The prognosis of advanced gastric cancer and gastroesophageal junction (GEJ) adenocarcinoma is poor. Although che-motherapy prolongs patient survival and improves quality of life to a greater extent best supportive care compared to, the median over-survival of patients with advanced gastric cancer is limited to approximately 7-10 months. With remarkable progress in the understand-ing of molecular mechanisms, molecular-targeted agents have been developed and evaluated in international randomized phaseⅢclini-cal trials. These agents may change the treatment mode of this disease. A ToGA study initially demonstrated that the trastuzumab, the monoclonal antibody of HER-2, as a molecular-targeted agent, in combination with chemotherapy, can prolong the overall survival of patients to 13.8 months. Several agents targeting angiogenesis, c-Met, PARP, and immunotherapy are currently subjected to clinical tri-als. This review summarizes the current status of molecular-targeted therapies for gastric cancer and GEJ adenocarcinoma.

BACKGROUND:Matrix attachment region(MAR)are DNA elements that bound to the nuclear matrices after chromatin digested with restriction endonuclease.Plenty of studies have shown that MAR considered as initiaI point of DNA replication or transcription of regulatory gene.Thereby,construction of MAR expression vector can elevate the overall level of transgene expression,enhance stability of exogenous gene.as welI as increase frequency of stable transfectant cells.OBJECTIVE:To construction pLXSN-CAT recombinant retrovirus vector that containing chloramphenicol acetyltransferase(CAT)via cloning MAR sequence of human.and to explore the influence of MAR on the gene expression.METHODS:An open experiment was performed at the Department of Biochemistry and Molecular Biology.Xinxiang Medical College from September 2007 to December 2007 The PLXSN-CAT vector of CAT was constructed by the laboratory.TaqDNA polymerase,T_4 DNA ligase,DNA Marker,restriction enzyme BamH I,agarose gel DNA purification kit,as well as plasmid purification kit were purchased fromTakara Biotechn0Iogy(Dalian)Co.,Ltd.The sequence of csp-B MAR was amplified by polymerase chain reaction(PCR)method applied to human DNA.The fragment was inserted into retrovirus vector PLXSN-CAT plasmid.The recombinant plasmid was verified by double digestion and DNA sequencing.RESULTS AND CONCLUSION:The length of specific fragment applied by PCR was 931 bp,and the recombinant plasmid PLXSN-CAT-MAR presented two bands:5.9 kb and 931 bp using respective restriction enzymes BamH I The sequence of MAR was confirmed by blasting to Genbank(serial numobr:M6271 6).It suggested that MAR had been cloned into PLXSN-CATR vector correctly.The recombinant retrovirus vector PLXSN-MAR was successfully constructed.

BACKGROUND: Matrix attachment region (MAR), a DNA sequence, is still bound to the nuclear matrices after chromatindigested with restriction endonuclease, not only affects expression of endogenous gene, but also overcames transgenic silence andimproves transcription and expression of exogenous gene. OBJECTIVE: To investigate the influence of ?-interferon MAR of CHO cells on the transgenic expression of chloramphenicolacetyltransferase (CAT). DESIGN, TIME AND SETTING: The opening experiment was performed at the Department of Biochemistry and MolecularBiology, Molecular Institute, Xinxiang Medical College from October 2006 to April 2007. MATERIALS: CHO cell lines were obtained from China Center for Type Culture Collection. The pCATG vector of CAT and G418screening markers were constructed by this laboratory. METHODS: Human ?-interferon MAR by PCR was digested with SacI/KpnI and BamHI/SalI, and was inserted into pCATGvector, which was propagated in Escherichia coli JM109, then extracted and purified followed by enzyme digestion analysis. Vectorof CAT expression cassette and human ?-interferon MAR by the two sides was successfully constructed, and christened aspCAT-MAR. Two methods were compared between CHO cells of pCATG transformation and CHO cells of pCATG-MARtransformation. After G418 selecting, genome DNA of cell lines of G418 was extracted, then primers for PCR to amplify the CATtarget gene fragment was designed. MAIN OUTCOME MEASURES: The activity of CAT was analyzed by ELISA method. It was also tested to see if thepCATG-MAR was stably integrated into genomic DNA in the transfected cells. RESULTS: CHO cells of pCATG transformation was screened to have 16 strains of positive cell, and CHO cells of pCATG-MARtransformation was screened to have 17 strains of positive cell. Human ?-interferon MAR could increase the CAT gene expressionby 2.8 fold. The coefficient of variation of CHO cells of pCATG transformation was 2.065 0, and coefficient of variation of CHOcells of pCATG-MAR transformation was 0.813 1. Genome DNA of stable transformation cell lines was amplified by a fragment of437 bp. The results confirmed the pCAT-MAR vector was stably integrated into genomic DNA. CONCLUSION: Human ?-interferon MAR can increase transgenic expression in CHO cells and decrease the transgenicexpression variation in different transfected cells.

