A self-sustained nanoplatform reverses TRAIL-resistance of pancreatic cancer through coactivating of exogenous and endogenous apoptotic pathway.

Since the 5-year survival rate of pancreatic cancer is only 10.0%, new therapies are urgently needed. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically on tumor cells, nevertheless its clinical application was seriously restricted by resistance and short in vivo half-life. Herein, a novel multifunctional R6ST protein equipped with cell penetrating peptides R6, intrinsic apoptosis inducing tetrapeptide AVPI and soluble TRAIL was designed and constructed. Then, it was recruited to prepare self-sustained nanoplatform (SSN) to reverse TRAIL-resistance of pancreatic cancer through simultaneously promoting extrinsic and intrinsic apoptotic pathway, as well to elongate circulation time. Once administrated, high tumor accumulation and cellular uptake of SSN were achieved through prolonged circulation time, targeting ability of soluble TRAIL to death receptors and positive-charged R6, and further enhanced through reversed upregulation of death receptors on TRAIL-resistant tumor cells by the cumulated artesunate released in cytoplasm as a positive feedback loop. Furthermore, this loop simultaneously promoted extrinsic apoptosis of TRAIL fragment via the upregulated death receptors on TRAIL-resistant pancreatic cancer cells and intrinsic apoptosis of AVPI tetrapeptide via the efficient accumulation and uptake of R6ST on SSN. Hence, SSN exhibited synergistic antitumor effect and provided a new strategy for TRAIL-resistant pancreatic cancer therapy.

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.

A novel TRAIL mutant-TRAIL-Mu3 enhances the antitumor effects by the increased affinity and the up-expression of DR5 in pancreatic cancer.

PURPOSE: Pancreatic cancer is a malignant tumor of the digestive system with poor prognosis and high mortality, and the treatment of pancreatic cancer still remains a major challenge. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis selectively in cancer cells while causing virtually no damage to normal cells, which is promising for cancer therapy. However, many primary tumors and cancer cell lines including various human pancreatic cancer cell lines were found to be resistant to TRAIL-induced apoptosis. Therefore, the purpose of the study was to improve antitumor effect of TRAIL on pancreatic cancer. METHODS: The 114-121 amino acid coding sequence "VRERGPQR" of wild type TRAIL protein that was selected changed into "RRRRRRRR", and the novel membrane-penetrating peptide-alike mutant protein was named TRAIL-Mu3. The antitumor effect of TRAIL-Mu3 was analyzed both in vitro and in vivo. Western blotting, immunofluorescence and flow cytometry were used to investigate the underlying mechanisms. RESULTS: TRAIL-Mu3 could enhance the antitumor effects on pancreatic cancer cell lines, and the antitumor effect of TRAIL-Mu3 was stronger than gemcitabine in vivo. The immunofluorescence results suggested that TRAIL-Mu3 could remarkably enhance the affinity to pancreatic cancer cells. The Western blot results showed that treatment with TRAIL-Mu3 caused a clear cleavage of caspase-3 and caspase-8. In addition, both the Western blot and flow cytometry suggested a significantly up-expression of DR5 in TRAIL-Mu3 group. CONCLUSIONS: Membrane-penetrating peptide-alike mutant-TRAIL-Mu3 induced pancreatic cancer cell death more efficiently than TRAIL, and this effect was supposed to be mediated by the increased affinity to cell membrane, the up-regulation of DR5 and the enhancement of activated caspase.

TRAIL and curcumin codelivery nanoparticles enhance TRAIL-induced apoptosis through upregulation of death receptors.

Active targeting nanoparticles were developed to simultaneously codeliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Curcumin (Cur). In the nanoparticles (TRAIL-Cur-NPs), TRAIL was used as both active targeting ligand and therapeutic agent, and Cur could upregulate death receptors (DR4 and DR5) to increase the apoptosis-inducing effects of TRAIL. Compared with corresponding free drugs, TRAIL-Cur-NPs group showed enhanced cellular uptake, cytotoxicity and apoptosis induction effect on HCT116 colon cancer cells. In addition, in vivo anticancer studies suggested that TRAIL-Cur-NPs had superior therapeutic effect on tumors without obvious toxicity, which was mainly due to the high tumor targeting and synergistic effect of TRAIL and Cur. The synergistic mechanism of improved antitumor efficacy was proved to be upregulation of DR4 and DR5 in tumor cells induced by Cur. Thus, the prepared codelivery nanoparticles may have potential applications in colorectal cancer therapy.

TRAIL mutant membrane penetrating peptide alike (TMPPA) TRAIL­Mu3 enhances the antitumor effects of TRAIL in vitro and in vivo.

