Smh-3 induces G2/M arrest and apoptosis through calcium‑mediated endoplasmic reticulum stress and mitochondrial signaling in human hepatocellular carcinoma Hep3B cells

  • Authors:
    • Chin-Yu Liu
    • Jai-Sing Yang
    • Shih-Ming Huang
    • Jo-Hua Chiang
    • Ming-Hua Chen
    • Li-Jiau Huang
    • Ho-Yu Ha
    • Shinji Fushiya
    • Sheng-Chu Kuo
  • View Affiliations

  • Published online on: December 5, 2012     https://doi.org/10.3892/or.2012.2166
  • Pages: 751-762
Metrics: HTML 0 views | PDF 0 views     Cited By (CrossRef): 0 citations

Abstract

In the present study, we investigated the antitumor effects of Smh-3 on the viability, cell cycle and apoptotic cell death in human hepatocellular carcinoma Hep3B cells in vitro. We also investigated the molecular mechanisms involved in the effects of Smh-3 on human hepatoma Hep3B cells, including the effects on protein and mRNA levels which were determined by western blotting and DNA microarray methods, respectively. The results demonstrated that Smh-3 induced growth inhibition, cell morphological changes and induction of G2/M arrest and apoptosis in Hep3B cells. DNA microarray assay identified numerous 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. Furthermore, Smh-3 inhibited CDK1 activity, mitochondrial membrane potential (ΔΨm) and increased the cytosolic Ca2+ release and caspase-4, caspase-9 and caspase-3 activities in Hep3B cells. Western blot analysis demonstrated that Smh-3 increased the protein levels of caspase-4 and GADD153 that may lead to ER stress and consequently apoptosis in Hep3B cells. Taken together, Smh-3 acts against human hepatocellular carcinoma Hep3B cells in vitro through G2/M phase arrest and induction of calcium-mediated ER stress and mitochondrial-dependent apoptotic signaling pathways.

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 (57).

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) (1012). 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 (1618).

Endoplasmic reticulum (ER) stress induces apoptotic cell death (1315). 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 (2426).

In our previous study, we designed and synthesized a series of 2-phenyl-4-quinolone compounds as novel antitumor agents (2730). 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 I

Genes 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.

Table I

Genes 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.

