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Potent anticancer effects of lentivirus encoding a Drosophila melanogaster deoxyribonucleoside kinase mutant combined
Nian-Qu Zhang1, Lei Zhao, Shuai Ma
1Department of Surgical Oncology, First Affiliated Hospital, Center of Experiment Technology and Medical Research, Lab 1, Cancer Institute, China Medical University, Shenyang, China.
Objective:
Deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) mutants have been reported to exert suicide gene effects in combined gene/chemotherapy of cancer. Here, we aimed to further evaluate the capacity of the mutanted enzyme and its potential for inhibiting cancer cell growth.
Methods:
We altered the sequence of the last 10 amino acids of Dm-dNK to perform site-directed mutagenesis and constructed active site mutanted Dm-dNK (Dm-dNKmut), RT-PCR and western bloting studies were used to reveal the expression of 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, cell cytotoxicity was assessed by MTT assays, cell proliferation using a hemocytometer and apoptosis induction by thenannexin-V-FITC labeled FACS method. In vivo, an animal study was set out in which BALB/C nude mice bearing tumors were treated with lentivirus mediated expression of Dm-dNKmut with the pyrimidine nucleoside analog brivudine (BVDU, (E)-5-(2-bromovinyl)-(2-deoxyuridine).
Results:
The Dm-dNKmut could be stably expressed in the cancer cell lines and retained its enzymatic activity. Moreover, the cells expressing Dm-dNKmut exhibited increased sensitivity in combination with BVDU, with induction of apoptosis in vitro and in vivo.
Conclusion:
These findings underlined the importance of BVDU phosphorylated by Dm-dNKmut in transduced cancer cells and the potential role of Dm-dNKmut as a suicide gene, thus providing the basis for future intensive research for cancer therapy.
Insights
Mutant Deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNKmut) shows potential as a suicide gene. This enzyme enhances cancer cell sensitivity to brivudine (BVDU), inducing apoptosis in vitro and in vivo for future cancer therapy.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) mutants have shown promise in cancer therapy.
- Further evaluation of the mutated enzyme's capacity and its potential for inhibiting cancer cell growth is warranted.
Purpose of the Study:
- To evaluate the capacity of a mutated Dm-dNK (Dm-dNKmut) enzyme.
- To assess its potential for inhibiting cancer cell growth, particularly in combination with chemotherapy.
Main Methods:
- Site-directed mutagenesis was used to create Dm-dNKmut.
- Lentivirus-mediated expression of Dm-dNKmut was confirmed in breast, gastric, and colorectal cancer cell lines.
- Enzyme activity, cytotoxicity, proliferation, and apoptosis were assessed in vitro and in vivo using brivudine (BVDU).
Main Results:
- Dm-dNKmut was stably expressed in cancer cell lines with retained enzymatic activity.
- Cells expressing Dm-dNKmut showed increased sensitivity to BVDU.
- Apoptosis was induced in cancer cells both in vitro and in vivo.
Conclusions:
- BVDU phosphorylation by Dm-dNKmut in transduced cancer cells is crucial.
- Dm-dNKmut holds potential as a suicide gene for cancer therapy.
- These findings provide a basis for further research in cancer treatment.
