Knockdown of Apolipoprotein E Enhanced Sensitivity of Hep3B Cells to Cardiac Steroids via Regulating Na⁺/K⁺-ATPase Signalosome Free

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 anticancer agents substantially increased (8–12). Our lab also tried to develop derivatives of BF as possible new anticancer 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 anticancer agents was hindered because the mechanisms of their anticancer effects had not been fully understood.

In the current 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 Supplementary Fig. 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 with other cell lines. Expression levels of subunits of 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.

OUA and DIG with a purity of 98% were purchased from the Sigma-Aldrich Chemical Co.. 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 mol/L as stock solution and stored at −20°C.

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 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 laboratory, cells were expanded according to the manufacturer'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, RPMI1640 medium for SMMC-7721 and BEL-7402 cells, and DMEM (high glucose) for L-02 cells, respectively. The media were supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.

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 treated with BF, BF211, OUA, or DIG at different concentrations, or 0.1% DMSO (solvent control) for 72 hours. After treatment, the cell viability was evaluated by checking the absorbance at 570 nm.

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 treated with BF, BF211 at different concentrations, or 0.1% DMSO (solvent control) for 24 or 48 hours. 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.

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 minutes followed by 40 cycles at 95°C for 15 seconds and 61°C for 25 seconds. Each sample was analyzed in triplicate and the mean threshold cycle (C_t) value was calculated. The relative expression level was calculated using the ΔΔC_t method. The mRNA expression of GAPDH was used as an internal control. Primer sequences for PCR analysis are listed in Supplementary Table S1.

Cells cultured on poly-d-lysine–coated coverslips were fixed with cold 4% paraformaldehyde for 20 minutes. After washing with PBS three times, cells were blocked for 2 hours with PBS contained 0.1% Tween-20 and 1% BSA. Subsequently, cells were incubated with the antibodies listed in the Supplementary Table S2 and the nuclei were stained with DAPI. The stained cells were then observed with a laser-scanning confocal microscope (Olympus FV1000).

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

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/; ref. 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/; ref. 17) was also used to predict physical or functional associations among these genes.

Total RNAs extraction and reverse transcription were conducted as described above. Semiquantitative 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 seconds, and 72°C for 22 seconds of 30 cycles and a final extension at 72°C for 5 minutes, with a final hold at 20°C in a thermal cycler. Primer sequences of the genes are listed in Supplementary Table S1.

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 wild-type, APOE knockdown (transfected with siRNA for APOE for 24 hours) 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).

The Western blotting assay was conducted as described before (15). Antibodies used in Western blotting assay were listed in Supplemental Table S2.

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 with 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.

Student 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.

As shown in Fig. 1A and 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 (Supplementary Fig. S2A and S2B). To check whether Hep3B cells were resistant to all chemotherapy agents, we also studied the sensitivity of Hep3B cells to cisplatin, a commonly used anticancer agent, and compared the results with SK-HEP-1 cells which exhibited high sensitivity to cardiac steroids. As shown in Supplementary 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.

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. 1D). These results showed that the low sensitivity of Hep3B cells may not be related to expression level of Na⁺/K⁺-ATPase.

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. Furthermore, colocalization 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.

Transcriptomic datasets 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 Supplementary Table 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. Furthermore, we found a possible interaction network of the 36 genes as predicted (Supplementary Fig. 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. 3C). 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.

As shown in Supplementary 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 and D, MTT assay results showed that the proliferation-inhibiting effects of BF or BF211 were enhanced in cells with APOE knockdown. Furthermore, 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 and 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.

As showed in Fig. 5A and 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 and D and Supplementary Fig. S5. As shown in Fig. 5C, in the negative control cells, BF treatment for 24 hours 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 hours did not induce considerable caspase-3 activation and PARP cleavage (Supplementary Fig. S5). But, after 48 hours of BF treatment, caspase-3 activation and PARP cleavage 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.

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 and C), the LDH release assay (Fig. 6D and E) and the apoptosis assay (Fig. 6F) all indicated that ATP1B1 knockdown increased the sensitivity of Hep3B cells to BF or BF211. Furthermore, as 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.

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 current 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. As 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. As 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 classic 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). As 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 current study, we found that the expression level of caveolin-1 was low in Hep3B cells. More importantly, colocalization 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 with 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.

Although 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). Furthermore, 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 anticancer therapy are recruiting participants. Understanding the mechanism of anticancer effects of cardiac steroids is necessary for the development of cardiac steroids as new anticancer 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.

No potential conflicts of interest were disclosed.

Conception and design: X. Liu, M. Liu, L. Hu, D.-A. Guo

Development of methodology: M. Liu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Liu, L.-X. Feng, P. Sun

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Liu, P. Sun, B. Jiang, M. Yang

Writing, review, and/or revision of the manuscript: X. Liu, M. Liu, L. Hu, D.-A. Guo

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W. Liu, M. Lei, W. Wu

Study supervision: X. Liu, T. Mi

Other (uploaded RNA sequencing data to public databases): T. Mi

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).

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