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.     

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.     

DNA Damage Clustering after Ionizing Radiation and Consequences in the Processing of Chromatin Breaks

Charged-particle radiotherapy (CPRT) utilizing low and high linear energy transfer (low-/high-LET) ionizing radiation (IR) is a promising cancer treatment modality having unique physical energy deposition properties. CPRT enables focused delivery of a desired dose to the tumor, thus achieving a better tumor control and reduced normal tissue toxicity. It increases the overall radiation tolerance and the chances of survival for the patient. Further improvements in CPRT are expected from a better understanding of the mechanisms governing the biological effects of IR and their dependence on LET. There is increasing evidence that high-LET IR induces more complex and even clustered DNA double-strand breaks (DSBs) that are extremely consequential to cellular homeostasis, and which represent a considerable threat to genomic integrity. However, from the perspective of cancer management, the same DSB characteristics underpin the expected therapeutic benefit and are central to the rationale guiding current efforts for increased implementation of heavy ions (HI) in radiotherapy. Here, we review the specific cellular DNA damage responses (DDR) elicited by high-LET IR and compare them to those of low-LET IR. We emphasize differences in the forms of DSBs induced and their impact on DDR. Moreover, we analyze how the distinct initial forms of DSBs modulate the interplay between DSB repair pathways through the activation of DNA end resection. We postulate that at complex DSBs and DSB clusters, increased DNA end resection orchestrates an increased engagement of resection-dependent repair pathways. Furthermore, we summarize evidence that after exposure to high-LET IR, error-prone processes outcompete high fidelity homologous recombination (HR) through mechanisms that remain to be elucidated. Finally, we review the high-LET dependence of specific DDR-related post-translational modifications and the induction of apoptosis in cancer cells. We believe that in-depth characterization of the biological effects that are specific to high-LET IR will help to establish predictive and prognostic signatures for use in future individualized therapeutic strategies, and will enhance the prospects for the development of effective countermeasures for improved radiation protection during space travel.     

Isolation and Functional Characterization of Single Domain Antibody Modulators of Caspase-3 and Apoptosis

Apoptosis, or programmed cell death, is an essential process affecting homeostasis of cell growth, development, and the elimination of damaged or dangerous cells. Inappropriate cell death caused by oxidative stress has been implicated in the development of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and stroke. On the other hand, a defect in the cell death process leads to the development of cancer. For example, the main player of apoptosis, p53, is defective in many of the human cancers. Apoptosis is regulated by the interplay of pro-apoptotic and anti-apoptotic proteins from the Bcl-2 family and caspases. In particular, specific modulators of the activity of Caspase 3 could be very important for the development of therapies for diseases such as neurodegeneration and cancer. In this study, two V_HHs specific to Caspase 3 (VhhCasp31 and VhhCasp32) were isolated from a heavy chain antibody variable domain (V_HH) phage display library and tested for their apoptosis-modulating effects. While VhhCasp31 was found to be antagonistic towards Caspase 3, VhhCasp32 was agonistic. Furthermore, when expressed as intrabodies in SHSY-5Y neuroblastoma cells, VhhCasp31 rendered cells resistant to oxidative-stress-induced apoptosis, whereas VhhCasp32 resulted in apoptosis. These V_HH antagonist and agonist of apoptosis could have potential for the development of therapeutics for neurodegenerative diseases and cancer, respectively.     

Interruptin C,a Radioprotective Agent,Derived from Cyclosorus terminans Protect Normal Breast MCF-10A and Human Keratinocyte HaCaT Cells against Radiation-Induced Damage

Radiotherapy is a common method to treat cancers, with the goal of maximizing the dose to tumors while minimizing the dose to normal tissues. Radioprotectors can reduce the toxicity to normal tissues during radiotherapy. Several plant-derived compounds can function as radioprotectors by scavenging free radicals. We investigated the radioprotective activity of interruptin C from the fern Cyclosorus terminans. The molecular mechanism of interruptin C’s activity in X-ray-irradiated cells was evaluated. Superoxide dismutase activity was examined to investigate the antioxidant enzyme activity. Clonogenic cell survival was also investigated following radiation exposure. DNA damage and cell cycle progression were detected using micronuclei formation assays. DNA repair after irradiation was analyzed in a γH2AX assay. The levels of the proteins related to the radioprotective responses were analyzed by Western blotting. Interruptin C increased the antioxidant enzyme activity and significantly decreased the DNA damage by reducing the γH2AX foci and micronucleus formation in irradiated MCF-10A normal breast and HaCaT human keratinocyte cells. The apoptotic protein levels decreased, whereas the antiapoptotic protein levels increased. Interruptin C pretreatment increased the survival rate of irradiated MCF-10A and HaCaT cells. Moreover, the compound did not promote the survival of MDA-MB-231 and Hs578T breast cancer cells. Therefore, interruptin C may exert radioprotective activity without enhancing cancer cell proliferation.     

