Radiosensitization of metformin in pancreatic cancer cells via abrogating the G2 checkpoint and inhibiting DNA damage repair.

Recent evidences have demonstrated the potential of metformin as a novel agent for cancer prevention and treatment. Here, we investigated its ability of radiosensitization and the underlying mechanisms in human pancreatic cancer cells. In this study, we found that metformin at 5 mM concentration enhanced the radiosensitivity of MIA PaCa-2 and PANC-1 cells, with sensitization enhancement ratios of 1.39 and 1.27, respectively. Mechanistically, metformin caused abrogation of the G2 checkpoint and increase of mitotic catastrophe, associated with suppression of Wee1 kinase and in turn CDK1 Tyr15 phosphorylation. Furthermore, metformin inhibited both expression and irradiation-induced foci formation of Rad51, a key player in homologous recombination repair, ultimately leading to persistent DNA damage, as reflected by γ-H2AX and 53BP1 signaling. Finally, metformin-mediated AMPK/mTOR/p70S6K was identified as a possible upstream pathway controlling translational regulation of Wee1 and Rad51. Our data suggest that metformin radiosensitizes pancreatic cancer cells in vitro via abrogation of the G2 checkpoint and inhibition of DNA damage repair. However, the in vivo study is needed to further confirm the findings from the in vitro study.

[Therapeutic schemes for refractory ascites of advanced schistosomiasis: a clinical control study].

OBJECTIVE: To explore the therapeutic schemes for refractory ascites of advanced schistosomiasis. METHODS: The advanced schistosomiasis patients with refractory ascites were randomly divided into 4 groups: a conventional group, high-dose albumin group, high-dose diuretic group, and comprehensive group, and the course of the treatment was 4 weeks. The abdominal circumference, urine volume, and weight changes were observed daily, and B-ultrasound, liver function, and renal function were performed weekly. RESULTS: In the total effective rates, recurrence rates and A/G and renal function changes, the high-dose albumin group and comprehensive group were superior to the conventional group and high-dose diuretic group (P < 0.01). The death rate of the comprehensive group was the lowest among the 4 groups. CONCLUSION: The therapeutic scheme of the comprehensive group is optimum.

Oral delivery of tumor-targeting Salmonella exhibits promising therapeutic efficacy and low toxicity.

Tumor-targeting bacteria have been developed as powerful anticancer agents. Salmonella typhimurium VNP20009, a representative tumor-targeting strain, has been systemically administered as a single-agent therapy at doses of 1 x 10(6) to 3 x 10(6) colony-forming unit (cfu)/mouse, or in combination with other antitumor agents at doses of 1 x 10(4) to 2 x 10(5) cfu/mouse. Recently, we reported that oral delivery of VNP20009 at the dose of 1 x 10(9) cfu/mouse induced significant anticancer effects comparable to that induced by systemic administration of this strain at 1 x 10(4) cfu/mouse. To further address the efficacy and safety of oral administration of bacteria, here we performed a systemically comparative analysis of anticancer efficacy and toxicity of VNP20009 administered: (i) orally at a dose of 1 x 10(9) cfu/mouse (VNP9-oral); (ii) intraperitoneally at a dose of 1 x 10(4) cfu/mouse (VNP4-i.p.); or (iii) intraperitoneally at a dose of 1 x 10(6) cfu/mouse in tumor-free and tumor-bearing murine models. The results showed that VNP9-oral, similar to VNP4-i.p., induced significant tumor growth inhibition whereas VNP6-i.p. induced better anticancer effect in the B16F10 melanoma model. Among three treatments, VNP9-oral induced the mildest and reversible toxicity whereas VNP6-i.p. resulted in the most serious and irreversible toxicities when compared to other two treatments. Moreover, the combination of VNP9-oral with a low dose of chemotherapeutics produced comparable antitumor effects but displayed significantly reduced toxicity when compared to VNP6-i.p. The findings demonstrated that oral administration, as a novel avenue in the application of bacteria, is highly safe and effective. Moreover, the present preclinical study should facilitate the optimization of bacterial therapies with improved anticancer efficacy and reduced adverse effects in future clinical trials.

Oral delivery of tumor-targeting Salmonella for cancer therapy in murine tumor models.

Tumor-targeting bacteria have been investigated intensively in recent years as anticancer agents. To ensure the reliability of infection, bacteria have conventionally been injected intravenously or intraperitoneally into animals or humans. However, systemic infection of bacteria is rather inconvenient and carries the risk of obvious toxicity. Here we tested whether Salmonella typhimurium VNP20009, a tumor-targeting strain, could be administrated orally for tumor therapy. Tumor-targeting potential, antitumor effects, as well as toxicity of orally administrated VNP20009 were investigated in this study. Oral delivery of VNP20009 not only exhibited high tumor-targeting potential, but also led to a significant anticancer effect by delaying tumor growth and prolonging survival in murine tumor models. As part of combination therapy, orally administrated bacteria notably improved the antitumor effect of cyclophosphamide. In vitro and in vivo studies showed that VNP20009 significantly induced tumor cell apoptosis. No obvious toxicity was observed during the treatments with oral inoculation of VNP20009. Comparative analysis of toxicity in tumor-bearing and tumor-free mice further revealed that orally administrated Salmonella had high safety compared to conventional systemic infection of bacteria. The findings indicated that oral administration of tumor-targeting bacteria is effective and safe. This approach provides a novel avenue in the application of bacteria as a potential antitumor agent.

