Subcellular proteomics: Determination of specific location and expression levels of lymphatic metastasis associated proteins in hepatocellular carcinoma by subcellular fractionation

Biomedicine & Pharmacotherapy 65 (2011) 407–416

Original article

Subcellular proteomics: Determination of specific location and expression levels of lymphatic metastasis associated proteins in hepatocellular carcinoma by subcellular fractionation Asma Saleem Qazi, Mingzhong Sun, Yuhong Huang, Yuanyi Wei, Jianwu Tang * Dalian Medical University, Pathology department, 9-western section, Lvshun south street, Dalian, P.R. P.C. 116044, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 1 April 2011 Accepted 23 April 2011 Available online 12 June 2011

Background: Subcellular fractionation and proteomics form an ideal partnership when it comes to specific location and analysis of intracellular organelles and expression levels of multiprotein complexes. Lymphatic metastasis is the major complicated system involving multiple factors. However, to date lymphatic metastatic mechanism is poorly understood. Aim: To specifically locate expression site by subcellular fractionation, based on expression levels, interpret the involvement of different lymphatic metastasis associated proteins in hepatocellular carcinoma cell lines with different lymphatic metastasis potential. Method: Mouse hepatocellular cell lines Hca-F and Hca-P are used to evaluate the location and expression levels of some lymphatic metastasis-associated proteins in the cell by using subcellular fractionation kit and Western blot analysis. The proteins under studies were Gelsolin, JNK and Annexin 7. Results: Gelsolin was sequestered in cytoplasm, membrane and cytoskeleton in F-cells but in P-cells, it was found in cytoplasm and cytoskeleton .JNK was located in nuclear fraction and cytoskeleton in F and P cells, Annexin7 was in cytoplasm with its two isoforms only at this location, cell membrane and cytoskeleton in F and P cells. With the high expression level of Gelsolin, JNK and Annexin 7 in Hca-F cell line than Hca-P cell line. Conclusion: With subcellular fractionation specific location of Gelsolin, JNK and Annexin 7 at various cell sites during lymphatic metastasis were determined. High expression levels were found in high lymphatic metastasis potential cell lines which indicate their roles according to different expression sites in the disease. ß 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Comparative proteomics Hepatocellular carcinoma Lymphatic metastatis Metastatic associated proteins Subcellular location

1. Introduction Present estimates of the number of genes in the human genome expressed in a particular cell type reach 10,000. However, the number of proteins in the entire human body is expected to be many times higher. Thousands of chemical modifications occur after proteins are created that alter their enzymatic activity, binding ability, how long they remain active, and so on [1]. These modifications and the still-underestimated rate of alternative splicing give rise to a human proteome size that is likely to be significantly larger than the number of estimated genes. Because of the limited resolution power of separation technologies presently applied in proteomics research, additional fractionation steps are required [2]. Therefore, proteomics research has become increasingly aware of techniques for analyzing subcellular proteomes of reduced complexity. Subcellular fractionation, allowing the

* Corresponding author. Tel.: +86 4 11 86 11 00 02; fax: +86 4 11 86 11 88 66. E-mail address: [email protected] (J. Tang). 0753-3322/$ – see front matter ß 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2011.04.028

separation of organelles based on their physical properties, was initially applied to separate organelles derived from rat liver [3]; one major limitation in the successful fractionation of cells is the production of an ideal homogenate, that is, the release of organelles and other cellular constituents as a free suspension of intact, individual components [4]. Very often, cytoplasmic aggregates or cytoskeletal elements with nucleus are observed, to ensure the reliable fractionation and to overcome these problems, we have used subcellular fractionation kit in this research. HCC is a phenotypically and genetically heterogeneous polyclonal disease and resistant to most conventional chemotherapy. Ninety percent of malignant tumors are carcinomas, and lymph nodes are often the first organ to develop metastasis [5]. HCC is one of the globe’s most common types of cancer and one of the most fatal [6]. At present, HCC is largely a Third World disease, especially Southeast Asia and Africa, China alone accounts for more than 50%of the world HCC cases [7]. Lymphatic metastasis is a complex process involving multiple genes and their products. However, molecular mechanism of metastasis remains poorly understood [8]. A mouse hepatocellular cell line Hca-F with high lymphogenous metastatic

