Recombinant protein TRAIL-Mu3 enhances the antitumor effects in pancreatic cancer cells by strengthening the apoptotic signaling pathway.

Pancreatic cancer is a highly malignant type of cancer and its treatment remains a major challenge. The novel recombinant protein TNF-related apoptosis-inducing ligand (TRAIL)-Mu3 has been shown to exert stronger tumor inhibitory effects in colon cancer in vitro and in vivo compared with TRAIL. The present study investigated the antitumor effects of TRAIL-Mu3 on pancreatic cancer cells, and the possible mechanisms were further examined. Compared with TRAIL, TRAIL-Mu3 exhibited significantly higher cytotoxic effects on pancreatic cancer cell lines. The inhibitory effect of TRAIL-Mu3 on the viability of PANC-1 cells was shown to be a caspase-dependent process. The affinity of TRAIL-Mu3 to PANC-1 cell membranes was significantly enhanced compared with TRAIL. In addition, TRAIL-Mu3 upregulated death receptor (DR) expression in PANC-1 cells and promoted the redistribution of DR5 in lipid rafts. Western blotting results demonstrated that TRAIL-Mu3 activated the caspase cascade in a faster and more efficient manner compared with TRAIL in PANC-1 cells. Therefore, TRAIL-Mu3 enhanced the antitumor effects in pancreatic cancer cells by strengthening the apoptotic signaling pathway. The present study indicated the potential of TRAIL-Mu3 for the treatment of pancreatic cancer.

TRAIL inhibits HBV replication and expression by down-regulating liver-enriched transcription factors.

BACKGROUND AND STUDY AIMS: To investigate the role of low-concentration TRAIL on HBV replication and expression. MATERIAL AND METHODS: MTT assay was performed to determine the minimum concentrations of TRAIL protein in HepG2 cell apoptosis. HepG2 cells were transfected by HBV replication plasmid pHBV4.1. After the treatment with low concentration of TRAIL, the culture supernatant was collected to detect HBsAg and HBeAg by ELISA. Proteins were extracted from the resulted cells, followed by total RNA and HBV DNA intermediate replication. Southern Blot and Northern Blot were carried out to detect HBV RNA and HBV DNA replication intermediates, respectively. RT-PCR and Western Blot were carried out to detect gene and protein expressions for HNF4α, PPARα, and RXRα, respectively. RESULTS: 50 ng/ml of TRAIL protein led to significant decline on the secretions of HBsAg and HBeAg. Expression levels of HBV RNA and HBV DNA replication intermediates were significantly decreased too. In addition, gene and protein expressions of HNF4α, PPARα and RXRα also dropped, especially for PPARα whose expressions significantly decreased. CONCLUSION: TRAIL could inhibit HBV replication and expression by downregulating the expressions of liver-enriched transcription factors HNF4α, PPARα, and RXRα.

Expression of TRAIL in liver tissue from patients with different outcomes of HBV infection.

OBJECTIVES: Hepatitis B virus (HBV) infection triggers the production of TRAIL, suggesting that TRAIL may play a role in liver injury after HBV infection. However, it remains unclear whether TRAIL expression in liver tissue correlates with the extent of liver injury caused by HBV infection. The aim of this article was to investigate the correlation of TRAIL expression and disease severity. METHODS: Liver biopsy specimens were collected from 71 patients with different outcomes of HBV infection, including 25 cases of chronic hepatitis B (CHB), 18 cases of severe hepatitis B (SHB), and 28 cases of liver cirrhosis (LC). Besides, specimens from 33 healthy individuals without detectable liver diseases were used as negative control (NC). The expression of TRAIL was measured by immunohistochemistry. RESULTS: Expression of TRAIL in the HBV-infected patients was higher than that in the NC (P<0.001). Among the patients, TRAIL expression in the ones with CHB was significantly higher than that in NC (P<0.001). However, there was no statistically significant difference between patients with SHB and NC or between the ones with LC and NC (P=0.067 and P=0.178, respectively). Moreover, TRAIL expression in patients with CHB was higher than that in patients with SHB or LC (P<0.001 for both), whereas no statistically significant difference was observed between patients with SHB and the ones with LC (P=0.511). CONCLUSION: TRAIL is involved in the inflammatory and immunoregulatory response after HBV infection. However, there was no significant correlation between expression of TRAIL and the extent of liver injury.

[Effects of different Chinese medicine therapeutic methods on the ventricular remodeling and the heart function in chronic heart failure rats of Shen-yang deficiency syndrome].

