Effect of specific hTERT RNA interference on biological characteristics of colon carcinoma in vivo and in vitro

AIM: To investigate the effect of specific hTERT RNA interference on biological characteristics of colon carcinoma in vivo and in vitro. METHODS: A small interference RNA (siRNA) targeting to hTERT mRNA (pU6-hTERT-siRNA) was constructed. The siRNA was transfected into LoVo colon cancer cells in vivo and in vitro with Lipofectamine^TM2000. The groups of non-specific siRNA (pU6-hTERT) and non-treatment were designed as negative control and blank control,respectively. The cell growth in vitro was detected by MTT method. The effect of pU6-hTERT-siRNA on xenografts in nude mice was observed by determining the tumor size. The mRNA expression of hTERT in vitro and in vivo was detected by FQ-PCR quantitatively. The protein level of hTERT was determined by Western blotting. RESULTS: The inhibition rate of cell growth in vitro 72 h after transfection with recombinant plasmids containing hTERT-target sequences was 42.1%, significantly higher than that in control group (3.2%, P<0.01). The size of xenografts in pU6-hTERT-siRNA group was (85.9±18.7)mm³, significantly smaller than that in control group and blank group , P<0.01. The mRNA expression and the protein level of hTERT were both specifically inhibited by pU6-hTERT-siRNAs in LoVo colon cancer cells and xenografts (P<0.01). No difference between control group and blank group was observed (P>0.05).CONCLUSION: hTERT expression in LoVo colon cancer cells is inhibited significantly in vivo and in vitro by using plasmid-based siRNA. Down-regulation of hTERT expression distinctly inhibits the growth of LoVo colon cancer cells in vitro or subcutaneously transplanted in athymic mice.     

Effects of changing PKC activity on proliferation and telomerase expression in human hepatocellular carcinoma cells

AIM: To investigate whether protein kinase C (PKC) is involved in the proliferation and the telomerase expression in human hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cells (BEL-7402) were treated with exogenous phorbol-12-myristate-13-acetate (PMA, PKC activator) and staurosporine (SP, PKC inhibitor) for 48 hours. The techniques of cell culture and the telomeric repeat amplification protocol silver staining in combination with computer image scanning system in vitro were used to observe the variations of the growth and the telomerase expression. RESULTS: The proliferative potential of BEL-7402 cells was decreased by the action of PMA as well as SP, and the telomerase expression was also inhibited by PMA and SP. CONCLUSION: Our findings suggest that the proliferation of human hepatocellular carcinoma cells and the telomerase expression may be related to PKC.     

姬松茸多糖抗肿瘤的实验研究

应用细胞培养技术 ,观察了姬松茸多糖对K5 6 2和HL - 6 0细胞系的影响。研究结果表明 ,实验组活细胞数、集落形成明显减少 ,荧光显微镜下所见凋亡细胞显著增加。提示姬松茸多糖具有抗肿瘤效果。为了探讨姬松茸多糖抗肿瘤的机理 ,本实验还应用反义寡聚脱氧核糖核酸技术 ,进一步观察了姬松茸多糖与人端粒酶反义核酸协同诱导HL - 6 0细胞系凋亡的作用 ,实验结果表明 ,姬松茸多糖与反义寡核苷酸联合使用 ,可促进HL- 6 0细胞凋亡。     

Antitumor effect of allicin on esophageal cancer EC109 cells compared with chemotherapeutics in vitro

AIM: To investigate the inhibitory effect of allicin on human esophageal cancer cell line EC109 and its possible mechanism by comparison with chemotherapeutic drugs. METHODS: The EC109 cells were treated with different concentrations of allicin, 5-fluorouracil and cisplatin. The cell viability was detected by CCK8 assay and the activity of lactic dehydrogenase (LDH) was analyzed at 6 h, 12 h, 24 h, 48 h and 72 h. The apoptosis of the EC109 cells induced by Z-VAD-FMK, allicin, allicin+Z-VAD-FMK, 5-fluorouracil and cisplatin was analyzed by flow cytometry with Annexin V-FITC and PI double staining. The enzyme activity changes of caspase-3, caspase-8 and caspase-9 were detected by spectrophotometry. RESULTS: Allicin had inhibitory effect on the growth of the EC109 cells and killed them in concentration-dependent and time-dependent manners. LDH activity was decreased compared with 5-fluorouracil and cisplatin. The increased activity of caspase-3 and caspase-8 in allicin-treated cells was statistically significant, but caspase-9 activity changed without statistical significance. CONCLUSION: Allicin inhibits the growth of EC109 cells in concentration-and time-dependent manners through extrinsic apoptosis pathways activated by caspase-8, and the side effects are weaker compared with 5-fluorouracil and cisplatin.     

