Knockdown of HMGB1 improves apoptosis and suppresses proliferation and invasion of glioma cells

Background

The purposes of this study were to explore the effects of high mobility group protein box 1 (HMGB1) gene on the growth, proliferation, apoptosis, invasion, and metastasis of glioma cells, with an attempt to provide potential therapeutic targets for the treatment of glioma.

Methods

The expressions of HMGB1 in glioma cells (U251, U-87MG and LN-18) and one control cell line (SVG p12) were detected by real time PCR and Western blotting, respectively. Then, the effects of HMGB1 on the biological behaviors of glioma cells were detected: the expression of HMGB1 in human glioma cell lines U251 and U-87MG were suppressed using RNAi technique, then the influences of HMGB1 on the viability, cycle, apoptosis, and invasion abilities of U251 and U-87MG cells were analyzed using in a Transwell invasion chamber. Also, the effects of HMGB1 on the expressions of cyclin D1, Bax, Bcl-2, and MMP 9 were detected.

Results

As shown by real-time PCR and Western blotting, the expression of HMGB1 significantly increased in glioma cells (U251, U-87MG, and LN-18) in comparison with the control cell line (SVG p12); the vitality, proliferation and invasive capabilities of U251 and U-87MG cells in the HMGB1 siRNA-transfected group were significantly lower than those in the blank control group and negative control (NC) siRNA group (P<0.05) but showed no significant difference between the blank control group and NC siRNA group. The percentage of apoptotic U251 and U-87MG cells was significantly higher in the HMGB1 siRNA-transfected group than in the blank control group and NC siRNA group (P<0.05) but was similar between the latter two groups. The HMGB1 siRNA-transfected group had significantly lower expression levels of Cyclin D1, Bcl-2, and MMP-9 protein in U251 and U-87MG cells and significantly higher expression of Bax protein than in the blank control group and NC siRNA group (P<0.05); the expression profiles of cyclin D1, Bax, Bcl-2, and MMP 9 showed no significant change in both blank control group and NC siRNA group.

Conclusions

HMGB1 gene may promote the proliferation and migration of glioma cells and suppress its effects of apoptosis. Inhibition of the expression of HMGB1 gene can suppress the proliferation and migration of glioma cells and promote their apoptosis. Our observations provided a new target for intervention and treatment of glioma.     

Suppression of STIM1 inhibits human glioblastoma cell proliferation and induces G0/G1 phase arrest

Background

Depletion of calcium (Ca²⁺) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca²⁺ entry (SOCE) pathway which sustains long-term Ca²⁺ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca²⁺ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear.

Methods

Expression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo.

Results

We discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21^Waf1/Cip1_, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model.

Conclusion

Our findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma.     

MiR-125b acts as an oncogene in glioblastoma cells and inhibits cell apoptosis through p53 and p38MAPK-independent pathways

Background:

We have recently identified miR-125b upregulation in glioblastoma (GMB). The aim of this study is to determine the correlation between miR-125b expression and malignant grades of glioma and the genes targeted by miR-125b.

Methods:

Real-time PCR was employed to measure the expression level of miR-125b. Cell viability was evaluated by cell growth and colony formation in soft-agar assays. Cell apoptosis was determined by Hoechst 33342 staining and AnnexinV-FITC assay. The Luciferase assay was used to confirm the actual binding sites of p38MAPK mRNA. Western blot was used to detect the gene expression level.

Results:

The expression level of miR-125b is positively correlated with the malignant grade of glioma. Ectopic expression of miR-125b promotes the proliferation of GMB cells. Knockdown of endogenous miR-125b inhibits cell proliferation and promotes cell apoptosis. Further studies reveal that p53 is regulated by miR-125b. However, downregulation of the endogenous miR-125b also results in p53-independent apoptotic pathway leading to apoptosis in p53 mutated U251 cells and p53 knockdown U87 cells. Moreover, p38MAPK is also regulated by miR-125b and downregulation of miR-125b activates the p38MAPK-induced mitochondria apoptotic pathway.

Conclusion:

High-level expression of miR-125b is associated with poor outcomes of GMB. MiR-125b may have an oncogenic role in GMB cells by promoting cell proliferation and inhibiting apoptosis.     

