OR Oncology Reports 1021-335X 1791-2431 D.A. Spandidos 10.3892/or.2012.2166 or-29-02-0751 Articles Smh-3 induces G2/M arrest and apoptosis through calcium-mediated endoplasmic reticulum stress and mitochondrial signaling in human hepatocellular carcinoma Hep3B cells LIUCHIN-YU1 YANGJAI-SING2 HUANGSHIH-MING1 CHIANGJO-HUA4 CHENMING-HUA1 HUANGLI-JIAU1 HAHO-YU2 FUSHIYASHINJI5 KUOSHENG-CHU13 Graduate Institute of Pharmaceutical Chemistry, Taichung 404 Department of Pharmacology, China Medical University, Taichung 404 Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404 Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan, R.O.C. Department of Kampo Pharmaceutical Sciences, Nihon Pharmaceutical University, Saitama 362-0806, Japan Correspondence to: Professor Sheng-Chu Kuo, Graduate Institute of Pharmaceutical Chemistry, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan, R.O.C., E-mail: sckuo@mail.cmu.edu.tw 2 2013 05 12 2012 29 2 751 762 21 09 2012 02 11 2012 Copyright © 2013, Spandidos Publications 2013 This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

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.

 Smh-3 G2/M arrest apoptosis endoplasmic reticulum stress Hep3B cells 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 Ca<sup>2+</sup> 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 G<sub>2</sub>/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.

 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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Smh-3 decreases the percentage of Hep3B cell viability. (A) The chemical structure of Smh-3 (2-(3-(methylamino)phenyl)-6-(pyrrolidin-1-yl)quinolin-4-one). (B) Cells were incubated with various concentrations (0, 50, 100, 200 and 300 nM) of Smh-3 for 24 and 48 h and cell viability was determined using an MTT exclusion method. The total number of viable cells was counted using MTT analysis as described in Materials and methods. Each data point is the means ± SD of 3 experiments. *P<0.05.

Smh-3 affects cell cycle distribution, cell morphology and CDK1 activity in Hep3B cells. (A) Cells were treated with 0, 50, 100 and 200 nM Smh-3 for 24 h and then harvested for analysis of cell cycle distribution using flow cytometry. Bar graph represents the distribution of Hep3B cells in different phases of the cell cycle. (A) Cells cultured with or without 100 nM of Smh-3 for 24 h were examined for changes in cell morphology and were photographed using a phase-contrast microscope as described in Materials and methods. (C) Cells were treated with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h and then harvested for analysis of CDK1 activity. Each data point is the means ± SD of 3 experiments. *P<0.05.

Effects of Smh-3 on ER stress in Hep3B cells. (A) Cells were treated with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h and the intracellular Ca2+ levels were measured using flow cytometric analysis. (B) Cells were treated with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h and the whole cell lysates were subjected to caspase-4 activity assay. Each data point is the means ± SD of 3 experiments. *P<0.05. (C) Cells were treated with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h, and whole cell lysate were prepared and the caspase-4 and GADD153 protein levels were estimated by western blotting as described in Materials and methods.

Effects of Smh-3 on the mitochondrial-dependent apoptotic pathway in Hep3B cells. (A) Cells were treated with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h, and the mitochondrial membrane potential (ΔΨm) was measured by staining with DiOC6. Cells were treated with 0, 50, 100, 200 and 300 nM of Smh-3 for 24 h, and the whole cell lysates were subjected to caspase-9 (B) and caspase-3 (C) activity assay as described in Materials and methods. Each data point is the means ± SD of 3 experiments. *P<0.05.

A proposed model and the overall possible signaling pathways involved in the Smh-3-induced apoptosis in Hep3B cells.

