Effects and mechanism of ginsenoside Rh2 on the proliferation and apoptosis of human glioma cells / 中国药房

ABSTRACT OBJECTIVE To investigate the effects and mechanism of ginsenoside Rh2 on the proliferation and apoptosis in human glioma U87 and U251 cells. METHODS Using human glioma U87 and U251 cells as subjects， the proliferation and apoptosis， as well as the expression of histone deacetylase 1（HDAC1） protein and apoptosis-related proteins [B cell lymphoma-2（Bcl-2）， Bcl-2-associated X protein （Bax） and cleaved caspase-3] were detected after being treated with different concentrations of ginsenoside Rh2. RESULTS The concentrations of 10，20，30，40，50，60，70，80 μmol/L ginsenoside Rh2 could generally significantly increase the proliferation inhibition rate of U87 and U251 cells （P＜0.05 or P＜0.01）， and the half inhibitory concentrations of this component after 48 hours of action were 51.34 and 55.84 μmol/L， respectively；30，50 μmol/L ginsenoside Rh2 could increase the total apoptotic rate of both types of cells， reduced the protein expressions of HDAC1 and Bcl-2， and increased the protein expressions of Bax and cleaved caspase-3 significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Ginsenoside Rh2 has a significant inhibitory effect on the proliferation of glioma cells and promotes the apoptosis of cells， which may be through reducing the expression of HDAC1 protein and activating the Bcl-2 family protein-mediated apoptosis pathway.

Therapeutic efficacy of 166Holmium siloxane in microbrachytherapy of induced glioblastoma in minipig tumor model.

Glioblastoma is considered the most common malignant primary tumor of central nervous system. In spite of the current standard and multimodal treatment, the prognosis of glioblastoma is poor. For this reason, new therapeutic approaches need to be developed to improve the survival time of the glioblastoma patient. In this study, we performed a preclinical experiment to evaluate therapeutic efficacy of 166Ho microparticle suspension administered by microbrachytherapy on a minipig glioblastoma model. Twelve minipigs were divided in 3 groups. Minipigs had injections into the tumor, containing microparticle suspensions of either 166Ho (group 1; n = 6) or 165Ho (group 2; n = 3) and control group (group 3; n = 3). The survival time from treatment to euthanasia was 66 days with a good state of health of all minipigs in group 1. The median survival time from treatment to tumor related death were 8.6 and 7.3 days in groups 2 and control, respectively. Statistically, the prolonged life of group 1 was significantly different from the two other groups (p < 0.01), and no significant difference was observed between group 2 and control (p=0.09). Our trial on the therapeutic effect of the 166Ho microparticle demonstrated an excellent efficacy in tumor control. The histological and immunohistochemical analysis showed that the efficacy was related to a severe 166Ho induced necrosis combined with an immune response due to the presence of the radioactive microparticles inside the tumors. The absence of reflux following the injections confirms the safety of the injection device.

The Paracrine Effect of Hypoxic and Normoxic Cancer Secretion on the Proliferation of Brain Endothelial Cells (bEnd.3).

BACKGROUND: This study aimed to investigate the disruption of cell cycle phases of bEnd.3 cells exposed to cancer paracrine secretion. Cancer cells have been reported to use the secretion of paracrine factors to compromise the endothelial barrier to prepare for their passage into the parenchyma. As cancer cells are known to act differently under conditions of hypoxia, we investigated how conditional media (CM) derived from breast and glioblastoma cells incubated under conditions of normoxia and hypoxia would affect proliferation of brain endothelial cells (bEnd.3). METHODS: Brain endothelial cells (bEnd.3) were cultivated with normoxic and hypoxic CM generated from breast cancer MCF7 cells and glioblastoma U-87 cells. Cell proliferation was evaluated using the trypan blue exclusion assay and phases of the cell cycle were evaluated using flow cytometry. RESULTS: bEnd.3 proliferations was suppressed more aggressively with hypoxic CM after 72 and 96 h; cell cycle analysis showed that paracrine treatment tended to prevent BECs from entering the G2 phase, thus suppressing cell division. CONCLUSIONS: MCF7 and U-87 cells induce suppressed proliferation of BECs deferentially under hypoxia by blocking cell cycle progression to the G2 phase.

