression in transfected cells was detected by RT-PCR, and the cell growth inhibition and apoptosis in the absence or presence of irradiation were assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT-PCR. MTT showed that p53 gene alone induced strong inhibitory effect on the growth of U251 cells (inhibition rate (IR), (79.60 +/- 5.69)%). The killing effect of irradiation alone on U251 cells was not strong (IR: (17.06 +/- 4.35)% (17.39 +/- 1.67)% (18.73 +/- 4.68)%) and increased with the irradiation doses (3, 6, 9 Gy). When combined treatment of wild-type p53 gene transfection and irradiation was used, the effect was significantly increased (IR:(80.60 +/- 5.35)%. (90.30 +/- 1.67)%, (91.30 +/- 2.01)%). The apoptosis rate of U251 cells induced by p53 gene transfection was 17.38%. The rate induced by irradiation increased (4.61%, 4.84%, 5.40%) with the irradiation doses (3, 6, 9 Gy). The apoptosis rate was also significantly increased (17.80%, 20.03%, 22.34%) after combined treatment of p53 and irradiation with different doses (3, 6, 9 Gy). It is concluded that wild-type p53 gene and irradiation could result in synergistic inhibitory effect on the growth of human glioma cells.

The effects of wild-type p53 gene transfection on the growth and chemotherapeutic sensitivity of human glioma cells.

To evaluate the effects of wild-type p53 gene on the growth and chemotherapeutic sensitivity of human glioma cells, plasmid PC53-SN3 carrying wild-type p53 gene was transfected into U251 cells. p53 gene expression in transfected cells was detected by RT-PCR, the cell growth inhibition and apoptosis in either the absence or the presence of cisplatin was assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT-PCR. MTT showed that p53 gene by itself induced strong inhibition effect on the growth of U251 cells [inhibition rate, IR (79.60 +/- 5.69)%]. The killing effects of cisplatin by itself on U251 cells was not strong [IR (19.40 +/- 6.69)%, (24.41 +/- 2.68)%, (51.84 +/- 13.38)%, (66.22 +/- 5.02)%] and increased with the increase of cisplatin concentration (1, 2, 4, 8 micrograms/ml). When combined treatment of wild-type p53 gene transfection and cisplatin was used, that was significantly increased [IR (91.64 +/- 1.00)%, (94.98 +/- 1.67)%, (95.32 +/- 2.01)%, (95.65 +/- 1.00)%]. The apoptosis rate of U251 cells induced by p53 gene transfection was 17.38%. That induced by cisplatin increased (5.71%, 5.93%, 6.27%, and 6.81%) with the increase of cisplatin concentration (1, 2, 4, 8 micrograms/ml). The apoptosis rate was also significantly increased (23.50%, 23.54%, 23.89%, and 28.88%) after combined treatment of p53 and cisplatin with different concentration (1, 2, 4, 8 micrograms/ml). It is concluded that wild-type p53 gene and cisplatin could result in synergistic inhibition effects on the growth of human glioma cells.

Sensitivity of human glioma cells to cytotoxic lymphocytes and ligand — toxin conjugates

Sensitivity of human glioma cells to cytotoxic lymphocytes and ligand — toxin conjugates