Knockdown of Apolipoprotein E Enhanced Sensitivity of Hep3b Cells

Author Manuscript Published OnlineFirst on August 9, 2016; DOI: 10.1158/1535-7163.MCT-15-0961 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Knockdown of apolipoprotein E enhanced sensitivity of Hep3B cells

to cardiac steroids via regulating Na+/K+-ATPase signalosome

Miao Liu, Li-Xing Feng, Peng Sun, Wang Liu, Tian Mi, Min Lei, Wanying Wu,

Baohong Jiang, Min Yang, Lihong Hu*, De-An Guo*, and Xuan Liu*

Authors’ Affiliation: Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, P.R. China

Corresponding Author: Lihong Hu, De-An Guo, and Xuan Liu. Shanghai Institute

of Materia Medica, Chinese Academy of Sciences, 501 Hai-Ke Road, Shanghai

201203, P.R. China. Tel/Fax: 86-21-50272789. E-mail: [email protected] (Lihong

Hu), [email protected] (De-An Guo), [email protected] (Xuan Liu).

Running Title: APOE knockdown sensitized Hep3B cells to cardiac steroids.

Key Words: apolipoprotein E, Hep3B cells, cardiac steroids, Na+/K+-ATPase

signalosome

Abbreviations:

BF, bufalin; OUA, ouabain; DIG, digitoxin; APOE, apolipoprotein E; STR, DNA

profiling; LDH, lactate dehydrogenase; FPKM, fragments per kilobase of exon per

Downloaded from mct.aacrjournals.org on September 24, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 9, 2016; DOI: 10.1158/1535-7163.MCT-15-0961 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

million fragments mapped; STRING, Search Tool for the Retrieval of Interacting

Genes; TBST, Tris-buffer saline containing 0.1% (v/v) Tween-20; PRNP, prion

protein；FBLN1, fibulin 1; COL18A1, collagen, type XVIII, α1; NID1, nidogen 1;

BCAM, basal cell adhesion molecule; APOC1, apolipoprotein C-I; C3, complement

component 3; CTSB, cathepsin B; PI3K, phosphatidylinositol-4,5-bisphosphate

3-kinase.

Financial Support: This work was supported in part by supports received by X. Liu

from the Shanghai Science & Technology Support Program (13431900401), the

Shanghai Science & Technology Innovation Action Program (15140904800), the

National Nature Science Foundation of China (81373964) and the National Science &

Technology Major Project of China (2014ZX09301-306-03).

Disclosure of Potential Conflicts of Interest: The authors declare that they have no

conflicts of interest.

Word Count (excluding references): 4998

Total Number of Figures and Tables: 6 figures and 0 tables.

Supplemental Materials: 5 supplemental figures and 1 supplemental document

(including 3 supplemental tables and supplemental figure legends).

Abstract This study compared the sensitivity of human hepatoma Hep3B, SK-HEP-1,

SMMC-7721 and BEL-7402 cells to cardiac steroids including bufalin (BF), a bufalin

derivative (BF211), ouabain (OUA) and digitoxin (DIG). Hep3B cells exhibited

relatively low sensitivity to cardiac steroids. Expression levels of subunits of

Na+/K+-ATPase were high in Hep3B cells. However, co-localization of

Na+/K+-ATPase and caveolin was nearly undetectable in Hep3B cells. By using

RNA-Seq technology, we found a total of 36 genes to be differentially expressed

between Hep3B cells and SK-HEP-1 cells, which are highly sensitive to cardiac

steroids. Our bioinformatics analysis determined that these genes were mostly

comprised of extracellular space, protein binding and extracellular region. Among

these 36 genes, apolipoprotein E (APOE) played a critical role, since knockdown

APOE expression induced co-localization of Na+/K+-ATPase and caveolin and

increased sensitivity of Hep3B cells to both proliferation-inhibiting and cytotoxic

effects of BF or BF211. Also, the effects of BF on PI3K/AKT/GSK3β and apoptosis

signal cascades were enhanced in APOE knockdown cells. The results of our study

confirmed the role of Na+/K+-ATPase signalosome in cytotoxicity of cardiac steroids

and suggested that APOE regulated the sensitivity of cells to cardiac steroids by

affecting formation and function of Na+/K+-ATPase signalosome. In addition,

intercellular interaction with high level of Na+/K+-ATPase β1 subunit may be also a

factor in the low sensitivity of Hep3B cells to cardiac steroids.