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-change Gene symbol mRNA description
Biological process: angiogenesis
1.73 RBPJ Homo sapiens mRNA for H-2K binding factor-2
1.52 GPI Homo sapiens glucose phosphate isomerase
−1.64 SH2D2A Homo sapiens SH2 domain containing 2A
−1.74 RNH1 Homo sapiens ribonuclease/angiogenin inhibitor 1
−1.79 RTN4 Homo sapiens reticulon 4
−1.87 VEGFA Homo sapiens vascular endothelial growth factor A
Biological process: apoptosis and anti-apoptosis
1.73 PERP Homo sapiens PERP, TP53 apoptosis effector
1.61 BTG1 Homo sapiens B-cell translocation gene 1
1.59 DBH Homo sapiens dopamine β-hydroxylase
1.58 PARP11 Homo sapiens poly(ADP-ribose)polymerase family, member 11
1.58 SEMA3A Homo sapiens sema domain
−1.51 ABR Homo sapiens active BCR-related gene
−1.51 BLOC1S2 Homo sapiens biogenesis of lysosomal organelles complex-1
−1.51 BCL2L1 Homo sapiens BCL2-like 1
−1.52 CARD17 Homo sapiens caspase recruitment domain family, member 17
−1.53 YARS Homo sapiens tyrosyl-tRNA synthetase
−1.54 USP17L2 Homo sapiens ubiquitin specific peptidase 17-like 2
−1.56 APH1B Homo sapiens anterior pharynx defective 1 homolog B
−1.62 IHH Homo sapiens Indian hedgehog homolog
−1.75 CIDEB Homo sapiens cell death-inducing DFFA-like effector b
−1.85 BCL6 Homo sapiens B-cell CLL/lymphoma 6, transcript variant 1
Biological process: autophagy
1.97 ATG12 Homo sapiens ATG12 autophagy related 12 homolog
1.85 CLEC4F Homo sapiens C-type lectin domain family 4, member F
1.64 ACP2 Homo sapiens acid phosphatase 2, lysosomal
1.55 HPS1 Homo sapiens Hermansky-Pudlak syndrome 1
1.53 NEU1 Homo sapiens sialidase 1
1.51 GABARAP Homo sapiens GABA receptor-associated protein
−1.62 CHIT1 Homo sapiens chitinase 1
−1.68 VPS53 Homo sapiens vacuolar protein sorting 53 homolog
−2.12 SYT3 Homo sapiens synaptotagmin III
Biological process: calcium-mediated signaling
1.68 CCL3 Homo sapiens chemokine ligand 3
1.66 STC1 Homo sapiens stanniocalcin 1
1.53 CHRNA10 Homo sapiens cholinergic receptor
1.53 GRIN1 Homo sapiens glutamate receptor
1.50 ETNK2 Homo sapiens ethanolamine kinase 2
−1.50 CX3CL1 Homo sapiens chemokine ligand 1
−1.52 PLCG2 Homo sapiens phospholipase C, γ 2
−1.55 NMUR1 Homo sapiens neuromedin U receptor 1
−1.64 CALCB Homo sapiens calcitonin-related polypeptide β
−2.43 KCNA5 Homo sapiens potassium voltage-gated channel
Biological process: cell adhesion
2.50 PVRL2 Homo sapiens poliovirus receptor-related 2
1.71 SYMPK Homo sapiens symplekin
1.68 SIGLEC5 Homo sapiens sialic acid binding Ig-like lectin 5
1.63 LPXN Homo sapiens leupaxin
Biological process: cell adhesion
1.63 CDH6 Homo sapiens cadherin 6
1.56 GPR56 Homo sapiens G protein-coupled receptor 56
−1.50 PVRL4 Homo sapiens poliovirus receptor-related 4
−1.51 SIGLEC14 Homo sapiens sialic acid binding Ig-like lectin 14
−1.51 CPXM2 Homo sapiens carboxypeptidase X, member 2
−1.52 MMRN1 Homo sapiens multimerin 1
−1.53 NCAM1 Homo sapiens neural cell adhesion molecule 1
−1.54 PCDHB4 Homo sapiens protocadherin β 4
−1.54 PCDHB14 Homo sapiens protocadherin β 14
−1.58 PCDHB13 Homo sapiens protocadherin β 13
−1.58 PXN Homo sapiens paxillin
−1.61 HSPB11 Homo sapiens heat shock protein family B
−1.61 ITGA7 Homo sapiens integrin, α 7
−1.62 SIRPG Homo sapiens signal-regulatory protein γ
−1.62 NINJ2 Homo sapiens ninjurin 2
−1.62 NEO1 Homo sapiens neogenin homolog 1
−1.68 CYR61 Homo sapiens cysteine-rich, angiogenic inducer, 61
Biological process: cell cycle and mitosis