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potential (above 70%), and its syngenetic cell line Hca-P with low lymphogenous metastatic potential (below 30%) have been successfully set up in our lab from hepatocarcinomas in mice [9,10]. Hca-F and Hca-P metastasize only to lymph nodes, and not to other organs, which have been proved to be the ideal models for studying lymphatic metastasis for hepatocarcinoma [11]. The proteome analysis at the level of subcellular structures (that can be enriched by subcellular fractionation) represents an analytical strategy that combines classic biochemical fractionation methods and tools for the comprehensive identification of proteins. Among the key potentials of this strategy is the capability to screen not only for previously unknown gene products but also to assign them, along with other known, but poorly characterized gene products, to particular subcellular structures. Furthermore, the analysis at the subcellular level is a prerequisite for the detection of important regulatory events such as protein translocation in comparative studies. Our group has engaged in the research for molecular mechanism of lymphatic metastasis of HCC during the past decade. We have already screened out the lymphatic metastasis-associated candidates and found higher expression of some genes and proteins including Gelsolin, JNK, Annexin 7 in Hca-F than in Hca-P by cDNA microarray as well as by 2 –DIGE, mass spectrometry and liquid chromatography [12] and constructed a subtracted cDNA library in these cell lines using subtractive suppressive hybridization [13]. We have compared Hca-F cells and Hca-P cells between the expression of different genes by gene chip assays [14] and obtained the lymphatic metastasis associated proteins by using quantitative proteomics techniques [15]. With these technologies, we found Gelsolin gene in Hca-F cells 1.9 times higher than in Hca-P cells and the difference of Gelsolin protein in Hca-F cells and Hca-P cells is 1.7 times [13–15]. At both protein and gene level of Gelsolin in Hca-F and Hca-P cell lines, the results showed higher expression of Gelsolin in Hca-F cell line in liver ascites and can promote tumor cell migration and invasion in lymph node metastasis of liver cancer (Wang Shaoqing; unpublished data). In fact, in vivo and in vitro studies showed that not only invasion, migration and proliferation rather regulation of apoptosis also has an important role in tumor metastasis. We also found that JNK1 expression was much higher in HcaF than in Hca-P cell lines at both gene and protein levels. JNK1 protein has the same origin with mouse and humans in structure and function. Therefore, it is important to study the relationship between JNK1 and lymphatic metastasis of mouse hepatocellular carcinoma. In vitro, by Transwell technique, our results showed that the ability of migration and invasion of HcaF cells decreased significantly after inhibition of JNK1 expression by RNA interference. This indicated that JNK1 could regulate behaviors of migration and invasion in lymphatic metastasis of HCC cells [16]. Our group findings for Annexin 7 showed its overexpression in Hca-F cells than that of Hca-P cells at both gene and protein level, indicating its role in the proliferation, inhibition of tumor cell apoptosis, enhanced tumor cell migration and invasion. (Wang Zhiqiang; unpublished data). Annexin 7 expression in cancer tissues was 53.3%, which was significantly higher than that 25.0% in normal samples while significantly lower than that 76.7% in lymphatic metastasis in primary gastric cancer tissues [17]. The repression of Annexin A7 inhibits the mobility and invasion abilities of Hca-F cell, increases the apoptosis rate of Hca-F cell [18]. In this study, we have focused on Gelsolin, JNK and Annexin-7 subcellular location, compared their expression levels in the cells statistically and by Western blot analysis. Based on this data, we tried to figure out their involvement in lymphatic metastasis and its progression.

2. Materials and methods 2.1. Animals and cell lines Inbred 615 mice were provided by the animal facility of Dalian Medical University. Mouse hepatocarcinoma cell lines Hca-F, F sh RNA (F cells transfected with shRNA targeting Annexin 7, down regulation), Hca-P and P c DNA3.1 (P cells transfected by P cDNA3.1-Annexin7, up regulation) were established and stored by our department. To establish a mouse hepatic cancer cell line Hca-F transfected with shRNA, three sh RNA were designed and inserted into the pSilencer vector to silence Annexin7 gene. The most effective p Silencer –shRNA vector was selected based on the result of RT-PCR and Western blot. The Hca-F cells were transfected with the most effective P Silencer –shRNA and transfectants were selected by 400 mg/ml G418 (Geneticin). To construct PcDNA 3.1 – Annexin 7and to transfect P-cells stably, Annexin 7 gene was amplified by PCR. Bam H1 and EcoR1 enzymes were used to digest the Annexin 7 gene and PcDNA3.1 plasmid. This plasmid was transfected in P cells stably. The effectiveness was checked by genome DNA checkup and Western blot analysis. 2.2. Cell culture Hca-F and Hca-P cells were injected at 2  106 tumor cells in 0.1 ml cell suspension into inbred Chinese 615 mice and were grown into mouse abdominal cavity for 7 days. These cells were drawn and injected again in other 615 mice and allowed to grow for 5 days. Two passages were done in order to harvest large number of cells in around 2 weeks. The cells in ascites were drawn and seeded into vials for culture in 90% RPMI 1640 medium supplemented with gentamicin/streptomycin 100 U/ml, 10% fetal bovine serum (Gibco.) for 24 h in a 5%CO2 atmosphere at 37 8C in a humidified atmosphere than regular passages were done in order to grow large amount of cells in vitro. Cell viability was determined by Trypan blue exclusion test. Hca-F and Hca-P cells were inoculated (2  106 cells/mouse) into the left foot pad of each mouse among 10 mice in a group of 20 inbred 615 mice. On the 28th day of postinoculation, the mice were euthanized and their lymph nodes were collected and stained by HE and examined under microscope. Therefore, the lymph node metastasis rate was calculated. 2.3. Subcellular fractionation Before fractionation, cell suspension was made so that each aliquot will have 3–5  106 cells/frozen cell pellet. S-PEK (cat. no. 539790) was used for the differential extraction of proteins from mammalian cells according to their subcellular localization. The extraction procedure provided four fractions with decreased proteome complexity. Cell suspensions were washed with wash buffer (kit component no. KP 31250) and centrifuged twice according to manual’s instruction. 2.4. Subcellular protein extraction from culture cell suspension Before the beginning of extraction procedure, all buffers were mixed well by vortexing and kept on ice during the whole procedure. Cell suspension was transferred to 4 ml centrifuged tube and pellet by centrifugation at 1500 rpm for 10 min at 4 8C. Supernatant was aspirated and discarded. Pellet was washed twice with 2 ml ice cold wash buffer, resuspended and incubated for 5 min at 4 8C with gentle agitation, centrifuged in cold at 4 8C at 1500 rpm for 10 min. 1 ml extraction buffer 1 was mixed with 5 ml protease inhibitor cocktail and added to pellet, incubated for10 min at 4 8C, centrifuged at 2900 rpm for 10 min in cold at

