The zinc-finger protein KCMF1 is overexpressed during pancreatic cancer development and downregulation of KCMF1 inhibits pancreatic cancer development in mice.

Potassium channel modulatory factor 1 (KCMF1) was found upregulated in a differential screen in the metaplastic epithelium in the pancreas of transforming growth factor (TGF)-alpha transgenic mice. Expression analysis indicated broad overexpression in human cancer tissues. Therefore, we investigated the hypothesis that KCMF1 promotes metaplastic changes and tumor development. KCMF1 represents an evolutionarily highly conserved protein with a 95% identity between human and zebrafish. KCMF1 is expressed during embryonic development and in the majority of adult tissues investigated. Upregulation of nuclear KCMF1 expression is evident in preneoplastic lesions and in several epithelial malignancies, such as pancreatic cancer in mice and humans. In cell culture and in the chicken chorioallantoic membrane model, KCMF1 enhances proliferation, migration and invasion of HEK-293 and Panc1 cells. In crossbreeding experiments, KCMF1-knockdown gene trap mice showed a reduced number and size of premalignant lesions and absence of pancreatic cancer formation in TGF-alpha transgenic mice. This effect is related to the decreased expression of G1 to S cell-cycle regulators such as cyclin D and cyclin-dependent kinase (CDK) 4. Our data support the hypothesis that KCMF1 mediates pro-oncogenic functions in vitro and in vivo and downregulation of KCMF1 results in the inhibition of pancreatic cancer formation in mice. These effects are mediated through downregulation of cell-cycle control genes such as cyclin D and CDK4.

Expression of MTA1 promotes motility and invasiveness of PANC-1 pancreatic carcinoma cells.

The human metastasis-associated protein 1 (MTA1) is a constituent of the nucleosome-remodelling and -deacetylation complex. Its expression has been correlated with the invasion and metastasis of epithelial neoplasms. To address the functional consequences of MTA1 expression in pancreatic carcinoma cells, we have established PANC-1 pancreatic carcinoma cells that stably express MTA1 as an enhanced green fluorescent fusion protein (EGFP-MTA1). Here, we demonstrate that heterologous expression of EGFP-MTA1 markedly enhanced the cellular motility and the invasive penetration of epithelial barriers by the cells. Expression of EGFP-MTA1 had no effect on substrate-independent growth, but reduced substrate-dependent cell proliferation. In addition, the organisation of the cytokeratin filament system and the localisation of the actin cytoskeleton-associated protein IQGAP1 were distinctly altered in EGFP-MTA1-expressing cells. These results indicate that enhanced expression of MTA1 promotes the acquisition of an invasive, metastatic phenotype, and thus enhances the malignancy of pancreatic adenocarcinoma cells by modulation of the cytoskeleton.

Sodium butyrate and tributyrin induce in vivo growth inhibition and apoptosis in human prostate cancer.

Histone deacetylase inhibitors (HDACs) are known to exhibit antiproliferative effects on various carcinoma cells. In this study, the in vivo efficiency of two HDACs, sodium butyrate and tributyrin, on prostate cancer growth inhibition were investigated. To gain an insight into the possible underlying pathways, cell culture experiments were performed focusing on the expression of p21, Rb and c-myc. For in vivo testing, prostate cancer cell lines (PC3 and TSU-Pr1) were seeded on the chorioallantois membrane (CAM) and implanted in a xenograft model using nude mice. Standard Western blot analysis was performed for protein expression of p21, Rb and c-myc in HDAC-treated vs untreated prostate cancer cells. Both sodium butyrate and tributyrin had a considerable treatment effect on microtumours on the chicken egg at already very low concentrations of 0.1 mM. Tributyrin-treated tumours showed the strongest effect with 38% apoptotic nuclei in the prostate cancer cell line PC3. In the mouse model, there was almost no difference between sodium butyrate and tributyrin. In untreated animals the tumours were almost double the size 4 weeks after implantation. Tumours of the treatment groups had a significantly lower percentage of Ki-67-positive-stained nuclei. As demonstrated by Western blot analysis, these effects seem to be independent of p53 status and a pathway via p21-Rb-c-myc is possibly involved. In this study we have demonstrated a substantial in vivo treatment effect, which can be induced by the application of sodium butyrate or the orally applicable tributyrin in human prostate cancer. The given results may provide the rationale to apply these drugs in well-controlled clinical trials in patients being at high risk of recurrence after specific therapy or in patients with locally or distant advanced prostate cancer.

Chorioallantoic membrane assay: vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute model.

