Karonudib is a promising anticancer therapy in hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is generally caused by viral infections or consumption of mutagens, such as alcohol. While liver transplantation and hepatectomy is curative for some patients, many relapse into disease with few treatment options such as tyrosine kinase inhibitors, for example, sorafenib or lenvatinib. The need for novel systemic treatment approaches is urgent. Methods: MTH1 expression profile was first analyzed in a HCC database and MTH1 mRNA/protein level was determined in resected HCC and paired paracancerous tissues with polymerase chain reaction (PCR) and immunohistochemistry. HCC cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA. 8-oxoG was measured by the modified comet assay. The effect of MTH1 inhibition on tumor growth was explored in HCC xenograft in vivo models. Results: MTH1 protein level is elevated in HCC tissue compared with paracancerous liver tissue and indicates poor prognosis. The MTH1 inhibitor Karonudib (TH1579) and siRNA effectively introduce toxic oxidized nucleotides into DNA, 8-oxoG, and kill HCC cell lines in vitro. Furthermore, we demonstrate that HCC growth in a xenograft mouse model in vivo is efficiently suppressed by Karonudib. Conclusion: Altogether, these data suggest HCC relies on MTH1 for survival, which can be targeted and may open up a novel treatment option for HCC in the future

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

Representative images of “Comets” and the corresponding intensity profiles, showing (i) ~ 5% Tail DNA damage, typical of the NSCLC cells treated with no siRNA or scramble siRNA, and analysed by regular Fpg-modified alkaline comet assay (0.8 U Fpg/gel); and (ii) comets showing ~ 10% tail DNA, typical of the NSCLC cells treated with MTH1 siRNA. Superimposed on the Comet images are the image analysis software (Komet 5.5, Andor Technology) determined boundaries demarcating the ‘Comet head’ (pink circle) and ‘tail extent’ (vertical orange line) (Barber RC, Hickenbotham P, Hatch T, Kelly D, Topchiy N, Almeida GM, et al. Radiation-induced transgenerational alterations in genome stability and DNA damage. Oncogene. 2006;25(56):7336–7342). % tail DNA = 100 - % head DNA; % head DNA = (integrated optical head intensity / (integrated optical head intensity + integrated optical tail intensity)) × 100. (PDF 1431 kb

A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family

The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality

Targeting of mutant hogg1 in mammalian mitochondria and nucleus: effect on cellular survival upon oxidative stress

BACKGROUND: Oxidative damage to mitochondrial DNA has been implicated as a causative factor in a wide variety of degenerative diseases, aging and cancer. The modified guanine, 7,8-dihydro-8-oxoguanine (also known as 8-hydroxyguanine) is one of the major oxidized bases generated in DNA by reactive oxygen species and has gained most of the attention in recent years as a marker of oxidative DNA injury and its suspected role in the initiation of carcinogenesis. 8-hydroxyguanine is removed by hOgg1, a DNA glycosylase/AP lyase involved in the base excision repair pathway. METHODS: We over-expressed wild type and R229Q mutant hOGG1 in the nucleus and mitochondria of cells lacking mitochondrial hOGG1 expression through an expression vector containing nuclear and mitochondrial targeting sequence respectively. We used quantitative real time PCR to analyze mtDNA integrity after exposure to oxidative damaging agents, in cells transfected with or without mitochondrially-targeted mutant hogg1. RESULT: Over-expression of wild type hOgg1 in both nucleus and mitochondria resulted in increased cellular survival when compared to vector or mutant over-expression of hOGG1. Interestingly, mitochondrially-targeted mutant hogg1 resulted in more cell death than nuclear targeted mutant hogg1 upon exposure of cells to oxidative damage. Additional we examined mitochondrial DNA integrity after oxidative damage exposure using real-time quantitative PCR. The presence of mutant hogg1 in the mitochondria resulted in reduced mitochondrial DNA integrity when compared to the wild type. Our work indicates that the R229Q hOGG1 mutation failed to protect cells from oxidative damage and that such mutations in cancer may be more detrimental to cellular survival when present in the mitochondria than in the nucleus. CONCLUSION: These findings suggest that deficiencies in hOGG1, especially in the mitochondria may lead to reduced mitochondrial DNA integrity, consequently resulting in decreased cell viability

Pivotal Role of Inosine Triphosphate Pyrophosphatase in Maintaining Genome Stability and the Prevention of Apoptosis in Human Cells

Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer

Balancing repair and tolerance of DNA damage caused by alkylating agents

Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for a favourable response of an organism to alkylating agents. Furthermore, the response of an individual to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity