Intron-specific shRNA-mediated downregulation of survivin and promotion of apoptosis in HeLa cells.

Overexpression of the survivin gene contributes to tumorigenesis; it has been recognized as an important target for cancer therapy. In the present study, survivin expression was suppressed using recombinant plasmid mediated short hairpin RNAs (shRNAs) that were constructed to target exonic or intronic sequences of the survivin gene. In addition, a negative control shRNA was constructed. HeLa cells were transfected with specific shRNA constructs and the blocking efficiency of each shRNA was assessed at the mRNA and protein levels; and the five shRNA constructs with higher blocking efficiency were selected. Cell apoptosis was assessed by flow cytometry (FCM) following Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Hoechst staining was used to detect the morphological diversity of the nuclei in apoptotic cells. The results demonstrated that survivin expression was effectively reduced by the transfection of shRNAs in HeLa cells. In addition, the apoptotic rates of the shRNA-treated groups were significantly increased compared with the negative control group according to the FCM results. The nuclei of HeLa cells exhibited apoptotic characteristics in the shRNA-treated groups as identified by Hoechst staining. Survivin-targeting shRNAs effectively downregulated the expression of the gene and markedly increased the apoptotic rate of HeLa cells. Data from the present study also indicated that the intron-specific shRNA demonstrate a high efficiency of inhibition of survivin expression and were able to induce cell apoptosis of HeLa cells through RNAi, potentially providing novel target sites for tumor therapy. In conclusion, the present study suggests that intron-specific blocking of survivin by RNAi may provide a tool for anticancer therapy.

Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α.

The aim of the present study was to construct a fast-acting, eukaryotic expression vector in eukaryotic cells based on transmembrane-tumor necrosis factor­α (TM­TNF­α) structure. Two types of recombinant eukaryotic expression vectors were constructed, pcDNA3.1-TM-enterokinase-TNF­α and pcDNA3.1­TM­Factor Xa­TNF­α, according to the TNF­α transmembrane segments. Following the generation of these vectors, mouse embryonic 3T3 fibroblasts were transfected and reverse transcription­polymerase chain reaction and western blotting analyses were used to analyze mTNF­α mRNA and protein expression levels, respectively, in total cellular protein extracts and extracellular fluid. The biological activity of TNF-α in the extracellular fluid was then measured using an MTT assay. The vectors were successfully constructed, and mRNA and fusion proteins were detected in the 3T3 cells. Among the fusion proteins, the one observed in pcDNA3.1-TM-FactorXa-TNF-α-transfected 3T3 cells remained as a transmembrane protein. In addition, treatment of L929 cells with TNF­α derived extracellular fluid samples from pcDNA3.1­TM­FactorXa­TNF­α­transfected 3T3 cells was associated with a dose­dependent reduction in in cell­specific activity. The results indicate that proteins expressed using pcDNA3.1­TM­FactorXa­TNF­α vectors form transmembrane proteins. In addition, the results indicate that, only when coupled with FactorXa activity, the extracellular region of TM­TNF­α forms s­TNF­α, and the controlled expression of the fusion protein is initiated.

Construction of the POT1 promoter report gene vector, and the effect and underlying mechanism of the POT1 promoter in regulating telomerase and telomere length.

By using human genomic DNA as a template to clone protection of telomere 1 (POT1) promoter gene segments and construct the POT1 promoter luciferase report gene vector (pGL3-Control-POT1-promoter), the association between POT1, and the regulation of telomerase and telomere length was investigated. In the present study, two recombinant luciferase report gene vectors were constructed, which included different regions of the POT1 promoter. The plasmids were transformed into DH5α and the positive clones were obtained. The two plasmids termed as pGL3-Control-POT1-promoter-1 and pGL3-Control-POT1-promoter-2, were confirmed using restriction enzyme analysis and sequencing. They were separately and transiently transfected into four types of human tumor cells (A549, H460, HepG2 and HeLa). The transcriptional activities of the POT1 promoter were verified using the dual-luciferase assay. The relative expression of POT1 and human telomerase reverse transcriptase (hTERT), and telomere length were analyzed using quantitative polymerase chain reaction in the four types of non-transfected tumor cells. Using SPSS software, correlations between POT1 promoter activity, and POT1 expression, hTERT expression and telomere length were analyzed. Two POT1 promoter fragments (POT1-promoter-1 and -2) were successfully constructed into the pGL3-Control luciferase report gene vector. POT1-promoter-1 exhibited significantly stronger transcription activity compared with POT1-promoter-2. The results of the partial correlation and linear regression analyses were similar: POT1 promoter activity was identified to be significantly and positively correlated with POT1 expression and telomere length (partial correlation coefficients, both P<0.05; linear regression, both P<0.01). However, POT1 promoter activity and hTERT expression were significantly negatively correlated (both P<0.05). The results obtained in the present study suggest that the POT1 promoter influences telomere length. Furthermore, these data indicated that POT1 promoter activity and POT1, as well as telomere length, may be a useful biomarker for tumor detection and future patient prognosis.

