Effect of fucoxanthin on growth and apoptosis of HSC-T6 cells

AIM: To explore the effect of fucoxanthin (Fu) on the growth and apoptosis of HSC-T6 cells. METHODS: HSC-T6 cells were divided into blank control group, negative control group and drug groups (treated with different concentrations of Fu). The cell viability was detected by CCK-8 assay at 24 h, 48 h and 72 h after Fu treatment. The cell cycle distribution and apoptotic rate were analyzed by flow cytometry. The protein expression of Bcl-2 and Bax were detected by Western blot. RESULTS: Compared with blank control group, the viability of HSC-T6 cells was inhibited by Fu at concentrations of 15~75 μmol/L in a dose- and time-dependent manner (P < 0.01). The cell ratio of G₁ phase was significantly decreased (P < 0.01) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.01) in 60 μmol/L Fu group after 24 h. The cell ratio of G₁ phase was significantly decreased (P < 0.05) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.05) in 15 μmol/L and 30 μmol/L Fu groups in a dose-dependent manner after 48 h. The early cell apoptotic rates and total cell apoptotic rates were significantly increased in the Fu treatment groups in a dose-dependent manner (P < 0.05). The protein expression of Bax was significantly increased in the Fu treatment groups and the protein expression of Bcl-2 was significantly decreased in 30 μmol/L and 60 μmol/L Fu groups (P < 0.05).CONCLUSION: Fu inhibits the growth of HSC-T6 cells possiblely via arresting the cell cycle at S phase and G₂ phase. The apoptosis of HSC-T6 cells induced by Fu might be via down-regulating the protein expression of Bcl-2 and up-regulating the protein expression of Bax.     

Effect of down-regulation of X-box binding protein 1 gene expression on viability and apoptosis of glioma cells

AIM: To investigate the effect of down-regulation of X-box binding protein 1 (XBP1) expression on the viability and apoptosis of glioma cells. METHODS: The mRNA expression of XBP1 in the glioma tissues was detected by qPCR. Small interfering RNA (siRNA) interfering with XBP1 expression (XBP1-siRNA) was transfected into human brain glioma U251 cells. At the same time, control group (the cells without special treatment) and negative control (NC-siRNA) group (transfected with siRNA without any interference) were set up. The mRNA expression of XBP1 in the 3 groups 48 h after transfection was detected by qPCR. The protein levels of XBP1, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1 (cyclin D1), phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt (p-Akt) were determined by Western blot. The cell viability was measured by CCK-8 assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The expression level of XBP1 in the glioma tissues was significantly higher than that in the tumor adjacent tissues (P<0.05). The XBP1 expression at mRNA and protein levels was significantly decreased in the cells transfected with XBP1-siRNA (P<0.05). No statistically significant difference of the cell viability, cell cycle, apoptotic rate and the protein levels of PCNA, Bcl-2, Bax, cyclin D1, PI3K and p-Akt between NC-siRNA group and control group was observed. Compared with control group, the cell viability, S-phase cells and the protein levels of PCNA, Bcl-2, cyclin D1, PI3K, and p-Akt in XBP1-siRNA group were decreased significantly, and the apoptotic rate, G₀/G₁-phase cells and Bax protein expression were significantly increased (P<0.05). CONCLUSION: Down-regulation of XBP1 gene expression in brain glioma cells reduces the viability of cancer cells, blocks the cells in G₁ phase and promote apoptosis. The mechanism is related to the inhibition of PI3K/Akt signaling pathway.     

Effects of human xeroderma pigmentosum group D gene on proliferation of human vascular smooth muscle cells induced by interleukin-6

