Smh-3 induces G2/M arrest and apoptosis through calcium‑mediated endoplasmic reticulum stress and mitochondrial signaling in human hepatocellular carcinoma Hep3B cells
- Authors:
- Published online on: December 5, 2012 https://doi.org/10.3892/or.2012.2166
- Pages: 751-762
Abstract
Introduction
Cancer is the major cause of mortality in human populations worldwide, and human hepatocellular carcinoma is one of the most lethal types of cancers (1,2). Typical treatment approaches to human hepatocellular carcinoma include hepatic resection, chemotherapy, percutaneous ablation and transcatheter arterial chemoembolization and transplantation, yet patient outcomes are not satisfactory (3,4). Currently, investigators are focusing on new agents and novel targets for human hepatocellular carcinoma treatment (5–7).
Caspases are important proteases in cells. Following apoptotic stimuli, caspases can stimulate intracellular cascades and activate downstream caspase members (8,9). Several apoptotic stimuli have been reported that include extrinsic pathways (receptor-ligand interaction) and intrinsic pathways (mitochondrial-involved) (10–12). In the intrinsic apoptosis pathway, caspase-9 acts as a major initiator caspase, while in the extrinsic pathway, caspase-8 is a major initiator caspase (16–18).
Endoplasmic reticulum (ER) stress induces apoptotic cell death (13–15). Recent studies have identified ER as a third pathway implicated in apoptosis. ER has several biological functions including protein folding, protein trafficking and regulation of the intracellular calcium concentration in apoptosis (15,19,20). When ER disrupts the biological function, the unfolded protein response is triggered and this response occurs through the activation of ER stress sensor proteins, including inositol-requiring enzyme 1 (IRE1), GADD153 and activating transcription factor 6 (ATF-6) (10,11,21). The ubiquitin-proteasome system plays an important role in the degradation of unfolded proteins (22,23). The continued increase of unfolded proteins in the ER lumen disrupts Ca2+ homeostasis in the ER and ultimately leads to apoptosis. The major initiator caspase is caspase-4 in human cells or caspase-12 in murine cells (24–26).
In our previous study, we designed and synthesized a series of 2-phenyl-4-quinolone compounds as novel antitumor agents (27–30). 2-(3-(Methylamino)phenyl)-6-(pyrrolidin-1-yl)quinolin-4-one (Smh-3) (Fig. 1A) is a candidate exhibiting the most potential for antitumor activities. We demonstrated that Smh-3 induces G2/M phase arrest and mitochondrial-dependent apoptotic cell death through inhibition of CDK1 and AKT activity in HL-60 human leukemia cells (31). However, neither the cytotoxic effects of Smh-3 on human hepatocellular carcinoma cells, nor the molecular mechanisms underlying its anticancer activity have been investigated. Therefore, this study investigated the molecular mechanisms of the antitumor effects of Smh-3 on Hep3B cells in vitro.
Materials and methods
Materials, chemicals and reagents
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], potassium phosphate, trypan blue, propidium iodide (PI), Triton X-100, Tris-HCl and ribonuclease-A were obtained from Sigma-Aldrich Corp. (St. Louis, MO). 3′-Dihexyloxacarbocyanine iodide (DiOC6), RPMI-1640 medium, L-glutamine, fetal bovine serum (FBS), Trypsin-EDTA, penicillin, nitrocellulose membrane and the iBlot Dry Blotting system were obtained from Invitrogen Life Technologies (Carlsbad, CA). Caspase-4 activity substrate (Ac-LEVD-pNA) was purchased from BioVision (Mountain View, CA) and caspase-3 and -9 activity assay kits were purchased from R&D Systems (Minneapolis, MN). Primary antibodies (anti-caspase-4, anti-GADD153 and anti-β-actin) and second antibodies for western blotting were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture
The human hepatocellular carcinoma Hep3B cell line was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The Hep3B cells were incubated in 5% CO2 at 37°C in DMEM medium with 2 mM L-glutamine, supplemented with 10% heat-inactivated FBS and 1% antibiotic/antimycotic (100 units/ml penicillin and 100 μg/ml streptomycin) (32).