Phagocytosis of viable apoptotic cells inhibits the activation of T lymphocytes

AIM: To investigate whether viable apoptotic cells and phagocytosis of them affect the activation of T lymphocytes. METHODS: Ultraviolet irradiation was used to induce apoptotic cells in vitro and the model of phagocytosis of these cells was established. Cytokine TGF_β1 was detected by ELISA. The rate of apoptotic cells and phagocytosis of them were assessed by flow cytometry. Furthermore, flow cytometry was also employed to examine the expression of activation signs, such as CD69, CD25, CD71, of T lymphocytes under the intervention of apoptotic cells and macrophage which ingested apoptotic cells, to reflect whether the apoptotic cells and the phagocytosis of these cells could influence the activation of lymphocytes stimulated by Con A. RESULTS: Ingestion of apoptotic cells increased TGF_β1 secretion. Only the macrophages that had ingested apoptotic cells could suppress the activation of lymphocytes. The expression of the markers of lymphocytes activation such as CD69, CD25, CD71 had been restrained. These inhibition effects were abolished by monoclonal anti-TGF_β1 antibody. CONCLUSION: The macrophages that have ingested apoptotic cells inhibit expression of CD69, CD25 and CD71 of T lymphocytes stimulated by ConA. This effect is dependent on the increase in TGF_β1 secretion in local site.     

Effect of hTERT antisense oligodeoxynucleotide on Cis-diamminedichicloroplatinum-induced apoptosis in Jurkat cells

AIM:To explore the effect of hTERT antisense phosphorothioate oligodeoxynucleotide (ASODN) on apoptosis induced by chemotherapeutic drugs in Jurkat cell lines. METHODS:Cell viability was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation, DNA gel electrophoresis and flow cytometry analysis. RESULTS:The survival rates of Jurkat cells cultured with daunorubicin, vincristin, and etoposide, respectively were similar with that cultured with those chemotherapic drugs plus hTERT ASODN. The survival rates of Jurkat cells cultured with cis-diamminedichicloroplatinum(DDP) added 24 hours later were higher than that cultured with hTERT ASODN and DDP added 24 hours later. The survival rates of Jurkat cells cultured with DDP were similar with that cultured with hTERT SODN and DDP. In morphological observation of apoptotic cells using Giemsa staining, cells displayed classic apoptotic changes treated with DDP or DDP combined with hTERT ASODN or SODN at 48 hours. Agarose gel electrophoresis of genomic DNA from Jurkat cells treated with ASODN and DDP combination for 48 hours showed typical DNA "ladder". Neither the DNA from Jurkat cells treated with SODN plus DDP nor the DNA from the cells treated with DDP alone showed ladder pattern. Apoptosis rates of Jurkat cells treated with DDP for 48 hours after 24 hours of exposure to ASODN significantly increased. There was significant difference in the percentage of apoptotic Jurkat cells between hTERT ASODN plus DDP and SODN plus DDP or DDP alone, respectively. CONCLUSION:The hTERT ASODN complementary to the translation initiation region of hTERT mRNA enhanced DDP-induced apoptosis in Jurkat cells.     

Effect of inhibited ubiquitin-proteasome pathway on growth and activity of telomerase in esophageal carcinoma cells

AIM: To investigate the effect on growth and activity of telomerase in esophageal carcinoma cells by inhibiting ubiquitin-proteasome pathway(UPP). METHODS: The esophageal carcinoma cell strain Eca9706 was treated with MG-132 to inhibit its UPP specially. The effect of growth suppression on cells was evaluated with MTT assay, morphologic changes of cells were observed under microscope, cell cycle and apoptosis were detected by flow cytometry (FCM). DNA fragment analysis was used to confirm the presence of apoptosis. The activity of telomerase was detected. RESULTS: MG-132 had obvious inhibitory effect on the growth of Eca9706 cells in a dose and time-dependent manner. Obvious pathologic change of cells were observed under microscope, cells became round, small and exfoliating. The FCM analysis showed that the ratio of esophageal carcinoma cells of G₁ phase increased and a obviously apoptotic sub-G₁ peak was found. Agarose electrophoresis showed marked ladder. The activity of telomerase was obviously inhibited. CONCLUSIONS: MG-132 significantly inhibits the growth and the activity of telomerase of Eca 9706 cells. These findings indicate that inhibiting UPP is a new strategy for the treatment of esophageal carcinoma.     