AIM: To investigate the effects of human xeroderma pigmentosum group D (XPD) gene on the proliferation of human vascular smooth muscle cells (VSMCs) induced by interleukin-6 (IL-6). METHODS: Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into VSMCs by liposome, and then these cells were incubated with IL-6 at 1×10⁵ U/L for 48 h. The cells were divided into 6 groups: blank control group; pEGFP-N2 group; pEGFP-N2/XPD group; IL-6 group; IL-6 + pEGFP-N2 group; IL-6 + pEGFP-N2/XPD group. The expression of green fluorescent protein was observed under fluorescence microscope. The cell growth was detected by MTT method. The cell cycle and apoptosis rate were examined by flow cytometre. The expression levels of XPD, Bcl-2, Bax and wild type P53 (wt-P53) were detected by RT-PCR and Western blotting.RESULTS: Green fluorescence was observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2, indicating successful transfection MTT results showed that the transfection of pEGFP-N2/XPD inhibited the cell growth, and reduced the positive effects of IL-6 on VSMCs growth. Flow cytometry results showed that the transfection of pEGFP-N2/XPD increased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, decreased the cell numbers in S phase, and reduced the effects that IL-6 decreased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, and increased the cell numbers in S phase. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, Bax and wt-P53, decreased the expression of Bcl-2, and reduced the effects that IL-6 decreased the expression of Bax and wt-P53, and increased the expression of Bcl-2. CONCLUSION: XPD gene inhibits VSMCs proliferation, promotes VSMCs apoptosis, and reduces the effects that IL-6 promotes VSMCs proliferation and inhibits VSMCs apoptosis. Therefore, XPD gene is likely to be potential molecular target for treatment of atherosclerosis.     

Knockdown of astrocyte elevated gene-1 by siRNA inhibits cell proliferation and induces apoptosis in human neuroblastoma cell line

AIM: To investigate the effect of siRNA-induced astrocyte elevated gene-1 ( AEG-1 ) down-regulation on the proliferation, apoptosis and cell cycle of neuroblastoma cells. METHODS: An siRNA targeting to AEG-1 mRNA (AEG-1 siRNA) was constructed and transfected into neuroblastoma cells with Lipofectamine 2000. A non-specific siRNA (control siRNA) and non-treatment were used as negative control and blank control,respectively . The cell proliferation was detected by MTT method and colony formation assay. The apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Compared with control groups, the expression of AEG-1 mRNA was evidently declined in the cells transfected with AEG-1 siRNAs (P<0.05). AEG-1 siRNA significantly decreased the cell proliferation. After treated with AEG-1 siRNA for 48 h, the percentage of apoptotic cells was significant increased and the cell cycle was arrested in G₀/G₁ phase compared with the control cells (P<0.05). CONCLUSION: The mRNA expression of AEG-1 is down-regulated by AEG-1 siRNA in neuroblastoma cells. Knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibits cell proliferation and apoptosis, and induces cell arrest in G₀/G₁ phase of the cell cycle.     

Effects of CENP-W down-regulation on human glioma U87 cells

AIM: To study the effect of centromere protein W (CENP-W) down-regulation on human glioma U87 cells.METHODS: Small interfering RNA (siRNA) was used to inhibit the expression of CENP-W in the U87 cells. The interference effect of siRNA was evaluated by RT-qPCR and Western blot. The proliferation of the cells was analyzed by MTT assay, BrdU staining and colony formation experiment. Transwell chamber assay was used to detect the invasion ability of the cells. The cell migration ability was measured by a scratch test. The changes of the cell cycle distribution and apoptosis were analyzed by flow cytometry.RESULTS: The results of MTT assay, colony formation experiment and BrdU staining showed that the cell proliferation and colony formation abilities in experimental group were significantly lower than those in control group and negative control group. The results of Transwell and scratch experiments showed that the migration and invasion abilities in experimental group were weaker than those in blank control group and negative control group. The results of flow cytometry analysis showed that the cell cycle distribution in experimental group was arrested in G₀/G₁ phase. The percentage of apoptotic cells in experimental group was higher than that in control group (P<0.05).CONCLUSION: Down-regulation of CENP-W expression inhibits the proliferation, migration and invasion of human glioma cells and promotes the apoptosis of the cells, suggesting that CENP-W may be a potential target of gene therapy for human glioma.     

Expression of histone chaperone ASF1B in prostate cancer cells and its effect on cell viability in vitro