Introduction

Hepatocellular carcinoma is one of the most aggressive malignancies (1, 2).

The percentage of patients benefited from surgical treatment is low and the efficacy of

chemotherapy agents such as doxorubicin, cisplatin, and 5-fluorouracil is limited (3).

Huachansu injection, derived from a water-soluble extract of the traditional Chinese

medicine ChanSu, is commonly used in China to treat patients with hepatocellular

carcinoma (4, 5). ChanSu is obtained from skin and parotid venom secretion glands of

toads (6). Cardiac steroids such as bufalin (BF) are the active components of ChanSu

(7). In the last 20 years, interest of developing cardiac steroids into anti-cancer agents

substantially increased (8-12). Our lab also tried to develop derivatives of BF as

possible new anti-cancer agents (13). Synthesis of promising BF derivatives such as

BF211 (Patent Publication Number CN 102532235 B) had been reported (14).

However, the research and development of cardiac steroids as new anti-cancer agents

was hindered because the mechanisms of their anti-cancer effects had not been fully

understood.

In the present study, we studied possible factors that contribute to the sensitivity

of hepatoma cells to cardiac steroids to understand the mechanisms of cytotoxicity of

cardiac steroids. We tested the cytotoxicity of representative cardiac steroids such as

BF, BF211, OUA and DIG, whose structures were shown in Supplemental Figure S1,

in 4 types of hepatoma cell lines and one type of embryonic liver cell line.

Interestingly, one of the cell lines, Hep3B, exhibited lower sensitivity to cardiac

steroids when compared to other cell lines. Expression levels of subunits of 4

Na+/K+-ATPase, the direct target of cardiac steroids, in Hep3B cells and other cells

were compared. RNA-Seq technology was also used to compare the expression

profiles of Hep3B cells and SK-HEP-1 cells, which is the cell line with the highest

sensitivity. In total, we found 36 genes that were differentially expressed between

Hep3B and SK-HEP-1 cells. APOE (apolipoprotein E) protein might be an important

factor in regulating the sensitivity of cells to cardiac steroids, which was confirmed by

examining the cytotoxicity of cardiac steroids and signal cascades in Hep3B cells

transfected with siRNAs for the APOE gene. Finally, we also observed the role of the

Na+/K+-ATPase β1 subunit in the sensitivity of Hep3B cells.

Materials and methods

Chemicals

OUA and DIG with a purity of 98% were purchased from the Sigma-Aldrich

Chemical Co. (St. Louis, MO, U.S.A). BF with a purity of 98% was isolated from

ChanSu (13) and BF211 with a purity of 98% was synthesized by a structure

modification of BF (14). Stock solutions of the chemicals were prepared in DMSO to

the concentration of 0.1 M as stock solution and stored at -20°C.

Cell culture

The human hepatoma cell lines Hep3B, SK-HEP-1, SMMC-7721 and BEL-7402

were purchased from the Cell Resource Center of Shanghai Institutes for Biological

Sciences, Chinese Academy of Sciences (Shanghai, P.R. China) in 2012. Human

embryo liver L-02 cells were purchased from the BioHemes Company (Wuxi, P.R.

China) in 2015. The suppliers declared that the cells passed the test of DNA profiling

(STR) before sending to us. No authentication was done by the authors. In our lab,

cells were expanded according to the supplier’s protocols and then cryopreserved in

liquid nitrogen. For each experiment, cells were thawed and further cultured for at

least 3 passages before subjecting to treatments. Furthermore, cells were passaged for

fewer than 6 months after resuscitation. Cell culture mediums used for the cell lines

were MEM for Hep3B and SK-HEP-1 cells, RPMI-1640 medium for SMMC-7721

and BEL-7402 cells, DMEM (high glucose) for L-02 cells, respectively. The mediums

were supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 100

μg/mL streptomycin.

MTT Assay

Cell proliferation was assessed using MTT assay (15). Briefly, the Hep3B,

SK-HEP-1, SMMC-7721 , BEL-7402 and L-02 cells were seeded into 96-well plates

at a density of 7 × 103, 3 × 103, 5 × 103, 5 × 103 and 4 × 103 cells/well, respectively,

and allowed to grow overnight before treatment with BF, BF211, OUA or DIG at

different concentrations, or 0.1% DMSO (solvent control) for 72 h. After treatment,

the cell viability was evaluated by checking the absorbance at 570 nm.

