Apoptotic induction with bifunctional E.coli cytosine deaminase-uracil phosphoribosyltransferase mediated suicide gene therapy is synergized by curcumin treatment in vitro.
Development of novel suicide gene therapy vector with potential application in cancer treatment has a great impact on human health. Investigation to understand molecular mechanism of cell death is necessary to evaluate the therapeutic application of suicide vectors. For example, the bifunctional E.coli cytosine deaminase & uracil phosphoribosyltransferase fusion (CD-UPRT) gene expression is known to sensitize a wide range of cells toward nontoxic prodrug 5-flurocytosine (5-FC) by converting it to toxic compounds, but the exact pathway of cell death is yet to be defined. Herein, we investigated the mechanism of cell death by 5-FC/CD-UPRT suicide system in both cancer and non-cancer cells and found that the optimum 5-FC concentration led to programmed cell death in vitro. The CD-UPRT expression of transfected cells was measured by the RT-PCR analysis. Biochemical assays, such as mitochondrial activity (MTS) and lactate dehydrogenase (LDH) measurements exhibited cell death. Microscopic experiments showed characteristic onset of apoptosis which was further supported by internucleosomal DNA cleavage of BrdU labeled cellular DNA, appearance of characteristic laddering of chromosomal DNA and involvement of caspase pathway. Furthermore, the 5-FC/CD-UPRT-mediated apoptosis was potentiated with addition of a known anticancer agent curcumin. Our in vitro studies confirmed synergistic induction of apoptotic pathway in the combination treatment. Therefore, combination of 5-FC/CD-UPRT with curcumin could be a potential chemosensitization strategy for cancer treatment.
Gopinath
P
P
Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039, India.
Ghosh
Siddhartha Sankar
SS
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003474:Curcumin / Q000494:pharmacology*; D043525:Cytosine Deaminase / Q000378:metabolism*; D053938:DNA Fragmentation / Q000187:drug effects; D004357:Drug Synergism; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D005437:Flucytosine / Q000494:pharmacology; D020869:Gene Expression Profiling; D018014:Gene Transfer Techniques; D041722:Genes, Transgenic, Suicide; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D010430:Pentosyltransferases / Q000378:metabolism*
D049452:Green Fluorescent Proteins; D005437:Flucytosine; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D020169:Caspases; D043525:Cytosine Deaminase; D003474:Curcumin