Meta-analysis of simultaneous versus staged resection for synchronous colorectal liver metastases.

AIM: There is no clear consensus on the optimal timing of surgical resection for synchronous colorectal liver metastases (SCLM). This study is a meta-analysis of the available evidence. METHODS: Systematic review and meta-analysis of trials comparing outcomes following simultaneous resection with staged resection for SCLM published from 1990 to 2010 in PubMed, Embase, Ovid and Medline. Pooled odds ratios (OR) or weighted mean differences (WMD) with 95% confidence intervals (95% CI) were calculated using either the fixed effects or random effects model. RESULTS: Nineteen non-randomized controlled trials (NRCT) studies were included in this analysis. These studies included a total of 2724 patients: 1116 underwent simultaneous resection and 1608 underwent staged resection. Meta-analysis showed that shorter hospital stay (P < 0.001) and lower total complication rate (P < 0.001) were observed in patients undergoing simultaneous resection group. The overall survival rate in the simultaneous resection group did not statistically differ with that in the staged resection group at 1 year (P = 0.13), 3 years (P = 0.26), 5 years (P = 0.38), as well as the 1, 3 and 5 years disease-free survival rates (respectively, P = 0.55; P = 0.16; P = 0.12). No significant difference was noted between the two groups in terms of mortality (P = 0.16), intraoperative blood loss (P = 0.06) and recurrence (P = 0.47). CONCLUSION: Simultaneous resection is safe and efficient in the treatment of patients with SCLM while avoiding a second laparotomy. In selected patients, simultaneous resection might be considered as the preferred approach. However, the findings have to be carefully interpreted due to the lower level of evidence and the existence of heterogeneity.

[Expression of multiple tumor suppressor gene p16 and its relationship with prognosis of gastric cancer patients].

OBJECTIVE: To investigate the relationship between p16 expression and cell proliferation and prognosis in gastric cancer patients. METHODS: Gastric cancer cell lines SGC-7901, MKN45, MKN28, human embryonic kidney cell line HEK293, human fibroblast cell line MRC-5, and surgical specimens of gastric carcinoma and adjacent normal gastric mucosa from 65 patients were included in this study. RT-PCR, MTT and FCM assays were used to detect p16 expression in gastric cancer cell lines and surgical specimens of gastric cancer. MTT assay was used to determine cancer cell viability and FCM to detect cell cycle. Kaplan-Meier survival curve and Log-Rank statistics were used to analyze the relationship between p16 expression and survival of petients with gastric cancer. RESULTS: Gastric cancer cell lines were mostly negative for p16 expression, and p16 was re-expressed after the cells transfected with p16 gene by adenovirus AdCMV-p16. p16 re-expression resulted in the decrease of cancer cell viability and cancer cell cycle arrest with increased G(1) phase and decreased S phase. p16 expression in cancer specimens was 32.3% (21/65), significantly lower than the 81.5% (53/65) in normal mucosa (χ(2) = 32.124, P < 0.001). The disease-free survival was significantly shorter in p16-negative patients than that in p16-positive patients (P < 0.01), but not the overall survival (P > 0.05). p16 expression was significantly correlated with differentiation and lymph node metastasis, but not significantly correlated with sex, age, tumor size or invasion depth of the gastric cancer. CONCLUSIONS: p16 gene is important for cancer cell proliferation. The inactivation gives cancer cells a high activity for proliferation and metastasis, and then influences the disease-free survival of gastric cancer patients.

Hepatocellular carcinoma and hepatic adenocarcinosarcoma in a patient with hepatitis B virus-related cirrhosis.

The authors present the case of a 48-year-old man with hepatitis B cirrhosis, who developed two primary malignant liver tumors that were morphologically distinct from each other. The first tumor was a hepatocellular carcinoma and the second tumor, detected 17 months later was a hepatic carcinosarcoma with cholangiocarcinomatous and sarcomatous components, without any hepatocellular carcinoma component. Clonality studies using microsatellite-based loss of heterozygosity (LOH) demonstrated different LOH patterns existed between the hepatocellular carcinoma and the hepatic carcinosarcoma, indicative of different clonal origins. The authors discuss the histogenesis, histopathologic diagnosis, and clinical behavior of hepatic carcinosarcoma.

