Synergistic Antitumor Effects of Paclitaxel Combined with PDE5 Inhibitor Avanafil on Glioma U373 Cells

beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38) can effectively galactosylate the GlcNAcbeta1-->6Man arm of the highly branched N-glycans that are characteristic of glioma. Previously, we have reported that the expression of GalT V is increased in the process of glioma. However, currently little is known about the role of GalT V in this process. In this study, the ectopic expression of GalT V could promote the invasion and survival of glioma cells and transformed astrocytes. Furthermore, decreasing the expression of GalT V in glioma cells promoted apoptosis, inhibited the invasion and migration and the ability of tumor formation in vivo, and reduced the activation of AKT. In addition, the activity of GalT V promoter could be induced by epidermal growth factor, dominant active Ras, ERK1, JNK1, and constitutively active AKT. Taken together, our results suggest that GalT V functioned as a novel glioma growth activator and might represent a novel target in glioma therapy.

This study analyzed the influence of p120-catenin (catenin [cadherin-associated protein], delta 1 [CTNND1]) on the malignant characteristics of glioma and elucidated the potential underlying mechanism. The p120 expression level was assessed in the brain tissues of 42 glioma patients and 10 patients with epilepsy by using the immunohistochemical method. Meanwhile, quantitative polymerase chain reaction (QT-PCR) technology was employed to assess the expression of p120 in the brain tissues of 71 glioma patients and 13 epilepsy patients. LN229, U251, and U87 glioma cells were used for in vitro analysis and categorized into four treatment groups : siRNA-blank control (BC) group (no RNA sequence was transfected), siRNA-negative control (NC) group (transfected control RNA sequences with no effect), and siRNA-1 and siRNA-2 groups (two p120-specific interfering RNA transfection). p120 expression in these treatment groups was quantified by western blotting assay. The migratory and invasive capabilities of glioma cells were studied by wound healing assay and Transwell invasion assay, respectively, under different treatment conditions. MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) assay and cell cycle and apoptosis assay were used to determine glioma cell proliferation and apoptosis, respectively. Enzymelabeled assay was performed to measure intracellular calcium ion concentration. Immunofluorescence assay was performed for determining microtubule formation and glioma cell distribution. Brain tissues of the glioma group exhibited a remarkable increase in the p120 expression level as compared to brain tissues of the nontumor group (p<0.05). Furthermore, a strong positive correlation was noted between the malignancy degree in glioma brain tissues and p120 expression in Western blotting (r=0.906, p<0.0001) and QT-PCR (F=830.6, p<0.01). Compared to the BC and NC groups, the siRNA transfection groups showed a significant suppression in p120 expression in glioma cells (p<0.05), with a marked attenuation in the invasive, migratory, and proliferative capabilities of glioma cells as well as an increase in apoptotic potential (p<0.05). Enzyme-labeled assay showed a remarkable increase in calcium concentration in glioma cells after siRNA treatment. Immunofluorescence assay revealed that the microtubule formation ability of glioma cells reduced after siRNA treatment. p120 has a pivotal involvement in facilitating glioma cell invasion and proliferation by potentially modulating these processes through its involvement in microtubule formation and regulation of intracellular calcium ion levels.