Tumor-targeting Salmonella typhimurium improves cyclophosphamide chemotherapy at maximum tolerated dose and low-dose metronomic regimens in a murine melanoma model.

Chemotherapy for cancer is partly limited by the inability of drugs to act on poorly vascularized or avascularized areas of tumors. Tumor-targeting bacteria are capable of preferentially replicating in these poorly perfused regions. Some strains have been combined with chemotherapeutic agents and the results have been promising. However, no systematic work has been carried out to test the effect of bacteria on clinical modes of chemotherapy, such as standard maximum tolerated dose (MTD) and novel low-dose metronomic (LDM) chemotherapy. Here Salmonella typhimurium VNP20009 was combined with cyclophosphamide (CTX) at both MTD and LDM schedules in a murine melanoma model. The results showed that VNP20009 significantly improved the effects of all forms of CTX treatments. The combination of VNP20009 and CTX led to a more significant decrease in tumor microvessel density and serum vascular endothelial growth factor (VEGF) level, compared with either treatment alone. Furthermore, combination therapy remarkably increased the number of bacteria within tumors when compared with bacteria treatment alone. These findings suggest that tumor-targeting bacteria, in conjunction with CTX at standard MTD and LDM regimens, might be of clinical value for the treatment of melanoma.

Phylogenetic analysis of eight genes of H9N2 subtype influenza virus: a mainland China strain possessing early isolates' genes that have been circulating.

H9N2 subtype influenza virus has become worldwide and prevalent in China. Previous studies illustrated that at least three sublineages had been established in terrestrial poultry of Eurasian avian. In this presentation, eight full-length genes of an H9N2 strain, A/Chicken/Shanghai/F/98 (Ck/SH/F/98) were obtained. Sequence analysis and phylogenetic studies were conducted by comparing eight genes with those of all the available H9N2 strains from the GenBank. The results showed that four genes (HA, NA, M and NS genes) of Ck/SH/F/98 were incorporated into the sublineage represented by the early mainland China strain, Ck/BJ/1/94. However, the other four of RNP genes of Ck/SH/F/98 did not show close relationship with those of the three known sublineages' viruses. Therefore, Ck/SH/F/98 was a natural reassortant between different sublineages. In addition, comparison showed that Ck/SH/F/98 could be a putative precursor of a later isolate from southern China, Dk/ST/1605/01, with at least six genes of both closely related, indicating genes of Ck/SH/F/98 and early isolates had ever been circulating. Further comparison in terms of molecular markers of species specificity of HA1 revealed that DK/ST/1605/01 also resembled Ck/SH/F/98 more than a common earlier duck strain. The results supported the idea of two-way transmission between terrestrial and aquatic birds that emphasized the importance to raise concerns on the natural evolution of all the eight genes of H9N2 avian influenza viruses.

[Generation of attenuated H5N1 and H5N2 subtypes of influenza virus recombinants by reverse genetics system].

The HA connecting peptide at cleavage site, PQRERRKKR / GL, of an H5N1 subtype avian influenza virus (AIV) was replaced with PQRESR / GL, and then the modified HA gene was cloned into the transcription/expression vector, pHW2000, constructing a plasmid named pHW524-HA. The NA (N1) gene from the H5N1 virus and the NA (N2) gene from an H9N2 AIV were also cloned into pHW2000 separately, resulting in plasmids pHW506-NA and pHW206-NA. With the organization of pHW524-HA, pHW506-NA or pHW206-NA, and six plasmids containing internal genes from A/WSN/33 backbone virus, two transfectants, H5N1/WSN and H5N2/WSN, were subsequently generated by eight-plasmid system. After 15 consecutive passages in embryonated eggs, the two recombinants grew up to high titers of 1:2(9) in hemagglutination test with no changes in nucleotide sequences of the surface genes detected. Both the recombinant viruses belonged to mildly pathogen when evaluated by the pathogenicity test in six-week-old SPF chickens. H5N2/WSN recombinant virus was obviously less pathogenic than H5N1/WSN virus for embryonated chicken eggs. This presentation showed that the reverse genetics system is a very useful tool for studying the construction and function of individual genes and for the generation of virus as vaccine candidate.

[Construction of recombinant fowlpox virus expressing chicken IL-2 and assay of biologic activity of the product in vitro].

In order to determine the adjuvant effects of the chicken IL-2 (ChIL-2) on new generation vaccines, ChIL-2 gene was amplified from ConA-stimulated chicken spleen cells by RT-PCR and was directionally inserted into fowlpox virus (FPV) transferring vector p1175 under the control of FPV early/late promoter (PE/L), resulting in recombinant transferring vector p1175IL2. Then the p1175IL2 plasmid was transfected into chicken embryo fibroblasts (CEF) pre-infected with wild type FPV to generate recombinant fowlpox virus expressing ChIL-2 (rFPV-IL2). By selection of blue plaques on the CEF, overlaid with agar containing X-gal, rFPV-IL2 was obtained and purified. The supernatant from CEF monolayer infected with rFPV-IL2 (M.O.I2.0) after 72 hours was detected for the production of ChIL-2 by XTT/PMS colorimetric assay. About 3.6 x 10(5) u/mL of specific ChIL-2 activity was determined. The results show that rFPV-IL2 can express ChIL-2 effectively. rFPV-IL2 provides us with an effective tool for studying avian immunology as well as a potential vaccine-enhancing agent.