Matrix metalloproteinase-mediation of tumor targeting human recombinant tumor necrosis factor-α fusion protein.

The aim of the present study was to use genetic engineering in order to establish an efficient tumor necrosis factor (TNF)-α fusion protein with low toxicity, which may be used to target tumors. Four types of matrix metalloproteinase (MMP)-mediated tumor targeting human recombinant TNF-α (rhTNF-α) fusion protein vectors were constructed. These were subsequently introduced into Escherichia coli. rhTNF-α fusion protein with a glutathione S-transferase (GST)-tag was purified using GST resin affinity chromatography, and GST-tags were digested using factor Xa. The cytotoxic effects of the fusion protein on L929 cells were determined using MTT assays. At a concentration of 1 pM, the GST-tagged fusion protein exerted no cytotoxic effects on the cells, compared with the negative control cells (P=0.975>0.05). However, at a concentration of 1000 pM, the deblocking fusion protein exerted greater cytotoxic effects on L929 cells, compared with positive control cells (P<0.05). Treatment with the fusion protein also induced cell apoptosis in the nasopharyngeal cancer cell line, CNE-2Z, which secretes high levels of MMP-1. In conclusion, the results of the present study suggested that MMP-mediated rhTNF-α fusion protein induces CNE-2Z cells apoptosis. rhTNF-α exhibits high efficacy and tumor cell targeting capability, with low toxicity effects on healthy cells.

Antitumor effect and mechanism of action of a tumor-targeting recombinant human tumor necrosis factor-α fusion protein mediated by urokinase.

The aim of this study was to investigate the effect of the tumor­targeting recombinant human tumor necrosis factor (rhTNF)­α fusion protein mediated by urokinase on Sl80 tumor­bearing mice, as well as to explore its mechanisms of action. Furthermore, the study aimed to observe the effect of the protein on liver and kidney function. rhTNF­α fusion protein prokaryotic expression vectors were constructed using genetic engineering techniques, and were introduced into Escherichia coli. Expression of the fusion protein was induced, and it was then separated and purified in order to determine its cytotoxic activity on L929 cells. Kunming mice were randomly divided into four groups after being inoculated with S180 tumor cells. The groups were then injected with saline (control group, group S), or saline with 0.1 µg/ml fusion protein (low dose group, group L), 0.2 µg/ml fusion protein (middle dose group, group M) or 0.3 µg/ml (high dose group, group H). The mice were sacrificed after 12 days and liver [mg/kg; (liver weight/body weight) x 1,000] and kidney [mg/kg; (kidney weight/body weight) x 1,000] indices, tumor weight, the percentage reduction in mean tumor size, and the levels of alanine transaminase (ALT), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in each group of mice were determined. In addition, the levels of urokinase­type plasminogen activator (uPA), the expression of bcl­2, bax and vascular endothelial growth factor (VEGF), and the percentage of apoptotic cells were measured with an enzyme­linked immunosorbent assay, streptavidin­biotin complex of immunohistochemistry and terminal deoxynucleotidyl transferase­mediated dUTP nick end labeling, respectively. The fusion protein significantly inhibited the growth of S180 tumor cells in vivo in a dose­dependent manner. With an increase in the dose of fusion protein, ALT, uPA, bcl­2 and VEGF levels decreased, and ALB levels increased. However, liver and kidney indices and bax expression were not significantly altered. Cr and BUN levels did not change significantly in the low and middle dose groups, but did increase in the high dose group. Compared with the control group, the percentage of apoptotic cells in the high­dose group was significantly higher. In conclusion, the fusion protein significantly inhibited S180 tumor growth in a mouse model, possibly by reducing the levels of uPA, bcl­2 and VEGF. There was a mildly toxic effect on the kidneys with the high dose, but a protective effect in the liver.

