| Background:It is now known that the potential of a tumor cell to metastasize depends on its interactions with the homeostatic factors that promote tumor-cell growth, survival, angiogenesis, invasion and metastasis. Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide with about 600,000 patients dying from the disease annually. The geographic areas at highest risk are located in Eastern Asia, Middle Africa, and some countries of Western Africa. Hepatocellular carcinoma is a highly vascularized tumor, which makes vascular targeting approaches appealing for the treatment of HCC. It is phenotypically and genetically heterogenous polyclonal disease and resistant to most conventional chemotherapy. Development of lymph node metastasis is a complex process involving multiple genes. The high mortality and poor prognosis caused by lymph node metastasis in cancer patient and the uncertain mechanism are always the prominent problems in the field of oncology therefore it is very important to understand the exact mechanism of metastasis, cancer markers involved in this process and their expressions at specific sites in the host cells. Hca-F and Hca-P are a pair of syngenetic mouse hepatocarcinoma ascites cell lines, showing a specific potential of lymphogenetic metastasis when inoculated subcutaneously in 615 mice. Hca-F has high lymphatic metastatic potential (>70%) and Hca-P has low lymphatic metastatic potential (< 30%) Hca-F and Hca-P metastasize only to lymph nodes, and not to other organs, which have been proved the ideal models for studying lymphatic metastasis for hepatocarcinoma.The targeting of proteins to particular sub cellular 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 has been a tendency to focus on sub cellular proteomes, concerning specific sub cellular compartments and macromolecular structures of the cell, the separation of the protein mixture into organelles or other multi-protein complex.Fractions prior to a proteomics analysis can increase the probability of detecting low-copy number proteins. Sub cellular proteome research cannot only provide information about sub cellular location of certain proteins and imply their function, but also about the whole-protein components of the specific sub cellular fractions (organelles or other multi-protein complexes) and then help understand their structures and biological functions.The differentially identified proteins by sub cellular fractionation may seems to involve in nucleic acid binding, oxidoreductse and cytoskeleton proteins in cellular integrity, signal transduction pathways, cell structure and motility, nucleoside, nucleotide and nucleic acid metabolism, regulation of actin cytoskeleton, focal adhesion, antigen processing and presentation etc.We have analyzed and compared some metastasis-associated biomarkers and their expression levels statistically. We have also located their sub cellular expression sites by fractionating the cells into their compartment. Comparison of each metastasis-associated biomarker was made among the different metastatic potential cell lines. Results of these studies revealed higher expression of each metastasis -associated biomarker in high lymphogeneous metastatic potential cell line than in low lymphogeneous metastatic potential cell line. Therefore, these metastasis-associated biomarkers can be correlated with lymph node metastasis of mouse hepatocarcinoma.Objectives1.To fractionate the cells into their sub cellular compartment with their complete cellular integrity.2.To observe the expression level of each lymphatic metastatic biomarker which are Gelsolin, JNK and Annexin 7.3.To compare the expression levels of these biomarkers statistically among the different metastatic potential cell lines and by up regulating and down regulating their expression levels in these cell lines. 4.To correlate the potential of these cancer biomarkers and their involvement in lymphatic metastasis due to the change of the level of their expression in hepatocarcinoma ascites cell lines.Method1.Hca-F and Hca- P tumor cells were injected into inbred 615 mice and were grown into mouse abdominal cavity. Two passages were done to harvest large number of cells further the cells were seeded into vials for culture under suitable growth conditions for 24h in a 5%CO2 atmosphere at 37℃in a humidified atmosphere. Cell viability was determined by Trypan blue exclusion test.2.Hca-F and Hca-P cells were inoculated (2×106 cells/mouse) into the left footpad of each mouse in a group of 20 inbred 615 mice. On the 28th day post inoculation, 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.3.The cells were washed and centrifuged in a refrigerated centrifuge. The cell pellet was transferred into other reaction tube and washed three times with washing buffer provided in the kit. Each time the pellet is mixed in the rotatory shaker at 4℃and centrifuged in a refrigerated centrifuge. Cells were fractionated using sub cellular proteome extraction kit. The extraction procedure provided four fractions with decreased proteome complexity. For fractionation, cells suspension was aliquoted and each aliquote has 3-5 x 106 cells/frozen cell pellet. The four fractions obtained after fractionation were cytoplasm, membranes and membrane bound organelles, nuclear material and cytoskeletal protein.4. Concentration of protein was determined by Bradford assay using bovine serum albumin (BSA) as standard.Expression levels of three lymphatic metastatic associated biomarkers were analyzed by western blot analysis and compared statistically. The metastatic associated proteins compared were Gelsolin, JNK and Annexin7.5.The western blot 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 and applied with respective antibodies for each protein under study. After extensive washings, an ECL western blotting kit detected bands. For each fraction different loading control protein were used depending on their specific sub cellular site of expression. 