AIM To investigate the expression of histone chaperone anti-silencing function 1B （ASF1B） in prostate cancer cells and its effect on cell viability in vitro. METHODS Human prostate cancer PC-3 cells were used, and knockdown of ASF1B was conducted by small interfering RNA （siRNA） transfection into the cells. The cells were divided into control group, siRNA negative control vector （mock） group and siRNA-ASF1B group. The viability of the PC-3 cells treated with ASF1B-siRNA for 12, 24 and 48 h was measured by MTT assay. Flow cytometry was used to analyze cell apoptosis and cell cycle distribution. The mRNA expression of apoptosis-related molecules was detected by RT-qPCR, and the expression levels of MAPK/JNK/ERK signaling pathway-related proteins were determined by Western blot. RESULTS The protein level of ASF1B in the normal cells （benign prostatic hyperplasia） was significantly lower than that in the PC-3 cells （P<0.01）. Compared with control group and mock group, the protein expression level of ASF1B in the PC-3 cells transfected with siRNA-ASF1B plasmid and the viability of the PC-3 cells were significantly decreased （P<0.01）. The apoptotic rate of the PC-3 cells transfected with siRNA-ASF1B plasmid was significantly higher than that in control group （P<0.01）, and the cell cycle was arrested at G₁ phase. The mRNA levels of p53, caspase-3, Bax and PARP-1 in the PC-3 cells transfected with siRNA-ASF1B plasmid were up-regulated compared with those in control group and Mack group （P<0.01）. In addition, the protein levels of MAP2K4 and p-JNK in the PC-3 cells in siRNA-ASF1B group were significantly higher than those in mock group （P<0.01）, while the protein level of p-ERK was significantly lower than that in mock group （P<0.01）. CONCLUSION ASF1B silencing induces G₁ arrest and promotes apoptosis of PC-3 cells. Activating MAPK/JNK/ERK signaling pathway may be a possible contributor to the anti-prostate cancer effect of siRNA-ASF1B.     

Effect of sodium selenite on proliferation of endometrial cancer cells

AIM: To investigate the effect and mechanism of sodium selenite (Na₂SeO₃) on the proliferation of endometrial cancer cells. METHODS: Endometrial cancer Ishikawa cells and HEC-1A cells were treated with Na₂SeO₃. The effect of Na₂SeO₃ on cell proliferation was determined by MTT assay. The effects of Na₂SeO₃ on cell cycle distribution and apoptosis were tested by flow cytometric analysis. The expression of cyclin A was detected by Western blotting. RESULTS: Na₂SeO₃ inhibited the proliferation of Ishikawa cells and HEC-1A cells. For Ishikawa cells, IC₅₀ was 3.26 μmol/L, and for HEC-1A cells, IC₅₀ was 4.77 μmol/L. After treated with Na₂SeO₃, the cells in G₀/G₁ phase were reduced and the cells in S phase and G₂/M phase were increased. Na₂SeO₃ also increased the percentage of apoptosis cells. The result of Western blotting showed that the expression of cyclin A was increased. CONCLUSION: Na₂SeO₃ inhibits the proliferation of endometrial cancer Ishikawa cells and HEC-1A cells via up-regulating the expression of cyclin A, arresting cell cycle and inducing apoptosis.     

Effect of 7-hydroxyisoflavone on HCT116 cells proliferation by Id1

AIM:To investigated the effect of 7-hydroxyisoflavone (7-HIF) on the proliferation, apoptosis and stem-like cell feature of colorectal cancer cells. METHODS:The effect of 7-HIF on the proliferation of HCT116 cells was detected by WST-1 assay and colony formation assay. The effects of 7-HIF on the cell cycle distribution and apoptosis in the HCT116 cells were analyzed by flow cytometry. The expression of cell-cycle related proteins and the stemness related proteins was determined by Western blot. RESULTS:After treated with 7-HIF (200 μmol/L), the viability of HCT116 cells was inhibited, and the size and number of the colony were decreased as compared with control group (P<0.05). The G₀/G₁ phase of the cell cycle was increased. The proportion of S phase was decreased and the cells were mainly arrested in G₀/G₁ phase. The apoptotic rate of HCT116 cells was 21.4%, which was significantly higher than that in the control group (1.1%). The results of Western blot revealed that the expression of inhibitor of differentiation 1(Id1) was significantly decreased (P<0.05). The expression of cell cycle markers cyclin D1 and cyclin E, the proliferative markers survivin and PCNA, and stem cell markers CD133, ALCAM and EpCAM were all down-regulated by 7-HIF treatment (P<0.05). CONCLUSION:7-HIF inhibits the proliferation and induces the apoptosis of colorectal cancer cells, and inhibits the stem-like cell feature, which may be related to Id1 inhibition.     

