Tumour irradiation combined with vascular-targeted photodynamic therapy enhances antitumour effects in pre-clinical prostate cancer.

BACKGROUND: There is a need to improve the treatment of prostate cancer (PCa) and reduce treatment side effects. Vascular-targeted photodynamic therapy (VTP) is a focal therapy for low-risk low-volume localised PCa, which rapidly disrupts targeted tumour vessels. There is interest in expanding the use of VTP to higher-risk disease. Tumour vasculature is characterised by vessel immaturity, increased permeability, aberrant branching and inefficient flow. FRT alters the tumour microenvironment and promotes transient 'vascular normalisation'. We hypothesised that multimodality therapy combining fractionated radiotherapy (FRT) and VTP could improve PCa tumour control compared against monotherapy with FRT or VTP. METHODS: We investigated whether sequential delivery of FRT followed by VTP 7 days later improves flank TRAMP-C1 PCa tumour allograft control compared to monotherapy with FRT or VTP. RESULTS: FRT induced 'vascular normalisation' changes in PCa flank tumour allografts, improving vascular function as demonstrated using dynamic contrast-enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumour growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP, and improved overall survival. CONCLUSION: Combining FRT and VTP may be a promising multimodal approach in PCa therapy. This provides proof-of-concept for this multimodality treatment to inform early phase clinical trials.

Renal biopsies from donors with acute kidney injury show different molecular patterns according to the post-transplant function.

The utilization of kidneys from donors with acute kidney injury (AKI) is often limited by unpredictable post-transplantation outcomes. The aim of our study was to identify protein mediators implicated in either recovery or failure of these organs. Forty kidney biopsies from donors with (20) and without AKI (20) were selected and then subdivided according to the post-transplant outcome defined as a threshold of 45 ml/min for the eGFR at 1 year from transplantation. Tissue homogenates were analysed by western blot to assess how the levels of 17 pre-selected proteins varied across the four groups. Samples from AKI kidneys with a poor outcome showed a fourfold increase in the levels of PPARg and twofold reduction of STAT1 compared to the other groups (p < 0.05). On the contrary, antioxidant enzymes including TRX1 and PRX3 were increased in the AKI kidneys with a good outcome (p < 0.05). An opposite trend was observed for the detoxifying enzyme GSTp which was significantly increased in the AKI group with poor versus good outcome (p < 0.05). The importance of lipid metabolism (PPARg) and inflammatory signals (STAT1) in the function recovery of these kidneys hints to the therapeutical targeting of the involved pathways in the setting of organ reconditioning.

Identification of local and circulating cancer stem cells in human liver cancer.

UNLABELLED: Increasing evidence has revealed the importance of cancer stem cells (CSCs) in carcinogenesis. Although liver CSCs have been identified in hepatocellular carcinoma (HCC) cell lines, no data have shown the presence of these cells in human settings. The present study was designed to delineate CSCs serially from HCC cell lines, human liver cancer specimens to blood samples, using CD90 as a potential marker. The number of CD90(+) cells increased with the tumorigenicity of HCC cell lines. CD45(-)CD90(+) cells were detected in all the tumor specimens, but not in the normal, cirrhotic, and parallel nontumorous livers. In addition, CD45(-)CD90(+) cells were detectable in 90% of blood samples from liver cancer patients, but none in normal subjects or patients with cirrhosis. A significant positive correlation between the number of CD45(-)CD90(+) cells in the tumor tissues and the number of CD45(-)CD90(+) cells in the blood samples was identified. CD90(+) cells sorted from cell lines and CD45(-)CD90(+) cells from the tumor tissues and blood samples of liver cancer patients generated tumor nodules in immunodeficient mice. Serial transplantation of CD90(+) cells from tumor xenografts generated tumor nodules in a second and subsequently third batch of immunodeficient mice. Treatment of CD90(+) CSCs with anti-human CD44 antibody induced cell apoptosis in a dose-dependent manner. CONCLUSION: Identification of CD45(-)CD90(+) CSCs in both tumor tissues and circulation suggests that CD45(-)CD90(+) could be used as a marker for human liver cancer and as a target for the diagnosis and therapy of this malignancy.

High doses of tyrosine kinase inhibitor PTK787 enhance the efficacy of ischemic hypoxia for the treatment of hepatocellular carcinoma: dual effects on cancer cell and angiogenesis.

