- Email: [email protected]
Ech1 is a potent suppressor of lymphatic metastasis in hepatocarcinoma
Biomedicine & Pharmacotherapy 67 (2013) 557–560
Available online at
www.sciencedirect.com
Original article
Ech1 is a potent suppressor of lymphatic metastasis in hepatocarcinoma Jun Zhang a, Mingzhong Sun b, Rongkuan Li c, Shuqing Liu d, Jun Mao a, Yuhong Huang a, Bo Wang a, Li Hou a, Mohammed Mohammed Ibrahim a, Jianwu Tang a,* a Department of Pathology, Dalian Medical University, Key Laboratory of Tumor Metastasis of Liaoning Province University, 9, West Lvshun Southern Road, Dalian, 116044 Liaoning, China b Department of Biotechnology, Dalian Medical University, 116044 Dalian, China c Department of Gastroenterology and Hepatology, the Second Affiliated Hospital of Dalian Medical University, 116044 Dalian, China d Department of Biochemistry and Molecular Biology, Dalian Medical University, 116044 Dalian, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 February 2013 Accepted 10 March 2013
We have previously demonstrated that Ech1 is involved in the lymphatic metastasis of tumors in vitro. Here, we gain an insight into the role that Ech1 is playing in Hca-F cell. The expression of Annexin A7, Gelsolin and Clic1 genes, which were also relevant to tumor lymphatic metastasis, had been inhibited due to downregulation Ech1 gene by Western blot analysis. And downregulated of Ech1 inhibits the metastasic capability of Hca-F cells to peripheral lymph nodes in vivo. Our work indicates although the involvement of Ech1 in tumor metastasis development and progression, but the subcellular location of Ech1 has not much contribution to that. ß 2013 Published by Elsevier Masson SAS.
Keywords: Enoyl coenzyme A hydratase 1 (Ech1) Lymphatic metastasis Subcellular location Hepatocarcinoma
1. Introduction Metastasis has been regarded as a hallmark of malignancy that distinguishes invasive tumors from benign ones and constitutes a major cause of cancer mortality, and lymph node metastasis (LNM) is recognized as an important factor involved in the tumor malignancy progression. The mechanisms of tumor-associated lymphatic metastasis have been extensively studied recently [1– 3]. Enoyl coenzyme A hydratase 1 (Ech1) is the second enzyme in the pathway of fatty acid degradation via b-oxidation. A variety of studies have demonstrated that Ech1 may be associated with tumor progression [4–7]. Hca-F, a mouse hepatocarcinoma cell line with a high rate of lymphatic metastasis (> 75%), has proved to be an ideal model for studying the process of lymphatic metastasis [8–12]. We used gene chip and proteomics technique found that Ech1, Annexin, Clic1, Gelsolin were upregulated in the Hca-F cell [6–12], and then downregulated expression of Ech1 in Hca-F cell, which could inhibited proliferation of the Hca-F cells, increased the ratio of HcaF cells in S phase to G1 phase and decreased the adhesion and migration capacities of Hca-F cells [13]. Our current work is to gain an insight into the role that Ech1 is playing in Hca-F cell:
* Corresponding author. Tel.: +86 411 86110298; fax: ++86 411 86118866. E-mail address: [email protected] (J. Tang). 0753-3322/$ – see front matter ß 2013 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.biopha.2013.03.018
we got the Ech1 downregulated cell, and observe the expression of Annexin, Clic1, Gelsolin; we put Ech1 downregulated cell to inject into the mice with observation of the subsequent effects; we observe subcellular location of Ech1.
