In Silico Investigation of the Biological Implications of Complex DNA Damage with Emphasis in Cancer Radiotherapy through a Systems Biology Approach

Different types of DNA lesions forming in close vicinity, create clusters of damaged sites termed as “clustered/complex DNA damage” and they are considered to be a major challenge for DNA repair mechanisms resulting in significant repair delays and induction of genomic instability. Upon detection of DNA damage, the corresponding DNA damage response and repair (DDR/R) mechanisms are activated. The inability of cells to process clustered DNA lesions efficiently has a great impact on the normal function and survival of cells. If complex lesions are left unrepaired or misrepaired, they can lead to mutations and if persistent, they may lead to apoptotic cell death. In this in silico study, and through rigorous data mining, we have identified human genes that are activated upon complex DNA damage induction like in the case of ionizing radiation (IR) and beyond the standard DNA repair pathways, and are also involved in cancer pathways, by employing stringent bioinformatics and systems biology methodologies. Given that IR can cause repair resistant lesions within a short DNA segment (a few nm), thereby augmenting the hazardous and toxic effects of radiation, we also investigated the possible implication of the most biologically important of those genes in comorbid non-neoplastic diseases through network integration, as well as their potential for predicting survival in cancer patients.     

Differential response induced by exposure to low-dose ionizing radiation in SHSY-5Y and normal human fibroblast cells

Radiation therapy has been used in the treatment of a wide variety of cancers for nearly a century and is one of the most effective ways to treat cancer. Low-dose ionizing radiation (IR) can interfere with cell division of cancer and normal cells by introducing oxidative stress and injury to DNA. The differences in the response to IR-induced DNA damage and increased reactive oxygen species between normal human fibroblasts (NHFs) and cancerous SHSY-5Y cells were considered. H2AX staining and comet assays revealed that NHF cells responded by initiating a DNA repair sequence whereas SHSY-5Y cells did not. In addition, NHF cells appeared to quench the oxidative stress induced by IR, and after 24 h no DNA damage was present. SHSY-5Y cells, however, did not repair their DNA, did not quench the oxidative stress, and showed characteristic signs that they were beginning to undergo apoptosis. These results indicate that there is a differential response between this cancerous and normal cell line in their ability to respond to low-dose IR, and these differences need to be exploited in order to treat cancer effectively. Further study is needed in order to elucidate the mechanism by which SHSY-5Y cells undergo apoptosis following radiation and why these normal cells are better equipped to deal with IR-induced double-strand breaks and oxidative stress.     

Enhanced radiation-induced cell killing by Herbimycin A pre-treatment

Herbimycin A (HA), as in Geldanamycin, binds to conserved pockets of heat shock protein 90 (Hsp90) and inhibits its chaperone functions. Hsp90 plays an integral role in cancer cell growth and survival, because it maintains the stability of several key proteins by its chaperone's activity. It is known that some of the proteins associated with radiation responses are functionally stabilized by Hsp90. In this study, we investigated the effect of HA on radiosensitivity in human cancer cells and the mechanism related to the sensitization. In order to gain a mechanistic insight of this sensitization, we examined repair of DNA double-strand breaks (DSBs) in irradiated human cancer cells pre-treated with HA, as unrepaired DSBs are thought to be the main cause of radiation-induced cell death. Cellular radiosensitivity was determined by clonogenic assay, and the DSB rejoining kinetics was examined by constant field gel electrophoresis. SQ-5, a lung squamous carcinoma cell line, showed synergistic increase in radiosensitivity when cells were pre-treated with HA. In addition, HA significantly inhibited repair of radiation-induced DSBs. These results suggest that the combination of HA and ionizing radiation may be a useful therapeutic strategy for treating certain cancer cells.     

Targeting Mechanisms of the DNA Damage Response (DDR) and DNA Repair by Natural Compounds to Improve cAT-Triggered Tumor Cell Death

Recently, we identified secalonic acid F (SA), 5-epi-nakijiquinone Q (NQ) and 5-epi-ilimaquinone (IQ) as natural compounds (NC) affecting mechanisms of the DNA damage response (DDR). Here, we further characterized their effects on DDR, DNA repair and cytotoxicity if used in mono- and co-treatment with conventional anticancer therapeutics (cAT) (cisplatin (Cis), doxorubicin (Doxo)) in vitro. All three NC influence the phosphorylation level of selected DDR-related factors (i.e., pCHK1, pKAP1, pP53, pRPA32) in mono- and/or co-treatment. Both SA and NQ attenuate the Cis- and Doxo-induced G2/M-phase arrest and effectively stimulate caspase-mediated apoptosis. Notably, SA impacts DNA repair as reflected by enhanced steady-state levels of Cis-(1,2-GpG)-DNA adducts and Doxo-induced DNA double-strand breaks (DSB). Moreover, SA decreased the mRNA and protein expression of the homologous recombination (HR)-related DSB repair factors RAD51 and BRCA1. Both SA and NQ promote Cis- and Doxo-induced cytotoxicity in an additive to synergistic manner (CI ≤ 1.0). Summarizing, we conclude that SA promotes cAT-driven caspase-dependent cell death by interfering with DSB repair and DDR-related checkpoint control mechanisms. Hence, SA is considered as the most promising lead compound to evaluate its therapeutic window in forthcoming pre-clinical in vivo studies.     