Celastrol blocks cell cycle of A549 cells by regulating miR-17-5p/miR-155-5p and targeting cyclin D1

AIM: To investigate the effect of celastrol on the cell cycle of human lung adenocarcinoma A549 cells and to probe into its mechanisms.METHODS: A549 cells were exposed to celastrol at gradient concentrations. The cell viability and apoptosis were detected by MTT assay and flow cytometry, respectively, and the median lethal concentration (LC₅₀) of celastrol was screened. The A549 cells were treated with celastrol at LC₅₀, and the cell cycle was detected by flow cytometry. The expression of cyclin D1 was determined by Western blot, and the expression of microRNA (miR)-17-5p and miR-155-5p was detected by real-time PCR. The correlation between cyclin D1 and miR-17-5p or miR-155-5p was predicted by bioinformatics software. After miR-17-5p mimics/miR-155-5p mimics/mutant-miR-17-5p/mutant-miR-155-5p and pcDNA-GFP-cyclin D1-3'UTR were cotransfected into the A549 cells, the changes of GFP expression were evaluated by fluorescence microscopy and flow cytometry. Finally, after miR-17-5p mimics or miR-155-5p mimics were transfeced into the A549 cells, the expression of miR-17-5p and miR-155-5p was detected by real-time PCR, and the protein level of cyclin D1 was determined by Western blot. RESULTS: With the increasing concentration of celastrol, the viability inhibition rate and apoptotic rate of the A549 cells were increased, indicating that celastrol effectively inhibited the growth of A549 cells and induced apoptosis. The LC₅₀ of celastrol was almost 3 μmol/L. After treatment with celastrol at LC₅₀, the A549 cell cycle was arrested at G₁ phase, the protein expression of cyclin D1 was down-regulated (P<0.01), and the expression levels of miR-17-5p and miR-155-5p were significantly increased (P<0.01). The results of bioinformatics software prediction indicated that there were binding sites for miR-17-5p and miR-155-5p in the 3'-UTR of cyclin D1. After cotransfected with miR-17-5p or miR-155-5p and pcDNA-GFP-cyclin D1-3'UTR into the A549 cells, the expression of GFP declined (P<0.05). After miR-17-5p or miR-155-5p mimics were transfected into A549 cells, the results of real-time PCR showed this treatment significantly increased the miRNA expression (P<0.01), and the results of Western blot showed the transfection inhibited cyclin D1 expression (P<0.01).CONCLUSION: Celastrol blocks the A549 cells at G₁ phase, inhibits the viability and induces apoptosis, which may be caused by up-regulating the expression of miR-17-5p and miR-155-5p, and then down-regulating cyclin D1 expression. This study provides a new theoretical basis for the treatment of non-small-cell lung cancer with celastrol.     

Repressive effect of miRNA-363 via SOX4 on human osteosarcoma cell growth and apoptosis

AIM: To detect the expression of miRNA-363 and SOX4 in osteosarcoma tissues and to investigate the effect of miRNA-363 on the viability and apoptosis of human osteosarcoma cell line MG-63.METHODS: Real-time PCR was used to detect the expression level and the relationship of miRNA-363 and SOX4 mRNA in the osteosarcoma tissues and the corresponding paratumorous tissues collected from 63 patients. The expression levels of miRNA-363 and SOX4 in osteosarcoma cell line MG-63 after transfected with miRNA-363 mimics were measured. The cell viability was measured by CCK-8 assay. Flow cytometry was used to monitor the changes of cell cycle and apoptosis. The changes of SOX4 and miRNA-363 expression levels in the MG-63 cells after transfection with SOX4 siRNA or pcDNA/SOX4 was detect by real-time PCR.RESULTS: The expressed level of miRNA-363 was lower, and the expression level of SOX4 was higher in the osteosarcoma tissues than those in the adjacent normal tissues. A significantly negative correlation between the expression levels of miRNA-363 and SOX4 was observed. The expression of miRNA-363 in the MG-63 cells after transfection with miRNA-363 mimics was significantly up-regulated, while the expression of SOX4 in the MG-63 cells was significantly down-regulated, with significant difference as compared with the cells transfected with miRNA-NC and control cells. The viability of MG-63 cells was inhibited, the cell cycle was arrested in G₀/G₁ phase, and the cell apoptosis was increased by transfection with miRNA-363 mimics. The relative protein expression levels of SOX4 in SOX4 siRNA group and pcNDA/SOX4 group were significantly different from those in negative control group, but the relative expression levels of miRNA-363 had no significant difference. Over-expression of SOX4 restored the viability of the MG-63 cells reduced by miR-363.CONCLUSION: The expression level of miRNA-363 is low in human osteosarcoma tissue. miRNA-363 may inhibits the viability of osteosarcoma cell line MG-63 and promotes cell apoptosis in vitro via inhibiting the SOX4 expression.     

