Novel melatonin-trientine conjugate as potential therapeutic agents for Alzheimer's disease.

Researchers continue to explore drug targets to treat the characteristic pathologies of Alzheimer's disease (AD). Some drugs relieve the pathological processes of AD to some extent, but the failed clinical trials indicate that multifunctional agents seem more likely to achieve the therapy goals for this neurodegenerative disease. Herein, a novel compound named melatonin-trientine (TM) has been covalently synthesized with the natural antioxidant compounds melatonin and the metal ion chelator trientine. After toxicological and pharmacokinetic verification, we elucidated the effects of intraperitoneal administration of TM on AD-like pathology in 6-month-old mice that express both the ß-amyloid (Aß) precursor protein and presenilin-1 (APP/PS1). We found that TM significantly decreased Aß deposition and neuronal degeneration in the brains of the APP/PS1 double transgenic mice. This result may be due to the upregulation of iron regulatory protein-2 (IRP2), insulin degrading enzyme (IDE), and low density lipoprotein receptor related protein 1 (LRP1), which leads to decreases in APP and Aß levels. Additionally, TM may promote APP non-amyloidogenic processing by activating the melatonin receptor-2 (MT2)-dependent signaling pathways, but not MT1. In addition, TM plays an important role in blocking γ-secretase, tau hyperphosphorylation, neuroinflammation, oxidative stress, and metal ion dyshomeostasis. Our results suggest that TM may effectively maximize the therapeutic efficacy of targeting multiple mechanisms associated with AD pathology.

Inducing effects of hepatocyte growth factor on the expression of vascular endothelial growth factor in human colorectal carcinoma cells through MEK and PI3K signaling pathways.

BACKGROUND: Vascular endothelial growth factor plays a key role in human colorectal carcinoma invasion and metastasis. However, the regulation mechanism remains unknown. Recent studies have shown that several cytokines can regulate the expression of vascular endothelial growth factor in tumor cells. In this study, we investigated whether hepatocyte growth factor can regulate the expression of vascular endothelial growth factor in colorectal carcinoma cells. METHODS: Hepatocyte growth factor and vascular endothelial growth factor in human serum were measured by ELISA. The mRNA level of vascular endothelial growth factor was analyzed by reverse transcription-PCR. Western blot assay was performed to evaluate levels of c-Met and several other proteins involved in the MAPK and PI3K signaling pathways in colorectal carcinoma cells. RESULTS: Serum hepatocyte growth factor and vascular endothelial growth factor were significantly increased in colorectal carcinoma subjects. In vitro extraneous hepatocyte growth factor markedly increased protein and mRNA levels of vascular endothelial growth factor in colorectal carcinoma cells. Hepatocyte growth factor induced phosphorylation of c-Met, ERK1/2 and AKT in a dose-dependent manner. Specific inhibitors on MEK and PI3K inhibited the hepatocyte growth factor-induced expression of vascular endothelial growth factor in colorectal carcinoma cells. CONCLUSION: This present study indicates that hepatocyte growth factor upregulates the expression of vascular endothelial growth factor in colorectal carcinoma cells via the MEK/ERK and PI3K/AKT signaling pathways.

Tamoxifen can reverse multidrug resistance of colorectal carcinoma in vivo.

AIM: To investigate the effect of tamoxifen (TAM) on multidrug resistance (MDR) of colorectal carcinoma in vivo and its relationship with estrogen receptor (ER). METHODS: Multidrug resistance was determined by means of semi-quantitative retro-transcription polymerase chain reaction (RT-PCR) to test mdr1 gene mRNA and ER expression was studied by immunohistochemistry. Tumor tissues from three cases of human colon carcinoma, which had mdr1(+)/ER(+), mdr1(+)/ER(-), mdr1(-) expressions, were planted subcutaneously in the neck of nude mice to establish three xenograft models. These models were subdivided into four subgroups randomly: Doxorubicin (DOX)-treated group, TAM-treated group, DOX and TAM group and control group. The dimensions of these xenografts were measured after each course of treatment and the xenografts were removed at the end of the experiments for measurements of weight and the variation of mdr1 mRNA level with RT-PCR. In each course, TAM (15 mg/(kg/d)) was administrated orally per day in the first seven days and DOX (3.6 mg/kg) was injected peritoneally on the first day. Data was evaluated by q and t tests. RESULTS: In the animal models with mdr1(-) tumor, the weights and volumes of the planted tumor in DOX group ((39.1+/-2.29) mg, (31.44+/-1.61) mm(3)) and TAM and DOX group ((38.72+/-2.56) mg, (31.31+/-1.74) mm(3)), which were lesser than that of control group ((45.48+/-3.92) mg, (36.42+/-2.77)mm(3), P = 0.037, P = 0.016 respectively) significantly. In the animal models with mdr1(+)/ER(+) tumor, the weights and volumes of planted tumor were not affected by DOX or TAM treatment; however, in TAM and DOX group ((425.5+/-28.58) mg, (340.35+/-22.28) mm(3)), they were significantly less than that of control group ((634.23+/-119.41) mg, (507.45+/-93.34) mm(3), P = 0.022, P = 0.045 respectively), which are similar to that in the models with mdr1(+)/ER(-) tumor. No significant changes were found in the expressive level of mdr1 mRNA following these treatments. CONCLUSION: The expression of mdr1 gene corresponds to the sensitivity of colon cancer to anti-tumor drugs in vivo. TAM can reverse the MDR of colorectal carcinoma in nude mice, which is independent of the expression of ER; however, no change was observed in the expressive level of mdr1 mRNA.

Laparoscopic versus conventional open resection of rectal carcinoma: A clinical comparative study.

