ABSTRACT
Aim To study the protection and possible mechanism of 5-hydroxy-1 H-indazole against 1-methyl-4-phenylpyridinium iodide ( MPP+)-induced SH-SY5 Y cell apoptosis. Methods An apoptotic model was es-tablished in human neuroblastoma SH-SY5 Y by MPP+in vitro. MTT analysis was used to evaluate the protec-tive effect of 5-hydroxy-1H-indazole. Immunochemistry and Hoechst33258 nuclear staining were used to ob-serve the neuroprotection and anti-apoptosis of 5-hy-droxy-1H-indazole. Western blot was used to detect the levels of P-tau ( Ser396 ) closely related to neuronal apoptosis and its upstream kinases:P-GSK-3β and CDK5 . Results MPP+ induced activation of GSK-3β, increase of activity of CDK5 , tau hyperphosphory-lation and neuronal cell apoptosis. However,5-hydrox-y-1 H-indazole reduced the activities of GSK-3β and CDK5,then decreased the level of tau hyperphosphory-lation and inhibited MPP+-induced SH-SY5 Y cells ap-optosis. Conclusions 5-hydroxy-1H-indazole could attenuate MPP+-induced SH-SY5 Y neuronal cell apop-tosis. Possible mechanism is that 5-hydroxy-1H-in-dazole inhibits GSK-3βand CDK5 two signal transduc-tion pathways to lower the level of tau phosphorylation, then plays a role of neuroprotection.
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<p><b>OBJECTIVE</b>To investigate the effect of RbAp48 knockdown on the migration and invasion of human cervical cancer cells and explore the mechanism.</p><p><b>METHODS</b>A small interference RNA (siRNA) was used to knock down the expression of RbAp48 in MS751 cells. The changes in cell migration and invasion were evaluated using wound healing assay and Transwell assay, respectively, and the expressions of RbAp48, vimentin, N-cadherin, E-cadherin, Snail, Twist, MMP-2 and TIMP-2 were determined with Western blotting.</p><p><b>RESULTS</b>After siRNA-mediated RbAp48 knockdown, MS751 cells showed a significantly reduced expression of RbAp48 with significantly suppressed cell migration and invasion (P<0.01). RbAp48 knockdown induced obvious down-regulation of the expressions of interstitial cell phenotype proteins vimentin, N-cadherin, and MMP-2 and up-regulation of epithelial cell phenotype proteins E-cadherin and TIMP-2, suggesting the inhibition of epithelial- mesenchymal transition of the cells. The expressions of Snail and Twist were significantly down-regulated in the cells following RbAp48 knockdown.</p><p><b>CONCLUSION</b>Knockdown of RbAp48 can significantly inhibit epithelial-mesenchymal transition and suppress the migration and invasion of cervical cancer cell line MS751, the mechanism of which may involve the down-regulation of Snail and Twist expressions.</p>
Subject(s)
Female , Humans , Antigens, CD , Metabolism , Cadherins , Metabolism , Cell Line, Tumor , Cell Movement , Down-Regulation , Epithelial-Mesenchymal Transition , Gene Knockdown Techniques , Matrix Metalloproteinase 2 , Metabolism , Neoplasm Invasiveness , Nuclear Proteins , Metabolism , RNA, Small Interfering , Retinoblastoma-Binding Protein 4 , Genetics , Snail Family Transcription Factors , Tissue Inhibitor of Metalloproteinase-2 , Metabolism , Transcription Factors , Metabolism , Twist-Related Protein 1 , Metabolism , Up-Regulation , Uterine Cervical Neoplasms , Pathology , Vimentin , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of silencing Bmi-1 expression in reversing cisplatin resistance in human lung cancer cells and explore the possible mechanisms.</p><p><b>METHODS</b>Cisplatin-resistant A549/DDP cells with small interference RNA (siRNA)-mediated Bmi-1 expression silencing were examined for cisplatin sensitivity using MTT assay and alterations in cell cycle distribution and apoptosis with flow cytometry, and the changes in cell senescence was assessed using β-galactosidase staining. The protein expressions of Bmi-1, P14(ARF), P16(INK4a), P53, P21, Rb and ubi-H2AK119 in the cells were determined with Western blotting.</p><p><b>RESULTS</b>A549/DDP cells showed significantly higher Bmi-1 expression than A549 cells. After siRNA-mediated Bmi-1 silencing, A549/DDP cells showed significantly enhanced cisplatin sensitivity with an increased IC50 from 40.3±4.1 µmol/L to 18.3±2.8 µmol/L (P<0.01) and increased cell percentage in G0/G1 phase from (48.9±2.3)% to (78.7±7.6)% (P<0.01). Silencing Bmi-1 did not cause significant changes in the cell apoptosis rate but induced obvious senescence phenotype in A549/DDP cells with down-regulated expression of ubi-H2AK119 and up-regulated expressions of P14(ARF), P16(INK4a), P53, P21 and Rb.</p><p><b>CONCLUSION</b>Silencing Bmi-1 by RNA interference can induce cell senescence and resensitize A549/DDP cells to cisplatin possibly by regulating INK4a/ARF/Rb senescence pathway.</p>
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Apoptosis , Cell Cycle , Cell Line, Tumor , Cisplatin , Pharmacology , Drug Resistance, Neoplasm , Gene Silencing , Lung Neoplasms , Genetics , Polycomb Repressive Complex 1 , Genetics , RNA, Small InterferingABSTRACT
Aim To study the protective effect of 6-Hydroxy-1 H-indazole via inhibition of Tau phosphoryla-tion on MPP +-induced SH-SY5Y cells apoptotsis. Methods An apoptotic model induced by 1 -methy-4-phenylpridnium ion (MPP +)was established in cul-tured human neuroblastoma SH-SY5Y in vitro.MTT a-nalysis was used to evaluate the protective effect of pre-treatment of cells with 6-Hydroxy-1 H-indazole for 2 h. Hoechst33258 staining was used to observe apoptotic situation.Immunohistochemistry was used to detect the p-Tau (Ser396)expression.Results The viability of cells exposed to 200 μmol · L -1 MPP + for 48h was (47.80 ±0.84)% (P <0.01 ),MPP + induced phos-phorylation of Tau at Ser396 and neuronal apoptosis, while 6-Hydroxy-1 H-indazole inhibited MPP +-induced cellular apoptosis,increased the number of TH-positive cells via inhibiting Tau phosphorylation.Conclusion These results indicate that Tau may be a new target used to cure neurodegenerative disorders such as PD.
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Aim To study the effect of chromomycin on MPP~+-induced dopaminergic neuronal apoptosis.Methods An apoptosis model induced by 1-methyl-4-phenylpridnium ion(MPP~+)was established in cultured fetal mesencephalic neurons in vitro.Dopaminergic neuronal apoptosis was detected by Hoechst 33258 staining and phospho-Tau levels were detected by immunofluorescence.Results 10 μmol·L~(-1) MPP~+ hyperphosphorylated Tau at Ser396 and induced dopaminergic neuronal apoptosis.Chromomycin increased the number of TH-positive cells via inhibiting Tau phosphorylation.Conclusion These results indicate thatchromomycin inhibits Tau phosphorylation at Ser396 and therefore protects dopaminergic mesencephalic neurons from apoptosis induced by MPP+,which suggests that Chromomycin may be used to cure neurodegenerative disorders such as Parkinson's disease in clinic.