Blockade of Nuclear Factor-Κb (NF-Κb) Pathway Using Bay 11-7082 Enhances Arsenic Trioxide-Induced Antiproliferative Activity in U87 Glioblastoma Cells.

Background: Glioblastoma (GBM), the most aggressive and common form of glioma, accounts for over 13,000 death per year in the United States which indicates the importance of developing novel strategies for the treatment of this fatal malignancy. Although Arsenic trioxide (ATO) hinders the growth and survival of GBM cells, the requirement of concentrations higher than 4 µM for triggering apoptotic cell death has questioned its safety profile. Since the NF-κB signaling pathway plays a crucial role in tumorigenesis and chemo-resistance, targeting this oncogenic pathway may sensitize GBM cells to lower concentrations of ATO. Methods: Anti-tumor effects of ATO as monotherapy and in combination with Bay 11-7082 were determined using MTT, crystal violet staining, Annexin V/PI staining and scratch assays. Quantitative reverse transcription-PCR (qRT-PCR) analysis was applied to elucidate the molecular mechanisms underlying the anti-tumor activity of this combination therapy. Results: Our results revealed that ATO and Bay 11-7082 synergistically inhibited the proliferation and survival of GBM cells. Also, it was revealed that NF-κB inhibition using Bay 11-7082 enhanced the inhibitory effects of ATO on migration of GBM cells via suppressing the expression of NF-κB target genes such as TWIST, MMP2, ICAM-1, and cathepsin B. Furthermore, combination treatment of GBM cells with ATO and Bay 11-7082 significantly induce apoptotic cell death coupled with downregulation of NF-κB anti-apoptotic target genes including Bcl-2 and IAP family members. Conclusion: Altogether, these findings suggest that combination therapy with ATO and Bay 11-7082 may be a promising strategy for the treatment of GBM.

The Effect of Normoxic and Hypoxic U-87 Glioblastoma Paracrine Secretion on the Modulation of Brain Endothelial Cells.

BACKGROUND: Glioblastoma multiforme (GBM) is a highly invasive brain tumour, characterized by its ability to secrete factors promoting its virulence. Brain endothelial cells (BECs) in the GBM environment are physiologically modulated. The present study investigated the modulatory effects of normoxically and hypoxically induced glioblastoma U-87 cell secretions on BECs. METHODS: Conditioned media (CM) were derived by cultivating U-87 cells under hypoxic incubation (5% O2) and normoxic incubation (21% O2). Treated bEnd.3 cells were evaluated for mitochondrial dehydrogenase activity, mitochondrial membrane potential (ΔΨm), ATP production, transendothelial electrical resistance (TEER), and endothelial tight-junction (ETJ) gene expression over 96 h. RESULTS: The coculture of bEnd.3 cells with U-87 cells, or exposure to either hypoxic or normoxic U-87CM, was associated with low cellular viability. The ΔΨm in bEnd.3 cells was hyperpolarized after hypoxic U-87CM treatment (p < 0.0001). However, normoxic U-87CM did not affect the state of ΔΨm. BEC ATP levels were reduced after being cocultured with U-87 cells, or with hypoxic and normoxic CM (p < 0.05). Suppressed mitochondrial activity in bEnd.3 cells was associated with increased transendothelial permeability, while bEnd.3 cells significantly increased the gene expression levels of ETJs (p < 0.05) when treated with U-87CM. CONCLUSIONS: Hypoxic and normoxic glioblastoma paracrine factors differentially suppressed mitochondrial activity in BECs, increasing the BECs' barrier permeability.

Inhibition of Methamphetamine-Induced Cytotoxicity in the U87-Cell Line by Atorvastatin-Conjugated Carbon Nanotubes.

