Pim-1 kinase inhibitor SMI-4a inhibits proliferation and induces apoptosis in U937 cells

AIM: To study the growth-inhibiting and proapoptotic effects of Pim-1 kinase inhibitor SMI-4a on human acute myeloid leukemia cell line U937.METHODS: The effect of SMI-4a on U937 cell viability was measured by CCK-8 assay. The apoptotic rate was assessed by flow cytometry with Annexin V-PI staining and by fluorescence microscopy with Hoechst 33342 staining. Methylcellulose was used to assess colony formation ability of the cells. The expression of β-catenin in the cell cytosol and nucleus was detected by Western blot, and the expression of apoptosis-related proteins in the U937 cells was also examined. Intracellular distribution of β-catenin was detected by the method of immunofluorescence.RESULTS: SMI-4a inhibited the viability of U937 cells. Annexin V-PI staining showed that SMI-4a induced apoptosis in dose-and time-dependent manners. Hoechst 33342 staining also verified the apoptosis. SMI-4a significantly inhibited the colony formation capacity of the U937 cells. The results of Western blot demonstrated that SMI-4a upregulated the expression of PARP and Bax, downregulated the expression of Bcl-2 and change the distribution of β-catenin in intracellular compartment. Immunofluorescence observation found that SMI-4a decreased the expression level of β-catenin in the U937 cells.CONCLUSION: SMI-4a induces U937 cell apoptosis through regulating the expression of apoptosis-related genes.     

Effect of Ginsenoside Rh2 on apoptosis of human osteosarcoma cell line MG-63

AIM: To investigate the effect of Ginsenoside Rh2(Rh2) on the apoptosis of human osteosarcoma cell line MG-63.METHODS: The cell viability was determined by MTT assay. MG-63 cell apoptotic rate was examined by flow cytometry with Annexin V-PI double staining. The expression of Bcl-2, Bax, cytochrome C(Cyt C) and cleaved caspase-3 were measured by Western blot.RESULTS: Rh2 enhanced the apoptosis of MG-63 cells in a dose-dependent manner. Furthermore, after treatment with Rh2, the release of mitochondrial Cyt C and Bax expression were increased, while Bcl-2 and the ratio of Bcl-2/Bax were decreased as compared with control group(P<0.05). The protein level of cleaved caspase-3 was also increased(P<0.05).CONCLUSION: Ginsenoside Rh2 accelerates the apoptosis of MG-63 cells through mitochondria-dependent pathway, suggesting that Rh2 is a novel approach for the treatment of osteosarcoma.     

Dengue virus type 2 induces apoptosis of RAW264.7 cells and effect of apoptosis on virus replication

AIM:To explore the apoptotic pathway in dengue virus type 2 (DENV2)-infected RAW264.7 cells and to analyze the effect of apoptosis on virus replication. METHODS:RAW264.7 cells were infected with DENV2. MTT assay was used to detect the cell viability. Apoptosis was assessed by Hoechst 33342 staining and Annexin V-FITC/PI staining. The expression levels of caspase-3 and caspase-8 were determined by Western blotting. The activity of caspase-9 was measured with a colorimetric kit. Mitochondrial membrane potential was evaluated using JC-1 fluorescent staining. TCID50 was used to estimate the infectious virion concentration after using Z-VAD-FMK to inhibit apoptosis. RESULTS:The viability of RAW264.7 cells decreased after DENV2 infection at 24 h, 36 h and 48 h. Karyopyknosis in dengue virus-infected cells was observed. The protein levels of caspase-3 and caspase-8 and the activity of caspase-9 increased in the apoptotic cells after dengue virus infection. Mitochondrial membrane potential was reduced after dengue virus infection. There was a higher virion concentration in the cell culture medium after inhibition of apoptosis. CONCLUSION: Dengue virus induces apoptosis of RAW264.7 cells. Apoptotic inhibition of RAW264.7 cells facilitates the production of dengue virus.     

