HIF-1α facilitates glioma proliferation and invasion by activating pyroptosis signaling axis.

BACKGROUND: HIF-1α is thought to be a novel regulator which contributes to carcinogenesis. However, the mechanism underlying the effect of HIF-1α in gliomas remains largely unknown. METHODS: In the research, we demonstrate that HIF-lα mRNA and protein levels are elevated in glioma cells. The colony formation assays, transwell assays, and wound-healing assays showed that overexpression of HIF-1α promoted proliferation and invasion of glioma cells. RESULTS: Overexpression of HIF-lα also increased the expression of inflammatory factors related to pyrolysis (TNF-α, IL-10, and IL-1ß) and protein related to pyrolysis signal pathway (NLRP3, ASC, caspase-1, GSDMD, and GSDME). CONCLUSIONS: Therefore, we speculate that HIF-1α promotes the proliferation and invasion of glial cells by regulating pyrolysis pathway. These results might provide a novel strategy and target for treatment of glioma.

[Evodiamine Promotes Apoptosis of Glioma SHG-44 Cells and Its Mechanism].

Objective To explore the role of evodiamine in promoting the apoptosis of glioma SHG-44 cells and its mechanism.Methods The in vitro cultured glioma SHG-44 cells were divided into control group and evodiamine group(which was further divided into three subgroups according to the glycoside concentrations).Cell viability was determined by CCK-8 method,cells apoptosis rate by flow cytometry,and nucleus apoptosis by Hoechst 33258 nuclear staining.Cell morphological changes were observed by transmission electron microscope.Protein expressions of Cleaved Caspase-3 and Cleaved Caspase-9 were detected by Western blot analysis.Results Evodiamine significantly inhibited the proliferation of glioma SHG-44 cells.The apoptosis rate of Glioma cells increased in a dose-dependent manner as the evodiamine concentration increased.Evodiamine promoted the expressions of cleaved Caspase-3 and cleaved Caspase-9.Conclusion Evodiamine inhibits glioma cell proliferation by changing the expressions of cleaved Caspase-3 and cleaved Caspase-9.

STAT1 determines aggressiveness of glioblastoma both in vivo and in vitro through wnt/ß-catenin signalling pathway.

Glioblastoma is one of the most malignant tumors and causes the high mortality in cancer patients. Currently, there is no highly efficient therapy against glioblastoma. Therefore, searching for a new molecular target to anti-glioblastoma therapy is urgent and necessary. In this study, we elucidated the role of Signal transducer and activator of transcription 1 (STAT1) in proliferation, migration and apoptosis of glioblastoma cells. We found that STAT1 downregulation could weaken the aggressiveness of glioblastoma cells. Besides, the glioblastoma growth in vivo was also inhibited with the STAT1 downregulation by shRNA as well as by pharmacological stimulation withSTAT1inhibitors. This negative regulation of tumor growth was accompanied by the inhibition in epithelial-mesenchymal transition (EMT), whereas the STAT1 overexpression promoted EMT. Furthermore, the involvement of wnt/ß-catenin was observed in STAT1 downregulation mediated weakness in glioblastoma aggressiveness since application of activator wnt agonist 1 could counteract the inhibitory effect induced by STAT1 downregulation. Collectively, this work provided the evidence to support the conclusion that STAT1 can regulate the glioblastoma growth and migration, potentially serving as a therapeutic target against glioblastoma. SIGNIFICANCE OF THE STUDY: Glioblastoma is one of the most malignant tumors with very high mortality. Until now, there is no efficient therapy against glioblastoma. In this study, we found downregulation of Signal transducer and activator of transcription 1 (STAT1) could weaken the aggressiveness of glioblastoma cells through inhibition in epithelial-mesenchymal transition, mediated through wnt/ß-catenin signalling pathway. Thus, this work supported the regulatory role of STAT1 in glioblastoma growth and migration. This potentially serves as a new therapeutic target against glioblastoma.

Effect of microRNA-128 on cisplatin resistance of glioma SHG-44 cells by targeting JAG1.