In biological systems, carbon nanotubes can enhance the biological effects of drugs and reduce their side effects. Methamphetamine (METH) is a stimulant drug that induces cell death in various cell types, primarily neural cells. On the other hand, specific doses of atorvastatin (ATO) can stimulate cell growth and inhibit cell death in different cell lines. This study aimed to investigate the improvement effect of ATO@single-walled carbon nanotube (SWCNT) on METH-induced cell cytotoxicity in the U87 glioblastoma cell line. In this study, cells were cultured in 10 mM of METH during the cell treatment with 0-10 nM of ATO and ATO@SWCNT. The conjugated drugs to SWCNT as Van der Waals were detected using field emission scanning electron microscopy, Fourier transform-infrared spectroscopy, and other analyses. Then, the in vitro proliferating of ATO@SWCNT was explored against glioblastoma cells compared to pure ATO. This examine was performed using methyl thiazole tetrazolium approach, terminal deoxynucleotidyl transferase deoxy uridine-triphosphate nick end labeling assay, caspase-3 method, lactate dehydrogenase assay, and RH-123 assay with 10 mM METH. The results obtained from transmission electron microscopy analysis showed the average size of 50 nm for ATO@SWCNT. This study indicated that U87 cells, which were exposed to METH and suffered cell death, were severely reduced in the presence of ATO, especially ATO@SWCNT (for its anti-apoptotic effect), but they survived. This study suggests that ATO, which was primarily used to reduce blood lipids, can significantly reduce brain cell death. The findings of this study indicate that by using SWCNT, more drugs can reach the target cells. This method reduces the total amount of required medication and shows a more beneficial therapeutic effect.

Knockdown of Annexin-A1 Inhibits Growth, Migration and Invasion of Glioma Cells by Suppressing the PI3K/Akt Signaling Pathway.

ANXA1, which can bind phospholipid in a calcium dependent manner, is reported to play a pivotal role in tumor progression. However, the role and mechanism of ANXA1 involved in the occurrence and development of malignant glioma are still not well studied. Therefore, we explored the effects of ANXA1 on normal astrocytes and glioma cell proliferation, apoptosis, migration and invasion and the underlying mechanisms. We found that ANXA1 was markedly up-regulated in glioma cell lines and glioma tissues. Down-regulation of ANXA1 inhibited normal astrocytes and glioma cell proliferation and induced the cell apoptosis, which suggested that the consequences of loss of Annexin 1 are not specific to the tumor cells. Furthermore, the siRNA-ANXA1 treatment significantly reduced tumor growth rate and tumor weight. Moreover, decreasing ANXA1 expression caused G2/M phase arrest by repressing expression levels of cdc25C, cdc2 and cyclin B1. Interestingly, ANXA1 did not affect the expressions of ß-catenin, GSK-3ß and NF-κB, the key signaling molecules associated with cancer progression. However, siRNA-ANXA1 was found to negatively regulate phosphorylation of AKT and the expression and activity of MMP2/-9. Finally, the decrease of cell proliferation and invasiveness induced by ANXA1 down-regulation was partially reversed by combined treatment with AKT agonist insulin-like growth factor-1 (IGF-1). Meanwhile, the inhibition of glioma cell proliferation and invasiveness induced by ANXA1 down-regulation was further enhanced by combined treatment with AKT inhibitor LY294002. In summary, these findings demonstrate that ANXA1 regulates proliferation, migration and invasion of glioma cells via PI3K/AKT signaling pathway.

HSP60 participates in the anti-glioma effects of curcumin.

The chaperone protein heat shock protein 60 (HSP60) is considered a tumor promoter in several types of primary human tumors, where it orchestrates a broad range of survival programs. Curcumin (CCM) is well-established to exhibit several anticancer properties with an excellent safety profile. Our previous study showed that CCM suppresses extracellular HSP60 expression, which is typically released by activated microglia, and acts as an inflammatory factor by binding to Toll-like receptor 4 (TLR-4) on the cell membrane. The present study assessed whether CCM exerted its anti-neuroglioma effects on U87 cells via inhibition of HSP60/TLR-4 signaling, similar to that in microglia. The results demonstrated that CCM significantly inhibited the viability and invasive capacity of neuroglioma U87 cells as evidenced by a Cell Counting Kit-8 assay. Western blotting and ELISA results showed that CCM decreased the expression of HSP60 and its transcriptional factor, heat shock factor 1, and reduced HSP60 release. Accordingly, TLR-4, as the target of HSP60, and its downstream signaling proteins myeloid differentiation primary response 88 (MYD88), NF-κB, inducible nitric oxide synthase and cytokines IL-1ß and IL-6 were downregulated by CCM. The expression levels of apoptotic factors associated with NF-κB activation, including TNF-α and caspase-3 were increased in U87 cells by CCM treatment, while p53 expression, a tumor suppressor, was shown to be decreased. Based on the results of the present study, CCM may exert its anti-tumor effects in U87 cells by inhibiting the HSP60/TLR-4/MYD88/NF-κB pathway and inducing tumor cell apoptosis. Thus, CCM may be used as a potential therapy for the clinical treatment of neuroglioma.