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4 8C. Supernatant was collected in separate tube (fraction 1). Extraction buffer 2 with 5 ml protease inhibitor cocktail was mixed with pellet, resuspended and incubated for 30 min at 4 8C and then centrifuged at 7840 rpm for 10 min at 4 8C. Supernatant (fraction 2) is collected separately. In the pellet 500 ml of extraction buffer 3 with 5 ml protease inhibitor cocktail and 1.5 ml Benzonase nuclease was mixed and incubated for 10 min at 4 8C. Mixture was centrifuged at 8720 rpm for 10 min at 4 8C, supernatant (fraction 3) was collected separately. 500 ml of extraction buffer 4 with 5 ml protease inhibitor cocktail was added to pellet. Residual particles were suspended by pipetting (fraction 4). 2.5. Determination of protein concentration The concentration of protein in each fraction was calculated by Bradford assay using bovine serum albumin as standard. Subsequent measurements were taken at 595 nm with a spectrophotometer. Standard curve were drawn (Fig. 1) which provided the relative measurement of protein concentration. The OD value was obtained by Bio-SenSC300 (ShanFu Bio-Tech Co. Ltd, Shanghai, P. R. China). 2.6. Western blot This analysis was carried to evaluate proteins level in each fraction. The extracted proteins were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis, blotted on polyvinylidene difluoride membranes (Invitrogen), applied with antibodies for Gelsolin (Epitomics, 1:1000), JNK (Bio World Technology, 1:400) and Annexin 7 (SIGMA ALDRICH, 1:1500) after extensive washings bands were detected by an ECL Western blotting kit (Beyotime ECL) and analyzed by (Auto Chemi system Bio Imaging UVP). For each fraction, loading control proteins used were: for fraction 1 GAPDH (KANG CHEN), for fraction 2 pancadherin (Abcam), for fraction 3 c-jun (Abcam) and for fraction 4 bactin (Santa Cruz).

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2.7. Statistical analysis The data obtained was analyzed statistically by SPSS. Version 13.0 for Windows1 (Snow Panther, SPSS Inc, IL, USA) using independent t-test, paired t-test and one-way ANOVA at (P < 0.05). Variables were expressed as Mean  S.D. 3. Results 3.1. Analysis of metastatic rate of Hca-F and Hca-P cells The implanted tumors both in Hca-F and in Hca-P tumorbearing mice were palpable on the 7th day postinoculation. The rate of tumor formation in both groups was 100%. On the 28th day postinoculation, eight out of 10 mice in case of F cells and two out of 10 in case of P cells developed lymph node metastasis indicating the metastasis rate as 80% in F-cells and 20% in P-cells. 3.2. Extraction of subcellular fractions The extraction procedure using S-PEK provided four fractions of cell material. Extraction buffer I released cytosolic proteins (fraction 1). Subsequently, membranes and membrane organelles were solubilized by Extraction buffer II with nucleus and cytoskeleton integrity (fraction 2). Nuclear proteins were extracted with Extraction buffer III (fraction 3). Components of the cytoskeleton were finally separated with Extraction buffer IV (fraction 4). 3.3. Gelsolin expression at protein level The relative protein expression level in Hca-F cells showed higher expression of Gelsolin in cytosol, membrane fraction and cytoskeleton (Fig. 2) which were 84%, 19% and 69% compared with Hca- P cells for cytosol and cytoskeleton which were 76% and 36%