Down-regulated BARF1 expression induces EBV-positive gastric carcinoma cell apoptosis via activating caspase-dependent mitochondrial pathway

AIM: To investigate the effects of BARF1 down-regulation on EBV-positive gastric carcinoma cell apoptosis, and the molecular mechanisms by BARF1 silencing-mediated apoptosis. METHODS: After NUGC3 and SNU719 cells were transfected with NCsiRNA and siRNA, respectively, the protein levels of BARF1, Bcl-2, Bax, cytochrome C, caspase 3 and capase 9 were detected by Western blot, and the mRNA expression of BARF1, Bcl-2 and Bax was determined by RT-PCR. The cell viability was measured by the method of Trypan blue exclusion and the cell apoptosis was analyzed by flow cytometry analysis with Annexin V-FITC/PI staining. The expression of the apoptosis-related proteins in the cells transfected with siRNA and NCsiRNA was examined by human apoptosis antibody arrays. Mitochondrial membrane potential was determined by flow cytometry. The interaction between Apaf-1 and caspase 9 was confirmed by immunoprecipitation. RESULTS: Compared with untreated and NCsiRNA groups, BARF1 gene silencing significantly inhibited the cell viability, induced apoptosis, and reduced the mitochondrial membrane potential in the NUGC3 and SNU719 cells transfected with siRNA. BARF1 gene silencing up-regulated the expression of pro-apoptotic proteins and down-regulated the expression of anti-apoptotic proteins, and the Bcl-2/Bax ratio was significantly decreased. In BARF1 gene silencing cells, the caspase inhibitor z-VAD-fmk inhibited BARF1 silencing-mediated apoptosis, and significantly increased the levels of cleaved caspase 3 and caspase 9. The concentration of cytochrome C significantly increased as compared with NCsiRNA group, and Apaf-1 interacted with caspase 9 in the cytoplasm. CONCLUSION: BARF1 silencing induces apoptosis via the mitochondrial pathway through regulating the expression of Bcl-2 and Bax proteins in a caspase-dependent manner in the NUGC3 and SNU719 cells.     

Combination of sorafenib and daunorubicin has antileukemic activity on K562 cells

AIM: To investigate the synergetic inhibitory effect of sorafenib and daunorubicin (DNR) on K562 and U937 cells. METHODS: The inhibitory rate of sorafenib or daunorubicin alone, and the combined inhibitory rate of sorafenib and IC10 daunorubicin were measured by MTT assay. Apoptotic rate of single drug or combination was assessed by flow cytometry (Annexin Ⅴ/PI staining) and Hoechst 33258 staining assay. p-ERK1/2 level was detected by Western blotting after the cells were treated with sorafenib, daunorubicin and U0126 or combinations. Synergistic or antagonistic effect of proliferation and apoptosis on K562 and U937 was estimated according to the Jins Method. RESULTS: Combination of sorafenib and DNR showed synergistic growth inhibition (q>1.15, P<0.01) and synergistic promotion of apoptosis (q>1.15, P<0.05) in K562 and U937 cells. The level of p-ERK1/2 in K562 cells was obviously higher than that in U937 cells (P<0.01). p-ERK1/2 expression was completely inhibited in sorafenib or U0126 treated K562 cells for 24 h. Combination of U0126 with DNR inhibited the proliferation of K562 cells synergistically. CONCLUSION: Combination of sorafenib with DNR showed synergistic cell growth inhibition and promotion of apoptosis in K562 and U937 cells. U937 cells were more sensitive to DNR than K562 cells while K562 cells were more sensitive to sorafenib. Sorafenib enhances the anti-leukemic activity of DNR in K562 and U937 cells via down-regulation of p-ERK1/2 expression.     