ObjectiveRho-associated coiled-coil kinase 2 (ROCK2) is an attractive therapeutic target because it is overexpressed in many malignancies, including glioma. Therefore, we designed the current study to determine whether the downregulation of ROCK2 would sensitize the cytotoxic effect of temozolomide (TMZ) in U251 cells.MethodsGlycol-polyethyleneimine (PEG-PEI) was used to deliver siROCK2 to U251 cells, and the physical characteristics of the PEG-PEI/siROCK2 complex (referred to as the siROCK2 complex) were investigated. The transfection efficiency and cell uptake were determined by flow cytometry (FCM) and confocal laser microscopy (CLSM), respectively. U251 cells were then treated with 100 μM TMZ, siROCK2 complexes or their combination. The apoptosis rate and cell migration were measured by FCM and wound-healing assay, respectively. The levels of Bax, Bcl-2, cleaved caspase-3, MMP-2, and MMP-9 were detected to analyze the degrees of apoptosis and migration.ResultsOur results revealed that the characteristics of the siROCK2 complexes depended closely on the N/P ratios. PEG-PEI served as a good vector for siROCK2 and exhibited low cytotoxicity toward U251 cells. The CLSM assay showed that the siROCK2 complexes were successfully uptaken and that both the protein and mRNA levels of ROCK2 were significantly suppressed. Furthermore, the combination treatment induced a higher apoptosis rate and markedly increased the gap distance of U251 cells in the wound-healing assay. Levels of the proapoptotic proteins Bax and cleaved caspase-3 were significantly increased, whereas levels of the antiapoptotic protein Bcl-2 and the migration-related proteins MMP-2 and MMP-9 were significantly reduced by the combination treatment compared with either treatment alone.ConclusionsIn conclusion, our results demonstrate that the combination of TMZ and siROCK2 effectively induces apoptosis and inhibits the migration of U251 cells. Therefore, the combination of TMZ and siROCK2 complex is a potential therapeutic approach for human glioma.

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

Nectin-like Molecule 1 Inhibits the Migration and Invasion of U251 Glioma Cells by Regulating the Expression of An Extracellular Matrix Protein Osteopontin

IntroductionChlorogenic acid (CGA) is a type of polyphenolic substance that is widely extracted from many traditional Chinese medicines (eg, Lonicera japonica Thunb, Eucommia ulmoides Oliver) and exhibits a wide range of anti-tumor effects. However, the potential molecular mechanisms of CGA in glioma U373 cells remain unclear.MethodsNetwork pharmacology analysis was used to explore the potential therapeutic targets of CGA in glioma. Human glioma U373 cells were treated with different concentrations of CGA for 24 h. CCK-8 assays were used to detect the inhibitory rate of cell growth. Annexin V-FITC/PI staining and Hoechst 33342 staining were used to detect apoptosis. PI staining was used to investigate cell-cycle progression. Wound healing assays and transwell assays were used to detect the cell migration and invasion, respectively. Western blotting and immunohistochemistry were used to measure protein levels in vitro and in vivo.ResultsThe proliferation of U373 cells was significantly inhibited by CGA in a dose- and time-dependent manner. CGA significantly arrested the cell cycle of U373 cells in the G2/M phase and induced apoptosis. Moreover, CGA significantly suppressed the migration and invasion of U373 cells. Additionally, we found that CGA inhibited the growth of U373 cells in vivo. Furthermore, network pharmacology analysis suggested that the anti-tumor effects of CGA on U373 cells were associated with the down-regulation of the SRC/MAPKs signaling pathway.DiscussionThe present study indicated that CGA had anti-glioma effects on U373 cells by down-regulating SRC/MAPKs signal pathway.