Lactate dehydrogenase (LDH) release assay

LDH release of cells was measured using the CytoTox 96 Non-Radioactive

Cytotoxicity Assay (Promega). Briefly, cells were seeded into 96-well plates and

incubated overnight before treatment with BF, BF211 at different concentrations, or

0.1% DMSO (solvent control) for 24 or 48 h. After treatment, medium from each well

was collected to measure the amount of released LDH according to the

manufacturer’s instructions. Cells exposed to a lysis buffer (9% Triton X-100) were

used as positive control. Cell death was represented by percentage of released LDH in

the medium over total cellular LDH, LDH release of positive control cells.

RNA extraction and real-time PCR analysis

Total RNAs were extracted using the TRIzol reagent (Invitrogen) and reverse

transcribed using a PrimeScript RT reagent Kit with a gDNA Eraser (TaKaRa)

according to the manufacturer’s instructions. Real-time PCR amplifications were

performed using the SYBR Premix Ex TaqTM II (TaKaRa), and the thermal profile

was 95 °C for 2 min followed by 40 cycles at 95 °C for 15 sec and 61 °C for 25 sec.

Each sample was analyzed in triplicate and the mean threshold cycle (Ct) value was

calculated. The relative expression level was calculated using the ΔΔCt method. The

mRNA expression of GAPDH was used as an internal control. Primer sequences for

PCR analysis are listed in Supplemental Table S1.

Laser scanning confocal microscopy

Cells cultured on Poly-D-lysine-coated cover slips were ﬁxed with cold 4%

Paraformaldehyde for 20 min. After washing with PBS three times, cells were

blocked for 2 h with PBS contained 0.1% Tween-20 and 1% BSA. Subsequently, cells

were incubated with the antibodies listed in the Supplemental Table S2 and the nuclei

were stained with DAPI. The stained cells were then observed with a laser-scanning

confocal microscope (Olympus FV1000, Japan).

RNA-Seq

The RNA-Seq was serviced by the Encode Genomics Bio-Technology Co., Ltd.

(Suzhou, China). Briefly, Hep3B cells and SK-HEP-1 cells in the exponential phase

were harvested in Trizol. The total RNA was converted into a cDNA library of

template molecules suitable for subsequent cluster generation and validated using an

Agilent 2100 Bioanalyzer. Each library was sequenced using the 2 × 100 bp

paired-end Illumina/Solexa platform and averaged 70 million reads. The obtained

sequence reads were aligned to the genome (hg19 download from UCSC).

Cufflinks1.1.0 was used to identify differentially expressed genes and measured

transcript abundances with FPKM (fragments per kilobase of exon per million

fragments mapped). For differential expression tests, Cuffdiff from Cufflinks software

was applied over FPKM values. Significantly differentially expressed genes were

selected using multiple-test adjusted p-values.

Bioinformatics analysis

To obtain more information about significantly differentially expressed genes and

to assign statistical significance to our analysis, the gene list was submitted to

BioProfiling.de (http://mips.helmholtz-muenchen.de/proj/ppispider/) (16) and used

ProfCom_GO for grouping genes into functional classes. In addition, the STRING

(Search Tool for the Retrieval of Interacting Genes) database（http://string.embl.de/）

(17) was also used to predict physical or functional associations among these genes.

Confirmation of differentially expressed genes found in RNA-seq using

semi-quantitative reverse-transcription-PCR

Total RNAs extraction and reverse transcription were conducted as described

above. Semi-quantitative PCR amplifications were performed using the Premix Ex

TaqTM (TaKaRa). The thermal profile for cDNA synthesis was 98 °C for 15 seconds,

61°C for 10 sec, and 72 °C for 22 sec of 30 cycles and a final extension at 72°C for 5

min, with a final hold at 20°C in a thermal cycler. Primer sequences of the genes are

listed in Supplemental Tab. S1.

Influence of APOE knockdown on Hep3B cells

Validated siRNA for APOE (Sigma-Aldrich) was transfected into Hep3B cells by

using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s

protocol. Expression levels of APOE protein in wide type, APOE knockdown

(transfected with siRNA for APOE for 24 h) and negative control cells (transfected

with scrambled negative control siRNA) were checked using Western blotting assay.