The aim of the present study was to prepare a tumor necrosis factor ligand superfamily member 10 (TRAIL) mutant membrane penetrating peptide alike (TMPPA), TRAIL­Mu3, and to investigate its antitumor effects in colorectal cancer in vitro and in vivo. The pMD19/TRAIL plasmid with designed primers was amplified to construct the target gene; it was ligated with an expression plasmid and the expression was confirmed. Subsequently, TRAIL­Mu3 was purified and further confirmed by western blot analysis. Immunofluorescence analysis was used to detect the distribution of TRAIL­Mu3 in colorectal cancer cells. In addition, the present study investigated the antitumor effects of TRAIL­Mu3 on colorectal cancer in vitro and in vivo. A novel TMPPA, TRAIL­Mu3, was synthesized in the present study. Following a series of detection experiments, it was confirmed that the TRAIL­Mu3 gene was obtained and was able to express TRAIL­Mu3 successfully. The immunofluorescence analysis demonstrated that TRAIL­Mu3 exhibited a markedly enhanced affinity to the colorectal cancer cell surface. In addition, TRAIL­Mu3 exerted stronger antitumor effects, compared with TRAIL, on colorectal cancer in vitro and in vivo.

Mapping from frame-driven to frame-free event-driven vision systems by low-rate rate coding and coincidence processing--application to feedforward ConvNets.

Event-driven visual sensors have attracted interest from a number of different research communities. They provide visual information in quite a different way from conventional video systems consisting of sequences of still images rendered at a given "frame rate." Event-driven vision sensors take inspiration from biology. Each pixel sends out an event (spike) when it senses something meaningful is happening, without any notion of a frame. A special type of event-driven sensor is the so-called dynamic vision sensor (DVS) where each pixel computes relative changes of light or "temporal contrast." The sensor output consists of a continuous flow of pixel events that represent the moving objects in the scene. Pixel events become available with microsecond delays with respect to "reality." These events can be processed "as they flow" by a cascade of event (convolution) processors. As a result, input and output event flows are practically coincident in time, and objects can be recognized as soon as the sensor provides enough meaningful events. In this paper, we present a methodology for mapping from a properly trained neural network in a conventional frame-driven representation to an event-driven representation. The method is illustrated by studying event-driven convolutional neural networks (ConvNet) trained to recognize rotating human silhouettes or high speed poker card symbols. The event-driven ConvNet is fed with recordings obtained from a real DVS camera. The event-driven ConvNet is simulated with a dedicated event-driven simulator and consists of a number of event-driven processing modules, the characteristics of which are obtained from individually manufactured hardware modules.

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.

[Construction and expression of protein self-splicing prokaryotic expression vector pTWIN1- AcAPc2].

To express recombinant Ancylostoma caninum anticoagulant peptide-c2 (AcAPc2), a whole cDNA fragment encoding AcAPc2 was achieved by ligation- PCR and inserted into prokaryotic expression vector pTWIN1 for constructing the specific self-splicing prokaryotic expression vector, pTWIN1-AcAPc2; positive recombinants were transformed into E. coli ER2566 for expression research. The recombinant protein, AcAPc2-intein2-CBD, was soluble and expressed in E. coli ER2566 (about 30.1% fusion protein in total protein). AcAPc2-intein2-CBD was characterized to be 41 KD by SDS-PAGE and identified by Western-blot. The recombinant fusion protein was purified to a efficiently high degree by chitin affinity chromatography. After the process of specific self-splicing induced by beta-Mercaptoethanol, the target protein, AcAPc2, was obtained, characterized to be 21 KD by SDS-PAGE and migrated as a dimmer. Molecular weight of AcAPc2 conformed to native dimmer. Bio-information analysis indicated relationship between secondary construction of AcAPc2 and biologic function. These findings greatly facilitate the purification of AcAPc2 and are very important for the additional studies on its anti-coagulation mechanism and its clinical application as anti-coagulation medicine.

[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.

[Cloning and sequence analysis of the molecular-adjuvant gene of the complement C3d of mouse].

OBJECTIVE: Considering that the complement C3d plays a stronger positive role in the control of immunologic system, we constructed the recombinant plasmid of the molecular-adjuvant gene of the complement C3d in order to enhance the immunologic efficacy of the gene vaccine. METHODS: The main points were amplifying the segment of complement C3d gene by using of PCR; inserting it into PGEM-T; detecting it by digesting with Eco RI and sequencing; the whole length being 897 bp; then sub-cloning it into pVAX1. RESULTS: The DNA sequencing confirmed the C3d sequence. CONCLUSION: We have successfully cloned the gene of C3d and constructed the recombinant plasmid of pVAX1 C3d.

[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.