Effect of homocysteine on the apoptosis of cultured human umbilical vein endothelial cells

AIM: To investigate the effect of homocysteine(Hcy) on the apoptosis of endothelial cells (EC). METHODS: First-passaged human umbilical vein endothelial cells (hUVEC) were cultured with M199 containing 3 mmol/L Hcy. hUVEC apoptosis was detected as follow: demonstration of nuclear changes by Hoechst 33258 staining, agarose gel electrophoresis of DNA fragments, detection of apoptotic cells by flow cytometry following Annexin V-PI doubled stain, Western blot for P53 and Bax protein detection and colorimetry detecting caspase-3 activity. RESULTS: Compared with control, homocysteine induced characteristic apoptotic changes in hUVEC. The chromosomal DNA of hUVEC appeared "DNA ladder" by agarose gel electrophoresis. Apoptotic cells were increased significantly (P<0.01, n=3). Hcy promoted the expression of protein Bax, P53 (P<0.01, n=3) and enhanced the activity of caspase 3 (P<0.05, n=3). CONCLUSION: Homocysteine induces apoptosis in cultured hUVEC.     

Hexokinase 2 inhibits LPS-induced mitochondria-dependent apoptosis in human lung epithelial BEAS-2B cells

AIM: To explore the effect of hexokinase 2 (HK2) on lipopolysaccharide (LPS)-induced apoptosis of human lung epithelial BEAS-2B cells and the underlying mechanisms.METHODS: BEAS-2B cells were treated with LPS to induce cell injury, and the cell viability was examined by CCK-8 assay. Hoechst 33342 staining and Annexin V/PI double staining were used to analyze the apoptosis. The apoptotic pathway was identified by the specific inhibitor for caspase-8 or caspase-9. The releases of key mediators in mitochondrial apoptosis pathway were examined by Western blot. The effects of HK2 in these process were confirmed by HK2 over-expression followed by LPS treatment.RESULTS: CCK-8 assay showed that LPS treatment decreased the viability of BEAS-2B cells in a dose/time-dependent manner (P<0.01). The apoptosis of BEAS-2B cells was manifested by Hoechst 33342 and Annexin V/PI double staining. Pretreatment with z-LEHD-fmk, but not z-IETD-fmk, reversed the decreased cell viability under LPS stimulation. HK2 down-regulation was involved in LPS-induced apoptosis of the BEAS-2B cells. After HK2 over-expression, the cell viability was increased after LPS treatment. Releases of cytochrome C and apoptosis-inducing factor from mitochondrion to cytoplasm during apoptosis were also inhibited by HK2 over-expression.CONCLUSION: Hexokinase 2 inhibits LPS-induced mitochondria-dependent apoptosis in human lung epithelial cells.     

Tanshinone IIA prevents high glucose-induced human umbilical vein endothelial cell apoptosis

AIM: To investigate the effect of tanshinone IIA on the apoptosis of human umbilical vein endothelial cells(HUVECs) after high glucose treatment.METHODS: The cell viability was determined by MTT assay. The cell apoptotic rate was examined by flow cytometry with Annexin V-FITC/PI double staining. The expression of Bcl-2 and Bax, and the release of mitochondrial cytochrome C(Cyt C) were analyzed by Western blotting.RESULTS: Tanshinone IIA significantly inhibited high glucose-induced decrease in cell viability and increased the cell apoptosis. Additionally, after tanshinone IIA treatment, Bax expression and the release of mitochondrial Cyt C were significantly inhibited, while Bcl-2 expression was increased.CONCLUSION: Tanshinone IIA prevents high glucose-induced endothelial cell apoptosis via mitochondria-dependent pathway.     

Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the apoptosis and molecular mechanism of human hepatocellular carcinoma HepG2 cells induced by ginsenoside Rh4. METHODS: Human hepatocellular carcinoma HepG2 cells were treated with ginsenoside Rh4 at doses of 10, 20 and 40 μmol/L, and the inhibitory effect of ginsenoside Rh4 on HepG2 cell viability was measured by MTT assay. The apoptotic rate of HepG2 cells was analyzed by flow cytometry. The morphological changes of the HepG2 cells were observed by Hoechst 33258 and TUNEL staining. The expression of apoptosis-related proteins Bax, Bcl-2, caspase-3 and caspase-9 was determined by Western blot. RESULTS: Ginsenoside Rh4 promoted apoptosis of HepG2 cells in a dose-dependent manner. TUNEL and Hoechst 33258 staining showed that the cells appeared obvious shrinking, swelling and rupture after treated with ginsenoside Rh4 for 24 h. The results of Western blot showed that with the increasing concentrations of ginsenoside Rh4, the expression of pro-apoptotic proteins Bax, cleaved caspase-3 and caspase-9 increased, while anti-apoptotic protein Bcl-2 decreased gradually. CONCLUSION: Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells, and the main mechanism may be related to down-regulation of Bcl-2 and up-regulation of Bax, cleaved caspase-3, and caspase-9.     

Effect of ruthenium-polypyridine complex on apoptosis of gastric cancer SGC-7901 cells

AIM:To study the effect of ruthenium-pyridine complex Ru1 on apoptosis of gastric cancer SGC-7901 cells. METHODS:MTT assay and crystal violet staining method were used to detect the viability and cell number of SGC-7901 cells treatment with Ru1. Annexin V-FITC and PI staining was performed to test the apoptosis rate of SGC-7901 cells. The protein expression of Bax and Bcl-2 was detected by Western blot. RESULTS:The results of MTT assay and crystal violet staining showed that the ruthenium-pyridine complexes significantly reduced the viability and cell number of SGC-7901 cells. Treatment with Ru1 for 24 h significantly increased the apoptotic rate of SGC-7901 cells (P<0.05). Ru1 up-regulated the expression of Bax protein and down-regulated the expression of Bcl-2 protein in the SGC-7901 cells (P<0.05). CONCLUSION:Ru1 induces apoptosis of SGC-7901 cells by affecting the expression of Bax and Bcl-2 proteins.     

Reversal of apoptosis resistance of doxorubicin-resistant human myelogenous leukemia cell line K562/DOX by a cyclosporin D analogue PSC833

AIM:To study the reversal effect of a cyclosporin D analogue PSC833 on multidrug resistance of doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells. METHODS:The reversal effects of PSC833 on resistance to doxorubicin (DOX)/vincristine (VCR) in K562/DOX cells were observed by MTT assay. The cell cycle analysis was performed by flow cytometry. Annexin V/PI staining was used to identify PSC833-induced apoptosis in K562/ DOX cells. These cells underwent incubation with DCFH-DA, JC-1 and Fluo-3/AM followed by flow cytometry for the measurement of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and intracellular calcium, respectively. The protein levels of cytochrome C (Cyt C), Bcl-2, Bax, and cleaved caspase-3 were detected by Western blotting. RESULTS:The DOX/VCR-induced cytotoxicity was significantly potentiated by PSC833. PSC833 arrested the cells in G2/M phase and increased the apoptosis induced by DOX in K562/DOX cells. During the apoptosis, the level of ROS and intracellular calcium increased, while the level of ΔΨm decreased. Furthermore, the release of Cyt C, activation of caspase-3, up-regulation of Bax and down-regulation of Bcl-2 were observed in K562/DOX cells treated with PSC833 and DOX. CONCLUSION: The reversal effect of PSC833 on multidrug resistance in K562/DOX cells is associated with the induction of apoptosis through a mitochondria-dependent pathway.     

T0901317 induces apoptosis of human breast cancer MDA-MB-231 cells by up-regulating LXRα expression

AIM: To investigate the pro-apoptotic effect of T0901317, an artificial agonist of liver X receptor α (LXRα), on human breast cancer MDA-MB-231 cells and its mechanism. METHODS: MDA-MB-231 cells were treated with different concentrations (0, 10, 20 and 40 μmol/L) of T0901317 for different time (0, 12, 24 and 48 h). The cell apoptosis was determined by Annexin V/propidium iodide staining and Hoechst 33342 staining. The expression of apoptosis-related proteins, such as Bcl-2, caspase 3 and cleaved caspase-3, and LXRα was determined by Western blot. The mRNA expression of Bcl-2 and LXRα was analyzed by RT-qPCR. RESULTS: T0901317 induced the cell apoptosis in a dose-and time- dependent manner. The expression of cleaved caspase-3 and LXRα was up-regulated, but Bcl-2 was down-regulated by T0901317. The mRNA expression of Bcl-2 was down-regulated, while LXRα was up-regulated by T0901317.CONCLUSION: T0901317 up-regulates LXRα expression and induces the apoptosis of MDA-MB-231 cells.     

