The safety, feasibility and oncological outcomes of laparoscopic completion total gastrectomy for remnant gastric cancer: a prospective study with 3-year follow-up (FUGES-004 study).

BACKGROUND: The efficacy of laparoscopic completion total gastrectomy (LCTG) for remnant gastric cancer (RGC) remains controversial. METHODS: The primary outcome was postoperative morbidity within 30 days after surgery. Secondary outcomes included 3-year disease-free survival (DFS), 3-year overall survival (OS), and recurrence. Inverse probability treatment weighted (IPTW) was used to balance the baseline between LCTG and OCTG. RESULTS: Final analysis included 46 patients with RGC who underwent LCTG at the FJMUUH between June 2016 and June 2020. The historical control group comprised of 160 patients who underwent open completion total gastrectomy (OCTG) in the six tertiary teaching hospitals from CRGC-01 study. After IPTW, no significant difference was observed between the LCTG and OCTG groups in terms of incidence (LCTG vs. OCTG: 28.0% vs. 35.0%, P=0.379) or severity of complications within 30 days after surgery. Compared with OCTG, LCTG resulted in better short-term outcomes and faster postoperative recovery. However, the textbook outcome rate was comparable between the two groups (45.9% vs. 32.8%, P=0.107). Additionally, the 3-year DFS and 3-year OS of LCTG were comparable to those of OCTG (DFS: log-rank P=0.173; OS: log-rank P=0.319). No significant differences in recurrence type, mean recurrence time, or 3-year cumulative hazard of recurrence were observed between the two groups (all P>0.05). Subgroup analyses and concurrent comparisons demonstrated similar trends. CONCLUSIONS: This prospective study suggested that LCTG was non-inferior to OCTG in both short- and long-term outcomes. In experienced centers, LCTG may be considered as a viable treatment option for RGC.

DPY30 promotes colorectal carcinoma metastasis by upregulating ZEB1 transcriptional expression.

DPY30 belongs to the core subunit of components of the histone lysine methyltransferase complex, which is implicated in tumorigenesis, cell senescence, and other biological events. However, its contribution to colorectal carcinoma (CRC) progression and metastasis has yet to be elucidated. Therefore, this study aimed to investigate the biological function of DPY30 in CRC metastasis both in vitro and in vivo. Herein, our results revealed that DPY30 overexpression is significantly positively correlated with positive lymph nodes, epithelial-mesenchymal transition (EMT), and CRC metastasis. Moreover, DPY30 knockdown in HT29 and SW480 cells markedly decreased EMT progression, as well as the migratory and invasive abilities of CRC cells in vitro and lung tumor metastasis in vivo. Mechanistically, DPY30 increased histone H3K4me3 level and promoted EMT and CRC metastasis by upregulating the transcriptional expression of ZEB1. Taken together, our findings indicate that DPY30 may serve as a therapeutic target and prognostic marker for CRC.

DPY30 Promotes Proliferation and Cell Cycle Progression of Colorectal Cancer Cells via Mediating H3K4 Trimethylation.

DPY30, a core subunit of the SET1/MLL histone H3K4 methyltransferase complexes, plays an important role in diverse biological functions through the epigenetic regulation of gene transcription, especially in cancer development. However, its involvement in human colorectal carcinoma (CRC) has not been elucidated yet. Here we demonstrated that DPY30 was overexpressed in CRC tissues, and significantly associated with pathological grading, tumor size, TNM stage, and tumor location. Furthermore, DPY30 knockdown remarkably suppressed the CRC cell proliferation through downregulation of PCNA and Ki67 in vitro and in vivo, simultaneously induced cell cycle arrest at S phase by downregulating Cyclin A2. In the mechanistic study, RNA-Seq analysis revealed that enriched gene ontology of cell proliferation and cell growth was significantly affected. And ChIP result indicated that DPY30 knockdown inhibited H3 lysine 4 trimethylation (H3K4me3) and attenuated interactions between H3K4me3 with PCNA, Ki67 and cyclin A2 respectively, which led to the decrease of H3K4me3 establishment on their promoter regions. Taken together, our results demonstrate overexpression of DPY30 promotes CRC cell proliferation and cell cycle progression by facilitating the transcription of PCNA, Ki67 and cyclin A2 via mediating H3K4me3. It suggests that DPY30 may serve as a potential therapeutic molecular target for CRC.