This current study intends to investigate the effect of microRNA-128 (miR-128) on cisplatin (DDP) resistance in glioma SHG-44 cells. SHG-44/DDP cells were transfected with miR-128 antisense oligonucleotide (ASO) and assigned into blank, resistance, NC, anti-miR-128, miR-128 mimic, si-JAG1, and anti-miR-128 + si-JAG1 groups. qRT-PCR and Western blotting were employed for determining expression of miR-128, JAG1, Bax and Bcl-2. MTT assay, Giemsa staining, and flow cytometry were applied to detect DDP resistance, cellular morphology, and cell cycle, respectively. JAG1 is targeted and negatively regulated by miR-128. In in vitro experiments, compared with the blank group, the rest groups exhibited declined miR-28 and Bax expression, lowered cell inhibition rate and apoptosis rate, but elevated JAG1 and Bcl-2 expression with cells arrested in the S phase. Compared with the resistance group, the anti-miR-128 group showed decreasedBax expression along with a lowered cell inhibition rate and apoptosis rate, but increased JAG1 and Bcl-2 expression with reduced cells arrested in the S phase; while the miR-128 mimic group showed an opposite trend; the si-JAG1 group showed decreased Bcl-2 expression and reduced cells in the S phase. In in vivo experiments, compared with the resistance group, the tumor growth rate, tumor volume, and weight as well as JAG1 expression accelerated in the anti-miR-128 group; whereas the miR-128 mimic and si-JAG1 groups exhibited an opposite trend. Our findings demonstrated that miR-128 ASO transfection might down-regulate the expression of miR-128 in SHG-44/DDP and up-regulate the DDP resistance in SHG-44/DDP cells, providing a potential treatment target for glioma.

Mechanism of Oridonin-induced Apoptosis of Glioma SHG44 Cells / 中国医科大学学报

Objective To study the molecular mechanism of oridonin-induced apoptosis of ghoma SHG44 cells.Methods A growth curve was plotted using CCK-8 colorimetric method with different concentrations of oridonin (0,1.25,2.5,5,10,20,and 40 μmol/L)to observe its effect on the growth of SHG44 cells.Hoechst33258 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to examine the changes in cell morphology and flow cytometry was used to detect cell apoptosis.Western blotting was used to analyze the expression of apoptosis-related proteins (Caspase-3,cleaved Caspase-3,Bax,and Bcl-2)in SHG44 cells.Results SHG44 cell proliferation was significantly suppressed after 24 and 48 h Oridonin treatment,with a half-maximal inhibitory concentration of 7.865 and 4.74 μmol/L,respectively.Hoechst33258 and TUNEL staining showed changes in cell morphology such as shrinkage and nucleus fragmentation and morphogenesis,which are indicative of apoptosis.Western blotting analysis showed that oridonin inhibited the expression of Bcl-2 and activated the expression of Caspase-3 and Bax.Conclusion Oridonin can inhibit the proliferation and induce the apoptosis of SHG44 cells by regulating the expression of apoptosis-related proteins.

Schidandrin B kills tumor cells by initiating apoptosis in glioma SHG-44 cells.

OBJECTIVE: To observe the proliferation inhibition, cell cycle, and apoptosis of human glioma cell line SHG-44 treated with different concentration of Schidandrin B and explore the effect of Schidandrin B on glioma SHG-44 cells. METHODS: Glioma SHG-44 cells were treated with Schidandrin B (0, 50, 100 or 200 µg/mL) for 24, 48, 72 and 96 h, and cells were treated with vehicle as control. Viability of cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis; cell cycle was examined with flow cytometry assay; apoptosis was detected with annexin V assay. Bax and caspase-3 proteins expression were checked by Western blot. RESULTS: MTT analysis showed that viability of glioma SHG-44 cells significantly decreased after exposure to Schidandrin B for the indicated time. Flow cytometry revealed that the number of cells in the sub G1 phase was increased, however, the number of cells in G0/G1, S and G2/M phases were decreased after treatment with 50, 100 or 200 µg/mL Schidandrin B, compared with the respective control group. Annexin V analysis confirmed that apoptosis rates of the control group, 50, 100, and 200 µg/mL Schidandrin B group were 1.76%±0.47%, 13.98%±5.05%, 19.64%±5.53% and 63.28%±6.88% respectively, apoptotic rate increased significantly with dose-dependent manner, and apoptosis of cells were observed under the inverted microscope after 100 µg/mL Schidandrin B treatment. Bax and caspase-3 protein were highly expressed in Schidandrin B group compared with the control group. CONCLUSION: The apoptosis could be induced by different concentration of Schidandrin B on glioma SHG-44 cells, and the mechanism may be directly excited by Schidandrin B in glioma SHG-44 cells through activating mitochondrial pathway.