Determination of cell morphology and the percentage of viable cells
For analysis of cell morphological changes, cells treated with Smh-3 (100 nM) in the well were examined and photographed under a phase-contrast microscope at a magnification of ×400. The quantitative analysis of cell viability was performed by MTT assay. Cells (1×104 cells/well) on 96-well plates were exposed to Smh-3 (0, 50, 100, 200 and 300 nM) and 0.1% DMSO as a vehicle control. After a 24- and 48-h incubation, 100 ml MTT (0.5 mg/ml) solution was added to each well, and the plate was incubated at 37°C for 4 h. Then, 0.04 N HCl in isopropanol was added, and the absorbance at 570 nm was measured for each well. All results were representative of 3 independent experiments (33,34).
DNA content and cell cycle distribution analysis
Hep3B cells were incubated with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h. For determination of cell cycle phase and apoptosis, cells were fixed gently in 70% ethanol at −20°C overnight, and then re-suspended in PBS containing 40 μg/ml PI, 0.1 mg/ml RNase and 0.1% Triton X-100 in a dark room. Cell cycle distribution and apoptotic nuclei were determined by flow cytometry (31,35,36).
CDK1 kinase assay
CDK1 kinase activity was analyzed according to the protocol outlined for the CDK1 kinase assay kit (Medical & Biological Laboratories International, Nagoya, Japan). In brief, the ability of the cell extract prepared from each treatment to phosphorylate its specific substrate, MV peptide, was measured as previously described (31,33).
Caspase activity assay
Hep3B cells were incubated with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h. Cells were lysed in lysis buffer [50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 10 mM EGTA, 10 mM digitonin and 2 mM DTT]. Approximately 50 μg of cytosol proteins was incubated with caspase-4 (BioVision), caspase-9 and caspase-3-specific substrates (R&D System) for 1 h at 37°C. The caspase activity was determined by measuring OD405 as previously described (31,33,37).
Assay of intracellular Ca2+ levels
Hep3B cells were treated with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h. Cells were harvested, washed twice and re-suspended in 3 mg/ml of Fluo-3/AM (Calbiochem; La Jolla, CA) at 37°C for 30 min and analyzed by flow cytometry (Becton-Dickinson FACSCalibur) (38,39).
Determination of mitochondrial membrane potential (ΔΨm)
Hep3B cells were treated with 0, 50, 100, 200 and 300 nM of Smh-3. The cells were harvested and washed twice, resuspended in DiOC6 (4 mmol/l) and incubated for 30 min before being analyzed by flow cytometry (Becton-Dickinson FACSCalibur) (38,39).
Western blot assay
Hep3B cells were placed into 75-T flask. Cells in each well were treated without and with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h. Cells were collected and total protein from each treatment was extracted and placed into buffer (PRO-PREP™ protein extraction solution, Korea) and centrifuged at 12,000 rpm for 10 min at 4°C. The quantitated total protein from each treatment was determined by Bradford assay. Proteins from each treatment were resolved on an SDS polyacrylamide gel through electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were incubated with a blocking buffer of 5% non-fat dry milk in Tris-buffered saline containing Tween-20 for 1 h at room temperature and then incubated with the specific primary antibodies (anti-GADD153 and anti-caspase-4). The membranes were washed and then treated by appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using an ECL detection kit (GE Healthcare, Princeton, NJ) (33,40).
cDNA microarray analysis
Hep3B cells were treated with or without 100 nM of Smh-3 for 24 h. Then cells from each treatment were harvested, and the total RNA was extracted using the Qiagen RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA). The isolated total RNA was used for cDNA synthesis and labeling and microarray hybridization. The fluorescence-labeled cDNA were then hybridized to their complements on the chip (Affymetrix GeneChip Human Gene 1.0 ST array, Affymetrix, Santa Clara, CA, USA). Finally the resulting localized concentrations of fluorescent molecules were detected and quantitated (Asia Bio-Innovations Corp.). The resulting data were analyzed using Expression Console software (Affymetrix) with default RMA parameters. Genes regulated by citosol were determined to have a 1.5-fold change in expression (41).
Statistical analysis
Significance of the mean values between the Smh-3-treated group and control group was obtained using the Student’s t-test. Data were expressed as the means ± SD. P<0.05 was considered to indicate a statistically significant difference (33,40).
