Effects of RUNX3 siRNA on the growth and drug sensitivity of SH-SY5Y cells

AIM: To investigate the effects of RUNX3 gene on the growth and drug sensitivity of SH-SY5Y cells.METHODS: The siRNA plasmid of RUNX3 was constructed and transfected into SH-SY5Y cells. Stable transfectants were identified by RT-PCR and Western blotting. The growth curve, cell cycle distribution, drug sensitivity assay and accumulation of adriamycin in cells were detected by MTT assay and flow cytometry. The expressions of cyclin D1, CDK4, CDK6, p21, p27, Bcl-2, Bax, P-gp and MRP were analyzed by Western blotting. RESULTS: mU6pro-RUNX3 siRNA was successfully constructed and transfected into SH-SY5Y cells. Down-regulation of RUNX3 significantly promoted the cellular proliferation, inhibit the drug sensitivity and intracellular adriamycin accumulation of cells, compared with that in the controls (P<0.05). The expressions of P-gp, Bcl-2 and cyclin D1 in transfected cells were increased, while p21 decreased.CONCLUSION: RUNX3 might play important roles in the development of neuroblastoma.     

Effects of TSG101 siRNA on the growth and drug sensitivity of SH-SY5Y cells

AIM:To investigate the effects of TSG101 siRNA on the growth and drug sensitivity of human neuroblastoma cell line SH-SY5Y.METHODS:The small interfering RNA eukaryotic expression vector specific to human TSG101 gene was constructed by gene recombination,then transfected into SH-SY5Y cells.Stable transfectants were obtained by G418 screening and further identified by RT-PCR and Western blotting analysis.The growth curve was made using MTT assay.Cell cycle distribution of the transfected cells was studied by flow cytometry and the proliferative indexes were calculated.The apoptosis after CDDP treatment was detected by DNA ladder and Annexin V/propidium iodide binding analyses.The expression of Bcl-2,Bax,P-gp and MRP were analyzed by Western blotting.RESULTS:mU6pro-TSG101 siRNA was successfully constructed and transfected into SH-SY5Y cells.As detected by MTT and flow cytometry,down-regulation of TSG101 significantly suppressed the proliferation of SH-SY5Y cells with a G1 cell cycle arrest,compared with that in control (P<0.05).As detected by DNA ladder and Annexin V/propidium iodide binding analyses,down-regulation of TSG101 significantly enhanced the sensitivity of SH-SY5Y cells to CDDP-induced apoptosis,compared with that in control (P<0.05).The expression of P-gp and Bcl-2 in transfected cells were decreased as compared with that in the control,while MRP and Bax were not.CONCLUSIONS:Down-regulation of TSG101 suppresses the proliferation of SH-SY5Y cells,and enhances the sensitivity of SH-SY5Y cells to conventional chemotherapeutic agents to a degree,suggesting TSG101 may be useful for gene therapy in the future.     

Protective effect of osthole on SH-SY5Y cells transfected with APP595/596 gene

AIM: To explore the protective effect of osthole on the SH-SY5Y cells transfected with APP595/596 gene, and to investigate the molecular mechanism. METHODS: The SH-SY5Y cells were transfected with APP595/596 gene in vitro for establishing a cell model to study the pathogenic role of amyloid β-protein (Aβ). The cell viability was detected by CCK-8 assay. The release of lactate dehydrogenase (LDH) was determined by the colour reaction of diaphorase-INT. The cell apoptotic rate was analyzed by flow cytometry. The expression of β-site APP cleaving enzyme 1(BACE1) at mRNA and protein levels was detected by RT-PCR and Western blot. The expression of Aβ was measured by the technique of immunofluorescence cytochemistry and Western blot. RESULTS: Treatment with osthole inhibited the LDH release, and increased the viability of the cells. The percentage of apoptotic cells was also significantly decreased. Osthole also inhibited the expression of BACE1 at mRNA and protein levels and the protein expression of Aβ. CONCLUSION: Osthole has protective effect on SH-SY5Y cells transfected with APP595/596 gene. The mechanism may be association with inhibiting the mRNA and protein expression of BACE1.     