Fold-changeGene symbolmRNA description
Biological process: angiogenesis
1.73RBPJHomo sapiens mRNA for H-2K binding factor-2
1.52GPIHomo sapiens glucose phosphate isomerase
−1.64SH2D2AHomo sapiens SH2 domain containing 2A
−1.74RNH1Homo sapiens ribonuclease/angiogenin inhibitor 1
−1.79RTN4Homo sapiens reticulon 4
−1.87VEGFAHomo sapiens vascular endothelial growth factor A
Biological process: apoptosis and anti-apoptosis
1.73PERPHomo sapiens PERP, TP53 apoptosis effector
1.61BTG1Homo sapiens B-cell translocation gene 1
1.59DBHHomo sapiens dopamine β-hydroxylase
1.58PARP11Homo sapiens poly(ADP-ribose)polymerase family, member 11
1.58SEMA3AHomo sapiens sema domain
−1.51ABRHomo sapiens active BCR-related gene
−1.51BLOC1S2Homo sapiens biogenesis of lysosomal organelles complex-1
−1.51BCL2L1Homo sapiens BCL2-like 1
−1.52CARD17Homo sapiens caspase recruitment domain family, member 17
−1.53YARSHomo sapiens tyrosyl-tRNA synthetase
−1.54USP17L2Homo sapiens ubiquitin specific peptidase 17-like 2
−1.56APH1BHomo sapiens anterior pharynx defective 1 homolog B
−1.62IHHHomo sapiens Indian hedgehog homolog
−1.75CIDEBHomo sapiens cell death-inducing DFFA-like effector b
−1.85BCL6Homo sapiens B-cell CLL/lymphoma 6, transcript variant 1
Biological process: autophagy
1.97ATG12Homo sapiens ATG12 autophagy related 12 homolog
1.85CLEC4FHomo sapiens C-type lectin domain family 4, member F
1.64ACP2Homo sapiens acid phosphatase 2, lysosomal
1.55HPS1Homo sapiens Hermansky-Pudlak syndrome 1
1.53NEU1Homo sapiens sialidase 1
1.51GABARAPHomo sapiens GABA receptor-associated protein
−1.62CHIT1Homo sapiens chitinase 1
−1.68VPS53Homo sapiens vacuolar protein sorting 53 homolog
−2.12SYT3Homo sapiens synaptotagmin III
Biological process: calcium-mediated signaling
1.68CCL3Homo sapiens chemokine ligand 3
1.66STC1Homo sapiens stanniocalcin 1
1.53CHRNA10Homo sapiens cholinergic receptor
1.53GRIN1Homo sapiens glutamate receptor
1.50ETNK2Homo sapiens ethanolamine kinase 2
−1.50CX3CL1Homo sapiens chemokine ligand 1
−1.52PLCG2Homo sapiens phospholipase C, γ 2
−1.55NMUR1Homo sapiens neuromedin U receptor 1
−1.64CALCBHomo sapiens calcitonin-related polypeptide β
−2.43KCNA5Homo sapiens potassium voltage-gated channel
Biological process: cell adhesion
2.50PVRL2Homo sapiens poliovirus receptor-related 2
1.71SYMPKHomo sapiens symplekin
1.68SIGLEC5Homo sapiens sialic acid binding Ig-like lectin 5
1.63LPXNHomo sapiens leupaxin
Biological process: cell adhesion
1.63CDH6Homo sapiens cadherin 6
1.56GPR56Homo sapiens G protein-coupled receptor 56
−1.50PVRL4Homo sapiens poliovirus receptor-related 4
−1.51SIGLEC14Homo sapiens sialic acid binding Ig-like lectin 14
−1.51CPXM2Homo sapiens carboxypeptidase X, member 2
−1.52MMRN1Homo sapiens multimerin 1
−1.53NCAM1Homo sapiens neural cell adhesion molecule 1
−1.54PCDHB4Homo sapiens protocadherin β 4
−1.54PCDHB14Homo sapiens protocadherin β 14
−1.58PCDHB13Homo sapiens protocadherin β 13
−1.58PXNHomo sapiens paxillin
−1.61HSPB11Homo sapiens heat shock protein family B
−1.61ITGA7Homo sapiens integrin, α 7
−1.62SIRPGHomo sapiens signal-regulatory protein γ
−1.62NINJ2Homo sapiens ninjurin 2
−1.62NEO1Homo sapiens neogenin homolog 1
−1.68CYR61Homo sapiens cysteine-rich, angiogenic inducer, 61
Biological process: cell cycle and mitosis
2.13CNNM3Homo sapiens cyclin M3
1.84RAB11BHomo sapiens RAB11B, member RAS oncogene family
1.80CDKN3Homo sapiens cyclin-dependent kinase inhibitor 3
1.6440789Homo sapiens septin 3
1.59MGC16703Homo sapiens tubulin, α pseudogene
1.55KRT18Homo sapiens keratin 18
1.52ARL8BHomo sapiens ADP-ribosylation factor-like 8B