Inhibition of NLRP3 attenuates sodium dextran sulfate-induced inflammatory bowel disease through gut microbiota regulation.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic, life-threatening inflammatory disease of gastrointestinal tissue characterized by inflammation of the gut. Recent studies have shown that gut microbiota is involved in the pathophysiology of IBD. However, it is unknown whether direct inhibition of NLR family pyrin domain containing 3 (NLRP3) inflammasome regulates IBD and alters gut microbiota. METHODS: Here, the NLRP3 expression was evaluated in the colon of IBD subjects. Then, we investigated the effects of NLRP3 inhibition by MCC950 on the gut microbiota and IBD-like symptoms induced by dextran sulfate sodium (DSS). RESULTS: Firstly, NLRP3 and IL-1ß levels were increased in patients with IBD as compared with healthy individuals. Then, the animal experiment showed that NLRP3 inhibition by MCC950 significantly attenuated IBD-like symptoms such as diarrhea and colonic inflammation in DSS-induced mice. In addition, NLRP3 inhibition inhibited NLRP3/ASC/caspase-1/IL-1ß signaling pathway in the colon, which was over-activated by DSS. Furthermore, MCC950 increased the abundance of phylum Firmicutes, decreased the abundance of phylum Bacteroidetes, and increased the Firmicutes/Bacteroidetes ratio, indicating that the inhibition of NLRP3 inflammasome could regulate the abundance of intestinal flora. According to correlation analysis, NLRP3 might produce its functional role in the regulation of oxidation indicators by changing the gut microbiota composition, especially the phylum Bacteroidota, genus Lactobacillus and species Lactobacillus reuteri. CONCLUSIONS: This study suggests that NLRP3 inflammasome inhibition attenuates IBD-like symptoms by regulating gut microbiota, and provides a basis for the clinical application of NLRP3 as a target for the treatment of IBD.

Conditional survival and recurrence of remnant gastric cancer after surgical resection: A multi-institutional study.

The present study was designed to evaluate the dynamic survival and recurrence of remnant gastric cancer (RGC) after radical resection and to provide a reference for the development of personalized follow-up strategies. A total of 298 patients were analyzed for their 3-year conditional overall survival (COS3), 3-year conditional disease-specific survival (CDSS3), corresponding recurrence and pattern changes, and associated risk factors. The 5-year overall survival (OS) and the 5-year disease-specific survival (DSS) of the entire cohort were 41.2% and 45.8%, respectively. The COS3 and CDDS3 of RGC patients who survived for 5 years were 84.0% and 89.8%, respectively. The conditional survival in patients with unfavorable prognostic characteristics showed greater growth over time than in those with favorable prognostic characteristics (eg, COS3, ≥T3: 46.4%-83.0%, Δ36.6% vs ≤T2: 82.4%-85.7%, Δ3.3%; P < 0.001). Most recurrences (93.5%) occurred in the first 3 years after surgery. The American Joint Committee on Cancer (AJCC) stage was the only factor that affected recurrence. Time-dependent Cox regression showed that for both OS and DSS, after 4 years of survival, the common prognostic factors that were initially judged lost their ability to predict survival (P > 0.05). Time-dependent logistic regression analysis showed that the AJCC stage independently affected recurrence within 2 years after surgery (P < 0.05). A postoperative follow-up model was developed for RGC patients. In conclusion, patients with RGC usually have a high likelihood of death or recurrence within 3 years after radical surgery. We developed a postoperative follow-up model for RGC patients of different stages, which may affect the design of future clinical trials.

Development and External Validation of Web-Based Models to Predict the Prognosis of Remnant Gastric Cancer after Surgery: A Multicenter Study.

