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ORIGINAL ARTICLE
Year : 2016  |  Volume : 53  |  Issue : 2  |  Page : 216-219
 

Expression of Raf kinase inhibitor protein in human hepatoma tissues by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight methods


1 Department Medical Technology, Fooyin University, Kaohsiung City, Taiwan
2 Department of Hepato-Gastroenterology, Center-of-Hepato-Gastroenterology, Yuan's General Hospital, Kaohsiung City, Taiwan
3 Department of Biomedical Engineering, I-Shou University, Taiwan

Date of Web Publication6-Jan-2017

Correspondence Address:
H R Chang
Department of Biomedical Engineering, I-Shou University
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.197730

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 » Abstract 

Purpose: Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. To reduce the mortality and improve the effectiveness of therapy, it is important to search for changes in tumor-specific biomarkers whose function may involve in disease progression and which may be useful as potential therapeutic targets. Materials and Mehtods: In this study, we use two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to observe proteome alterations of 12 tissue pairs isolated from HCC patients: Normal and tumorous tissue. Comparing the tissue types with each other, 40 protein spots corresponding to fifteen differentially expressed between normal and cancer part of HCC patients. Results: Raf kinase inhibitor protein (RKIP), an inhibitor of Raf-mediated activation of mitogen-activated protein kinase/extracellular signal-regulated kinase, may play an important role in cancer metastasis and cell proliferation and migration of human hepatoma cells. RKIP may be considered as a marker for HCC, because its expression level changes considerably in HCC compared with normal tissue. In addition, we used the methods of Western blotting and real time-polymerase chain reaction to analysis the protein expression and gene expression of RKIP. The result showed RKIP protein and gene expression in tumor part liver tissues of HCC patient is lower than peritumorous non-neoplastic liver tissue of the corresponding HCC samples. Conclusion: These results strongly suggest that RKIP may be considered to be a marker for HCC and RKIP are down-regulated in liver cancer cell.


Keywords: Hepatocellular carcinoma, matrix-assisted laser desorption/ionization time-of-flight, Raf kinase inhibitor protein, two-dimensional polyacrylamide gel electrophoresis


How to cite this article:
Tsao D A, Shiau Y F, Tseng C S, Chang H R. Expression of Raf kinase inhibitor protein in human hepatoma tissues by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight methods. Indian J Cancer 2016;53:216-9

How to cite this URL:
Tsao D A, Shiau Y F, Tseng C S, Chang H R. Expression of Raf kinase inhibitor protein in human hepatoma tissues by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight methods. Indian J Cancer [serial online] 2016 [cited 2020 Aug 7];53:216-9. Available from: http://www.indianjcancer.com/text.asp?2016/53/2/216/197730



 » Introduction Top


Numerous genes associated with tumorigenesis have been identified. However, the vast majority of cancer deaths are due to metastatic disease, the identification of genes involved in metastasis has lagged behind. Hepatocellular carcinoma (HCC) is known as a common malignant tumor and accounts for 80-90% of primary liver tumors and is one of the most common and devastating malignant diseases world-wide. It is known that HCC develops from chronic inflammatory liver disease due to chronic hepatitis B or C infection.[1],[2] In addition, development of HCC is linked to cirrhosis.[3],[4] The chronic inflammation and cell damage in a cirrhotic liver may have provided the proliferative stimuli to promote the hepatocarcinogenesis.[5] Accordingly, to understand the mechanisms of hepatocarcinogenesis, analysis of proteome alteration is required.

Two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), enable the study of cancer proteomics the analysis of the abundance of total protein in human tumors.[6],[7],[8],[9] The value of this technique for detecting novel cancer-related proteins and for classifying human cancer (e.g. lung cancer, colon cancer, breast cancer, liver cancer etc.,) has been exhibited by several other studies.[6],[7],[8],[9] Using the technology, the change in Raf kinase inhibitor protein (RKIP) expression levels in tissue samples from HCC patients can also be demonstrated in the study.

The RKIP is a widely expressed modulator of the Raf kinase cascade and other signaling pathways.[10],[11],[12] RKIP was identified as an inhibitor of the mitogen-activated protein kinases (MAPK) signaling pathway.[11],[12],[13],[14],[15] Overexpression of RKIP suppressed MAPK signaling and down-regulation of RKIP had the opposite effect.