Hesperetin attenuates hypoxia/reoxygenation-induced apoptosis in H9c2 cells via inhibition of calcium overload

AIM: To investigate the effect of hesperetin on hypoxia/reoxygenation (H/R)-induced apoptosis in the H9c2 cells and to clarify the underlying mechanism. METHODS: The H/R model was established and the H9c2 cells were pretreated with hesperetin for 4 h. The cell viability and cell damage were measured by CCK-8 assay and lactate dehydrogenase (LDH) detection. The apoptosis was analyzed by Hoechst 33258 staining and flow cytometry. The intracellular calcium fluorescence intensity was measured by fluorescence microscopy and flow cytometry. The calcium-ATPase activity and the level of adenosine triphosphate (ATP) were measured by ELISA. The mitochondrial membrane potential was measured by JC-1 staining. The protein expression levels of Bcl-2, Bax and cytochrome C (Cyt-C) were determined by Western blot. RESULTS: Hesperetin reduced the apoptosis of the H9c2 cells induced by H/R, decreased intracellular Ca²⁺ fluorescence intensity, elevated Ca²⁺-ATPase activity, inhibited the mitochondrial membrane potential depolarization and increased the level of ATP (P<0.05). In addition, hesperetin significantly reduced the release of Cyt-C protein from mitochondria to cytoplasma and increased the Bcl-2/Bax ratio (P<0.05). After using the calcium ion inhibitor nimodipine, the percentage of the cells with mitochondrial membrane depolarization was decreased, the ATP level was increased and the protein expression of mitochondrion-related apoptosis molecules were decreased (P<0.05). CONCLUSION: Hesperetin reduces the apoptosis of the H9c2 cells induced by H/R, which may be related to inhibition of calcium overload and improvement of mitochondrial function.     

Effects of matrine on apoptosis and related protein expression in human medulloblastoma D341 cells

AIM: To investigate matrine-induced apoptosis of human medulloblastoma D341 cells and the expression of Bax, Bcl-2, serine/threonine kinase Akt and phosphorylated Akt (p-Akt) in vitro. METHODS: D341 cells were divided into experimental groups (added with matrine at different concentrations) and control group (under the same conditions without matrine). The proliferation of D341 cells was analyzed by CCK-8 assay. Apoptosis was detected by Annexin V-FITC/PI double staining and the expression of Bax, Bcl-2, Akt and p-Akt was detected by Western blotting. RESULTS: Matrine significantly inhibited the proliferation of D341 cells and increased the apoptosis in a dose- and time-dependent manner. The cell apoptosis was characterized by chromatin condensation with margination of chromatin to the nuclear membrane, increased when and larger cytoplasmic vacuoles, and formation of apoptotic body after treatment with matrine. The expression of Bax increased, while the expression of Bcl-2 and p-Akt decreased when the drug concentration gradually increased. CONCLUSION: Matrine induces the apoptosis of human medulloblastoma D341 cells in vitro by activation of Bax, down-regulation of Bcl-2 and reduction of p-Akt expression level in the PI3K/Akt signaling pathway.     