BACKGROUND: Remnant gastric cancer (RGC) is a rare malignant tumor with poor prognosis. There is no universally accepted prognostic model for RGC. METHODS: We analyzed data for 253 RGC patients who underwent radical gastrectomy from 6 centers. The prognosis prediction performances of the AJCC7th and AJCC8th TNM staging systems and the TRM staging system for RGC patients were evaluated. Web-based prediction models based on independent prognostic factors were developed to predict the survival of the RGC patients. External validation was performed using a cohort of 49 Chinese patients. RESULTS: The predictive abilities of the AJCC8th and TRM staging systems were no better than those of the AJCC7th staging system (c-index: AJCC7th vs. AJCC8th vs. TRM, 0.743 vs. 0.732 vs. 0.744; P>0.05). Within each staging system, the survival of the two adjacent stages was not well discriminated (P>0.05). Multivariate analysis showed that age, tumor size, T stage, and N stage were independent prognostic factors. Based on the above variables, we developed 3 web-based prediction models, which were superior to the AJCC7th staging system in their discriminatory ability (c-index), predictive homogeneity (likelihood ratio chi-square), predictive accuracy (AIC, BIC), and model stability (time-dependent ROC curves). External validation showed predictable accuracies of 0.780, 0.822, and 0.700, respectively, in predicting overall survival, disease-specific survival, and disease-free survival. CONCLUSIONS: The AJCC TNM staging system and the TRM staging system did not enable good distinction among the RGC patients. We have developed and validated visual web-based prediction models that are superior to these staging systems.

Preoperative radiotherapy improves survival in rectal signet-ring cell carcinoma-a population-based study.

BACKGROUND: To investigate the clinical value of preoperative and postoperative radiotherapy (RT) in patients with rectal signet-ring cell carcinoma (SRCC). METHODS: Using the Surveillance, Epidemiology, and End Results program patients with stage II-III rectal SRCC were retrospectively included between 1988 and 2012. Univariate and multivariate Cox regression analyses were performed to analyze the effect of preoperative and postoperative RT on cause-specific survival (CSS). RESULTS: A total of 292 patients were included: 138 patients received preoperative RT, 101 patients received postoperative RT, and 53 patients underwent surgery alone. Overall, 5- and 10-year CSS was 43.8 and 37.6%, respectively. Preliminary survival analysis demonstrated that preoperative RT improved CSS versus surgery alone, especially in patients with stage III disease. Multivariate analysis demonstrated that preoperative RT was independent predictors for CSS in stage III rectal SRCC. CSS in preoperative and postoperative RT groups was comparable. CONCLUSIONS: Preoperative RT significantly improved survival outcomes in patients with stage III rectal SRCC.

Lymph node dissection improved survival in patients with metastatic thoracic esophageal cancer: An analysis of 220 patients from the SEER database.

BACKGROUND: To assess the clinical value of lymph node dissection and lymph node status in patients with metastatic thoracic esophageal cancer (MTEC). METHODS: The Surveillance Epidemiology and End Results (SEER) database was used to identify patients with MTEC who had undergone esophagectomy from 2004 to 2012. Kaplan-Meier survival analysis and Cox proportional hazard regression were used to identify factors significantly associated with overall survival. RESULTS: A total 220 eligible patients were identified, 162 (73.6%) of which underwent lymph node dissection. The 1-year, 3-year, and 5-year overall survival rates were 55.0%, 17.9%, and 9.2%, respectively; the median survival time was 13 months. Lymph node dissection was an independent prognostic factor of overall survival (hazard ratio: 0.527, 95% confidence interval: 0.377-0.736, p < 0.001). Patients who had undergone lymph node dissection had better overall survival than those who did not (1-year, 62.8% vs. 33.7%; 3-year, 21.4% vs. 7.9%). In patients who had undergone lymph node dissection, multivariate analysis determined that nodal stage was an independent prognostic factor. However, the extent of lymph node dissection was not associated with overall survival. CONCLUSIONS: Lymph node dissection improves survival in patients with MTEC who undergo esophagectomy, and the current lymph node staging can be used as a prognostic factor in patients with MTEC.

Correlation Between Interleukin-1ß-511 C/T Polymorphism and Gastric Cancer in Chinese Populations: A Meta-Analysis.

