title: Raft-dependent endocytosis of autocrine motility factor/phosphoglucose isomerase: a potential drug delivery route for tumor cells.
abstract: BACKGROUND
  Autocrine motility factor/phosphoglucose isomerase (AMF/PGI) is the extracellular ligand for the gp78/AMFR receptor overexpressed in a variety of human cancers. We showed previously that raft-dependent internalization of AMF/PGI is elevated in metastatic MDA-435 cells, but not metastatic, caveolin-1-expressing MDA-231 cells, relative to non-metastatic MCF7 and dysplastic MCF10A cells suggesting that it might represent a tumor cell-specific endocytic pathway.
  
  
  RESULTS
  Similarly, using flow cytometry, we demonstrate that raft-dependent endocytosis of AMF/PGI is increased in metastatic HT29 cancer cells expressing low levels of caveolin-1 relative to metastatic, caveolin-1-expressing, HCT116 colon cells and non-metastatic Caco-2 cells. Therefore, we exploited the raft-dependent internalization of AMF/PGI as a potential tumor-cell specific targeting mechanism. We synthesized an AMF/PGI-paclitaxel conjugate and found it to be as efficient as free paclitaxel in inducing cytotoxicity and apoptosis in tumor cells that readily internalize AMF/PGI compared to tumor cells that poorly internalize AMF/PGI. Murine K1735-M1 and B16-F1 melanoma cells internalize FITC-conjugated AMF/PGI and are acutely sensitive to AMF/PGI-paclitaxel mediated cytotoxicity in vitro. Moreover, following in vivo intratumoral injection, FITC-conjugated AMF/PGI is internalized in K1735-M1 tumors. Intratumoral injection of AMF/PGI-paclitaxel induced significantly higher tumor regression compared to free paclitaxel, even in B16-F1 cells, known to be resistant to taxol treatment. Treatment with AMF/PGI-paclitaxel significantly prolonged the median survival time of tumor bearing mice. Free AMF/PGI exhibited a pro-survival role, reducing the cytotoxic effect of both AMF/PGI-paclitaxel and free paclitaxel suggesting that AMF/PGI-paclitaxel targets a pathway associated with resistance to chemotherapeutic agents. AMF/PGI-FITC uptake by normal murine spleen and thymus cells was negligible both in vitro and following intravenous injection in vivo where AMF/PGI-FITC was selectively internalized by subcutaneous B16-F1 tumor cells.
  
  
  CONCLUSIONS
  The raft-dependent endocytosis of AMF/PGI may therefore represent a tumor cell specific endocytic pathway of potential value for drug delivery to tumor cells.
authors.affiliation: Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Liliana D
authors.initials: LD
authors.lastname: Kojic
authors.forename: Sam M
authors.initials: SM
authors.lastname: Wiseman
authors.forename: Fariba
authors.initials: F
authors.lastname: Ghaidi
authors.forename: Bharat
authors.initials: B
authors.lastname: Joshi
authors.forename: Hinyu
authors.initials: H
authors.lastname: Nedev
authors.forename: H Uri
authors.initials: HU
authors.lastname: Saragovi
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Nabi
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage*; D018938:Caco-2 Cells; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D016503:Drug Delivery Systems / Q000379:methods*; D004705:Endocytosis / Q000502:physiology*; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism*; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D021962:Membrane Microdomains / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D017239:Paclitaxel / Q000008:administration & dosage / Q000737:chemistry; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D018121:Receptors, Cytokine; C555000:AMFR protein, human; C555002:Amfr protein, mouse; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases; D005956:Glucose-6-Phosphate Isomerase; D017239:Paclitaxel