Effects of edaravone on high glucose-induced apoptosis in SH-SY5Y cells via regulating microRNA-25

AIM: To observe the effects of edaravone on high glucose-induced apoptosis of SH-SY5Y cells and its potential mechanism. METHODS: The SH-SY5Y cells were cultured in the DMEM medium with 100 mmol/L glucose and 100 μmol/L edaravone for 24 h. The viability of the SH-SY5Y cells was detected by MTT assay. The levels of ROS in the cells were determined by DCFH-DA fluorescent probing. The apoptotic rates of the cells were analyzed by flow cytometry. The protein expression of Bax and Bcl-2 in the cells were detected by Western blot. The expression levels of micro-RNA-25 (miR-25) were determined by real-time PCR. To further clarify the target sites of edaravone on inhibiting apoptosis induced by high glucose, miR-25 inhibitor was applied to the SH-SY5Y cells and the activity of caspase-3 was measured.RESULTS: Compared with control group, the cell viability was decreased significantly in model group, and the ROS level was increased significantly. The protein expression of Bax was up-regulated significantly, while the expression levels of Bcl-2 and miR-25 were significantly down-regulated. Compared with model group, the cell viability was increased significantly in edaravone group. The ROS level was decreased significantly. Meanwhile, the expression of Bax was down-regulated, while the expression of Bcl-2 and miR-25 was up-regulated with statistical significance. The caspase-3 activity of the cells incubated with 100 mmol/L glucose and miR-25 inhibitor was increased. However, no alteration of caspase-3 activity with edaravone added simultaneously was observed. CONCLUSION: Edaravone inhibits the apoptosis of SH-SY5Y cells induced by high glucose with the potential target site of miR-25.     

Effect of sorcin expression on sensitivity of human glioma cells to cisplatin

AIM：To investigate the effect of sorcin expression on the sensitivity of human glioma cells to cisplatin. METHODS：pSilencerTM 3.1-H1-sorcin siRNA recombinant plasmid was constructed, and transfected into human glioma U251 cells. RT-PCR and Western blotting were used to analyze the expression of sorcin at mRNA and protein levels after transfection. The viability of U251 cells was measured by MTT assay. The protein expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) in U251 cells was detected by Western blotting. RESULTS：The plasmid pSilencerTM 3.1-H1-sorcin siRNA was successfully constructed, and was confirmed by restriction enzyme digestion and sequence analysis. The expression of sorcin at mRNA and protein levels was significantly decreased after sorcin siRNA was transfected into U251 cells (P<0.05). Inhibition of sorcin expression significantly decreased the viability of U251 cells treated with cisplatin (P<0.05), and the expression of P-gp and MRP1 proteins was also inhibited (P<0.05). CONCLUSION：Inhibition of sorcin expression increases the sensitivity of U251 cells to cisplatin by decreasing the expression of resistance-related proteins P-gp and MRP1, suggesting that sorcin may be associated with the resistance of glioma cells to cisplatin.     

Effects of BCL-3 gene silencing on the proliferation and drug sensitivity of human colorectal cancer cell line RKO with high expression of BCL-3

AIM：To construct lentiviral vectors for RNA interference (RNAi) of BCL-3 gene, and to detect the changes of biological behaviors and drug sensitivity of colorectal cancer cells after BCL-3 gene silencing. METHODS：The expression of BCL-3 in five human colorectal cancer cell lines was detected by RT-PCR and Western blotting. Lentiviral vectors for RNAi of BCL-3 gene were constructed and transfected into the human colorectal cancer cell line with high expression of BCL-3, and then the silencing effect was detected by Western blotting. After BCL-3 gene silencing, the change of cell proliferation was detected by MTT assay and soft agar colony formation assay, and the change of drug sensitivity was detected by MTT assay. RESULTS：BCL-3 was highly expressed in human colorectal cancer cell line RKO. Lentiviral vectors for RNAi of BCL-3 gene were successfully constructed, and Western blotting showed that BCL-3-shRNA2 could efficiently inhibit the expression of BCL-3 protein in RKO cells. After BCL-3 gene silencing, the proliferation ability and colony formation rate of RKO cells were decreased, and the median inhibitory concentration of oxaliplatin for RKO cells also decreased significantly. CONCLUSION: Inhibition of BCL-3 gene expression decreases the proliferation ability of human colorectal cell line RKO with high expression of BCL-3, and enhances the sensitivity of RKO cells to oxaliplatin.     