Inhibitory effect and induction of apoptosis of caffeic acid Ge on growth of U14 in mice

AIM: To evaluate the antitumor effect of caffeic acid Ge on U14 tumor bearing mice. METHODS: The tumor inhibitory ratios of caffeic acid Ge on the growth of U14 in mice was observed. Apoptosis morphological transformation of U14 cells induced by caffeic acid Ge was detected by electronic scan microscope and MG-P staining. Alteration of cell cycle was analyzed by flow cytometry. Apoptosis-related protein levels of Bax and Bcl-2 were determined by immunity histochemistry technology. MTT assay was applied to study the antitumor activities of caffeic acid Ge in U14 cell lines in vitro. RESULTS: Tumor inhibitory rates in caffeic-acid Ge groups were 38.50%, 47.17% and 64.02% (from low dose to high dose) (P<0.01). Caffeic acid Ge induced apoptosis in U14 cells detected by MG-P staining, histopathology and electronic microscopy, the typical apoptosis characteristics in morphology were observed. Flow cytometry results indicated that most of the U14 cells treated with caffeic acid Ge were arrested at the sub-G₀-G₁ phase and the U14 cells were blocked in S phase. The results of immunity histochemistry indicated that the expression of Bcl-2 protein was down-regulated by caffeic acid Ge while Bax protein was up-regulated in U14 tumor tissue (P<0.05). The MTT results suggested that cafffeic acid Ge exhibited high antiproliferative activity in U14 cell lines, and the IC₅₀ for 48 h was 48.57 mg/L. The above results showed that caffeic acid Ge exhibited high antiproliferative activity in vitro. CONCLUSION: Caffeic acid Ge has antiproliferative and proapoptotic effect on U14 tumor cells in vivo and in vitro. Caffeic acid Ge increases the expression of Bax and decreases the expression of Bcl-2 to induce apoptosis of U14 cells. This is one of the possible mechanisms of antitumor.     

Advanced glycation end products induce rat chondrocyte injury by modulating oxidative stress

ATM: To explore the possibility that advanced glycation end products (AGEs) induces rat chondrocyte injury by modulating oxidative stress. METHODS: Primarily cultured rat chondrocytes were identified. The viability of the chondrocytes was measured by CCK-8 assay. The intracellular levels of reactive oxygen species (ROS) were detected by DCFH-DA staining. The number of apoptotic cells was determined by Hoechst 33342 nuclear staining and flow cytometry. RT-PCR was performed to measure the mRNA levels of Bax, Bcl-2, caspase-3, MMP3, MMP13 and COL2 in the chondrocytes. Western blotting was used to evaluate the protein levels of cleaved caspase-3, MMP3, MMP13 and COL2. RESULTS: Compared with control group, the intracellular levels of ROS in the chondrocytes treated with AGEs were significantly increased (P<0.05), and pretreatment with N-acetyl-L-cysteine (NAC) suppressed the formation of ROS (P<0.05). Besides, NAC inhibited AGEs-induced apoptosis of the chondrocytes, as indicated by reduceing the levels of Bax/Bcl-2 and caspase-3, decreased the expression of MMP3 and MMP13, and reduced the loss of COL2.CONCLUSION: AGEs induce chondrocyte injury by activating oxidative stress.     

Effects of up-regulation of c-myc expression on U937 cell line

AIM:To establish a human monocytic　leukemia cell line U937 stably expressing c-myc gene and to investigate the biological characteristics of this cell line. METHODS:The recombinant plasmid MSCV-c-myc-IRES-GFP (MMIG) was constructed. MMIG and MSCV-IRES-GFP (MIG) were used to package the viruses for infecting U937 cells. Fluorescence-activated cell sorter (FACS) was used for sorting U937/GFP and U937/MYC cells. The GFP-positive cells were detected by fluorescence microscopy and FACS. The protein expression of c-Myc, survivin, X-linked inhibitor of apoptosis protein (XIAP) and Bcl-2 was detected by Western blotting. Cell proliferation was evaluated by MTT assay. Propidium iodide (PI) staining was used to determine the cell cycle distribution. Self-renewal ability was observed by colony- forming assay. RESULTS:The GFP expression in the cells infected with MIG or MMIG virus was observed under fluorescence microscope. The green fluorescent rate of the cells infected with MIG was 26.0%, while that of the cells infected with MMIG was 27.7%. The protein expression of c-Myc in MMIG-infected U937 cells was higher than that in MIG-infected cells. After sorting, the green fluorescent rates of U937/GFP and U937/MYC cells reached 98.7% and 93.7%, respectively. The protein expression of c-Myc in U937/MYC cells was higher than that in U937/GFP cells. In addition, survivin, a downstream protein of c-Myc, was up-regulated, while the protein expression of XIAP and Bcl-2 remained unchanged. Cell cycle analysis showed that the percentage of the cells in S phase increased in U937/MYC cells. Moreover, the proliferation and colony-forming ability of U937/MYC cells were also enhanced. CONCLUSION: U937/MYC cell line stably expressing c-myc gene was successfully established. c-Myc may increase cell viability via enhancing the expression of anti-apoptotic protein survivin, the cell cycle transition and the self-renewal ability.     