[Establishment and characterization of a human gallbladder carcinoma cell line EH-GB1 originated from a metastatic tumor].

OBJECTIVE: To establish a human gallbladder carcinoma cell line derived from a metastatic gallbladder carcinoma and identify its biological characteristics. METHODS: Tissue samples were separated from the surgical specimen obtained from a patient with metastatic carcinoma and single-cell suspension was prepared. Then the cells were cultured in DMEM medium supplemented with 15% fetal bovine serum. The morphology of tumor cells was observed under an electron microscope. The cell growth curve was plotted. The tumorigenicity of the cell line was studied by subcutaneous inoculation in SCID mice. The cells were infected by lentiviral vector carrying fluorescent report genes (lenti-GFP and lenti-Red2) separately for expressions of GFP and Red2, respectively. RESULTS: A novel metastatic gallbladder carcinoma cell line was successfully established and named "EH-GB1". It could be passaged for over 20 generations with typical malignant epithelial morphology and a stable growth cycle of 24 h. Tumors were formed in all of the 10 SCID mice inoculated with EH-GB1 cells subcutaneously, and the tumor cells were tumor marker CA19-9-positive. Continuous expressions of fluorescent report genes were observed in EH-GB1 cells infected by lenti-GFP and lenti-Red2. CONCLUSION: EH-GB1 cells might be the first stable cell line of human gallbladder carcinoma established from a metastatic focus of gallbladder carcinoma. This cell line with continuous expressions of GFP and Red2 might be a novel and perfect experimental model for clinical and basic research on gallbladder carcinoma.

[Construction of a RU486 inducible recombinant adenoviral vector carrying murine interleukin-12 gene and experimental treatment of colonic carcinoma].

OBJECTIVE: To construct a RU486 inducible recombinant adenovirus of murine IL-12 protein and study its effect and safety on colonic cancer. METHODS: The replication-defective recombinant adenovirus were produced after cotransfection of shutter vector pDC-RUmIL-12 and adenovirus DNA helper plasmid pBHGloxDeltaE1, 3Cre into HEK293 cells. The recombined adenovirus was purified by CsCl density gradient centrifugation and its titer was determined by end point dilution assay. Expression of this regulatable recombinant adenovirus vector in infected C26 colonic carcinoma cells was tested by ELISA kit in vitro. The tumor model was established by hypodermic inoculation of C26 cells. Sixty tumor-bearing mice were randomly divided into 4 groups: Ad-buffer group; Ad-RUmIL-12 group; Ad-RUmIL-12 + RU486 group and Ad-mIL-12 group, and the treatment effects and side effects were evaluated. RESULTS: The adenoviral vector containing murine IL-12 gene was identify by PCR. The viral titer of Ad-RUmIL-12 was 4.62 x 10(10) pfu/ml. The expression of IL-12 protein was induced by the RU486 and the highest expression (516 +/- 43) pg/ml whereas no significant IL-12 protein was detected without inducer or getting rid of the inducer [(38 +/- 3) pg/ml and (42 +/- 5) pg/ml respectively]. The tumor size increased rapidly in group Ad-buffer and group Ad-RUmIL-12 (P > 0.05). Administration of Ad-RUmIL-12 + RU486 and Ad-mIL-12 were showed to delay markedly the growth of transplanted C26 tumor (P > 0.05). Significantly necrosis was observed in both Ad-mIL-12 and Ad-RUmIL-12 + RU486 experimental groups, but the level of the serum alanine transaminase and the rate of side effect was higher in Ad-mIL-12 group (4/15 and 10/15 respectively, P < 0.05). CONCLUSION: A RU486 regulatable recombinant adenoviral vector containing IL-12 gene was successfully constructed. The expression of vector Ad-RUmIL-12, regulated by inducer RU486 in vivo, can obviously improve safety in tumor treatment and provide a good primer for further researches on in vivo gene therapy.

[Detection of multiple gene mutations in non-small-cell lung cancer by suspension microarray].

