Correlation between the progressive cytoplasmic expression of a novel small heat shock protein (Hsp16.2) and malignancy in brain tumors

<p>Abstract</p> <p>Background</p> <p>Small heat shock proteins are molecular chaperones that protect proteins against stress-induced aggregation. They have also been found to have anti-apoptotic activity and to play a part in the development of tumors. Recently, we identified a new small heat shock protein, Hsp16.2 which displayed increased expression in neuroectodermal tumors. Our aim was to investigate the expression of Hsp16.2 in different types of brain tumors and to correlate its expression with the histological grade of the tumor.</p> <p>Methods</p> <p>Immunohistochemistry with a polyclonal antibody to Hsp16.2 was carried out on formalin-fixed, paraffin-wax-embedded sections using the streptavidin-biotin method. 91 samples were examined and their histological grade was defined. According to the intensity of Hsp16.2 immunoreactivity, low (+), moderate (++), high (+++) or none (-) scores were given.</p> <p>Immunoblotting was carried out on 30 samples of brain tumors using SDS-polyacrylamide gel electrophoresis and Western-blotting.</p> <p>Results</p> <p>Low grade (grades 1–2) brain tumors displayed low cytoplasmic Hsp16.2 immunoreactivity, grade 3 tumors showed moderate cytoplasmic staining, while high grade (grade 4) tumors exhibited intensive cytoplasmic Hsp16.2 staining. Immunoblotting supported the above mentioned results. Normal brain tissue acted as a negative control for the experiment, since the cytoplasm did not stain for Hsp16.2. There was a positive correlation between the level of Hsp16.2 expression and the level of anaplasia in different malignant tissue samples.</p> <p>Conclusion</p> <p>Hsp16.2 expression was directly correlated with the histological grade of brain tumors, therefore Hsp16.2 may have relevance as becoming a possible tumor marker.</p

Desethylamiodarone-A metabolite of amiodarone-Induces apoptosis on T24 human bladder cancer cells via multiple pathways.

Bladder cancer (BC) is a common malignancy of the urinary tract that has a higher frequency in men than in women. Cytostatic resistance and metastasis formation are significant risk factors in BC therapy; therefore, there is great interest in overcoming drug resistance and in initiating research for novel chemotherapeutic approaches. Here, we suggest that desethylamiodarone (DEA)-a metabolite of amiodarone-may have cytostatic potential. DEA activates the collapse of mitochondrial membrane potential (detected by JC-1 fluorescence), and induces cell death in T24 human transitional-cell bladder carcinoma cell line at physiologically achievable concentrations. DEA induces cell cycle arrest in the G0/G1 phase, which may contribute to the inhibition of cell proliferation, and shifts the Bax/Bcl-2 ratio to initiate apoptosis, induce AIF nuclear translocation, and activate PARP-1 cleavage and caspase-3 activation. The major cytoprotective kinases-ERK and Akt-are inhibited by DEA, which may contribute to its cell death-inducing effects. DEA also inhibits the expression of B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) and reduces colony formation of T24 bladder carcinoma cells, indicating its possible inhibitory effect on metastatic potential. These data show that DEA is a novel anti-cancer candidate of multiple cell death-inducing effects and metastatic potential. Our findings recommend further evaluation of its effects in clinical studies

Correlation between the progressive cytoplasmic expression of a novel small heat shock protein (Hsp16.2) and malignancy in brain tumors-2

<p><b>Copyright information:</b></p><p>Taken from "Correlation between the progressive cytoplasmic expression of a novel small heat shock protein (Hsp16.2) and malignancy in brain tumors"</p><p>http://www.biomedcentral.com/1471-2407/7/233</p><p>BMC Cancer 2007;7():233-233.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2234428.</p><p></p>ar Hsp16.2 immunoreactivity, whereas no immunoreactivity in the cytoplasm. . Pilocytic astrocytoma. Strong intranuclear immunopositivity but no intracytoplasmic staining was detected. . Grade 2 astrocytoma shows intensive intranuclear labeling as well as mild cytoplasmic staining. . Grade 3 astrocytoma exhibits strong Hsp 16.2 positivity intranuclearly and moderate Hsp16.2 positivity in the cytoplasm. Grade 1 meningeoma showing high expression of Hsp16.2 intranuclearly and mild expression in the cytoplasm. . Grade 3 meningeoma displayed strong intranuclear and moderate cytoplasmic staining for Hsp16.2. Grade 2 ependymoma with strong intranuclear and moderate cytoplasmic immunopositivity. Grade 3 oligodendroglioma exhibiting intensive intranuclear and moderate cytoplasmic immunoreactivity. . Grade 4 glioblastoma showing strong Hsp16.2 positivity intranuclearly and intracytoplasmically alike. . Grade 4 PNET with intensive staining in the nucleus as well as in the cytoplasm