Roles for RKIP in diverse biological processes, including nervous and cardiac functions, membrane biogenesis and spermatogenesis, have been suggested.[10],[11],[12] RKIP may play an important role in prostate cancer metastasis and cell proliferation and migration of human hepatoma cells.[16],[17]

Although the molecular mechanism by which RKIP inhibits the extracellular signal-regulated kinases/MAPK signaling pathway has been partially delineated, little is known about the role of RKIP in human hepatoma tissues by 2DE and MALDI-TOF methods. In this study, we used 2-DE and MALDI-TOF MS to identify the alteration of RKIP and used Western blotting and real time-polymerase chain reaction (RT-PCR) to determine the expression of RKIP that are related to human hepatoma.


 » Materials and Methods Top


HCC tissues processing

Processing of HCC tissues for proteomic analysis in our laboratory has been approved by the local scientific ethics committee. All patients had given their informed consent for the study. HCCs and matched peritumorous non-neoplastic liver tissues derived from 42 patients were obtained from the center of hepatogastroenterology, Yuan's General, Kaohsiung, Taiwan. Surgical specimens were immediately snap-frozen and stored in liquid nitrogen.

Protein extraction and preparation

HCCs and matched peritumorous non-neoplastic liver tissues homogenized on ice in lysis buffer consisting of 8M urea, 2% (w/v) CHAPS, 0.002% Bromophenol Blue. Sample was incubated at 4°C for 15 min and centrifuged at 4°C for 30 min at 12000 rpm to remove deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and any particulate materials (tissue and cell debris). Precipitate the supernatant 2 h at −20°C with acetone/100% (w/v) trichloroacetic acid. The protein concentration of the sample was measured on a Model 590 Microplate Reader using a Protein Assay Kit (Bio-Rad Laboratories) with bovine serum albumin as standard. The final protein solution was a store at −20°C until further use.

Two-dimensional polyacrylamide gel electrophoresis

Appropriate amount of protein sample was mixed with rehydration buffer (8M urea, 2% (w/v) CHAPS, 0.5% (w/v) IPG buffer, 65 mM dithiothreitol (DTT) and a trace of bromophenol blue), and loaded in the isoelectric focusing (IEF) focusing tray, then gently placed non-linear pH 3-10NL IPG strips gel (Amersham Biosciences) side down onto the rehydration solution and overlay each IPG with 2 ml mineral oil to prevent evaporation during the rehydration process. IEF was carried out on an IPGphorII electrophoresis unit (Amersham Biosciences) at 100 V for 30 min, 250V for 30 min, 500V for 30 min, 1000V for 30 min, 4000V for 30 min, 6000V for 7 h 30 min (45 KV/h) and 7000V for 60 min in a gradient mode at 18°C. The IPG strip after IEF was equilibrated for two times 15 min under gentle agitation in equilibration solution I including 0.065M DTT and equilibration solution I including 0.014M iodoacetamide. After equilibration, the gel strip was placed on the upper edge of the 12.5% polyacrylamide gel. The electrophoresis gel was run at voltage 250V, 200 mA for approximately 6 h. The electrophoresis was stopped when the bromophenol blue front had traversed the gel.

Gel staining and MALDI-TOF MS analysis

The gel were stained with silver nitrate and scanned with an UMAX Scanner. Gel containing protein spots selected for identification were re-swelled in water. The spots could excise from the gel. Protein was subjected to trypsin digestion and subsequent identification by MALDI-TOF MS (Micro flex, Bruker). The MS data was submitted to the ProFound-Peptide Mapping facility.

RT-PCR

Total tissue RNA was isolated from HCCs and matched peritumorous non-neoplastic liver tissues derived from 42 patients using TRIzol reagent (Life Technologies). Reverse transcribed complementary DNA was performed using superscript II RT RNase H-reverse transcriptase and random primer (Life Technologies, Inc.). Quantity and quality of messenger RNA (mRNA) from all samples were certified by RT-PCR amplification of the β-actin gene. Amplification of RKIP transcripts was done using Perkin-Elmer GeneAmp PCR system 9600 (Norwalk, Conn., USA) and oligonucleotide primers (RKIP [5′]: 5′-ATGCCGGTGGACCTCAGCAAG-3′; RKIP [3′] 5′-CTACTTCCCAGACAGCTGCTC-3′). Thermal conditions of the system were as follows: 94°C for 2 min; 30 cycles at 94°C for 15 s, 55°C for 30 s, 72°C for 2 min; one cycle at 72°C for 10 min. The resulting PCR products (20 μl) were resolved in a 2% agarose gel and visualized under ultraviolet light after ethidium bromide staining.