Establishment of human brain glioma cell SHG-44 subcutaneous model in nude mice and observation of growth characteristics / 中华行为医学与脑科学杂志

Objective To establish nude mouse model with human brain glioma SHG-44 and understand its growing characteristics in vivo.Methods The 4-week-old male mice were randomly divided into high density cell suspension inoculation group(n=10),low density cell suspension group(n=10),the tumor tissue mass vaccination group(n=10)and the blank control group with normal saline injection(n=10).The SHG-44 human brain glioma cell suspension was injected into the subcutaneous of the nude mice' s armpit.The tumor tissue was cut into 1 mm3 after tumor tissue growth and formation,and re-inoculated into the subcutaneous of the new nude mice' s armpits.Apart from daily observation,the long and short diameters of tumor were recorded every 5 days after graft.All the mice were sacrificed at 60 days and the tumor tissues were harvested for pathological examination.Results With a longer incubation period and slower growth rate,the tumor formation rate in high density cell suspension inoculation group and low density cell suspension group was lower compared with that in the tumor tissue mass vaccination group.Around day 20,grafted tumor appeared remarkably big((41.51 ±6.42)mm3) with good morphology.On day 50,the tumor derived from group the tumor tissue mass vaccination group((565.69± 123.36)mm3) showed a bigger size in comparison with that from high density cell suspension inoculation group((203.85±104.63) mm3) and low density cell suspension group ((153.02± 31.76) mm3,all P＜0.05).The tumors in three groups were well defined with a rich vascularity and no apparent invasion was observed.The positive expression of GFAP and S-100 in a large body of tumor cells was observed under optical microscope.Conclusion With a shorter incubation period and faster growth,the mouse tumor models established with tissue pieces from the tumor-bearing mice are much better compared to those with cell suspension.

DAPT suppresses the proliferation of human glioma cell line SHG-44.

OBJECTIVE: To explore the suppressing effect of γ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism. METHODS: The SHG-44 cell was treated by DAPT with different concentration. The proliferation of cells was detected by MTT assay; cell cycle and TSC of CD133(+) were determined by flow cytometry analysis technique; the key factor in Notch signaling pathway (Notch-1, Delta-1, Hes-1) was measured by reverse transcriptase-polymerase chain reaction and western blotting. RESULTS: DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05). And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner. DAPT increased the rate of cells in G0/G1 phase of SHG-44 cells, while it decreased the rate of cells in S phase. TSC of CD133(+) was significantly reduced after DAPT treated SHG-44 cells. The expression of protein and mRNA of Notch-1, Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses. CONCLUSIONS: DAPT can downregulate these key factor in Notch signaling pathway, reduce the TSC of CD133+ and inhibit the proliferation of SHG-44 cells.

DAPT suppresses the proliferation of human glioma cell line SHG-44

OBJECTIVE@#To explore the suppressing effect of γ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.@*METHODS@#The SHG-44 cell was treated by DAPT with different concentration. The proliferation of cells was detected by MTT assay; cell cycle and TSC of CD133(+) were determined by flow cytometry analysis technique; the key factor in Notch signaling pathway (Notch-1, Delta-1, Hes-1) was measured by reverse transcriptase-polymerase chain reaction and western blotting.@*RESULTS@#DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05). And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner. DAPT increased the rate of cells in G0/G1 phase of SHG-44 cells, while it decreased the rate of cells in S phase. TSC of CD133(+) was significantly reduced after DAPT treated SHG-44 cells. The expression of protein and mRNA of Notch-1, Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.@*CONCLUSIONS@#DAPT can downregulate these key factor in Notch signaling pathway, reduce the TSC of CD133+ and inhibit the proliferation of SHG-44 cells.