Effects of baicalin on CA46 cell proliferation inhibition and apoptosis induction

AIM: To investigate the effects of baicalin on proliferation inhibition and apoptosis induction in human Burkitt lymphoma cell line CA46 and to explore its underlying mechanisms. METHODS: CA46 cells were exposed to baicalin at different dosages and its proliferation inhibition was detected by MTT assay. The ability of baicalin to induce CA46 cell apoptosis was examined by Annexin V-FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation. The mRNA expressions of c-myc and bcl-2 were detected by RT-PCR, and the protein expressions of c-Myc, Bcl-2, caspase-3 precursor (procaspase-3) and poly ADP-ribose polymerase (PARP) were detected by Western blotting. RESULTS: Baicalin remarkably inhibited the CA46 cell proliferation, with an IC50 value of 10 μmol/L. Apoptosis was remarkably induced by baicalin in a dose-dependent manner, and its earlier and later stages were detected by annexin V-FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation, respectively. Furthermore, RT-PCR showed that the mRNA expressions of c-myc and bcl-2 in treated CA46 cells decreased in a time-dependent manner. Western blotting showed that the protein expressions of c-Myc, Bcl-2, procaspase-3 and PARP (116 kD) in baicalin treated CA46 cells were down-regulated in a time-dependent manner, while the expression of PARP(85 kD) was up-regulated. CONCLUSION: Baicalin efficiently induces proliferation inhibition and apoptosis in CA46 cells, which may be related with the down-regulation of c-Myc and Bcl-2 expressions, as well as the up-regulation of caspase-3 activity.     

Protective effect of NAD(P)H:quinone oxidoreductase 1 on dopaminergic cells

AIM: To determine the influence of NAD(P)H:quinone oxidoreductase 1 (NQO1) on dopamine-induced toxicity in dopaminergic cells.METHODS: MTT assay was used to determine the toxic curve of dopamine in SH-SY5Y cells. Lipofection was applied to transfect SH-SY5Y cells with an NQO1 expression plasmid. The endogenous and transfected NQO1 expression was detected by immunofluorescence staining and Western blotting. The content of cellular quinone protein was measured by nitroblue tetrazolium (NBT) method. RESULTS: Dopamine reduced SH-SY5Y cell proliferation in a dose-dependent manner, which was correlated with an increase in the content of quinone protein. Increased expression of NQO1 by transient transfection or by phase II enzyme inducer sulforaphane treatment alleviated dopamine-induced toxicity and reduced the content of cellular quinone protein. CONCLUSION: Increased NQO1 expression protects SH-SY5Y cells against cytotoxicity caused by dopamine.     

Rapamycin reduces SH-SY5Y cell damage induced by oxygen-glucose deprivation

AIM: To observe the effect of rapamycin (Rapa) on human neuroblastoma SH-SY5Y cell injury induced by oxygen-glucose deprivation (OGD), and to explore the role of autophagy in this process. METHODS: The SH-SY5Y cells were randomly divided into 4 groups:normal control group:the cells were cultured without OGD treatment; Rapa group:the cells were pretreated with Rapa for 1 h; OGD group:the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 1% O₂, 94% N₂ and 5% CO₂ for 12 h; Rapa+OGD group:the cultured cells were treated with Rapa for 1 h, and then were given the same treatments as those in OGD group. The cell viability was assessed by MTT assay. The degree of the cell damage was evaluated by determining the leakage of lactate dehydrogenase (LDH). The enzyme activity of caspase-3 was detected. TUNEL staining were used to detect the variation of cell apoptosis. The protein levels of apoptosis-related proteins Bax and Bcl-2, autophagy-related protein beclin-1 and autophagy marker protein LC3B were determined by Western blot. RESULTS: Compared with OGD group, the viability of the SH-SY5Y cells was significantly increased, and the activity of caspase-3 was significantly reduced in Rapa+OGD group (P<0.05). The SH-SY5Y cell injury was apparent after OGD with a great increase in the apoptotic rate (P<0.05). Compared with OGD group, the apoptotic rate significantly decreased in Rapa+OGD group (P<0.05). Compared with control group, the protein level of Bcl-2 was significantly decreased (P<0.05) and the protein level of Bax was significantly increased in OGD group. Compared with OGD group, the levels of Bcl-2, beclin-1 and LC3B-Ⅱ were significantly increased and the protein level of Bax was significantly increased in Rapa+OGD group (P<0.05). CONCLUSION: Rapamycin has a protective effect on in vitro cultured SH-SY5Y cells injured by OGD. The mechanism may be related to the promotion of autophagy.     