OBJECTIVE: To construct a high-throughput suspension microarray for detecting the hotspot gene mutations of p53, p16, retinoblastoma (Rb) and epidermal growth factor receptor (EGFR) and to investigate the significance of this multimarker panel in molecular diagnosis of non-small-cell lung cancer (NSCLC). METHODS: The specific probes of normal or mutated sequences targeting the hotspot mutation sites of p53, p16, Rb and EGFR were designed and immobilized to carboxylated Luminex microspheres (micro-beads). Genomic DNA was extracted from 65 specimens of cancer tissues and 20 adjacent normal lung tissues. p53, p16, Rb and EGFR genes were amplified by PCR, hybridized with the specific probes on the beads and measured using Luminex 100. RESULTS: The single gene mutations of p53, p16, Rb or EGFR in NSCLC specimens were 53.8% (35/65), 20.0% (13/65), 7.7% (5/65) or 35.4% (23/65) respectively. The para-tumor normal tissue specimens were 5.0% (1/20), 5.0%(1/20), 0 and 0 respectively. For combined detections of four genes, the sensitivity, specificity and accuracy were 81.5% (53/65), 90.0% (18/20) and 83.5%(71/85) respectively. The mutation rates of this panel in stage I, stage II and stage III were 78.3% (18/23), 80.0% (16/20) and 86.4% (19/22) respectively. CONCLUSIONS: A high-throughput suspension microarray with a higher specificity and sensitivity has been built. It may be used to simultaneously detect the gene mutations of p53, p16, Rb or EGFR in NSCLC specimens. This suspension microarray is helpful to improve the sensitivity of molecular diagnosis of NSCLC and guide the molecular targeting therapy of NSCLC.

Telomerase-specific oncolytic virotherapy for human hepatocellular carcinoma.

AIM: To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted in telomerase-positive hepatocellular carcinoma. METHODS: CNHK300, ONYX-015 (55 kDa protein deleted adenovirus) and wtAd5 (wild type adenovirus 5) were compared, and virus proliferation assay, cell viability assay, Western blot and fluorescence microscopy were used to evaluate the proliferation and cytolysis selectivity of CNHK300. RESULTS: The replicative multiples in Hep3B and HepG II after 48 h of CNHK300 proliferation were 40625 and 65326 fold, respectively, similar to that of wtAd5. However, CNHK300 exhibited attenuated replicative ability in normal fibroblast cell line BJ. CNHK300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection (MOI), but could not affect growth of normal cells even at a high MOI. CONCLUSION: CNHK300 is a cancer-selective replication-competent adenovirus which can cause oncolysis of liver cancer cells as well as wtAd5 (wild type adenovirus 5), but had severely attenuated replicative and cytolytic ability in normal cells. This novel strategy of cancer treatment offers a promising treatment platform.

Antitumor activity of an hTERT promoter-regulated tumor-selective oncolytic adenovirus in human hepatocellular carcinoma.

AIM: To construct a tumor-selective replication-competent adenovirus (RCAd), SG300, using a modified promoter of human telomerase reverse transcriptase (hTERT). METHODS: The antitumor efficacy of SG300 in hepatocellular carcinoma was assessed in vitro and in vivo. In vitro cell viability by MTT assay was used to assess the tumor-selective oncolysis and safety features of SG300, and in vivo antitumor activity of SG300 was assessed in established hepatocellular carcinoma models in nude mice. RESULTS: SG300 could lyse hepatocellular carcinoma cells at a low multiplicity of infection (MOI), but could not affect growth of normal cells even at a high MOI. Both in Hep3B and SMMC-7721 xenograft models of hepatocellular carcinoma, SG300 had an obvious antitumor effect, resulting in a decrease in tumor volume. Its selective oncolysis to tumor cells and safety to normal cells was also superior to that of ONYX-015. Pathological examination of tumor specimens showed that SG300 replicated selectively in cancer cells and resulted in apoptosis and necrosis of cancer cells. CONCLUSION: hTERT promoter-regulated replicative adenovirus SG300 has a better cancer-selective replication-competent ability, and can specifically kill a wide range of cancer cells with positive telomerase activity, and thus has better potential for targeting therapy of hepatocellular carcinoma.

[CNHK200-hA-a gene-viral therapeutic system and its antitumor effect on lung cancer].