Fig. 1. The standard curve of concentration of protein samples. (a) The optical density curve of F-cell and determination of protein concentration using linear equation and its coefficient. This graph was drawn using MS Excel1 2007 software. The graph was plotted against O.D. (x-axis) and volume of protein sample (y-axis); (b) The optical density curve of P-cells for determination of protein concentration using coefficient of linear equation. This graph was drawn using MS Excel1 2007 software. The graph was plotted against O.D. (x-axis) and volume of protein sample (y-axis); (c) The optical density curve of F shRNA-cells and determination of protein concentration using linear equation and its coefficient. This graph was drawn using MS Excel1 2007 software. The graph was plotted against O.D. (x-axis) and volume of protein sample (y-axis); (d) The optical density curve of P cDNA3.1-cells and determination of protein. Concentration using linear equation and its coefficient. This graph was drawn using MS Excel1 2007 software. Graph was plotted against O.D. (x-axis) and volume of protein sample (y-axis).

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respectively (Table 1). Yet in P cells, expression was not detected on membranes. The values were calculated by taking mean three times of six values for each fraction and analyzed at P < 0.05. Statistically, expression levels of both cell lines and among different fractions of cells for each cell line were compared. When values were compared among different cell lines, they were found as 0.84  0.05, 0.69  0.061 for F-cells and 0.76  0.05, 0.36  0.049 for P-cells for fraction 1 and 4, respectively. Fraction 2 was not compared between two cell lines as Gelsolin expression was not found in P-cells. When the comparison was made among the four fractions for each cell line, then the values were 0.80  0.063,0.19  0.047 and 0.53  0.018 for fraction 1, fraction 2 and fraction 4, respectively. Gelsolin expression was found highest in fraction 1 among all the fractions expressed and expression was higher in Hca-F than in Hca-P cell line. It was found at three locations in a cell although fraction 2 has shown slight expression only in Hca-F (Table 2). 3.4. JNK expression at protein level Expression levels of JNK in Hca-F cells were 31% and 86% and in Hca-P cells were 22% and 74% in nuclear material and cytoskeleton, respectively (Table 1). Hca-F cells showed slightly higher expression than Hca-P cells (Fig. 3). Values were calculated by taking mean three times of six values and were analyzed at P < 0.05 between the Hca-F and Hca-P cell lines and among the fractions of cells.

Statistical values calculated were 0.31  0.05 and 0.86  0.073 for F-cells and 0.22  0.027 and 0.74  0.069 for P cells in fraction 3 and 4, respectively. Among the fractions of each cell line the values were 0.28  0.017and 0.79  0.026 for fraction 3 and fraction 4, respectively. JNK expression was higher in fraction 4 than in fraction 3 among the fractions and Hca-F than in Hca-P among the cell lines. JNK expression was found at two subcellular locations which are cytoskeleton and nucleus (Table 2). Statistical analysis has indicated that the expression level of JNK was higher in Hca-F than in Hca-P. 3.5. Annexin 7 expression at protein level Annexin 7 was detected in cytosol, on membranes and cytoskeleton. The expression level was highest in Hca-F cells and lowest in Hca-P cells among all four cell lines analyzed (Figs. 4 and 5). These were Hca-F, F shRNA, Hca-P and P cDNA 3.1. Expression levels were 73%, 62.5%, 48% and 59.8% (47 KDa) and 9.9%, 4%, 2% and 2.86% (51 KDa) for two isoforms in fraction 1, respectively. Fraction 2 has 36%, 28%, 22% and 25% whereas fraction 4 has 53%, 48%, 39% and 43%, respectively (Table 1) . The statistical comparison was made at P < 0.05 between the four cell lines analyzed for Annexin 7 and among the four fractions of cells. The values were 0.099  0.015, 0.02  0.005, 0.04  0.009 and

Fig. 2. (a) Gelsolin expression in cytosolic, membraneous and cytoskeletal fractions 1,2 and 4 respectively of F-cells; (b) Gelsolin expression in cytosolic, and cytoskeletal fractions 1 and 4, respectively of P-cells; (c) GAPDH internal standard for fraction 1; (d) pan-cadherin internal standard for fraction 2; (e) c-jun internal standard for fraction 3; (f) b-actin internal standard for fraction 4.

