Fluoxetine Induces Apoptosis through Extrinsic/Intrinsic Pathways and Inhibits ERK/NF-κB-Modulated Anti-Apoptotic and Invasive Potential in Hepatocellular Carcinoma Cells In Vitro

Abstract

The aim of the present study was to verify the effects of fluoxetine on dysregulation of apoptosis and invasive potential in human hepatocellular carcinoma (HCC) SK-Hep1 and Hep3B cells. Cells were treated with different concentrations of fluoxetine for different times. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assays were used for testing the effects of fluoxetine on cell viability. The regulation of apoptosis signaling, and anti-apoptotic, proliferation, and metastasis-associated proteins after fluoxetine treatment were assayed by flow cytometry and Western blotting assay. The detection of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation after fluoxetine treatment was performed by NF-κB reporter gene assay. The results demonstrated that fluoxetine significantly reduced cell viability, cell migration/invasion, NF-κB, extracellular signal-regulated kinases (ERK) activation, and expression of anti-apoptotic (Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (C-FLIP), Myeloid cell leukemia-1 (MCL-1), X-Linked inhibitor of apoptosis protein (XAIP), and Survivin), proliferation (Cyclin-D1), angiogenesis (vascular endothelial growth factor (VEGF)), and metastasis-associated proteins (matrix metalloproteinase-9 (MMP-9)). Fluoxetine also significantly induced apoptosis, unregulated extrinsic (activation of first apoptosis signal protein and ligand (Fas/FasL), and caspase-8) and intrinsic (loss of mitochondrial membrane potential (ΔΨm) pathways and increased Bcl-2 homologous antagonist killer (BAK) apoptosis signaling. Taken together, these results demonstrated that fluoxetine induced apoptosis through extrinsic/intrinsic pathways and diminished ERK/NF-κB-modulated anti-apoptotic and invasive potential in HCC cells in vitro.

Induction of apoptosis through extrinsic/intrinsic pathways and suppression of ERK/NF‐κB signalling participate in anti‐glioblastoma of imipramine

“…The assessment of in vitro invasion and migration activities were carried out using matrigel‐coated or uncoated transwell cell culture chambers (8 μm pore size) as described previously . Briefly, U‐87 MG and GBM8401 cells (3 × 10 6 cells/well) in serum‐free medium were maintained in a 10‐cm dish and were incubated with imipramine (0, 40 and 80 µmol/L) for 48 hours.…”

Hyperforin induces apoptosis through extrinsic/intrinsic pathways and inhibits EGFR/ERK/NF‐κB‐mediated anti‐apoptotic potential in glioblastoma

“…For cleaved‐caspase‐3 (FITC) and annexin‐V (PE) analysis, cells were harvested and stained by cleaved‐caspase‐3 and annexin‐V dye for 30 minutes 19 . For subG1 analysis, the cells were fixed by 70% ethonal at −20°C overnight and stained with staining solution [40 μg/mL PI, 100 μg/mL RNase and 1% Triton X‐100 in PBS] at 37°C for 1 hour in dark 20 . Finally, subG1 population, cleaved caspase‐3 and Annexin‐V analyzed with a flow cytometer (BD Biosciences, FACS Calibur, San Jose, California) and quantified by FlowJo software (version 7.6.1; FlowJo LLC, Ashland, Oregon).…”

Hyperforin Induces Apoptosis Through Extrinsic/Intrinsic Pathways and Inhibits NF-ĸB-modulated Survival and Invasion Potential in Bladder Cancer

“…Extrinsic apoptosis analysis. Approximately 5×10 5 cells/well of TSGH-8301 cells in 6-well plates were incubated with 0, 10 and 20 μM of hyperforin for 48 h. The cells were trypsinized, harvested, and stained with FAS, FASL and cleaved caspase-8, as described in previous studies (12,13). The results of above staining were measured using the FlowJo 7.6.1 software.…”

“…Approximately 5×10 5 cells/well of TSGH-8301 cells in 6-well plates were incubated with 0, 10 and 20 μM of hyperforin for 48 h. The cells were trypsinized, harvested, and were stained with Fluo-3/AM (2.5 μg/ml) and 500 μL of DiOC 6 (4 μmol/l) for 30 minutes to measure the changes of intracellular Ca 2+ level, and mitochondrial membrane potential (ΔΨm) levels, respectively. The results of above staining were measured using the FlowJo 7.6.1 software (13).…”