OBJECTIVE: To develop a novel vector system, which combines the advantages of the gene therapy, antiangiogenic therapy and virus therapy, and to observe its effect on lung cancer. METHODS: Human angiostatin gene hA(k1-5) was inserted into the genome of the replicative virus specific for the tumor cells by virus recombination technology. The expression of hA(k1-5), its effect on tumor growth in vitro and in vivo were studied. RESULTS: A new kind of gene-viral vector system, designated as CNHK200-hA(k1-5), in which the E1b55 000 gene was deleted but the E1a gene of adenovirus preserved, was constructed. The novel vector system possessed the same property as the replicative virus ONYX-015, which replicates in p53- tumor cells but not in normal cells, thus specifically kills tumor cells. In vitro, CNHK200-hA and Ad-hA both could kill A549 tumor cells but the latter needed 100 times more MOI to achieve the same amplitude of cell killing. In vivo, the therapeutic effect of CNHK200-hA on human lung cancer A549 xenograft in nude mice was significantly better than that of Ad-hA and that of tumor-replicative virus ONYX-015. CONCLUSION: CNHK200-hA(k1-5), a novel vector is constructed in which the angiostatin gene is inserted into the genome of the replicative adenovirus cytotoxic to p53-negative tumor cells. It has the advantages of specific tumor targeting, high level gene expression in tumor cells, and potent tumoricidal activity.

[hTERT promoter regulated replication-selective adenovirus CNHK300 in treatment of hepatocellular carcinoma].

OBJECTIVE: To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted at telomerase-positive hepatocellular carcinoma. METHODS: Human liver cancer cell line HepGII and Hep3B, human embryonic kidney cell line 293, and normal human fibroblasts of the line BJ were cultured and added with adenoviruses CNHK300, ONYX-015 (55 000 protein deleted adenovirus), or wtAd5 (wild type 5) with different multiplicity of infection (MOI) for 7 days. 293 cells were used to measure the titer of the filial generation virus from different cells. The cell survival rate was calculated by MTT method 2, 4, 6, and 8 days after. Different cells were added with CNHK300 virus and then the E1A protein in the cytoplasm was measured by western blotting. Fluorescence microscopy was used to observe the CNHK300-EGFP proliferation after the cells were cultured and added with the virus for 1.5 hours. RESULTS: The replicative viruses CNHK300 and wtAd5 proliferated rapidly in HepGII and Hep3B cells since 24 hours after inoculation and proliferated 40625 and 65326 times respectively with a proliferation potential similar to that of the wild-type adenovirus and much higher than that of the ONYX-015 virus. CNHK300 of the MOI of 0.0002 killed half of the cancer cells, especially those of the line Hep3B, within 5 approximately 6 days, and CNHK300 virus of the MOI of 0.5 pfu/cell killed almost all the HepGII cells in the 8th day, with a killing power lower than that of the wild-type virus and higher than that of the ONYX-015 cells. The IC(50) was as low as MOI of 0.002 pfu/cell for the Hep3B cell and was as high as MOI of 100 pfu/cell for the BJ cell. CNHK300 was a less powerful killer of fibroblasts than wild-type virus. E1A expression was shown by western blotting in 293 cells and CNHK300-infected liver cancer cells, but not in the CNHK300-infected normal human fibroblasts. Fluorescence microscopy showed only isolated fluorescence-positive fibroblasts till the 10th day of infection, but obvious proliferation of CNHK300-EGFP virus since the 3rd day and fluorescence-positive cells in sheets by the 7th day, however, the fluorescent intensity was weakened since the 10th day. CONCLUSION: Tumor-selective adenovirus CNHK300 replicates in telomerase-positive liver cancer cells efficiently as well as wtAd5 and causes oncolysis, but has severely attenuated proliferation and cytolysis in normal cells.

[Treatment of hepatocellular carcinoma by transfecting interleukin-12 and interleukin-2 fusion gene intrasplenically, an experimental study].

