Artikel
The Effect of Src Kinase Inhibition on 5-Fluorouracil Chemosensitivity Is Related to Thymidylate Synthase Expression in Human Pancreatic Carcinoma Cells
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Veröffentlicht: | 20. März 2006 |
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Gliederung
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5-Fluorouracil (5-FU) is a widely used chemotherapeutic agent that inhibits cancer cell growth and initiates apoptosis by targeting thymidylate synthase (TS) leading to direct incorporation of 5-FU metabolites into DNA and RNA. Over-expression of TS is strongly associated with the resistance of tumor cells to TS inhibitors. Attempts to modulate 5-FU cytotoxicity have focused primarily on enhancing TS inhibition. We tested the hypothesis that Src tyrosine kinase inhibition could augment the chemosensitivity of 5-FU-resistant human pancreatic cancer cells to 5-FU. The role of the c-src proto-oncogene product was implicated in our study by the observation that the efficacy of cytotoxic agents in chemoresistant tumors is significantly enhanced by the co-administration of Src kinase inhibitors.
Pancreatic carcinoma cells AsPC and AsPCres, a stably 5-FU-resistant subline of AsPC, were exposed to combinations of 5-FU and Src tyrosine kinase inhibitors PP2 or AZM475271 (AZM475271 was kindly provided by Astra Zeneca). Src expression, phosphorylation, and activity were analyzed by immunoblotting and ELISA-based Src kinase inhibition test. Cellular proliferation was quantified by MTT Viability Assay Kit. Quantification of apoptosis was performed using propidium iodide staining for cell cycle analysis by FACS. Expression of TS protein was analyzed by Western Blot. The therapeutic efficacy of 5-FU in combination with PP2 was also tested in nude mice orthotopically xenografted with AsPCres cells. TS and Src expression was determined using IHC.
After repeated treatment of AsPC cells with 5-FU, the IC50 value increased 4-fold, compared to the untreated ASPC cell line. Combination therapy reflected the chemotherapeutic sensitivity of this cell line towards 5-FU. TS protein was overexpressed in AsPCres cells compared to the sensitive ASPC cells (2-fold), however, following treatment with Src kinase inhibitors the TS over-expression decreased significantly. Src kinase inhibition augmented 5-FU-induced apoptosis in AsPCres in vitro and in vivo. In the orthotopic pancreatic cancer nude mouse model PP2 in combination with 5-FU substantially decreased tumor growth and inhibited metastasis.
5-FU resistance has been shown to be reversed via TS regulation by targeting Src tyrosine kinase. Collectively, our findings make a strong augment for pursuing a strategy combining 5-FU with Src inhibitors, at least in pancreatic cancer cells displaying resistance to this chemotherapeutical agent.