In vivo antitumor activity of Euphorbia lathyris ethanol extract in colon cancer models.

Euphorbia lathyris seeds have been used to treat various medical conditions. We previously reported that ethanolic extract from the defatted seed of Euphorbia lathyris (EE) (variety S3201) possesses a potent in vitro antitumor activity against colon cancer (CRC) cell lines. However, the effects of EE on CRC in vivo models and its possible preventive activity have not been elucidated. The aim of this study is to develop an in vivo study to corroborate its efficacy. For this purpose, two tumor induction models have been developed. In orthotopic xenograft model, it has been shown that EE reduces tumor size without hematological toxicity. The ethanolic extract induced an intense apoptosis in tumors mediated by caspase 3. Using the Azoxymethane/Dextran Sulfate Sodium model, a reduction of dysplastic polyps has been demonstrated, showing its preventive power. Furthermore, EE promoted the presence of an eubiotic microbiotal environment in the mucosa of the colon and induced an increase in antioxidant enzyme activity. This fact was accompanied by a modulation of cytokine expression that could be related to its protective mechanism. Therefore, although further experiments will be necessary to determine its applicability in the treatment of CRC, ES could be a new prevention strategy as well as treatment for this type of tumor, being a powerful candidate for future clinical trials.

Tumour malignancy loss and cell differentiation are associated with induction of gef gene in human melanoma cells.

BACKGROUND: Gene therapy is a new method used to induce cancer cell differentiation. Our group previously showed that transfection of the gef gene from Escherichia coli, related to cell-killing functions, may be a novel candidate for cancer gene therapy. Its expression leads to cell cycle arrest unrelated to the triggering of apoptosis in MS-36 melanoma cells. OBJECTIVES: To determine the basis of the antiproliferative effect of the gef gene in this cell line. METHODS: Transmission electron microscopy, apoptosis analysis by confocal microscopy, flow cytometry and immunocytochemical analysis were used. RESULTS: Ultrastructural analysis showed a strikingly different morphology after treatment with dexamethasone and expression of the gef gene, with large accumulations of pigment throughout the cell cytoplasm and presence of melanosomes in different stages of development. High mitochondrial turnover and myeloid bodies, characteristics of neurone cells, were also observed. In addition, both immunocytochemical and indirect immunofluorescence analysis demonstrated a significant decrease in HMB-45, Ki-67 and CD44 antigen expression and an increase in S100 and p53 expression in gef gene-transfected MS-36 melanoma cells that were correlated with the duration of dexamethasone treatment. In the present work, we report that gef gene not only reduces cell proliferation in transfected melanoma MS-36TG cell line but also induces morphological changes clearly indicative of melanoma cell differentiation and a reduction in tumour malignancy. CONCLUSIONS: These findings support the hypothesis that the gef gene offers a new approach to differentiation therapy in melanoma.

Induction of drug resistance in embryonal rhabdomyosarcoma treated with conventional chemotherapy is associated with HLA class I increase.

Effectiveness of conventional cytotoxic treatment of rhabdomyosarcoma (RMS) may be limited by the development of multidrug resistance (MDR) mediated by mdr1 gene. This gene codes for P-glycoprotein (P-gp) which has been related to a immunoregulatory function. Modulation of HLA expression by P-gp has been described in different types of tumor cells including RMS. However, very little is known about biological implications of the P-gp expression in RMS patients treated with conventional chemotherapy. In order to study the problem, we used embryonal RMS tissue samples from treated patients. Our results indicated that positive RMS samples to mdr1 show higher HLA class I expression than those which were negative to mdr1 PCR, what indicates a significant correlation between the expression of both molecules. In addition, we developed two resistant RMS cell lines (A-204-1 and 2) using similar concentrations of actinomycin D as are plasma levels in clinical situation. Both resistant cell lines showed mdr1 expression and an increase of HLA class I expression which was dose-dependent. These results demonstrated that conventional chemotherapy of embryonal RMS is able to induce resistance which can modulate HLA class I expression and suggest that immunological studies of these tumors may be necessary to the design new specific therapeutic strategies.

Non-viral and viral vectors for gene therapy.

Human gene therapy can be defined as the delivery of genetic material into a patient's cells with a therapeutic aim. The success or failure of gene therapy depends on the development and efficiency of the transfection of viral and non-viral vectors. Viral vectors typically offer higher transduction efficiency and long-term gene expression, but may be associated with toxicity, immunogenicity, restricted target cell specificity and high cost. Non-viral methods have become widespread because of their relative safety, capacity to transfer large genes, site-specificity and their non-inflammatory, non-toxic and non-infectious properties. However, the clinical usefulness of non-viral methods is limited by their low transfection efficiency and relatively poor transgene expression. In this review, we describe the progress made in the development of gene delivery technology and its possible application in clinical trials.

Cancer gene therapy: strategies and clinical trials.