Glucosylceramide synthase upregulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway in leukemia multidrug-resis-tant cell line

AIM: To investigate whether glucosylceramide synthase (GCS) regulates apoptosis-related gene bcl-2 expression via MEK/ERK signaling pathway, thus enhancing drug resistance of K562/A02 human leukemia multidrug resistant cell line. METHODS: siRNA targeting GCS was transfected into K562/A02 cells. Bcl-2, p-ERK and total ERK expression at mRNA and protein levels after GCS knockdown were detected by real-time PCR and Western blotting. After exposed to MEK-ERK pathway inhibitor U0126, the expression of Bcl-2 at mRNA and protein levels also was analyzed by real-time PCR and Western blotting, respectively. The viability of the cells was evaluated by CCK-8 assay. RESULTS: The expression of GCS and Bcl-2, as well as MEK/ERK signaling were significantly inhibited in K562/A02 cells by GCS siRNA transfection compared with negative control group. Inactivation of MEK/ERK signaling due to U0126 treatment decreased Bcl-2 mRNA and protein levels in a concentration-dependent manner, and sensitized K562/A02 cells to adriamycin. CONCLUSION: GCS may affect the expression of apoptosis-related gene bcl-2 by MEK/ERK signaling pathway, thus regulating multidrug resistance of human leukemia K562/A02 cells.     

Cytotoxicity of lidocaine on SH-SY5Y cells

AIM: To observe the effects of lidocaine on SH-SY5Y cells and to investigate the neural cytotoxicity of lidocaine. METHODS: Cultured SH-SY5Y cells in vitro were divided into 4 groups: in group C (control group), SH-SY5Y cells were cultured under normal conditions; in group L1, SH-SY5Y cells were cultured with 0.5% lidocaine for 10 min; in group L2, SH-SY5Y cells were cultured with 1% lidocaine for 10 min; in group L3, SH-SY5Y cells were cultured with 2% lidocaine for 10 min. Cell morphology, cell viability and apoptosis were detected to evaluate the effects of lidocaine on the cells. RESULTS: SH-SY5Y cells in group C showed dendritic protrusions, enlarged nervous process and dense network. However, SH-SY5Y cells in the groups of L1, L2 and L3 showed the disappearance of dendritic protrusions, cell shrinkage, cell size reduction and round shape. Compared with SH-SY5Y cells in group C, the cell viability significantly decreased in the groups of L1, L2 and L3 with the increase in the concentration of lidocaine. Apoptotic rate of SH-SY5Y cells in group C was between 5.9% and 6.3%, and it increased with the increases in the concentration of lidocaine. CONCLUSION: Lidocaine at concentrations of 0.5%, 1%, 2% can damage SH-SY5Y cells, and this injury become obviously serious with the increase in the concentrations of lidocaine, indicating that we should use the minimum effective concentration of lidocaine in clinic to prevent the harmful effect of the drug.     

