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01.12.2018 | Research article | Ausgabe 1/2018 Open Access

BMC Cancer 1/2018

MTH1 deficiency selectively increases non-cytotoxic oxidative DNA damage in lung cancer cells: more bad news than good?

Zeitschrift:
BMC Cancer > Ausgabe 1/2018
Autoren:
Hussein H. K. Abbas, Kheloud M. H. Alhamoudi, Mark D. Evans, George D. D. Jones, Steven S. Foster
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12885-018-4332-7) contains supplementary material, which is available to authorized users.

Abstract

Background

Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells. However, findings from previous MTH1 studies have been contradictory, meaning the relevance of MTH1 in cancer is still to be determined. Here we ascertained the role of MTH1 specifically in lung cancer cell maintenance, and the potential of MTH1 inhibition as a targeted therapy strategy to improve lung cancer treatments.

Methods

Using siRNA-mediated knockdown or small-molecule inhibition, we tested the genotoxic and cytotoxic effects of MTH1 deficiency on H23 (p53-mutated), H522 (p53-mutated) and A549 (wildtype p53) non-small cell lung cancer cell lines relative to normal MRC-5 lung fibroblasts. We also assessed if MTH1 inhibition augments current therapies.

Results

MTH1 knockdown increased levels of oxidatively damaged DNA and DNA damage signaling alterations in all lung cancer cell lines but not normal fibroblasts, despite no detectable differences in reactive oxygen species levels between any cell lines. Furthermore, MTH1 knockdown reduced H23 cell proliferation. However, unexpectedly, it did not induce apoptosis in any cell line or enhance the effects of gemcitabine, cisplatin or radiation in combination treatments. Contrastingly, TH287 and TH588 MTH1 inhibitors induced apoptosis in H23 and H522 cells, but only increased oxidative DNA damage levels in H23, indicating that they kill cells independently of DNA oxidation and seemingly via MTH1-distinct mechanisms.

Conclusions

MTH1 has a NSCLC-specific p53-independent role for suppressing DNA oxidation and genomic instability, though surprisingly the basis of this may not be reactive-oxygen-species-associated oxidative stress. Despite this, overall our cell viability data indicates that targeting MTH1 will likely not be an across-the-board effective NSCLC therapeutic strategy; rather it induces non-cytotoxic DNA damage that could promote cancer heterogeneity and evolution.
Zusatzmaterial
Additional file 1: Endogenous MTH1 levels are similar in H23, H522, A549 and MRC-5 cell lines. MTH1 band intensities in no siRNA samples were normalized to corresponding α-Tubulin loading control bands (see Fig. 1 for representative Western blot images). Mean values and SD were calculated from the normalised values of independent experiments. Numbers of independent experiments (n) are indicated. Error bars represent SD. (PDF 221 kb)
Additional file 5: MTH1 knockdown with another siRNA similarly does not induce apoptosis in H23 cells. a Western blots to determine MTH1 protein levels in H23 cell cultures grown in media without transfection reagent (no siRNA), or following transfection with MTH1 siRNA (ThermoFisher Scientific, S194633, oligonucleotide 5′- > 3′ sequences were sense UUAACUGGAUGGAAGGGAAtt and antisense AUCCAGUUAAUUCCAGAUGaa) or scramble siRNA. Representative day 4 blot shown. Day 4 MTH1 band intensities were normalized to corresponding α-Tubulin loading control bands, and then siRNA samples were normalised to corresponding no siRNA bands. Numbers of independent experiments (n) are indicated. Mean values were calculated from the normalised values of the independent experiments. Error bars represent SD. Asterisks represent a significant difference between MTH1 siRNA and corresponding no siRNA normalised values (**P < 0.01, *P < 0.05). b Apoptosis assay to determine cell viability of H23 cells cultured for 4 days in media without transfection reagent (no siRNA), or following transfection with MTH1 siRNA (S194633) or scramble siRNA. Harvested cells were dual stained with annexin V-FITC/PI and assessed by flow cytometry to detect both early and late apoptosis. 2 days after transfection, 0.01 μM gemcitabine (Gem) or 5 μM cisplatin (Cis) were added to the appropriate cultures for the remaining 48 h. Positive control of 48 h VP-16 treatment (+ve) also included (n = 1). (PDF 436 kb)
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