Effect of ABCE 1 gene silencing on proliferation and migration of human bladder cancer cell line T24

AIM: To investigate the effect of small interfering RNA (siRNA)-mediated ABCE1 knockdown on the survival, cell cycle and invasion of human bladder cancer cell line T24. METHODS: The siRNA against ABCE1 was constructed and transfected into the T24 cells with Lipofectamine^TM 2000. The expression of ABCE1 was detected by RT-PCR and Western blot. Flow cytometry was used to detect the cell cycle. The effects of ABCE1 gene silencing on proliferation, migration and invasion of T24 cells were evaluated by CCK-8 assay, wound-healing assay and Transwell invasion assay, respectively. RESULTS: Compared with control group and blank group, the mRNA and protein levels of ABCE1 were significantly decreased in experimental group after transfected with ABCE1 siRNA. The cell cycle was arrested at G₀/G₁ phase and the cell number in S phase was decreased in the T24 cells. Compared with control group and blank group, the proliferation of T24 cells in experimental group was inhibited significantly, and the migration and invasion abilities of T24 cells in experimental group decreased significantly. CONCLUSION: Knockdown of ABCE1 gene may decrease migration, invasion and proliferation abilities in T24 cells.     

Berberine enhances mitomycin C-induced cell cycle arrest and apoptosis of T24 bladder cancer cells

AIM: To observe the effects of the combination of berberin (Ber) and mitomycin C (MMC) on the cell cycle arrest and apoptosis of T24 bladder cancer cells and the underlying mechanisms. METHODS: The T24 cells were exposed to MMC in the presence or absence of difference concentrations of Ber. The viability of the T24 cells was determined by CCK-8 assay. The cell cycle distribution was detected by flow cytometry. The apoptosis was analyzed by flow cytometry with Annexin V-FITC/PI staining, and the protein expression levels of cyclin D1, survivin, CDK2, CDK4, p21 and p27 were determined by Western blot. RESULTS: CCK-8 experiments showed that Ber enhanced the inhibitory effect of MMC on the viability of T24 cells. The results of flow cytometry showed that Ber also enhanced the blockade effect of MMC on T24 cells in G₀/G₁ phase (P<0.05). Compared with the MMC group, Ber increased the expression of p21 and p27 up-regulated by MMC, and decreased the expression of cynlin D1, CDK2 and CDK4 (P<0.05). Meanwhile, Ber promoted MMC to inhibit the expression of survivin (P<0.05). Ber increased the apoptosis of T24 cells induced by MMC (P<0.05). CONCLUSION: Ber significantly enhances the inhibitory effect of MMC on the viability of T24 cells. The mechanism may be related to up-regulation of p21 and p27, thereby inhibiting the expression of cyclin D1, CDK-2 and CDK-4. At the same time, Ber inhibits the protein expression of survivin, which eventually leads to cell arrest in G₀/G₁ phase and promotes apoptosis.     

RNAi-mediated knocking-down of LBH gene promotes cell cycle progress in 16HBE cells

AIM: To investigate the effect of small interfering RNA(siRNA) on limb-bud and heart(LBH) gene expression in 16HBE cells, and further to observe the change of 16HBE cell cycle after knocking down of LBH gene. METHODS: Synthetical siRNA targeting LBH gene was transfected into 16HBE cells by the method of lipofectamine 2000. The mRNA expression of LBH was examined by real time RT-PCR. The cell cycle was assayed by flow cytometry. The expression of cyclin E1 and E2 was also detected by real time RT-PCR and Western blotting.RESULTS: The expression of LBH gene decreased by 86% in 16HBE cells transfected with 50 nmol/L of siRNA for 48 h. After transfected with siRNA for 48 h, 16HBE cells in G₁ phase decreased by 9.28% and the cells in S phase increased by 14.08%. The expression level of cyclin E2 in 16HBE cells transfected with siRNA was twice higher than that of the negative control cells.CONCLUSION: Sequence-specific siRNA targeting LBH is capable of suppressing LBH gene expression in 16HBE cells. Down-regulation of LBH expression in 16HBE cells may promote the progress of cell cycle, and up-regulation of cyclin E2 expression plays a role in the process of G₁/S phase.     

