Potent Anticancer Effects of Lentivirus Encoding a Drosophila Melanogaster Deoxyribonucleoside Kinase Mutant Combined with Brivudine

Abstract

<br/><b>Objective</b>: Deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) mutants have been reportedto exert suicide gene effects in combined gene/chemotherapy of cancer. Here, we aimed to further evaluate thecapacity of the mutanted enzyme and its potential for inhibiting cancer cell growth. <br/><b>Methods</b>: We altered thesequence of the last 10 amino acids of Dm-dNK to perform site-directed mutagenesis and constructed activesite mutanted Dm-dNK (Dm-dNKmut), RT-PCR and western bloting studies were used to reveal the expressionof lentivirus mediated Dm-dNKmut in a breast cancer cell line (Bcap37), a gastric cancer cell line (SGC7901)and a colorectal cancer cell line (CCL187). [3H]-labeled substrates were used for enzyme activity assays, cellcytotoxicity was assessed by MTT assays, cell proliferation using a hemocytometer and apoptosis inductionby thenannexin-V-FITC labeled FACS method. In vivo, an animal study was set out in which BALB/C nudemice bearing tumors were treated with lentivirus mediated expression of Dm-dNKmut with the pyrimidinenucleoside analog brivudine (BVDU, (E)-5-(2-bromovinyl)-(2-deoxyuridine). <br/><b>Results</b>: The Dm-dNKmut couldbe stably expressed in the cancer cell lines and retained its enzymatic activity. Moreover, the cells expressingDm-dNKmut exhibited increased sensitivity in combination with BVDU, with induction of apoptosis in vitroand in vivo. <br/><b>Conclusion</b>: These findings underlined the importance of BVDU phosphorylated by Dm-dNKmutin transduced cancer cells and the potential role of Dm-dNKmut as a suicide gene, thus providing the basis forfuture intensive research for cancer therapy.