OBJECTIVE: To study the inhibitory effect of retroviral packaging cells injected intrasplenically encoding mouse interleukin-12 (mIL-12) and human interleukin-2 (hIL-2) fusion gene on the growth of hepatocellular carcinoma. METHODS: The retroviral vectors encoding mIL-12 gene, hIL-2 gene, and mIL-12 and hIL-2 genes, GCIL12EXPN, GCXEIL2PN, and GCIL12EIL2PN were constructed and then transfected into the retroviral packaging cells PA317 to construct cells PA317-GCIL12EXPN, PA317-GCXEIL2PN, and PA317-GCIL12EIL2PN. Rat hepatocellular carcinoma cells CBRH3 were implanted into the livers of Wistar rats to establish hepatoma animal model. Then the rats were divided into 5 groups to be injected intrasplenically with normal saline one day after the implantation (0.8 ml/rat, group I, n = 10), blank vector PA317-GCXEXPN one day after the implantation (10(7) cells/rat, group II, n = 10), PA317-GCIL12EXPN containing IL-2 gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group III, n = 40), PA317-GCXEIL2PN containing mIL-12 gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group IV, n = 40), and PA317-GCIL12EIL2PN containing IL-12-IL-2 fusion gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group V, n = 40) respectively. The rats surviving longer than 2 months were re-injected with hepatocellular carcinoma cells. The therapeutic effect, immune function and toxic effect were evaluated. CT was conducted on the liver before and after the experiment. Laparotomy was performed 3 and 7 days after treatment to resect some of the carcinoma tissues to undergo pathological examination and OX8 immunohistostaining. Serum mIL-12 and hIL-2 were detected one day before and 3, 7, 30, and 60 days after treatment. RESULTS: The average survival times of the rats treated with IL-12-IL-2 fusion gene at the first, third, fifth and seventh day after tumor implantation were 53.3 +/- 3.7 days, 49.3 +/- 4.2 days, 31.0 +/- 2.1 days, and 24.3 +/- 1.4 days respectively, longer than those treated with IL-2 gene (25.0 +/- 2.5 days, 23.5 +/- 2.0 days, 18.3 +/- 2.4 days, and 12.0 +/- 1.8 days respectively, P < 0.001), and those treated with IL-12 gene (39.0 +/- 4.8 days, 32.0 +/- 3.9 days, 23.0 +/- 2.5 days, and 19.4 +/- 2.1 days respectively, P < 0.001). Long survival (>or= 60 days) rate in the rats treated with IL-12-IL-2 fusion gene on the first and third day was 30%. The serum mIL-12 and hIL-2 levels in these rats remained high on the 60th day after treatment. The pathological study showed that the number of infiltrating lymphocytes in liver tumor tissues was increased in the IL-12-IL-2 fusion gene treatment group. CONCLUSION: The retroviral packaging cell line injected intrasplenically encoding mIL-12 and hIL-2 fusion gene inhibits the growth of hepatocellular carcinoma significantly in rats. The therapeutical efficacy of early administration is superior to that of late one.

[Treatment of hepatocellular carcinoma with a novel gene-viral therapeutic system CNHK300-murine endostatin].