Let-7b expression determines response to chemotherapy through the regulation of Cyclin D1 in Glioblastoma

Background

Glioblastoma is the most common type of primary brain tumors. Cisplatin is a commonly used chemotherapeutic agent for Glioblastoma patients. Despite a consistent rate of initial responses, cisplatin treatment often develops chemoresistance, leading to therapeutic failure. Cellular resistance to cisplatin is of great concern and understanding the molecular mechanisms is an utter need.

Methods

Glioblastoma cell line U251 cells were exposed to increasing doses of cisplatin for 6 months to establish cisplatin-resistant cell line U251R. The differential miRNA expression profiles in U251 and U251R cell lines were identified by microarray analysis and confirmed by Q-PCR. MiRNA mimics were transfected into U251R cells, and cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis.

Results

U251R cells showed 3.1-fold increase in cisplatin resistance compared to its parental U251 cells. Microarray analysis identified Let-7b and other miRNAs significantly down-regulated in U251R cells compared to U251 cells. Transfection of Let-7b mimics greatly re-sensitized U251R cells to cisplatin, while transfection of other miRNAs has no effect or slightly effect. Cyclin D1 is predicted as a target of Let-7b through bioinformatics analysis. Over-expression of Let-7b mimics suppressed cyclin D1 protein expression and inhibited cyclin D1-3’-UTR luciferase activity. Knockdown of cyclin D1 expression significantly increased cisplatin-induced G1 arrest and apoptosis.

Conclusions

Collectively, our results indicated that cisplatin treatment leads to Let-7b suppression, which in turn up-regulates cyclin D1 expression. Let-7b may serve as a marker of cisplatin resistance, and can enhance the therapeutic benefit of cisplatin in glioblastoma cells.     

Elevated expression of chloride intracellular channel 1 is correlated with poor prognosis in human gliomas

Background

Chloride intracellular channel 1 (CLIC1) is expressed ubiquitously in human tissues and is involved in the regulation of cell cycle, cell proliferation and differentiation. Recent studies have shown that CLIC1 is highly expressed in several human malignant tumors. However, its roles in human gliomas are still unclear. The aim of this study was to investigate the clinicopathological significance and prognostic value of CLIC1 expression in human gliomas.

Methods

CLIC1 expression in human gliomas and nonneoplastic brain tissues was measured by real-time quantitative RT-PCR assay and immunohistochemistry. Its association with clinicopathological factors or prognosis in patients with gliomas was statistically analyzed.

Results

The expression of CLIC1 at both mRNA and protein levels was significantly increased in high-grade (Grade III~IV) glioma tissues compared with that in low-grade (Grade I~II) and nonneoplastic brain tissues, and was up-regulated with ascending tumor World Health Organization (WHO) grades. The elevated expression of CLIC1 protein was also significantly correlated with low Karnofsky performance score (KPS) (P=0.008). Moreover, both univariate and multivariate analysis shown that high CLIC1 expression was significantly associated with poor prognosis in patients with gliomas (P<0.001 and P=0.01, respectively). In particular, the elevated CLIC1 expression also correlated with shorter overall survival in different glioma subgroups stratified according to the WHO grading.

Conclusions

Our data provide the first evidence that CLIC1 expression might play an important role in the regulation of aggressiveness in human gliomas. The elevated expression of CLIC1 might represent a valuable prognostic marker for this disease.     

TGM2 inhibition attenuates ID1 expression in CD44-high glioma-initiating cells

Background

CD44 is a molecular marker associated with cancer stem cell populations and treatment resistance in glioma. More effective therapies will result from approaches aimed at targeting glioma cells high in CD44.

Methods

Glioma-initiating cell lines were derived from fresh surgical glioblastoma samples. Expression of tissue transglutaminase 2 (TGM2) was attenuated through lentivirus-mediated short hairpin RNA knockdown. MTT assay [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was used to evaluate the growth inhibition induced by TGM2 inhibitor. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling was used to evaluate cell apoptosis following TGM2 inhibition. CD44⁺ glioma stem cells were sorted by flow cytometry. A nude mice orthotopic xenograft model was used to evaluate the in vivo effect of TGM2 inhibitor.