OBJECTIVE: To observe the effects of warming yang method, nourishing yin method, activating blood method, and the combined treatment method on the ventricular remodeling (VR) and the heart function of heart failure (HF) rats of Shen-yang deficiency syndrome (SYDS). METHODS: The Sprague-Dawley (SD) HF rat model of SYDS was established by continuously intravenous dripping adriamycin after economizing bilateral thyroid tissues. Rats were then randomly divided into the model group (administered with normal saline at the daily dose of 6 mL/kg by gastrogavage), the warming yang group (administered with Wenyang Jianxinling Extractum at the daily dose of 6 mL/kg by gastrogavage), the activating blood group (administered with Ligusticum Wallichii and Salvia Miltiorrhiza Extractum at the daily dose of 6 mL/kg by gastrogavage), the nourishing yin group (administered with Radix Ophiopogonis and Rhizoma Anemarrhenae Extractum at the daily dose of 6 mL/kg by gastrogavage), and the combined treatment group (administered with Yin-Yang Supplementing Extractum at the daily dose of 6 mL/kg by gastrogavage), 10 in each group. Another 10 SD rats were taken as the normal control group. They ate food and drank water freely. All rats were intervened for four successive weeks. The left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), maximal rate of left ventricular pressure of development ( +dp/dtmax), and maximal rate of left ventricular pressure of decline (-dp/dtmax) were observed. The systolic blood pressure (SBP) and the diastolic blood pressure (DBP) were determined. The mean arterial pressure (MAP) was calculated. The heart rate (HR) was recorded. Then all rats were killed and their hearts were taken out to weigh the heart mass (HM), the left ventricular mass (LVM), the right ventricular mass (RVM). The heart mass index (HMI) and the left ventricular mass index (LVMI) were calculated. The mRNA expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP1) in the myocardium were detected using RT-PCR. The serum levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), MMP-9, and TIMP-1 were assayed by double antibody sandwich ELISA. RESULTS: Compared with the normal control group, HR, LVEDP, HM, LVM, HMI, RVM, LVMI, NT-proBNP, MMP-9, and MMP-9 mRNA significantly increased, but SBP, DBP, MAP, LVSP, +/- dp/dtmax, TIMP-1, and TIMP-1 mRNA significantly decreased in the model group with statistical difference (P<0.05). Compared with the model group, SBP and DBP, +/-dp/dtmax increased,while LVEDP, NT-proBNP, and MMP-9 decreased in the warming yang group. HR, LVEDP, HM, LVM, HMI, RVM, LVMI, MMP-9 mRNA, NT-proBNP, and MMP-9 significantly decreased, while TIMP-1 mRNA increased in the activating blood group. HR, LVEDP, +/- dp/dtmax, LVMI, NT-proBNP, and MMP-9 decreased, while TIMP-1 increased in the combined treatment group, showing statistical difference (P<0.05). Compared with the warming yang group, SBP and +dp/dtmax decreased in the nourishing yin group; HR and MMP-9 mRNA decreased in the activating blood group, HR decreased in the combined treatment group, all showing statistical difference (P<0.05). There was no statistical difference in the rest indices (P>0.05). CONCLUSION: Activating blood method and combined treatment method could regulate the expressions of MMP-9 and TIMP1 mRNA of HF rats of SYDS, effectively ameliorate the VR, and improve the HF.

[Immune response enhanced by genes encoding IFN-alpha 8 and T alpha 1 co-inoculated with HBV DNA vaccine].

OBJECTIVES: To evaluate if the humoral immune response of hepatitis B DNA vaccine pVAX1-S2S could be enhanced by Talpha1 and/or IFNa expression plasmid co-inoculated. METHODS: The following mammalian expression recombinant plasmids were constructed: the plasmid pVAX1-S2S expressing hepatitis B surface antigen S2S, the plasmid pVAX1-T/I co-expressing thymosin a and IFNalpha, the plasmid pVAX1-I/S2S co-expressing IFNalpha and S2S. These plasmids were inoculated intramuscularly into several BALB/c mice groups in different combinations. In the co-immunization group 1 (pVAX1-I/S2S), each mouse was inoculated with 100 microg of pVAX1-I/S2S; in the co-immunization group 2 (pVAX1-S2S) each mouse was co-inoculated with pVAX1-S2S and 50 microg of pVAX1-TI; in the control group each mouse was inoculated with 100 microg of pVAX1-S2S. All the immunizations were boosted at 2 and 4 week intervals; then the serum samples were collected to detect the anti-HBs and anti-preS2 strengths. RESULTS: 3, 5 and 8 weeks after the first inoculation, the positive rates of anti-HBs were 12.5%, 12.5%, 62.5% respectively in the co-immunization group 1 and 25%, 50%, 50% in the co-immunization group 2, while those in the control group were 0, 25%, 37.5%. The titers of anti-preS2 in co-immunization group 2 was 5 times higher than those in the other two groups. CONCLUSION: The data shows that Talpha1 and/or IFNalpha expression plasmid co-inoculated with pVAX1-S2S might act as an adjuvant to enhance the humoral immune response induced by pVAX1-S2S.

Production of native protein by using Synechocystis sp. PCC6803 DnaB mini-intein in Escherichia coli.

To directly express native recombinant proteins in Escherichia coli, a new expression vector pSB was constructed using Ssp DnaB mini-intein. Using the vector, native proteins could be produced with the help of C-terminal self-cleavage of the intein. In this study, we cloned hIFNalpha-4 gene into pSB and used E. coli strain Origami B (DE3) as the host. Expression experiments were carried out both in Shake flasks and a 5 L bioreactor. The results indicated hIFNalpha-4 could be expressed in the form of soluble protein with correct folding in E. coli. The maximal hIFNalpha-4 content was 21.7% of total protein, and the antiviral activity of the protein was 1.2x10(8 )IU mg(-1). Overall, good effects were achieved with this system. This intein-mediated protein expression system opens up a useful method for production of native recombinant protein in E. coli.