Effect of ClC-3 siRNA on cell cycle of HeLa cells

AIM: To investigate the roles of ClC-3 chloride channels in the regulation of cell cycle and the relationship between ClC-3 chloride channels and the cell cycle regulators, such as cyclin D1, cyclin-dependent kinase (CDK)4, CDK6, P21 and P27 in the HeLa cells.METHODS: ClC-3 genes were silenced by the siRNA technique in the HeLa cells. The transfection efficiency of ClC-3 siRNA was detected by real-time PCR. The cell cycle distribution was analyzed by the flow cytometry. The protein expression of ClC-3, P21, P27, CDK4, CDK6 and cyclin D1 was determined by Western blot.RESULTS: ClC-3 was knocked down by ClC-3 siRNA in the HeLa cells. Transfection of the cells with ClC-3 siRNA arrested the cells at G₀/G₁ phases, decreased the expression of cyclin D1, CDK4 and CDK6, and increased the expression of P21 and P27.CONCLUSION: ClC-3 plays an important role in the cell cycle of HeLa cells through the G₁-S transition point. ClC-3 may regulate the cell cycle progression by up-regulation of cyclin D1, CDK4 and CDK6 expression and/or by down-regulation of P21 and P27 expression.     

PPARγ mediates effects of diosgenin on proliferation and apoptosis in human glioblastoma U87MG cells

AIM:To investigate the effect of diosgenin (Dio) on the proliferation, apoptosis and expression of peroxisome proliferator-activated receptor γ (PPARγ) in human glioblastoma U87MG cells and its possible mechanism. METHODS:Human astrocytes (HA) and U87MG cells were cultured in vitro and treated with Dio (0, 10, 20, 30, 40 and 50 μmol/L) and GW9662 (5 μmol/L) for 48 h, and then the cell viability was detected by CCK-8 assay. Cell colony formation assay was used to assess the proliferation potential. Flow cytometry was used to analyze the cell cycle distribution and apoptosis. The mRNA expression level of PPARγ was measured by RT-PCR. Western blot was used to determine the protein levels of PPARγ, cyclin D1, cyclin E1, Bcl-2 and Bax. RESULTS:Dio had no significant influence on the viabi-lity of HA (P>0.05). However, Dio remarkably reduced the viability of U87MG cells in a dose-dependent manner (P<0.05) with IC₅₀ of 24.31 μmol/L. Meanwhile, Dio remarkably diminished colony formation ability (P<0.05), induced G₀/G₁ phase arrest of the cell cycle and apoptosis (P<0.05), up-regulated the expression of PPARγ at mRNA and protein levels, increased the protein level of Bax (P<0.05), and down-regulated the protein levels of cyclin D1, cyclin E1 and Bcl-2 (P<0.05) in a dose-dependent manner. However, these effects induced by Dio were inhibited by GW9662 (P<0.05), a specific inhibitor of PPARγ. CONCLUSION:Dio may inhibit proliferation and induce apoptosis in human glioblastoma U87MG cells most likely via up-regulating the expression of PPARγ, and then down-regulating the protein levels of cyclin D1, cyclin E1 and Bcl-2, and up-regulating the protein level of Bax.     

Expression of eEF1A2 in hepatocellular carcinoma

AIM: To study the expression of eukaryotic elongation factor 1A2 (eEF1A2) in the hepatocellular carcinoma (HCC) tissues and the effects of eEF1A2 over-expression on the biological behaviors of the HCC cells. ME-THODS: The expression of eEF1A2 at mRNA and protein levels in the HCC tissues and matched liver tissues from 62 HCC patients, and 20 normal liver tissues were detected by the methods of real-time PCR and immunohistochemical staining, respectively. The mRNA and protein expression of eEF1A2 in the HCC cells was also determined by real-time PCR and Western blot, respectively. The lentivirus containing eEF1A2 gene was constructed, and was used to infect the HCC cells with low eEF1A2 expression. The expression of eEF1A2 at mRNA and protein levels in the infected cells was detected by real-time PCR and Western blot, respectively. The cell activity, cell cycle and mRNA expression of albumin were measured by MTT assay, DNA ploid analysis and real-time PCR, respectively.RESULTS: The mRNA expression levels and protein expression positive rates of eEF1A2 in the 62 cases of HCC tissues, were significantly higher than those of 62 matched liver tissues and 20 normal liver tissues (P<0.01). eEF1A2 mRNA and protein were highly expressed in SMMC-7721 cells and BEL-7402 cells, and expressed in SK-HEP-1 cells at low level. The expression of eEF1A2 at mRNA and protein levels in the SK-HEP-1 cells was significantly enhanced by infection of GV287-eEF1A2 expression lentivirus.Compared with negative control group (transfected with negative control lentivirus), the cell activity in eEF1A2 over-expression group (transfected with GV287-eEF1A2 expression lentivirus) was significantly enhanced, the mRNA expression of albumin was remarkably reduced, and the cells in G₀/G₁ phase were significantly decreased with increased percentage of the cells in S and G₂/M phases.CONCLUSION: eEF1A2 is selectively over-expressed in human HCC cancer tissues. eEF1A2 might be a putative oncoprotein in HCC. eEF1A2 over-expression has noticeable effects on the HCC cell proliferation enhancement, differentiation inhibition, and cell cycle acceleration through the G₀/G₁ phase to S phase and G₂/M phases.     

