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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 9  |  Page : 473-479

Modulation of the gamma-secretase activity as a therapy against human hepatocellular carcinoma


1 Department of Microbiology and Immunology, Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Medical School, Southeast University, Nanjing, Jiangsu Province, China
2 Department of Microbiology and Immunology, Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Medical School, Southeast University, Nanjing; Department of Clinical Laboratory Science, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi, Jiangsu Province, China
3 Department of Cell Biology, College of Life Science, Beijing Normal University, Beijing, China

Date of Web Publication29-Jun-2018

Correspondence Address:
Yuqing Shen
Department of Microbiology and Immunology, Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Medical School, Southeast University, 87 Dingjiaqiao Road, Nanjing, Jiangsu Province 210009
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.174542

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 > Abstract 

Objective: Hepatocellular carcinoma (HCC) is the fifth most common tumor worldwide. The discovery of new therapies against HCC is highly dependable on finding molecules which play essential roles in cancer development. The objective of this study was to evaluate the activity of gamma secretase (γ-secretase), and the antitumor effects of a γ-secretase inhibitor (GSI) in HCC.
Methods: The expression of presenilin 1 (PS1), a core component of γ-secretase, was examined by Western blot. Activity of γ-secretase was measured by a luciferase-based reporter system, and cancer cells were transfected either with PS1 dominant negative mutant (PS1D385A) or treated with GSI.
Results: Expression of PS1 was increased in HCC tissue and several HCC cell lines, which were accompanied by elevated γ-secretase activity. Cell colony formation and cell proliferation were decreased upon treatment with GSI but not with PS1D385A transfection.
Conclusion: GSIs may be appealing candidates for the development of new therapies against HCC.

Keywords: Cell proliferation, gamma-secretase, hepatocellular carcinoma, liver, presenilin 1


How to cite this article:
Shen Y, Yin Y, Peng Y, Lv D, Miao F, Dou F, Zhang J. Modulation of the gamma-secretase activity as a therapy against human hepatocellular carcinoma. J Can Res Ther 2018;14, Suppl S2:473-9

How to cite this URL:
Shen Y, Yin Y, Peng Y, Lv D, Miao F, Dou F, Zhang J. Modulation of the gamma-secretase activity as a therapy against human hepatocellular carcinoma. J Can Res Ther [serial online] 2018 [cited 2019 Sep 17];14:473-9. Available from: http://www.cancerjournal.net/text.asp?2018/14/9/473/174542


 > Introduction Top


Gamma secretase (γ-secretase) belongs to the family of intramembrane cleaving protease. By catalyzing the final cleavage to produce of Aβ peptides, γ-secretase draws a tremendous attraction for its involvement in the pathology of Alzheimer' disease.[1] It is shown that γ-secretase is composed of four proteins: Presenilins (PSs), nicastrin (NCT), anterior-pharynx defective-1 (APH 1), and PSEN enhancer 2 (PEN2), with PSs harboring the catalytic activity.[2] The multiprotein complex can have different composition since there are two homologous of PSs (PS1 and PS2) and two of APH 1. They are present in different tissues and may provide heterozygous biochemical properties.[3]

Up to now, there are more than 90 substrates described for γ-secretase including Notch, E-cadherin, and epithelial cell adhesion molecular.[4] Because of its various substrates, γ-secretase is considered to be involved in several processes in health and also in disease, for example, cancer. Partial loss of γ-secretase activity in the skin can result in the formation of epidermal hyperplasia and skin tumors in adult mice.[5] Indeed, by reducing the level of γ-secretase, the risk for development of hyperproliferation of granulocyte increases.[6] However, upregulation of NCT has been shown in invasive breast cancer, PS1 in human malignant melanoma, and PS2 in brain tumors.[7],[8],[9] Therefore, γ-secretase can act not only as a tumor promoter but also as a tumor suppressor in different cell types. Thus, understanding the exact activity of γ-secretase in a specific cell context is very important for the development of novel therapeutically approaches.

Hepatocellular carcinoma (HCC) is the fifth most common tumor worldwide. Curative resection, liver transplantation, radiofrequency ablation, transarterial chemoembolization, and radioembolization are beneficial for the patient in the early or intermediate stage of disease.[10] For patients with advanced HCC, sorafenib, a small molecule inhibitor of several tyrosine protein kinases designed to inhibit cancer angiogenesis is the only approved therapy.[11] Novel systemic molecular targeted agents including doxorubicin, octreotide, and oxaliplatin, tegafur/uracil, cisplatin and gemcitabine, and a monoclonal antibody inhibiting the insulin-like growth factor-1 receptor and their combinations with sorafenib are emerging.[12],[13],[14] Although all of these studies report some survival advantage over sorafenib alone, the overall survival time of the patients with advanced disease is still shorter than a year.[15],[16] Since hepatocarcinogenesis is the result of genetic alterations with aberrant activation of multiple signaling cascades, the discovery of novel molecular therapies that target specific signaling pathways is urgently needed.