Results
Smh-3 decreases the percentage of Hep3B viable cells
To investigate the effect of Smh-3 on cell proliferation, Hep3B cells were treated with 0, 50, 100, 200 and 300 nM of Smh-3 for 48 h. The cell viability following each treatment was analyzed by MTT assay. As shown in Fig. 1B, Smh-3 inhibited Hep3B cell growth in a dose- and time-dependent manner. The half maximal inhibitory concentration IC50 following a 48-h treatment of Smh-3 was 68.26±3.24 nM.
Smh-3 induces G2/M arrest and decreases CDK1 activity and apoptosis in Hep3B cells
Smh-3 induced cell morphological changes and decreased the cell numbers of Hep3B cells (Fig. 2B). Mitotic and apoptotic cells appeared smaller, round and blunt in size following exposure to Smh-3. To investigate the cell cycle distribution of Hep3B cells following Smh-3 treatment, cells were stained with propidium iodide (PI). Flow cytometry revealed that Smh-3 treatment (0, 50, 100 and 200 nM) of Hep3B cells significantly increased the G2/M cell population at 48 h (Fig. 2A). Furthermore, Smh-3 treatment increased the sub-G1 cell population at 48 h in a concentration-dependent manner. These data suggest that Smh-3 effectively induces G2/M arrest and promotes cell death. We examined the CDK1 activity in Smh-3-treated Hep3B cells. Treatment with 0, 50, 100, 200 and 300 nM Smh-3 caused a significant decrease in CDK1 activity (Fig. 2C). Our results suggest that the downregulation of CDK1 activity plays an important role in G2/M phase arrest in Smh-3-treated Hep3B cells.
cDNA microarray analysis
The microarray analysis indicated 192 genes were upregulated and 278 genes were downregulated in Hep3B cells following treatment with Smh-3. Moreover, the mRNA descriptions of the genes are listed in Table I. DNA microarray assay revealed that many differentially expressed genes related to angiogenesis, autophagy, calcium-mediated ER stress signaling, cell adhesion, cell cycle and mitosis, cell migration, cytoskeleton organization, DNA damage and repair, mitochondrial-mediated apoptosis and cell signaling pathways were present in the Hep3B cells following Smh-3 treatment.
 | Table IGenes exhibiting more than 1.5-fold changes in mRNA levels in Hep-3B cells following a 24-h treatment with Smh-3 as identified using DNA microarray. |
Effects of Smh-3 on ER stress in Hep3B cells
Our previous research demonstrated that Smh-3 acts against HL-60 leukemia cells in vitro via G2/M phase arrest, downregulation of AKT activity and induction of mitochondrial-dependent apoptotic pathways (31). To confirm the possibility that the Smh-3-induced apoptosis could be related to contributions from the ER stress signal pathways, Hep3B cells were treated with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h. The intracellular Ca2+ release, caspase-4 activity and the levels of ER stress-associated proteins were examined. The quantities and results are shown in Fig. 3. Results from the flow cytometric assay indicated that Smh-3 induced the production of intracellular Ca2+ release (Fig. 3A). To evaluate whether or not Smh-3-induced apoptosis is involved in activation of caspase-4, we determined the caspase-4 activity using caspase colorimetric analysis. Smh-3 at concentrations of 0, 50, 100, 200 and 300 nM stimulated caspase-4 activity in a concentration-dependent manner Fig. 3B. It was reported that GADD153 is a hallmark of ER stress (42,43). Smh-3-treated Hep3B cells were harvested for western blot analysis of the expression levels of the ER stress pathway-related GADD153 and caspase-4 proteins. Smh-3 promoted the protein levels of GADD153 and caspase-4 (Fig. 3C). Based on these results, we suggest that Smh-3-induced apoptosis in Hep3B cells may be mediated through the ER stress-dependent apoptotic signaling pathway.
Effects of Smh-3 on the loss of ΔΨm level, caspase-9 and caspase-3 activities in Hep3B cells
To confirm the possibility that Smh-3-induced apoptosis is related to contributions from the mitochondrial signal pathways, Hep3B cells were treated with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h, and changes in ΔΨm, caspase-9 and caspase-3 activities were examined; the quantities and results are shown in Fig. 4. Results from the flow cytometric assay indicated that Smh-3 decreased the level of ΔΨm (Fig. 4A). To evaluate whether or not Smh-3-induced apoptosis is involved in activation of caspase-9 and caspase-3, we detected the caspase-9 and caspase-3 activities using caspase colorimetric analysis. Concentrations of 0, 50, 100, 200 and 300 nM of Smh-3 stimulated caspase-9 activity (Fig. 4B) and caspase-3 activity (Fig. 4C) in a concentration-dependent manner.