miR-9 and let-7g enhance the sensitivity to ionizing radiation by suppression of NFκB1

The activation of nuclear factor-kappa B1 (NFkB1) in cancer cells may confer resistance to ionizing radiation (IR). To enhance the therapeutic efficiency of IR in lung cancer, we screened for microRNAs (miRNAs) that suppress NFkB1 and observed their effects on radiosensitivity in a human lung cancer cell line. From time series data of miRNA expression in γ-irradiated H1299 human lung cancer cells, we found that the expression of miR-9 was inversely correlated with that of NFκB1. Overexpression of miR-9 down-regulated the level of NFκB1 in H1299 cells, and the surviving fraction of γ-irradiated cells was decreased. Interestingly, let-7g also suppressed the expression of NFκB1, although there was no canonical target site for let-7g in the NFκB1 3' untranslated region. From these results, we conclude that the expression of miR-9 and let-7g could enhance the efficiency of radiotherapy for lung cancer treatment through the inhibition of NFκB1.     

recA-dependent DNA repair in UV-irradiated Escherichia coli

UV-radiation-induced lesions in DNA result in the formation of excision gaps, daughter-strand gaps (DSG) and double-strand breaks (DSB), which are repaired by several different mechanisms. Postreplication repair. The recA gene is a master gene that controls all of the pathways of postreplication repair. The repair of DSG proceeds by one pathway that is also recF dependent, and one pathway that is constitutive and independent of the recF and recBC genes. A small fraction of the recF recB-independent repair of DSG is dependent upon the umuC gene, and may define an error-prone pathway of postreplication repair. Unrepaired DSG can be converted to DSB, which are normally repaired by the RecBCD pathway. However, in the recBC sbcB background, these DSB are repaired by a recF-dependent process. The RecF pathways of postreplication repair appear to utilize DNA containing a single-stranded region (either a gap or a DSB with a single-stranded end), while the RecBCD pathway appears to utilize the blunt ends of duplex DNA to promote the recombinational repair of DSB. The polA gene (especially the 5'----3' exonuclease activity of DNA polymerase I) functions in pathways of postreplication repair (both for the repair of DSG and DSB) that are largely independent of the recF gene. Nucleotide excision repair. The repair of excision gaps is independent of the recA gene in cells with unreplicated chromosomes, but is recA dependent in cells with partially replicated chromosomes at the time of UV irradiation. This recA-dependent repair of excision gaps appears to be analogous to the recF- and recB-dependent pathways of postreplication repair, i.e. the RecF pathway repairs DNA gaps, and the RecBCD pathway repairs the DSB that arise at unrepaired gaps.     

Molecular Characterization of DNA Double Strand Breaks with Tip‐Enhanced Raman Scattering

DNA double strand breaks (DSBs) are deadly lesions that can lead to genetic defects and cell apoptosis. Techniques that directly detect DNA DSBs include scanning electron microscopy, atomic force microscopy (AFM), and fluorescence based approaches. While these techniques can be used to identify DSBs they provide no information on the molecular events occurring at the break. Tip‐enhanced Raman scattering (TERS) can provide molecular information from DNA at the nanoscale and in combination with AFM provides a new way to visualize and characterize the molecular structure of DSBs. DSBs result from cleavage at the 3’‐ and 5’‐bonds of deoxyribose upon exposure to UVC radiation based on the observation of P? O? H and methyl/methylene deformation modes enhanced in the TERS spectra. It is hypothesized that strand fragments are hydrogen‐terminated at the lesion, indicating the action of free radicals during photon exposure.     

