Kim Ryungsa, Murakami Shigeru, Toge Tetsuya
Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan.
Int J Oncol. 2003 Apr;22(4):835-41.
Human thymidine phosphorylase (dThdPase) is an angiogenic factor identical to platelet-derived endothelial cell growth factor (PD-ECGF). Thymidine phosphorylase is also a converting enzyme of the prodrug 5'-deoxy-5-fluorouridine (5'-DFUR) to 5-fluorouracil (5-FU) in tumors. To assess the role of dThdPase in targeting chemotherapy, we examined the relationship between the expression of dThdPase and the sensitivity of 5'-DFUR in cancer cell lines, and also examined whether transfection of dThdPase cDNA enhanced the drug-sensitivity to 5'-DFUR with or without angiogenesis in breast cancer cells. Thirteen human cancer cell lines consisting of 4 breast cancer, 6 gastric cancer, and 3 colon cancer cell lines were used. Expression of dThdPase was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). In vitro drug-sensitivity was assessed by MTT assay, and anti-tumor effect in vivo was assessed using nude mouse xenografts. Intratumoral microvessel density was evaluated by immunohistochemical staining to factor VIII related antigen. Transfection of dThdPase cDNA was performed using pcDNA3 expression vector encoding its cDNA by the lipofection method. An inverse relationship between the expression of dThdPase and the IC50 values of 5'-DFUR was observed (p=0.1278, rho=-0.440) in the 13 cancer cell lines. Transfection of dThdPase cDNA into MCF-7 breast cancer cells resulted in an approximately 2.6- and 10-fold increase of the expression of dThdPase mRNA and its enzyme activity, respectively, compared to the control vector alone. The sensitivity to 5'-DFUR in the transfected cells was increased approximately 20-fold compared to the parent cells and control vector alone, and the sensitivity to 5-FU was also somewhat increased. In contrast, the sensitivity to ADM, CDDP, and VP-16 was not different between the transfected and control cells. In nude mice xenografts of the transfected cells, treatment with 5'-DFUR had a significant anti-tumor effect compared to those of the untreated transfected cells and control vector alone treated with 5'-DFUR (p<0.01). Intratumoral microvessel density in the transfected cells was not significantly increased with or without treatment with 5'-DFUR compared to control vector alone. The high expression of dThdPase was correlated with an increase in the sensitivity to 5'-DFUR in gastrointestinal and breast cancer cell lines. The introduction of dThdPase cDNA in breast cancer cells enhanced the sensitivity to 5'-DFUR without an increase of tumor angiogenesis, and targeting chemotherapy of dThdPase may be a good tumor-specific and personalized therapy for improving the poor prognosis of cancer patients who show high expressions of dThdPase.
人胸苷磷酸化酶（dThdPase）是一种血管生成因子，与血小板衍生的内皮细胞生长因子（PD - ECGF）相同。胸苷磷酸化酶也是肿瘤中前药5'-脱氧-5-氟尿苷（5'-DFUR）转化为5-氟尿嘧啶（5-FU）的转化酶。为了评估dThdPase在靶向化疗中的作用，我们研究了dThdPase的表达与癌细胞系中5'-DFUR敏感性之间的关系，还研究了在有无血管生成的情况下，转染dThdPase cDNA是否能增强乳腺癌细胞对5'-DFUR的药物敏感性。使用了由4种乳腺癌、6种胃癌和3种结肠癌细胞系组成的13种人类癌细胞系。通过逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）评估dThdPase的表达。通过MTT法评估体外药物敏感性，使用裸鼠异种移植评估体内抗肿瘤作用。通过免疫组织化学染色评估肿瘤内微血管密度以检测VIII因子相关抗原。使用编码其cDNA的pcDNA3表达载体通过脂质转染法进行dThdPase cDNA的转染。在13种癌细胞系中观察到dThdPase的表达与5'-DFUR的IC50值呈负相关（p = 0.1278，rho = -0.440）。与单独的对照载体相比，将dThdPase cDNA转染到MCF-7乳腺癌细胞中导致dThdPase mRNA的表达及其酶活性分别增加约2.6倍和10倍。与亲代细胞和单独的对照载体相比，转染细胞对5'-DFUR的敏感性增加了约20倍，对5-FU的敏感性也有所增加。相反，转染细胞与对照细胞对阿霉素（ADM）、顺铂（CDDP）和依托泊苷（VP-16）的敏感性没有差异。在转染细胞的裸鼠异种移植中，与未处理的转染细胞和单独用5'-DFUR处理的对照载体相比，用5'-DFUR处理具有显著的抗肿瘤作用（p <0.01）。与单独的对照载体相比，无论是否用5'-DFUR处理，转染细胞中的肿瘤内微血管密度均未显著增加。dThdPase的高表达与胃肠道和乳腺癌细胞系对5'-DFUR敏感性的增加相关。在乳腺癌细胞中引入dThdPase cDNA可增强对5'-DFUR的敏感性，而不会增加肿瘤血管生成，并且dThdPase的靶向化疗可能是一种良好的肿瘤特异性和个性化治疗方法，可改善dThdPase高表达的癌症患者的不良预后。