miR-9-3p inhibits glioma cell proliferation and apoptosis by directly targeting FOXG1.

There is accumulating evidence indicating that microRNA (miR)-9-3p expression is abnormal in patients with glioma; however, the role of miR-9-3p in glioma remains unclear. In the present study, reverse transcription-quantitative PCR and immunohistochemical assays were conducted to assess miR-9-3p and forkhead box G1 (FOXG1) expression, respectively. A luciferase reporter assay was performed to confirm the target of miR-9-3p. Moreover, cell counting kit-8 and flow cytometry assays were used to assess proliferation and apoptosis, respectively. The present study demonstrated that miR-9-3p is significantly downregulated, and FOXG1 is significantly upregulated, in patients with glioma. miR-9-3p overexpression inhibited proliferation and increased the apoptosis of both U87MG and TG-905 cells. In addition, FOXG1 was identified as a direct target of miR-9-3p, and FOXG1 silencing enhanced the inhibitory effect of miR-9-3p on proliferation and apoptosis in U87 MG and TG-905 cells. In conclusion, the present results suggest that miR-9-3p may suppress malignant biological properties by targeting FOXG1. Thus, miR-9-3p may serve as a diagnostic target and novel prognostic marker in patients with glioma.

SSADH Variants Increase Susceptibility of U87 Cells to Mitochondrial Pro-Oxidant Insult.

Succinate semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme, encoded by ALDH5A1, mainly involved in γ-aminobutyric acid (GABA) catabolism and energy supply of neuronal cells, possibly contributing to antioxidant defense. This study aimed to further investigate the antioxidant role of SSADH, and to verify if common SNPs of ALDH5A1 may affect SSADH activity, stability, and mitochondrial function. In this study, we used U87 glioblastoma cells as they represent a glial cell line. These cells were transiently transfected with a cDNA construct simultaneously harboring three SNPs encoding for a triple mutant (TM) SSADH protein (p.G36R/p.H180Y/p.P182L) or with wild type (WT) cDNA. SSADH activity and protein level were measured. Cell viability, lipid peroxidation, mitochondrial morphology, membrane potential (ΔΨ), and protein markers of mitochondrial stress were evaluated upon Paraquat treatment, in TM and WT transfected cells. TM transfected cells show lower SSADH protein content and activity, fragmented mitochondria, higher levels of peroxidized lipids, and altered ΔΨ than WT transfected cells. Upon Paraquat treatment, TM cells show higher cell death, lipid peroxidation, 4-HNE protein adducts, and lower ΔΨ, than WT transfected cells. These results reinforce the hypothesis that SSADH contributes to cellular antioxidant defense; furthermore, common SNPs may produce unstable, less active SSADH, which could per se negatively affect mitochondrial function and, under oxidative stress conditions, fail to protect mitochondria.

Effects of progesterone on the cell number of gliomaspheres derived from human glioblastoma cell lines.