2. Materials and methods 2.1. Cell line culture and animals The mouse hepatocarcinoma cell line Hca-F, with an LNM rate over 75%, and Ech1 stably downregulation of Hca-F cell (FEch1 downregulated cell) was established and maintained in our laboratory. Inbred Chinese 615-mice (aged 6–8 weeks, weighing 18–22 g) were provided by the Experimental Animal House of our university. The Hca-F cell cultured in 90% RPMI 1640 supplemented with penicillin/streptomycin and 10% fetal bovine serum (Gibco, USA), and FEch1 downregulated cell cultured in 90% RPMI 1640 supplemented with penicillin/streptomycin 10% fetal bovine serum and neomycin (400 mg/mL) at 37 8C with 5% CO2. 2.2. Tumorigenicity assay in 615-mice Twenty inbred 615-mice were equally divided into two groups. Hca-F and FEch1 downregulated Cell (1 106) were inoculated into the feet pad of 615-mice subcutaneously. On the 28th day of postinoculation, the mice were euthanized and their lymph nodes
J. Zhang et al. / Biomedicine & Pharmacotherapy 67 (2013) 557–560
558
were isolated, weighed, sectioned, and stained with hematoxylin and eosin. The primary tumor, popliteal, inguinal and iliac lymph node were removed, weighted, and fixed in neutral-buffer formalin and embedded in paraffin, sections, then cut and stained with hematoxylin-eosin. The existence of metastatic foci was examined under microscope in sections. Anyone of popliteal lymph node, iliac lymph node, inguinal lymph node that had metastatic foci (secondary tumors) microscopically was considered as metastases. Then lymphatic metastasis rates were calculated.
separately. Then, 500 uL extraction buffer 4 with 5 uL protease inhibitor cocktail was added to pellet. Residual particles were suspended by pipetting (fraction IV). Different fraction we choose different internal control, we use GAPDH (Kangcheng; 1:7500) as cytosolic proteins internal control, ATP (Proteintech; 1:1200) as membranes and membrane organelles proteins internal control, c-Jun (Abcam; 1:1000) as nuclear protein internal control, and vimentin (Sigma; 1:2000) as cytoskeleton proteins internal control.
2.3. Western blot (WB) analysis
3. Results
Equal amounts of protein from Hca-F and FEch1 downregulated cell were separated for different expression using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). These were incubated with a monoclonal antibody against Annexin A7 (sigma; 1:15,000), Clic1 (Santa Cruz; 1:200), and Gesolin (BD Biosciences; 1:1000) in 5% dried milk for 1 h, then incubated with horseradish peroxidase-linked antibodies (1:5000 dilution) for 1 h at room temperature. Enhanced chemiluminescence (ECL; GE Healthcare, USA) was performed to visualize the protein bands.
3.1. Expression of Annexin A7 is upregulated in the FEch1 cell line
2.4. Subcellular protein extraction from culture cell suspension According to ProteoExtract Subcellular Proteome Extraction Kit (Merk), Cell suspension was made so that each aliquot will have 4 106 cells/cell pellet, all buffers were mixed well by vortexing and kept on ice during the whole procedure. Cell suspension was transferred to centrifuged tube and pellet by centrifugation at 200 g 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 200 g for 10 min, supernatant was aspirated and discarded. One milliliter extraction buffer mixed with 5 uL protease inhibitor cocktail and added to pellet, incubated for 10 min at 4 8C with gentle agitation, centrifuged at 1000 g for 10 min at 4 8C. Supernatant was collected in separate tube (fraction I). Extraction buffer 2 with 5 uL protease inhibitor cocktail was mixed with pellet, resuspended and incubated for 30 min at 4 8C, and centrifuged at 5500 g for 10 min at 4 8C. Suspernant (fraction II) is collected separately. In the pellet 500 uL Extraction buffer 3 with 5 uL inhibitor cocktail and 1.5 uL Benzonase nuclease was mixed and incubated for 10 min at 4 8C, mixture was centrifuged at 6800 g for 10 min at 4 8C, supernatant (fraction III) was collected
downregulated
WB results indicated Annexin A7 in FEch1 downregulated cell upregulated 11% compared to Hca-F cell (Fig. 1). 3.1.1. Expression of Clic1 is upregulated in the FEch1 downregulated cell line WB results indicated Clic1 in FEch1 downregulated cell is upregulated 69% compared to Hca-F cell (Fig. 1). 3.1.2. Expression of Gelsolin is upregulated in the FEch1 downregulated cell line WB results indicated Gelsolin in FEch1 downregulated cell is upregulated 13% compared to Hca-F cell (Fig. 1). 3.2. Downregulated of Ech1 inhibits the metastasic capability of Hca-F cells to peripheral lymph nodes in vivo To evaluate whether downregulated of Ech1 expression could inhibit the metastatic capability of Hca-F cell to peripheral lymph nodes in vivo, Hca-F and FEch1 downregulated cell were injected into footpad of 615-mice, respectively. After cells inoculation in mice for 28 days, a significant reduction of tumor lymph node metastasis was observed. Overall, there were eight out of 10 (8/ 10) mice transplanted with Hca-F cells show metastasis to lymph nodes, while in the other group, significantly fewer mice has lymph node metastasis (4/9,) transplanted with FEch1 downregulated cell (Fig. 2). 3.3. Ech1 expression at protein level of subcellular fraction The extraction procedure provided four fractions of cell material. Fraction I extraction of proteins is from cytosolic proteins,
Fig. 1. Western blotting analysis of expressions of Annexin A7, Clic1, Gelsolin in normal Hca-F and FEch1
downregulated
cell.