A previous report demonstrated the inhibitory properties of difluorophenylacetyl- alanyl- phenylglycine-t -butyl-ester (DAPT), a type of γ-secretase inhibitor (GSI), on the growth of an HCC cell line.[17] Our group has also shown that GSI decreases cell viability in HCC cell lines.[18] However, the activity of γ-secretase has never been investigated in HCC and whether the GSI-dependent effects on cell proliferation are mediated by γ-secretase inhibition is currently unknown.

In this study, we found that the expression of PS1 and the level of γ-secretase activity were increased in HCC tissue and several HCC cell lines. Diminished γ-secretase activity was achieved by transfect the cancer cell with PS1 dominant negative (DN) mutant (PS1D385A) or treatment with GSI. Cell colony formation and cell proliferation were only decreased by treatment of GSI, with obviously γ-secretase inhibition. However, cell adherence was not affected by downregulation of γ-secretase activity.


 > Methods Top


Cell lines and reagents

DAPT were purchased from Calbiochemical Corporation (Calbiochemical Co., Los Angeles, California, USA) and dissolved in dimethyl sulfoxide (DMSO). The peripheral nontumor cell line (QSG7701) and five human HCC cell lines (HepG2, BEL7402, BEL7404, SMMC7721, and BEL7405) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco-BRL, Grand Island, New York, USA) and supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL) in a 5% CO2 incubator at 37°C. All cell lines were obtained from the cell bank of the Type Culture Collection of the Chinese Academy of Science (Shanghai, China).

Expression vector construction and stable cell transfection

Expression vectors of the pAG3zeo-PS1D385A (kindly provided by Dr. Huaxi Xu, Burnham Institute, USA) were subcloned into the pcDNA3.1(−)/myc-His A vector (Invitrogen). PcDNA3.1-PS1D385A or pcDNA3.1 mock vectors were transfected into HCC cell line BEL-7404 cells using the FuGENE HD transfection reagent (Roche, Nutley, New Jersey, USA). For stable cell population selection, 24 h after transfection, cells were replated in RPMI-1640 (Gibco-BRL) with 10% (vol/vol) fetal calf serum and 400 μg/ml G418 (Sigma, St. Louis, MO, USA). G418-resistant clones were selected and expanded. The protein levels of PS1D385A in these cells were checked by Western blot analysis.

Luciferase reporter assay

One day before transfection, 2 × 105 cells were seeded in each well of a six-well plate. Using the FuGENE 6 (Roche), the cells were co-transfected with pMst-GV-APP and pG5E1B-luc (kindly provided by Professor Thomas Sudhof, University of Texas Southwestern, USA) accompanied by pRL-SV40 (Promega), which served as a control to determine transfection efficiency. After 48 h of transfection, the cells were lysed, and luciferase activity was assayed with the Promega Dual-Glo assay kit using a TD 20/20n luminometer (Turner Biosystems, Sunnyvale, CA, USA). The data represented the average of three independent experiments and were shown with the standard error.

Western blot analysis

The whole-cell lysates were obtained by protein lysis buffer treatment with protease inhibitors. The protein concentration was quantified by the Bradford assay (Pierce, Rockford, Illinois, USA) using bovine serum albumin (BSA) as the standard. After centrifugation, 20 mg of total protein was denatured at 100°C for 5 min in a protein sample buffer, separated on a 10% polyacrylamide gel electrophoresis (PAGE) under denaturing conditions (sodium dodecyl sulfate [SDS]-PAGE), and transferred to a nitrocellulose membrane (Roche) for immunoblotting. Immunoblot analysis was carried out using the primary antibody, horseradish peroxidase-conjugated secondary antibodies and developed using the enhanced chemiluminescence kits (Amersham Biosciences, Little Chalfont, UK).

Analysis of cell growth in vitro

The in vitro growth rates of all cell lines were measured using the cell counting kit (CCK)-8 (Dojindo, Kamimashiki-gun Kumamoto, Japan). Briefly, cells were seeded in 96-well plates at 1500 cells per well. On the day for detection, the culture medium was replaced with an equal volume of a fresh medium that contained 10% CCK8 of 5 ml/mg stock. Plates were incubated at 37°C for 2 h, and the cell proliferation rate was determined by measuring the absorbance at 450 nm using the Microplate Reader (Thermo Fisher, Waltham, Massachusetts, USA). For GSI treatment, 24 h after seeding, the cells were treated with DAPT at different concentrations.