Discussion
There are no reports concerning the effects of Smh-3 on apoptosis and associated gene expression in Hep3B cells. The present study is the first to show that Smh-3 induces a cytotoxic effect which includes induction of G2/M phase arrest and apoptosis and changes in the expression of associated gene in Hep3B cells. In previous studies, we showed that Smh-3 triggered apoptosis in HL-60 human leukemia cells (31); however, the involvement of ER stress in Smh-3-induced apoptosis in cancer cells is still unclear. Our results demonstrated that Smh-3 treatment increased the protein levels of caspase-4 and GADD153 in Hep3B cells (Fig. 3B and C). Our novel findings suggest that these events demonstrate that the ER stress apoptotic pathway is involved in the Smh-3 effects in vitro. It has been reported that cellular organelles such as mitochondria, ER, lysosomes and Golgi apparatus are also major targets of apoptotic initiation (44,45). Many chemotherapy agents that strongly affect the function of the ER are identified as strong inducers of GADD153. This suggests that increased intracellular Ca2+ induces mitochondrial swelling (39,46). Following mitochondrial permeabilization, cytochrome c, Apaf-1, procaspase-9, Endo G and AIF are released into the cytosol, activating caspase-3 via caspase-9 (47,48). Smh-3 induced the activation of caspase-9 and caspase-3 after a 48-h treatment (Fig. 4B and C), suggesting that Smh-3 possibly activates the mitochondrial signaling pathway. Our results demonstrated that Smh-3 decreased the ΔΨm after a 24 h treatment with Smh-3 (Fig. 4A), and then promoted caspase-9 and caspase-3 activities in Hep3B cells (Fig. 4B and C). In addition, caspase-8 activity exhibited no significant increase in the Smh-3-treated Hep3B cells (data not shown). Based on the above evidence, Smh-3-stimulated apoptotic cell death is involved in the crosstalk between the ER and mitochondria.
Based on the change in gene expression profile in Smh-3-treated Hep3B cells by DNA microarray, we found that cellular and molecular responses to Smh-3 treatment are multifaceted and are likely to be mediated via a variety of regulatory pathways. Smh-3 regulated the expression of important genes that control cell growth, angiogenesis, autophagy, calcium-mediated ER stress signaling, cell adhesion, cell cycle and mitosis, cell migration, cytoskeleton organization, DNA damage and repair, mitochondrial-mediated apoptosis, transcription and translation and cell signaling pathways (Table I). Regulation of these genes may be responsible for inhibiting the proliferation of Hep3B cells. It was reported that cyclins associate with cyclin-dependent protein kinases (CDKs) and CDK inhibitor (CKI) to control the process of the cell cycle. The CDK inhibitor (CKI) has been demonstrated to arrest the cell cycle and inhibit the cell growth of cancer cells (33,40,46). From a gene expression profile, we found that Smh-3 altered the expression of cyclin and cyclin-dependent kinase inhibitors and cytoskeleton organization genes including CNNM3, CDKN3, RPRM, CCNG1, ACTB, ACTG1, TUBA1B, TUBB2C and MAP1B, suggesting a change in cyclin, cyclin-dependent kinase inhibitors, and microtubule interaction which could finally lead to cell cycle G2/M arrest (Fig. 2B).
In conclusion, the molecular signaling pathways involved in Smh-3 effects on Hep-3B cells are summarized in Fig. 5. Based on these data, further detailed investigations including anti-metastasis, anti-angiogenesis and autophagy induction studies are required in order to establish cause and effect relationships between these altered genes and the outcome of human hepatocellular carcinoma patients.
Acknowledgements
The study was supported by research grants from the National Science Council of P.R. China awarded to S.-C.K. (NSC 100-2320-B-039-001) and Taiwan Department of Health, China Medical University Hospital Cancer Research Center of Excellence (DOH101-TD-C-111-005).
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