The present study aimed to investigate the therapeutic efficacy of combining vascular endothelial growth factor (VEGF) receptor blockade using tyrosine kinase inhibitor PTK787 with hypoxia for the treatment of hepatocellular carcinoma (HCC). The in vivo effects of the treatments were determined in a rat orthotopic HCC model, in which hypoxia was generated by hepatic artery ligation (HAL). Compared with HAL alone, PTK787 combined with HAL significantly prolonged the animal survival, reduced the tumor size, induced more tumor tissue necrosis and apoptosis, and down-regulated the expression of von Willebrand factor. The mechanism was explored in vitro using murine HCC and endothelial cell lines, respectively. PTK787 combined with hypoxia decreased the expression of VEGF and VEGF receptors in both cell lines and suppressed the cell viability by induction of cell cycle arrest and promotion of apoptosis. Up-regulation of cleaved form caspase-9 and down-regulation of Bcl-2 and cyclin D1 were detected with the combined treatment. Hypoxia sensitized endothelial cells to the inhibitory effect of PTK787 on forming tubular-like structure. The motility of tumor cells was inhibited by hypoxia and the combined approach, with down-regulation of Rac1, Rho, and phosphorylated Akt expression. However, in the endothelial cells, the combined treatment inhibited the hypoxia-enhanced cell motility, with suppressed Rac1, Rho, and phosphorylated Akt expression. In conclusion, PTK787 combined with hypoxia achieved a better therapeutic efficacy than hypoxia alone through enhancing hypoxia-induced antitumor cell effect and preventing the activation of angiogenic process.

Therapeutic potential of cardiotrophin 1 in fulminant hepatic failure: dual roles in antiapoptosis and cell repair.

HYPOTHESIS: Administration of cardiotrophin 1 (CT-1) can treat experimental fulminant hepatic failure (FHF). DESIGN: Rat model with FHF induced by D-galactosamine (D-gal). SETTING: Fulminant hepatic failure is a rapidly progressive disease that lacks effective nonsurgical treatment. Cardiotrophin 1 is a member of the interleukin 6 family that can protect cells from damage in some animal disease models. ANIMALS: A rat model of FHF was induced by an intraperitoneal injection of D-gal (1.4 g/kg of body weight). Cardiotrophin 1 was administered at different time points after D-gal injection. RESULTS: Administration of CT-1 at 12 and 18 hours had a survival rate of 80% (12/15) and 70% (7/10), respectively, which was significantly higher than that of nontreatment (28% [5/18]). In addition, improvement of liver histologic findings, shortening of activated clotting time, and decrease in serum levels of total bilirubin and alanine aminotransferase were detected with CT-1 treatment. Administration of CT-1 decreased apoptotic cells and increased Ki-67 cells in the liver tissues. In vitro, CT-1 administration significantly decreased apoptotic cells and sequentially down-regulated the expression of proapoptotic molecules and up-regulated the expression of antiapoptotic molecules at different culture periods. D-galactosamine culture induced morphologic damage in a hepatocyte cell line, which was greatly improved by CT-1 administration. In addition, CT-1-treated cells demonstrated increased expression of glycoprotein 130 and up-regulation of cyclin D1 and heat shock protein 90. CONCLUSION: Cardiotrophin 1 may improve the outcome of D-gal-induced FHF through its effects on antiapoptosis and cell repair.

Significance of the serum brain-derived neurotrophic factor and platelets in hepatocellular carcinoma.

The present study investigated the significance of the serum brain-derived neurotrophic factor (BDNF) and platelets in relation to the clinicopathological features of hepatocellular carcinoma (HCC) patients. Localization of the BDNF expression in human HCC tissues was performed by immunohistochemistry. The measurement of soluble BDNF in the serum was performed by enzyme-linked immunosorbent assay. BDNF was expressed in the cytoplasm of the tumor cells. A positive correlation between the tissue and serum levels of BDNF was identified in the HCC patients. The serum levels of BDNF were positively correlated with the platelet counts in the HCC patients. A higher level of serum BDNF was significantly correlated with a tumor size >5 cm, poorly differentiated HCC, the presence of microsatellite tumor nodules, and the absence of cirrhosis in the non-tumorous tissues. A higher level of the serum BDNF/platelet ratio was associated with a poorer disease-free survival after hepatic resection. This study suggested that the tumor cell was a source of serum BDNF in HCC. A higher serum BDNF level was associated with a more advanced tumor status in the HCC patients. The interaction between serum BDNF and platelets might play an important role in HCC tumor progression.

Brain-derived neurotrophic factor promotes tumorigenesis via induction of neovascularization: implication in hepatocellular carcinoma.