In recent years, great advances have been made in developing novel therapeutic systems based on the introduction of genetic material into damaged cells and designed to correct the error underlying the disease or destroy the pathological cell. One of the main applications of this new approach, known as gene therapy, is the treatment of malignant pathological tumours, in which classic treatments with radiotherapy, chemotherapy and surgery are only palliative. Strategies developed to date include the use of suicide genes, immunity-enhancing genes, apoptosis-inducing genes or genes that inhibit the neovascularization of the tumour, and the blocking of mutated tumour suppressor genes or their restoration in the tumour cell. The effectiveness shown in cell culture and animal experiments and some promising results in clinical trials suggest that gene therapy will help to improve the prognosis of cancer patients and may become the treatment of choice.

Modulation of Ki-67 expression and morphological changes induced by gef gene in MCF-7 human breast cancer cells.

New therapeutic strategies are required to overcome the limitations of conventional breast cancer treatment. Suicide gene therapy offers a potential approach to this type of tumour, since systems based on the use of prodrugs may present some drawbacks related to toxicity, drug release and bioavailability. The gef gene has cell-killing functions in Escherichia coli and does not depend on the use of a prodrug for its action, making it an attractive target for suicide gene therapy. We created a gef-overexpressing human breast cancer cell line (MCF-7TG) by transfecting the gef gene under the control of a pMAMneo promotor. Dexamethasone-induction of gef gene expression in MCF-7TG cells produced a significant decrease in Ki-67 expression, which is a known proliferation marker. In addition, annexin-V-FITC and propidium iodide assays showed the presence of apoptotic cell death, which was confirmed by scanning electron microscopy. The most significant finding was the presence of "craters" in the cell membrane, as previously described in other apoptotic breast cancer cells. These results demonstrate the ability of the gef gene to down regulate Ki-67 expression and induce apoptosis in a breast cancer cell line, suggesting its potential application as a new gene therapy strategy for this type of tumor.

Tissue Specific Promoters in Colorectal Cancer.

Colorectal carcinoma is the third most prevalent cancer in the world. In the most advanced stages, the use of chemotherapy induces a poor response and is usually accompanied by other tissue damage. Significant progress based on suicide gene therapy has demonstrated that it may potentiate the classical cytotoxic effects in colorectal cancer. The inconvenience still rests with the targeting and the specificity efficiency. The main target of gene therapy is to achieve an effective vehicle to hand over therapeutic genes safely into specific cells. One possibility is the use of tumor-specific promoters overexpressed in cancers. They could induce a specific expression of therapeutic genes in a given tumor, increasing their localized activity. Several promoters have been assayed into direct suicide genes to cancer cells. This review discusses the current status of specific tumor-promoters and their great potential in colorectal carcinoma treatment.

Production and characterization of a new monoclonal antibody, GR-ICOR-2, recognizing sarcomeric actin: analysis of the expression in the developing chick heart.

We produced and characterized a specific monoclonal antibody (mAB) designated GR-ICOR-2. This mAb recognizes sarcomeric actin molecules (43 kDa) and was used in an immunohistochemical analysis of staining patterns in Hamburger and Hamilton's stages 18, 22 and 25 (HH 18, 22 and 25) embryonic chick hearts. Staining showed a mainly cytoplasmic distrubition in three regions: the atrioventricular (AV) canal cushion tissue, the primitive ventricle, and conal crests. In addition, this mAb-cross-reacted with rabbit and human cardiac and skeletal muscle tissue; but not with smooth muscle tissue.

Modulation of contractile proteins in embryonic and fetal chick cardiac cells by phorbol ester, gamma-interferon, 5-azacytidine and diacylglycerols.

We studied changes in the concentration of tropomyosin, actin, desmin and vimentin in cultured myocardiocytes from Hamburger and Hamilton's stages 29 and 39 chick embryos (HH29 and HH39) (1), treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), 5-azacytidine (AZA), gamma interferon (INF) and diacylglycerols (DAG). In embryonic myocardiocytes at HH29, the first three agents modified the intracellular distribution of the thin filament proteins tropomyosin and actin, increasing their cytoplasmic concentration and decreasing their cytoskeletal concentration. The concentration of the intermediate filament proteins desmin and vimentin increased in both subcellular fractions after treatment with these drugs. In fetal myocardiocytes at HH39, total protein content decreased after treatment with these drugs. Cytoplasmic and cytoskeletal concentrations of actin and tropomyosin decreased to different degrees after treatment with TPA, AZA or DAG in HH39 myocardiocytes. TPA, AZA and DAG decreased desmin in the cytoplasmic and cytoskeletal fractions. These findings suggest that the drugs tested alter the normal protein composition in cultured myocardiocytes, and have different effects depending on the developmental stage in which the embryo is treated.

Expression of epidermal growth factor receptor in chick embryo myocardiocytes: relation with desmin expression during cardiac development.

The epidermal growth factor receptor is related with processes of cell replication and differentiation. We used the intermediate filament protein desmin as a marker to study the relation between muscle cell differentiation and modifications in the expression of this receptor during heart development in the chick embryo. Epidermal growth factor receptor was expressed as early as Hamburger and Hamilton's stage 17, when myocardiocytes are still poorly differentiated and desmin-negative. Expression became steadily weaker as the heart matured, and decreased after Hamburger and Hamilton's stage 25, a key stage in heart maturation characterized by a sharp increase in desmin expression. Our findings suggest that in the chick embryo, the expression of epidermal growth factor receptor becomes steadily weaker as myocardiocyte differentiation progresses.