Thiazine, a 6-membered distinctive heterocyclic motif with sulfur and nitrogen atoms, is one of the heterocyclic compounds that functions as a core scaffold in a number of medicinally significant molecules. Small thiazine-based compounds may operate simultaneously on numerous therapeutic targets and by employing a variety of methods to halt the development, proliferation, and vasculature of cancer cells. We have, herein, reported a series of substituted 1,4 benzothiazines as potential anticancer agents for the treatment of lung cancer. In order to synthesize 2,3-disubstituted-1,4 benzothiazines in good yield, a facile green approach for the oxidative cycloaddition of 2-amino benzenethiol and 1,3-dicarbonyls employing a catalytic amount of ceric ammonium nitrate has been devised. All the molecules have been characterized by spectral analysis and tested for anticancer activity against the A-549 lung cancer cell line using various functional assays. Further in silico screening of compound 3c against six crucial inflammatory molecular targets, such as Il1-α (PDB ID: 5UC6), Il1- β (PDB ID: 6Y8I), Il6 (PDB ID: 1P9M), vimentin (PDB ID: 3TRT), COX-2 (PDB ID: 5KIR), Il8 (PDB ID: 5D14), and TNF-α (PDB ID: 2AZ5), was done using AutoDock tool. Among the synthesized compounds, propyl 3-methyl-3,4-dihydro-2H-benzo[b][1,4]thiazine-2- carboxylate (3c) was found to be most active based on cell viability assays using A-549 lung cancer cell line and was found to effectively downregulate various pro-inflammatory genes, like Il1-α, Il1-β, Il6, vimentin, COX-2, Il8, and TNF-α in vitro. The ability of the molecule to effectively suppress the proliferation and migration of lung cancer cells in vitro has been further demonstrated by the colony formation unit assay and wound healing assay. Molecular docking analysis showed the maximal binding affinity (- 7.54 kcal/mol) to be exhibited by compound 3c against IL8. A green unconventional route for the synthesis of 2,3-disubstituted-1,4 benzothiazines has been developed. All the molecules were screened for their activity against lung cancer and the data suggested that the presence of an additional unbranched alkyl group attached to the thiazine ring increased their activity. Also, in vitro and in silico modeling confirmed the anti-cancer efficiency of compound 3c, encouraging the exploration of such small molecules against cancer.

Background Glioma is one of the most malignant tumors, which leads to high mortality in cancer patients. At present, there is no effective therapy for glioma. Therefore, it is urgent and necessary to find new molecular targets for anti-glioma therapy. Objective The present study aimed to investigate the role of signal transducer and activator of transcription 1 (STAT1) in the development and progression of human glioma and related mechanisms. Methods According to the instructions of Lipofectamine TM2000 transfection reagent, we transiently transfected the plasmid pcDNA3.1-STAT1 into glioma U251 cells. Then STAT1 expression in glioma U251 and LN382 cells was detected by Western blot. MTT was performed to assay the proliferative activity of U251 cells after STAT1 transduction, flow cytometry was used to detect cell cycle and apoptosis indicators, cell migration indicator was determined by Wound healing, and Western blot was used for detecting the expression level and change trend of p53, p21, bcl-2, Caspase-8, Cyclin A and Cyclin E in transfected cells. Results Overexpressed STAT1 significantly inhibited U251 cell proliferation and promoted U251 cell apoptosis. Meanwhile, high expression of STAT1 can increase the expression of p53, p21, and Caspase-8 while inhibiting the expression of bcl-2, Cyclin A, and Cyclin E. Conclusion Highly expressed STAT1 inhibits the proliferative activity of human glioma U251 cells and can promote tumor cell apoptosis and block cell cycle progression while regulating the expression of various signal transduction molecules. Thus, STAT1 has a critical function in the development and progression of glioma and is a novel target for glioma therapy.

Arenobufagin, isolated from toad venom, inhibited epithelial-to-mesenchymal transition and suppressed migration and invasion of lung cancer cells via targeting IKKβ/NFκB signal cascade