Results

TGM2 was highly expressed in CD44-high glioblastoma tissues and tumor-derived glioma-initiating cell lines. TGM2 knockdown impaired cell proliferation and induced apoptosis in CD44-high glioma-initiating cell lines. Further studies indicated that expression of inhibitor of DNA binding 1 protein (ID1) is regulated by TGM2 and might be an important mediator for TGM2-regulated cell proliferation in CD44-high glioma-initiating cell lines. TGM2 inhibitor reduces ID1 expression, suppresses cell proliferation, and induces apoptosis in CD44-high glioma-initiating cell lines. Furthermore, TGM2 is highly expressed in CD44⁺ glioma stem cells, while pharmacological inhibition of TGM2 activity preferentially eliminates CD44⁺ glioma stem cells. Consistently, TGM2 inhibitor treatment reduced ID1 expression and induced apoptosis in our orthotopic mice xenograft model, which can be translated into prolonged median survival in tumor-bearing mice.

Conclusions

TGM2 regulates ID1 expression in glioma-initiating cell lines high in CD44. Targeting TGM2 could be an effective strategy to treat gliomas with high CD44 expression.     

Nitroxoline induces apoptosis and slows glioma growth in vivo

Background

Nitroxoline is an FDA-approved antibiotic with potential antitumor activity. Here we evaluated whether nitroxoline has antiproliferative properties on glioma cell growth in vitro and in vivo using glioma cell lines and a genetically engineered PTEN/KRAS mouse glioma model.

Methods

The effect of nitroxoline treatment on U87 and/or U251 glioma cell proliferation, cell-cycle arrest, invasion, and ability to induce an apoptotic cascade was determined in vitro. Magnetic resonance imaging was used to measure glioma volumes in genetically engineered PTEN/KRAS mice prior to and after nitroxoline therapy. Induction of apoptosis by nitroxoline was evaluated at the end of treatment using terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL).

Results

Nitroxoline inhibited the proliferation and invasion of glioblastoma cells in a time- and dose-dependent manner in vitro. Growth inhibition was associated with cell-cycle arrest in G₁/G₀ phase and induction of apoptosis via caspase 3 and cleaved poly(ADP-ribose) polymerase. In vivo, nitroxoline-treated mice had no increase in tumor volume after 14 days of treatment, whereas tumor volumes doubled in control mice. Histological examination revealed 15%–20% TUNEL-positive cells in nitroxoline-treated mice, compared with ∼5% in the control group.

Conclusion

Nitroxoline induces apoptosis and inhibits glioma growth in vivo and in vitro. As an already FDA-approved treatment for urinary tract infections with a known safety profile, nitroxoline could move quickly into clinical trials pending confirmatory studies.     

MiR-124-5p inhibits the growth of high-grade gliomas through posttranscriptional regulation of LAMB1

Background

Glioblastoma multiforme (GBM) is the most aggressive form of human brain tumor. It was previously shown that high levels of laminin-8 expression were a predictor of tumor recurrence and patient survival. It is thus important to elucidate the mechanism by which laminin-8 expression is regulated and determine how this contributes to glioma progression. This study investigated the mechanism of regulation of LAMB1, which encodes the β1 chain of laminin-8, in glioma cells lines and in a mouse model of GBM.

Methods

The expression levels of LAMB1 and miR-124-5p were examined in glioma cell lines (U87 and U251) and GBM tissue samples by quantitative PCR and Western blotting. The potential regulation of LAMB1 by miR-124-5p was investigated by assessing the effects of restored miR-124-5p expression on cell proliferation, colony formation, and tumor growth and angiogenesis. The effects of inhibiting LAMB1 on tumor growth and angiogenesis were also assessed.

Results

The upregulation of LAMB1 expression was highly correlated with the downregulation of miR-124-5p. LAMB1 protein expression was suppressed by miR-124-5p. The restoration of miR-124-5p expression suppressed glioma growth by inhibiting angiogenesis, effects that were also observed upon LAMB1 knockdown.

Conclusions

The findings indicate that miR-124-5p functions as a tumor suppressor and could serve as a molecular marker for glioma diagnosis and as a potential therapeutic target in GBM treatment.     