Inhibition of K562 cell growth by antisense drug targeting with VEGF mRNA in vitro

AIM: To investigate inhibition of K562 cell growth by antisense drug targeted VEGF mRNA. METHODS: X7, 20-mer antisense sequences were selected, synthesized and modified with phosphorothioate. The drug was transfected into K562 cells in the present of lipofection. Cell growth was assayed by trypan blue dye exclusion assay and MTT. The level of VEGF protein in the media was determined by ELISA. The morphology of apoptotic cells were observed by Giemsa staining, and the propotion of apoptotic cells was detected by flow cytometry. RESULTS: The antisense drug inhibited growth of K562 and downregulated expression of VEGF protein significantly, compared with Scrambed control group and showed dose-dependent relation. Signs of apoptosis of K562 cells were not observed. CONCLUSION: Inhibition of K562 cell proliferation, but not cells apoptosis induction is the mechanism of inhibing growth of K562 cells by antisense drug targeted VEGF mRNA. At same time, VEGF has function of promoting K562 cell proliferation, and VEGF mRNA may be a new target attached by drugs.     

Influence of hypoxia-inducible factor-1 alpha on lung cancer cell A549 growth in vitro and in vivo

AIM: To study the role of hypoxia-inducible factor-1alpha(HIF-1α) on lung cancer cells A549 growth in vitro and in vivo. METHODS: To observe the growth rate of A549 cells after HIF-1α transfected, A549 cells (1×10⁶/mouse) were inoculated subcutaneously into 20 nude mice, which were randomly divided into two groups: the control group (group A, n=10), the HIF-1α transfected group(group B, n=10). The weights of subcutaneous tumor were detected. The resected specimens were made into paraffin-embedded sections. The proliferating cell nuclear antigen (PCNA) was identified by immunohistochemistry(ISH). The expressions of HIF-1α、 apoptosis-related protein survivin and bcl-2 were analyzed by Western blot. RESULTS: The growth rates of the HIF-1α transfected lung cancer cells A549 were significantly increased, and more importantly, the HIF-1α transfected lung cancer cells A549 was able to enhance lung cancer growth in nude mice(P＜0.05). The PCNA were increased significantly in group B, compared with group A. The expressions of HIF-1α, survivin and bcl-2 in group B were increased significantly than that of group A. CONCLUSIONS: HIF-1α increases lung cancer cells A549 growth in vitro and in vivo and its mechanism may be due to promotion of proliferation and inhibition of apoptosis.     

Role of cyclooxygenase-2 in injury induced by hypoxia/reoxygenation in cultured rat cortical neurons

AIM: To observe the role of cyclooxygenase-2 (COX-2) in injury induced by hypoxia and reoxygenation in cultured rat cortical neurons and protective effects of COX-2 specific inhibitor NS398.METHODS: Primary rat cortical neuronal cells were cultured. Experiments were divided into control group, hypoxia/reoxygenation group and hypoxia/reoxygenation with COX-2 inhibitor group. Cell viability was measured by MTT assay. COX-2 protein expression was examined by Western blotting. Apoptosis was measured by DNA agarose electrophoresis.RESULTS: The expression levels of COX-2 increased significantly after neurons were treated with hypoxia and reoxygenation, compared with control group and hypoxia/reoxygenation with COX-2 inhibitor group (P＜0.05). COX-2 specific inhibitor NS398 protected neurons from death (P＜0.05 and P＜0.01), DNA fragmentation analysis showed DNA fragmentation was inhibited significantly by NS398.CONCLUSION: COX-2 is involved in the pathogenesis of neuron apoptosis induced by hypoxia/reoxygenation. COX-2 specific inhibitor significantly protects cortical neurons against hypoxia/reoxygenation injury and inhibits apoptosis induced by hypoxia.     