Vacuum-UV and Low-Energy Electron-Induced DNA Strand Breaks – Influence of the DNA Sequence and Substrate

DNA is effectively damaged by radiation, which can on the one hand lead to cancer and is on the other hand directly exploited in the treatment of tumor tissue. DNA strand breaks are already induced by photons having an energy below the ionization energy of DNA. At high photon energies, most of the DNA strand breaks are induced by low-energy secondary electrons. In the present study we quantified photon and electron induced DNA strand breaks in four different 12mer oligonucleotides. They are irradiated directly with 8.44 eV vacuum ultraviolet (VUV) photons and 8.8 eV low energy electrons (LEE). By using Si instead of VUV transparent CaF₂ as a substrate the VUV exposure leads to an additional release of LEEs, which have a maximum energy of 3.6 eV and can significantly enhance strand break cross sections. Atomic force microscopy is used to visualize strand breaks on DNA origami platforms and to determine absolute values for the strand break cross sections. Upon irradiation with 8.44 eV photons all the investigated sequences show very similar strand break cross sections in the range of 1.7–2.3×10⁻¹⁶ cm². The strand break cross sections for LEE irradiation at 8.8 eV are one to two orders of magnitude larger than the ones for VUV photons, and a slight sequence dependence is observed. The sequence dependence is even more pronounced for LEEs with energies <3.6 eV. The present results help to assess DNA damage by photons and electrons close to the ionization threshold.     

Phenylpropanoids in radioregulation: double edged sword

Radiotherapy, frequently used for treatment of solid tumors, carries two main obstacles including acquired radioresistance in cancer cells during radiotherapy and normal tissue injury. Phenylpropanoids, which are naturally occurring phytochemicals found in plants, have been identified as potential radiotherapeutic agents due to their anti-cancer activity and relatively safe levels of cytotoxicity. Various studies have proposed that these compounds could not only sensitize cancer cells to radiation resulting in inhibition of growth and cell death but also protect normal cells against radiation-induced damage. This review is intended to provide an overview of recent investigations on the usage of phenylpropanoids in combination with radiotherapy in cancer treatment.     

Induction of persistent double strand breaks following multiphoton irradiation of cycling and G1-arrested mammalian cells-replication-induced double strand breaks

DNA double strand breaks (DSBs) are amongst the most deleterious lesions induced within the cell following exposure to ionizing radiation. Mammalian cells repair these breaks predominantly via the nonhomologous end joining pathway which is active throughout the cell cycle and is error prone. The alternative pathway for repair of DSBs is homologous recombination (HR) which is error free and active during S- and G2/M-phases of the cell cycle. We have utilized near-infrared laser radiation to induce DNA damage in individual mammalian cells through multiphoton excitation processes to investigate the dynamics of single cell DNA damage processing. We have used immunofluorescent imaging of gamma-H2AX (a marker for DSBs) in mammalian cells and investigated the colocalization of this protein with ATM, p53 binding protein 1 and RAD51, an integral protein of the HR DNA repair pathway. We have observed persistent DSBs at later times postlaser irradiation which are indicative of DSBs arising at replication, presumably from UV photoproducts or clustered damage containing single strand breaks. Cell cycle studies have shown that in G1 cells, a significant fraction of multiphoton laser-induced prompt DSBs persists for > 4 h in addition to those induced at replication.     

Molecular Regulation of UV‐Induced DNA Repair

Ultraviolet (UV) radiation from sunlight is a major etiologic factor for skin cancer, the most prevalent cancer in the United States, as well as premature skin aging. In particular, UVB radiation causes formation of specific DNA damage photoproducts between pyrimidine bases. These DNA damage photoproducts are repaired by a process called nucleotide excision repair, also known as UV‐induced DNA repair. When left unrepaired, UVB‐induced DNA damage leads to accumulation of mutations, predisposing people to carcinogenesis as well as to premature aging. Genetic loss of nucleotide excision repair leads to severe disorders, namely, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS), which are associated with predisposition to skin carcinogenesis at a young age as well as developmental and neurological conditions. Regulation of nucleotide excision repair is an attractive avenue to preventing or reversing these detrimental consequences of impaired nucleotide excision repair. Here, we review recent studies on molecular mechanisms regulating nucleotide excision repair by extracellular cues and intracellular signaling pathways, with a special focus on the molecular regulation of individual repair factors.     

Immunocytochemical Localization of XRCC1 and γH2AX Foci Induced by Tightly Focused Femtosecond Laser Radiation in Cultured Human Cells

To assess the prospects for using intense femtosecond laser radiation in biomedicine, it is necessary to understand the mechanisms of its action on biological macromolecules, especially on the informational macromolecule—DNA. The aim of this work was to study the immunocytochemical localization of DNA repair protein foci (XRCC1 and γH2AX) induced by tightly focused femtosecond laser radiation in human cancer A549 cells. The results showed that no XRCC1 or γH2AX foci tracks were observed 30 min after cell irradiation with femtosecond pulses of 10¹¹ W∙cm⁻² peak power density. An increase in the pulse power density to 2 × 10¹¹ W∙cm⁻² led to the formation of linear tracks consisting both of XRCC1 and γH2AX protein foci localized in the places where the laser beam passed through the cell nuclei. A further increase in the pulse power density to 4 × 10¹¹ W∙cm⁻² led to the appearance of nuclei with total immunocytochemical staining for XRCC1 and γH2AX on the path of the laser beam. Thus, femtosecond laser radiation can be considered as a tool for local ionization of biological material, and this ionization will lead to similar effects obtained using ionizing radiation.     