The abdominoperineal excision (APE) is still one of the standard operations for low rectal cancer. The exralevator APE can reduce the positive rate of circumferential margin and perforation rate of rectal cancer, but the incidence of postoperative complications is relatively high. With the continuous development of minimally invasive surgery, the transperineal minimally invasive Abdominoperineal excision (Tpm-APE) is proposed. Compared with traditional APE, the Tpm-APE has potential technical advantages, but there is a lack of large sample and multicenter clinical research evidence. The authors share the design and results of an international multicenter clinical study to investigate the clinical practice of Tpm-APE in the treatment of low rectal cancer.

Objective: This study aims to analyze the therapeutic effect and prognostic factors of carcinoma of parotid gland (CPG). Methods: Data on 103 CPG patients were retrospectively analyzed. The patients were divided into the simple surgery group (Group One) and post-operative radio-chemotherapy group (Group Two). Kaplan-Meier survival analysis, Log-rank test, and Cox re-gression analysis were employed to analyze the five-year overall survival. Chi-square test was applied to compare the local control rate and recurrence-free survival. Logistic regression analysis was used to determine the correlation between all factors and the local control rate. Results:For all patients, the five-year local control rate, five-year recurrence-free survival rate, and five-year overall survival rate were 71.49%, 69.61%, and 76.10%respectively. The five-year local control ratio (81.96%vs. 61.90%), five-year recurrence-free surviv-al (78.69%vs. 59.52%), and five-year overall survival (88.12%vs. 68.50%) were significantly improved in Group Two compared with Group One. The logistic regression analysis showed that the therapeutic method, T staging, as well as pN(+) neck and tumor differentia-tion were significantly correlated to the five-year local control rate and five-year recurrence-free survival (P<0.01). Cox regression anal-ysis showed that therapeutic method, T stage, as well as pN(+) neck and tumor differentiation were significantly correlated to the five-year overall survival (P<0.01). Conclusion:Post-operative radio-chemotherapy can improve the local control and overall survival rates. This therapeutic method is applicable to patients with T3-4 tumors, as well as pN(+) neck and middle-low differentiation.

Objective To explore the effects of silencing Livin gene by RNA interference mediated by lentiviral vector on colorectal cancer HT-29 cell xenograft growth and sensitivity to radiotherapy in nude mice.Methods BALB/c nude mice models were established by subcutaneously inoculating differently treated HT-29 cells into nude mice and the tumor growth situation of tumors was observed by measuring the volume of tumors and the weight of the nude mice at different time points after cell seeding.Livin expression was detected by RT-PCR and immunohistochemistry,respetively.Apoptosis rate was detected by TUNEL.Normal saline,lentivirus carring unrelated sequences,lentivirus caning Livin shRNA were injected intratumorally.All the nude mice were given 10 Gy of 6 MV X-ray irradiation.The changes of mice weight and the tumor volume were measured at different time points and the weight and tumor growth curves were drawn.Results The inhibition rate of tumor volume was(50.04 ± 0.07)％ and the tumor weight of the RNA interfering group was significantly less than that in experimental group compared to the blank and negative groups(F=4.85,P＜0.05),and the inhibition rate of tumor weight was(50.27 ±0.17)％.Relative Livin mRNA expression level in the RNA interfering experimental group was(17.75 ±0.08)％,and was significantly lower than that of the blank group(67.60 ± 0.05)％ and the negative group(68.54 ± 0.03)％(F=89.97,P＜0.01).Livin protein expression level in the RNA inferring group was also significantly lower[(36.00 ± 3.40)％ versus(85.00 ± 3.15)％,(80.33 ± 3.08)％,F=107.32,P＜0.01].The apoptosis rate in the RNA interfering experimental group was significantly higher than that in the blank and the negative groups[(23.67 ± 2.25)％ versus(5.00 ± 1.50)％,(8.33 ± 1.82)％,F=56.94,P＜0.01].Combined with radiotherapy,the tumor volume at different groups had significant difference(F=10.70,P＜0.01),and RNA interfering group was significantly less than negative group and blank group(F=7.01-9.32,P＜0.01).Conclusions Silencing of Livin gene expression by lentiviral vector-mediated RNA interference could inhibit the growth of colorectal HT-29 cell xenograft and increase the sensitivity of the transplanted tumors to radiotherapy.

Objective To investigate whetherthe extract of HUANGPI inhibitthe secretion of TNF-αvia TLR4/MyD88/TRAF6 signaling pathway. Methods ELISA assay was performed to determine TNF-α level in cell culture medium. MTT assay was used to detect the effects of the extract of HUANGPI and LPS on the viabilities of RAW 264.7 cells. Proteinexpressions of TLR4 and TRAF6 were detected by Western blotting assay. Results The extract of HUANGPI inhibited the secretion of TNF-αin a dose-dependent manner. Compared to LPS group , were TNF-αwas significantly suppressed in the cells in LPS+MyD88 inhibitor group , LPS+extract group and LPS+extract+MyD88 inhibitor group,with the corresponding reductions of TLR4 and TRAF6 protein expression at74% and21%,70% and27%,44% and8.5%, respectively. Conclusion MYD88-dependent signaling pathway might be involved in the mechanism underlying the effect of the extract of HUANGPI on suppressing LPS-induced inflammation.