Effects of salidroside on glioma formation and growth inhibition together with improvement of tumor microenvironment

Objective：To test the effects of salidroside on formation and growth of glioma together with tumor microenvironment.Methods：Salidroside extracted from Rhodiola rosea was purified and treated on human glioma cells U251 at the concentration of 20 μg/mL.3-（4,5-dimethylthiazol-2-yl）-2,5-dephenyltetrazolium bromide （MTT） assay for cytotoxicity and flow cytometry （FCM） for cell cycle analysis were performed.Then for in vivo study,xenotransplantation tumor model in nude mice was generated and treated with salidroside at the concentration of 50 mg/kg.d for totally 20 d.Body weight and tumor size were detected every 2 d after the treatment.The levels of 8-isoprostane,superoxide dismutase （SOD） and malondialdehyde （MDA）,special markers for oxidative stress,were detected while immunofluoresence staining was performed for astrocyte detection.Results：For in vitro study,salidroside could decrease the viability of human glioma cells U251 and the growth of U251 cells at G0/G1 checkpoint during the cell cycle.For in vivo study,salidroside could also inhibit the growth of human glioma tissue in nude mice.The body weight of these nude mice treated with salidroside did not decrease as quickly as control group.In the tumor xenotransplantation nude mice model,mice were found of inhibition of oxidative stress by detection of biomarkers.Furthermore,overgrowth of astrocytes due to the stimulation of oxidative stress in the cortex of brain was inhibited after the treatment of salidroside.Conclusions：Salidroside could inhibit the formation and growth of glioma both in vivo and in vitro and improve the tumor microenvironment via inhibition of oxidative stress and astrocytes.     

ProBDNF and its receptors are upregulated in glioma and inhibit the growth of glioma cells in vitro

Background

High-grade glioma is incurable, with a short survival time and poor prognosis. The increased expression of p75 neurotrophin receptor (NTR) is a characteristic of high-grade glioma, but the potential significance of increased p75NTR in this tumor is not fully understood. Since p75NTR is the receptor for the precursor of brain-derived neurotrophic factor (proBDNF), it is suggested that proBDNF may have an impact on glioma.

Methods

In this study we investigated the expression of proBDNF and its receptors p75NTR and sortilin in 52 cases of human glioma and 13 cases of controls by immunochemistry, quantitative real-time PCR, and Western blot methods. Using C6 glioma cells as a model, we investigated the roles of proBDNF on C6 glioma cell differentiation, growth, apoptosis, and migration in vitro.

Results

We found that the expression levels of proBDNF, p75NTR, and sortilin were significantly increased in high-grade glioma and were positively correlated with the malignancy of the tumor. We also observed that tumors expressed proBDNF, p75NTR, and sortilin in the same cells with different subcellular distributions, suggesting an autocrine or paracrine loop. The ratio of proBDNF to mature BDNF was decreased in high-grade glioma tissues and was negatively correlated with tumor grade. Using C6 glioma cells as a model, we found that proBDNF increased apoptosis and differentiation and decreased cell growth and migration in vitro via p75NTR.

Conclusions

Our data indicate that proBDNF and its receptors are upregulated in high-grade glioma and might play an inhibitory effect on glioma.     

Somatostatin and opioid receptors do not regulate proliferation or apoptosis of the human multiple myeloma U266 cells

Background

opioid and somatostatin receptors (SSTRs) that can assemble as heterodimer were individually reported to modulate malignant cell proliferation and to favour apoptosis. Materials and methods: SSTRs and opioid receptors expression were examined by RT-PCR, western-blot and binding assays, cell proliferation was studied by XTT assay and propidium iodide (PI) staining and apoptosis by annexin V-PI labelling.

Results

almost all human malignant haematological cell lines studied here expressed the five SSTRs. Further experiments were conducted on the human U266 multiple myeloma cells, which express also μ-opioid receptors (MOP-R). XTT assays and cell cycle studies provide no evidence for a significant effect upon opioid or somatostatin receptors stimulation. Furthermore, neither direct effect nor potentiation of the Fas-receptor pathway was detected on apoptosis after these treatments.

Conclusion

these data suggest that SSTRs or opioid receptors expression is not a guaranty for an anti-tumoral action in U266 cell line.     

Functional analysis of a novel glioma antigen,EFTUD1

Background

A cDNA library made from 2 glioma cell lines, U87MG and T98G, was screened by serological identification of antigens by recombinant cDNA expression (SEREX) using serum from a glioblastoma patient. Elongation factor Tu GTP binding domain containing protein 1 (EFTUD1), which is required for ribosome biogenesis, was identified. A cancer microarray database showed overexpression of EFTUD1 in gliomas, suggesting that EFTUD1 is a candidate molecular target for gliomas.

