Cytotoxic activity of proteins isolated from extracts of Corydalis cava tubers in human cervical carcinoma HeLa cells

<p>Abstract</p> <p>Background</p> <p><it>Corydalis cava </it>Schweigg. & Koerte, the plant of numerous pharmacological activities, together with the studied earlier by our group <it>Chelidonium majus </it>L. (Greater Celandine), belong to the family <it>Papaveraceae</it>. The plant grows in Central and South Europe and produces the sizeable subterraneous tubers, empty inside, which are extremely resistant to various pathogen attacks. The <it>Corydalis sp. </it>tubers are a rich source of many biologically active substances, with the extensive use in European and Asian folk medicine. They have analgetic, sedating, narcotic, anti-inflammatory, anti-allergic and anti-tumour activities. On the other hand, there is no information about possible biological activities of proteins contained in <it>Corydalis cava </it>tubers.</p> <p>Methods</p> <p>Nucleolytic proteins were isolated from the tubers of <it>C. cava </it>by separation on a heparin column and tested for DNase activity. Protein fractions showing nucleolytic activity were tested for cytotoxic activity in human cervical carcinoma HeLa cells. Cultures of HeLa cells were conducted in the presence of three protein concentrations: 42, 83 and 167 ng/ml during 48 h. Viability of cell cultures was appraised using XTT colorimetric test. Protein fractions were separated and protein bands were excised and sent for identification by mass spectrometry (LC-ESI-MS/MS).</p> <p>Results</p> <p>The studied protein fractions showed an inhibiting effect on mitochondrial activity of HeLa cells, depending on the administered dose of proteins. The most pronounced effect was obtained with the highest concentration of the protein (167 ng/ml) - 43.45 ± 3% mitochondrial activity of HeLa cells were inhibited. Mass spectrometry results for the proteins of applied fractions showed that they contained plant defense- and pathogenesis-related (PR) proteins.</p> <p>Conclusions</p> <p>The cytotoxic effect of studied proteins toward HeLa cell line cells has been evident and dependent on increasing dose of the protein. The present study, most probably, represents the first investigations on the effect of purified PR proteins from tuber extracts of a pharmacologically active plant on cell lines.</p

An efficient 3D cell culture method on biomimetic nanostructured grids.

Current techniques of in vitro cell cultures are able to mimic the in vivo environment only to a limited extent, as they enable cells to grow only in two dimensions. Therefore cell culture approaches should rely on scaffolds that provide support comparable to the extracellular matrix. Here we demonstrate the advantages of novel nanostructured three-dimensional grids fabricated using electro-spinning technique, as scaffolds for cultures of neoplastic cells. The results of the study show that the fibers allow for a dynamic growth of HeLa cells, which form multi-layer structures of symmetrical and spherical character. This indicates that the applied scaffolds are nontoxic and allow proper flow of oxygen, nutrients, and growth factors. In addition, grids have been proven to be useful in in situ examination of cells ultrastructure

P1 promoter IGF-1 polymorphism and IGF-1, IGF-R, LSF, and TSG 101 expression profile in endometriosis

Objectives: The presence of the endometrium outside the uterine cavity affects about 10% of women of childbearing age. Studies of the progression of endometriosis to cancer have been supported by numerous evidences of gene expression or gene defect caused by oxidative stress and inflammation. We decided to check the expression of selected factors responsible for the proliferation, as in the stages of neoplasia. Material and methods: A group of 80 women with ovary localization of endometriosis was qualified for research. The control group was 90 patients with ovarian simplex or follicular cysts. The DNA isolation, immunohistochemical analysis of IGF 1, IGF–R, TSG 101, and LSF expressions with a quantitative scoring of slides and electron microscopy was performed. Results: The IGF-1-immunopositive cells in the reference group were in statistically significantly higher number compared to the cells forming the foci of endometriosis (p = 0.0282). However, the number of IGF-R-immunopositive cells was comparable to the endometriosis (p = 0.1264). In the control group, the number of LSF-immunopositive cells was statistically significantly higher in comparison to endometriosis foci (p = 0.000001), but the number of TSG 101-immunositive cells was comparable to endometriosis foci (p = 0.3834). A weak negative correlation between the number of cells expressing the TSG 101 factor and the IGF-1 receptor was found in the endometriosis group (r = –0.26, p = 0.0196). The analysis of CA single nucleotide polymorphism in the DNA isolated from both groups showed a comparable incidence of MSS and MSI-L genotypes (chi2 p = 0,9160). Conclusions: How these factors affect the development of endometriosis and whether they could be helpful in the diagnosis requires further research

HeLa cells stained with Hoechst 33342 and propidium iodide after 24 (A), 48 (B) and 72 (C) hours of 3D culture on the nanostructure grids.

<p>Original magnification x200. A fragment of the spherical 3D structure of cell growing on nanostructured grids visualized after 48 hours. Original magnification x400 (D).</p

Light microscopy image of nanostructured grid.

<p>Original magnification x200 (A). Scanning electron microscopy image of nanostructured microfiber. Original magnification x500 (B).</p

Cells growing on the scaffold visualized using light microscopy after 48 hours of incubation.

<p>Original magnification x200 (A). Scanning electron microscopy image of HeLa cells on the nanostructured grid fibers after 48 hours of incubation. Original magnification x100 (B).</p

Transmission electron microscopy images of HeLa cells on the nanostructure grid fibers after 48 h culture.

<p>Original magnification x6000.</p