1.52RPRMHomo sapiens reprimo, TP53 dependent G2 arrest mediator candidate
−1.50HAUS6Homo sapiens HAUS augmin-like complex, subunit 6
−1.52HSPA8Homo sapiens heat shock 70 kDa protein 8
−1.55ACLYHomo sapiens ATP citrate lyase
−1.57CCNG1Homo sapiens cyclin G1
−1.62CHEK2Homo sapiens CHK2 checkpoint homolog
−1.63TAF1Homo sapiens TAF1 RNA polymerase II
−1.73PHF16Homo sapiens PHD finger protein 16
−1.87DUSP1Homo sapiens dual specificity phosphatase 1
−1.89PISDHomo sapiens THAP domain containing, apoptosis associated protein 1
−1.96CLPXHomo sapiens non-SMC condensin I complex, subunit H
−2.10SLC25A26Homo sapiens septin 2
Biological process: cell migration
−1.54PLXNB1Homo sapiens plexin B1
−1.56CLIC4Homo sapiens chloride intracellular channel 4
−1.66RNF20Homo sapiens ring finger protein 20
−1.72S100A2Homo sapiens S100 calcium binding protein A2
−1.73SEMA3CHomo sapiens sema domain, immunoglobulin domain, short basic domain
Biological process: cytoskeleton organization and mitosis
2.19TPM3Homo sapiens tropomyosin 3
1.63CHD2Homo sapiens chromodomain helicase DNA binding protein 2
1.59CORO2AHomo sapiens coronin, actin binding protein, 2A
1.59ARHGAP26Homo sapiens Rho GTPase activating protein 26
1.59DSTNHomo sapiens destrin (actin depolymerizing factor)
1.57ACTBHomo sapiens actin
Biological process: cytoskeleton organization and mitosis
1.57ACTG1Homo sapiens actin, γ 1
1.56TUBA1BHomo sapiens tubulin, α 1b
1.51TUBB2CHomo sapiens tubulin, β 2C
1.50KIF21AHomo sapiens kinesin family member 21A
−1.51KIF19Homo sapiens kinesin family member 19
−1.51MC1RHomo sapiens melanocortin 1 receptor
−1.51RUVBL1Homo sapiens RuvB-like 1
−1.53NDE1Homo sapiens nudE nuclear distribution gene E homolog 1
−1.58C2CD3Homo sapiens C2 calcium-dependent domain containing 3
−1.59NXF5Homo sapiens nuclear RNA export factor 5
−1.70BICD2Homo sapiens bicaudal D homolog 2
−1.76CYP1A1Homo sapiens cytochrome P450, family 1, subfamily A, polypeptide 1
−1.88MAP1BHomo sapiens microtubule-associated protein 1B
−1.95KRT1Homo sapiens keratin 1
Biological process: DNA damage and repair
2.53NPM1Homo sapiens nucleophosmin
1.93DDIT3Homo sapiens DNA-damage-inducible transcript 3
1.80BRCC3Homo sapiens BRCA1/BRCA2-containing complex, subunit 3
1.56FTOHomo sapiens fat mass and obesity associated
1.52HIPK1Homo sapiens homeodomain interacting protein kinase 1
−1.54MRPS11Homo sapiens mitochondrial ribosomal protein S11
−1.54GNL1Homo sapiens guanine nucleotide binding protein-like 1
−1.57NAA10Homo sapiens N(α)-acetyltransferase 10, NatA catalytic subunit
−1.58MORF4L2Homo sapiens mortality factor 4 like 2
−1.59ATRXHomo sapiens α thalassemia/mental retardation syndrome X-linked
−1.59RUVBL2Homo sapiens RuvB-like 2
−1.63NCOA6Homo sapiens nuclear receptor coactivator 6
−1.72POLBHomo sapiens polymerase, β
−1.83ATMINHomo sapiens ATM interactor
−2.03CIRBPHomo sapiens cold inducible RNA binding protein
−2.78SGK3Homo sapiens serum/glucocorticoid regulated kinase family, member 3
Biological process: DNA transcription and replication
1.96GLI1Homo sapiens GLI family zinc finger 1
1.58CDC7Homo sapiens cell division cycle 7 homolog
1.53SETHomo sapiens SET nuclear oncogene
−1.55MCM6Homo sapiens minichromosome maintenance complex component 6
−1.59THRAHomo sapiens thyroid hormone receptor, α
−1.67NOBOXHomo sapiens NOBOX oogenesis homeobox
Biological process: ER function and ER stress
1.78NECAB1Homo sapiens N-terminal EF-hand calcium binding protein1
1.77EFCAB4BHomo sapiens EF-hand calcium binding domain 4B
1.71CHRNA7Homo sapiens cholinergic receptor, nicotinic, α 7
1.65RYR1Homo sapiens ryanodine receptor 1
1.63GRIN2AHomo sapiens glutamate receptor