Methods

EFTUD1 expression in glioma cell lines and glioma tissue was assessed by Western blot, quantitative PCR, and immunohistochemistry. The effect on ribosome biogenesis, cell growth, cell cycle, and induction of apoptosis and autophagy in glioma cells during the downregulation of EFTUD1 was investigated. To reveal the role of autophagy, the autophagy-blocker, chloroquine (CQ), was used in glioma cells downregulating EFTUD1. The effect of combining CQ with EFTUD1 inhibition in glioma cells was analyzed.

Results

EFTUD1 expression in glioma cell lines and tissue was higher than in normal brain tissue. Downregulating EFTUD1 induced G1 cell-cycle arrest and apoptosis, leading to reduced glioma cell proliferation. The mechanism underlying this antitumor effect was impaired ribosome biogenesis via EFTUD1 inhibition. Additionally, protective autophagy was induced by glioma cells as an adaptive response to EFTUD1 inhibition. The antitumor effect induced by the combined treatment was significantly higher than that of either EFTUD1 inhibition or CQ alone.

Conclusion

These results suggest that EFTUD1 represents a novel therapeutic target and that the combination of EFTUD1 inhibition with autophagy blockade may be effective in the treatment of gliomas.     

Monocarboxylate transporters (MCTs) in gliomas: expression and exploitation as therapeutic targets

Background

Gliomas exhibit high glycolytic rates, and monocarboxylate transporters (MCTs) play a major role in the maintenance of the glycolytic metabolism through the proton-linked transmembrane transport of lactate. However, their role in gliomas is poorly studied. Thus, we aimed to characterize the expression of MCT1, MCT4, and their chaperone CD147 and to assess the therapeutic impact of MCT inhibition in gliomas.

Methods

MCTs and CD147 expressions were characterized by immunohistochemistry in nonneoplastic brain and glioma samples. The effect of CHC (MCT inhibitor) and MCT1 silencing was assessed in in vitro and in vivo glioblastoma models.

Results

MCT1, MCT4, and CD147 were overexpressed in the plasma membrane of glioblastomas, compared with diffuse astrocytomas and nonneoplastic brain. CHC decreased glycolytic metabolism, migration, and invasion and induced cell death in U251 cells (more glycolytic) but only affected proliferation in SW1088 (more oxidative). The effectiveness of CHC in glioma cells appears to be dependent on MCT membrane expression. MCT1 downregulation showed similar effects on different glioma cells, supporting CHC as an MCT1 inhibitor. There was a synergistic effect when combining CHC with temozolomide treatment in U251 cells. In the CAM in vivo model, CHC decreased the size of tumors and the number of blood vessels formed.

Conclusions

This is the most comprehensive study reporting the expression of MCTs and CD147 in gliomas. The MCT1 inhibitor CHC exhibited anti-tumoral and anti-angiogenic activity in gliomas and, of importance, enhanced the effect of temozolomide. Thus, our results suggest that development of therapeutic approaches targeting MCT1 may be a promising strategy in glioblastoma treatment.     

SIRT1 is required for oncogenic transformation of neural stem cells and for the survival of “cancer cells with neural stemness” in a p53-dependent manner

Background

Cancer stemness, observed in several types of glioma stem cells (GSCs), has been demonstrated to be an important barrier for efficient cancer therapy. We have previously reported that cancerous neural stem cells (F3.Ras.CNSCs), derived from immortalized human neural stem cells by a single oncogenic stimulation, form glial tumors in vivo.

Method

We searched for a commonly expressed stress modulator in both F3.Ras.CNSCs and GSCs and identified silent mating type information regulation 2, homolog (SIRT1) as a key factor in maintaining cancer stemness.

Result

We demonstrate that the expression of SIRT1, expressed in “cancer cells with neural stemness,” is critical not only for the maintenance of stem cells, but also for oncogenic transformation. Interestingly, SIRT1 is essential for the survival and tumorigenicity of F3.Ras.CNSCs and GSCs but not for the U87 glioma cell line.

Conclusion

These results indicate that expression of SIRT1 in cancer cells with neural stemness plays an important role in suppressing p53-dependent tumor surveillance, the abrogation of which may be responsible not only for inducing oncogenic transformation but also for retaining the neural cancer stemness of the cells, suggesting that SIRT1 may be a putative therapeutic target in GSCs.