PURPOSE: Chorioallantoic membranes have been used as a reliable biomedical assay system for many years. Chicken eggs in the early phase of breeding are between in vitro and in vivo systems but may provide an immunodeficient, vascularized test environment. We tested this model as an in vivo system for prostate cancer research. MATERIALS AND METHODS: Single cell suspensions of LNCaP, PC-3 and Tsu-Pr1 human prostatic cancer cell lines as well as 2 immortalized normal human prostate epithelial cell lines were inoculated on the chorioallantoic membrane of fertilized chicken eggs on day 5 or 6 of breeding. Tumor growth and viability of the embryo was evaluated by stereo microscopy. At day 10 the membranes were removed and embedded in paraffin. Cell morphology was assessed after hematoxylin and eosin staining. Cellular expression of cytokeratin, prostate specific antigen and androgen receptor as well as apoptosis induction was confirmed by immunohistochemistry. RESULTS: Three days after tumor cell inoculation on the extraembryonic vascular system of the chorioallantoic membrane cell growth and formation of 3-dimensional tumors became apparent in 100% of inoculated membranes. Strong neo-angiogenesis was detected next to the established tumors and tumor cells invading the stroma of the chorioallantoic membrane. Cytokeratin expression as well as prostate specific antigen and androgen receptor in LNCaP cells confirmed the human prostate tumor origin. Assessment of quantitative in vivo apoptosis induction in LNCaP cells after intravenous injection of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate confirmed the model as a versatile in vivo system. CONCLUSIONS: The well vascularized chorioallantoic membrane of bred chicken eggs is a suitable system for early in vivo cancer research. Reliable growth of prostate cancer cell lines is feasible and allows the evaluation of proliferation and apoptosis induction after intravascular or topic application of anticancer drugs. Exploitation of this assay enables a substantial reduction in or substitution for subsequent animal experiments.

Methylene blue mediated photodynamic therapy in experimental colorectal tumors in mice.

Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for the topical treatment during photodynamic therapy (PDT). The therapeutic response of MB+ was investigated in vivo by local injection of MB+ in a xenotransplanted subcutanous tumor (adeno-carcinoma, G-3) in female nude mice. MB+ in a concentration of 1% was applied both undiluted and diluted to 0.1 and 0.01% with isotonic sodium chloride. Treatment with 1% MB+ and subsequent irradiation at 662 nm with 100 J/cm2 led to complete tumor destruction in 79% of the treated animals. A decrease of the fluence rate from 100 to 50 mW/cm2 increased the phototoxic response as well as fractionated light application. Small sensitizer concentrations reduced the PDT effect significantly. It seems that the light induced reaction of MB+ could be correlated with the rapid production of reactive oxygen species. Below a threshold dose of MB+ oxidative damage of the tissue is prevented. However, above this dose, as a point of no return, MB+ acts as an extremely potent oxidant.

Photodynamic therapy of experimental colonic tumours with 5-aminolevulinic-acid-induced endogenous porphyrins.

5-Aminolevulinic acid (5-ALA) is a precursor in the biosynthesis of haem. External application of 5-ALA leads to the formation of protoporphyrin IX, the last intermediate product before haem, which is an effective sensitiser. The 5-ALA-induced endogenous photosensitisation of tumour cells has been exploited for photodynamic therapy (PDT). Experimental human G-3 colonic tumours were transplanted into nude mice, and ten mice were treated by PDT. Ten animals served as controls. We measured a fluorescence intensity of the tumour that was about eight times higher than in the surrounding tissue; a good correlation between the fluorescence intensity and the photodynamic effect was found. Tumour growth was inhibited significantly after PDT, two tumours being destroyed completely after the second PDT treatment. In addition, on-line fluorescence detection during PDT showed a change in the intensity and the fluorescence spectrum of protoporphyrin IX caused by photobleaching and the formation of photoproducts.

Photodynamic activity of liposome-delivered Cd-texaphyrin using tumor-bearing nude mice.

Photodynamic effects in bladder-tumor bearing nude mice induced by a krypton ion laser (752.5 nm) after i.v. application of liposome-delivered cadmium texaphyrin were investigated. Cd-texaphyrin possesses strong absorption transitions in the far red spectral region (around 760 nm) and a high quantum efficiency of singlet oxygen production. No therapeutic effects were obtained using irradiation 24 hours after administration of the sensitizer, whereas phototreatment 2 hours after application led to significant tumor reduction. This corresponds well with studies on fluorophore-labeled liposomes indicating highest tumor accumulation 1-2 hours after administration. However, the induced cytotoxic effects were lower than those of HpD.