J. Zhang et al. / Biomedicine & Pharmacotherapy 67 (2013) 557–560
559
Fig. 2. Primary tumor and regional LNM of implanted tumor of Hca-F and FEch1 downregulated cell. A. Primary tumor of Hca-F. B. Regional LNM of implanted tumor of Hca-F. C. Primary tumor of FEch1 downregulated. D. Regional LNM of implanted tumor of FEch1 downregulated.
fraction II is from membranes and membrane organelles, fraction III is from nuclear protein, fraction IV is from components of cytoskeleton. Ech1 was found expressed almost at the same location although the expression level one is normal and the other is downregulation. Ech1 expression was found in cytosol, membrane fraction, and Ech1 expression was higher in cytosol than other fractions both in Hca-F cell and Ech1 downregulated Hca-F cell (Fig. 3).
Fig. 3. Ech1 expression in subcellular protein extraction from cells. For all blots, lane 1: cytoplasmic protein of Hca-F; lane 2: membrane/organelle proteins of Hca-F; lane 3: nuclear proteins of Hca-F; lane 4: cytoskeletal proteins of Hca-F; lane 5: cytoplasmic protein of FEch1 downregulated; lane 6: membrane/organelle proteins of FEch1 downregulated; lane 7: nuclear proteins of FEch1 downregulated; lane 8: cytoskeletal proteins of FEch1 downregulated, Proteins were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies to the indicated markers. GAPDH as cytosolic proteins internal control, ATP as membranes and membrane organelles proteins internal control, c-Jun as nuclear protein internal control, and vimentin as cytoskeleton proteins internal control.
4. Discussion Tumor metastasis is the major cause of tumor related death, Hca-F is a hepatocarcinoma cell line presenting highly lymph node metastasis, we study from analysis of genome to the analysis of proteome, and got Ech1 gene candidates concordantly expressed increase both at mRNA and protein level in the Hca-F cell highly lymph node metastasis. Our previous work has demonstrate that Ech1 is involved in the lymphatic metastasis in vitro and was confirmed in tissue from patients with gastric carcinoma with lymph node metastasis. Our current work observed when Ech1 downregulated, we monitor the expression of LNM related protein Anxa7, Clic1, Gsn, which is also candidates concordantly expressed increase both at mRNA and protein level in the Hca-F cell, is upregulated, that maybe indicated they together in the web involved in tumor lymph node metastasis, but the mechanism is still poorly understood. Our previous study on Ech1 has shown that it was involved in the tumor malignancy progression. To further explore the function of Ech1 in vivo, animal model were set by our lab to detect Lymph node metastases rate specifically. Histological analysis revealed that there were obvious morphological changes between the downregulation of Ech1 expression and Hca-F cell, Ech1 involved in lymph node metastasis rate were decreased markedly after down-regulating Ech1 in Hca-F cells from 80% to 45%. As described above, silencing of Ech1 in Hca-F cell could inhibit tumor metastatic potential to peripheral lymph nodes. Ech1 maybe showed suppressor role in hepatocarcinoma. The targeting of protein to specific subcellular structures is an important principle of the functional organization of cells at the molecular level. Expression of different location might depend upon their specific function, type, and stage of cancer [14]. In turn, knowledge about the subcellular location of a protein is a characteristic that may provide a hint as to the function of the
560
J. Zhang et al. / Biomedicine & Pharmacotherapy 67 (2013) 557–560
protein. Our previously demonstrated the subcellular location of Annexin A7 has positive involvement in hepatocellular carcinoma metastasis [15]. Unfortunately the current work indicates the involvement of Ech1 in tumor metastasis development and progression, but the subcellular location of Ech1 has not much contribution to that. However, future studies are required to further evaluate the complete mechanisms of Ech1 to suppress lymphatic metastasis, our present study highlights a potentially important role for Ech1 in tumor progress. Ech1 may serve as a novel therapeutic target for hepatocarcinoma. 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. 81071725) and the Educational Department of Liaoning Province (Nos. 20082250103 and 2009S028). References [1] Kopfstein L, Veikkola T, Djonov VG, Baeriswyl V, Schomber T, Strittmatter K, et al. Distinct roles of vascular endothelial growth factor-D in lymphangiogenesis and metastasis. Am J Pathol 2007;170(4):348–61. [2] He Y, Rajantie I, Pajusola K, Jeltsch M, Holopainen T, Yla-Herttuala S, et al. Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels. Cancer Res 2005;65(11):4739–46.