Cytotoxicity of BF or BF211 on APOE knockdown cells was checked and compared

with the negative control cells. Briefly, APOE knockdown cells or negative control

cells were treated with BF or BF211 at different concentrations for the indicated time

periods. Then, cell viability and cell death were checked using MTT and LDH assay

as described above.

The expression levels of subunits of Na+/K+-ATPase and caveolins in APOE

knockdown cells and negative control cells were checked using real-time PCR

analysis as described above. The subcellular location of Na+/K+-ATPase α1 subunit

and caveolin-1 was observed using Laser scanning confocal microscopy as described

above. Apoptosis in cells treated with BF was measured using an Alexa Fluor 488

Annexin V/Dead Cell Apoptosis kit (v13245, Life Technologies). The DNA

histogram of cells treated with BF was observed using flow cytometry analysis (18).

Western blotting assay

The Western blotting assay was conducted as described before (15). Antibodies

used in Western blotting assay were listed in Supplemental Tab. S2.

Checking the influence of ATP1B1 knockdown on Hep3B cells

Validated siRNA for ATP1B1 (Sigma-Aldrich) was transfected into Hep3B cells

by using Lipofectamine RNAiMAX (Invitrogen). Scrambled negative control siRNA

(Sigma-Aldrich) was used as the negative control. Cytotoxicity of BF or BF211 on

ATP1B1 knockdown cells was checked using MTT, LDH release assay and flow

cytometry analysis of apoptosis and then was compared to the negative control cells.

The subcellular location of E-cadherin in ATP1B1 knockdown cells and negative

control cells was observed using the Laser scanning confocal microscopy as described

above.

Statistical analysis

Student’s t-test was used to evaluate the differences between the treated and the

control groups. The data are expressed as the mean ± SEM, and results from a

minimum of three independent experiments were used for the statistical analysis.

Results

Hep3B cells exhibited lower sensitivity to cardiac steroids compared with other

hepatoma cells and embryo liver L-02 cells

As shown in Fig.1 A-B, among the 5 types of cells, Hep3B cells exhibited the

lowest sensitivity to BF or BF211. Similar results were found for the treatment of

OUA, DIG (Supplemental Fig. S2A-B). To check whether Hep3B cells were resistant

to all chemotherapy agents, we also studied the sensitivity of Hep3B cells to cisplatin,

a commonly used anti-cancer agent, and compared the results to SK-HEP-1 cells

which exhibited high sensitivity to cardiac steroids. As shown in Supplemental Fig.

S2C, the sensitivity of Hep3B cells to cisplatin was similar to or even higher than the

sensitivity of SK-HEP-1 cells. These results suggested that the low sensitivity of

Hep3B cells to cardiac steroids was not based on deficiency in cell death signaling

and execution pathways but might be related to specific response to cardiac steroids.

Expression levels of subunits of Na+/K+-ATPase in different cell lines

As shown in Fig. 1C, the mRNA expression levels of Na+/K+-ATPase α1

(ATP1A1), α2 (ATP1A2), and β1 (ATP1B1) subunit in Hep3B cells were significantly

higher than other hepatoma cells as well as L-02 cells. The protein expression levels

of Na+/K+-ATPase α1 and Na+/K+-ATPase β1 in the cell lines were further

confirmed using Western blotting analysis (Fig.1 D). These results showed that the

low sensitivity of Hep3B cells may not be related to expression level of

Na+/K+-ATPase.

Co-localization of Na+/K+-ATPase and caveolin in Hep3B cells and SK-HEP-1

cells

Caveolins were markers of caveolae and were the partners of Na+/K+-ATPase in

forming the Na+/K+-ATPase signalosome (19). As shown in Fig. 2A, expression

levels of caveolin-1 and caveolin-2 in Hep3B cells were lower (0.03 and 0.40 fold)

than that of SK-HEP-1 cells. The expression level of caveolin-3 was higher (2.28 fold)

than that of SK-HEP-1 cells. Immunocytochemical staining of the Na+/K+-ATPase

α1 subunit and caveolin-1 was used to determine the subcellular localization of

Na+/K+-ATPase and caveolins. As shown in Fig. 2B, the level of the

Na+/K+-ATPase α1 subunit in Hep3B cells was stronger but the level of caveolin-1

was lower compared with the SK-HEP-1 cells, which was consistent with results of

the RT-PCR assay and Western blotting assay. Further, co-localization of the

Na+/K+-ATPase α1 subunit and caveolin-1 on the plasma membrane, which indicated

possible formation of the Na+/K+-ATPase signalosome, was observed in SK-HEP-1

cells but barely observed in Hep3B cells.

