Danthron, an Anthraquinone Derivative, Induces DNA Damage and Caspase Cascades-Mediated Apoptosis in SNU-1 Human Gastric Cancer Cells through Mitochondrial Permeability Transition Pores and Bax-Triggered Pathways

Abstract

Anthraquinones have been shown to induce apoptosis in different types of tumor cells, but the mechanisms of danthron-induced cytotoxicity and apoptosis in human gastric cancer cells have not been adequately explored. This study investigated the roles of caspase cascades, ROS, DNA damage, mitochondrial disruption, and Bax and Bcl-2 proteins in danthron-induced apoptosis of SNU-1 human gastric cancer cells, a commonly used cell culture system for in vitro studies. Cells were incubated with different concentrations of danthron in a time- and/or dose-dependent manner. Cell morphological changes (shrinkage and rounding) were examined by a phase-contrast microscope, whereas cell viability and apoptotic populations were determined by flow cytometric analysis using propidium iodide (PI) and annexin V-FITC staining. The fluorescent DAPI nucleic acid stain and Comet assay were applied to detect danthron-induced chromatin condensation (an apoptotic characteristic) and DNA damage. Increasing the levels of caspase-3, -8, and -9 activities was involved in danthron-induced apoptosis, and they could be attenuated by inhibitors of specific caspases, indicating that danthron triggered the caspase-dependent apoptotic pathway. Further studies with flow cytometric analyses indicated that cellular levels of ROS, cytosolic Ca(2+), and mitochondrial permeability transition (MPT) pore opening were increased, but the level of mitochondrial membrane potential (ΔΨ(m)) was decreased. Also, the ratio of Bax/Bcl-2 levels and other proapoptotic proteins associated with modulating the ΔΨ(m) were up-regulated. Apoptotic signaling was also stimulated after exposure to danthron and determined by Western blotting and real-time PCR analyses. In summary, it is suggested that danthron-induced apoptotic cell death was involved in mitochondrial depolarization, which led to release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G) and caused the activation of caspase-9 and -3 in SNU-1 human gastric cancer cells.

Gallic acid suppresses the migration and invasion of PC-3 human prostate cancer cells via inhibition of matrix metalloproteinase-2 and -9 signaling pathways

“…These cells were analyzed with a flow cytometer (BD FACSCalibur, San Jose, CA, USA) equipped with an argon ion laser at 488 nm wavelength (24,25).…”

“…The invasion assay of PC-3 cells was performed by using Matrigel (BD Biosciences, Bedford, MA, USA)-coated transwell cell culture chambers (8 µm pore size, EMD Millipore, Temecula, CA, USA) as described previously (31). PC-3 cells (10 4 cells/0.4 ml) were cultured for 24 h in serum-free RPMI-1640 medium and then placed in the upper chamber of the transwell insert (5x10 4 cells/well) and treated with 0.5% DMSO (as a control) or GA (25,50 or 100 µM). However, the RPMI-1640 medium containing 10% FBS was placed in the lower chamber.…”

“…The supernatant was collected and total protein was quantitated using a Bio-Rad protein assay kit (Hercules, CA, USA) with bovine serum albumin (BSA) as the standard. Protein abundance of MMP-2, MMP-9, SOS1, GRB2, PKC, NF-κB p65, JNK1/2, ERK1/2, p38, PI3K, AKT, p-AKT (Thr308) and p-AKT (Ser473) (Santa Cruz Biotechnology, Inc.) were determined by SDS-PAGE and Western blotting as previously described (24)(25)(26). NIH Image J software was used to determine the band intensity from Western blot analysis.…”

“…6 cells/well) were placed in 6-well plates and incubated without and with GA (25 or 50 µM) for 24 h. Cells were harvested and total RNA was extracted from each treatment by using the Qiagen RNeasy Mini Kit (Qiagen, Inc., valencia, CA, USA) as described previously (24,25). RNA samples were reverse-transcribed at 42˚C with High Capacity cDNA Reverse Transcription Kit for 30 min according to the protocol of the supplier (Applied Biosystems, Carlsbad, CA, USA).…”

Cellular Caspases: New Targets for the Action of Pharmacological Agents

“…The release is promoted by Bcl-2 family proteins (Вах-protein). The flavoprotein AIF released from the mitochondrial intermembrane region is an apoptosis effector that would then act independently of caspases [24,29].…”