AIMS: The malignancy of the Glioblastomas (GBM), the most frequent and aggressive brain tumors, have been associated with the presence of glioma stem cells (GSCs) which can form gliomaspheres (GS) in vitro. Progesterone (P) increases the proliferation, migration, and invasion of GBM cell lines through the interaction with its intracellular receptor (PR). However, it is unknown if the PR is expressed and the possible effects of P in the formation/differentiation of GS. MAIN METHODS: GS were grown from U251 and U87 cell lines by selective culture with serum-free neural stem cell medium. GSCs were identified by the detection of Sox2, Ki67, Nestin, CD133, and CD15 by immunofluorescence. Additionally, the relative expression of PROM1, NES, SOX2, OLIG2, EZH2, BMI1 and PR genes was evaluated by RT-qPCR. The GS were treated with P, and the number of cells was quantified. By RT-PCR the ßIII-TUB and GFAP differentiation genes were evaluated. KEY FINDINGS: GS were maintained until passage four. The expression of all GSCs markers was significantly higher in GS as compared with the basal culture of U251 and U87 cells. We demonstrated for the first time that PR is expressed in GS and this expression was higher as compared with the U251 and U87 cells in basal conditions. Also, we observed that P increased the number of cells derived primary gliomaspheres (GS1) from the U251 line, as well as the expression of the neuronal differentiation marker ßIII-TUB. SIGNIFICANCE: These results suggest the participation of P in the growth of GSCs.

Apoptosis induction effect of Apocynum venetum polyphenol on human U87 glioma cells via NF-κB pathway.

Aim: Apocynum venetum polyphenol (AVP) was used in in vitro glioma cells culture to prove the growth inhibitory effect of AVP on human U87 glioma cells via NF-κB pathway. Materials & methods: The MTT assay, DAPI morphology, quantitative PCR and western blot experiments were used for determination in vitro. Results & conclusion: AVP can also induce U87 cancer cells apoptosis illustrated by DAPI morphology. AVP could enhance the mRNA and protein expression of IκB-α, TNF-α, TRAIL, caspase-3 and caspase-9 in U87 cancer cells and reduce those of NF-κBp65, cIAP-1, cIAP-2, TGF-ß2, CyclinD1, VEGF and IL-8. After ammonium pyrrolidine dithiocarbamate (PDTC) treatment, the NF-κBp65 expression was reduced in U87 cells, and AVP could raise these effects. The results of HPLC indicate that AVP mainly contains six constituents. The growth inhibitory effects of AVP on U87 glioma cells are predominantly from these natural active constituents.

Tibolone Effects on Human Glioblastoma Cell Lines.

BACKGROUND: Ovarian steroid hormones are involved in modulating the growth of glioblastomas (the most common, aggressive, and lethal brain tumor) through the interaction with their intracellular receptors. Activation of sex hormone receptors is involved in glioblastomas progression. Tibolone (TIB) is a selective tissue estrogenic activity regulator widely prescribed to treat menopausal symptoms and to prevent bone lost. The effects of TIB on the growth of glioblastoma are unknown. AIM OF THE STUDY: To evaluate the effects of TIB on cell number, migration, and invasion of two derived human glioblastoma cell lines (U251 MG and U87), as well as the role of this steroid in estrogen and progesterone receptors activity and content. METHODS: U251-MG and U87 human glioblastoma cell lines were grown with different doses of TIB. The number of cells was determined and migration and invasion tests were carried out. Protein expression was performed by Western blot. RESULTS: We observed that TIB (10 nM) increased the number of cells by inducing proliferation with no effects on cell migration or invasion. The increase in cell proliferation induced by TIB was blocked by estrogen (ERs) or progesterone receptor (PRs) antagonists, ICI 182, 780 and RU 486, suggesting that these receptors mediate proliferating actions of TIB; TIB also modified the content of ERs and PRs by increasing ER-α, ER-ß, and PR-B, while decreased PR-A. CONCLUSION: Our results suggest that TIB increases cell number and proliferation of human glioblastoma cells through the regulation of ERs and PRs actions and content.

[Effects of FPR2 gene silencing on the proliferation, migration and invasion of human glioma U87 cells].