[3] Shayan R, Achen MG, Stacker SA. Lymphatic vessels in cancer metastasis: bridging the gaps. Carcinogenesis 2006;27(9):1729–38. [4] Yokoyama Y, Tsuchida S, Hatayama I, Satoh K, Narita T, Rao MS, et al. Loss of peroxisomal enzyme expression in preneoplastic and neoplastic lesions induced by peroxisome proliferators in rat livers. Carcinogenesis 1992;13:265–9. [5] Yokoyama Y, Kuramitsu Y, Takashima M, Iizuka N, Toda T, Terai S, et al. Proteomic profiling of proteins decreased in hepatocellular carcinoma from patients infected with hepatitis C virus. Proteomics 2004;4:2111–6. [6] Nishigaki R, Osaki M, Hiratsuka M, Toda T, Murakami K, Jeang KT, et al. Proteomic identification of differentially-expressed genes in human gastric carcinomas. Proteomics 2005;5:3205–13. [7] Vallat L, Magdele´nat H, Merle-Be´ral H, Masdehors P, Potocki de Montalk G, Davi F, et al. The resistance of B-CLL cells to DNA damage-induced apoptosis defined by DNA microarrays. Blood 2003;101:4598–606. [8] Cui X, Tang JW, Song B, Wang B, Chen S, Hou L. High expression of osteoglycin decreases gelatinase activity of murine hepatocarcinoma Hca-F cells. World J Gastroenterol 2009;15:6117–22. [9] Zhang YH, Wang SQ, Sun CR, Wang M, Wang B, Tang JW. Inhibition of JNK1 expression decreases migration and invasion of mouse hepatocellular carcinoma cell line in vitro. Med Oncol 2011;28(4):966–72. [10] Song B, Tang JW, Wang B, Cui XN, Hou L, Sun L, et al. Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip. World J Gastroenterol 2005;14:1463–72. [11] Sun MZ, Liu S, Tang JW, Wang Z, Gong X, Sun C, et al. Proteomics analysis of two mice hepatocarcinoma ascites syngeneic cell lines with high and low lymph node metastasis rates provide potential protein markers for tumor malignancy attributes to lymphatic metastasis. Proteomics 2009;9:3285–302. [12] Liu S, Sun MZ, Tang JW, Wang Z, Sun C, Greenaway FT. High-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry, two-dimensional difference in-gel electrophoresis and gene microarray identification of lymphatic metastasis-associated biomarkers. Rapid Commun Mass Spectrom 2008;22:3172–8. [13] Zhang J, Song M, Wang J, Sun M, Wang B, Li R, et al. Enoyl coenzyme A hydratase 1 is an important factor in the lymphatic metastasis of tumors. Biomed Pharmacother 2011;65(3):157–62. [14] Al-Marzoqee FY, Khoder G, Al-Awadhi H, John R, Beg A, Vincze A, et al. Upregulation and inhibition of the nuclear translocation of Oct4 during multistep gastric carcinogenesis. Int J Oncol 2012;41(5):1733–43. [15] Qazi AS, Mingzhong S, Yuhong H, Yuanyi W, Jianwu T. Subcellular proteomics: determination of specific location and expression levels of lymphatic metastasis associated proteins in hepatocellular carcinoma by subcellular fractionation. Biomed Pharmacother 2011;65:407–16.