ABSTRACTBackground and AimGlioma is a highly aggressive malignancy of the central nervous system with a poor prognosis. The nucleoside‐binding oligomerization domain‐containing protein 1 (NOD1)/receptor‐interacting protein 2 (RIP2) pathway is involved in various inflammatory responses and is closely associated with microglial polarization. Microglial M2 polarization alters the glioma microenvironment, promoting tumor growth. This study aimed to investigate the role of the NOD1/RIP2 pathway in glioma progression.MethodsWe explored the mechanism of NOD1/RIP2 in glioma progression through bioinformatics analysis, clinical sample evaluation, and in vivo and in vitro experiments. Bioinformatics analysis was conducted to assess NOD1 expression in glioma tissues. Multiparameter MRI and histologic analyses were performed on human tissues, and the correlation between the relative apparent diffusion coefficient (rADC) and NOD1 expression was analyzed. C6 and U251 glioma cells were treated with ML130, a NOD1 inhibitor, and assessed using 5‐ethynyl‐2′‐deoxyuridine (EdU), plate cloning, Transwell, and wound healing assays. Key molecules of the NOD1/RIP2 pathway were examined through immunofluorescence and Western blotting. Quantitative real‐time polymerase chain reaction (qRT‐PCR) was used to detect Arg1 and CD206 expression in BV2 mouse microglia cultured with C6‐derived conditioned medium (CM). The changes in glioma cell biologic behavior were assessed using C6/BV2‐derived CM through EdU, wound healing, and Transwell assays. Finally, the role and mechanism of NOD1 in glioma growth were evaluated using a rat glioma model.ResultsBioinformatics analysis showed that NOD1 was highly expressed in glioma tissues and strongly correlated with glioma grade. Human brain glioma samples exhibited increased expression of NOD1, RIP2, Iba1, interleukin‐1β, and CD206, with higher expression in high‐grade gliomas compared to low‐grade gliomas. The NOD1 expression was negatively correlated with rADC values. Treatment with ML130 inhibited glioma cell proliferation, migration, and invasion while reducing NOD1 and RIP2 expression. The expression levels of Arg1 and CD206 in BV2 cells cultured with C6‐derived CM decreased in both ML130 and GSK‐583 groups, while the expression levels increased in the ie‐DAP group. Compared with the control group, the expressions of Arg1 and CD206 in microglia in the GSK‐583 + ML130 and GSK‐583 + ie‐DAP groups were inhibited, and the inhibitory effect in the GSK‐583 + ML130 group was more significant. Furthermore, after culturing with C6/BV2 derived CM, the activity of C6 cells was significantly inhibited in the ML130 group, while the activity increased in the ie‐DAP group. The activity of C6 cells in the GSK‐583 + ML130 and GSK‐583 + ie‐DAP groups was significantly decreased, and this effect was more pronounced in the GSK‐583 + ML130 group. ML130‐treated glioma‐bearing rats exhibited reduced tumor growth, suppressed NOD1/RIP2 pathway activation, and inhibited microglial M2 polarization. However, the results observed in the ie‐DAP group were opposite.ConclusionsNOD1 is an effective predictor of preoperative glioma grade and prognosis. It facilitates glioma progression by promoting microglial M2 polarization through the NOD1/RIP2 pathway.

Objective To investigate the effect of galangin on proliferation and apoptosis of glioma cells in vitro. Methods (1) The glioma cells U87 and U251were divided into blank control group, DMSO group, 100, 200, 300 and 400 μmol/L galangin treatment groups. MTT was used to study the effects of drugs on the proliferation of U251 and U87 cells. (2) Hoechest staining was used to observe cell apoptosis in the presence of different concentrations of galangin (0, 100 and 200 μmol/L). (3) Flow cytometry was employed to detect the apoptosis of U251 and U87 cells in the presence of different concentrations of galangin (100 and 200 μmol/L). (4) Western blotting was employed to detect the expressions of apoptosis-related protein β-Catenin, B-cell lymphoma-2 (Bcl-2), Bcl-2 related protein gene (Bax), cleaved-caspase-3, cleaved-caspase-9 and poly (ADP-ribose) polymerase (PARP) in the presence of different concentrations of galangin. Results (1) The proliferation of U251 and U87 cells was obviously inhibited after 100, 200, 300 and 400 μmol/L galangin treatments, and dose-effect relation was noted. The concentrations of galangin at half rate of inhibition (IC50) were 281, 321, 276 and 229 μmol/L in U251 cells, and 289.4, 261.1, 247.4 and 225.3 μmol/L in the U87 cells after 100, 200, 300 and 400 μmol/L galangin treatments for 24 h. (2) Under the action of galangin, corresponding increase in apoptosis rates of U251 and U87 cells was noted following the increase of galangin concentrations (0, 100 and 200 μmol/L), with significant differences (P<0.05). (3) The detection of cell apoptosis by flow cytometry found similar changes. (4) Western blotting results indicated that galangin at the concentration of 0, 100 and 200 μmol/L could significantly decrease the expressions of apoptosis-related protein β-Catenin and Bcl-2, and increase the Bax, cleaved-caspase-3 and cleaved-caspase-9, and cleaved-PARP expressions; significant differences were noted between each two concentrations (P<0.05). Conclusion Galangin can inhibit proliferation of glioma cells U251 and U87, and induce mitochondrial pathway of apoptosis via Wnt/β-Catenin signaling. Key words: Glioma; Wnt/β-Catenin signaling; Galangin; Apoptosis; Mitochondrial pathway

