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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.
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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.
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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.
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Humans , Antineoplastic Agents , Pharmacology , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Cell Shape , Dipeptides , Pharmacology , Glioma , Signal TransductionABSTRACT
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.
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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.
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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.
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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.
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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.
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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