6. Statistical comparison was made using SPSS for each lymphatic metastasis associated protein under study and for better contrast the comparison was made among cell lines with high and low lymphatic metastasis potential.7.Annexin7 was further analyzed and compared by enhancing annexin7 i.e., up regulation and by silencing annexin7 i.e., down regulation of the protein expression. To establish a mouse hepatic cancer cell line Hca-F transfected with sh RNA (short hairpin RNA) three sh RNA were designed and inserted into the p Silencer vector to silence Annexin7 gene. The most effective p Silencer-sh RNA 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-sh RNA and transfectants were selected by 400μg/μl G 418. To construct P cDNA 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 P cDNA 3.1 plasmid. This plasmid was transfected in P cells stably.Results1.The implanted tumors both in Hca-F tumor-bearing mice and in Hca-P tumor-bearing mice were palpable on the 7th day post-inoculation. The rate of tumor formation in both groups was 100%. The p Silencer -shRNA vector -Annexin 7 to Hca-F cells and P cDNA 3.1-Annexin 7 to Hca-P cells was transfected successfully.2.The extraction procedure using Proteome Extraction Kit (S-PEK) provided four fractions of cell material. Extraction bufferⅠreleased cytosolic proteins (fraction 1). Subsequently, Extraction bufferⅡwith nucleus and cytoskeleton integrity (fraction 2) solubilizes membranes and membrane organelles. Nuclear proteins are extracted with Extraction bufferⅢ(fraction 3). Components of the cytoskeleton are finally separated and solubilized with Extraction bufferⅣ(fraction 4).3.The relative protein expression level of Gelsolin was determined by western blot analysis. The expression levels were calculated by taking mean values thrice of six values for each fraction. Hca-F cells showed higher expression of Gelsolin than in Hca-P cell lines among each fraction of its expression. Data was found significant when analyzed statistically. When standard deviation and mean values were compared for Gelsolin’s expression level in different cell lines, they were found as 0.84±0.05 for F-cells and 0.77±0.05 for P-cells. F-cells have little higher expression as compared to P-cells in fraction 1. Gelsolin expression was not found in fraction 2 of P-cells.When fraction 4 is compared among the two cell lines the values were 0.69±0.061 for F- cells and 0.36±0.049 for P-cells. When the comparison was made among the three fractions of Gelsolin expression 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 for F cells. Among the two fractions of Gelsolin expression for P-cells, the values of expression levels were 0.70±0.05 and 0.32±0.045 for fraction 1 and fraction 4. Statistically, all the data was analyzed and found significant at P<0.05.4. Expression level of JNK was determined by western blot analysis in Hca-F and Hca-P cell line.In both the hepatocarcinoma cell lines JNK was expressed in nuclear fraction 3 and cytoskeletal fraction 4. Expession was found higher in fraction 4 than in fraction 3 and in F-cells than in P-cells. Statistical values calculated were 0.31±0.051 for F-cells and 0.22±0.027 for P cells in case of fraction 3 and 0.86±0.073 for F-cells and 0.74±0.069 for P-cells in case of fraction 4. Among the two fractions of JNK expression in F-cells, the values of expression levels were 0.30±0.025 and 0.80±0.05 and 0.26±0.03 for fraction 3 and 0.70±0.05 for fraction 4 in fractions of JNK expression in P cells. Statistically, all the data was analyzed and found significant at P< 0.05.5.Annexin 7 was detected in cytosolic fraction 1, membranous fraction 2 and cytoskeletal fraction 4. The expression levels were detected by western blot analysis. The expression level was found highest in Hca-F cells compared to P cell line. Hca-P showed minimum expression level among all four cell lines analyzed. These were Hca-F, F shRNA-ANX 7, Hca-P and P cDNA 3.1-ANX 7. The statistical comparison was made between the four cell lines analyzed for Annexin 7 and among the four fractions of each cell line. The values were 0.099±0.015,0.02±0.005,0.04±0.009 and 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 cytosolic fraction 1 of F-cells, P-cells, F shRNA-ANX 7 and P cDNA 3.1-ANX7 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-ANX 7 and P cDNA 3.1-ANX 7 respectively. When the data was compared among the three fractions of its expression in F-cells the values were obtained among the two band’s expression level in fraction 1 as 0.061±0.005 for 51 KDa and 0.76±0.069 for 47 KDa,0.28±0.080 for fraction 2 and 0.45±0.096 for fraction 4. When the data was compared among the three fractions of its expression in P-cells the values were 0.032±0.005 for 51 KDa isoform in fraction 1 and 0.45±0.01 for 47 KDa in fraction 1,0.20±0.01 for fraction 2 and 0.32±0.024 for fraction 4. All the data was found significant at P<0.05.6.Gelsolin and Annexin 7 were found expressed almost at same locations with the different expression levels. JNK and Annexin7 almost have same molecular weight but their sites of expression were different.Conclusion1.All the results lead to conclusion that there might be a positive correlation with the lymphatic metastasis protein Gelsolin, JNK and Annexin 7 under this study and lymphatic metastasis progression and development. All were expressed at high levels in lymphatic metastasis cell lines with high potential of metastasis with some differences in their expression levels indicating their strong involvement in tumor metastasis.2.Expression at different sub cellular location might link their different roles in development of tumor metastasis and indicated their different distribution patterns.3.Annexin 7 two isoforms were detected only in cytoplasm but not in other fractions of its expression might be due to location of expression. Both the isoform were observed in liver cells.4. All three proteins are expressed more in Hca-F than in Hca-P cell lines.The ability of metastasis of mouse hepatocarcinoma cell line Hca-F was reduced by silencing Annexin7 after its down regulation and increased by enhancing Annexin 7 in mouse hepatocarcinoma cell line Hca-P proved that this lymphatic metastasis associated tumor protein played important role in hepatocarcinoma and showed close relationship in development and progression of lymphatic metastasis of hepatocarcinoma.5.Gelsolin has been found with distribution pattern at different sub cellular location and with increased expression levels in high metastatic potential cell lines. It was found sharing the same expression site as that of Annexin 7.Both of them are calcium dependent phospholipid binding proteins.6. JNK was also observed expressing at higher levels in high metastatic potential cell line. Although it has almost the same molecular weight with Annexin 7 yet it was not found showing much similarities with annexin 7 expression. Sub cellular fractionation will provide prognostic tools for metastatic risk and in anti cancer campaign. |
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