The effect of DEA on the expression of BMI1 and on the cell cycle in T24 cells.

<p>(A) T24 cells were exposed to increasing concentrations of DEA for 24-hour intervals. Equal amounts of lysate protein were subjected to gel electrophoresis. Expression levels of BMI1 were monitored by immunoblot assay. GAPDH was used as loading control. The results are presented as representative immunoblots and their densitometric analysis in bar diagram. Data represent mean ± SD of three independent experiments: **p < 0.01 and ***p < 0.001 compared to the corresponding control group. (B) T24 cells were treated with 10 μM of DEA for 24 hours. Cells were harvested, fixed with ethanol and stained with propidium iodide. DNA content was determined using the Muse^™ Cell Analyzer. Graphs demonstrate the percentage of G0/G1phase (dark gray bars), S phase (striped bars) and G2/M phase (white bars). Untreated cells served as controls. Each column represents the average obtained from three independent experiments. Data are presented as the mean ± SD, ***p < 0.001 compared to control.</p

Effect of DEA on activation of apoptosis in T24 cells.

<p>T24 cells were treated with increasing concentrations of DEA for 24 hours to induce apoptosis, then stained with the Muse^™ Annexin V & Dead Cell Reagent, and acquired on the Muse^™ Cell Analyzer. (A) Graphs demonstrate the percentage of living (dark gray bars), early apoptotic (striped bars), late apoptotic (white bars) and total apoptotic cells (light gray bars). Untreated cells served as controls. The results are mean ± SD of three independent experiments performed in at least quadruplicate: *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the corresponding control group. In order to investigate the fragmentation of nuclei in T24 cells we treated them with increasing concentrations of DEA for 24 hours. Apoptosis was assessed by Hoechst 33342 staining. (B) Quantification of apoptotic cells was performed by taking the images in random fields and counting cells with strong fluorescence, and condensed or fragmented nuclei. (C) The rate of nuclear fragmentation is also presented in bar diagrams. Each column represents the average obtained from three independent experiments. Data are presented as the mean ± SD, **p < 0.01, ***p < 0.001 compared to control.</p

Structure of desethylamiodarone.

<p>Structure of desethylamiodarone.</p

Effect of DEA on the AIF translocation to the nucleus.

<p>In order to investigate the Ca²⁺-dependent nuclear translocation of AIF, we treated T24 cells with increasing concentrations of DEA for 24 hours, then we isolated the nuclei. Equal amounts of nuclear fraction protein lysates were subjected to gel electrophoresis. The expression level of AIF was monitored by immunoblot assay. HH1 was used as loading control. The results are presented as representative immunoblots (A) and densitometric analysis of immunoblots in bar diagrams (B). The results are mean ± SD of three independent experiments: **p < 0.01 and ***p < 0.001 compared to the corresponding control group.</p

Effect of DEA on cell viability and on colony formation of T24 cells.

<p>(A) T24 cells were exposed to increasing concentrations of DEA for 24 (dark grey bars) and 48 hours (light gray bars). Untreated cells served as controls. Data represent means ± SD of three independent experiments performed in at least quadruplicate: *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the corresponding control group. (B) For the colony formation assay T24 cells were exposed to increasing concentrations of DEA for 7 days. The results are presented as representative images of the colony formation assay. The colony-forming abilities are also presented in bar diagrams (C). Untreated cells served as controls. The results are mean ± SD of three independent experiments performed in at least quadruplicate: *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the control group.</p