Western blotting

Tissues were lysed in lysis buffer (20 mM Tris-HCl (pH 7.4), 2 mM ethylene glycol tetraacetic acid, 5 mM ethylenediaminetetraacetic acid, 500 μM sodium orthovanadate (Na3 VO4), 1% triton X-100, 0.1% sodium dodecyl sulfate (SDS), 10 μg/ml aprotinin [sigma], 10 μg/ml leupeptin [sigma], 1 mM phenylmethylsulfonyl fluoride [sigma]) for 20 min on ice. The lysates were centrifuged at 12,000 rpm for 10 min at 4°C, the resulting supernatant was isolated, the concentration of protein was measured and the pellet was discarded. The cell supernatants were diluted with SDS sample buffer (0.125M Tris-HCl, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, pH 6.8), and boiling water bath for 5 min before electrophoresis. Aliquots corresponding to 50 μg of protein were applied to 12% SDS-polyacrylamide gels. Western blotting of proteins consisted of overnight electro blotting the proteins from the polyacryamide gel onto nitrocellulose filter paper. After blocking with 5% non-fat dried milk (2 h at room temperature), the nitrocellulose sheet incubated with a 1:500 dilution of rabbit polyclonal immunoglobulin G (IgG) (Santa Cruz biotechnology, Inc.) raised against RKIP and detected with a 1:3000 dilution of horse radish peroxidase-conjugated goat antibody to rabbit IgG (Santa Cruz biotechnology, Inc.). For quantification, the visualized films were recorded on a digital imaging system (Alpha Imager 2000, Alpha Innotech Corp., San Leandro, CA) and analyzed in a densitometrical analysis system (Apha Ease version 3.23, Alpha Innotech Corp.).


 » Results Top


Differentially protein expression between normal and cancer part of HCC patient by 2D and MALDI-TOF

Proteomic of 12 pairs of HCC tissues were obtained by 2-DE [Figure 1]. Patient 21 as representation, 2-D map of peritumorous non-neoplastic liver tissue (A) and tumorous part (B) were shown. 40 protein spots corresponding to 15 differentially expressed between normal and cancer part of HCC patients. 10 proteins were up-regulated and five proteins down-regulated.
Figure 1: Two-dimensional polyacrylamide gel electrophoresis (2-DE) map of human liver proteins obtained from liver tissue. 2-DE was performed on an immobilized pH 3-10 strip, followed by the second-dimensional separation on 12.5% polyacrylamide gels. The separated proteins were stained with silver nitrate (a) 2-DE map of peritumorous non-neoplastic liver tissue of hepatocellular carcinoma (HCC) patient is shown and its protein spots are labeled. (b) The protein 2-DE map of tumorous tissue of HCC patient and its protein spots are labeled

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Verify the differentially expression of RKIP in HCC by Western blotting

We conducted Western blotting to analysis the RKIP protein differential expression in liver cancer. As shown in [Figure 2], RKIP protein expression in tumor part liver tissues of HCC patient number 21 and 42 is lower than peritumorous non-neoplastic liver tissue of the corresponding HCC samples. Western blotting further validated down-regulated expression of candidate protein RKIP in tumor tissues.
Figure 2: Verify the differentially expression in hepatocellular carcinoma by Western blotting. (a) Equal quantities of total protein extract from peritumorous non-neoplastic liver tissue and tumorous liver tissue of HCC patients were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter membranes. The membranes were probed with anti-Raf kinase inhibitor protein antibodies. Lanes represent the level of RKIP (23 KDa). N: peritumorous non-neoplastic liver tissue; T: tumorous liver tissue; 21, 42: patient number. (b) Lanes represent the level of β-actin (42 KDa). N: peritumorous non-neoplastic liver tissue; T: tumorous liver tissue; 21, 42: patient number. (c) Quantitation of RKIP expression. For quantification, the visualized films were recorded on a digital imaging system and analyzed in a densitometrical analysis system

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Validation the decreased expression of RKIP in HCC by RT-PCR