OBJECTIVE: To investigate the anti-tumor effects of a novel gene-viral therapeutic system CNHK300-murine endostatin (CNHK300-mE) in hepatocellular carcinoma (HCC). METHODS: A novel gene-viral therapeutic system named CNHK300-mE was constructed by employing the human telomerase reverse transcriptase (hTERT) promoter to drive the expression of adenovirus E1A gene and cloning the therapeutic gene murine endostatin (mE) into the adenovirus genome. Hepatocellular cells of the HepGII and Hep3B lines and normal fibroblasts of the MRC-5 line were cultured and infected with the viruses CNHK300-mE, ONYX-015, replicative adenovirus without therapeutic gene, and Ad-mE, non-replicative adenovirus with the same therapeutic gene. Ninety-six hours after the infection, tissue culture infectious dose 50 method was used to detect the titer of virus in the supernatants. MTT method was used to examine the cytolytic capability. The expression of E1A and mE were examined by Western blotting. ELISA assay was used to detect the transgene expression of mouse endostatin. Healthy nude Balb/c mice were injected with hepatic cancer cells of the SMMC 7221 line. Forty mice with tumors 5 approximately 8 mm in diameter were randomly divided into 4 groups of 20 mice: CNHK300-mE group (CNHK300-mE was injected into the tumor once every other day for 5 times), Ad-mE group (Ad-mE was injected), ONYX-015group (ONYX-015 was injected), and control group (diluent of virus was injected). 3, 7, 14, 21, and 28 days after the initial injection the size of tumor was examined. 48 hours after the finish of the whole course of treatment, the mice were killed. ELISA was used to detect the expression of mE in blood. The growth of tumor was examined by HE staining, The angiogenesis in the tumor was observed by immunohistochemistry with von Willebrand factor and The proliferation of transplanted tumor was observed by immunohistochemistry with adenovirus envelop protein hexon. RESULTS: Ninety-six hours after the infection of the cells by CNHK300-mE virus was replicated by 6329 +/- 1830 and 25 136 +/- 6890 times in the HepGII and Hep3B cells respectively, 3296 and 12 824 times higher than in the MRC-5 cells respectively. The replication multiples of ONYX-015 virus in the HepGII and Hep3B cells were 2040 +/- 450 and 3980 +/- 740 times respectively, both significantly lower than those of CNHK300-mE virus (both P < 0.05). However, no remarkable replication of Ad-mE virus was seen in the Western blotting showed the expression of therapeutic gene mE in HepGII and Hep3B cells infected with CNHK300-mE on Ad-mE. Hep3B cells, the band of CNHK300-mE being thicker than that of Ad-mE and the band of Ad-mE being similar to that of CNHK300-mE in the MRC-5 cells. ELISA showed that the expression of mE protein in the HepGII cells infected by CNHK300-mE virus increased time-dependently during the period of 7 days after virus infection, significantly higher than the expression in the HepGII cells infected by Ad-mE virus (P < 0.05). The tumors of the CNHK300-mE virus-infected mice were significantly smaller than those of the Ad-mE and ONYX-015-infected mice (both P < 0.01). ELISA showed that the mE protein content in the blood of the CNHK300mE-infected mice was significantly higher than that of the Ad-mE group (P < 0.05). Hexon immunohistochemistry showed patchy and diffuse positive staining related to apoptosis and necrosis of tumor cells in the transplanted tumors of the CNHK300-mE virus-infected mice, however, only sporadic positive staining was seen in the Ad-mE virus-infected mice. CONCLUSION: Being capable of specifically replicating in the telomerase-positive HCC cells and mediating effective expression of therapeutic gene in vitro and in vivo, the novel gene-viral therapeutic system CNHK300-mE holds potential for treatment of HCC.

Antiangiogenesis gene armed tumor-targeting adenovirus yields multiple antitumor activities in human HCC xenografts in nude mice.

AIM: Gene therapy represents a promising therapeutic strategy for hepatocellular carcinoma (HCC). To improve the ratio of killing efficacy on tumor cells to side-effect on normal cells, we constructed an oncolytic adenovirus vector, AdSu-hE, expressing the human endostatin (hE) gene, in which the chimeric promoter of human epidermal growth factor receptor 2 enhancer and human telomerase reverse transcriptase promoter was used to control the adenoviral E1a gene. METHODS: Tumor-selective replication of adenovirus AdSu-hE and its concomitant expression of endostatin were measured by 50% tissue culture infective dose method, fluorescent protein expression, Western blot and enzyme linked immunosorbent assay in cancer and normal cell lines. The antitumor efficacy was observed in nude mice bearing human HCCs. RESULTS: The oncolytic adenovirus AdSu-hE replicated restrictedly in telomerase-positive cancer cells and resulted in oncolysis, but did not replicate in normal cell lines. Along with virus replication, AdSu-hE mediated 5-fold increased expression of endostatin in tumor cells compared with that in normal cells. Moreover, AdSu-hE expressed more endostatin in cancer cells than the non-replicative adenovirus vector Ad-hE. In vivo administration of the oncolytic adenovirus AdSu-hE into HCC-bearing nude mice produced a significant tumor reduction by synergistic effects of virus oncolysis and endostatin antiangiogenesis. CONCLUSION: The oncolytic virus with antiangiogenesis gene driven by the chimeric promoter has an improved killing efficacy on tumor cells, and may be useful for cancer gene therapy.

E1B-55kD-deleted oncolytic adenovirus armed with canstatin gene yields an enhanced anti-tumor efficacy on pancreatic cancer.