Objective To study the effect of survivin on proliferation and invasion of glioma cells treated by bevacizumab (Bev). Methods The human spongioblastoma cell line U87 was routinely cultured in vitro; the growth rates of U87 cells after 0, 2, 4, 6, 8 and 10 mg/mL Bev treatment for 2, 4 and 6 d were determined by MTT assay. The specific shRNA vectors (pRNAT, pRNAT-survivin shRNA, pRNAT-NS si) were transfected into U87 cells; U87, U87/sur(-), U87/pCtrl and U87/NS si cells were cultured for one d, and then, they were divided into 6 groups: U87 cells without Bev treatment, U87/sur(-) cells without Bev treatment, U87 cells with Bev treatment, U87/sur(-) cells with Bev treatment, U87/pCtrl cells with Bev treatment and U87/NS si cells with Bev treatment; 6 mg/mL Bev was given to each Bev treatment group. The cell invasion capacity was determined by Traswell assay; the cell movement and migratory capacities were detected by wound-healing assay, and the cell proliferation was determined by MTT assay. Results The cells treated by Bev at concentrations of 0, 2, 4, or 6 mg/mL exhibited similar viability (P>0.05), while the cells treated by Bev at concentrations of 8 or 10 mg/mL showed significantly decreased viability as compared with cells treated by Bev at concentration of 0 mg/mL (P<0.05). As compared with U87 cells with Bev treatment, U87/sur(-) cells with Bev treatment had significantly decreased viability (P<0.05); U87/sur(-) cells with Bev treatment had significantly decreased viability as compared with U87/sur(-) cells without Bev treatment (P<0.05); as compared with that in the U87 cells without Bevb treatment, the migration distance in the U87 cells with Bev treatment was significantly longer (P<0.05). As compared with that in the U87 cells without Bev treatment, the migration distance in the U87/sur(-) cells with Bev treatment was significantly shorter (P<0.05); as compared with that in the U87 cells without Bev treatment, the number of cells invading into the lower chamber in the U87 cells with Bev treatment was significantly larger (P<0.05); as compared with that in the U87 cells without Bev treatment, the the number of cells invading into the lower chamber in the U87/sur(-) cells with Bev treatment was significantly smaller (P<0.05). Conclusion Down-regulation of survivin could suppress glioma cells invasion induced by Bev treatment, and synergistic effect is observed between down-regulation of survivin and Bev treatment in suppressing the viability of glioma cells. Key words: Glioma; Bevacizumab; Survivin; Proliferation; Invasion

