Protein array technology not only identifies a large number of proteins but also determines their expression levels. In the present study, antibody array analysis is used to decipher the proteins involved in hesperidin-induced cell death in HepG2 cells. Altered proteins in hesperidin treated cells were compared with that of untreated control cells by using a RayBio® Label-based (L series) human antibody array kit. The identified proteins were further confirmed using western blot analysis. STRING software based analysis was used to determine the protein-protein interactions. Many proteins related to signal transduction, cellular mechanisms, cell growth and proliferation regulatory proteins were identified. Among the proteins identified Hsp90, Smac/DIABLO, Prdx6 and FRK were significantly reduced in hesperidin treated cells. To the best of the authors' knowledge, the present study is the first to use antibody array for identifying proteins marker in hesperidin-induced cell death in HepG2 cells. The present study provides a novel insight into the anticancer mechanism of hesperidin.
Hesperidin is a flavone glycoside that is abundantly present in citrus fruits. Many have reported the anticancer activity of hesperidin on malignant cancer cells such as breast and prostate cancer (
Proteomic profiling has become a promising technology and been widely used for clinical biomarker identification, pathogenesis investigation, new drug discovery, pharmacological research and toxicological examination (
The antibody array analysis identified many proteins that are signal transduction proteins, structural proteins, mitochondrial proteins and proteins related to cellular metabolism. The STRING database was used to identify the protein-protein interactions of the proteins identified by antibody array. As most of the biological functions are transmitted via proteins, identification of proteins in hesperidin induced cell death will provide a better understanding on the anticancer mechanism of hesperidin.
Dulbecco's modified Eagle's medium (DMEM) was purchased from HyClone; GE Healthcare Life Sciences (Chalfont, UK). Antibiotics (streptomycin/penicillin) and fetal bovine serum (FBS) were obtained from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Goat anti-rabbit IgG (no. ADI-SAB-300, 1:2,000) and horseradish peroxidase-conjugated goat anti-mouse IgG (no. ADI-SAB-100-J, 1:2,000) were both purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). The RayBio® Label-based (L series) human L-507 antibody array 1000 membrane kit was purchased from RayBiotech (Norcoss, GA, USA). Anti-Prdx6 (ab59543, 1:1,000) and Anti-Frk (ab64914, 1:1,000) were purchased from Abcam (Cambridge, UK). Anti-Smac (no. 15108S, 1:1,000) and Anti-Hsp90 (no. 4877S, 1:1,000) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA).
HepG2 cells were obtained from Korean Cell Line Bank (Seoul, Korea) and were maintained in DMEM supplemented with 10% heat inactivated FBS and 1% penicillin/streptomycin at 37°C in a 5% CO2 incubator. Cells were treated with vehicle alone (1% DMSO) or 1 mM hesperidin dissolved in 1% DMSO.
An antibody array was carried out according to the manufacturer's instructions. Overnight grown cells were treated/untreated with 1 mM hesperidin. Following a 24 h incubation, cells were trypsinized and dialyzed at 4°C. The dialyzed samples were then biotin-labeled using 1X labeling reagent provided in the assay kit. The membranes are then blocked in blocking buffer at room temperature with gentle shaking for 1 h. After aspirating the blocking buffer, 8 ml of the diluted sample is placed on each membrane and incubated at RT for 2 h with gentle shaking. The membranes are then washed with 1X washing buffer for 3 times. After washing, 1X horseradish peroxidase-conjugated streptavidin was added to each membrane and incubated at room temperature for 2 h with gentle shaking. After washing, the membranes were placed in the detection buffer for 2 min and then the signals were detected using a ChemiDoc imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). By comparing the signal intensities quantified by densitometry, relative expression levels of target proteins were made.
Protein-protein interactions were analyzed using Search Tool for the Retrieval of Interacting Genes (STRING database, version 10;
Overnight grown HepG2 cells incubated with or without 1 mM hesperidin for 24 h at 37°C were lysed overnight with radioimmunoprecipitation assay lysis buffer containing protease inhibitor cocktail and EDTA. The extracts were then centrifuged at 14,500 × g for 30 min at 4°C to remove debris. The protein concentration in the supernatants was determined using a Bradford protein assay kit (Bio-Rad Laboratories, Inc.), according to the manufacturer's instructions. After being boiled with loading buffer, 50 µg protein samples were separated by 12% SDS-PAGE and then transferred onto polyvinylidene difluoride membrane, which was blocked with 5% non-fat milk for 1 h. Then, membranes were incubated with primary antibodies at 4°C overnight. After washing five times, the membranes were incubated with respective horseradish peroxidase-conjugated secondary antibodies at 37°C for 3 h. Western blot analyses were developed with enhanced chemiluminescence kit (GE Healthcare Life Sciences). Normalization was ensured by β-actin and each band was quantified using ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA).
