Statistical analysis All the statistical results are presented as the mean??sd for at least three separate experiments. that YC-1 caused DNA fragmentation, a late stage feature of apoptosis. In addition, YC-1 increased the activities of caspase-9 and caspase-3, disrupted the mitochondrial membrane potential (AYm) and stimulated ROS production in CAR cells. The protein levels of cytochrome c, Bax and Bak were elevated while Bcl-2 protein expression was attenuated in YC-1-treated CAR cells. In summary, YC-1 suppressed the viability Rabbit Polyclonal to GUF1 of cisplatin-resistant CAR cells through inhibiting cell proliferation, arresting cell cycle at G0/G1 phase and triggering mitochondria-mediated apoptosis. Our results provide evidences to support the potentially therapeutic application of YC-1 on fighting against drug resistant oral cancer in the future. Cell Death Detection kit, Fluorescein (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany) according to the protocol by the manufacturer [101C104]. 2.8. Assays for caspase-3 and caspase-9 activities CAR cells (2??105 cells/ per well) were seeded into 6-well plates and incubated with 0, 25, 50, 75 and 100 of YC-1 for 48?h. At the end of the treatment, cells were harvested and cell lysates were assessed in accordance with the manufacturers instruction provided in the caspase-3 and caspase-9 Colorimetric Assay kits (R&D Systems Inc.). Cell lysate protein was then incubated for 1?h at 37?C with specific caspase-3 substrate (DEVD-pNA) or caspase-9 substrate (LEHD-pNA) in the reaction buffer (provided in MPC-3100 the kits). The OD405 of the released pNA in each sample was measured as previously described [86, 105]. 2.9. Detection of ROS generation and mitochondrial membrane potential (m) CAR cells (2??105 cells/ per well) were seeded into 6-well plates and incubated with 0, 25, 50, 75 and 100 of YC-1 for 48?h. At the end of the treatment, cells were harvested and incubated with 10?M H2DCFDA and 4 nM DiOC6 at 37?C for 30?min for H2O2 detection and Am, respectively. The mean fluorescence intensity (MFI) was quantified by BD CellQuest Pro software (BD Biosciences, San Jose, CA, USA) after analysis by flow cytometry [86, 105, 106]. 2.10. Statistical analysis All the statistical results are presented as the mean??sd for at least three separate experiments. Statistical analysis of data was done using one-way ANOVA followed by Students t-test. ***[48] reported that YC-1 inhibited cell proliferation, induced apoptotic cell death, and increased sensitivity to cisplatin in UM-1- and CAL 27-cisplatin resistance cells. However, the molecular mechanisms of YC-1-induced cell cycle arrest and death in cisplatin resistant oral cancer cells are not yet fully understood. In MPC-3100 this study, our results showed that 25-100 of YC-1 significantly inhibited the proliferation of cisplatin-resistant CAR cells (Fig. 1, Fig. 2 and Supplementary video). YC-1 treatment increased the number of cells in the G0/ G1 phase, suggesting that YC-1 caused MPC-3100 growth inhibition by promoting G0/G1 phase arrest in CAR cells (Fig. 3). The significant DNA fragmentation and caspase-3/ -9 activation in YC-1 treated cells (Fig. 4B, C, and D) indicate MPC-3100 that YC-1 can induce caspase- dependent apoptosis in CAR cells. Our findings provide MPC-3100 new insights addressing the anti-cancer activity of YC-1 in cisplatin-resistant CAR cells at the molecular levels. Once the mitochondrial apoptotic signaling is provoked, changes in the mitochondrial membrane permeability would lead to the loss of mitochondrial membrane potential. In addition, the mitochondrial outer membrane becomes leaky and releases the proapoptotic proteins; including cytochrome Apaf-1 and AIF) were observed after YC-1 treatment (Fig. 5)..