Targeting antitumor effect of rhTNF-α fusion protein mediated by matrix metalloproteinase-2.

The aim of this study was to examine the tumor therapy, targeting effects and side effects of tumor-targeting rhTNF-α fusion protein mediated by matrix metalloproteinase-2 in an animal model in order to provide experimental data for future development of drugs. The median lethal dose (LD50) was obtained from acute toxicity experiments. The A549 lung cancer xenograft model was established, and then randomly divided into the saline, standard substance, and low-, middle- and high-dose fusion protein experiment groups. Each group was administered drugs for 18 days. The length and width of the xenografts were measured every three days, after which the xenograft growth curve was drawn. The mice were sacrificed in each group following treatment and the tumor volume and weight were measured. The targeting, effectiveness and toxicity of the transformed fusion protein, and pathological changes of tumor and organ tissues were examined by hematoxylin and eosin (H&E) staining. Additionally, biochemical markers were used to detect damage of various organs after protein processing. Cell apoptosis and angiogenesis were determined using terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) testing and immunohistochemistry, respectively, in different dose groups. Tumor growth was markedly retarded in the high-dose experimental and standard hTNF-α groups with antitumor rates of 85.91 and 72.25%, respectively, as compared with the control group. Furthermore, the tumor tissue showed obvious apoptosis (the apoptotic index was 78.78 and 66.65%, respectively) and pathological changes in the high-dose experimental and standard hTNF-α groups. Tumor angiogenesis in each fusion protein group was inhibited (P<0.01) and the biochemical markers of various organs were greatly reduced in the high-dose experimental group (P<0.05). This finding indicated that slight toxic effects of fusion proteins were evident for the heart, liver and kidney. The reforming fusion protein can therefore target tumor tissues and efficiently kill tumor cells, with few side effects.

Fused polypeptide with DEF induces apoptosis of lung adenocarcinoma cells.

To analyze the effects of a new unknown peptide DEF on the growth of tumor cells, a fused polypeptide TAT-DV1-DEF was designed and synthesized. The lung adenocarcinoma cell line GLC-82 treated with TAT- DV1-DEF was analyzed with a cell counting kit 8, and the location of polypeptides in cells was observed under laser confocal microscopy. The efficiency of polypeptide transfection and changes in nuclear morphology were analyzed by flow cytometry and fluorescence microscopy, respectively. Finally, the mechanism of tumor cell growth inhibition was evaluated by Western blotting. We found that TAT-DV1-DEF could significantly inhibit the growth of the lung adenocarcinoma cell line GLC-82, but not the normal human embryonic kidney cell line HEK-293. Polypeptides were found to be mostly localized in the cytoplasm and some mitochondria. The efficiency of polypeptide transfection in the two cell types was approximately 99%. Apoptotic nuclei were observed under fluorescence microscopy upon treatment with polypeptides and DAPI staining. Western blot analyses indicated that the polypeptide inhibition of tumor cell growth was apoptosis dependent. In the present study, we demonstrated that fused polypeptides could induce apoptosis of the lung adenocarcinoma cell line GLC-82, indicating that the new unknown peptide DEF has antitumor effects.

Expression of hPOT1 in HeLa cells and the probability of gene variation of hpot1 Exon14 in endometrial cancer are much higher than in other cancers.

