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 1 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). 2 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. 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. 3 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. 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 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. 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. 5 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. 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.