2.13 CNNM3 Homo sapiens cyclin M3
1.84 RAB11B Homo sapiens RAB11B, member RAS oncogene family
1.80 CDKN3 Homo sapiens cyclin-dependent kinase inhibitor 3
1.64 40789 Homo sapiens septin 3
1.59 MGC16703 Homo sapiens tubulin, α pseudogene
1.55 KRT18 Homo sapiens keratin 18
1.52 ARL8B Homo sapiens ADP-ribosylation factor-like 8B
1.52 RPRM Homo sapiens reprimo, TP53 dependent G2 arrest mediator candidate
−1.50 HAUS6 Homo sapiens HAUS augmin-like complex, subunit 6
−1.52 HSPA8 Homo sapiens heat shock 70 kDa protein 8
−1.55 ACLY Homo sapiens ATP citrate lyase
−1.57 CCNG1 Homo sapiens cyclin G1
−1.62 CHEK2 Homo sapiens CHK2 checkpoint homolog
−1.63 TAF1 Homo sapiens TAF1 RNA polymerase II
−1.73 PHF16 Homo sapiens PHD finger protein 16
−1.87 DUSP1 Homo sapiens dual specificity phosphatase 1
−1.89 PISD Homo sapiens THAP domain containing, apoptosis associated protein 1
−1.96 CLPX Homo sapiens non-SMC condensin I complex, subunit H
−2.10 SLC25A26 Homo sapiens septin 2
Biological process: cell migration
−1.54 PLXNB1 Homo sapiens plexin B1
−1.56 CLIC4 Homo sapiens chloride intracellular channel 4
−1.66 RNF20 Homo sapiens ring finger protein 20
−1.72 S100A2 Homo sapiens S100 calcium binding protein A2
−1.73 SEMA3C Homo sapiens sema domain, immunoglobulin domain, short basic domain
Biological process: cytoskeleton organization and mitosis
2.19 TPM3 Homo sapiens tropomyosin 3
1.63 CHD2 Homo sapiens chromodomain helicase DNA binding protein 2
1.59 CORO2A Homo sapiens coronin, actin binding protein, 2A
1.59 ARHGAP26 Homo sapiens Rho GTPase activating protein 26
1.59 DSTN Homo sapiens destrin (actin depolymerizing factor)
1.57 ACTB Homo sapiens actin
Biological process: cytoskeleton organization and mitosis
1.57 ACTG1 Homo sapiens actin, γ 1
1.56 TUBA1B Homo sapiens tubulin, α 1b
1.51 TUBB2C Homo sapiens tubulin, β 2C
1.50 KIF21A Homo sapiens kinesin family member 21A
−1.51 KIF19 Homo sapiens kinesin family member 19
−1.51 MC1R Homo sapiens melanocortin 1 receptor
−1.51 RUVBL1 Homo sapiens RuvB-like 1
−1.53 NDE1 Homo sapiens nudE nuclear distribution gene E homolog 1
−1.58 C2CD3 Homo sapiens C2 calcium-dependent domain containing 3
−1.59 NXF5 Homo sapiens nuclear RNA export factor 5
−1.70 BICD2 Homo sapiens bicaudal D homolog 2
−1.76 CYP1A1 Homo sapiens cytochrome P450, family 1, subfamily A, polypeptide 1
−1.88 MAP1B Homo sapiens microtubule-associated protein 1B
−1.95 KRT1 Homo sapiens keratin 1
Biological process: DNA damage and repair
2.53 NPM1 Homo sapiens nucleophosmin
1.93 DDIT3 Homo sapiens DNA-damage-inducible transcript 3
1.80 BRCC3 Homo sapiens BRCA1/BRCA2-containing complex, subunit 3
1.56 FTO Homo sapiens fat mass and obesity associated
1.52 HIPK1 Homo sapiens homeodomain interacting protein kinase 1
−1.54 MRPS11 Homo sapiens mitochondrial ribosomal protein S11
−1.54 GNL1 Homo sapiens guanine nucleotide binding protein-like 1
−1.57 NAA10 Homo sapiens N(α)-acetyltransferase 10, NatA catalytic subunit
−1.58 MORF4L2 Homo sapiens mortality factor 4 like 2
−1.59 ATRX Homo sapiens α thalassemia/mental retardation syndrome X-linked
−1.59 RUVBL2 Homo sapiens RuvB-like 2
−1.63 NCOA6 Homo sapiens nuclear receptor coactivator 6
−1.72 POLB Homo sapiens polymerase, β
−1.83 ATMIN Homo sapiens ATM interactor
−2.03 CIRBP Homo sapiens cold inducible RNA binding protein
−2.78 SGK3 Homo sapiens serum/glucocorticoid regulated kinase family, member 3
Biological process: DNA transcription and replication
