Downregulation of importin-9 protects MCF-7 cells against apoptosis induced by the combination of garlic-derived alliin and paclitaxel.

Numerous studies on the biological mechanism of breast cancer have identified a number of potential therapeutic molecular targets. In this context, one type of potential candidates appears to be agents that target the actin cytoskeleton of cancer cells or regulate actin cytoskeleton dynamics. The aim of the present study was to study the impact of altered actin transport between the cytoplasm and nucleus by the downregulation of importin-9 (IPO9) in breast adenocarcinoma MCF-7 cells exposed to an apoptosis-inducing combination of garlic-derived S-allyl-L-cysteine sulfoxide (alliin) and paclitaxel (PTX). The expression of IPO9 was downregulated by the transfection of non-aggressive breast cancer MCF-7 cells with siRNA against IPO9. The altered expression of IPO9 and cofilin-1 (CFL1) was examined using western blotting. Moreover, the effect of the downregulation of IPO9 on cell death induced by the combination of PTX and alliin was also investigated. The alterations of IPO9 and CFL1 levels were also related with F-actin organizational changes and F-actin fluorescence intensity in the nuclear/perinuclear area of the cells. The results presented here indicate that alliin and PTX act synergistically to promote and potentiate apoptosis in MCF-7 cells. Furthermore, using RNA interference technique, we showed that downregulation of IPO9 expression was correlated with a significant reduction in the apoptotic cell population as well as with a decrease in F-actin content in whole cells, and in the cortical and nuclear/perinuclear areas of the cells. Simultaneously, the downregulation of IPO9 was also accompanied by the increased post-translational expression of CFL1. Furthermore, the data obtained in the present study allow us to conclude that CFL1 itself does not translocate actin into the cell nucleus but this transport requires the functional expression of IPO9.

Tropomyosin-1 protects transformed alveolar epithelial cells against cigaret smoke extract through the stabilization of F-actin-dependent cell-cell junctions.

The aim of the study was to estimate the effect of tropomyosin-1-based structural stabilization of F-actin in transformed human alveolar epithelial line H1299 cells subjected to high oxidative stress induced by cigaret smoke extract. We demonstrated here that cigaret smoke extract induces cell shrinking and detachment as a consequence of F-actin cytoskeleton degradation in H1299 cells not overexpressing tropomyosin-1. Furthermore, the treatment of these cells with cigaret smoke extract resulted in the loss of peripheral localization of ZO-1 and initiated apoptosis. In contrast, structural stabilization of F-actin, by overexpression of tropomyosin-1, preserved cell to cell interactions through the attenuation of cortical actin organization into thin fibers and thus protected these cells against oxidative stress-induced degradation of actin cytoskeleton and cell death. In conclusion, we suggest that structural stabilization of thin cortical F-actin fibers increases link between tight junctions proteins and actin cytoskeleton and thus protects H1299 cells against cigaret smoke extract.

Green tea extract induces protective autophagy in A549 non-small lung cancer cell line.

BACKGROUND AND OBJECTIVES: For many decades, polyphenols, including green tea extract catechins, have been reported to exert multiple anti-tumor activities. However, to date the mechanisms of their action have not been completely elucidated. Thus, the aim of this study was to assess the effect of green tea extract on non-small lung cancer A549 cells. MATERIAL AND METHODS: A549 cells following treatment with GTE were analyzed using the inverted light and fluorescence microscope. In order to evaluate cell sensitivity and cell death, the MTT assay and Tali image-based cytometer were used, respectively. Ultrastructural alterations were assessed using a transmission electron microscope. RESULTS: The obtained data suggested that GTE, even at the highest dose employed (150 µM), was not toxic to A549 cells. Likewise, the treatment with GTE resulted in only a very small dose-dependent increase in the population of apoptotic cells. However, enhanced accumulation of vacuole-like structures in response to GTE was seen at the light and electron microscopic level. Furthermore, an increase in the acidic vesicular organelles and LC3-II puncta formation was observed under the fluorescence microscope, following GTE treatment. The analysis of the functional status of autophagy revealed that GTE-induced autophagy may provide self-protection against its own cytotoxicity, since we observed that the blockage of autophagy by bafilomycin A1 decreased the viability of A549 cells and potentiated necrotic cell death induction in response to GTE treatment. CONCLUSION: Collectively, our results revealed that A549 cells are insensitive to both low and high concentrations of the green tea extract, probably due to the induction of cytoprotective autophagy. These data suggest that a potential utility of GTE in lung cancer therapy may lie in its synergistic combinations with drugs or small molecules that target autophagy, rather than in monotherapy.

Hyperthermia induces cytoskeletal alterations and mitotic catastrophe in p53-deficient H1299 lung cancer cells.

Hyperthermia is used in cancer therapy, however much remains to be discovered regarding its mechanisms of action at the cellular level. In this study, the effects of hyperthermia on cell death, survival, morphology and the cytoskeleton were investigated in a non-small cell lung cancer cell line, H1299. Despite the fact that this cell line is widely used in research, it has not yet been tested for heat shock sensitivity. Cells were given a 30-min heat shock at 43.5°C and 45°C and left to recover at 37°C for 24 and 48 h. 24 h after heat shock treatment, we monitored changes in the organization of the cytoskeleton using immunofluorescence microscopy. The number of actin stress fibers was significantly reduced, microtubules formed a looser meshwork, a portion of the cells possessed multipolar mitotic spindles, whereas vimentin filaments collapsed into perinuclear complexes. 48 h following heat stress, most of the cells showed recovery of the cytoskeleton, however we observed a considerable number of giant cells that were multinucleated or contained one enlarged nucleus. The data obtained by MTT assay showed a dose-dependent decrease of cell viability, while flow cytometric analysis revealed an increase in the number of cells with externalized phosphatidylserine. The results suggest that one of the modes of heat-induced cell death in H1299 cells is mitotic catastrophe, which probably ends in apoptosis.

Ciprofloxacin is a potential topoisomerase II inhibitor for the treatment of NSCLC.

Lung cancer is one of the most common tumors and its treatment is still inefficient. In our previous work we proved that ciprofloxacin has a different influence on five cancer cell lines. Here, we aimed to compare the biological effect of ciprofloxacin on cell lines representing different responses after treatment, thus A549 was chosen as a sensitive model, C6 and B16 as highly resistant. Three different cell lines were analyzed (A549, B16 and C6). The characterization of continuous cell growth was analyzed with the Real-Time Cell Analyzer (RTCA)-DP system. Cytoskeletal changes were demonstrated using immunofluorescence. The cell cycle was analyzed using flow cytometry. Ciprofloxacin was cytostatic only against the A549 cell line. In the case of other tested cell lines a cytostatic effect was not observed. Cytoskeletal analysis confirms the results obtained with RTCA-DP. A549 cells were inhibited in the G2/M phase suggesting a mechanism related to topoisomerase II inhibition. The biological effects of ciprofloxacin support the hypothesis that this drug can serve as an adjuvant treatment for lung cancer, due to its properties enabling topoisomerase II inhibition.