Objective: To investigate the effects of formyl peptide receptor 2 (FPR2) silencing on the proliferation, migration and invasion of human glioma U87 cells and its possible mechanisms. Methods: The expression of FPR2 was detected in normal glial cells, glioma cells, normal brain tissues and glioma tissues using Western blot and immunohistochemistry staining. A synthesized siRNA duplex was employed to inhibit FPR2 in human glioma cells (U87). The knockdown efficiency was evaluated by real-time reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot. MTT, transwell assays and flow cytometry analyses were used to determine the cell proliferation, migration, invasion and apoptotic rates of U87 cells, respectively. Mice xenograft experiments were used to observe the effect of FPR2 silencing on the tumorigenesis of U87 cells in vito. Western blot and enzyme-linked immunosorbent assays were performed to detect the expression and release of cell cycle and migration-related proteins. Results: The expression of FPR2 was significantly higher in glioma cell lines and glioma tissues than that in normal glial cells and brain tissues. Compared with blank control and negative control, FPR2 mRNA and protein levels in siRNA group were significantly downregulated. The cell proliferation inhibitory rates in FPR2 siRNA group were (23.1±5.1)%, (39.6±5.6)% and (44.4±6.7)% at 24 h, 48 h and 72 h, respectively, which were significantly increased than those in negative control group [(3.2±0.6)%, (5.7±0.8)% and (7.9±0.9)%, respectively; P<0.05]. The apoptosis rate in FPR2 siRNA group was (17.4±2.1)%, which was significantly elevated than that in the negative control group with (5.4±0.5)% and blank control group with (3.8±0.3)% (all P<0.05). In addition, the numbers of migrated cells were 108.7±9.5 in FPR2 siRNA group, which was significantly lower than that in blank control group 312.9±17.5 and negative control group (304.4±15.7, all P<0.05). Likewise, the numbers of invaded cells were 19.3±3.2 in FPR2 siRNA group, which was significantly lower than that in blank control group 106.9±8.5 and negative control group (102.4±7.4, all P<0.05). Moreover, the growth of FPR2 siRNA transfected U87 cells in vivo was remarkably decreased comparing with the negative group (P<0.05). Furthermore, the expression of cyclin D1 and VEGF in FPR2 silencing U87 cells was suppressed mainly through ß-catenin signaling pathway. Conclusions: FPR2 silencing by siRNA can inhibit the growth, migration and invasion ability, but promote the apoptosis of U87 cells. The possible mechanisms might be associated with the inhibitory expression of cyclin D1 and VEGF.

Lauryl Gallate Induces Apoptotic Cell Death through Caspase-dependent Pathway in U87 Human Glioblastoma Cells In Vitro.

BACKGROUND/AIM: The treatment of human glioma tumor is still an unmet medical need. Natural products are always promising resources for discovery of anticancer drugs. Lauryl gallate (LG) is one of the derivatives of gallic acid, widely present in plants, that has been shown to induce anticancer activities in many human cancer cell lines; however, it has not been studied in human glioma cell lines. Thus, the effects of LG on human glioblastoma U87 cells were investigated in the present in vitro study. MATERIALS AND METHODS: Cell morphology and viability were examined by phase-contrast microscopy. Annexin V/Propidium iodide (PI) double staining were performed and assayed by flow cytometry to confirm that viable cell number reduction was due to the induction of apoptosis. Furthermore, U87 cells were exposed to LG in various concentrations and were analyzed by caspase activity assay. To further confirm that LG induced apoptotic cell death, the expression of apoptosis-associated proteins in LG-treated U87 cells was tested by western blot. RESULTS: LG induced morphological changes and decreased viability in U87 cells. Annexin V/PI double staining revealed that LG induced apoptotic cell death in U87 cells in a dose-dependent manner. The increased activities of caspase-2, -3, -8 and -9 demonstrated that LG induced U87 cell apoptosis through a caspase-dependent pathway. In terms of molecular level, LG increased pro-apoptotic proteins Bax and Bak and decreased anti-apoptotic protein Bcl-2 in U87 cells. Furthermore, LG also suppressed the expression of p-Akt, Pak1, Hif-1α and Hif-2α, ß-catenin and Tcf-1 in U87 cells. CONCLUSION: These results suggest that LG induced apoptotic cell death via the caspase-dependent pathway in U87 cells.

Human corticotrophin releasing factor inhibits cell proliferation and promotes apoptosis through upregulation of tumor protein p53 in human glioma.

