Lentivirus-mediated expression of Drosophila melanogaster deoxyribonucleoside kinase driven by the hTERT promoter combined with gemcitabine: A potential strategy for cancer therapy

Abstract

In contrast to other enzymes, Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) has a broad substrate specificity and high catalytic rate when transferred in human cells. This makes it a promising therapeutic agent when administered together with several cytotoxic nucleoside analogs, such as gemcitabine 2',2'-difluoro-deoxycytidine (dFdC). Therefore, lentiviral vectors, which potentially allow stable long-term transgene expression, are good candidates for gene delivery vehicles. In the present study, we successfully developed a lentivirus-mediated transgene expression system of Dm-dNK under the control of hTERT promoter against the breast cancer cell line (Bcap37), the gastric cancer cell line (SGC7901) and the normal fibroblast cell line (WI-38). Moreover, we also analyzed its targeted cytotoxicity in vitro with treatment of the prodrug dFdC. Bcap37 tumor growth was inhibited in nude mice. Both cancer cell lines exhibited apparent cytotoxicity when infected with recombinant lentivirus constructs expressing Dm-dNK. In contrast, lentivirus-infected WI-38 cells exhibited less cytotoxicity. These data suggested that Dm-dNK was sensitive to dFdC, and it resulted in synergistic growth inhibition and apoptosis induction in vitro. In addition, Lenti-hTERT-dNK/dFdC also suppressed tumor growth in vivo. Our results suggest that the Lenti-hTERT-dNK/dFdC system is a safe and feasible treatment strategy in the development of suicide gene therapy.

Recombinant deoxyribonucleoside kinase from Drosophila melanogaster can improve gemcitabine based combined gene/chemotherapy for targeting cancer cells

“…The truncated variant A significant increase in the sensitivity of transfected cell lines to gemcitabine was observed, as shown by a decrease in IC 50 compared with control group. The IC 50 and folds of higher than previously reported for human uterine sarcoma cell line Messa 10K, where a full-length DmdNK was transfected to sensitize human cancer cell lines to various nucleosides [9,[41][42][43][50][51][52]. A possible explanation for the increased sensitivity to gemcitabine is the lower K m value of DmdNKΔC20 for this analog and unique interaction of fluoride atoms of gemcitabine [40].…”

“…DmdNK and its improved variants, produced by site-directed mutagenesis, have been tested for their optimum application in suicide gene therapy [38][39][40]. Both viral (adeno-and lentivirus-based vectors) and non-viral delivery systems have been used for transfecting different cancer cells with DmdNK and its mutants, to study the combined cytotoxic effect of gene/prodrug and to reverse drug resistance in various cancer cell lines [9,[41][42][43]. To the best of our knowledge, DmdNKΔC20 has not been reported neither for sensitizing human cancer cell lines to gemcitabine nor for the reversal of gemcitabine resistance in drug-resistant cancer cells so far.…”

Therapeutic strategies for targeting telomerase in cancer

“…A novel replication adenovirus, Ad‐hTERT‐E1A, in which the promoter of hTERT controls the selective replication of vectors in various tumor cells has been reported . In addition, Zhang et al successfully developed a Lenti‐hTERT‐dNK/2′,2′‐difluoro‐deoxycytidine (dFdC) system, which suppressed Bcap37 tumor growth and exhibited apparent cytotoxicity in vitro, as well as less cytotoxicity in normal fibroblast cell lines (WI‐38). Furthermore, Yu et al constructed a Lenti‐hTERT‐CD/GFP/5‐flucytosine (5‐FC) system that significantly reduced cell proliferation by prodrug 5‐FC in the telomerase‐positive tumor cells.…”

Efficacy of lentivirus‑mediated Drosophila�melanogaster deoxyribonucleoside kinase combined with (E)‑5‑(2‑bromovinyl)‑2'‑deoxyuridine or 1‑β‑D‑arabinofuranosylthymine therapy in human keloid fibroblasts

“…Notably, the lentiviral vector has been identified to facilitate the incorporation of target genes into the host genome for efficient and stable expression of target genes in dividing and non-dividing cells (11,12). In the present study, a lentivirus vector was used to express the Dm-dNK suicide gene in keloid fibroblasts (KF).…”