1.96 GLI1 Homo sapiens GLI family zinc finger 1
1.58 CDC7 Homo sapiens cell division cycle 7 homolog
1.53 SET Homo sapiens SET nuclear oncogene
−1.55 MCM6 Homo sapiens minichromosome maintenance complex component 6
−1.59 THRA Homo sapiens thyroid hormone receptor, α
−1.67 NOBOX Homo sapiens NOBOX oogenesis homeobox
Biological process: ER function and ER stress
1.78 NECAB1 Homo sapiens N-terminal EF-hand calcium binding protein1
1.77 EFCAB4B Homo sapiens EF-hand calcium binding domain 4B
1.71 CHRNA7 Homo sapiens cholinergic receptor, nicotinic, α 7
1.65 RYR1 Homo sapiens ryanodine receptor 1
1.63 GRIN2A Homo sapiens glutamate receptor
1.61 SNAP25 Homo sapiens synaptosomal-associated protein
1.53 ALG13 Homo sapiens asparagine-linked glycosylation 13 homolog
1.53 MLEC Homo sapiens malectin
1.51 CYP2B6 Homo sapiens cytochrome P450, family 2, subfamily B, polypeptide 6
−1.50 UBQLN4 Homo sapiens ubiquilin 4
−1.50 FITM1 Homo sapiens fat storage-inducing transmembrane protein 1
Biological process: ER function and ER stress
−1.55 SEC22B Homo sapiens SEC22 vesicle trafficking protein homolog B
−1.55 DERL1 Homo sapiens Der1-like domain family, member 1
−1.63 FOXRED2 Homo sapiens FAD-dependent oxidoreductase domain containing 2
−1.63 CYP4A22 Homo sapiens cytochrome P450, family 4, subfamily A, polypeptide 22
−1.69 PLA2G2A Homo sapiens phospholipase A2, group IIA
−1.72 HSP90B2P Homo sapiens heat shock protein 94b
−2.36 NOX4 Homo sapiens NADPH oxidase 4
−4.84 S100A7 Homo sapiens S100 calcium binding protein A7
Biological process: mitochondrial function
1.91 ACAD8 Homo sapiens 5-methyltetrahydrofolate-homocysteine methyltransferase reductase
1.74 ACOT2 Homo sapiens ATP synthase, H+ transporting, mitochondrial F0 complex
1.73 SPNS1 Homo sapiens MpV17 mitochondrial inner membrane protein
1.72 NMRAL1 Homo sapiens NmrA-like family domain containing 1, mitochondrial
1.69 ACSS2 Homo sapiens NADH dehydrogenase 1 β subcomplex, 4, 15 kDa
1.68 SAT2 Homo sapiens cytochrome P450, family 2, subfamily D, polypeptide 6
1.56 MTRR Homo sapiens cytochrome b-561
1.54 ATP5J Homo sapiens potassium inwardly-rectifying channel
1.52 MPV17 Homo sapiens pyruvate dehydrogenase phosphatase regulatory subunit
1.50 LONP1 Homo sapiens solute carrier family 3, member 1
−1.50 NDUFB4 Homo sapiens thymidine phosphorylase
−1.54 CYP2D6 Homo sapiens dynamin 1-like
−1.55 CYB561 Homo sapiens solute carrier family 25, member 30
−1.57 KCNJ11 Homo sapiens MOCO sulphurase C-terminal domain containing 2
−1.58 PDPR Homo sapiens cytochrome c oxidase subunit VIb polypeptide 1
−1.59 SLC3A1 Homo sapiens phosphatidylserine decarboxylase
−1.62 TYMP Homo sapiens ClpX caseinolytic peptidase X homolog
−1.62 DNM1L Homo sapiens solute carrier family 25, member 26
−1.64 SLC25A30 Homo sapiens yrdC domain containing
−1.65 MOSC2 Homo sapiens surfeit 1
−2.10 COX6B1 Homo sapiens cytochrome P450, family 27, subfamily C, polypeptide 1
Biological process: cell signal transduction
2.54 ZCCHC8 Homo sapiens Rho GTPase activating protein 12
2.18 DHX35 Homo sapiens cannabinoid receptor 2
2.12 DGCR14 Homo sapiens guanine nucleotide binding protein
2.08 RPS13 Homo sapiens calcitonin-related polypeptide α
1.97 SNRPG Homo sapiens opsin 4
1.83 RBMY1A1 Homo sapiens growth factor receptor-bound protein 10
1.83 ARHGAP12 Homo sapiens ribosomal protein S6 kinase, 90 kDa, polypeptide 3
1.80 CNR2 Homo sapiens prolactin
1.78 GNAO1 Homo sapiens ras homolog gene family, member G