BACKGROUND Several studies have indicated that interleukin (IL)-1ß-511 C/T polymorphism may contribute to individual susceptibility to gastric cancer, but the results vary among regions and races. No relevant meta-analysis has been conducted in a Chinese population. Therefore, we performed the current meta-analysis to investigate the possible correlation between IL-1ß-511 C/T polymorphism and gastric cancer susceptibility in Chinese subjects. MATERIAL AND METHODS PubMed, EmBase, Cochrane Library, Chinese Biology Medicine (CBM), Chinese National Knowledge Infrastructure (CNKI), and Wanfang databases were searched for case-control studies published before 21 January 2015 and investigating a correlation between IL-1ß-511 C/T polymorphism and gastric cancer susceptibility. Two investigators independently screened the studies, extracted data, and evaluated the quality of included studies with the Newcastle-Ottawa scale. Meta-analysis was conducted with STATA 12.0. RESULTS A total of 27 articles from 28 case-control studies were collected. Meta-analysis showed that IL-1ß-511C/T polymorphism was related to increased susceptibility to gastric cancer in Chinese subjects [T vs. C: OR=1.21, 95%CI (1.07-1.37), P<0.01; TT vs. CC: OR=1.41, 95%CI (1.11-1.80), P<0.01; CT vs. CC: OR=1.26, 95% CI (1.05-1.50), P<0.01; TT+CT vs. CC: OR=1.31, 95%CI (1.08-1.58), P<0.01; and TT vs. CT+CC: OR=1.24, 95%CI (1.05-1.47), P<0.01]. Subgroup analysis showed a significant correlation between IL-1ß-511C/T polymorphism and susceptibility to gastric cancer in residents of southern China and in patients with intestinal-type gastric cancer, but not in residents of northern China or in patients with diffuse gastric cancer. Moreover, H. pylori-infected subjects carrying T (CT+TT) exhibited a relatively higher risk of GC [OR=2.4, 95% CI (1.2-5.1), P=0.02]. CONCLUSIONS IL-1ß-511C/T polymorphism is significantly associated with increased susceptibility to gastric cancer in residents of southern China and in intestinal-type gastric cancer. We also found a synergistic interaction between IL-1ß-511C/T polymorphism and H. pylori infection in the development of GC.

IL-1ß induced RXRα overexpression through activation of NF-κB signaling in gastric carcinoma.

BACKGROUND: Abnormal expression of Retinoid X Receptor α (RXRα) seems to be a frequent incident in a variety of cancers. However, the expression pattern and the mechanisms in gastric carcinoma (GC) remain unclear. METHODS: In GC tissues and cell lines, the expression levels of RXRα mRNA and protein were detected by Q-PCR and Western blot, respectively; the localization of RXRα was evaluated by immunohistochemistry (IHC) or immunocytochemistry (ICC). The effect of IL-1ß on RXRα expression and localization was detected by Western blot and ICC. Nuclear factor-κB (NF-κB) pathway was assessed via Western blot. RESULTS: RXRα expression was markedly elevated at both mRNA and protein levels in GC tissues and cell lines (all P<0.05). The abnormal overexpression of RXRα was predominantly visualized in cytoplasm. IL-1ß significantly induced cytoplasmic expression of RXRα in a time-dependent manner. Co-incubation with IL-1ß enhanced phospho-IKKα (p-IKKα) expression and this effect could be inhibited by the specific inhibitor for NF-κB (all P<0.01). CONCLUSIONS: IL-1ß upregulated RXRα through activation of NF-κB signaling and these suggested a possible clinic significance of retinoid receptor expression in the diagnosis and treatment of GC.

Influence of different treatment modalities on survival of patients with low-grade endometrial stromal sarcoma: A retrospective cohort study.