To investigate the expression of hPOT1 in the HeLa cell line and screen point mutations of hpot1 in different tumor tissues a two step osmotic method was used to extract nuclear proteins. EMSA was performed to determine the expression of hPOT1 in the HeLa cell line. PCR was also employed to amplify the exon14 sequence of the hpot1 gene in various of cancer tissues. A SV gel and PCR clean-up system was performed to enrich PCR products. DNAStar was used to analyse the exon14 sequence of the hpot1 gene. hPOT1 was expressed in the HeLa cell line and the signal was gradually enhanced as the amount of extracted nuclear proteins increased. The DNA fragment of exon14 of hpot1 was successfully amplified in the HeLa cell line and all cancer tissues, point mutations being observed in 2 out of 3 cases of endometrial cancer (66.7%) despite the hpot1 sequence being highly conserved. However, the sequence of hpot1 exon14 do not demonstrate point mutations in most cancer tissues. Since hPOT1 was expressed in HeLa cell and the probability of gene point variants was obviously higher in endometrial cancer than other cancers, it may be involved in the pathogenesis of gynecological cancers, especially in cervix and endometrium.

[Adenosine receptors agonists mitigated PAH of rats induced by chronic hypoxia through reduction of renin activity/angiotensin II levels and increase of inducible nitric oxide synthase-nitric oxide levels].

OBJECTIVE: Recent studies showed that adenosine played important roles in vasodilation. This study aimed to investigate the effects of adenosine, its A1 and A2b receptor agonists on pulmonary artery hypertension (PAH) induced by chronic hypoxia in rats by continuously subcutaneous administration with an osmotic pump for 14 days, and to see if rennin angiotensin system and inducible nitric oxygen synthase (iNOS)/nitric oxide (NO) mediate the effects. METHOD: Fifty-six male SD rats were randomly assigned to seven groups. Each group included eight rats. They were normoxic group, hypoxic group, adenosine-treated group [adenosine was administered at a dose of 150 µg(kg·min) under the hypoxic condition], adenosine A1 receptor agonist CPA-treated group [CPA was administered at a dose of 20 µg/(kg·min) under the hypoxic condition], CPA plus selective adenosine A1 antagonist DPCPX-treated group [CPA and DPCPX were administered simultaneously under the hypoxic condition, the dose of CPA was the same as the above, and the dose of DPCPX was 25 µg/(kg·min)], adenosine A2b receptor agonist NECA-treated group [NECA was administered at a dose of 30 µg/(kg·min) under the hypoxic condition], NECA plus selective adenosine A2b receptor antagonist MRS-treated group[ NECA and MRS1754 were administered simultaneously under the hypoxic condition, the dose of NECA was the same as the above, and the dose of MRS1754 was 50 µg/(kg·min)]. Osmotic pumps containing adenosine or selective adenosine A1 receptor agonist (CPA), or nonselective but potent adenosine A2b receptor agonist (NECA) were placed subcutaneously 7 days after hypoxia and continuously administered the agents for 14 days.Mean pulmonary artery pressure (mPAP) was detected after administration of the agents. Then blood samples were taken from heart for measurement of renin activity, angiotensin II (AngII) and endothelin-1 (ET-1) concentration by radioimmunoassay, NO by measuring nitrate. Small pulmonary arteries were prepared for immunoreactivity staining of proliferating cell nuclear antigen (PCNA) and iNOS. RESULT: (1) Chronic hypoxia induced PAH [mPAP: (31.38 ± 3.42) mm Hg]. Adenosine or CPA or NECA administered for 14 days by subcutaneous route attenuated the mPAP [(21.17 ± 3.56) mm Hg, (22.88 ± 2.95) mm Hg, (19.81 ± 2.39) mm Hg, respectively], which showed significant difference when compared with hypoxia group (P < 0.05 respectively). (2) Plasma rennin activity and AngII level in hypoxia group [(2.51 ± 0.25) ng/(ml·h), (83.01 ± 9.38) pg/ml] were significantly higher than that in normoxic group (P < 0.05, respectively).(3) Adenosine treatment decreased the rennin activity and AngII level when compared with hypoxic group(P < 0.05, respectively);CPA and NECA attenuated respectively the rennin activity and AngII level of rats induced by chronic hypoxia (P < 0.05, respectively). (4) Adenosine administration for 14 days attenuated the wall thickness induced by chronic hypoxia (P < 0.05). CPA showed no effect on wall thickness, but NECA significantly attenuated the wall thickness (P < 0.05). (5) The number of iNOS staining positive cells in small pulmonary artery was higher in hypoxia group than in that in normoxic rats (23.75 ± 7.91 vs. 8.00 ± 2.20, P < 0.05). Adenosine or CPA, or NECA administration increased respectively the iNOS expression in rats treated with chronic hypoxia. Chronic hypoxia caused significant decrease of nitric oxide level. Adenosine treatment increased the nitric oxide level in rats treated with chronic hypoxia. CPA and NECA also increased respectively the nitric oxide level in rats treated with chronic hypoxia. Chronic hypoxia caused significant increase of ET-1 level. The ET-1 level in rats treated with adenosine, CPA or NCEA respectively were lower than that in chronic hypoxia rats (P < 0.05). (6) Adenosine treatment partially attenuated the number of PCNA-positively stained cells. NECA treatment also attenuated the PCNA expression, but CPA showed no effect. CONCLUSION: Adenosine and its agonists CPA, NECA administered continually by subcutaneous route attenuate mPAP of rats induced by chronic hypoxia. CPA attenuates mPAP through reduction of RA/AngII activity and balance of NO/ET-1 level. NECA attenuates mPAP by inhibiting PCNA expression and proliferation of mooth muscle of pulmonary artery.

