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We investigated the effects of combined targeted gene therapy on recurrence and metastasis after liver cancer resection in nude mice. Twenty BALB/C mice were randomly divided into control and treatment groups with 10 mice in each group and a male/female ratio of 1:1. Luciferase gene-labeled human primary hepatic carcinoma cell line MHCC97-H was then used to prepare a carcinoma model. An optical in vivo imaging technique (OIIT) was used 10 days later to detect the distribution of tumor cells, followed by partial liver resection and gene therapy. In the treatment group, 100 mL phosphate-buffered saline (PBS) containing 1 x 1012 rAAV/AFP/IL-24 gene viral vectors was injected into liver sections and peritumoral posterior peritoneal tissues; in the control group, the same amount of PBS containing 1 x 1012 empty viral vectors was injected at the same sites. OIIT was then used to detect the in vivo tumor metastasis 21 days later. Luciferase gene-labeled human primary hepatic carcinoma cell line MHCC97-H successfully infected 20 nude mice, and OIIT showed that the two groups exhibited metastasis after local tumor resection, but there were more tumor cells in the control group (P < 0.05). rAAV/AFP/IL-24 gene therapy can inhibit recurrence after liver cancer resection.

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that has a central role in the regulation of tumour metabolism under hypoxic conditions. HIF-1α stimulates glycolytic energy production and promotes tumour growth. Sirtuins are NAD(+)-dependent protein deacetylases that regulate cellular metabolism in response to stress; however, their involvement in the hypoxic response remains unclear. In this study, it is shown that SIRT2-mediated deacetylation of HIF-1α regulates its stability in tumour cells. SIRT2 overexpression destabilized HIF-1α under hypoxic conditions, whereas HIF-1α protein levels were high in SIRT2-deficient cells. SIRT2 directly interacted with HIF-1α and deacetylated Lys709 of HIF-1α. Deacetylation of HIF-1α by SIRT2 resulted in increased binding affinity for prolyl hydroxylase 2, a key regulator of HIF-1α stability, and increased HIF-1α hydroxylation and ubiquitination. Moreover, a pharmacological agent that increased the intracellular NAD(+)/NADH ratio led to the degradation of HIF-1α by increasing SIRT2-mediated deacetylation and subsequent hydroxylation. These findings suggest that SIRT2-mediated HIF-1α deacetylation is critical for the destablization of HIF-1α and the hypoxic response of tumour cells.

Over 90% of human cervical carcinoma is associated with high risk human papillomavirus (HPV), and a number of biological effects that could contribute to cancer suppression by ω3-polyunsaturated fatty acids (ω3-PUFAs) have been reported. However, the anti-cancer effect of ω3-PUFAs on cervical cancer has been not known yet. In this study, we report inhibitory mechanisms of ω3-PUFAs on cell growth and invasion in cervical cancer. DHA inhibited the cell growth in a dose- and time-dependent manner. In flow cytometry analysis, cell cycle of DHA-treated HeLa cells was arrested in G2/M phase and SubG1 cells also increased. Moreover, apoptotic cell death was confirmed by TUNEL assay, the induction of PARP cleavage and down-regulation of Bcl-2. The invasiveness of cells was significantly inhibited by DHA treatment in vitro transwell assay as well. The MMP-9 and MMP-2 promoter activities were decreased after DHA treatment. Cox-2 and VEGF promoter activities were also inhibited by DHA. Furthermore, DHA decreased the levels of reporter activity of NF-κB, which is transcription factor that regulates MMPs, Cox-2 and VEGF expression. In in vivo experiments, when human papillomavirus type 16 (HPV16)-transformed mouse TC-1 cells were injected into the tail vein of Fat1 mice (Fat1 transgenic mice express a Caenorhabditis elegans ω3-desaturase converting ω6- to ω3-PUFAs endogenously.) and WT (wild type) mice, lung metastasis of TC-1 cells was dramatically inhibited in Fat1 transgenic mice compared to WT mice. Taken together, these findings provide evidence that ω3-PUFAs may inhibit metastasis as well as cell growth and invasion through suppression of MMPs/COX-2/VEGF expression by inhibition of NF-κB expression in cervical cancer cells, indicating that the utilization of ω3-PUFAs may represent a potential effective therapy for the chemoprevention and treatment of human cervical cancer. This work was supported by basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2010-0016447 and R13-2007-020-01000-0), Korea.