BACKGROUND: To assess the efficacy of different treatment modalities on the outcome of patients with low-grade endometrial stromal sarcoma (LG-ESS). METHODS: Patients with LG-ESS who received hysterectomy from March 1991 to December 2013 were retrospectively analyzed. The associations between clinicopathologic variables and disease free survival (DFS) were evaluated. RESULTS: One hundred and fourteen patients met the eligibility requirements. All patients received hysterectomy as the main treatment, 17.5% (20/114) of patients received ovarian preservation, and 62.3% (71/114) of patients received lymphadenectomy. Fifty-six patients received chemotherapy, 36 patients received radiotherapy, and 11 patients received endocrine therapy. The median follow-up duration was 40 months. The 5-year and 10-year DFS rates were 91.8% and 77.4%, respectively. The 5-year and 10-year overall survival rates were 96.7% and 96.7%, respectively. Univariate analyses showed that there were no risk factors associated with DFS. Lymphadenectomy, lymph node status, ovarian preservation, chemotherapy, radiotherapy, and endocrine therapy had no significant effect on DFS. CONCLUSIONS: Hysterectomy has been the mainstay of treatment for LG-ESS. The optimal treatment strategy in LG-ESS remains to be determined.

Research of shRNAmir inhibitory effects towards focal adhesion kinase expression in the treatment of gastric cancer.

Gastric cancer is the fourth most common type of malignant tumor, with a poor prognosis. Focal adhesion kinase (FAK) mediates the crosslink of intracellular signaling networks, playing a key role in cell migration and invasion. The aim of the present study was to investigate the effects of FAK interference on the proliferation ability, invasion and metastasis of gastric cancer cells. The FAK-RNAi lentiviral vector was infected into SGC7901 gastric cancer cells in order to observe the in vivo situations of tumor growth and metastasis before and after the FAK interference. The growth of SGC7901 gastric cancer cells in the interference group was significantly inhibited compared with that of the negative control (P<0.05) and the blank control groups (P<0.05), and the FAK expression significantly decreased (P<0.05). The in vitro invasion and metastasis experiments showed that the cell invasion and metastasis abilities of the interference group significantly decreased when compared with those of the negative control (P<0.05) and blank control groups (P<0.05). In the nude mouse subcutaneous tumor transplantation model, the mean ± standard deviation tumor weight of the interference group (1.474±0.9840 g) was lower than that of the negative control (3.134±0.3299 g) and blank control (2.68±0.12 g) groups (P<0.05). In the nude mice, the liver and peritoneal metastasis rates of the interference group were significantly lower than those of the negative control (P<0.05) and the blank control groups (P<0.05), and the FAK mRNA of the interference group significantly reduced (P<0.05). In conclusion, FAK interference could effectively suppress the proliferation, invasion and metastasis of transfected SGC7901 gastric cancer cells, and could inhibit the growth and distant metastasis of gastric cancer in nude mice.

[Study on early postoperative nutritional support in elderly patients with gastric cancer].

OBJECTIVE: To explore the optimal postoperative nutritional support in elderly patients with gastric cancer. METHODS: One hundred and twenty elderly patients with gastric cancer undergoing radical gastrectomy were prospectively enrolled from January 2010 to March 2013 and randomly divided into total parenteral nutrition group(TPN, n=40), early total enteral nutrition group (TEN, n=40) and enteral plus parenteral nutrition group(EN+PN, n=40). Clinical charasteristics including treatment tolerance, nutritional indexes, immune indexes, time to first flatus, incidence of postoperative infection and anastomotic leakage, were analyzed and compared. RESULTS: Treatment tolerance in EN+PN group(97.5%, 39/40) was significantly higher than that in TPN group(82.5%, 33/40) and TEN group(80.0%, 32/40)(both P<0.05). The nutritional indices, including prealbumin, albumin, transferrin, body mass index, and the incidence of anastomotic leakage were similar in the 3 groups(P>0.05). The immune indices, including CD3, CD4, CD4/CD8, were significantly reduced after operation in each group. However, they were significantly higher in EN+PN group and TEN group than those in TPN group(both P<0.05). Furthermore, compared to the TPN group, the incidence of postoperative infection(surgical site infection, pulmonary infection, abdominal infection) was significantly lower and time to first flatus was significantly shorter in EN+PN group and TEN group. CONCLUSIONS: Early enteral nutrition after gastric cancer surgery is safe, simple and feasible. EN plus PN is the best way to administer postoperative nutritional support in elderly patients with gastric cancer.