Effects of high-energy-pulse-electron beam radiation on biomacromolecules

To study the molecular mechanism of high mutation frequency induced by high-energy-pulse-electron (HEPE) beam radiation, the effects of HEPE radiation on yeast cells, plasma membrane, plasmid DNA, and protein activity were investigated by means of cell counting, gel electrophoresis, AO/EB double fluorescent staining, etc. The results showed that the viability of yeast cells declined statistically with increase of absorbed doses. The half lethal dose (LD50) was 134 Gy. HEPE beam radiation had little influence on the function of plasma membrane and protein, while it could induce much DNA damage of single strand breaks (SSB) and double strand breaks (DSB) that were required for gene mutation. The G-value for DSB formation of HEPE beam radiation in aqueous solution was 5.7 times higher than that caused by 60Co gamma rays. HEPE can be a new effective method for induced mutation breeding and deserves further research in the future.     

Phospholipase D inhibitor enhances radiosensitivity of breast cancer cells

Radiation and drug resistance remain the major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Dysregulation of phospholipase D (PLD) has been found in several human cancers and is associated with resistance to anticancer drugs. In the present study, we evaluated the effects of PLD inhibition on cell survival, cell death and DNA damage after exposure to ionizing radiation (IR). Combined IR treatment and PLD inhibition led to an increase in the radiation-induced apoptosis of MDA-MB-231 metastatic breast cancer cells. The selective inhibition of PLD1 and PLD2 led to a significant decrease in the IR-induced colony formation of breast cancer cells. Moreover, PLD inhibition suppressed the radiation-induced activation of extracellular signal-regulated kinase and enhanced the radiation-stimulated phosphorylation of the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Furthermore, PLD inhibition, in combination with radiation, was very effective at inducing DNA damage, when compared with radiation alone. Taken together, these results suggest that PLD may be a useful target molecule for the enhancement of the radiotherapy effect.     

Cancer‐Targeted Selenium Nanoparticles Sensitize Cancer Cells to Continuous γ Radiation to Achieve Synergetic Chemo‐Radiotherapy

Cancer radiotherapy with ¹²⁵I seeds demonstrates higher long‐term efficacy and fewer side effects than traditional X‐ray radiotherapy owing to its low‐dose and continuous radiation but is still limited by radioresistance in clinical applications. Therefore, the design and synthesis of sensitizers that could enhance the sensitivity of cancer cells to ¹²⁵I seeds is of great importance for future radiotherapy. Selenium nanoparticles (SeNPs) have been found to exhibit high potential in cancer chemotherapy and as drug carriers. In this study, we found that, based on the Auger‐electron effect and Compton effect of Se atoms, cancer‐targeted SeNPs in combination with ¹²⁵I seeds achieve synergetic effects to inhibit cancer‐cell growth and colony formation through the induction of cell apoptosis and cell cycle arrest. Detailed studies on the action mechanisms reveal that the combined treatments effectively activate intracellular reactive oxygen species (ROS) overproduction to regulate p53‐mediated DNA damage apoptotic signaling pathways and mitogen‐activated protein kinase (MAPK) phosphorylation and to prevent the self‐repair of cancer cells simultaneously. Taken together, the combination of SeNPs with ¹²⁵I seeds could be further exploited as a safe and effective strategy for next‐generation cancer chemo‐radiotherapy in clinical applications.     

Exploiting the nucleotide substrate specificity of repair DNA polymerases to develop novel anticancer agents

The genome is constantly exposed to mutations that can originate during replication or as a result of the action of both endogenous and/or exogenous damaging agents [such as reactive oxygen species (ROS), UV light, genotoxic environmental compounds, etc.]. Cells have developed a set of specialized mechanisms to counteract this mutational burden. Many cancer cells have defects in one or more DNA repair pathways, hence they rely on a narrower set of specialized DNA repair mechanisms than normal cells. Inhibiting one of these pathways in the context of an already DNA repair-deficient genetic background, will be more toxic to cancer cells than to normal cells, a concept recently exploited in cancer chemotherapy by the synthetic lethality approach. Essential to all DNA repair pathways are the DNA pols. Thus, these enzymes are being regarded as attractive targets for the development of specific inhibitors of DNA repair in cancer cells. In this review we examine the current state-of-the-art in the development of nucleotide analogs as inhibitors of repair DNA polymerases.