Kojic Liliana D, Wiseman Sam M, Ghaidi Fariba, Joshi Bharat, Nedev Hinyu, Saragovi H Uri, Nabi Ivan R
Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada.
PLoS One. 2008;3(10):e3597. doi: 10.1371/journal.pone.0003597. Epub 2008 Oct 31.
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.
METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.
自分泌运动因子/磷酸葡萄糖异构酶（AMF/PGI）是gp78/AMFR受体的细胞外配体，在多种人类癌症中过表达。我们之前表明，相对于非转移性MCF7细胞和发育异常的MCF10A细胞，AMF/PGI依赖脂筏的内化在转移性MDA - 435细胞中升高，但在表达小窝蛋白-1的非转移性MDA - 231细胞中未升高，这表明它可能代表一种肿瘤细胞特异性的内吞途径。
方法/主要发现：同样，使用流式细胞术，我们证明相对于表达小窝蛋白-1的转移性HCT116结肠细胞和非转移性Caco - 2细胞，在表达低水平小窝蛋白-1的转移性HT29癌细胞中，AMF/PGI依赖脂筏的内吞作用增强。因此，我们利用AMF/PGI依赖脂筏的内化作为一种潜在的肿瘤细胞特异性靶向机制。我们合成了一种AMF/PGI - 紫杉醇偶联物，发现它在诱导易于内化AMF/PGI的肿瘤细胞的细胞毒性和凋亡方面与游离紫杉醇一样有效，相比之下，对AMF/PGI内化较差的肿瘤细胞则不然。小鼠K1735 - M1和B16 - F1黑色素瘤细胞内化异硫氰酸荧光素（FITC）偶联的AMF/PGI，并且在体外对AMF/PGI - 紫杉醇介导的细胞毒性高度敏感。此外，在体内瘤内注射后，FITC偶联的AMF/PGI在K1735 - M1肿瘤中被内化。即使在已知对紫杉醇治疗耐药的B16 - F1细胞中，瘤内注射AMF/PGI - 紫杉醇诱导的肿瘤消退也明显高于游离紫杉醇。用AMF/PGI - 紫杉醇治疗显著延长了荷瘤小鼠的中位生存时间。游离AMF/PGI发挥促生存作用，降低了AMF/PGI - 紫杉醇和游离紫杉醇的细胞毒性作用，这表明AMF/PGI - 紫杉醇靶向与化疗药物耐药相关的途径。正常小鼠脾脏和胸腺细胞对AMF/PGI - FITC的摄取在体外和体内静脉注射后都可以忽略不计，而AMF/PGI - FITC在皮下B16 - F1肿瘤细胞中被选择性内化。
结论/意义：因此，AMF/PGI依赖脂筏的内吞作用可能代表一种对肿瘤细胞给药具有潜在价值的肿瘤细胞特异性内吞途径。
