Enhancement of sensitivity to capecitabine in human renal carcinoma cells transfected with thymidine phosphorylase cDNA

Abstract

The purpose of the present study was to examine directly the role of thymidine phosphorylase (TP) in the sensitivity of renal cell carcinoma (RCC) to a novel fluoropyrimidine carbamate, capecitabine. TP cDNA-transfected RCC are used in these experiments to provide a basis for improved therapeutic benefit in chemoimmunotherapy. Human RCC line KU2 cells were transfected with pcDNA3.1/zeo(؉) with or without human TP cDNA by the lipofectin method. We established a clone transfected with pcDNA3.1/zeo(؉)/TP (KU2-TP15) and a clone transfected with pcDNA3.1/zeo(؉) as a control (KU2-C1). TP expression levels (mean ؎ SD) examined by enzyme-linked immunosorbent assay (ELISA) were 1.3 ؎ 0.14 U/mg protein in KU2, 1.6 ؎ 0.57 U/mg protein in KU2-C1 and 216 ؎ 25.6 U/mg protein in KU2-TP15. Immunohistochemical staining of subcutaneous tumors established in Balb/c nu/nu mice showed that KU2-TP15 was strongly positive for TP expression, whereas KU2 and KU2-C1 were negative. Sensitivities in vitro to 5-fluorouracil (5FU), 5-deoxy-5-fluorouridine (5DFUR) and capecitabine in KU2-TP15 were significantly enhanced compared with those in KU2 or KU2-C1. A moderate but statistically significant bystander effect was observed in vitro. KU2-TP15 tumors showed significant increase in the in vivo sensitivities to 5DFUR and capecitabine as compared with the vehicle alone while KU2-C1 tumors did not. The difference in tumor-free rate in mice bearing KU2-TP15 at 2 months after the cessation of treatment was statistically significant between the capecitabine treatment group and the controls, the 5FU treatment group and the 5DFUR treatment group. The present study clearly provides direct evidence for the role of TP in mediating the sensitivity of RCC to capecitabine. The conventional treatments with cytotoxic agents, hormones and cytokines for renal cell carcinoma (RCC) have been disappointing. 1-3 Therefore, it seems of value to explore a new regimen or a novel agent with high therapeutic index for the improvement of therapeutic efficacy against RCC. Recently, chemoimmunotherapy consisting of 5-fluorouracil (5FU) and interferon-␣ (IFN␣) with or without interleukin-2 (IL-2) has been reportedly useful in the treatment of RCC, with overall response rates of 30% to 48.6%. 4 -8 Capecitabine, a novel fluoropyrimidine carbamate, is an orally administered, tumor-selective cytotoxic agent that is converted to 5FU by three enzymes. 9 After oral administration, capecitabine is converted to 5Ј-deoxy-5-fluorocytidine (5ЈDFCR) by carboxylesterase mainly located in the liver, to 5Ј-deoxy-5-fluorouridine (5ЈDFUR) by cytidine deaminase in the liver and tumors and finally to 5FU and its active metabolites by thymidine phosphorylase (TP) in various tumors. It has been shown that TP expression or the TP/dihydropyrimidine dehydrogenase (DPD) ratio correlates well with the sensitivity to capecitabine in human cancer xenograft models, suggesting that levels of these enzymes would be a predictive factor for capecitabine. 10 The purpose of the present study was to examin...

Modulation of thymidine phosphorylase by neoadjuvant chemotherapy in primary breast cancer

“…Many basic and clinical studies have indicated that 5-FU-contaning regimens are more effective for TP-overexpressing tumour cells as compared with TP-less-expressing tumour cells (Fox et al, 1997;Evrard et al, 1999;Gasparini et al, 1999;Morita et al, 2001;Yang et al, 2002). Therefore, these two scenarios should be further studied.…”

“…Experimental studies showed that 5 0 -DFUR is much more active in TP-transfected cells than in mocktransfected cells (Patterson et al, 1995;Evrard et al, 1999). It is also true that 5 0 -DFUR is more effective for TP-overexpressing tumour xenografts than for tumour xenografts expressing normal or low levels of TP (Morita et al, 2001;Ishikawa et al, 1998). Furthermore, several preliminary studies also confirmed that TP expression in tumour cells was a predictive factor for favourable prognosis in cancer patients treated with 5 0 -DFUR (Yamamoto et al, 1996;Ishii et al, 1996;Koizumi et al, 1999;Nishimura et al, 2002).…”

Forced Expression of Cytidine Deaminase Confers Sensitivity to Capecitabine

“…We previously transfected TP cDNA into the human renal cancer cell line KU2 to examine its sensitivity to fluoropyrimidines, and showed that transfection of TP cDNA made the cells sensitive to 5′DFUR and capecitabine [36]. Our previous study suggests that capecitabine exerts an antitumor effect primarily on cells with high TP expression.…”

“…Several studies with the transfection of TP cDNA into various cancer cell lines showed that TP plays an important role in the activation of 5′DFUR [35]and capecitabine [36]. We previously transfected TP cDNA into the human renal cancer cell line KU2 to examine its sensitivity to fluoropyrimidines, and showed that transfection of TP cDNA made the cells sensitive to 5′DFUR and capecitabine [36].…”

Genetic factors influencing Pyrimidine-antagonist chemotherapy

“…A clear correlation was found between TP activity and in vitro sensitivity to the 5-FU analogue 5 0 -dFUR, and to a lesser extent to 5-FU itself. [76][77][78][79][80][81] Marchetti et al 76 also studied the effect of TP overexpression on in vivo tumour growth in a rat model. The impact on tumour growth turned out to be relatively modest and only involved the initial stages of tumour growth.…”