Tanino Tadatoshi, Nawa Akihiro, Kondo Eisaku, Kikkawa Fumitaka, Daikoku Tohru, Tsurumi Tatsuya, Luo Chenhong, Nishiyama Yukihiro, Takayanagi Yuki, Nishimori Katuhiko, Ichida Seiji, Wada Tetsuyuki, Miki Yasuyoshi, Iwaki Masahiro
School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan.
Pharm Res. 2007 Mar;24(3):555-65. doi: 10.1007/s11095-006-9171-6.
The aim of the study was to investigate whether 2'-ethylcarbonate-linked paclitaxel (TAX-2'-Et) circumvents P-glycoprotein (P-gp)-mediated cellular efflux and cytotoxicity enhanced by TAX-2'-Et activation within human culture cells transfected with a rabbit liver carboxylesterase (Ra-CES) cDNA.
TAX-2'-Et transport was characterized in a human colon carcinoma cell line (Caco-2) and paclitaxel (TAX)-resistant ovarian carcinoma cells (SKOV3/TAX60). Expression of P-gp, multidrug resistance protein (MRP) 2 and Ra-CES was detected by Western blotting. Cytotoxicity against Ra-CES-expressing cells and cellular amount of TAX produced were determined by MTT assay and using HPLC, respectively.
Unlike rhodamine123 and TAX, TAX-2'-Et did not exhibit polarized transport in the Caco-2 cells in the absence or presence of verapamil. P-gp levels were expressed much higher in the SKOV3/ TAX60 cells than in the Caco-2 cells. MRP2 protein was not detectable in the SKOV3/TAX60 cells. Uptake by the SKOV3/TAX60 cells was similar in quantity to the amount internalized by P-gp-negative SKOV3 cells. In the SKOV3/TAX60 cells, cellular uptake of TAX-2'-Et was not altered regardless of the absence or presence of verapamil. The cytotoxicity to the untransfected SKOV3 cells induced by TAX-2'-Et was significantly lower than that induced by TAX. In the Ra-CES-expressing SKOV3 line, the EC50 value of TAX (10.6 nM) was approximately four-fold higher than that of TAX-2'-Et (2.5 nM). Transfection of Ra-CES into another TAX-resistant ovarian carcinoma cells (KOC-7c) conferred a high level of TAX-2'-Et cytotoxicity via prodrug activation. The intracellular levels of TAX produced from TAX-2'-Et in the Ra-CES-positive KOC-7c cells significantly increased compared with the levels seen in exposure of the untransfected KOC-7c cells to TAX.
TAX-2'-Et can circumvent P-gp-associated cellular efflux of TAX. TAX-2'-Et is converted into TAX by the Ra-CES, supporting its potential use as a theoretical GDEPT strategy for cancer cells expressing high levels of P-gp. The TAX-2'-Et prodrug efficiently increased the amount of intracellular TAX, which mediates tumor cell death.
本研究旨在探讨2'-碳酸乙酯连接的紫杉醇（TAX-2'-Et）是否能规避P-糖蛋白（P-gp）介导的细胞外排作用，以及在转染兔肝脏羧酸酯酶（Ra-CES）cDNA的人培养细胞中TAX-2'-Et激活后细胞毒性是否增强。
在人结肠癌细胞系（Caco-2）和耐紫杉醇（TAX）的卵巢癌细胞（SKOV3/TAX60）中对TAX-2'-Et的转运进行表征。通过蛋白质印迹法检测P-gp、多药耐药蛋白（MRP）2和Ra-CES的表达。分别采用MTT法和高效液相色谱法测定对表达Ra-CES的细胞的细胞毒性以及产生的TAX的细胞量。
与罗丹明123和TAX不同，无论是否存在维拉帕米，TAX-2'-Et在Caco-2细胞中均未表现出极化转运。SKOV3/TAX60细胞中P-gp水平的表达远高于Caco-2细胞。在SKOV3/TAX60细胞中未检测到MRP2蛋白。SKOV3/TAX60细胞的摄取量与P-gp阴性的SKOV3细胞内化的量相似。在SKOV3/TAX60细胞中，无论是否存在维拉帕米，TAX-2'-Et的细胞摄取均未改变。TAX-2'-Et对未转染的SKOV3细胞的细胞毒性显著低于TAX诱导的细胞毒性。在表达Ra-CES的SKOV3细胞系中，TAX的半数有效浓度（EC50）值（10.6 nM）约为TAX-2'-Et（2.5 nM）的四倍。将Ra-CES转染到另一种耐TAX的卵巢癌细胞（KOC-7c）中通过前药激活赋予了高水平的TAX-2'-Et细胞毒性。与未转染的KOC-7c细胞暴露于TAX时相比，Ra-CES阳性的KOC-7c细胞中由TAX-2'-Et产生的TAX细胞内水平显著增加。
TAX-2'-Et可规避与P-gp相关的TAX细胞外排。TAX-2'-Et被Ra-CES转化为TAX，支持其作为一种针对高表达P-gp的癌细胞的理论基因导向酶解药物前体疗法（GDEPT）策略的潜在用途。TAX-2'-Et前药有效增加了介导肿瘤细胞死亡的细胞内TAX量。