Colony formation assay

Colony formation assay was determined by a two-layer agar system. Liver cancer cells were plated in a six-well plate at the density of 1.5 × 104 cells per well. Colonies were visualized daily. In DAPT experiments, cells were suspended in the presence of different concentration of DAPT or DMSO (solvent control) (Sigma, St. Louis, MO, USA) at a final concentration of 0.5%.

Adhesion assay

Ninety-six-well plates coated with collagen were blocked in 0.5% BSA in the DMEM medium. Then, 2 × 104 of cells were plated on collagen-treated plates and incubated at 37°C for 90 min in CO2 incubator. Cells were washed with phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde for 60 min. After washing with PBS, cells were stained with crystal violet (5 mg/ml in 2% ethanol). Then, 1% Triton X-100 was added, and the plate was read at 560 nm to detect adhesion of cells.

Hepatocellular carcinoma specimens

Sixteen fresh liver tissue samples were obtained from liver cancer patients in Zhongda Hospital affiliated to Southeast University (Jiangsu province, China) who had undergone surgical resection. The clinicopathologic parameters of all cases were listed in [Table 1]. The collection of tissues was approved by the Human Ethics Committee of Zhongda Hospital and followed the guidelines of the China Public Health Service. Surgically removed biopsies were immediately snap-frozen in liquid nitrogen and stored at −70°C for up to 6 months. All these samples were reviewed by a pathologist to confirm the diagnosis of HCC.
Table 1: Clinical parameters of hepatocellular carcinoma patients and the expression of presenilin 1 in hepatocellular carcinoma specimens

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Statistical analysis

Data are presented as a mean ± standard deviation. All comparisons between groups were performed using two-tailed paired Student's t-test. All P < 0.05 were considered significant.


 > Results Top


Increased expression of presenilin 1 in hepatocellular carcinoma tissue

The PS1 protein was detected in whole SDS-soluble extracts of noncancerous and cancerous tissues using a monoclonal antibody, generated against the N-terminals (residues 21-80) amino acids of human PS1. Immunoblot analysis revealed that PS1 accumulated only as a ~28 kDa fragment, while the ~43–45 kDa fragment which represents the full-length PS1 was not detectable [Figure 1]. These results supported the idea that PS1 is subjected to be processed endoproteolytically in vivo, and the ~28 kDa fragment is characterized as the N-terminal fragments (NTFs) of self-catalyzed PS1. PS1 was expressed in the nontumorous liver tissue. However, the expression levels of PS1 were dramatically increased in 13 out of the 16 samples of liver cancer tissues [Table 1], indicating enhanced PS1 expression compared with noncancerous tissue.
Figure 1: Increased expression of presenilin 1 in hepatocellular carcinoma tissues. The expression of presenilin 1 was detected in the tumor (T) and nontumorous liver tissues (N) by Western blot. Presenilin 1 accumulated as a ~28 kDa fragment characterized by the N-terminal fragments of self-catalyzed presenilin 1

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Enhanced presenilin 1 expression and gamma-secretase activity in hepatocellular carcinoma cell lines

The expression of PS1 protein was detected in human liver cell line QSG7701 and 5 HCC cell lines. Compared to the level of PS1 in the QSG-7701 cells, all cancer cell lines showed increased expression of NTF of PS1 while BEL7404 and HepG2 cells demonstrating the highest amount of expression [Figure 2]a. Although PSs have the central roles in catalytic activity, they absolutely require the cofactors to form a functional γ-secretase complex.[2] To complement our observation of enriched expression of PS1 in HCC cell lines, we examined the activity of γ-cleavage in those cells using a luciferase-based γ-secretase reporter system. The quantitative measuring system is based on a Gal4-BD, and a VP16AD being inserted to the C-terminal of the γ-secretase cleavage sites of APP695. Upon cleavage at the γ-secretase site, the intracellular domains of the reporter molecules translocate to the nucleus and activate transcription of the luciferase gene via binding to the upstream Gal4 binding sites. Thus, the monitored luciferase activity corresponds to the γ-secretase activity.[19] After transfection of the luciferase reporter system into the cell lines, a strong activation of the luciferase gene was observed in all the HCC cell lines except BEL7405 compared with that in QSG7701 cells [Figure 2]b. Our data indicated that the level of γ-secretase activity increases with the amount of endogenous PS1 protein expressed in those cancer cells.
Figure 2: Enhanced presenilin 1 expression and increased activity of gamma-secretase in hepatocellular carcinoma cell lines. (a) One human liver cell line and five hepatocellular carcinoma cell lines were analyzed for the expression of presenilin 1 by Western blot. (b) The activities of gamma-cleavage in those cells were measured using a luciferase-based gamma-secretase reporter system. Data are presented as a percentage of activity where 100% activity corresponds to the reporter gene activity obtained in the BEL7404 cells. The graph shows mean ± standard deviation of three independent experiments