PURPOSE: Brain-derived neurotrophic factor (BDNF) has emerged as a novel angiogenic factor, and yet its impact on tumorigenesis is unclear. This study aimed at investigating the roles of BDNF in angiogenesis and tumor development. EXPERIMENTAL DESIGN: BDNF was overexpressed in a mouse endothelial cell (EC) line by stable transfection, and angiogenic properties of the transfectants were assessed. Microarray analysis was employed to explore the molecular pathways. The impact of modulating BDNF levels in two mouse EC lines on tumorigenic potential of a transformed mouse liver cell line was evaluated by an in vivo cotransplantation model. BDNF and tropomyosin receptor kinase B (TrkB) protein levels were determined in 50 pairs of human hepatocellular carcinoma (HCC) tissues by Western blotting and immunohistochemistry. Survival analysis was carried out to determine their clinical significance. RESULTS: Overexpression of BDNF could promote EC proliferation, migration, invasion, and survival. Microarray and molecular studies showed that RhoA, caspase-9, caspase-3, growth arrest specific 6, and VEGF could mediate BDNF/TrkB-induced angiogenesis. The cotransplantation experiment showed that high BDNF-expressing ECs could facilitate tumor angiogenesis and growth, whereas knockdown of BDNF by short hairpin RNAs impaired such effects. Furthermore, examination on human HCC tissues revealed upregulation of BDNF and TrkB protein levels in 46.0% and 33.3% of the cases studied, respectively. Immunohistochemistry disclosed strong BDNF reactivity in both tumor and endothelial cells. High TrkB expression was associated with shorter overall survival. CONCLUSIONS: BDNF/TrkB system was crucial for tumor angiogenesis and growth, which may represent a potential target for antiangiogenic therapy in HCC.

Inhibition of mTOR enhances chemosensitivity in hepatocellular carcinoma.

The present study investigated the effect of mammalian target of rapamycin (mTOR) inhibition on HCC cells in vitro and in vivo, either alone or in combination with cytotoxic agents. In vitro, HCC cell lines were exposed to RAD001, an mTOR inhibitor, either alone or in combination with cisplatin. Alone, RAD001 suppressed cell proliferation in all cell lines tested, but did not induce apoptosis. RAD001 in combination with cisplatin induced a significant increase in the number of apoptotic cells, downregulated the expression of pro-survival molecules, Bcl-2, survivin and cyclinD1, and increased the cleavage of PARP, compared to RAD001 or cisplatin alone. Transfection of p53 into the Hep3B cell line increased the sensitivity of tumor cells to cisplatin. The suppression of HCC tumor growth in vivo was enhanced by RAD001 combined with cisplatin, accompanied by a significant increase in the number of apoptotic cells in tumor tissues. This study demonstrates that inhibition of mTOR suppresses tumor growth and sensitizes tumor cells to chemocytotoxic agents.

Induction of long-term liver allograft survival by delayed immunosuppression is dependent on interleukin-10.

This study aims to investigate the potential role of endogenous interleukin (IL)-10 in long-term liver allograft survival induced by delayed immunosuppression (FK506 days 2-7). Liver transplantation was performed by using Dark Agouti and Lewis rats as donors and recipients, respectively. The delayed immunosuppression protocol induced indefinite allograft survival. A transient upregulation of plasma IL-10 levels was detected in the nontreatment and FK506 treatment groups. Macrophages were found to be one of the major sources of IL-10 produced from the liver allografts. Administration of IL-10-neutralizing antibody shortened the long-term isograft survival and FK506-induced indefinite allograft survival, particularly in the FK506 group. Damaged liver graft histology and increase of plasma alanine aminotransferase levels were detected in the groups with IL-10 antibody treatment. In an ex vivo setting, IL-10 recombinant protein augmented the expression of Foxp3, downregulated the expression of IL-2 and interferon gamma, and induced the generation of CD4(+)CD25(+)Foxp3(+) and CD8(+)CD25(+)Foxp3(+) cells, but this effect was blocked by the administration of IL-10 antibody. Finally, administration of IL-10 recombinant protein after the decline of endogenous IL-10 levels improved allograft survival, and a 100% long-term allograft survival was achieved by the combination of IL-10 with low-dose FK506. In conclusion, the delayed immunosuppression could induce long-term liver allograft survival in the presence of endogenous IL-10 produced by the tissue macrophages. Supplementary exogenous IL-10 administration combined with low-dose immunosuppressive drug may be a useful strategy to induce long-term liver allograft survival.

Inhibition of Stat3 activity by YC-1 enhances chemo-sensitivity in hepatocellular carcinoma.

