However, the contrary results have been obtained by Khan et al

However, the contrary results have been obtained by Khan et al., who have revealed that the treatment with 10?M fisetin for 24?h decreased the viability of A549 cells by 25?%, as measured using MTT assay [3]. cell nuclei and mitotic spindles, the pattern of LC3-II immunostaining, the formation of autophagic vacuoles at the electron and fluorescence microscopic level, the disruption of cell membrane asymmetry/integrity, cell cycle progression and the expression level of LC3-II, Bax, Bcl-2 and caspase-3 mRNA. Results Here, we reported the first experimental evidence for the presence of synergism between fisetin and paclitaxel in the in vitro model of non-small cell lung cancer. This synergism was, at least partially, ascribed to the induction of mitotic catastrophe. The switch from the cytoprotective autophagy to the autophagic cell death was also implicated in the mechanism of the synergistic action of fisetin and paclitaxel in the A549 cells. In Lidocaine (Alphacaine) addition, we revealed that this synergism between fisetin and paclitaxel was cell line-specific as well as that fisetin synergizes with arsenic trioxide, but not with mitoxantrone and methotrexate in the A549 cells. Conclusions Our Lidocaine (Alphacaine) results provide rationale for further testing of fisetin in the combination with paclitaxel or arsenic trioxide to obtain detailed insights into the mechanism of their synergistic action as well as to evaluate their toxicity towards normal cells in an animal model in vivo. We conclude that Lidocaine (Alphacaine) this study is potentially interesting for the development of novel chemotherapeutic approach to non-small cell lung cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12935-016-0288-3) contains supplementary Lidocaine (Alphacaine) material, which is available to authorized users. for 8?min, resuspended in annexin binding buffer (ABB) and incubated with Annexin V Alexa Fluor 488 at room temperature (RT) in the dark for 20?min. Following the centrifugation at 300for 5?min, the cells were again resuspended in ABB and incubated with propidium iodide at RT in the dark for 5?min. The cells were analyzed using Tali image-based cytometer (Invitrogen/Life Technologies, Carlsbad, CA, USA). The data were quantified by FCS Express Research Edition software (version 4.03; De Novo Software, New Jersey, NJ, USA) and expressed as the percentage of cells in each population (viable Annexin V?/PI?; early apoptotic Annexin V+/PI?; late apoptotic Annexin V+/PI+; necrotic Annexin V?/PI+). The Lidocaine (Alphacaine) sum of the early and late apoptotic cells represented the total apoptosis. Cell cycle analysis For DNA content analysis, the Tali Cell Cycle Kit (Invitrogen/Life Technologies, Carlsbad, CA, USA) was used according to the manufacturers instructions. Briefly, the treated cells were harvested from 6-well plates by trypsinization, rinsed with PBS, fixed in ice-cold 70?% ethanol at 4?C, and left at ?25?C overnight. The next day, the cells were centrifuged at 1000for 5?min at 4?C and washed with PBS. After the centrifugation at 500for 10?min at 4?C, the cells were resuspended in the Tali Cell Cycle Solution containing propidium iodide (PI), RNase A, and Triton X-100. Following 30-min incubation at RT in the dark, the cells were analyzed using Tali image-based cytometer (Invitrogen/Life Technologies, Carlsbad, CA, USA) and the percentage of cells in each phase of the cell cycle was decided using FCS Express Research Edition software (version 4.03; De Novo Software, New Jersey, NJ, USA). Fluorescent staining of -tubulin and cell nuclei For spindle morphology assessment, the cells were seeded on glass cover slides in 12-well plates, permitted to adhere overnight and then treated with fisetin and/or paclitaxel. After the prefixation step with 1?mM bifunctional protein cross-linking reagent 3,30-dithiodipropionic acid (DTSP; Sigma-Aldrich, St. Louis, MO, USA) in Hanks balanced salt solution (HBSS; Sigma-Aldrich, St. Louis, MO, USA) for 10?min, the cells were extracted in TsB (0.5?% Triton X-100; Serva, Heidelberg, Germany) in microtubule stabilizing buffer (MTSB; 1?mM EGTA, 4?% poly(ethylene glycol), 10?mM PIPES; Sigma-Aldrich, St. Louis, MO, USA) made up of DTSP (dilution 1:50) for 10?min and rinsed with TsB for 5?min. Following the fixation of the cells with 4?% paraformaldehyde (Serva, Heidelberg, Germany) in MTSB for 15?min and three washing actions with PBS, the cells were incubated with 1?% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) diluted in tris-buffered saline (TBS; Sigma-Aldrich, St. Louis, MO, USA) for 15?min. -tubulin Rabbit Polyclonal to ABHD12 was labeled using the mouse monoclonal antibody specific for -tubulin (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:65 in 1?% BSA-PBS (1?h, a moist chamber). This was followed by rinsing the cells.