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Table 1 Expression levels of lymphatic metastasis associated proteins in each subcellular fraction. Proteins and cell lines

Fraction 1

Fraction 2

Fraction 3

Fraction 4

Gelsolin in Hca-F Gelsolin in Hca-P JNK in Hca-F JNK in Hca-P

84  5 76  5 – –

19  4.7 – – –

– – 31  5 22  2.7

69  6.1 36  4.9 86  7.3 74  6.9

Annexin7 in Hca-F 51 KDa 47 KDa

9.9  1.5 73  6.4

36  4.9

–

53  5.5

Annexin7 in Hca-P 51 KDa 47 KDa

2  0.5 48  3.5

22  1.8

–

39  2.2

Annexin7 in F sh RNA 51 KDa 47 KDa

4  0.9 62  5.5

28  3.3

–

48  4.2

Annexin7 in PcDNA 3.1 51 KDa 47 KDa

2.8  0.6 59  4.9

25  2.4

–

43  3.7

Formulae of cell expression level (%): % of mean O.D. of six values of each fraction in each cell line analyzed/mean O.D. of internal standard for each fraction. –: no band detection in these fractions; all values are presented as % mean  S.D.; fraction 1: cytoplasm, fraction 2: membranes, fraction 3: nuclear material, fraction 4: cytoskeleton.

Table 2 Location of proteins. Location of GELSOLIN

JNK

Annexin 7

In

F

P

F

P

F

P

F -shRNA

P-cDNA3.1

Cytoplasm Membranes Nuclear material Cytoskeleton

++++ + – +++

+++ – – ++

– – ++ ++++

– – + +++

+++ ++ – +++

++ + – ++

+++ ++ – ++

++ ++ – ++

++++: very high expression (80% and above);+++: high expression (50–80%); ++: moderate expression (30–50%); +: slight expression (30%and below); –: no expression.

0.028  0.006 for 51 KDa isoform and 0.73  0.064, 0.48  0.035, 0.62  0.055 and 0.59  0.049 for 47 KDa isoform in fraction 1 of Fcells, P-cells, F shRNA and P cDNA 3.1, respectively. 0.36  0.049, 0.22  0.018, 0.28  0.033 and 0.25  0.024 in fraction 2 and 0.53  0.055, 0.39  0.022, 0.48  0.042 and 0.43  0.037 for fraction 4 in F-cells, P-cells, F shRNA and P cDNA 3.1 respectively. When the data was compared among fractions of each cell line, the values were 0.60  0.015 for fraction 1,0.28  0.08 for fraction 2 and 0.45  0.09 for fraction 4. Among both isoforms, expressions were in fraction 1 0.31  0.051 for 51 KDa and 0.76  0.06 for 47 KDa. Statistical comparison indicated that expression of annexin 7 was higher in HCa-F than In F shRNA (annexin 7 expression was downregulated). When HCa-P and P cDNA 3.1 (annexin 7 expression was upregulated) were compared results have shown less expression in HCa-P. Among fractions fraction 1 has highest expression in all cell lines with two bands of different molecular weight (51 and 47 KDa) at P < 0.05, expression of 47 KDa band was found higher in all cell lines (Table 2). 3.6. Same expression sites Gelsolin and annexin 7 was found expressed almost at the same locations although the expression levels were different. In order to see their role and dependency, annexin 7 in F-cell lines was downregulated and Gelsolin expression level was checked. It was noted that increasing amount of protein will increase the expression level till 40–50 mg but diminished above 70 mg. similarly analysis was done also by up regulating annexin 7 expression in P cell lines protein expression was prominent till 20 mg but increasing beyond 35 mg, it was diminished. It is evident from this study that the two-tumor metastasis related protein

might have some relation till certain amount of protein concentration. JNK and annexin 7 almost have same molecular weight but their sites of expression are different. Although both are expressed at cytoskeleton in the up- and downregulated cell lines for annexin 7, JNK expression was also checked. It was noted that till 15–30 mg expression was higher but in the same cell lines, it was diminished after increasing amount of protein above 40 mg. This statistical comparison was also made between Gelsolin, JNK and Annexin 7, analyzed during this study using one way ANOVA at P < 0.05. The values (not shown in this data) were found not related to each other. 4. Discussion Metastasis is a complex series of steps in which cancer cells leave the original tumor site and migrate to other parts of the body via the bloodstream or the lymphatic system. A wide range of metastasis leads to the failure of treatment and the death of patients. Therefore, investigation of cancer metastasis, particularly the mechanisms, is important to improve the efficacy of the treatment [8]. Cancer cell expression is regulated by interactions of tumor cells with host microenvironment, both in primary and secondary lesions whether the specific environment of the lymph nodes influenced anchorage of carcinoma cells, remains unclear [19]. In this study, between two cell lines Gelsolin is expressed more in high metastatic potential cell line indicating its involvement in tumor metastasis progression. However, on membranes it is expressed in high potential cell line only, it might indicate that at later stages of disease, it is expressed at membranes and it affects cell migration and motility which can make important contribution to metastatic cascade.

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Fig. 3. (a) JNK expression in nuclear and cytoskeletal fractions 3 and 4, respectively of F-cells; (b) JNK expression in nuclear and cytoskeletal fractions 3 and 4, respectively of Pcells; (c) GAPDH internal standard for fraction 1; (d) pan-cadherin internal standard for fraction 2; (e) c-jun internal standard for fraction 3; (f) b-actin internal standard for fraction 4.