Available online at
www.sciencedirect.com
Original article
Ech1 is a potent suppressor of lymphatic metastasis in hepatocarcinoma Jun Zhang a, Mingzhong Sun b, Rongkuan Li c, Shuqing Liu d, Jun Mao a, Yuhong Huang a, Bo Wang a, Li Hou a, Mohammed Mohammed Ibrahim a, Jianwu Tang a,* a Department of Pathology, Dalian Medical University, Key Laboratory of Tumor Metastasis of Liaoning Province University, 9, West Lvshun Southern Road, Dalian, 116044 Liaoning, China b Department of Biotechnology, Dalian Medical University, 116044 Dalian, China c Department of Gastroenterology and Hepatology, the Second Affiliated Hospital of Dalian Medical University, 116044 Dalian, China d Department of Biochemistry and Molecular Biology, Dalian Medical University, 116044 Dalian, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 February 2013 Accepted 10 March 2013
We have previously demonstrated that Ech1 is involved in the lymphatic metastasis of tumors in vitro. Here, we gain an insight into the role that Ech1 is playing in Hca-F cell. The expression of Annexin A7, Gelsolin and Clic1 genes, which were also relevant to tumor lymphatic metastasis, had been inhibited due to downregulation Ech1 gene by Western blot analysis. And downregulated of Ech1 inhibits the metastasic capability of Hca-F cells to peripheral lymph nodes in vivo. Our work indicates although the involvement of Ech1 in tumor metastasis development and progression, but the subcellular location of Ech1 has not much contribution to that. ß 2013 Published by Elsevier Masson SAS.
Keywords: Enoyl coenzyme A hydratase 1 (Ech1) Lymphatic metastasis Subcellular location Hepatocarcinoma
1. Introduction Metastasis has been regarded as a hallmark of malignancy that distinguishes invasive tumors from benign ones and constitutes a major cause of cancer mortality, and lymph node metastasis (LNM) is recognized as an important factor involved in the tumor malignancy progression. The mechanisms of tumor-associated lymphatic metastasis have been extensively studied recently [1– 3]. Enoyl coenzyme A hydratase 1 (Ech1) is the second enzyme in the pathway of fatty acid degradation via b-oxidation. A variety of studies have demonstrated that Ech1 may be associated with tumor progression [4–7]. Hca-F, a mouse hepatocarcinoma cell line with a high rate of lymphatic metastasis (> 75%), has proved to be an ideal model for studying the process of lymphatic metastasis [8–12]. We used gene chip and proteomics technique found that Ech1, Annexin, Clic1, Gelsolin were upregulated in the Hca-F cell [6–12], and then downregulated expression of Ech1 in Hca-F cell, which could inhibited proliferation of the Hca-F cells, increased the ratio of HcaF cells in S phase to G1 phase and decreased the adhesion and migration capacities of Hca-F cells [13]. Our current work is to gain an insight into the role that Ech1 is playing in Hca-F cell:
* Corresponding author. Tel.: +86 411 86110298; fax: ++86 411 86118866. E-mail address: [email protected] (J. Tang). 0753-3322/$ – see front matter ß 2013 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.biopha.2013.03.018
we got the Ech1 downregulated cell, and observe the expression of Annexin, Clic1, Gelsolin; we put Ech1 downregulated cell to inject into the mice with observation of the subsequent effects; we observe subcellular location of Ech1.
2. Materials and methods 2.1. Cell line culture and animals The mouse hepatocarcinoma cell line Hca-F, with an LNM rate over 75%, and Ech1 stably downregulation of Hca-F cell (FEch1 downregulated cell) was established and maintained in our laboratory. Inbred Chinese 615-mice (aged 6–8 weeks, weighing 18–22 g) were provided by the Experimental Animal House of our university. The Hca-F cell cultured in 90% RPMI 1640 supplemented with penicillin/streptomycin and 10% fetal bovine serum (Gibco, USA), and FEch1 downregulated cell cultured in 90% RPMI 1640 supplemented with penicillin/streptomycin 10% fetal bovine serum and neomycin (400 mg/mL) at 37 8C with 5% CO2. 2.2. Tumorigenicity assay in 615-mice Twenty inbred 615-mice were equally divided into two groups. Hca-F and FEch1 downregulated Cell (1 106) were inoculated into the feet pad of 615-mice subcutaneously. On the 28th day of postinoculation, the mice were euthanized and their lymph nodes
J. Zhang et al. / Biomedicine & Pharmacotherapy 67 (2013) 557–560
558
were isolated, weighed, sectioned, and stained with hematoxylin and eosin. The primary tumor, popliteal, inguinal and iliac lymph node were removed, weighted, and fixed in neutral-buffer formalin and embedded in paraffin, sections, then cut and stained with hematoxylin-eosin. The existence of metastatic foci was examined under microscope in sections. Anyone of popliteal lymph node, iliac lymph node, inguinal lymph node that had metastatic foci (secondary tumors) microscopically was considered as metastases. Then lymphatic metastasis rates were calculated.