[Effect of compound salvia injection on nitrate ester tolerance].

OBJECTIVE: To investigate the effect and mechanism of Compound Salvia injection (CSI) on nitrate ester tolerance. METHODS: Eighty-four patients with coronary heart disease (CHD) were randomly divided into three groups, Group A treated with isosorbide dinitrate (ISD, 15 mg, 4 times per day) alone, Group B with ISD plus CSI and Group C with ISD plus vitamin C. The therapeutic course for all groups was 10 days. The tolerance to nitrate ester and blood pressure were monitored. Before and after treatment, the color Doppler ultrasonic apparatus was used to detect the baseline value of humeral arterial internal diameters (D0), the humeral arterial dilatory response under compression [D1, that is, the flow-mediated vasodilation (FMD)] and the vasodilatory response after sucking of nitroglycerin (D2). And the blood levels of endothelin-1 (ET-1), endothelial nitric oxide synthase (eNOS) mRNA expression were determined. The endothelial-dependent vasodilation (EDD) was expressed by (D1 - D0)/D0 x 100%, and the endothelial-independent vasodilation (EID) was expressed by (D2 - D0)/D0 x 100%. RESULTS: (1) The occurrence rate of nitrate tolerance in Group B and C (28.57% and 35.7%) was lower than that in Group A (64.29%), but insignificant difference was found between the former two. (2) After treatment, blood pressure increased in Group A to the level of pre-treatment, that in Group C also increased but still lower than that of pre-treatment, while insignificant increase was observed in Group B, comparison between Group B and C showed significant difference (P < 0.05). (3) After treatment, EID lowered in Group A, EDD increased in Group B and C (P < 0.05), EDD and EID in Group B and C were higher than those in Group A (P < 0.05), and EDD was higher in Group B than in Group C (P < 0.05). (4) After treatment, ET-1 mRNA expression lowered in Group B, eNOS mRNA expression increased in Group B and C, with significant difference as compared with those before treatment and those in Group A (P < 0.05), and eNOS mRNA expression in Group C was lower than that in Group B (P < 0.05). CONCLUSION: CSI could partially prevent the occurrence of tolerance to nitrate ester, with the effect better than vitamin C, the mechanism might be related with its regulation on eNOS, ET-1 mRNA expression and protection on vascular endothelial function.

[Effect of Xiangdan Injection on mRNA expression of endothelial vaso-active factors of patients with coronary heart disease and blood stasis].

OBJECTIVE: To evaluate the effect of Xiangdan Injection on mRNA expression of the endothelial vaso-active factors of patients with coronary heart disease and blood stasis. METHODS: Fifty-six patients were randomly divided into two groups:twenty-eight patients were treated according to the therapeutic guide for coronary heart disease as the control group and 28 were given the same treatment plus Xiangdan Injection as the treated group. The expressions of ET-1 and eNOS mRNA were examined with RT-PCR before experiment and ten days later. RESULTS: The positive rate of eNOS mRNA of the treated group increased, while the positive rate of ET-1 mRNA of the treated group decreased after ten day's treatment, with significant differences as compared with that before the experiment. Xiangdan Injection up-regulated the eNOS mRNA expression and suppressed the ET-1 mRNA expression. Changes of expression were not observed in the control group. CONCLUSION: Xiangdan Injection improves the endothelial function of patients with coronary heart disease and blood stasis by regulating the expressions of ET-1 and eNOS mRNA.

[Early indexes to predict the therapeutic effect of interferon on chronic hepatitis B].

OBJECTIVE: To summarize the clinical changing characters of the clinical markers after interferon treatment in chronic hepatitis B (CHB) and make out practical indexes to predict the effect. METHODS: 150 CHB patients were randomly divided into two groups: therapeutic group (90) and control group (60) in the prospective controlled trial. The levels of endogenous interferon before treatment, interferon antibody at the end of the second month and fourth month after treatment, alanine aminotransferase (ALT) and HBV DNA in the serum were detected. Then the data was analysed to find out indexes for predicting the effect. RESULTS: (1) The clearance rate of HBeAg had no significant difference in age except for 20 - 30 and 30 - 40 (t > 2.331 2, P < 0.01). (2) It was more effective if ALT level was higher than 400 U/L before treatment and it decreased more than 50% two months after treatment. (3) The patients whose HBV DNA was negative (dot hybridization) or less than 10(6) copies/ml before treatment had higher rate of HBeAg clearance. (4) There was no effect on patients whose interferon antibody turned positive at the end of the second month. (5)A predictive method of comprehensive factors was made out, whose sensitivity, specificity, and accuracy were 80%, 100% and 90%, respectively. CONCLUSION: The clinical characters of these Chinese patients are different from those of the westerners and the effects of interferon have close relation to the levels of ALT, HBV DNA and interferon antibody.