Effect of low molecular weight heparin on hypoxia-induced apoptosis in primary cultured neonate rat cerebral cortical neurons

AIM:To investigate the mechanism by which low molecular weight heparin (LMWH) inhibits apoptosis of primary cultured cerebral cortical neurons caused by hypoxia. METHODS:The anti-apoptosis effect of LMWH on primary cultured neurons was observed by methods of the primary culture of cerebral neurons of postnatal rats in free-serum with neurobasal medium supplied with 2% B27 supplement, hypoxic culture of neurons, Hoechst 33342 staining and immunocytochemistry. RESULTS:At concentrations of 250-500 U/L, LMWH reduced apoptosis rate of cerebral cortical neurons induced by hypoxia (P<0.05) and apoptosis rate was lower in LMWH groups than that in BDNF (50 μg/L) group (P<0.05). LMWH (500 U/L) increased Bcl-2 protein expression (P<0.05) and decreased Bax protein expression (P<0.01) in the cerebral cortical neurons induced by hypoxia, the ratio of Bcl-2/Bax was improved (P<0.01). The ratio of Bcl-2/Bax in LMWH (500 U/L) group was higher than that in normal control group, BDNF group and apoptosis positive group (P<0.05). CONCLUSION:LMWH at concentrations of 250-500 U/L is able to prevent cerebral cortical neurons from apoptosis in primary culture under hypoxia. The effect of anti-apoptosis may be related to up-regulation of Bcl-2 protein expression, down-regulation of Bax-2 protein expression, and increase in the ratio of Bcl-2/Bax.     

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.     

Berberine promotes apoptosis of human breast cancer cells by regulating miRNA-146a

ATM: To observe the effect of berberine on apoptosis of MCF-7 cells and its potential mechanism. METHODS: The MCF-7 cells were divided into control group and the groups with 3 different doses of berberine. The cell viability was detected by MTT assay, while the cell apoptosis was measured by Hoechst 33258 staining and flow cytometry assay. The protein levels of p-P65, Bax and Bcl-2 were Western blot. The levels of microRNA-146a(miRNA-146a) in the MCF-7 cells were detected by RT-qPCR. The miRNA-146a siRNA was transfected to the MCF-7 cells after an evaluation of transfection efficacy, which was co-incubated with berberine to observe its effects on the mRNA levels of Bax and Bcl-2. RESULTS: Compared with control group, the cell viabilities were decreased significantly in medium and high doses of berberine treatment groups with a dose-dependent manner (P<0.01). The cell apoptosis was increased significantly in medium and high doses of berberine treatment groups dose-dependently (P<0.05). The protein levels of Bax were up-regulated, while those of Bcl-2 and p-P65 were down-regulated significantly by the treatment of berberine (P<0.05). In addition, the miRNA-146a levels were increased significantly in medium and high doses of berberine treatment groups (P<0.05) and showed a dose-dependent manner. The mRNA levels of Bax were decreased, while the mRNA levels of Bcl-2 were increased after transfection with miRNA-146a siRNA and co-incubated with berberine.CONCLUSION: Berberine promotes apoptosis of MCF-7 cells. The mechanism may be related to inhibit the activity of NF-κB by incresing the levels of miRNA-146a.