In vitro single-strand DNA damage and cancer cell cytotoxicity effects of Temozolomide

Abstract

© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Temozolomide (TMZ) has been approved against certain tumors, but substantial evidence is lacking to demonstrate TMZ as a generator of single-strand break in vitro DNA and cell-based assay. Presently, we have attempted to elucidate detailed evidence that TMZ is able to generate extensive single-strand break on DNA substrates. The cell-based cytotoxicity and DNA content was evaluated for TMZ to corroborate its single-strand break activity. We employed plasmid and genomic DNA as a substrate during assessment of in vitro single-strand break potential of TMZ. Further, we tried to evaluate DNA fragmentation, DNA content, and overall cytotoxicity, and DNA ligase III protein level in HeLa and MCF-7 using MTT, Trypan blue exclusion, apoptotic DNA fragmentation, fluorescent microscopy, and immunoblot assay. Our observations indicate that TMZ is able to generate extensive single-strand breaks on different DNA substrate. For cytotoxicity assay, L189 augmented the effects of TMZ against HeLa cells from 36% to 50% cell death, while MCF-7 was resistant. However, we did not notice any apoptotic DNA fragments due to toxicity effects of TMZ in both cell types. Furthermore, we observed higher DNA ligase III among HeLa and MCF-7 in comparison with normal epithelial cells. We suggest differential response of HeLa and MCF-7 against the combinatorial effects of TMZ and L189, which may be due to their distinct single-strand break repair. The findings support combinatorial treatment using TMZ and repair protein inhibitors to enhance cytotoxicity.