Establishment of gastric cancerous multi-drug resistance cell stain BGC823/5-FU and its resistance mechanism

AIM: To establish the gastric cancerous multidrug resistance cell stain BGC823/5-FU and investigate the relationship between the resistance and the expression of apoptosis related protein Survivin, Bcl-2, Bax and caspase-3. METHODS: Human gastric cancer cell line BGC823 was induced into MDR cell line by intermittent administration of high dose of 5-FU. MTT assay was used to detect the sensitivity of these MDR cells to some chemotherapeutic agents. Flow cytometry was used to detect the expression of P-glucoprotein and the accumulative value of intracellular daunorubicin (DNR) in these MDR cells. Western blotting was used to detect the expression of Survivin, Bcl-2, Bax and caspase-3. RESULTS: The resistance cell stain BGC823/5-FU was established, which possessed the ability of 10.82 fold resistance to 5-FU and cross-resistance to adriamycin, mitomycin C and cisplatin. The expression of P-glucoprotein was higher in BGC823/5-FU cells than that in BGC823 cells, while the accumulative value of intracellular DNR was decreased in BGC823/5-FU cells. Compared with its parent cells, expressions of Bax and caspase-3 in BGC823/5-FU cells were significantly down-regulated, surviving and Bcl-2 were upregulated in BGC823/5-FU cells. CONCLUSION: Gastric cancer cell line BGC823 has been induced into MDR cell line BGC823/5-FU. P-glucoprotein, Survivin, Bcl-2/Bax ratio and caspase-3 may play an important role in MDR of BGC823/5-FU cells.     

Mechanism responsible for inhibitory effect of recombinant rat augmenter of liver regeneration on renal turbular epithelial cell apoptosis by gentamycin

AIM: To investigate the effects of recombinant rat augmenter of liver regeneration (rrALR) on apoptosis of renal tubular cells (NRK-52E cells) induced by gentamycin sulfate (GM). METHODS: The cultured NRK-52E cells were divided into four groups: normal control cells, cells with GM (GM 1.6 g/L) or GM and rrALR (15 mg/L or 25 mg/L) treatments. The apoptosis of cultured cells were assessed at 24 h, 48 h by AO/EB staining, DNA agarose gel electrophoresis analysis and flow cytometry using Annexin V-FITC and propidium iodide (PI) staining. The protein and mRNA expressions of Bcl-2 and Bax were detected by Western blotting and RT-PCR, respectively. RESULTS: (1) rrALR inhibited NRK-52E cells apoptosis induced by GM (P＜0.05). (2) rrALR promoted the expression of Bcl-2 protein and mRNA, but inhibited the Bax protein and mRNA expression (P＜0.05) in cultured NRK-52E cells in a dose-dependent manner. The value of Bcl-2/Bax increased. CONCLUSION: rrALR inhibits renal tubular epithelial cell apoptosis and ameliorates cell injury induced by nephrotoxic drug GM presumably via the regulation of Bcl-2 and Bax protein and mRNA expressions.     

Role of divalent metal transporter 1 in degeneration of dopaminergic neuron

AIM: To observe the expression of divalent metal transporter 1(DMT1) in SH-SY5Y cells with lactacystin-induced injury, and to investigate the possible role of DMT1 in the degeneration of dopaminergic neuron in Parkinson disease(PD). METHODS: An in vitro model of cell injury was established in SH-SY5Y cells induced by lactacystin. The protein expression of DMT1 was detected by immunofluorescence and Western blotting. Under the environment of high iron level, the cellular oxidative stress was observed by fluorescent probe DCFH-DA. The level of α-synuclein was determined by immunohistochemistry and Western blotting. RESULTS: The cell viability rate was reduced by lactacystin in a concentration-dependent pattern. Compared with the control, the protein level of DMT1 was obviously increased in lactacystin-treated cells. The arrangements of the changes from high to low in decreased cell viability, increased intracellular oxidative stress and increased aggregation of α-synuclein(43-55 kD) were Fe²⁺ treatment group > lactacystin treatment group > control group(P<0.05). CONCLUSION: Lactacystin up-regulates the protein expression of DMT1. By this way, the increased function of DMT1-mediated iron uptake may play an important role in iron or iron-catalyzed oxidative reactions to enhance α-synuclein aggregation, leading to the degeneration of neurons in PD.     