Corticotropin-releasing factor (CRF) and its receptors have been detected in numerous tumors and have an important role in tumorigenesis and proliferation. However, the role of these peptides has not been established in human glioma and malignant glioma cell lines. The present study evaluated for the first time, the expression of CRF receptor 1 (CRFR1) in 35 human glioma samples, 13 normal brain tissues and human U87 glioma cells using immunohistochemistry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Levels of CRFR1 were identified to be significantly increased in human glioma and U87 cells and higher levels of CRFR1 were observed in glioma tissues of higher grade. The biological functions of human CRF (hCRF) on U87 cells glioma cells were investigated by cell counting, a bromodeoxyuridine assay and flow cytometry. The U87 cells under hCRF treatment exhibited reduced proliferation, increased apoptosis and a cell cycle arrest in S and G2/M phase. The tumor protein p53 (p53) gene may participate in the activation of hCRF via CRFR1 in U87 cells, therefore p53 mRNA and protein were evaluated using RT-qPCR and western blot analysis. Finally, the present results suggest that hCRF inhibits proliferation and induces cell-cycle arrest and apoptosis in U87 cells via the CRFR1-mediated p53 signaling pathway. Therefore, the present study also suggests that hCRF may be used therapeutically, and CRFR1 may be a putative therapeutic target for human glioma.

Effects of FPR2 gene silencing on the proliferation, migration and invasion of human glioma U87 cells / 中华肿瘤杂志

Objective@#To investigate the effects of formyl peptide receptor 2 (FPR2) silencing on the proliferation, migration and invasion of human glioma U87 cells and its possible mechanisms.@*Methods@#The expression of FPR2 was detected in normal glial cells, glioma cells, normal brain tissues and glioma tissues using Western blot and immunohistochemistry staining. A synthesized siRNA duplex was employed to inhibit FPR2 in human glioma cells (U87). The knockdown efficiency was evaluated by real-time reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot. MTT, transwell assays and flow cytometry analyses were used to determine the cell proliferation, migration, invasion and apoptotic rates of U87 cells, respectively. Mice xenograft experiments were used to observe the effect of FPR2 silencing on the tumorigenesis of U87 cells in vito. Western blot and enzyme-linked immunosorbent assays were performed to detect the expression and release of cell cycle and migration-related proteins.@*Results@#The expression of FPR2 was significantly higher in glioma cell lines and glioma tissues than that in normal glial cells and brain tissues. Compared with blank control and negative control, FPR2 mRNA and protein levels in siRNA group were significantly downregulated. The cell proliferation inhibitory rates in FPR2 siRNA group were (23.1±5.1)%, (39.6±5.6)% and (44.4±6.7)% at 24 h, 48 h and 72 h, respectively, which were significantly increased than those in negative control group [(3.2±0.6)%, (5.7±0.8)% and (7.9±0.9)%, respectively; P<0.05]. The apoptosis rate in FPR2 siRNA group was (17.4±2.1)%, which was significantly elevated than that in the negative control group with (5.4±0.5)% and blank control group with (3.8±0.3)% (all P<0.05). In addition, the numbers of migrated cells were 108.7±9.5 in FPR2 siRNA group, which was significantly lower than that in blank control group 312.9±17.5 and negative control group (304.4±15.7, all P<0.05). Likewise, the numbers of invaded cells were 19.3±3.2 in FPR2 siRNA group, which was significantly lower than that in blank control group 106.9±8.5 and negative control group (102.4±7.4, all P<0.05). Moreover, the growth of FPR2 siRNA transfected U87 cells in vivo was remarkably decreased comparing with the negative group (P<0.05). Furthermore, the expression of cyclin D1 and VEGF in FPR2 silencing U87 cells was suppressed mainly through β-catenin signaling pathway.@*Conclusions@#FPR2 silencing by siRNA can inhibit the growth, migration and invasion ability, but promote the apoptosis of U87 cells. The possible mechanisms might be associated with the inhibitory expression of cyclin D1 and VEGF.