Effect of nicotine in vitro on expressions of apoptosis-related genes in human lung adenocarcinoma cells by cDNA microarray

AIM: To investigate effect of nicotine on growth of human lung adenocarnoma cells and expressions of apoptosis-related gene. METHODS: Lung adenocarcinoma cell line, SPC-A-1, was cultured in the presence of various concentrations (1-1 000 μg/L) of nicotine for 48 hours. MTT was applied to evaluate effect of nicotine in vitro on growth of SPC-A-1 cell line. After SPC-A-1 cells were treated with 100 μg/L for 48 hours, cDNA expression profile microarray was used to detect the expressions of 451 apoptosis-related genes in SPC-A-1 cell line. RESULTS: Significant proliferation in SPC-A-1 cells treated with nicotine (1-10 μg/L) was observed, but this effect decreased with increase in concentration of nicotine in culture. Growth inhibition rate of 1, 10, 100, 1 000 μg/L of nicotine was 27%, -40%, -40% and -93%. Microarray detection showed that significantly different expressions appeared in 80 of 451 apoptosis-related genes. 29 apoptosis-promoted genes and 26 apoptosis-inhibited genes were up-regulated significantly (CY3/CY5>2.0), and 25 genes were significantly down-regulated (CY3/CY5<0.5). CONCLUSION: Nicotine may promote growth of human lung adenocarcinoma cell through regulating many apoptosis-related gene expressions.     

Activation of caspase-3 during bacterial redox protein azurin -induced apoptosis in U2OS cells

AIM: To study whether caspase-3,8 is activated during azurin-induced apoptosis in U2OS cells. METHODS: AnnexinV /PI method was used to detect apoptosis. The changes of procaspase-3 were analyzed by Western blot, the changes of caspase-3 mRNA were detected by semi-quantitative RT-PCR, and caspase-3 relative activity was determined by colorimetric assay. RESULTS: After U2OS cells were treated with 0, 25, 50, 100, 200, 500 mg/L azurin for 24 h, respectively, the level of procaspase-3 protein decreased and the level of caspase-3 mRNA increased as azurin concentration increased. When the cells were treated with 100 mg/L azurin for 6, 12, 24, 48 h, respectively, the caspase-3 activity began to rise from 6 h,reached the peak at 24 h,and was still higher than the control group at 48 h (P<0.01). After the cells were treated with azurin at different concentrations for 24 h, caspase-3 activity increased in a concentration -dependent manner. CONCLUSION: Caspase-3 is activated and plays an important role in azurin-induced apoptosis in U2OS cells.     

Effect of Yigu capsule drug-containing serum on apoptosis and tartrate resistant acid phosphatase secretion in rat osteoclasts

AIM:The effects of Yigu capsule on tartrate resistant acid phosphatase (TRAP) secretion and apoptosis in rat osteoclasts were investigated in order to further explore its mechanism of preventing and treating osteoporosis.METHODS:(1) Twenty-month-old Sprague-daweley rats were randomly divided into two groups(Yigu capsule group and saline group), and the drug-containing serum and control serum were prepared. (2) The newborn Sprague-daweley rat osteoclasts were cultured with different concentrations of Yigu capsule drug-containing serum. At different time point, TRAP activity was measured and the survival osteoclast was counted under reverse microscope.The percentage of osteoclast apoptosis was observed under fluorescence microscope after acridine orange staining.RESULTS:TRAP activity was lower and the percentage of osteoclast apoptosis was higher in drug-containing serum group than in control group at 24, 48 and 72 h(P<0.01), respectively, and the survival osteoclasts were less in drug-containing serum group than in control groups at 24, 48 and 72 h(P<0.01).CONCLUSIONS:These data suggest that Yigu capsule drug-containing serum induces apoptosis and inhibits TRAP activity in osteoclasts, which may be one of the mechanisms of Yigu capsule preventing and treating osteoporosis.     

Effects of melatonin on apoptosis of lymphocytes induced by ionizing radiation in mice

AIM: To explore the effect of melatonin (MLT) on the apoptosis of thymocytes and splenocytes in mice induced by ionizing radiation and its mechanism. METHODS: The percentages of apoptotic bodies and the DNA lytic rates of thymocytes and splenocytes in mice in vitro and in vivo were detected with flow cytometry and fluorospectrophotometry, respectively. RESULTS: The apoptosis of mouse thymocytes and splenocytes in vitro increased with significant dose-dependence in 0.5-6.0 Gy X-irradiation. When MLT of 2 mmol·L^-1 was added into thymocytes or splenocytes in vitro before irradiation with 0.5-6.0 Gy X-rays, the percentages of apoptotic bodies and the DNA lytic rates all decreased significantly as compared with those in the irradiation group. The percentages of apoptotic bodies in these two kinds of cells were 86.25% and 89.22% of those in the irradiation group, respectively, and the DNA lytic rates were 87.23% and 89.16%, respectively. When MLT was injected into intraperitonium in mice 60 min before whole-body irradiation with 2 Gy X-rays, the percentages of apoptotic bodies and the DNA lytic rates were significantly lower than those in the irradiation group, and near or lower than those in the sham-irradiation group. MLT of 0.1-2.5 mg/kg decreased the lymphocyte apoptosis, but without significant dose-dependence. CONCLUSION: The protective effects of MLT on mouse lymphocytes damaged by irradiation in vivo were obvious than those in vitro.     