There are some evidences based on proteomic comparison about diminished expression of Gelsolin through a transcriptionindependent ubiquitination-dependent mechanism [20]. Gelsolin is secretory in nature, it is also evident that protein binding activate receptors which can bind with a chemokine or growth factor and leads to dynamic morphological changes in cytoskeleton via a variety of signal transduction pathways [21] but these mechanisms are only partly understood so far. Gelsolin is one of the most important cytoskeletal actin structure-regulating proteins, and its expression in almost all eukaryotic cells is testimony to its fundamental importance in maintaining an organized actin cytoskeleton [22] in a calcium-dependent manner. Properties of the cytoskeleton depend on filament length, flexibility, concentration and presence of cross-links. Proteins with the capacity to alter such properties are potentially important for regulating cellular morphology and function [22,23]. The physiological functions of the 90 KDa Gelsolin protein are regulated by the intracellular Ca2+ concentration and by its binding to diphosphoinositides, both of which serve to activate its actinsevering potential, capping the barbed ends, and promoting nucleation of polymerization [24]. It can be speculated with our studies that cytoplasmic Gelsolin has some role in cytoskeletal

reorganization. Importantly, later in the transformation sequence, Gelsolin expression increases, as suggested that high lymph node metastatic cancers react stronger toward anti-Gelsolin antibodies as compared with low lymph node metastatic cancers. This result is consistent with an earlier study of Thomson et al. [25], which already reported increased Gelsolin expression in lymph node positive cancers and would suggest that the expression of the Gelsolin-specific ubiquitin ligase active in the earlier phases of the transformation process, is lost at later stages. Nevertheless, the mechanisms mediating Gelsolin expression in different cancer cells are not very clear. Our findings about Gelsolin expression level are consistent with the previous observation that the overexpression of Gelsolin in primary and metastatic oral squamous cell carcinoma is often associated with a poor prognosis [26]. The over-expression of Gelsolin has also been reported in a series of human cancers. For example, higher levels of Gelsolin were significantly associated with poor survival or an increased risk of death in patients with lung cancer [27,28]. There are strong evidence that Gelsolin plays an important role in cellular proliferation and migration in cervical cancer and suggest that Gelsolin is a promising marker for cervical cancer screening and prognosis [29].

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Fig. 4. (a) Annexin 7 expression in cytosolic, membranous and cytoskeletal fractions 1,2 and 4, respectively of F-cells with its two isoforms 51 and 47 KDa; (b) Annexin 7 expression in cytosolic, membranous and cytoskeletal fractions 1,2 and 4 respectively of P-cells with its two isoforms 51 and 47 KDa; (c) GAPDH internal standard for fraction 1; (d) pan-cadherin internal standard for fraction 2; (e) c-jun internal standard for fraction 3; (f) b-actin internal standard for fraction 4.

In contrast, several laboratories have reported that low expression of Gelsolin protein has been detected in astrocytoma [30] in papillary renal cell carcinoma [31] in colorectal cancer [32] and gastric cancer [33]. JNKs (46 KDa) are important regulators of cell proliferation and apoptosis. JNKs have been proposed to function both as tumor suppressors and mediators of cancer cell proliferation depending on cell-type, stimulus, temporal aspects and context of other signaling pathways [34,35]. JNK is an important member of the MAPK superfamily. In this study, we focused on JNK’s subcellular location, its level of expression at that particular location and its role in lymphatic metastasis. Our results showed that the cellular location of JNK’s are nuclear material and cell cytoskeleton with higher level of expression in high metastatic potential cell line. Higher expression in Hca-F indicates that it might play a role in tumor progression or development as the metastatic potential of this cell line is quiet high . The possible evidences of its location in nucleus can be that JNK’s are involved in MAPK tyrosine and threonine phosphorylation [36] which potentiates c-Jun transcriptional activity [37]. In addition, JNK substrates have been identified in other cellular compartments so that the actions of JNK extend beyond transcriptional events. These nonnuclear JNK substrates include the microtubule-associated protein tau [38]. With this study, we got evidences of JNK expression in cytoskeleton where the expression

level is very high in both the cell lines used but still slightly higher in Hca-F cell line. JNK has been shown to be constitutively activated in many different types of tumor cell lines [39] and central to the apoptotic process in numerous cell types [40,41]. Some studies have demonstrated that activation of JNK could promote genesis of hepatic carcinoma by enhancing proliferation of hepatic cells [42]. It has been reported that inhibition of JNK1 could inhibit lymphatic metastasis in pancreatic carcinoma cell lines with ability of lymphatic metastasis by reducing lymphangiogenesis and expression of VEGF-C [43]. Another study showed that the JNK1 pathway was found to be significantly elevated in metastatic HCC cell lines occurring in portal vein when compared with primary HCC cell lines [44] JNK1 can regulate cellular migration because it is located in FAP which is an important signal transduction pathway. In focal adhesion plaque, the direct substrate of JNK1 is c-Jun. The activation of JNK1 can increase transcription of c-Jun and as a result, the transcription of activated protein 1 is increased [45] which plays an important role in the process of tumor metastasis including cellular motion of tumor, extracellular matrix degradation, abnormal adhesion and growth of vessels in metastasis [46]; these roles of JNK might support their expression at cytoskeleton as they are important in the maintenance of cellular structures. Some of these previous findings serve as a proof that JNK’s are involved in tumor progression, migration and development which support our finding for HCC.