separately. Then, 500 uL extraction buffer 4 with 5 uL protease inhibitor cocktail was added to pellet. Residual particles were suspended by pipetting (fraction IV). Different fraction we choose different internal control, we use GAPDH (Kangcheng; 1:7500) as cytosolic proteins internal control, ATP (Proteintech; 1:1200) as membranes and membrane organelles proteins internal control, c-Jun (Abcam; 1:1000) as nuclear protein internal control, and vimentin (Sigma; 1:2000) as cytoskeleton proteins internal control.
2.3. Western blot (WB) analysis
3. Results
Equal amounts of protein from Hca-F and FEch1 downregulated cell were separated for different expression using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). These were incubated with a monoclonal antibody against Annexin A7 (sigma; 1:15,000), Clic1 (Santa Cruz; 1:200), and Gesolin (BD Biosciences; 1:1000) in 5% dried milk for 1 h, then incubated with horseradish peroxidase-linked antibodies (1:5000 dilution) for 1 h at room temperature. Enhanced chemiluminescence (ECL; GE Healthcare, USA) was performed to visualize the protein bands.
3.1. Expression of Annexin A7 is upregulated in the FEch1 cell line
2.4. Subcellular protein extraction from culture cell suspension According to ProteoExtract Subcellular Proteome Extraction Kit (Merk), Cell suspension was made so that each aliquot will have 4 106 cells/cell pellet, all buffers were mixed well by vortexing and kept on ice during the whole procedure. Cell suspension was transferred to centrifuged tube and pellet by centrifugation at 200 g 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 200 g for 10 min, supernatant was aspirated and discarded. One milliliter extraction buffer mixed with 5 uL protease inhibitor cocktail and added to pellet, incubated for 10 min at 4 8C with gentle agitation, centrifuged at 1000 g for 10 min at 4 8C. Supernatant was collected in separate tube (fraction I). Extraction buffer 2 with 5 uL protease inhibitor cocktail was mixed with pellet, resuspended and incubated for 30 min at 4 8C, and centrifuged at 5500 g for 10 min at 4 8C. Suspernant (fraction II) is collected separately. In the pellet 500 uL Extraction buffer 3 with 5 uL inhibitor cocktail and 1.5 uL Benzonase nuclease was mixed and incubated for 10 min at 4 8C, mixture was centrifuged at 6800 g for 10 min at 4 8C, supernatant (fraction III) was collected
downregulated
WB results indicated Annexin A7 in FEch1 downregulated cell upregulated 11% compared to Hca-F cell (Fig. 1). 3.1.1. Expression of Clic1 is upregulated in the FEch1 downregulated cell line WB results indicated Clic1 in FEch1 downregulated cell is upregulated 69% compared to Hca-F cell (Fig. 1). 3.1.2. Expression of Gelsolin is upregulated in the FEch1 downregulated cell line WB results indicated Gelsolin in FEch1 downregulated cell is upregulated 13% compared to Hca-F cell (Fig. 1). 3.2. Downregulated of Ech1 inhibits the metastasic capability of Hca-F cells to peripheral lymph nodes in vivo To evaluate whether downregulated of Ech1 expression could inhibit the metastatic capability of Hca-F cell to peripheral lymph nodes in vivo, Hca-F and FEch1 downregulated cell were injected into footpad of 615-mice, respectively. After cells inoculation in mice for 28 days, a significant reduction of tumor lymph node metastasis was observed. Overall, there were eight out of 10 (8/ 10) mice transplanted with Hca-F cells show metastasis to lymph nodes, while in the other group, significantly fewer mice has lymph node metastasis (4/9,) transplanted with FEch1 downregulated cell (Fig. 2). 3.3. Ech1 expression at protein level of subcellular fraction The extraction procedure provided four fractions of cell material. Fraction I extraction of proteins is from cytosolic proteins,
Fig. 1. Western blotting analysis of expressions of Annexin A7, Clic1, Gelsolin in normal Hca-F and FEch1
downregulated
cell.