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Inhibition of the gamma-secretase activity decreases tumor growth in vitro

To understand the role of γ-secretase in liver tumor growth in vitro, BEL7404 cells were stably transfected with a DN form of PS1, PS1D385A. The mutation of the conserved transmembrane (TM) aspartate residues in PS1, Asp385 in TM7, was confirmed by reduced γ-secretase activity in mice.[20] Western blot analysis showed the expressed PS1D385A proteins in stable transfected BEL7404 cells E12 and G10 [Figure 3]a. The activation of the luciferase gene decreased up to 67% in these two cell lines compare with the activity in the BEL7404 cells [Figure 3]b. However, no significant difference in tumor growth and colony formation in soft agar was noticeable between the PS1D385A expressed cells and the nontransfected BEL7404 cells [Figure 3]c and [Figure 3]d.
Figure 3: Inhibition of the activity of gamma-secretase after transfection with a dominant negative form of presenilin 1, presenilin 1D385A. (a) The expression of presenilin 1 D385A was detected in the stable transfected cell lines by Western blot. The presenilin 1 D385A protein accumulated as a ~42 kDa fragment (full length) since the mutation-reduced gamma-secretase processing of itself. (b) The activities of gamma-cleavage in G10 and E12 cells were measured using a luciferase-based gamma-secretase reporter system. Data were presented as percent activity where 100% activity corresponds to the reporter gene activity obtained in the nontransfected BEL7404 cells. (c) Cell viabilities of G10, E12, and nontransfected BEL7404 cells were assayed for five consecutive days using the cell counting kit 8 methods. The results represent two independent experiments in triplicate. (d) G10, E12, and nontransfected BEL7404 cells were plated per well in six-well plates for soft agar colony assay, and experiments were performed in duplicates. Data are presented as percent value where 100% value corresponds to the colony obtained in the vehicle control

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Next, a high efficient GSI, DAPT was used to inhibit the γ-secretase activity in BEL7404 cells. The level of the reporter gene activity decreased with the amount of DAPT added into the culture medium. The activation of the luciferase gene decreased to 13% when 40 μM DAPT was used [Figure 4]a. Furthermore, our results evidenced that the growth of BEL7404 cells was decreased upon DAPT treatment, and a significant inhibition was obtained using 40 μM DAPT [Figure 4]b. To test whether γ-secretase affected anchorage-independent growth, cells were cultivated with different concentrations of DAPT in 0.3% soft agar for 4 weeks, and colonies were counted. DAPT resulted in decreased number of colonies in a dose-dependent manner [Figure 4]c. HepG2, another cell line showing decreased γ-secretase activity upon DAPT treatment, also showed a decline in cell proliferation and reduced colony formation in soft agar with the presence of DAPT [Figure 4]e, [Figure 4]f, [Figure 4]g. In addition, BEL7404 and HepG2 cells were plated on collagen with or without DAPT to perform adhesion assay. DAPT did not change the cell adhesion to collagen in either cell line [Figure 4]d and [Figure 4]h.
Figure 4: Inhibition of the activity of gamma-secretase by difluorophenylacetyl-alanyl-phenylglycine-t-butyl-ester (gamma-secretase inhibitor) diminishes tumor growth. (a) The activities of gamma-cleavage in difluorophenylacetyl-alanyl-phenylglycine-t-butyl-ester treated BEL7404 (a) and HepG2 (e) cells were measured using a luciferase-based gamma-secretase reporter system. Data are presented as percent activity where 100% activity corresponds to the reporter gene activity obtained in the vehicle control. (b) Cell viabilities of difluorophenylacetyl-alanyl-phenylglycine-t-butyl-ester treated BEL7404 (b) and HepG2 cells (f) were assayed for consecutive 5 days using the cell counting kit 8 methods. (c) Difluorophenylacetyl-alanyl-phenylglycine-t-butyl-ester treated BEL7404 (c) and HepG2 cells (g) cells were plated in six-well plates for soft agar colony assay, and experiments were performed in duplicates. Representative data of one experiment is shown. Data are presented as percent value where 100% value corresponds to the colony obtained in the vehicle control. (d) Difluorophenylacetyl-alanyl-phenylglycine-t-butyl-ester treated BEL7404 (d) and HepG2 cells (h) cells were plated on collagen-precoated plates for cell adhesion assay. Data are presented as percent value where 100% value corresponds to the order deviation value obtained in the vehicle control. *P < 0.05; **P < 0.01