Introduction: Reactive oxygen species (ROS) produced by docosahexaenoic acid (DHA) have an important function in cancer cell death. However, the exact mechanism of ROS production, after DHA stimulation, is not clearly understood. Here, we determined that elevated levels of ROS generated by mitochondrial respiration is directly associated with DHA-induced cervical cancer cell death. Material and Method: The effects of DHA on cell proliferation and cell cycle were examined by MTT assay and FACS. DHA-induced apoptosis was analyzed using the TUNEL assay, caspase activity assay, and western blot. Dihydroethidium (DHE) was used for reactive oxygen species (ROS) measurement in cytosol. MitoSox was performed for ROS measurement in mitochondria. Results and Discussion: The levels of caspase 3 activity, TUNELpositive staining cells and Sub-G1 portion were markedly increased in DHA-treated cancer cells, suggesting that apoptosis is responsible for the DHA-induced cervical cancer cell death. Furthermore, DHA was able to induce both mitochondrial complex I substrateand complex II substratesupported mitochondrial ROS production in isolated mitochondria from rodent liver. Meanwhile, a reduction in oxygen consumption rate and an increase in mitochondrial ROS production as measured by MitoSOX, were also observed in DHA-treated cancer cells, indicating that DHA can directly act on mitochondrial respiration and enhance ROS generation. The role of DHA-induced mitochondrial ROS production in apoptosis was further identified by the findings that DHA reduced the mitochondrial membrane potential, resulting in cardiolipin oxidation and cytochrome c release from mitochondria, and that N-acetylcysteine, an antioxidant almost completely blocked these processes as well as ROS production occurred in mitochondria and remarkably reversed the apoptotic cell death triggered by DHA. Conclusion: From the results presented here, we conclude that mitochondria actively participate in the DHA-induced apoptotic cell death by the generation of mitochondrial ROS. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (2011– 0006232 and 2011–0003060).

Recent molecular and clinical studies have shown that invasion and metastasis may occur very early in tumour development, thus emphasizing the potential importance of spesific and sensitive detection of circulating tumour cells (CTC). With the ability of refined technologies the identification of CTCs from peripheral blood is emerging as a useful tool for the detection of malignancy, monitoring disease progression and measuring response to therapy. The goal of this study was to identify optimal marker or marker combinations for detection of CTCs in the gastrointestinal malignancies using RT-PCR. Materials and Methods: To detect the presence of CTCs, we analyzed Cytokeratin 19 (CK19), Cytokeratin 20 (CK20) and Mucin 1 (MUC1) mRNA in the peripheral blood of 31 patients with gastrointestinal (gastric, stomach and colorectal) carcinoma and 30 healthy individuals. Results: In RT-PCR analysis of the peripheral blood, 77.4% (24/31), 58.06% (18/31) and 3.22% (1/31) of cancer patients were positive for MUC1, CK20 and CK19 mRNA respectively. The sensivity and specificity for any one of mRNA detected in peripheral blood is 83.3% and 66.6% respectively, with an acurracy of 59%. Conclusions: Our study suggest that MUC1 and CK20 mRNAs in the peripheral blood could be useful molecular markers for gastrointestinal tumours. Combination of these two tumour-spesific mRNA markers would increase the detection rate and may be clinically helpful in predicting the tumour presence and colorectal cancer metastasis.

Angiogenesis, the process of new blood vessel formation, is essential for tumor growth and metastasis. Although the protective role of ω3-PUFAs on cancer progression has been reported, the anti-angiogenic mechanisms remain to be further understood. The aim of this study is to investigate the anti-angiogenic mechanisms of ω3-PUFAs in pancreatic cancer. In angiogenesis protein array chip assay, only angiogenic ELR+ CXC chemokines expression, which activate endothelial cell migration and neutrophils recruitment, was significantly reduced by DHA treatment in SW1990 human pancreatic cancer cells. The reduced expression of ELR+ CXC chemokine was confirmed by RT-PCR. Chemotactic migration of HUVEC was significantly reduced in DHA-treated SW1990 conditioned medium (CM) compared to control CM. DHA also inhibited VEGF reporter activity in SW1990 cells. VEGF-induced endothelial cell proliferation and migration were markedly inhibited by DHA treatment. MMP-2 and MMP-9 promoter activities were reduced by DHA treatment, and ultimately attenuated the invasiveness of SW1990 cells as revealed by matrigel droplet assay. Moreover, NF-κB reporter activity, which is closely correlated with the expression of CXC chemokine, VEGF and MMPs, was reduced by DHA treatment. When mouse pancreatic cancer PANC02 cells were implanted into Fat-1 transgenic mice, tumor size and volume were dramatically reduced in Fat-1 mice compared to control mice. In immunohistochemical analysis of tumors of Fat-1 mice, apoptosis (TUNEL positive cells) was markedly increased and microvessel density (CD31 intensity) was significantly reduced in Fat-1 mice compared to control mice. In addition, VEGF-induced neovascularization to a subcutaneous matrigel plug was significantly inhibited in Fat-1 mice compared with control mice. Our finding suggests that ω3-PUFAs can suppress tumor angiogenesis through inhibition of ELR+ chemokines expression and VEGF signal in pancreatic cancer. Thus ω3-PUFAs supplementation may beneficial for chemoprevention and treatment of pancreatic cancer by anti-angiogenic effect. This work was supported by basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R13-2007-020-01000-0 and 2010-0016447), Korea.