Conditionally-replicating adenovirus (CRAd) therapy is currently being tested against pancreatic cancer and has shown some promise. To improve the efficacy, a novel virus CRAd-Cans was designed by deletion of E1B-55kDa gene for selective replication in tumor cells, as well as carrying a new angiogenesis inhibitor gene, canstatin. CRAd-Cans mediated higher expression of canstatin in BxPC-3 pancreatic cancer cell line compared to the replication-deficient adenovirus Ad5-Cans. The modified CRAd-Cans manifested the same selective replication and cytocidal effects in pancreatic cancer cells as ONYX-015 in vitro, yet showed greater reduction of tumor growth in nude mice with markedly prolonged survival rate in vivo (P<0.05), compared to that of either ONYX-015 or Ad5-Cans. Pathological examination revealed viral replication, decreased microvessel density and increased cancer cell apoptosis in CRAd-Cans-treated xenografts. The results suggest that the novel oncolytic virus CRAd-Cans, showing synergistic effects of oncolytic therapy and anti-angiogenesis therapy, is a new promising therapeutics for pancreatic cancer.

[Construction and expression of RU486-inducible eukaryotic vector carrying red fluorescent protein].

OBJECTIVE: To construct an inducible eukaryotic vector carrying red fluorescent protein (DsRed) and evaluate the regulation of DsRed gene expression in vitro. METHODS: The vector pRS17-RUDsRed containing DsRed gene, promoter and RU486-inducible system was constructed using molecular biological methods. To minimize potential interference, the two transcriptional elements were spaced with a 1.6 kb insulator. Fluorescence microscopy and flow cytometry were used to observe the activation of this regulatable vector after transfection in MFC cells. RESULTS: The vector was identified by digestion with different restriction enzymes, sequencing and PCR. In the absence of RU486, the cells transfected with the vector exhibited very low DsRed protein expression, and the addition of RU486 induced efficient DsRed expression in the cells. CONCLUSION: The RU486-inducible eukaryotic vector carrying DsRed protein allows effective regulation of the target gene expression in vitro, which provides a useful tool for gene regulation and gene therapy studies.

[Anti-tumor effect of gene-viral therapeutic system CNHK300-murine endostatin on nude mouse gastric cancer].

OBJECTIVE: To investigate the anti-tumor effect of a novel gene-viral therapeutic system CNHK300-murine endostatin (CNHK300-mE) on gastric cancer. METHODS: SGC-7901 gastric cancer cells (5 x 10(7) cells/mouse) were injected s.c. into the right flank of Balb/c nude mice, grown to 4-5 mm to demonstrate tumor take, and 10(9) pfu/100 microl CNHK300-mE virus was injected into tumors. Tumor sizes were measured with calipers every other day. Serum samples were obtained by retro-orbital puncture and level of endostatin expression in serum was quantitated by ELISA. Fifteen days after treatment, all mice were sacrificed and tumors were excised for immunohistochemical staining of PCNA, hexon and vWF. Tumor cell apoptosis was detected by TUNEL method. RESULTS: From the 7th day post-treatment, the bearing tumors of mice treated with CNHK300-mE were significantly smaller than those of control group treated with PBS. Seven days after treatment, expression of endostatin was (2115 +/- 770) ng/ml, significantly higher than that of control group. Immunohistochemical staining indicated that hexon was expressed in treated tumor cells, and PCNA LI (label index) [(55.0+/-1.4)% vs control (74.1 +/- 0.4)%, P<0.05], microvessel density (MVD) of CNHK300-mE treated tumors decreased significantly. Apoptosis obviously increased in tumor cells[(78.4 +/- 9.1)% vs control (15.2 +/- 0.5)%, P<0.01]. Apoptosis bodies and crystal grid were found in tumor cell nuclear by electron microscope. CONCLUSIONS: Gene-viral therapeutic system CNHK300-murine endostatin can replicate in gastric cancer cells. The mouse endostatin gene cloned into CNHK300-mE expressed in high level. CNHK300-mE may induce tumor cells apoptosis, reduce the expression of PCNA and efficiently suppress gastric cancer growth through inhibiting tumor angiogenesis.