Mechanistic Insights into Radioprotection by Caffeine and Understanding Impact of Caffeine on Nonhomologous End Joining: Implications in Cancer

Abstract

Maintenance of genome integrity is of utmost importance for the survival of any organism. Genetic instability is usually associated with various disorders, and it often leads to premature aging and predisposition to various types of cancer. Human genome is subjected to constant damage by several endogenous (ROS, replication stress, base mismatch, alkylation/hydrolysis of bases) and exogenous (naturally occurring isotopes, ionizing radiation, UV rays, chemical exposure) DNA damaging factors. Among various exogenous sources, radiation is the most commonly encountered and a significant contributor of DNA damage inside cells. Ionizing radiation can induce DNA damage in two ways: directly, wherein DNA breaks occur due to the linear transfer of energy to the phosphodiester bonds, or indirectly by radiolysis of water molecules, subsequently resulting in the production of free radicals or reactive oxygen species, which further induces DNA breaks. Caffeine (1, 3, 7-trimethylxanthine) is one of the most widely and commonly consumed stimulant across the world. Caffeine belongs to the methylxanthine class and has been the most extensively studied methylxanthine to date. Its role as an antioxidant has been extensively investigated over the past several years and it has been shown that caffeine can scavenge the reactive oxygen species (ROS) such as hydroxyl free radical and alkoxyl radical by radical adduct formation mechanism. Since free radicals and reactive oxygen species form one of the important intermediates in radiation-induced toxicity within cells, we aimed to explore that whether caffeine could impart protection against ionizing radiation induced DNA damage owing to its antioxidant property and could serve as a radioprotectant. In the first part of my study, we assessed the impact of caffeine on radiation-induced DNA damage using in vitro, ex vivo, and in vivo model systems. Several lines of experimentations performed in the presence of increasing doses of caffeine using single-stranded and double-stranded oligomeric DNA substrates as well as the plasmid DNA indicated that caffeine imparted reduced radiosensitivity when exposed to irradiation (IR). In-depth analysis revealed that the observed protection is due to reduced induction of single- and double-strand DNA breaks upon exposure to ionizing radiation. Using ex vivo model system we observed that the treatment of cells with caffeine prior to irradiation exposure resulted in reduction of DNA double-/single-strand breaks formation inside the cell. By using immunofluorescence and neutral comet assay, we observed that the level of DNA damage induced in caffeine treated mammalian cells is significantly less as compared to the untreated control upon exposure to IR. This suggests that presence of caffeine significantly reduces the level of DNA damage induced in mammalian cells upon IR treatment. We have also shown that caffeine can efficiently quench the reactive oxygen species (ROS) generated via H2O2 treatment or exposure to IR in mammalian cells. Finally, we showed that lower concentration 10 of caffeine protects the cell from the cytotoxic effects post irradiation owing to reduction of DNA strand breaks formation. Using in vivo model system, we further showed that caffeine treatment in mice extended their lifespan, as compared to untreated control upon exposure to irradiation. In our study, the extension of lifespan in caffeine-treated mice groups was achieved with a much lower dose than that used in a previous study, where a tenfold higher dose of caffeine was required to reduce radiosensitivity (George et al., 1999). Thus, the results from our study suggest that caffeine reduces the level of DNA damage induction upon IR exposure and therefore imparts protection to the genome. To counteract the threats posed by DNA damage and faithful transmission of genetic material, mammalian cells have evolved different mechanisms to repair the damaged DNA, collectively termed as the DNA damage response (DDR) – which helps to detect DNA lesions, signal their presence, and promote their repair. Various DNA repair mechanisms are employed by mammalian cells for the repair of the damaged DNA, including base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), and double-strand break repair pathways depending on the type of DNA damage. The DNA double-strand break repair mechanism encompasses homologous recombination (HR), nonhomologous end joining (NHEJ), and the recently discovered microhomology-mediated end joining (MMEJ) or Alt-NHEJ. HR requires genetic information from the homologous DNA sequence of the sister chromatid for repair and occurs in the S and G2 phases of the cell cycle. However, NHEJ involves the direct joining of the broken DNA ends and is error-prone but is active throughout the cell cycle. Alternative NHEJ (Alt-NHEJ), considered as a backup pathway to canonical NHEJ, utilizes small regions of microhomology (5–25 