Neuronal apoptosis and the chance of apoptosis-related proteins in ovariectomied rats with the effect of App17-mer peptide

AIM: To observe the expression of apoptosis-related proteins in hippocampal neurons of ovariectomized (OVX) rats and explore the neuroprotective mechanism of the App17-mer peptide. METHODS: Female Wistar rats were randomly divided into three groups. Bilaterally ovariectomized rats with injection of App 17P peptide (3.5 μg in 0.1 mL/per rat, three times a week) formed the experimental group (17P+ OVX group). Anti-AIF, Bcl-2 and Bax antibodies were applied in the immunohistochemistry experiment. TUNEL was employed to detect apoptosis. RESULTS: The number of apoptotic neurons was clearly higher in hippocampal and cortex in OVX group than that in OVX+17P group. Immunohistochemistry demonstrated the increased expression of AIF, Bax in hippocampal neurons of OVX group. OVX group showed a significantly reduced expression of Bcl-2 in hippocampal neurons. Hippocampal tissue from OVX group showed the increased expression of AIF,Bax ,and showed diminished expression of Bcl-2 , treating with App17-mer peptide normalized the expression of these proteins. CONCLUSIONS: The expression of apoptosis-related proteins were abnormal in the OVX rats. App17-mer peptide normalized these changes ; Estrogen deficiency induced neuronal apoptosis. App17-mer peptide diminished apoptosis.     

Significance of CK19 expression in detecting lymph node micrometastases in patients with laryngeal carcinoma

AIM: To evaluate the significance of cytokeratin19 (CK19) expression in diagnosing micrometastases in lymph nodes in patients with laryngeal carcinoma. METHODS: Forty cases of laryngeal carcinoma together with 163 lymph nodes were studied by the staining with hematoxylin and eosin(HE)and immunostaining with antibody against cytokeratin 19 (CK19) on histological sections of the primary tumor and regional lymph nodes. RESULTS: In all cases, CK19 was positively expressed in the primary tumor. Among 163 lymph nodes, metastases were confirmed by HE in 23 lymph nodes, and in 42 lymph nodes staining with immunohistochemistry, micrometastases were found in 19 lymph nodes. Micrometastases in the lymphonodes were significantly related to the T staging. CONCLUSION: Immunohistochemical staining is a valuable method for the detection of node micrometastases in patients with laryngeal carcinoma.     

Changes of MAPKs expression in rat hippocampal neurons after sleep deprivation

AIM: To explore the change and the possible role of MAPKs in rat hippocampus neuron after sleep deprivation. METHODS: The morphology of hippocampus neuron after sleep derivation was observed by TUNEL and HE staining, the activity of ERK was assayed by β-liquid scintillation counting and the expression of JNK was detected by Western blot. RESULTS: In paradoxical sleep deprivation (PSD) group, the number of apoptotic cells in hippocampus was increased. The scores of ERK activity were 1 764.00±941.56. Compared with control groups, the ERK activity was obviously decreased (P<0.05). The JNK expression was 87.5%, which was higher than that in control group. CONCLUSION: These results provide some important evidences that the sleep deprivation could cause changes in MAPKs activity, which may be related to the mechanism of hippocampus neuron apoptosis.     

Changes of serum T-PSA,F-PSA and F/T ratio in patients with prostate cancer (PCa) and its clinical significance

AIM: To evaluate the changes of serum total prostate specific antigen (T-PSA), free prostate specific antigen (F-PSA) and the ratio of F-PSA to T-PSA (F/T) in patients with prostate cancer and its clinical significance. METHODS: The concentrations of T-PSA and F-PSA in serum were measured by micropartical enzyme immunoassay (MEIA) using AxSYM System, and the F/T ratio was calculated. RESULTS: Before operation, the concentrations of T-PSA and F-PSA in patients with PCa were much higher and F/T ratio was significantly lower than that in patients with benign prostate hyperplasia (BPH). T-PSA and F-PSA levels decreased, but F/T ratio increased after operation in PCa and BPH. F/T ratio in 83.5% PCa and 6.5% BPH was less than 0.16. To diagnosis PCa, the sensitivity of F/T ratio was 83.5%, and the specificity was 86.7%. CONCLUSION: Serum T-PSA, F-PSA and F/T ratio are important parameters for the early diagnosis of prostate cancer.