Independent replicate antibody array experiments were performed and the RayBio® Human L-507 array analysis tool software, provided with the array, was used to analyze the signal intensities of the identified proteins. The data were normalized after background subtraction with the positive control densities. The positive control of the first sample is considered as 1 and the signal intensities of other samples are calculated according to the formula: Normalized signal intensity of particular spot = (signal intensity of particular spot) × (positive signal intensity on reference array/positive signal intensity on sample array).
Data are expressed as the mean ± standard deviation of a minimum of three replicates independent experiments. A Student's t-test was performed using SPSS version 10.0 for Windows (SPSS Inc., Chicago, IL, USA). P<0.05 was determined to indicate a statistically significant difference.
Antibody array analysis of HepG2 cells treated with or without hesperidin was carried out in order to identify the proteins involved in hesperidin induced cell death. As described in our previous study, paraptotic cell death was induced in HepG2 cells by treating with 1 mM hesperidin for 24 h (
The STRING database was used to analyze the protein-protein interactions. The
To validate the antibody array analysis results, western blot analysis was conducted using the same cell lysate that were used for antibody array analysis. Immunoblots of Hsp90, Prdx6, FRK and Smac were carried out. Hesperidin significantly reduced the protein expression of Hsp90, Prdx6, FRK and Smac (
In the present study, using antibody array techniques identified the proteins involved in hesperidin-induced cell death. Antibody array analysis has the advantaged of measuring multiple proteins in a parallel manner. A total of 19 proteins were identified and STRING analysis of these proteins indicated Hsp90 to be an important node within the entire network. Hsp90 is usually overexpressed in cancer cells and is considered as a potential target in cancer therapeutics. It is a chaperone protein required for the stability of various client proteins and many of these proteins are involved in cell signaling, proliferation and survival (
Interestingly, protein expression of Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low pI) was significantly reduced in hesperidin-treated HepG2 cells. It is a pro-apoptotic mitochondrial protein and negatively regulates the function of inhibitors of apoptosis proteins (IAPs). It promotes apoptosis by activating caspases (
In our previous study, it was observed that oxidative stress induced by hesperidin leads to paraptotic cell death in HepG2 cells (
Among the proteins identified during antibody array analysis, FRK (Fyn-related tyrosine kinase) expression was also downregulated. FRK belongs to Src non-receptor tyrosine kinase family (
To the best of the authors' knowledge, the present study is the first study on using antibody array for protein profiling of paraptosis cell death induced by hesperidin. In the present study, many target proteins were identified and have also demonstrated their interactions with each other. From the protein-protein interaction generated by STRING Hsp90 was identified to be an important node in hesperidin-induced cell death. As a chaperon protein, Hsp90 is required for the stability of various client proteins and many of which are involved in cell signaling, proliferation and survival. As a result, inhibiting Hsp90 may play an important role in hesperidin-induced cell death. As in the authors' previous study, it has been determined that hesperidin induced cell death is paraptosis (
The present study was supported by a grant from the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (grant nos. 2012M3A9B8019303 and 2017R1A2B4003974).
Antibody array analysis of HepG2 cells. (A) Altered protein spots detected during paraptosis. HepG2 cells were treated with or without 1 mM hesperidin and subjected to RayBio® Label-based array. Protein spots: 1, ErbB3; 2, Csk; 3, IGFIIR; 4, Prdx6; 5, calbindin; 6, Apex1; 7, BNIP2; 8, FOXN3; 9, FRK; 10, livin; 11, Itk; 12, Hsp70; 13, Hsp90; 14, Midkine; 15, S100A4; 16, S100A6; 17, ROS; and 18, Smac. (B) Signal intensities of the identified proteins. ERbB3, Receptor tyrosine-protein kinase erbB-3; IGFIIR, insulin-like growth factor type II receptor; Prdx6, peroxiredoxin 6; Apex1, DNA-(apurinic or apyrimidinic site) lyase; BNIP2, BCL2/adenovirus E1B 19 kDa protein-interacting protein 2; FOXN3, forkhead box protein N3; FRK, Fyn related tyrosine kinase; Itk, tyrosine-protein kinase; HSP, heat shock protein; ROS, proto-oncogene receptor tyrosine kinase; Smac, second mitochondria derived activator of caspases.