1.77 CALCA Homo sapiens leukocyte-associated immunoglobulin-like receptor 2
1.75 OPN4 Homo sapiens CD79b molecule, immunoglobulin-associated β
1.74 GRB10 Homo sapiens leukocyte immunoglobulin-like receptor
1.74 RPS6KA3 Homo sapiens CD28 molecule
1.69 PRL Homo sapiens A kinase anchor protein 3
1.69 RHOG Homo sapiens RAB6C
1.68 LAIR2 Homo sapiens RAB41
1.66 CD79B Homo sapiens RAB15
1.66 LILRB2 Homo sapiens ras homolog gene family, member V
1.65 CD28 Homo sapiens RAB, member of RAS oncogene family-like 2A
Biological process: cell signal transduction
1.65 AKAP3 Homo sapiens RAB, member of RAS oncogene family-like 2B
1.65 RAB6C Homo sapiens RAB43, member RAS oncogene family
1.63 RAB41 Homo sapiens leukemia inhibitory factor
1.63 RAB15 Homo sapiens CD81 molecule
1.62 RHOV Homo sapiens CD74 molecule
1.59 RABL2A Homo sapienss transforming growth factor, β 3
1.59 RABL2B Homo sapiens sema domain
1.58 RAB43 Homo sapiens acylglycerol kinase
1.56 LIF Homo sapiens pleckstrin and Sec7 domain containing
1.56 CD81 Homo sapiens fibroblast growth factor binding protein 1
1.54 CD74 Homo sapiens olfactory receptor
1.53 TGFB3 Homo sapiens vomeronasal 1 receptor 3
1.53 SEMA4C Homo sapiens olfactory receptor
1.52 AGK Homo sapiens olfactory receptor
1.52 PSD Homo sapiens olfactory receptor
1.51 FGFBP1 Homo sapiens olfactory receptor
1.51 OR51A4 Homo sapiens taste receptor
1.51 VN1R3 Homo sapiens olfactory receptor
−1.50 OR5M10 Homo sapiens olfactory receptor
−1.51 OR52N1 Homo sapiens neuromedin B receptor
−1.51 OR1B1 Homo sapiens G protein-coupled receptor 120
−1.52 OR3A3 Homo sapiens olfactory receptor
−1.52 TAS2R1 Homo sapiens olfactory receptor
−1.53 OR3A1 Homo sapiens olfactory receptor
−1.53 OR1D5 Homo sapiens olfactory receptor
−1.53 NMBR Homo sapiens MAS-related GPR, member X2
−1.54 GPR120 Homo sapiens G protein-coupled receptor 119
−1.54 OR10C1 Homo sapiens olfactory receptor
−1.54 OR10H5 Homo sapiens G protein-coupled receptor 109B
−1.55 OR10G4 Homo sapiens G protein-coupled receptor 83
−1.56 OR4D5 Homo sapiens G protein-coupled receptor 39
−1.56 MRGPRX2 Homo sapiens olfactory receptor
−1.56 GPR119 Homo sapiens olfactory receptor
−1.57 OR7C2 Homo sapiens insulin receptor substrate 1
−1.57 GPR109B Homo sapiens insulin-like 3
−1.58 GPR83 Homo sapiens FYN binding protein
−1.58 GPR39 Homo sapiens dual specificity phosphatase 16
−1.59 OR2L13 Homo sapiens hypocretin receptor 1
−1.62 OR52A1 Homo sapiens secretogranin V
−1.63 IRS1 Homo sapiens GRB2-related adaptor protein
−1.63 INSL3 Homo sapiens regulatory factor X-associated ankyrin-containing protein
−1.66 FYB Homo sapiens ubiquitin specific peptidase 8
−1.69 DUSP16 Homo sapiens syndecan binding protein
−1.72 HCRTR1 Homo sapiens discoidin domain receptor tyrosine kinase 2
−1.72 SCG5 Homo sapiens AXL receptor tyrosine kinase
−1.74 GRAP Homo sapiens adenylate cyclase activating polypeptide 1
−1.75 RFXANK Homo sapiens EPH receptor A8
Biological process: cell signal transduction
−1.75 USP8 Homo sapiens TBC1D30 mRNA for TBC1 domain family, member 30
−1.75 SDCBP Homo sapiens Rho guanine nucleotide exchange factor
−1.77 DDR2 Homo sapiens RAS p21 protein activator 4
−1.78 AXL Homo sapiens RAS-like, family 12
−1.78 ADCYAP1 Homo sapiens mediator complex subunit 13
−1.82 EPHA8 Homo sapiens mitogen-activated protein kinase kinase 7
−1.92 TBC1D30 Homo sapiens dishevelled, dsh homolog 3
−2.28 ARHGEF10 Homo sapiens LIM domain binding 1
−3.04 RASA4 Homo sapiens coiled-coil domain containing 88C