Biorecognition and subcellular trafficking of HPMA copolymer-anti-PSMA antibody conjugates by prostate cancer cells.
Biorecognition and subcellular trafficking of HPMA copolymer-anti-PSMA antibody conjugates by prostate cancer cells.
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DOI：
10.1021/mp8002682
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发表时间：
2009-05
影响因子：
4.9
通讯作者：
Kopecek J
中科院分区：
文献类型：
--
作者：
Liu J;Kopecková P;Bühler P;Wolf P;Pan H;Bauer H;Elsässer-Beile U;Kopecek J
新一代抗前列腺特异性膜抗原（PSMA）的抗体已被证明能特异性结合活前列腺癌细胞表面的PSMA分子。为了探索抗PSMA抗体作为前列腺癌大分子药物靶向部分的潜力，合成了荧光标记的HPMA（N-（2-羟丙基）甲基丙烯酰胺）共聚物-抗PSMA抗体偶联物（P-anti-PSMA），并研究了其在C4-2前列腺癌细胞中的内吞和亚细胞运输机制。放射免疫测定显示，P-抗-PSMA的解离常数为C4-2前列腺癌细胞在低纳摩尔范围内，接近游离抗-PSMA的值。这表明抗PSMA与HPMA共聚物的缀合不损害它们的结合亲和力。P-抗-PSMA的内吞速率比含有非特异性IgG的对照HPMA共聚物缀合物的内吞速率快得多。网格蛋白介导的内吞作用和巨胞饮作用的选择性途径抑制剂抑制P-抗PMSA的内化。通过siRNA下调网格蛋白重链表达进一步证明了对网格蛋白介导的内吞作用的抑制。利用发动蛋白显性失活突变体Dyn K44 A阻断不依赖网格蛋白、小窝的内吞途径，发现部分P-anti-PSMA通过这一途径被C4-2细胞内吞。因此，多种受体介导的内吞途径，包括网格蛋白介导的内吞作用，巨胞饮作用和动力蛋白非依赖性内吞作用，参与了P-抗-PSMA的内化。估计每种途径参与P-抗PSMA内吞的程度。含有P-抗-PSMA的膜囊泡与过表达Rab 7（一种晚期内体定位蛋白）的膜囊泡快速共定位，表明部分P-抗-PSMA被转运至晚期内体。
A new generation of antibodies against the prostate specific membrane antigen (PSMA) has been proven to bind specifically to PSMA molecules on the surface of living prostate cancer cells. To explore the potential of anti-PSMA antibodies as targeting moieties for macromolecular therapeutics for prostate cancer, fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymer - anti- PSMA antibody conjugates (P-anti-PSMA) were synthesized and the mechanisms of their endocytosis and subcellular trafficking in C4-2 prostate cancer cells were studied. Radioimmunoassays showed the dissociation constants of P-anti-PSMA for C4-2 prostate cancer cells in the low nanomolar range, close to values for free anti-PSMA. It indicated that conjugation of anti-PSMA to HPMA copolymers did not compromise their binding affinity. The rate of endocytosis of P-anti-PSMA was much faster than that of control HPMA copolymer conjugates containing non-specific IgG. Selective pathway inhibitors of clathrin-mediated endocytosis and of macropinocytosis inhibited the internalization of P-anti-PMSA. Inhibition of clathrin-mediated endocytosis was further evidenced by down-regulation of clathrin heavy chain expression by siRNA. Using a dominant-negative mutant of dynamin (Dyn K44A) to abolish the clathrin-, caveolae-independent endocytic pathway, we found that some of P-anti-PSMA adopted this pathway to be endocytosed into C4-2 cells. Thus multiple receptor-mediated endocytic pathways, including clathrin-mediated endocytosis, macropinocytosis, and dynamin-independent endocytosis, were involved in the internalization of P-anti-PSMA. The extent of the participation of each pathway in P-anti-PSMA endocytosis was estimated. Membrane vesicles containing P-anti-PSMA rapidly co-localized with membrane vesicles overexpressing Rab7, a late endosome localized protein, demonstrating that a part of P-anti-PSMA was transported to late endosomes.
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