Adenovirus-mediated tissue-targeted expression of the CDglyTk gene for the treatment of breast cancer.

The aim of this study was to evaluate the selective killing efficacy of adenovirus (Ad)-mediated double suicide genes driven by the kinase domain-containing receptor (KDR) promoter in human breast cancer cells and vascular endothelial cells. Two Ad-mediated double suicide gene systems [with the two suicide genes, thymidine kinase (TK) and cytosine deaminase (CD)] with the KDR promoter (Ad-KDRP-CDglyTK) and the cytomegalovirus (CMV) promoter (Ad-CMV-CDglyTK) were established and transfected into the KDR-expressing MCF7 human breast cancer, EC304 human vascular endothelial and LS174T human colon carcinoma, which does not express KDR, cell lines. The selective killing efficiency and specificity of the double suicide gene system were measured in vitro by the analysis of cellular proliferation and assayed in vivo by subcutaneous injection of MCF7 cells into nude mice. The microvessel density (MVD) in the transplanted tumor was determined by immunohistochemical staining of CD34 cells. Our results showed that the transgenic CDglyTK genes were expressed in three cell lines (MCF7, ECV304 and LS174T) infected with Ad-CMV-CDglyTK. However, of the cells infected with Ad-KDRP-CDglyTK, the transgenic CDglyTK gene was only expressed in the KDR-expressing MCF7 and ECV304 cells, but not in the KDR-deficient LS174T cells. Cell proliferation was significantly reduced in a dose-dependent manner by pre-treatment with ganciclovir (GCV) and 5-fluorocytosine (5-FC) in MCF7 and ECV304 cells with transfected KDRP-CDglyTK genes and the three cell lines transfected with the CMV-CDglyTK genes. Similar results were not observed in the LS174T cells with transfected KDRP-CDglyTK genes. The results of this study show that the tumor-targeted expression of CDglyTK driven by the KDR promoter has a high specificity and performance. The killing effect of the CD/TK fusion gene in the target cells was significantly increased compared with the single suicide gene. The cell cycle of MCF7 and ECV304 cells transfected with KDRP-CDglyTK genes was arrested at the S phase following treatment with the prodrugs. The tumors formed by the MCF7 cells with the double suicide gene system were much smaller and the MVD of the tumor tissue was significantly decreased compared with the control. This study demonstrates that tumor­targeted expression of the CDglyTK gene driven by the KDR promotor may be a novel strategy for the gene therapy of human breast cancer.

[Specific killing effects of combination of recombinant adenovirus containing double suicide gene driven by KDR promoter and survivin antisense oligonucleotide on colorectal cancer cells and vascular endothelial cells].

OBJECTIVE: To evaluate the specific killing effects of combination of recombinant adenovirus mediated double suicide gene driven by KDR promoter and survivin antisense oligonucleotide(ASODN) on colorectal cancer cells and vascular endothelial cells. METHODS: The 293 packaging cells were transfected with the plasmids of pAdEasy-CDglyTK and the recombinant adenovirus were generated. The KDR expressive cells of SW620, ECV304 were infected with adenovirus, meanwhile survivinASODN was transferred into the same cells. The infection rate of adenovirus and transfection efficiency of survivinASODN were observed and the expression of CDglyTK was detected by RT-PCR. The expression of survivin was measured by Western blot. The killing effects and bystander effects on SW620, ECV304 were examined through MTT method. RESULTS: The cells which were infected with the adenovirus mediated double suicide gene could be transfected with the survivin ASODN and the infection rate was not affected as well as the transfection efficiency. The high expression of CDglyTK gene was found in SW620, ECV304 cells infected with recombinant adenovirus and survivin ASODN decreased the survivin protein level. The survival rate of gene therapy group was significantly lower than that of negative group. The combination of survivin ASODN and AdKDR-CDglyTK gene therapy showed significantly lower survival rate of SW620 and ECV304 cells as compared with the AdKDR-CDglyTK or survivin ASODN used alone (P<0.05). The survival rate was slightly lower in GCV 100 microg/ml, 5-FC 2000 microg/ml than that AdKDR-CDglyTK used alone (P>0.05). The combined therapy of AdKDR-CDglyTK and survivin ASODN showed synergistic killing efficacy and more significant bystander effects. CONCLUSION: The combined gene therapy of AdKDR-CDglyTK system and survivin ASODN has stronger specific killing effects on colorectal cancer cells and vein endothelial cells.

