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

Abstract

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.