Salinomycin inhibited proliferation and induced apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP

AIM: To investigate the effect of salinomycin on the proliferation and apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP. METHODS: The inhibitory effect of salinomycin on the growth of A549/DDP cells was tested by MTT method in vitro . The apoptosis and mitochondrial membrane potential (ΔΨ_m) of A549/DDP cells were assayed by flow cytometry. The activity of caspase-3, 8 and 9 was determined by the method of colorimetry. The levels of cytochrome C, Bcl- 2, Bax, β-catenin, and phosphorylated low-density lipoprotein receptor-related protein 6(p-LRP6) were measured by Western blotting. RESULTS: Salinomycin inhibited the growth of A549/DDP cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L decreased ΔΨ_m level, and increased reactive oxygen species (ROS), cytochrome C and cytosolic Ca²⁺ release in the cells. Salinomycin also increased the acti-vity of caspase-3, 8, and 9 in the cells, reduced the ratio of Bcl-2/Bax, and decreased the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin depresses the cell growth by inhibiting Wnt signaling, and induces the apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP via mitochondria-dependent and Bcl-2/Bax pathways.     

Expression of SCD-1 in cervical carcinoma and effect of its down-regulation on proliferation and apoptosis of cervical carcinoma cells

AIM:To examine the expression of stearoyl-CoA desaturase-1 (SCD-1) in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki, and to investigate the effect of down-regulation of SCD-1 on the proliferation and apoptosis of cervical carcinoma cells. METHODS:The expression of SCD-1 was detected by Western blotting in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki. SCD-1 siRNA and control siRNA were utilized to transfect CaSki cells by Lipofectamine 2000, and SCD-1 protein level was determined by Western blotting after transfection. Furthermore, CCK-8 and flow cytometry were utilized to investigate the changes of cell proliferation and apoptosis after transfection with SCD-1 siRNA in CaSki cells. Subsequently, the activities of caspase-3 and caspase-9 were analyzed by Caspase-Glo3/7 and 9 detection kit after transfection with SCD-1 siRNA in CaSki cells. Finally, the protein expression of Bcl-2 and Bax was detected by Western blotting. RESULTS:The protein expression of SCD-1 in cervical squamous cell carcinoma tissues was significantly higher than that in normal cervical tissues, and the protein expression of SCD-1 in the 3 cervical carcinoma cell lines was obviously higher than that in normal cervical tissues, in which CaSki cells displayed the highest SCD-1 protein level. In addition, the protein expression of SCD-1 in SCD-1 siRNA group was significantly lower than that in untreated group and control siRNA group. Compared with untreated group and control siRNA group, the proliferation of CaSki cells was markedly inhibited in SCD-1 siRNA group. Early apoptotic rate in SCD-1 siRNA group was evidently higher than that in untreated group and control siRNA group. The activities of caspase-3 and caspase-9, and the level of Bax protein were significantly elevated, and the protein level of Bcl-2 was obviously reduced after transfection with SCD-1 siRNA in CaSki cells. CONCLUSION: SCD-1 may play an important role in the occurrence and development of cervical carcinoma, and its down-regulation, which mediates cell proliferation inhibition and apoptosis, may be tightly associated with the activities of caspase-3 and caspase-9, and the protein expression of Bcl-2 and Bax.