[Specific killing effects of combination of double suicide gene and survivin antisense oligonucleotide on breast tumor cells and vein endothelial cells].

OBJECTIVE: To evaluate the selectively killing effects of combination of adenovirus mediated double suicide gene driven by kinase-domain insert containing receptor (KDR) promoter and survivin antisense oligonucleotide on breast tumor cells and vein endothelial cells. METHODS: Human embryonal kidney cells 293 were transfected with the plasmids of pAdEasy-KDR-CDglyTK and the adenovirus was generated. The KDR expressive cells of MCF-7, ECV304 were infected by adenovirus and survivin ASODN was transferred into the same cells meanwhile. The infection rates of adenovirus and transfection efficiency of survivin ASODN were observed and the expression of CDglyTK was detected by RT-PCR. The expression of survivin was measured by Western blot. The killing effects and bystander effects on cells were assessed by MTT assay. RESULTS: The cells infected by the adenovirus mediated double suicide gene could be transfected with the survivin ASODN and the infection rate was not affected as well as the transfection rate. The high expression of CDglyTK gene was found in the two kinds of cells and survivin ASODN decreased the survivin protein level. When survivin ASODN was transferred into MCF-7, ECV304 cells, the survival rates were 56.4% +/- 3.8% and 55.9% +/- 3.6% respectively. The combination of survivin ASODN and AdKDR-CDglyTK gene therapy showed significantly lower survival rate comparing with using each treatment alone (P < 0.05) and the survival rate decreased gradually with the increasing of the concentration of GCV and 5-FC. But the survival rate for combined gene therapy group was slightly lower in GCV 100 microg/ml, 5-FC 2000 microg/ml than that of AdKDR-CDglyTK group (P > 0.05). The combination of survivin ASODN and AdKDR-CDglyTK therapy showed synergistic killing efficacy and more significant bystander effects. CONCLUSION: The combined therapy with AdKDR-CDglyTK system and survivin ASODN shows obvious killing effects on breast tumor cells and vein endothelial cells.

[Targeted killing of colorectal tumor cells by lentiviral constructs containing CD/TK suicide genes and KDR promoter].

OBJECTIVE: To investigate the selective killing of colorectal tumor cells by lentivirus-mediated double suicide gene under the regulation of KDR promoter. METHODS: 293T packaging cells were transfected with the plasmid FGW-KDRP-CD/TK to obtain the infectious viruses. KDR-expressing LoVo cells and LS174T cells that did not produce KDR were transfected with the recombinant virus, and the transfection efficiency was evaluated by the fluorecence microscope. RT-PCR was employed to examine the expression of CDglyTK. After treatment of the cells with 5-FC and GCV, the killing effects on the two cell lines were evaluated. RESULTS: The recombinant construct showed similar infection rate of the two cell lines. RT-PCR demonstrated that CDglyTK gene was expressed only in LoVo cells infected with FGW-KDRP-CD/TK but not in LS147T cells, and the sensitivity of the two cell lines to the prodrugs was significantly different (P<0.001). The killing effect of the double suicide gene was much stronger than that of single suicide gene administered (P<0.001). CONCLUSION: The double suicide gene driven by KDR promoter has specific killing effect on the KDR-expressing colorectal tumor cells.

[Effect of KDR recombinant adenovirus containing double suicide gene on proliferation of human stomach adneocarcinoma SCG7901 cells].