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Fig. 5. (a) Annexin 7 expression in cytosolic, membranous and cytoskeletal fractions 1,2 and 4, respectively of F-shRNA cells with its two isoforms of protein 51 and 47 KDa; (b) Annexin 7 expression in cytosolic, membranous and cytoskeletal fractions 1,2 and 4, respectively of PcDNA 3.1 cells with its two isoform of protein 51 and 47 KDa; (c) GAPDH internal standard for fraction 1; (d) pan-cadherin internal standard for fraction 2; (e) c-jun internal standard for fraction 3; (f) b-actin internal standard for fraction 4.

The annexins are a multigene family of calcium dependent phospholipid binding proteins. The different members of this family shows a specific cell and tissue type pattern of expression. Functions of the annexins include vesicle aggregation, ion channel regulation as well as roles in cell cycle regulation, cell signaling, cell differentiation and as extracellular receptors. Several of the annexins have been linked to the development and progression of several different types of cancer, there appears to be tumor-type-specific alterations in the expression of individual annexins [47]. This study is at cellular level and Annexin 7 is sequestered in cytoplasm, membranes, membrane bound organelles and cytoskeleton. Both the isoforms of this protein are expressed only in cytoplasm in HCC cell lines. This indicates that both forms of Annexin 7 (47 and 51 KDa) is present in liver cancer cells. There are evidences of the expression of both the forms in red blood cells [44], brain and heart cells as well [48]. Absence of 51 KDa isoform in membranes and cytoskeleton may linked to specific pattern of expression. There are evidences of expression of 51 KDa isoform only in skeletal muscle cells [44] yet 47 KDa isoform is mostly and usually expressed out of the two isoforms. The 47 KDa isoform has been identified and proposed to be a key component in the process of the Ca2+-dependent vesicle release, a process with which cells might protect themselves against an attack for example of complement components [49].

Some annexins show increased expression in specific types of tumors, while others show loss of expression. Mechanistic studies relating the changes in annexin expression to tumor cell function, particularly tumor invasion and metastasis, angiogenesis and drug resistance, are now emerging, hence the annexins may also be useful biomarkers in the clinic [50]. Our data also indicates different expression pattern of Annexin 7 depending on their specific location in the cell. In the cell line with high metastatic potential, the expression was highest among all cell lines analyzed. We have further investigated F cells by transfecting shRNA which targets Annexin 7 expression i.e., by downregulating the expression of Annexin 7 in high metastatic potential cell line and it was observed that expression was decreased as compared to Hca-F cell indicating that Annexin 7 is involved in metastasis as downregulation causes the expression to decrease. For further comparison, P cells were transfected with P cDNA 3.1 to increase the expression of Annexin 7 in low metastatic potential cell line. In the Annexin 7 transfected P cell line, the expression was higher than the P-cell line so upregulation increases the expression. This indicates the involvement of Annexin 7 in tumor metastasis development and progression in HCC. It was observed in this study that Annexin 7 was expressed in cytoskeleton. The possible explanation for this might be that the