Genes differentially expressed between Hep3B cells and SK-HEP-1 cells

Transcriptomic data sets were donated to NCBI SRA database (accession number:

SRX434118, SRX434119). By using cufflinks software, the RNA-seq reads mapped

to the human genome were assembled per gene and condensed into FPKM expression

values, which provided a sample-centered absolute measure of the expression level of

each gene in the studied cell population (20). As shown in Fig. 3A, summarized

FPKM expression values of Hep3B cells and SK-HEP-1 cells were represented on a

scatter plot (log10 scale of FPKM). This plot showed a strong linear correlation

between the two types of cells. Most of the genes run along the diagonal and can be

considered common genes, genes with similar expression levels in the two types of

cells. By setting the q-value<0.05, we detected 36 significantly differentially

expressed genes, as shown in the dispersed gene dots in Fig. 3A. The names and

FPKM values of the 36 proteins are shown in Supplemental Tab. S3. Enrichment

analysis results of molecular functions of the 36 genes (Fig. 3B) suggested that

extracellular space, protein binding and extracellular region were the first three

distinct cellular functions related to the 36 genes. Further, we found a possible

interaction network of the 36 genes as predicted (Supplemental Figure S3) and a close

interaction among 9 genes including APOC1, APOE, BCAM, C3, COL18A1, CTSB,

FBLN1, PRNP and NID1. APOE was located at the center of the interaction network.

The expression levels of the 9 genes were further confirmed using RT-PCR (Fig.3 C).

As shown in Fig. 3C, PCR analysis results of the expression levels of the 9 genes in

Hep3B cells and SK-HEP-1 cells were consistent with the results of RNA-seq.

Influence of APOE knockdown on sensitivity of Hep3B cells to cardiac steroids

and localization of Na+/K+-ATPase and caveolins

As shown in Supplemental Fig. S4, the protein expression level of APOE was

higher in Hep3B cells than in other hepatoma cells. The results were consistent with

the results of both the RNA-seq analysis and the RT-PCR analysis. Transfection of

siRNA for APOE silenced the expression of APOE in Hep3B cells (Fig. 4A). APOE

knockdown did not induce significant change in expression levels of the

Na+/K+-ATPase subunits or caveolins (Fig. 4B). But, as shown in Fig. 4C-D, MTT

assay results showed that the proliferation-inhibiting effects of BF or BF211 were

enhanced in cells with APOE knockdown. Further, results of the LDH release assay

showed that the cytotoxic effects of BF or BF211 were also enhanced in cells with

APOE knockdown (Fig. 4E-F). Notably, as shown in Fig. 4G, APOE knockdown cells

exhibited colocalization of the Na+/K+-ATPase α1 subunit and caveolin-1, which

indicated a possible formation of the Na+/K+-ATPase signalosome.

Influence of APOE knockdown on BF-induced apoptosis and cell cycle arrest and

activation of PI3K/AKT/GSK3β/caspase signal cascades

As showed in Fig. 5A-B, BF-induced apoptosis was enchanced in cells with

APOE knockdown. Results of checking the activation of the PI3K/AKT/GSK3β

pathway and caspase-dependent apoptosis in APOE knockdown cells and negative

control cells were shown in Fig. 5C, 5D and Supplemental Fig. S5. As shown in Fig.

5C, in the negative control cells, BF treatment for 24 h slightly inhibited the

PI3K/AKT/GSK3β pathway. While, BF treatment induced a strong decrease in

phosphorylation of PI3K and AKT, as well as in GSK3β in APOE knockdown cells.

BF treatment for 24 h did not induce considerable caspase-3 activation and PARP

cleavage (Supplemental Fig. S5). But, after 48 h of BF treatment, caspase-3 activation

and PARP celeavage could be observed, especially in APOE knockdown cells (Fig.