The present study investigated the effect of YC-1, a novel anti-cancer agent, on the chemo-sensitivity of hepatocellular carcinoma (HCC). YC-1 was administered with chemo-cytotoxic drug, cisplatin, both in vitro and in vivo. YC-1 alone downregulated the expression of phosphorylated form of signal transducers and activators of transcription 3 (P-Stat3[705]), a key mediator in chemo-resistance. When combined with cisplatin, YC-1 further promoted tumor cell apoptosis, decreased the expression of P-Stat3(705), Bcl-xL, CyclinD1 and survivin, and induced the cleavage of caspase 9 and PARP. Overexpression of Stat3 reversed YC-1 induced cell death. YC-1 inhibited Stat3 activity by enhancing the polyubiquitination of P-Stat3(705) induced by cisplatin. In the in vivo setting, YC-1 combined with cisplatin remarkably suppressed tumor growth in a HCC xenograft model, and this effect was also accompanied by YC-1 mediated downregulation of P-Stat3(705), Bcl-xL, Cyclin D1 and survivin, and induction of cleaved caspase 9 and PARP in the tumor tissues. In conclusion, the present study demonstrated a novel anti-cancer effect of YC-1 in enhancing chemo-sensitivity of HCC cells to cisplatin through a Stat3 dependent manner. This finding provides insight into design of a new therapeutic strategy to improve efficacy of chemotherapy in HCC patients.

Suppression of hypoxia inducible factor-1alpha (HIF-1alpha) by YC-1 is dependent on murine double minute 2 (Mdm2).

Inhibition of HIF-1alpha activity provides an important strategy for the treatment of cancer. Recently, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) has been identified as an anti-HIF-1alpha drug in cancer therapy with unclear molecular mechanism. In the present study, we aimed to investigate the effect and mechanism of YC-1 on HIF-1alpha in a hepatocellular carcinoma cell line under hypoxic condition, which was generated by incubating cells with 0.1% O(2). The phenotypic and molecular changes of cells were determined by cell proliferation assay, apoptosis assay, luciferase promoter assay, and Western blot analysis. YC-1 arrested tumor cell growth in a dose-dependent manner, whereas it did not induce cell apoptosis. Hypoxia-induced upregulation of HIF-1alpha was suppressed by YC-1 administration. YC-1 inhibited HIF-1alpha protein synthesis under normoxia and affected protein stability under hypoxia. YC-1 suppressed the expression of total and phosphorylated forms of murine double minute 2 (Mdm2), whereas this inhibitory effect was blocked by overexpression of Mdm2. In conclusion, YC-1 suppressed both protein synthesis and stability of HIF-1alpha in HCC cells, and its inhibitory effects on HIF-1alpha were dependent on Mdm2.

Neutron and X-ray diffraction and spectroscopic investigations of intramolecular [C-H...F-C] contacts in post-metallocene polyolefin catalysts: modeling weak attractive polymer-ligand interactions.

A family of Group 4 post-metallocene catalysts, supported by fluorine-functionalized tridentate ligands with the fluorine substituent in the locality of the metal center, is described. For the first time, the contentious C-H...F-C interaction has been characterized by a neutron diffraction study, which has allowed the position of the hydrogen atoms to be accurately determined. The nature of the weak intramolecular C-H...F-C contacts in these complexes in solution and the solid state was probed by using multinuclear NMR spectroscopy in tandem with neutron and X-ray crystallography. Evidence is presented to demonstrate that the spectroscopic C-H...F-C coupling occurs "through-space" rather than "through-bond" or by MF coordination. The titanium catalysts exhibit excellent activities and high co-monomer incorporation in olefin polymerization. The observed intramolecular C-H...F-C interactions are important with regards to potential applications in polyolefin catalysis because they substantiate the proposed ortho-F...H(beta) ligand-(polymer chain) contacts derived from DFT calculations for the remarkable fluorinated phenoxyimine Group 4 catalysts. Compared with agostic and co-catalyst...metal contacts, weak attractive noncovalent interactions between a polymer chain and a judiciously designed "active" ligand is a new concept in polyolefin catalysis.

Platelet activation during tumor development, the potential role of BDNF-TrkB autocrine loop.

Platelets are an important place for the storage of angiogenic factors, such as vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). The present study aims to investigate the interaction between BDNF-TrkB pathway and platelet activation during tumor development. In an orthotopic hepatocellular carcinoma (HCC) model, increased levels of serum and plasma BDNF were detected with tumor progression. Higher numbers of CD62P+ and TrkB+ platelets were found in the tumor-bearing rats. In the in vitro setting, tumor-conditioned-medium (TCM) and BDNF recombinant protein stimulated CD62P upregulation and subsequent BDNF release in the freshly isolated platelets, whereas this effect could be inhibited by TrkB blockade. TCM and BDNF culture augmented the expression of heat shock protein 90 (Hsp90) in the platelets, which could be reversed by TrkB blockade. In conclusion, this study suggested the presence of BDNF-TrkB autocrine loop in platelets and its importance in regulating platelet activation during tumor development.