J. Zhang et al. / Biomedicine & Pharmacotherapy 67 (2013) 557–560
559
Fig. 2. Primary tumor and regional LNM of implanted tumor of Hca-F and FEch1 downregulated cell. A. Primary tumor of Hca-F. B. Regional LNM of implanted tumor of Hca-F. C. Primary tumor of FEch1 downregulated. D. Regional LNM of implanted tumor of FEch1 downregulated.
fraction II is from membranes and membrane organelles, fraction III is from nuclear protein, fraction IV is from components of cytoskeleton. Ech1 was found expressed almost at the same location although the expression level one is normal and the other is downregulation. Ech1 expression was found in cytosol, membrane fraction, and Ech1 expression was higher in cytosol than other fractions both in Hca-F cell and Ech1 downregulated Hca-F cell (Fig. 3).
Fig. 3. Ech1 expression in subcellular protein extraction from cells. For all blots, lane 1: cytoplasmic protein of Hca-F; lane 2: membrane/organelle proteins of Hca-F; lane 3: nuclear proteins of Hca-F; lane 4: cytoskeletal proteins of Hca-F; lane 5: cytoplasmic protein of FEch1 downregulated; lane 6: membrane/organelle proteins of FEch1 downregulated; lane 7: nuclear proteins of FEch1 downregulated; lane 8: cytoskeletal proteins of FEch1 downregulated, Proteins were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies to the indicated markers. GAPDH as cytosolic proteins internal control, ATP as membranes and membrane organelles proteins internal control, c-Jun as nuclear protein internal control, and vimentin as cytoskeleton proteins internal control.
4. Discussion Tumor metastasis is the major cause of tumor related death, Hca-F is a hepatocarcinoma cell line presenting highly lymph node metastasis, we study from analysis of genome to the analysis of proteome, and got Ech1 gene candidates concordantly expressed increase both at mRNA and protein level in the Hca-F cell highly lymph node metastasis. Our previous work has demonstrate that Ech1 is involved in the lymphatic metastasis in vitro and was confirmed in tissue from patients with gastric carcinoma with lymph node metastasis. Our current work observed when Ech1 downregulated, we monitor the expression of LNM related protein Anxa7, Clic1, Gsn, which is also candidates concordantly expressed increase both at mRNA and protein level in the Hca-F cell, is upregulated, that maybe indicated they together in the web involved in tumor lymph node metastasis, but the mechanism is still poorly understood. Our previous study on Ech1 has shown that it was involved in the tumor malignancy progression. To further explore the function of Ech1 in vivo, animal model were set by our lab to detect Lymph node metastases rate specifically. Histological analysis revealed that there were obvious morphological changes between the downregulation of Ech1 expression and Hca-F cell, Ech1 involved in lymph node metastasis rate were decreased markedly after down-regulating Ech1 in Hca-F cells from 80% to 45%. As described above, silencing of Ech1 in Hca-F cell could inhibit tumor metastatic potential to peripheral lymph nodes. Ech1 maybe showed suppressor role in hepatocarcinoma. The targeting of protein to specific subcellular structures is an important principle of the functional organization of cells at the molecular level. Expression of different location might depend upon their specific function, type, and stage of cancer [14]. In turn, knowledge about the subcellular location of a protein is a characteristic that may provide a hint as to the function of the
560
J. Zhang et al. / Biomedicine & Pharmacotherapy 67 (2013) 557–560
protein. Our previously demonstrated the subcellular location of Annexin A7 has positive involvement in hepatocellular carcinoma metastasis [15]. Unfortunately the current work indicates the involvement of Ech1 in tumor metastasis development and progression, but the subcellular location of Ech1 has not much contribution to that. However, future studies are required to further evaluate the complete mechanisms of Ech1 to suppress lymphatic metastasis, our present study highlights a potentially important role for Ech1 in tumor progress. Ech1 may serve as a novel therapeutic target for hepatocarcinoma. 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. 81071725) and the Educational Department of Liaoning Province (Nos. 20082250103 and 2009S028). References [1] Kopfstein L, Veikkola T, Djonov VG, Baeriswyl V, Schomber T, Strittmatter K, et al. Distinct roles of vascular endothelial growth factor-D in lymphangiogenesis and metastasis. Am J Pathol 2007;170(4):348–61. [2] He Y, Rajantie I, Pajusola K, Jeltsch M, Holopainen T, Yla-Herttuala S, et al. Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels. Cancer Res 2005;65(11):4739–46.
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