1.61SNAP25Homo sapiens synaptosomal-associated protein
1.53ALG13Homo sapiens asparagine-linked glycosylation 13 homolog
1.53MLECHomo sapiens malectin
1.51CYP2B6Homo sapiens cytochrome P450, family 2, subfamily B, polypeptide 6
−1.50UBQLN4Homo sapiens ubiquilin 4
−1.50FITM1Homo sapiens fat storage-inducing transmembrane protein 1
Biological process: ER function and ER stress
−1.55SEC22BHomo sapiens SEC22 vesicle trafficking protein homolog B
−1.55DERL1Homo sapiens Der1-like domain family, member 1
−1.63FOXRED2Homo sapiens FAD-dependent oxidoreductase domain containing 2
−1.63CYP4A22Homo sapiens cytochrome P450, family 4, subfamily A, polypeptide 22
−1.69PLA2G2AHomo sapiens phospholipase A2, group IIA
−1.72HSP90B2PHomo sapiens heat shock protein 94b
−2.36NOX4Homo sapiens NADPH oxidase 4
−4.84S100A7Homo sapiens S100 calcium binding protein A7
Biological process: mitochondrial function
1.91ACAD8Homo sapiens 5-methyltetrahydrofolate-homocysteine methyltransferase reductase
1.74ACOT2Homo sapiens ATP synthase, H+ transporting, mitochondrial F0 complex
1.73SPNS1Homo sapiens MpV17 mitochondrial inner membrane protein
1.72NMRAL1Homo sapiens NmrA-like family domain containing 1, mitochondrial
1.69ACSS2Homo sapiens NADH dehydrogenase 1 β subcomplex, 4, 15 kDa
1.68SAT2Homo sapiens cytochrome P450, family 2, subfamily D, polypeptide 6
1.56MTRRHomo sapiens cytochrome b-561
1.54ATP5JHomo sapiens potassium inwardly-rectifying channel
1.52MPV17Homo sapiens pyruvate dehydrogenase phosphatase regulatory subunit
1.50LONP1Homo sapiens solute carrier family 3, member 1
−1.50NDUFB4Homo sapiens thymidine phosphorylase
−1.54CYP2D6Homo sapiens dynamin 1-like
−1.55CYB561Homo sapiens solute carrier family 25, member 30
−1.57KCNJ11Homo sapiens MOCO sulphurase C-terminal domain containing 2
−1.58PDPRHomo sapiens cytochrome c oxidase subunit VIb polypeptide 1
−1.59SLC3A1Homo sapiens phosphatidylserine decarboxylase
−1.62TYMPHomo sapiens ClpX caseinolytic peptidase X homolog
−1.62DNM1LHomo sapiens solute carrier family 25, member 26
−1.64SLC25A30Homo sapiens yrdC domain containing
−1.65MOSC2Homo sapiens surfeit 1
−2.10COX6B1Homo sapiens cytochrome P450, family 27, subfamily C, polypeptide 1
Biological process: cell signal transduction
2.54ZCCHC8Homo sapiens Rho GTPase activating protein 12
2.18DHX35Homo sapiens cannabinoid receptor 2
2.12DGCR14Homo sapiens guanine nucleotide binding protein
2.08RPS13Homo sapiens calcitonin-related polypeptide α
1.97SNRPGHomo sapiens opsin 4
1.83RBMY1A1Homo sapiens growth factor receptor-bound protein 10
1.83ARHGAP12Homo sapiens ribosomal protein S6 kinase, 90 kDa, polypeptide 3
1.80CNR2Homo sapiens prolactin
1.78GNAO1Homo sapiens ras homolog gene family, member G
1.77CALCAHomo sapiens leukocyte-associated immunoglobulin-like receptor 2
1.75OPN4Homo sapiens CD79b molecule, immunoglobulin-associated β
1.74GRB10Homo sapiens leukocyte immunoglobulin-like receptor
1.74RPS6KA3Homo sapiens CD28 molecule
1.69PRLHomo sapiens A kinase anchor protein 3
1.69RHOGHomo sapiens RAB6C
1.68LAIR2Homo sapiens RAB41
1.66CD79BHomo sapiens RAB15
1.66LILRB2Homo sapiens ras homolog gene family, member V
1.65CD28Homo sapiens RAB, member of RAS oncogene family-like 2A
Biological process: cell signal transduction
1.65AKAP3Homo sapiens RAB, member of RAS oncogene family-like 2B
1.65RAB6CHomo sapiens RAB43, member RAS oncogene family
1.63RAB41Homo sapiens leukemia inhibitory factor
1.63RAB15Homo sapiens CD81 molecule
1.62RHOVHomo sapiens CD74 molecule
1.59RABL2AHomo sapienss transforming growth factor, β 3
1.59RABL2BHomo sapiens sema domain
1.58RAB43Homo sapiens acylglycerol kinase