RT-PCR analysis was performed as shown in [Figure 3]. RKIP mRNA expression in tumor part liver tissues of HCC patient number 21 and 42 show distinctly lower than peritumorous non-neoplastic liver tissue. Therefore, RT-PCR result is consistent with the result from the Western blotting analysis RKIP mRNA expression was present in peritumorous non-neoplastic liver tissues in all the 42 HCC patients while it was significantly reduced in 30 (71%) of the corresponding HCC samples. These results strongly suggest RKIP down-regulated in liver cancer cell.
Figure 3: Agarose gel assessment of Raf kinase inhibitor protein gene expression from peritumorous non-neoplastic liver tissue and tumorous liver tissue of hepatocellular carcinoma patients. Agarose gel stained with ethidium bromide showing electrophoretic bands corresponding to amplification of RKIP (a) and β-actin (b). Lanes represent the level of RKIP (534 bp) and β-actin (600 bp). N: peritumorous non-neoplastic liver tissue; T: tumorous liver tissue; 21, 42: patient number; M: marker

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 » Discussion Top


We believe this is the first study to use 2-DE and MALDI-TOF MS observing RKIP alterations of two tissue types isolated from livers of HCC patients: Normal and tumorous tissue. We hypothesized that RKIP alteration may play a significant role in the molecular pathogensis of HCC.

Forty protein spots corresponding to fifteen differentially expressed between normal and cancer part of HCC patients. 10 proteins were up-regulated and five proteins down-regulated; they seem to play important roles in a variety of pathways including glycolysis, fatty acid transport, amino acid metabolism and stress. A remarkable finding is the down-regulation of RKIP protein in HCC. Raf pathway has been linked to carcinogenesis because they are involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation and differentiation. The identification of potential biomarkers such as of RKIP in our findings may provide further useful insights into the pathogenesis of HCC.

Biochemical techniques focused on changes in the proteome in benign and malignant conditions have been applied to the study of HCC. Although relatively little information is currently available about the human HCC proteome, the major goals of the analysis of HCC cancer are to better understand tumor biology, to define early detection biomarkers and predictors of tumor behavior and to identify potential new therapeutic targets.

The MAPK are important components of conserved signaling pathways controlling embryogenesis, cell differentiation, proliferation and death.[18],[19] The MAPK signaling cascades are activated by MAPK kinase (MAPKK), which is activated by a MAPKK kinase.[20]

A comparative proteomic approach has been used to identify and analyze proteins related to cancer. Proteome of clinical HCCs tissue samples and matched normal adjacent tissue samples were obtained by 2-DE. Two-dimensional electrophoretic technique that allows the investigator to survey 2,000 sports throughout the Proteome on one gel. Recent technological advancement in MALDI-TOF enables us to analyze biomolecules at a high sensitivity and efficiency. MALDI-TOF has been proved to be a versatile and effective method for the analysis of peptides and proteins.[21] The main advantages of MALDI include the determination of molecular weight of posttranslationally modified proteins, even mixtures of peptides and proteins can be subjected to this study and proteins with masses even greater than 100 kDa can be analyzed.[22] In addition, MALDI is now being widely used for peptide mapping and protein sequencing.[23] In this study, we chose MALDI-TOF because of its high sensitivity and its ability to process many samples quickly.

The alteration of some proteins had been studied in HCC tumors by 2-DE and MALDI-TOF MS.[9],[24],[25] However, there is no information on RKIP alteration in the HCC. RKIP revealed in the experiment can be used in the future for studies pertaining to hepatocarcinogenesis or as diagnostic markers and therapeutic targets for HCC.

The identified proteins, both up-regulated and down-regulated, may participate in the progression of malignant growth or in the maintenance of normal growth of liver cells. Of the 15 proteins that were selected, 10 proteins were up-regulated and five proteins down-regulated; they seem to play important roles in a variety of pathways including glycolysis, fatty acid transport, amino acid metabolism and stress. A remarkable finding is the down-regulation of RKIP protein in HCC. RKIP appears to be novel candidates for useful HCC markers. Raf pathway has been linked to carcinogenesis because they are involved in various cellular processes such as cell cycle regulation, apoptosis, proliferation and differentiation. Furthermore, RKIP may be considered to be potential biomarkers for HCC and provide further useful insights into the pathogenesis of HCC. Further, research on HCC may provide more insight with regard to its clinical stages and origin.

 
 » References Top

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