5D). Furthermore, as shown in Fig. 5E, BF induced siginificant increase in percentage

of cells at G2/M phase and cells at sub-G1 phase in cells with APOE knockdown.

Results of checking cell cycle regulators (Fig. 5F) suggested that CDC25C and CDC2

but not cyclin D1 might be involved in the G2/M arrest of BF-treated cells. The

decreasing effects of BF on CDC2 level were slight stronger in APOE knockdown

cells than that in negative control cells. These results indicated that APOE knockdown

enhanced sensitivity of Hep3B cells to cell apoptosis and cell cycle arrest induced by

BF.

The influence of Na+/K+-ATPase β1 knockdown on Hep3B cells

To study the role of the high level of Na+/K+-ATPase β1 (ATP1B1) in Hep3B

cells, the sensitivity of Hep3B cells with ATP1B1 knockdown was also studied.

Transfection of siRNA for ATP1B1 silenced the expression of Na+/K+-ATPase β1 in

Hep3B cells (Fig. 6A). Results of the MTT assay (Fig. 6B-C), the LDH release assay

(Fig. 6D-E) and the apoptosis assay (Fig. 6F) all indicated that ATP1B1 knockdown

increased the sensitivity of Hep3B cells to BF or BF211. Further, since the

Na+/K+-ATPase β1 subunit was closely related to intercellular junctions (21), the

location of E-cadherin, an adherens junction protein (22), was observed. As shown in

Fig. 6G, knockdown of ATP1B1 resulted in the disassembly of adherens junctions,

which could facilitate binding between BF and the Na+/K+-ATPase.

Discussion

Many prior studies reported the cytotoxicity of cardiac steroids in cancer cells

including hepatoma cells (23-25), but their mechanisms were not fully understood. In

the present study, we investigated the cytotoxicity of representative cardiac steroids

(BF, BF211, OUA and DIG) in 4 types of hepatoma cells and embryo liver L-02 cells.

Hep3B cells exhibited relatively low sensitivity to cardiac steroids. Understanding the

mechanism of low sensitivity of Hep3B cells is helpful to understanding the

cytotoxicity mechanism of cardiac steroids. Since Na+/K+-ATPase on the plasma

membrane is the accepted direct target of cardiac steroids, the expression of

Na+/K+-ATPase subunits in Hep3B cells was studied and compared with those of

SK-HEP-1 cells, which had a high sensitivity to cardiac steroids. Since the expression

levels of the subunits of Na+/K+-ATPase in Hep3B cells were high, we deduced that

the low sensitivity of Hep3B cells was not related to the expression level of

Na+/K+-ATPase.

Previous studies discovered the existence of two pools of Na+/K+-ATPase

within the plasma membrane: the classical pool of enzymes acting as an energy

transducing ion pump, and the signal transducing pool of enzymes that was restricted

to the caveolae, which formed the “Na+/K+-ATPase signalosome” (26). By binding

to Na+/K+-ATPase signalosome, cardiac steroids could activate multiple downstream

signal transduction pathways (27). The identification of a mutant α1 subunit of

Na+/K+-ATPase which had normal pumping function but was defective in signal

transduction supported the existence of two pools of Na+/K+-ATPase (28). Since

depletion of caveolae increased the pumping function of Na+/K+-ATPase but

suppressed signal transduction induced by cardiac steroids, the function of caveolae

Na+/K+-ATPase in signal transduction was confirmed (29). In the present study, we

found that the expression level of caveolin-1 was low in Hep3B cells. More

importantly, co-localization of the Na+/K+-ATPase α1 subunit and caveolin-1 was

observed in SK-HEP-1 cells, but was barely observed in Hep3B cells. Therefore,

Hep3B cells may be deficient in the Na+/K+-ATPase signalosome.