OBJECTIVE: To study the effect of adenovirus (Ad)-mediated fusion gene system driven by KDR promoter on the proliferation of human stomach adneocarcinoma SCG7901. METHODS: The KDR-expressing SCG7901 cells and HepG2 cells that did not express KDR were both transfected with AdEasy-KDR-CDglyTK followed by treatment with the prodrugs 5-FC and/or GCV at different concentrations. The killing effects of the transfection on the cells were evaluated. RESULTS: The expression of green fluorescent protein (GFP) was observed in 95% of the infected SCG7901 and HepG2 cells with the multiple of infection (MOI) of the Ads of 100. Transfection of SCG7901 and HepG2 cells did not produce significant changes in the cell growth, and the infected cells exhibited different sensitivities to the two prodrug: SCG7901 cells infected with rAd were highly sensitive to the prodrugs, but the infected HepG2 cells were not (P<0.01). The killing effect of CDglyTK fusion gene on the target cells was much stronger than that of either the single suicide gene (P<0.01). CONCLUSION: CDglyTK fusion gene system driven by KDR promoter selectively kills the KDR-CDglyTK SCG7901 cells and inhibits their proliferation.

[In vitro killing effect of adenovirus-mediated fusion gene system driven by KDR promoter on gastric cancer cells].

OBJECTIVE: To evaluate the killing effect of adenovirus(Ad)-mediated double suicide gene driven by kinase domain-containing receptor (KDR) promoter on gastric cancer MGC-803 cells. METHODS: The 293 packaging cells were transfected by the plasmids pAdEasy-KDR-CDglyTK to generate infectious viruses. The gastric cancer MGC-803 cells were infected by the Ad followed by treatment with 5-FC and/or ganciclovir at different concentrations. The cell-killing effects were evaluated and the bystander effects analyzed after coculture of the cells without AdKDR-CDglyTK infection with the infected cells at different ratios. The cell cycle distribution was detected by flow cytometry and the pathological changes of the cells were observed by electron microscopy. RESULTS: The infection rate of the resultant recombinant Ad in the cells increased gradually with increment of the multiplicity of infection (MOI) of the Ads. The killing effect of CD/TK fusion gene on the MGC-803 cells was much stronger than that of either of the single suicide gene (P<0.001), and considerable bystander effect was observed. The Ad infection caused MGC-803 cell growth arrest at G(1) phase with onset of apoptotic and necrotic morphologies of the cells as seen under electron microscope. CONCLUSION: The CD/TK fusion gene system driven by the KDR promoter possesses effective killing effect on the KDR-expressing gastric cancer MGC-803 cells.

[Adenovirus mediated double suicide gene under controlling by KDR promoter selectively kills vascular endothelial cells and colorectal tumor cells].

OBJECTIVE: To investigate the selectively killing effect of adenovirus (Ad) mediated double suicide gene under the regulation of KDR promoter on vascular endothelial cells and colorectal tumor cells. METHODS: 293 packaging cells were transfected with the plasmids of pAdEasy- KDR- CDglyTK and pAdEasy- CMV- CDglyTK and the infectious viruses were generated. The KDR expressive cells of ECV304,SW620 and the KDR inexpressive cells of LS174T were infected by two Ads. The infection rate was observed and the expression of CDglyTK was detected by RT- PCR. After treatment with different concentrations of 5- FC and GCV,the killing effect and bystander effect on ECV304,SW620 and LS174T were examined. RESULTS: The titers of these two purified Ads were 2.0 x 10(12 ) pfu/ml. There was no significant difference in infection rate between two recombinant Ads infecting various cells,and the infection rate increased in accordance with the enhancing titers of Ads. RT- PCR demonstrated that there existed the product of CDglyTK gene in all the cells infected by Ad- CMV- CDglyTK and the cells infected by Ad- KDR- CDglyTK except in the SL174T. The curative effect in this system on various cells was shown as follows: (1) All cells infected with Ad- CMV- CDglyTK and some cells of ECV304 and SW620 infected with Ad- KDR- CDglyTK were highly sensitive to the prodrugs,but there was no significant differences among them (P > 0.05); compared with ECV304 and SW620 cells,LS174T cells were not sensitive to the two prodrugs (P< 0.001). (2) The efficacy of double suicide gene was better than that of single suicide gene (P< 0.001). (3) The system had considerable bystander effect. CONCLUSION: The double suicide gene under the regulation of KDR promoter has specific killing effect on the KDR- expressing endothelial cells and colorectal tumor cells.