The effects of over-expression of ANXA10 gene on proliferation and apoptosis of hepato-cellular carcinoma cell line HepG2 were elucidated. The human ANXA10 gene was subcloned into the lentiviral vector, PGC-FU, to generate the lentiviral expression vector, PGC-FU-ANXA10. The corrected ANXA10 was confirmed by endoenzyme digestion, and sequencing. Recombinant lentiviruses were produced by 293T cells following the co-transfection of PGC-FU-ANXA10 with the packaging plasmids pHelper1.0 and pHelper2.0. The resulting recombinant lentiviruses carrying ANXA10 were then used to infect human embryonic kidney epithelial cells, and lentiviral particles were produced. The ANXA10 expression in 293T cells was detected by using fluorescent microscope and Western blotting. HepG2 cells were infected, and divided into PGC-Fu-ANXA10 group, PGC-Fu group and HepG2 cell group. The changes of ANXA10 mRNA and protein expression were detected by using RT-PCR and Western blotting respectively. Flow cytometry and MTT assay were performed to examine the changes in cell apoptosis and proliferation respectively. The recombinant PGC-FU-ANXA10 vector was successfully constructed, the ANXA10 protein was detected by using Western blotting, and virus titer was 2×10(8) TU/mL. The recombinant lentiviruses were effectively infected into HepG2 cells in vitro and the infection efficiency was 70%. At 72 h after infection, the ANXA10 mRNA and protein expression levels in PGC-Fu-ANXA10 group were significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05); the in vitro growth inhibition rate of HepG2 cells in PGC-Fu-ANXA10 group was 24.65%, significantly higher than that in PGC-Fu group and HepG2 cell group (P<0.05), but there was no significant difference between PGC-Fu group and HepG2 cell group; the apoptosis rate in PGC-Fu-ANXA10 group, PGC-Fu group and HepG2 cell group was (51.92±1.41)%, (19.00±1.12)% and (3.59±0.89)% respectively. The apoptosis rate in PGC-Fu-ANXA10 group was significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05). The recombinant lentiviruses PGC-FU-ANXA10 were constructed successfully and infected into HepG2 cells. The overexpression of ANXA10 gene can significantly inhibit proliferation and promote apoptosis of HepG2 cells in vitro.

The effects of over-expression of ANXA10 gene on proliferation and apoptosis of hepato-cellular carcinoma cell line HepG2 were elucidated. The human ANXA10 gene was subcloned into the lentiviral vector, PGC-FU, to generate the lentiviral expression vector, PGC-FU-ANXA10. The corrected ANXA10 was confirmed by endoenzyme digestion, and sequencing. Recombinant lentiviruses were produced by 293T cells following the co-transfection of PGC-FU-ANXA10 with the packaging plasmids pHelper1.0 and pHelper2.0. The resulting recombinant lentiviruses carrying ANXA10 were then used to infect human embryonic kidney epithelial cells, and lentiviral particles were produced. The ANXA10 expression in 293T cells was detected by using fluorescent microscope and Western blotting. HepG2 cells were infected, and divided into PGC-Fu-ANXA10 group, PGC-Fu group and HepG2 cell group. The changes of ANXA10 mRNA and protein expression were detected by using RT-PCR and Western blotting respectively. Flow cytometry and MTT assay were performed to examine the changes in cell apoptosis and proliferation respectively. The recombinant PGC-FU-ANXA10 vector was successfully constructed, the ANXA10 protein was detected by using Western blotting, and virus titer was 2×10(8) TU/mL. The recombinant lentiviruses were effectively infected into HepG2 cells in vitro and the infection efficiency was 70%. At 72 h after infection, the ANXA10 mRNA and protein expression levels in PGC-Fu-ANXA10 group were significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05); the in vitro growth inhibition rate of HepG2 cells in PGC-Fu-ANXA10 group was 24.65%, significantly higher than that in PGC-Fu group and HepG2 cell group (P<0.05), but there was no significant difference between PGC-Fu group and HepG2 cell group; the apoptosis rate in PGC-Fu-ANXA10 group, PGC-Fu group and HepG2 cell group was (51.92±1.41)%, (19.00±1.12)% and (3.59±0.89)% respectively. The apoptosis rate in PGC-Fu-ANXA10 group was significantly higher than in PGC-Fu group and HepG2 cell group (P<0.05). The recombinant lentiviruses PGC-FU-ANXA10 were constructed successfully and infected into HepG2 cells. The overexpression of ANXA10 gene can significantly inhibit proliferation and promote apoptosis of HepG2 cells in vitro.
Annexins ; genetics ; Apoptosis ; genetics ; Carcinoma, Hepatocellular ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics