Abstract
BackgroundAutocrine 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.Methodology/Principal FindingsSimilarly, 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/SignificanceThe raft-dependent endocytosis of AMF/PGI may therefore represent a tumor cell specific endocytic pathway of potential value for drug delivery to tumor cells.
Highlights
Endocytosis is the general mechanism by which cells regulate entry of external substances into the cell and represents an important route for delivery of targeted therapeutics for a variety of pathologies including cancer [1]
We show that raft-dependent uptake of Autocrine motility factor/phosphoglucose isomerase (AMF/PGI) is specific for gp78/AMFR-positive/Cav1-negative metastatic colon cancer cells, does not target normal immune cells and occurs in vivo in subcutaneous K1735-M1 and B16-F1 melanoma tumor models
We previously showed that gp78/AMFR expression was elevated in metastatic MDA-435 and MDA-231 cells relative to nonmetastatic MCF-7 and dysplastic MCF10A mammary tumor cells and that AMF/PGI-FITC uptake was selectively increased in MDA435 cells that express reduced levels of Cav1 [41]
Summary
Endocytosis is the general mechanism by which cells regulate entry of external substances into the cell and represents an important route for delivery of targeted therapeutics for a variety of pathologies including cancer [1]. Caveolae-mediated uptake is a well-characterized endocytic mechanism in endothelial cells [5], but whether other raft-dependent pathways represent selective portals into specific cell types, such as tumor cells, remains to be demonstrated. A novel promising target for anti-cancer agents is the receptor for autocrine motility factor/phosphoglucose isomerase (AMF/PGI), known as gp78/AMFR, that was recently identified as one of 189 genes mutated at significant frequency in breast and colorectal cancer [6]. 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
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