nucleotides) and is therefore referred to as microhomology-mediated end joining (MMEJ). This repair pathway is highly mutagenic and employs a different set of proteins as compared to classical NHEJ, including the MRN complex, PARP1, Pol θ, and Ligase III/XRCC1. Caffeine is an analogue of adenosine and has been extensively used as a tool to study DNA repair signalling. It has been reported that caffeine inhibits the in vitro protein kinase activity of ATM (Ataxia-telangiectasia-mutated) and ATR (Ataxia-telangiectasia and RAD3-related). It has also been demonstrated that caffeine treatment results in dose-dependent eviction of Rad51 from ssDNA and thereby inhibit the repair by homologous recombination. DNA-PKcs which is also a member of PI3KK family, a key protein involved in NHEJ, is shown to be inhibited in vitro by caffeine. Thus, although a few other studies suggest that caffeine could play a role in DNA repair inhibition, its effect on NHEJ is poorly understood. In the second part of my study, we investigated and tried to understand the impact of caffeine on NHEJ mediated repair and its effect on cancer. The results of our study showed that the exposure to the caffeine led to a significant reduction in DNA end joining when 11 oligomeric DNA substrate harbouring DSBs were incubated with cell-free extract prepared from rat testes or mammalian cancer cell lines. Different double-stranded DNA substrates containing compatible and non-compatible ends were used for the end-joining assays. Results showed that irrespective of DNA end sequences and the configuration of DNA ends a consistent inhibition of NHEJ was observed. Specifically, a dose-dependent decrease in the end joined product with the increasing concentration of caffeine was observed. Further, an extrachromosomal assay system was used to evaluate the effect of caffeine on NHEJ within mammalian cells. The results showed evidence for the inhibition of NHEJ at the intracellular level. Results of the in silico, biophysical and biochemical assays indicated that XRCC4 protein could be the target of caffeine when it inhibits the NHEJ. Thus, our results suggest that caffeine inhibits NHEJ-mediated repair of DSBs. Since caffeine is the most commonly consumed methylxanthine globally and more than half of the population consumes it in a day-to-day manner through various dietary sources, particularly through coffee, we were interested in investigating whether the caffeine in coffee decoction has the same inhibitory effect on NHEJ mediated repair mechanisms as purified caffeine. Similar results were observed in vitro and in vivo scenario when NHEJ assays were performed using coffee decoction, suggesting that caffeine in coffee decoction has an inhibitory effect on the NHEJ mediated repair. Based on the current findings revealing the inhibitory effect on NHEJ by caffeine, we were eager to investigate whether this inhibition of the double-strand break repair pathway will have any impact on combating cancer. The results of Trypan blue as well as the Alamar blue assays suggest that there was a dose-dependent increase in cell death upon treatment of caffeine as well as coffee decoction in various cell lines. The results of our study were comparable with that of previous studies. Using immunofluorescence assay, Propidium Iodide staining assay, JC1 assay as well as Annexin-PI assay, we showed that there is a dose-dependent increase in DNA breaks and, thereby leading to cell death when treated with increasing concentration of caffeine or coffee decoction. Further, using the mice tumor models, we showed that caffeine post-treatment (10 mg/Kg and 50 mg/Kg body weight) results in a significant decrease in the rate of tumor development as compared to the untreated control. Interestingly, in another experiment, when caffeine (20 mg/Kg body weight) was pre-treated for continuous 21 days prior to tumor development, we observed a significant reduction in the rate of tumor progression in the pre-treated animal group as compared to the untreated control group. Similar results were also observed when mice were pre-treated with 20 mg/Kg caffeine equivalent dose of coffee decoction. These results indicate that caffeine consumption could have a positive impact on reducing the risk of cancer development. Taken together, we characterized the property of cellular radioprotection mediated by an exogenous source, caffeine. In addition, our findings establish caffeine as a biochemical 12 inhibitor of NHEJ mediated repair. In alignment with the previous reported studies, we also demonstrated the anti-cancer potential of the world’s most commonly consumed neuro-stimulant, caffeine. In summary, it appears that intake of caffeine at very low dose (< 1mM) can protect human genome from radiation induced damage whereas its consumption at very high concentration may lead to inhibition of DNA repair resulting in the induction of DNA breaks. Although excess generation of DNA breaks are not good for a normal cell, but when present in a cancerous cell it may play a beneficial role in inducing the apoptosis.