To further clarify factors involved in the regulation of the Na+/K+-ATPase

signalosome, the gene expression profile of Hep3B cells was determined using

RNA-Seq and compared to the SK-HEP-1 cells. Interestingly, the 36 genes that

differentially expressed between the two cell lines were mostly associated with

extracellular space, protein binding and extracellular region, which may be related to

the binding between cardiac steroids and the membrane Na+/K+-ATPase. APOE, one

of the 36 genes, was found to have critical position in the interaction network. APOE

protein is a 34-kDa glycoprotein that serves as a ligand for low density lipoprotein

receptors and also appears to have a wide variety of functions in addition to lipid

transport (30). Our results showed that the expression level of APOE was high in

Hep3B cells and that the knockdown of APOE by siRNA increased the sensitivity of

Hep3B cells to cardiac steroids. Previous studies suggested that APOE impacted

subcellular distribution/interaction of caveolin 1 (31, 32) and may be involved in the

formation of lipid rafts, which is a necessary condition for caveolae (33). Therefore,

APOE may affect the formation of the Na+/K+-ATPase signalosome by impacting the

subcellular distribution of caveolin-1. Results from studying the expression and

distribution of Na+/K+-ATPase subunits and caveolins showed that APOE

knockdown had no significant influence on their expression levels but induced clear

co-localization of caveolin-1 and the Na+/K+-ATPase α1 subunit at the plasma

membrane. APOE knockdown improved the formation of Na+/K+-ATPase

signalosome.

Though the components in the Na+/K+-ATPase signalosome had not been fully

clarified, reported partners of the Na+/K+-ATPase signalosome included Src, PI3K

and EGFR (34-36). PI3K was a critical component of the signalosome and was bound

to a proline-rich region of the α-subunit of Na+/K+-ATPase (37). Binding between

Na+/K+-ATPase and cardiac steroids triggered various downstream signaling

cascades which affect cell growth, apoptosis, adhesion and motility (38). Among these

signaling cascades, the PI3K-AKT pathway was one of the most important oncogenic

pathways in human cancers, including hepatocellular carcinoma (39). Further, the

PI3K/AKT pathway was found to be involved in the cytotoxicity of cardiac steroids

including BF (40, 41). Our study showed that the inhibition of BF on the

PI3K/AKT/GSK3β pathway and activation of the apoptosis cascades were stronger in

Hep3B cells with APOE knockdown compared with the negative control cells.

Therefore, function of the Na+/K+-ATPase signalosome was enhanced in cells with

APOE knockdown.

The level of the Na+/K+-ATPase β1 subunit was found to be significantly higher

in Hep3B cells. The Na+/K+-ATPase β subunits were not active subunits of

Na+/K+-ATPase, but they function to maintain membrane integrity and intercellular

interactions (21, 42, 43). To activate the Na+/K+-ATPase signalosome, cardiac

steroids must bind to Na+/K+-ATPase on plasma membrane, a process that may be

influenced by intercellular interactions. Knockdown of the Na+/K+-ATPase β1

subunit induced disassembly of adherens junctions and increased sensitivity of Hep3B

cells, suggested the contribution of β1 subunit to regulating binding between BF and

the Na+/K+-ATPase. The functions of the Na+/K+-ATPase β1 subunit and

intercellular junctions in sensitivity of cells to cardiac steroids needs further study.

In summary, our study showed that deficiency in the Na+/K+-ATPase

signalosome was a factor in the low sensitivity of Hep3B cells to cardiac steroids.

Currently, several clinical trials using digoxin (NCT02138292, NCT01887288

NCT02212639) in anti-cancer therapy are recruiting participants. Understanding the

mechanism of anti-cancer effects of cardiac steroids is necessary for the development

of cardiac steroids as new anti-cancer agents. Our results confirmed the important role

of the Na+/K+-ATPase signalosome in cytotoxicity of cardiac steroids, and found the

contribution of APOE protein in regulating the formation and function of the

Na+/K+-ATPase signalosome.

References

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Figure Legends

Figure 1. A-B, Cell viability (MTT assay) of four types of hepatoma cells and one

type of embryo liver cells treated with various concentrations of BF or BF211 for 72 h.

Data presented was mean ± SEM of three independent experiments. Significantly 21

different between Hep3B cells and SK-HEP-1 cells (*p<0.05, **p<0.01). C, PCR

analysis of mRNA expression levels of Na+/K+-ATPase subunits in the cell lines.

Data presented was mean ± SEM of three independent experiments. Significantly

different vs Hep3B cells (*p<0.05, **p<0.01). D, Protein expression levels of

Na+/K+-ATPase α1 and Na+/K+-ATPase β1 in the cell lines.

Figure 2. Expression and localization of Na+/K+-ATPase and caveolins in Hep3B

and SK-HEP-1 cells. A, PCR analysis of mRNA expression levels of caveolin-1,

caveolin-2, caveolin-3 in Hep3B and SK-HEP-1 cells. Data presented was mean ±

SEM of three independent experiments. Significantly different vs Hep3B cells (*

p<0.05, **p<0.01). B, Immunocytostaining of Na+/K+-ATPase α1 subunit (red),

caveolin-1 (green) and nucleus (blue) in Hep3B and SK-HEP-1 cells. Representative

results of three independent experiments. Scale bar=20 µm.

Figure 3. Comparison of gene expression profiles of Hep3B cells and SK-HEP-1

cells and bioinformatic analysis results of the differentially expressed genes. A,

Scatter plots figure of gene expression profiles of Hep3B cells and SK-HEP-1 cells.

Spots marking in red or green were genes with higher or lower levels in Hep3B cells

compared with SK-HEP-1 cells. B, GO enrichment analysis of the 36 differentially

expressed genes. Full report on enriched categories of degree 0. C, RT-PCR analysis

results of expression levels of APOE, APOC1, C3, FBLN1, PRNP, CTSB, COL18A1,

NID1 and BCAM.

Figure 4. A, Protein levels of APOE in wild type, negative control and APOE

knockdown Hep3B cells. B, Levels of Na+/K+-ATPase subunits and caveolins in

APOE knockdown and the negative control Hep3B cells. C-D, Cell viability (MTT

assay) of APOE knockdown cells and the negative control cells treated with various

concentrations of BF or BF211 for 72 h. E-F, Cell death (LDH release assay) of APOE

knockdown cells and the negative control cells treated with various concentrations of

BF or BF211 for 24 or 48 h. Data presented was mean ± SEM of three independent

experiments. Significantly different vs negative control (*p<0.05, **p<0.01). G,

Immunocytochemical staining of Na+/K+-ATPase α1 subunit (red), caveolin-1 (green)

and nucleus (blue) in wild type, negative control and APOE knockdown cells.

Representative results of three independent experiments. Scale bar=20 µm.

Figure 5. A, Representative results of flow cytometry assay of apoptosis in negative

control and APOE knockdown Hep3B cells treated with various concentrations of BF

for 48 h. Representative results of three independent experiments. B, Quantification of

apoptotic cells as % over total number of cells in APOE knockdown cells and

negative cells treated with various concentrations of BF for 48 h. Data presented was

mean ± SEM of three independent experiments. Significantly different vs negative

control (*p<0.05, **p<0.01). C, Results of Western blotting assay of phosphorylation

of PI3K, AKT and GSK3β in negative control cells and APOE knockdown cells

treated with BF (50 nM) for 24 h. D, Results of Western blotting assay of PARP and

caspase-3 in wild type, negative control and APOE knockdown cells treated with BF

(50 nM) for 48 h. E, Results of flow cytometry analysis of the cell cycle phase

distribution of the negative control and APOE knockdown cells treated with various

concentrations of BF for 24 h. Data presented was mean ± SEM of three independent

experiments. Significantly different vs negative control (*p<0.05, **p<0.01). F,

Results of Western blotting assay of cyclin D1, CDC25C and CDC2 in negative

control and APOE knockdown cells treated with various concentrations of BF for 24

Figure 6. A, Protein expression levels of Na+/K+-ATPase β1 subunit in wild type,

negative control and ATP1B1 knockdown cells. B-C, Cell viability of ATP1B1

knockdown cells and negative cells treated with various concentrations of BF or

BF211 for 72 h. D-E, Cell death (LDH release assay) of ATP1B1 knockdown cells

and negative cells treated with various concentrations of BF or BF211 for 24 or 28 h.

F, Quantification of apoptotic cells as % over total number of cells in ATP1B1

knockdown cells and negative cells treated with various concentrations of BF for 48 h.

Data presented was mean ± SEM of three independent experiments. Significantly

different vs negative control (*p<0.05, **p<0.01). G, Immunocytochemical staining

of E-cadherin (green) and nucleus (blue) in wild type, negative control and ATP1B1

knockdown cells. Representative results of three independent experiments. Scale

bar=20 µm.

Knockdown of apolipoprotein E enhanced sensitivity of Hep3B cells to cardiac steroids via regulating Na+/K+-ATPase signalosome

Miao Liu, Li-Xing Feng, Peng Sun, et al.

Mol Cancer Ther Published OnlineFirst August 9, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-15-0961

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