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vesicle formation goes along with several other changes in the cell like cytoskeletal rearrangements and changes in the phospholipid orientation in the cellular membrane. So one of the functions seems to be the rearrangement of cell cytoskeleton and its orientation as Annexin 7 has so many functions it might be possible that with these arrangements it is involved in motility or some signal pathways. Furthermore, it is present in the cytoplasm where it is found on subcellular vesicle-like structures. This distribution appears to be independent of the tissue or cell type analyzed [51]. Annexins have been reported to bind directly to Factin, it might have a role in organizing the membrane cytoskeleton, controlling raft protein associations or influencing the ionic strength of cells on its own or by interfering with other signaling pathways. In this study, Annexin 7 expression was found on membranes, it is a Ca2+– and phospholipid-binding protein which is thought to be involved in membrane fusion processes and that Annexin A7 translocates to membranes in a Ca2+-dependent fashion and, when intracellular Ca2+ levels rise, sequentially redistributes to the plasma membrane as well as to intracellular vesicles [52]. The possible speculation for Annexin 7 in cell cytoplasm that it could be involved in the cytoplasmic Ca2+ homeostasis. It has been demonstrated that micromolar changes of the intracellular Ca2+ concentration exert a profound effect on the membrane properties that regulate cell deformability. Furthermore, the intracellular Ca2+ was shown to regulate the membrane stability through modulation of cytoskeletal protein interactions [52,53]. The expression and cellular localization of annexins have not received much previous study in cancer, with this investigation we tried to contribute a little in this field of research. The statistical comparison was done in three different ways: between cell lines with different metastatic potential for each protein under study, between different fractions of cells for each cell line and each protein under study, among the different proteins and their expression levels in each cell line. Statistical comparative analysis of our data is in support of high expression level of these proteins in HCC cell lines although the three proteins are involved in tumor metastasis yet they are independent of each other. The step from the analysis of the genome to the analysis of the proteome is not just a matter of numerical complexity in terms of variants of gene products that can arise from a single gene. A significant further level of complexity is introduced by the supramolecular organization of gene products because of protein–protein interactions or targeting of proteins to specific subcellular structures. The targeting of proteins to particular subcellular sites is an important principle of the functional organization of cells at the molecular level. In turn, knowledge about the subcellular localization of a protein is a characteristic that may provide a hint as to the function of the protein. There is currently no single proteome analysis strategy that can sufficiently address all levels of the organization of the proteome. This study provides the data of protein expression levels of tumor biomarkers and their respective subcellular location in the cells. Expression levels are compared using cell lines with high and low metastatic potential and with downregulation of high metastatic potential and upregulation of low metastatic potential. The difference in their metastasis potential is usually based on the difference in their phenotypes of gene expression and based on their location of expression. Still these procedures are at the nascent stage of understanding. Nonetheless, we anticipate further work to elucidate the tumor-specific action of these biomarkers that will provide useful tools for diagnosis, prognosis and therapy for cancer metastasis. The abundant proteomic information about proteins in the aimed tissues or cells of various tumors could help us find the key proteins related to cancer metastasis. Furthermore, it is useful to discover the cancer-specific protein markers for

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developing the new schemes for early prediction and diagnosis, even to explore the new drug targets for cancer therapy. 5. Conclusion Subcellular fractionation and purification of organelles had always been a challenge for cell biologists. Subcellular fractionation allows access to intracellular organelles and multiprotein complexes. Low abundant proteins and signaling complexes can be enriched, and at the same time complexity of the sample can be reduced. Analyzing subcellular fractions and organelles allows also tracking proteins that shuttle between different compartments. This is for the first time that we have observed one tumor metastasis related protein expression at different site within cell which is ensured by subcellular fractionation revealing different sites and expression levels will serve to find therapeutic targets. Annexin 7 shows high level of expression in high metastatic potential cell line. Downregulating its expression showed decreased expression. This shows its positive correlation in liver cancer. For further confirmation upregulation in low metastatic potential cell lines showed its increased level of expression. It has two isoforms when expressed at cytoplasm with higher level of 47 KDa expression. The expression was found at three different sites. Gelsolin and JNK were also found with high expression levels in high metastatic potential indicating positive correlation in progression of liver cancer. Expression at different subcellular location might depend upon their specific functions, type and stage of cancer. Increasing amount of protein above 70 mg will diminish the expression levels. Gelsolin and annexin share the same sites of expression. However, downregulation of annexin 7 caused an increase in gelsolin level within the limits 20–60 mg of protein, especially at membranes. Both might be serving as shuttling proteins during liver cancer. Importantly, subcellular fractionation is a flexible and adjustable approach that may be efficiently combined with other techniques to reveal hidden facts of the mysterious disease. Specific recognition of certain events such as those involved in protein sorting, vesicle targeting are required. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported by Grants from the National Natural Science Foundation of China [No. 30772468 and No. 81071725]; and the Educational Department of Liaoning Province [Nos. 2008225010-3 and 2007T024]. This work is also supported by grants from Liaoning Province (2009S028). We would like to thank Prof. Liu Qigui for helping in statistical interpretations, Mao Jun and Dr Qazi Basit for arranging materials and animal Center of Dalian medical university. References [1] Harrison PM, Kumar A, Lang N, Snyder M, Gerstein M. A question of size: the eukaryotic proteome and the problems in defining it. Nucleic Acids Res 2002; 30:1083–90. [2] Klose J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues: a novel approach to testing for induced point mutations in mammals. Humangenetik 1975;26:231–43. [3] Fleischer S, Kervina M. Subcellular fractionation of rat liver. Methods Enzymol 1974;31:6–41. [4] Gruenberg J, Howell KE. Fusion in the endocytic pathway reconstituted in a cell-free system using immuno-isolated fractions. Prog Clin Biol Res 1988;270: 317–31. [5] Sleeman JP. The lymph node as a bridgehead in the metastatic dissemination of tumors. Cancer Res 2000;157:55–81.

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