Radio-sensitization of SHG44 glioma cells by Aidi injection in vitro.

In this study, we aimed to explore the radio-sensitization of the SHG44 glioma cell line by Aidi injection and the possible mechanisms involved. The growth curve, cloning efficiency and divisional index of the SHG44 cell line were observed. The inhibition ratio was determined by MTT assay, the change in the cell cycle was analyzed by flow cytometry and the expression of cyclin B1 and Wee1 was detected by western blot analysis. The reproductive activity of the group treated with irradiation (IR) and Aidi injection was suppressed significantly, and the cloning efficiency and divisional index also declined. Aidi injection (15 µg/ml) induced G2/M phase arrest efficiently in the cell line after 48 h. The expression of cyclin B1 decreased in the group treated with IR and Aidi injection compared with either of those with IR or Aidi injection alone. The expression of Wee1 increased in the group treated with IR and Aidi injection compared with that in the groups treated with either IR or Aidi injection alone. In conclusion, Aidi injection is effective in radio-sensitization. The possible mechanisms involved may be associated with G2/M phase cell arrest, the downregulation of cyclin B1 and upregulation of Wee1 expression.

Establishment of SHG-44 cell line stably and highly expressing MCHR2 / 第二军医大学学报

Objective: To construct a eukaryotic expressing vector harboring human melanin-concentrating hormone receptor 2 (MCHR2) and to establish a SHG-44 cell line stably and highly expressing MCHR2. Methods: The full-length MCHR2 cDNA fragment was amplified from the human fetal brain cDNA library by PCR and was cloned into pcDNA3.1(+) to construct eukaryotic vector pcDNA3.1(+)/MCHR2; the latter was then transduced into SHG-44 cells by Lipofectamine™. After screening culture by G418, SHG-44 cells stably expressing MCHR2 were established. The transcription and expression of MCHR2 was identified by RT-PCR, Western blotting and immunofluorescence. Results: The full-length MCHR2 cDNA fragment was amplified and the eukaryotic expression vector pcDNA3.1(+)/MCHR2 was successfully constructed. The expression of MCHR2 was found positive by RT-PCR, Western blotting and immunofluorescence, indicating that the SHG-44 cell line stably and highly expressing MCHR2 was successfully established. Conclusion: The successful establishment of MCHR2-SHG-44 cell line provides a solid foundation for further study on MCHR2 function.

Role of endoplasmic reticulum stress in human brain gliomas cell apoptosis induced by proteasome inhibitor MG-132 / 肿瘤研究与临床

Objective To investigate the role of endoplasmic reticulum stress ( ERS) in human brain gliomas cell(SHG-44) apoptosis induced by proteasome inhibitor MG-132. Methods Human glioma cells were passage cultured. Glioma cells were treated by MG-132 with varying concentration(5, 10, 15 and 50 μmol/L) for 24 h. Compared with cells prior to the treatment (control group), cell viability was detected by MTT assay and the expression of ERS associated proteins GRP78 and apoptosis associated proteins Caspsse-12 was examined by PCR and Western-blotting. Results After MG-132 treatment for 24 h, SHG-44 cell viability was decreased significantly (39 %) (P <0.05), and continued to show a significant decline with the increasing concentration of MG-132 (P <0.05). RT-PCR results showed that the expression of ERS associated proteins GRP78 in SHG-44 cells were significantly increased after 5, 10, 15 and 50 μmol/L MG-132 treatment, and the expression of Caspase-12 was significantly increased after 5 μmol/L MG-132 treatment, slightly increased after 10 and 15 μmol/L treatment compared with that after 5 μmol/L treatment and reached the peak after 50 μmol/L treatment. Western-blotting results of GRP78 in SHG-44 cells were same as results of RT-PCR. Conclusion ERS may be involved in the apoptosis of gliomas cells induced by proteasome inhibitor MG-132.