1.56LIFHomo sapiens pleckstrin and Sec7 domain containing
1.56CD81Homo sapiens fibroblast growth factor binding protein 1
1.54CD74Homo sapiens olfactory receptor
1.53TGFB3Homo sapiens vomeronasal 1 receptor 3
1.53SEMA4CHomo sapiens olfactory receptor
1.52AGKHomo sapiens olfactory receptor
1.52PSDHomo sapiens olfactory receptor
1.51FGFBP1Homo sapiens olfactory receptor
1.51OR51A4Homo sapiens taste receptor
1.51VN1R3Homo sapiens olfactory receptor
−1.50OR5M10Homo sapiens olfactory receptor
−1.51OR52N1Homo sapiens neuromedin B receptor
−1.51OR1B1Homo sapiens G protein-coupled receptor 120
−1.52OR3A3Homo sapiens olfactory receptor
−1.52TAS2R1Homo sapiens olfactory receptor
−1.53OR3A1Homo sapiens olfactory receptor
−1.53OR1D5Homo sapiens olfactory receptor
−1.53NMBRHomo sapiens MAS-related GPR, member X2
−1.54GPR120Homo sapiens G protein-coupled receptor 119
−1.54OR10C1Homo sapiens olfactory receptor
−1.54OR10H5Homo sapiens G protein-coupled receptor 109B
−1.55OR10G4Homo sapiens G protein-coupled receptor 83
−1.56OR4D5Homo sapiens G protein-coupled receptor 39
−1.56MRGPRX2Homo sapiens olfactory receptor
−1.56GPR119Homo sapiens olfactory receptor
−1.57OR7C2Homo sapiens insulin receptor substrate 1
−1.57GPR109BHomo sapiens insulin-like 3
−1.58GPR83Homo sapiens FYN binding protein
−1.58GPR39Homo sapiens dual specificity phosphatase 16
−1.59OR2L13Homo sapiens hypocretin receptor 1
−1.62OR52A1Homo sapiens secretogranin V
−1.63IRS1Homo sapiens GRB2-related adaptor protein
−1.63INSL3Homo sapiens regulatory factor X-associated ankyrin-containing protein
−1.66FYBHomo sapiens ubiquitin specific peptidase 8
−1.69DUSP16Homo sapiens syndecan binding protein
−1.72HCRTR1Homo sapiens discoidin domain receptor tyrosine kinase 2
−1.72SCG5Homo sapiens AXL receptor tyrosine kinase
−1.74GRAPHomo sapiens adenylate cyclase activating polypeptide 1
−1.75RFXANKHomo sapiens EPH receptor A8
Biological process: cell signal transduction
−1.75USP8Homo sapiens TBC1D30 mRNA for TBC1 domain family, member 30
−1.75SDCBPHomo sapiens Rho guanine nucleotide exchange factor
−1.77DDR2Homo sapiens RAS p21 protein activator 4
−1.78AXLHomo sapiens RAS-like, family 12
−1.78ADCYAP1Homo sapiens mediator complex subunit 13
−1.82EPHA8Homo sapiens mitogen-activated protein kinase kinase 7
−1.92TBC1D30Homo sapiens dishevelled, dsh homolog 3
−2.28ARHGEF10Homo sapiens LIM domain binding 1
−3.04RASA4Homo sapiens coiled-coil domain containing 88C
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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February 2013
Volume 29 Issue 2

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APA
Liu, C., Yang, J., Huang, S., Chiang, J., Chen, M., Huang, L. ... Kuo, S. (2013). Smh-3 induces G2/M arrest and apoptosis through calcium‑mediated endoplasmic reticulum stress and mitochondrial signaling in human hepatocellular carcinoma Hep3B cells. Oncology Reports, 29, 751-762. https://doi.org/10.3892/or.2012.2166
MLA
Liu, C., Yang, J., Huang, S., Chiang, J., Chen, M., Huang, L., Ha, H., Fushiya, S., Kuo, S."Smh-3 induces G2/M arrest and apoptosis through calcium‑mediated endoplasmic reticulum stress and mitochondrial signaling in human hepatocellular carcinoma Hep3B cells". Oncology Reports 29.2 (2013): 751-762.
Chicago
Liu, C., Yang, J., Huang, S., Chiang, J., Chen, M., Huang, L., Ha, H., Fushiya, S., Kuo, S."Smh-3 induces G2/M arrest and apoptosis through calcium‑mediated endoplasmic reticulum stress and mitochondrial signaling in human hepatocellular carcinoma Hep3B cells". Oncology Reports 29, no. 2 (2013): 751-762. https://doi.org/10.3892/or.2012.2166