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 > Discussion Top


By virtue of its determining role in regulating cell proliferation, survival, and differentiation, Notch signaling pathway is probably one of the most well-studied γ-secretase-related functions in cancer development. Binding of Notch receptor to its ligand initiates the proteolytic cleavage by α-secretase and γ-secretase, thus releasing the intracellular domain of the Notch receptor (NICD). Subsequently, NICD translocates to the nucleus where it modulates gene expression.[21] Most of the developmental defects in skin observed in γ-secretase-deficient mice were related to Notch signaling pathway mutations.[22],[23] In the T-cell acute lymphoblastic leukemia, the majority of tumors acquired mutations that result in activation of Notch signaling pathway.[24] Therefore, they can be therapeutically targeted with GSIs.

However, the data regarding Notch involvement in HCC are still ambiguous, and the usage of GSIs to suppress HCC is largely ignored on the basis of a report that overexpression of Notch1-induced apoptosis in an HCC cell line.[25] In contrast, Notch signaling has been found to promote liver carcinogenesis in a genetically engineered mouse model, and its activation has been detected in one-third of human HCC samples.[26] Indeed, we also reported a high expression level of Notch receptors, ligands, and target molecules in HCC cell lines and tissues in a previous study.[18] Concomitant with our observations that the level of γ-secretase activity is increased in HCC and γ-secretase activity is reduced upon treatment with GSI, targeting Notch signaling pathway in liver cancer cells by GSIs might be a very promising treatment for HCC.

Due to the toxicity of nonselective inhibition of γ-secretase, clinical development of GSIs will require empirical testing with careful evaluation of benefit versus risk.[27] In this study, we found that the level of γ-secretase activity increased in HCC cell line BEL7404 and HepG2. DAPT, one of the GSIs, can obviously reduce the activity of γ-secretase, thus inhibiting cell colony formation and proliferation. It has been suggested that the original activity of γ-secretase could be a candidate biomarker for indicating the sensitivity of cancer cells to GSIs treatment. However, although the activity of γ-secretase can be evaluated in cancer cell lines by transfecting cells with luciferase-based γ-secretase reporter system, it is impossible to measure that in HCC samples in a similar way. Here, we show that the level of γ-secretase activity increased with more PS1 expression in 4 of 5 investigated HCC cell lines. In addition, 13 out of the 16 tumor samples showed enhanced PS1 expression, suggesting a high level of γ-secretase activity in the tumor tissues. However, the relationship between γ-secretase activity and PS1 expression in HCC tissues needs further investigation. Other suitable methods for γ-secretase activity examination should be developed or employed for clinical tissue samples.

In this study, cell colony formation and cell proliferation were decreased upon the inhibition of γ-secretase. However, the target gene of γ-secretase responsible for the phenotype remains still unknown. In another study, we showed that the expression of c-Myc, a Notch target gene, was decreased upon GSI-I treatment, and this suppression effect could be rescued by the exogenous expression of the constitutively active Notch1-ICN in BEL7404, providing the evidence that deregulation of c-Myc contributes to the suppressive effect of GSI-I on cell growth.[18] Upon observation that cell adherence was not affected by downregulation of γ-secretase activity, E-cadherin - another well-studied substrate of γ-secretase that mediates cell-cell contact - might not be functional in these HCC cell lines. The target genes of γ-secretase that might be responsible for the inhibitory property of GSI on the growth of liver cancer cells are currently under investigation. Although transfection with a DN form of PS1, PS1D385A can reduce the activity of γ-secretase in cancer cells, tumor growth, and colony formation in soft agar was not affected. Furthermore, compared with GSI treatment, the efficiency of inhibiting γ-secretase activity is much lower in PS1D385A transfected cells (87% vs. 33%). Therefore, we suspect that the major targets of γ-secretase which managed tumor cell growth are not affected by expression of PS1D385A.

In summary, this study provides evidence that γ-secretase activities are increased in HCC tissue and several HCC cell lines. Treatment with GSI decreases γ-secretase activity, thus inhibiting cell proliferation and anchorage-independent cell growth. Overall, our findings indicated that GSIs may be an appealing candidate for the development of new therapies against HCC.

Acknowledgments

We thank Dr. Francisco Javier Cubero (Department of Internal Medicine III, University Hospital RWTH Aachen, Aachen, Germany) for careful revision of the manuscript.

Financial support and sponsorship

This work was supported by National Nature Science Foundation of China (31100649).

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

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