AIM: To evaluate the feasibility of laparoscopic resection of rectal carcinoma and to compare the short-term outcome of laparoscopic procedure with conventional open surgery for rectal cancer. METHODS: Thirty-eight patients with rectal cancer were included in a prospective non-randomized study. The patients were assigned to laparoscopic (n=18) or open (n=18) colorectal resection. Case selection, surgical technique, and clinical and pathological results were reviewed. RESULTS: The operative time was longer in laparoscopic resection group (LAP) than in open resection group (189+/-18 min vs 146+/-22 min, P<0.05). Intraoperative blood loss and postoperative complications were less in LAP resection group than in open resection group. An earlier return of bowel motility was observed after laparoscopic surgery. The overall postoperative morbidity was 5.6% in the LAP resection group and 27.8% in open resection group (P<0.05). No anastomotic leakage was found in both groups. The pathologic examination showed that the length of the resected specimen, the mean number of harvested lymph nodes in laparoscopic resection group were comparable to those in open resection group. CONCLUSION: Laparoscopic total mesorectal excision (TME) for rectal cancer is a feasible but technically demanding procedure. The present study demonstrates the safety of the procedure, while oncologic results are comparable to the open surgery, with a favorable short-term outcome.

Effect of local CTLA4Ig gene transfection on acute rejection of small bowel allografts in rats.

AIM: To evaluate the local expression of CTLA4Ig gene in small bowels and its effect on preventing acute rejection of the small bowel allografts. METHODS: Groups of Wistar rats underwent heterotopic small bowel transplantation from SD rats. The recipients were randomly divided into experimental group (allografts were transfected with CTLA4Ig gene) and control group (non CTLA4Ig gene transfected). In the experimental group, the donor small bowels were perfused ex vivo with CTLA4Ig cDNA packaged with lipofectin vector via intra-superior mesenteric artery before transplantation, and the CTLA4Ig expression in the small bowel grafts post-transplantation was assessed by immunohistology. On d 3, 7 and 10 post-transplantation, the allografts in each group were harvested for the examination of histology and detection of apoptosis. RESULTS: Small bowel allografts treated with CTLA4Ig cDNA showed abundant CTLA4Ig expression after transplantation. Acute rejection of grade I on d 7 and grade II on d 10 after transplantation was noticed in the control allografts, and a dramatically increased number of apoptotic enterocytes in parallel to the progressive rejection could be recognized. In contrast, the allografts treated with CTLA4Ig cDNA showed nonspecific histological changes and only a few apoptotic enterocytes were found after transplantation. CONCLUSION: Local CTLA4Ig gene transfection of small bowel allograft is feasible, and the local CTLA4Ig expression in the allograft can prevent acute rejection after transplantation.

Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene.

AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were >15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P<0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P<0.05, P<0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was >15000 and 214.5+/-31.3 micromol.L(-1), P<0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. CONCLUSION: The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.

Semi-mature MyD88-silenced bone marrow dendritic cells prolong the allograft survival in a rat model of intestinal transplantation.

BACKGROUND: Semi-mature dendritic cells (DCs) may induce tolerance rather than immunity. However, little is known about the regulatory mechanism by which these DCs induce transplant tolerance. Myeloid differentiation factor 88 (MyD88) is a key adaptor of Toll-like receptor signaling, which plays a critical role in DC maturation. Activation of MyD88-silenced immature DCs results in the generation of semi-mature DCs. We explored the possibility of using these DCs to induce intestinal transplant tolerance in rats. METHODS: MyD88 expression was silenced in bone marrow DCs (F344 rats) using small interfering RNAs for 24 hours, at which point, lipopolysaccharide (LPS) was added to the culture for another 48 hours. These cells were analyzed for their in vitro and in vivo tolerizing capacities. RESULTS: Semi-mature DCs expressing moderate levels of MHC class II and low levels of co-stimulatory molecules were found to produce interleukin (IL)-10, while IL-12 production was decreased. In vitro co-culture with completely allogeneic T cells from Wistar rats led to a significant decrease in alloreactive T-cell responses. In vivo, the transfer of semi-mature DCs (1 × 10(6) cells) followed by the transplantation of fully mismatched intestinal grafts (F344 rats) led to significantly prolonged survival compared to rats receiving immature and mature DCs. Serum from semi-mature DC-treated rats contained lower concentrations of the pro-inflammatory cytokines IL-2 and interferon-γ 5 days after transplantation. CONCLUSION: Semi-mature DCs may promote inducible allograft tolerance and this study suggests a new strategy by which to facilitate the induction of transplant tolerance.

[Efficacy of antitumour colon cancer cell vaccine modified by Escherichia coli cytosine deaminase gene].

OBJECTIVES: To evaluate the efficacy of antitumour colon cancer cell vaccine modified by escherichia coli cytosine deaminase (EC-CD). METHODS: Mouse colon cancer cell vaccine CT26/CD was established by gene modification using retrovirus plasmid pLCDSN. Its tumorigenicity and effect on liver metastasis model established with wild-type colon cancer were evaluated. RESULTS: CT26/CD was established successfully and proliferated in medium containing 0.6 g/L G418 stably. EC-CD gene expression on these mutant cells was confirmed by RT-PCR. These mutant cells were more sensitive to 5-fluorocytosine (5-FC) compared with the wild-type ones (P=0.000), and presented excellent bystander effect. CT26/CD had the same tumorigenicity as its parental cells (P=0.892). CT26/CD, combined with the prodrug 5-FC, could inhibit tumor progress and live metastasis, and prolonged the survival of the liver metastasis model animals (P=0.000). CONCLUSION: The colon cancer cell vaccine modified by EC-CD presented anti-tumor effect in vivo, when treated with the prodrug.