All-trans retinoic acid (ATRA) is a derivative of vitamin A that can induce differentiation and apoptosis, as well as inhibit proliferation, in glioma cells. However, the effect of ATRA on the migration and invasiveness of glioma remains poorly understood. In addition, although it is universally accepted that ATRA can induce apoptosis and inhibit proliferation in glioma cells, the association between the concentration and effects of ATRA remain unclear. Therefore, the present study investigated the effects of ATRA treatment on the migration, invasion, apoptosis and proliferation of glioma cells. The U87 and SHG44 glioma cell lines were treated with various concentrations of ATRA, consisting of 0, 5, 10, 20 and 40 µmol/l. A scratch wound healing assay and a Matrigel invasion assay were used to investigate cell migration and invasion, respectively. Flow cytometry was performed to investigate apoptosis and cell cycle distribution. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to investigate the expression of matrix metalloproteinase (MMP)-2 and -9 in each cell treatment group. Following treatment with ATRA, the migration, invasion and proliferation of the glioma cells were significantly inhibited, and the apoptosis rate was significantly increased compared with that of the blank control group. Furthermore, a dose-effect association was identified between each effects and ATRA treatment. The mRNA and protein expression of MMP-2 in U87 glioma cells was not significantly affected following treatment with low concentrations of ATRA, consisting of 5 and 10 µmol/l ATRA, compared with the expression in the control group (P>0.05). However, treatment with high concentrations of ATRA, consisting of 20 and 40 µmol/l ATRA, significantly downregulated the expression levels of MMP-2 in U87 cells. In contrast to U87 cells, the administration of ATRA treatment to SHG44 glioma cells resulted in a significant and dose-dependent downregulation in MMP-2 mRNA and protein expression (P<0.01). In addition, significant downregulation of MMP-9 expression was identified in the two glioma cell lines (P<0.01). The results of the present study indicate that treatment with ATRA may inhibit migration, invasion and proliferation, and promote apoptosis in glioma cells. Furthermore, the current study indicates that the inhibition of glioma cell invasion by ATRA may be partially associated with its effect ability to downregulate MMP expression.

Glioma is one of the malignancy brain tumors, which deeply threaten the health of patients. Although the traditional therapies for glioma have improved, the outcome is still far from satisfactory. Bone Marrow Stromal Cells (BMSC)-based therapy provided novel insight in the treatment for glioma. However, the detailed molecular mechanism is still not clear. The aim of present study is to discover the novel factor in BMSC-based therapy for glioma. The cell proliferation and apoptosis were identified by using CCK-8 and flow cytometry. The invasion of glioma cells was examined by using Transwell assay and wound-healing assay respectively. qRT-PCR was used to examine the expression of miR-506. Western blot was used to examine the protein levels of CD63, TSG101, NUR77 and CXCR4. Our data suggested that BMSC-derived exosome inhibited the proliferation and contributed to apoptosis of human U87 cells after culturing with miR-506 mimic. Overexpression of miR-506 in BMSC-derived exosome inhibited the invasion of human glioma U87 cells, while these effects were deeply suppressed in the presence GW4869. Our present study demonstrated that BMSC inhibited the growth and metastasis of human glioma U87 cells through delivering exosomal miR-506, and provided the evidences to develop the BMSC-based therapy for glioma.

MicroRNAs (miRNAs) are novel small noncoding RNA molecules that regulate gene expression at the post transcriptional level. Compelling evidence shows that there are causative links between miRNAs deregulation and cancer development and progression. This study aims to explore the functions of miR-16-1 on proliferation, apoptosis, motility, and invasion of glioma cells. Quantitative real-time PCR (qRT-PCR) was performed to detect the expression of miR-16-1 in normal brain tissues and two glioma cell lines, including U251 and U87. CCK-8, Annexin V/FITC (fluorescein isothiocyanate), wound healing, and transwell assays were used to evaluate the functions of miR-16-1 that involves cell proliferation, apoptosis, motility, and invasion. In addition, we conducted qRT-PCR to examine mRNA expression levels of Zyxin, one of putative target genes of miR-16-1, in U251 glioma cells after transfecting with miR-16-1 mimics. As a result, miR-16-1 expression level was lower in U251 and U87 cells than normal brain tissues. After miR-16-1 was upregulated in U251 cells, cellular proliferation was notably attenuated but cell apoptosis was not significantly increased. Moreover, overexpression of miR-16-1 attenuated migration and invasion of glioma cells, and U251 cells transfected with miR-16-1 showed significantly lower endogenous mRNA levels of Zyxin than those transfected with nonspecific control miRNA or mock (P < 0.05). In summary, we demonstrated that miR-16-1 expression was markedly decreased in human glioma cell lines, and for the first time, described the roles of miR-16-1 in cellular proliferation, migration, and invasion abilities of high-invasive glioma cells, and suggested that Zyxin may be one of putative target genes of miR-16-1.