Relationship between of COX-2 expression and radiosensitivity of the irradiated human glioma SHG-44 cells / 肿瘤

Objective: To investigate the changes of radiosensitivity and the expression of cyclooxygenase 2 (COX-2) in the surviving progeny from the irradiated human glioma SHG-44 cells and to provide a theoretical basis for application of COX-2 inhibitors in clinic. Methods: Radiosensitivity of mother SHG-44 cells and that of surviving progeny from the irradiated human glioma SHG-44 cells were studied by measuring the colony-forming rate. The mRNA transcription and protein expression of COX-2 were detected by RT-PCR and immunohistochemical staining, respectively. Results: The radiosensitivity was decreased and the mRNA and protein expressions of COX-2 increased in the surviving progeny of the irradiated SHG-44 cells compared with mother SHG-44 cells. The expression levels of COX-2 were negatively correlated with the radiosensitivity in the irradiated SHG-44 cells. Conclusion: Radiation induced upregulation of COX-2 expression and downregulation of the radiosensitivity in the the surviving progeny from the irradiated human glioma SHG-44 cells. The elevated expression of COX-2 may be one of the reasons for the radioresistance of irradiated SHG-44 cells.

Tamoxifen inhibits cell proliferation and currents of sodium channel in glioma cell line SHG-44 / 基础医学与临床

Objective To explore the effects of tamoxifen on the proliferation of SHG-44 glioma cells and the currents of sodium channel. Methods The cell activity was detected by MTT. The alteration of cellular proliferation and apoptosis were dectected by flow cytometer. Whole-cell patch clamp technique was used to record the Na currents.Results After treatment with tamoxifen,the cells began aging and shedding and cell counting decreased.The cells in G2/M cell cycle were more than that in control and the apoptosis ration increased. Tamoxifen significantly decreased the amplitude of Na currents of SHG-44 cell line.This blocking effect was dose-dependent and voltage-dependent.When the holding potential was 0 mV, 8 ?mol/L tamoxifen could block this currents by 69%.The half inhibition concentration(IC50) was 5.54 ?mol/L. Conclusion Tamoxifen can inhibit SHG-44 glioma cells proliferation.The inhibion of sodium channel may be one of its mechanisms.

Inhibitory effect of particle ~(125)I on proliferation of intracranial glioma cells in rats / 吉林大学学报(医学版)

Objective To investigate the possibility and mechanism of ~ 125 I in treatment of glioma. Methods SHG-44 glioma cells were cultivated in vitro, the inhibitory effect of ~ 125 I on SHG-44 cell proliferation was determined by MTT method. The stereotactic method was used to establish the rat intracranial glioma model. The MRI was performed at 1st week after implantation and ~ 125 I was implanted in the glioma area, the MRI was performed to measure the diameter of tumor 2 weeks after implantation. The rats were killed after 2 weeks ,PCNA gene experession was determined with immunohistological method both in control and experiment group.Results one week after implantation the glioma grew,the results of MTT method showed the growth of the SHG-44 was inhibited, ~ 125 I inhibited the expression of PCNA gene and enlonged the rat survival period. Conclusion ~ 125 I can inhibit the growth of glioma ,the mechanism may be concerned with its inhibitory effect on PCNA gene expression.

STUDY ON GENE EXPRESSION AND REGULATION OF AROMATASE IN MALIGNANT GLIOMA CELL LINE SHG-44 / 解剖学报

Objective To explore the gene expression of aromatase and estrogen receptor (ER-?) in malignant glioma cell line SHG-44. Methods Cell culture, immunocytochemistry, in situ hybridization and RT-PCR techniques were used. Results Aromatase and estrogen receptor gene expressions were detected in SHG-44 cells.The aromatase gene in these cells was expressed by means of the multi promoters (1