[Human pot1 gene exon12 mutation screening in cultured human carcinoma cell strains (lines)].

OBJECTIVE: To screen the exon12 mutation of pot1 gene in cultured human carcinoma cell strains (lines). METHODS: The chromosomal DNA was extracted from 27 cultured carcinoma cell strains (lines). The exon 12 of pot1 gene was amplified by PCR, and the product was purified and screened. The screening results were compared with the data of GenBank and NCBI and the exon 12 mutations in cultured human carcinoma cell strains (lines) analyzed. RESULTS: The exon12 sequence of pot1 could be specifically amplified using the designed primers. Direct sequence analysis of the PCR products after purification showed that 4 of the 5 carcinoma cell lines of the female genital system such as Hela and HO8910-PM cells shared the same transition (G17722-->C) in exon12 of human pot1 gene resulting in a conversion of G1385-->C in the cDNA and amino acid change of Leu454-->Phe in the translated polypeptide. The rest of the 23 cell strains (lines) from different origins showed no such mutation. CONCLUSION: The exon12 (17,722 bp) is a mutant region specific for female genital system tumor.

[Experimental study of spirulina platensis in treating allergic rhinitis in rats].

OBJECTIVE: To determine the therapeutic effect of spirulina platensis in allergic rhinitis (AR). METHODS: Ovalbumin sensitized white rats used as AR animals were treated with spirulina platensis (SPP). At the end of the treatment, the differences in the behavior science were observed; the changes in the nasal mucosa and mast cell degranulation were studied pathologically; and the levels of serum histamine and total immunoglobulin (Ig) E were determined by enzyme-linked immune sorbent assay. RESULTS: The behavior science score of the SPP treatment group was lower than that of the negative control group (P < 0.01 ) ; inflammatory reaction of nasal mucosa in the SPP treatment group were remarkably relieved; the number of nasal mucosa mastocyte and mast cell degranulation in the SPP treatment group were lower than that of the negative control group (P <0.01 ). The levels of serum histamine and total IgE in the SPP treatment group were lower than that of the negative control group (P <0.01 ). It had no significant difference in the positive control group and the SPP treatment group and the blank control group (P > 0.05 ). CONCLUSION: Spirulina platensis can prevent and treat AR in rats, which implies the possibility of using spirulina platensis for AR patients in the future.

[Effects of polyporus polysaccharide on activity and mRNA expression of inducible nitric oxide synthase in peritoneal macrophages of mice].

OBJECTIVE: To study the mechanisms of the antitumor and immunoregulation functions of polyporus polysaccharide (PPS). METHODS: The production of nitric oxide (NO), the activity and mRNA expression of inducible nitric oxide synthase (iNOS) in peritoneal macrophages of mice administered with different dose of PPS were observed by Griess reaction, fluorimetry assay and RT-PCR, respectively. RESULTS: PPS could elevate the iNOS activity with dose-dependence and stimulate the iNOS mRNA expression of peritoneal macrophages in mice. CONCLUSION: The regulation of PPS on the production of NO in peritoneal macrophages of mice may occur at transcriptional level of iNOS. This indicates that the mechanism of PPS's antitumor and immunoregulation functions may be related to increasing NO output of macrophages through stimulating iNOS's denovo synthesis.