Tanshinone IIA inhibits doxorubicin resistance in gastric cancer cells

AIM: To investigate the inhibitory effect and the specific mechanism of tanshinone IIA on doxorubicin (DOX)-resistant gastric cancer cells. METHODS: The sensitivity of gastric cancer cells lines to DOX was determined by MTT assay. DOX-resistant gastric cancer cell lines were established by step selection with increasing concentrations of DOX. The cell cycle arrest, apoptosis and autophagy related-markers were analyzed by flow cytometry and Western blot. The expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multi-drug resistance-associated protein 1 (MRP-1) was determined by RT-qPCR and Western blot. RESULTS: DOX-sensitive cell lines SNU-719 and SNU-601 as well as the cell lines relatively resistant to DOX including SNU-638, SNU-668, SNU-216 and SNU-620 were identified according to the IC₅₀ values of DOX for different cell lines. Two DOX-resistant cell lines SNU-719R and SNU-601R were also established. Tanshinone IIA inhibited the expression of MRP-1 in DOX-resistant cell lines. Compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in cancer cells decreased the G₂/M phase cell number, increased the protein expression of p21, decreased the protein expressions of cyclin B1 and cyclin-dependent kinase 1 (CDK1) in the SNU-719 R cells and SNU-620 cells. In addition, compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in the cancer cells increased the protein expressions of p53, Bax and LC3B-II, decreased the protein expression of Bcl-2 and p62 (P<0.05). CONCLUSION: Tanshinone IIA is an effective drug in the inhibition of DOX resistance in gastric cancer.     

Adiponectin attenuates H2O2-induced SH-SY5Y cell injury and tau hyperphosphorylation via activating PP2A

AIM: To study the effects of adiponectin on H₂O₂-induced cell injury and tau hyperphosphorylation in human neuroblastoma SH-SY5Y cells. METHODS: Cell viability was determined by MTT assay. H₂O₂-induced cell injury and morphological changes in the SH-SY5Y cells with or without adiponectin treatment were observed. The level of tau phosphorylation as well as the activities of protein phosphatase 2A(PP2A) and of glycogen synthase kinase-3β(GSK-3β) were examined by Western blotting. RESULTS: Adiponectin significantly attenuated H₂O₂-induced cell injury(P<0.01). Adiponectin upregulated the activity of PP2A and decreased phosphorylation levels of tau under the stimulation with H₂O₂ (P<0.01). Okadaic acid, a specific inhibitor of PP2A, blocked the protective effects of adiponectin(P<0.01). Adiponectin increased the phosphorylation level of GSK-3β at Ser9 site under H₂O₂ stimulation(P<0.01). CONCLUSION: Adiponectin protects SH-SY5Y cells against H₂O₂-induced cell injury and decreases tau hyperphosphorylation by activating PP2A and inactivating GSK-3β.     

Effect of sodium ferulate on bcl-2 and bax gene expression of apoptotic hippocampal neurons induced by sodium ferulate

AIM: To investigate the protective effects of sodium ferulate (SF) on apoptosis in cultured hippocampal neurons induced by sodium nitroprusside (SNP), and the effect of SF on expression of bcl-2 and bax. METHODS: The primary cultured hippocampal neurons were exposed to 50 μmol SNP, a nitric oxide-donor, for 24 h after pretreatment with different concentrations of SF (10-160 μmol/mL) for 6 h. Then neuronal viability was tested by MTT assay. Fluorescent staining with Hoechst 33258 and agarose gel electrophoresis was used to analyze apoptosis. The expressions of bcl-2, bax mRNA and protein were tested by RT-PCR and Western blotting. RESULTS: Pretreatment with SF(10-160 μmol/L) for 6 h increased the survival rate of neurons. SF prevented the neuronal nuclei from shrinkage, condensation and cleavage and blocked neuronal nuclear DNA fragmentation induced by SNP. SF also increased the expressions of bcl-2 mRNA and Bcl-2 protein and decreased the expressions of bax mRNA and Bax protein. CONCLUSION: SF prevents the cultured hippocampal neurons against SNP neurotoxicity. The mechanism of protection is related to the increase in Bcl-2 level and the decrease in Bax level. As a result, the ratio of Bcl-2/Bax is changed.