Inhibitory effect of resveratrol on epithelial to mesenchymal transition of glioma U87 cells / 吉林大学学报(医学版)

Objective: To investigate the regulatory effects of resveratrol (Res) on the epithelial to mesenchymal transition (EMT), migration and invasion abilities of the glioma U87 cells, and to clarify the inhibitory effect of Res on EMT of the cells. Methods: The glioma U87 cells were divided into control group (no treatment), EMT group (EMT was induced with TGF-β1) and Res treatment group (EMT was treated with Res). The expression levels of mesenchymal markers (N-cadherin, β-catenin, Vimentin), EMT-inducing transcription factors (Snail, Slug, Twistl), matrix metalloproteinase (MMP)-2 and MMP-9 in the glioma U87 cells were measured by Western blotting method. Scratch-healing assay and Trans well assay were performed to examine the cell migration and invasion abilities. Results: Compared with control group, the protein expression levels of mesenchymal markers (N-cadherin, β-catenin, Vimentin), EMT-inducing transcription factors (Snail, Slug, Twistl), and MMP-2 and MMP-9 in EMT group were significantly increased (P<0. 05). Compared with EMT group, the expression levels of above proteins in Res treatment group were markedly down-regulated (P<0. 05 or P <0.01); the effect in 40 μmol · L1 Res treatment group was more obvious than that in 20 μmol · L-1 Res treatment group. The migration and invasion rates of the U87 cells in EMT group were obviously elevated compared with control group (P<0. 01), and the migration and invasion rates of the glioma U87 cells in Res treatment group were significantly lower than those in EMT group (P<0. 01). Conclusion: Res can inhibit EMT of the glioma U87 cells and reduce the migration and invasion abilities of the cells.

Cytotoxic and Apoptogenic Effects of Cyanidin-3-Glucoside on the Glioblastoma Cell Line.

OBJECTIVE: Glioblastoma multiforme (GBM) is the most prevalent and aggressive primary cerebral tumor. The median survival time is 15 months despite maximum treatment because the tumor is resistant to most therapeutic modalities. Several studies have indicated chemopreventive and chemotherapeutic activity of cyanidin-3-glucoside (C3G) as an anthocyanin component. We aimed to illustrate the cytotoxic and apoptogenic effects of C3G in the U87 cell line (human GBM cell line). METHODS: Cytotoxic activity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium assay after treatment with C3G at different concentrations in the U87 cell line. Cisplatin was used as a positive control for 24 and 48 hours. The percentage of apoptotic cells was determined using an Annexin V/propidium iodide assay, and the expression of bax, bcl2, and p53 genes was assessed using real-time polymerase chain reaction. RESULTS: Treatment of U87 cells with 40 µg/mL of C3G resulted in 32% apoptotic cells after 24 hours. To further confirm that C3G treatment induced apoptosis in U87 cells, RNA expression of bax, bcl2, and p53 genes was investigated after treatment. Real-time polymerase chain reaction indicated that the expression of bax and p53 increased, whereas the expression of bcl2 decreased. CONCLUSIONS: C3G had an apoptogenic effect in the GBM cell line. New information regarding the therapeutic effects of C3G in GBM could ultimately lead to the production of new drugs.

Analysis of the cytotoxicity of hierarchical nanoporous graphenic carbon against human glioblastoma grade IV cells.

A newly produced hierarchical, nanoporous carbon (HNC) material is studied for the first time in a biological model. The material consists of uniform particles and is characterized by a mean diameter <150 nm, a high specific surface area of 1,000 m2/g, well-developed porosity, and high electrical conductivity. These unique properties and ability to transfer charge create a possibility of employing HNC as a moderator of tumor cell growth. As the charge of HNC may interfere with cell membranes by adhesion and by bonding with cell receptors, it may block the supply of nutrients. The interactions of HNC with the U87 cells can also lead to the excessive generation of reactive oxygen species (ROS) and activate apoptotic mechanisms in cancer cells. The investigation was performed using U87 human glioblastoma and PCS-201-010 normal fibroblast cell lines, where cell morphology and ultrastructure, viability, ROS production, type of cell death, mitochondrial transmembrane potential, and the expression of genes engaged in apoptosis pathways are studied. The results demonstrate that cytotoxicity of HNC particles increases with concentration from 5 to 100 µg/mL by activation of apoptosis through the mitochondrial pathway, without inducing necrosis. Our research indicates the potential applicability of HNC in cancer therapy.