Human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) are considered effective molecular targets for current anticancer therapy. In this study, we investigated the therapeutic effects of targeting hTR and hTERT individually or in combination by recombinant adenovirus-delivered small interfering RNA (siRNA) in oral squamous cell carcinoma (OSCC) Tca8113. Further, we screened the optimal strategy for RNA interference. Our results show that these different recombinant adenoviruses specifically reduced the levels of hTR mRNA, hTERT mRNA, hTERT protein and telomerase activity in Tca8113 cells. Moreover, they successfully inhibited xenograft tumor growth in nude mice. The potency of their antitumor activities was ranked as follows: anti-hTR 4anti-hTR þ anti-hTERT 4anti-hTERT. Therefore, we demonstrated that the siRNA-expressing recombinant adenoviruses were an effective anticancer tool for treatment of OSCC. Furthermore, the anticancer effect of solely targeting hTR was more direct and efficient, compared with the effect of targeting hTR and hTERT in combination, or hTERT exclusively. The mechanism of this anticancer effect in OSCC was not only related to the inhibition of cell proliferation and the induction of cell apoptosis, but might also involve the inhibition of tumor angiogenesis.
Malignant ascites is common in various types of cancers and is difficult to manage. Vascular endothelial growth factor (VEGF) has a pivotal role in malignant ascites. The matrix protein of vesicular stomatitis virus (VSVMP) has been shown to inhibit host gene expression and induce the apoptosis of cancer cells. The present study was designed to determine whether VSVMP suppresses the formation of ascites in ascites-producing peritoneal carcinomatosis. BALB/c female mice, 6-8 weeks old, bearing peritoneal tumors of H22 or MethA cells received an intraperitoneal administration of 50 μg VSVMP/250 μg liposome complexes, 50 μg empty plasmid/250 μg liposome complexes or 0.9% NaCl solution, respectively, every 2 days for 3 weeks. Administration of VSVMP resulted in a significant inhibition in ascites formation, improvement in health condition and prolonged survival of the treated mice. Decreased peritoneum osmolarity and reduced tumor vascularity coincided with dramatic reductions in the VEGF level in ascites fluid and plasma. Examination of floating tumor cells collected from the peritoneal wash revealed an apparently increased number of apoptotic cells and profound downregulation of VEGF mRNA in the VSVMP-treated mice. Our data indicate for the first time that in BALB/c mice bearing H22 or MethA cell peritoneal tumors, VSVMP may inhibit VEGF production and suppress angiogenesis, consequently abolishing ascites formation.
Mouse PNAS-4 (mPNAS-4) has 96% identity with human PNAS-4 (hPNAS-4) in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data), it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2) and colon carcinoma cells (CT26) of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3). The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4) was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37°C, while it was almost exclusively expressed in soluble form at 20°C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28%. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.
Aim: To evaluate risk factors for venous thromboembolism (VTE) and deep vein thrombosis after living donor nephrectomy in a centre using extensive preoperative screening and perioperative venous duplex scan. Material and Methods: In 130 consecutive living kidney donors (laparoscopic 105, open 25) thrombophilia screening and pre-and postoperative ultrasonographies were performed. Donors were followed prospectively for at least 3 months. All donors received prophylaxis with the low molecular weight heparin enoxaparin and compression stockings. Donors with increased risk received the double dose of enoxaparin and the prophylaxis was continued for 6 weeks. Donors with venous thrombosis at discharge duplex also received prolonged prophylaxis. Results: The frequency of thrombophilia was similar to what can be expected in the Swedish population (four with factor V Leiden, one each with protein S defi ciency, prothrombin gene mutation and anticardiolipin antibodies). Preoperative duplex was normal. Three donors had small postoperative deep vein thrombosis. Twelve donors (9,2%) received an intensifi ed and prolonged prophylaxis. No further thromboembolic complications developed in three postoperative months. Conclusion: With the present protocol for preoperative evaluation, perioperative duplex screening and prophylaxis, the risk of postoperative venous thromboembolism is low after living donor nephrectomy. Given that 9,2% had risk factors or developed deep vein thrombosis and the extraordinary situation where an operation is performed on a healthy person who has no therapeutic benefi t and the low incidence of VTE in the present study, we recommend the presented approach to be implemented more broadly and that further studies should be performed in larger cohorts.
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