[Effect of adenosine on activity of transcription factor NF-kappa B and cytokines in myocardial tissue of experimental rats with pneumonia].

OBJECTIVE: Recent studies have shown that cytokines TNF-alpha and IL-6 play important roles in myocardial injury or dysfunction. Transcription nuclear factor (NF-kappa B) have been implicated in the regulation of a variety of cytokines in response to cellular defense. The authors observed the activity of NF-kappa B and cytokines TNF-alpha, IL-6 mRNA expression in myocardium to further investigate the mechanism of myocardial injury caused by infectious pneumonia. The therapeutic effect of exogenous adenosine was also studied by observing the influence on NF-kappa B and cytokines. METHODS: Thirty rats were divided into three experimental groups at random, each group had 10 rats. The model of pneumonia was induced by the injection of Staphylococcus aureus into the trachea of rats. Adenosine-treated rats were given daily slow intravenous injection of adenosine at a dose of 150 microg/kg.min for 3 days from the second day. All rats were killed on the fifth day. Myocardial tissues were preserved in liquid nitrogen for examination. Pathological examination of myocardium was done and TNF-alpha and IL-6 mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). NF-kappa B activity was measured by electrophoretic mobility shift assay (EMSA). RESULTS: (1) The myocardium in pneumonia group showed significant pathological lesion when compared with control group (P < 0.01). The pathological lesion of myocardium in adenosine-treated group significantly decreased when compared to pneumonia group (P < 0.05). (2) Significant increase of TNF-alpha and IL-6 mRNA expression was observed in myocardium of pneumonic rats when compared with control group (2.27 +/- 0.27 vs. 1.05 +/- 0.16; 1.89 +/- 0.31 vs. 1.12 +/- 0.25: P < 0.01, respectively). NF-kappa B activity of myocardium in pneumonia group was significantly higher than that in control group (13,033 +/- 1286 vs. 383 +/- 15: P < 0.01). (3) TNF-alpha and IL-6 mRNA expression was significantly decreased in adenosine-treated group when compared with pneumonia group (1.25 +/- 0.18 vs. 2.27; 1.31 +/- 0.25 vs. 1.89 +/- 0.31, P < 0.01, respectively). Comparing to that in pneumonia group, NF-kappa B activity of myocardium in adenosine-treated group was significantly decreased (4 487 +/- 562 vs. 13033 +/- 1286, P < 0.01), but it was still significantly higher than that in control group (4487 +/- 562 vs.383 +/- 15, P < 0.01). CONCLUSIONS: Increased activity of NF-kappa B and subsequent upregulation of TNF-alpha and IL-6 mRNA expression probably play a pivotal role in the mechanism of myocardial injury in rats with pneumonia. Exogenous adenosine can inhibit inflammatory change by lowering NF-kappa B activity and subsequent down-regulation of TNF-alpha and IL-6 expression. Our findings provide novel therapeutic evidence of adenosine in myocardial injury induced by pneumonia in clinic.

[Effect of kebimin decoction in treating allergic rhinitis and on blood levels of nitric oxide and superoxide dismutase].

OBJECTIVE: To observe the therapeutic effect of Kebimin decoction (KD) on allergic rhinitis (AR) and its effect on blood levels of nitric oxide (NO) and superoxide dismutase (SOD) activity. METHODS: Eighty-two AR patients were randomly divided into two groups, the treated group and the control group, 41 in each group. To the treated group, KD was given one dose per day for 10 days as one therapeutic course and 1-3 courses were given successively. The control group was treated with Xinfang Rhinitis capsule for 30 days. Blood levels were determined and compared before and after treatment. RESULTS: The total effective rate in the treated group was 93%, which was better than that in the control group (51%), the difference was significant (chi 2 = 17.704, P < 0.01). Serum level of NO was higher and that of SOD activity was lower in the AR patients than that in healthy persons (P < 0.01), KD could significantly lower the former and increase the latter (P < 0.01). CONCLUSION: The therapeutic effect of KD in treating AR was significant, its mechanism might be related with the lowering of NO and increasing of SOD activity in serum, as well as the scavenging of oxygen free radical.