Protein-protein interaction of the identified proteins. STRING database, version 10 (
Expression of proteins altered during paraptosis. HepG2 cells were untreated or treated with hesperidin for 24 h and whole cell lysate prepared from these cells were subjected to western blot analysis. Protein level of Prdx6, Hsp90, FRK and Smac/DIABLO are shown relative to the value for untreated control cells. Data represent the mean ± standard deviation of three replicates independent experiments. *P<0.05 vs. control. Prdx6, peroxiredoxin 6; HSP, heat shock protein; FRK, Fyn related tyrosine kinase.
Proteins identified by antibody array analysis. HepG2 cells were treated with/without 1 mM hesperidin and the extracted proteins were subjected to RayBio® Label-based antibody array.
Uniprot ID | Symbol | Protein name | P-value | Up/down regulation | Biological function |
---|---|---|---|---|---|
P26447 | S100A4 | S100A4 | 0.002 | ↑↑ | Calcium binding protein, promotes cell migration |
P05937 | – | Calbindin | 0.006 | ↑↑ | Calcium binding protein |
P08922 | ROS | Proto-oncogene receptor tyrosine kinase | 0.01 | ↑↑ | Cell growth, proliferation regulator, activates PI3k/Akt pathway, MAPK cascade |
P07900 | Hsp90 | Heat sock protein 90 | 0.03 | ↓↓ | Negative inhibitor of apoptosis, activates MAPK cascade |
P06703 | S100A6 | S100A6 | 0.03 | ↑↑ | Calcium binding protein |
P41240 | Csk | Tyrosine-protein kinase Csk | 0.04 | ↓↓ | Cell growth, differentiation, cell migration regulation |
P42685 | FRK | Fyn related tyrosine kinase | 0.05 | ↓↓ | Suppress growth and function during G1 and S phase of cell cycle |
P07948 | Lyn | Tyrosine-protein kinase Lyn | 0.05 | ↓↓ | Regulates activation of MAP kinase signaling cascade cascade, cell proliferation, survival, differentiation, migration, adhesion, degranulation, and cytokine release |
P30041 | Prdx6 | Peroxiredoxin 6 | 0.05 | ↓↓ | Cellular protection, cell survival |
O00409 | FOXN3 | Forkhead box protein N3 | 0.08 | ↓↓ | Transcriptional repressor, cell cycle arrest at G1 and G2 phase |
Q9GK35 | IGF-IIR | Insulin-like growth factor type II receptor | 0.08 | ↑↑ | Suppresses tumor growth |
P21860 | ErbB3 | Receptor tyrosine-protein kinase erbB-3 | 0.1 | ↑↑ | Activates MAPK and PI3/Akt pathway |
Q08881 | Itk | Tyrosine-protein kinase | 0.1 | ↓↓ | Itk phosphorylation leads to Ca2+ release from ER to the cytoplasm |
Q96CA5 | BRIC7 | Livin | 0.3 | ↑↑ | Inhibits apoptosis protein family member |
P27695 | Apex1 | DNA-(apurinic or apyrimidinic site) lyase | 0.5 | ↓↓ | Activator of transcriptional repression |
P0DMV8 | Hsp70 | Heat sock protein 70 | 0.5 | ↓↓ | Inhibits apoptosis, |
Q12982 | BNIP2 | BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 | 0.6 | ↓↓ | Suppresses cell death |
Q9NR28 | Smac | Second mitochondria derived activator of caspases | 0.6 | ↓↓ | Promotes apoptosis |
P21741 | – | Midkine | 0.9 | ↓↓ | Cell proliferation regulator, activates MAPK pathway |
List of predicted functional partners obtained from STRING database.
Protein | Function |
---|---|
SUGT1 | SGT1, suppressor of G2 allele of SKP1 ( |
STIP1 | Stress-induced-phosphoprotein 1. Mediates the association of the molecular chaperones Hsc70 and Hsp90 |
PTGES3 | Prostaglandin E synthase 3 (cytosolic). Molecular chaperone that localizes to genomic response elements in a hormone-dependent manner and disrupts receptor-mediated transcriptional activation, by promoting disassembly of transcriptional regulatory complexes |
CDC37 | Cell division cycle 37 homolog. Co-chaperone that binds to numerous kinases and promotes their interaction with the Hsp90 |
AHSA1 | AHA1, activator of heat shock 90 kDa protein ATPase homolog 1 |