Oxidation Chemistry of DNA and p53 Tumor Suppressor Gene

The chemistry of DNA and its repair selectivity control the influence of genomic oxidative stress on the development of serious disorders such as cancer and heart diseases. DNA is oxidized by endogenous reactive oxygen species (ROS) in vivo or in vitro as a result of high energy radiation, non-radiative metabolic processes, and other consequences of oxidative stress. Some oxidations of DNA and tumor suppressor gene p53 are thought to be mutagenic when not repaired. For example, site-specific oxidations of p53 tumor suppressor gene may lead to cancer-related mutations at the oxidation site codon. This review summarizes the research on the primary products of the most easily oxidized nucleobase guanine (G) when different oxidation methods are used. Guanine is by far the most oxidized DNA base. The primary initial oxidation product of guanine for most, but not all, pathways is 8-oxoguanine (8-oxoG). With an oxidation potential much lower than G, 8-oxoG is readily susceptible to further oxidation, and the products often depend on the oxidants. Specific products may control the types of subsequent mutations, but mediated by gene repair success. Site-specific oxidations of p53 tumor suppressor gene have been reported at known mutation hot spots, and the codon sites also depend on the type of oxidants. Modern methodologies using LC–MS/MS for codon specific detection and identification of oxidation sites are summarized. Future work aimed at understanding DNA oxidation in nucleosomes and interactions between DNA damage and repair is needed to provide a better picture of how cancer-related mutations arise.     

Cyclobutane Pyrimidine Dimer Density as a Predictive Biomarker of the Biological Effects of Ultraviolet Radiation in Normal Human Fibroblast

This study compared biological responses of normal human fibroblasts (NHF1) to three sources of ultraviolet radiation (UVR), emitting UVC wavelengths, UVB wavelengths, or a combination of UVA and UVB (solar simulator; emission spectrum, 94.3% UVA and 5.7% UVB). The endpoints measured were cytotoxicity, intra‐S checkpoint activation, inhibition of DNA replication and mutagenicity. Results show that the magnitude of each response to the indicated radiation sources was best predicted by the density of DNA cyclobutane pyrimidine dimers (CPD). The density of 6‐4 pyrimidine–pyrimidone photoproducts was highest in DNA from UVC‐irradiated cells (14% of CPD) as compared to those exposed to UVB (11%) or UVA–UVB (7%). The solar simulator source, under the experimental conditions described here, did not induce the formation of 8‐oxo‐7,8‐dihydroguanine in NHF1 above background levels. Taken together, these results suggest that CPD play a dominant role in DNA damage responses and highlight the importance of using endogenous biomarkers to compare and report biological effects induced by different sources of UVR.     

Never Cared for What They Do: High Structural Stability of Guanine-Quadruplexes in the Presence of Strand-Break Damage

DNA integrity is an important factor that assures genome stability and, more generally, the viability of cells and organisms. In the presence of DNA damage, the normal cell cycle is perturbed when cells activate their repair processes. Although efficient, the repair system is not always able to ensure complete restoration of gene integrity. In these cases, mutations not only may occur, but the accumulation of lesions can either lead to carcinogenesis or reach a threshold that induces apoptosis and programmed cell death. Among the different types of DNA lesions, strand breaks produced by ionizing radiation are the most toxic due to the inherent difficultly of repair, which may lead to genomic instability. In this article we show, by using classical molecular simulation techniques, that compared to canonical double-helical B-DNA, guanine-quadruplex (G4) arrangements show remarkable structural stability, even in the presence of two strand breaks. Since G4-DNA is recognized for its regulatory roles in cell senescence and gene expression, including oncogenes, this stability may be related to an evolutionary cellular response aimed at minimizing the effects of ionizing radiation.     

Antiproliferation- and Apoptosis-Inducible Effects of a Novel Nitrated [6,6,6]Tricycle Derivative (SK2) on Oral Cancer Cells

The benzo-fused dioxabicyclo[3.3.1]nonane core is the central framework in several natural products. Using this core, we had developed a novel nitrated [6,6,6]tricycle-derived compound containing an n-butyloxy group, namely, SK2. The anticancer potential of SK2 was not assessed. This study aimed to determine the antiproliferative function and investigated possible mechanisms of SK2 acting on oral cancer cells. SK2 preferentially killed oral cancer cells but caused no harmful effect on non-malignant oral cells. After the SK2 exposure of oral cancer cells, cells in the sub-G1 phase accumulated. This apoptosis-like outcome of SK2 treatment was validated to be apoptosis via observing an increasing annexin V population. Mechanistically, apoptosis signalers such as pancaspase, caspases 8, caspase 9, and caspase 3 were activated by SK2 in oral cancer cells. SK2 induced oxidative-stress-associated changes. Furthermore, SK2 caused DNA damage (γH2AX and 8-hydroxy-2′-deoxyguanosine). In conclusion, a novel nitrated [6,6,6]tricycle-derived compound, SK2, exhibits a preferential antiproliferative effect on oral cancer cells, accompanied by apoptosis, oxidative stress, and DNA damage.     

Deciphering UV-induced DNA Damage Responses to Prevent and Treat Skin Cancer

Ultraviolet (UV) radiation is among the most prevalent environmental factors that influence human health and disease. Even 1 h of UV irradiation extensively damages the genome. To cope with resulting deleterious DNA lesions, cells activate a multitude of DNA damage response pathways, including DNA repair. Strikingly, UV-induced DNA damage formation and repair are affected by chromatin state. When cells enter S phase with these lesions, a distinct mutation signature is created via error-prone translesion synthesis. Chronic UV exposure leads to high mutation burden in skin and consequently the development of skin cancer, the most common cancer in the United States. Intriguingly, UV-induced oxidative stress has opposing effects on carcinogenesis. Elucidating the molecular mechanisms of UV-induced DNA damage responses will be useful for preventing and treating skin cancer with greater precision. Excitingly, recent studies have uncovered substantial depth of novel findings regarding the molecular and cellular consequences of UV irradiation. In this review, we will discuss updated mechanisms of UV-induced DNA damage responses including the ATR pathway, which maintains genome integrity following UV irradiation. We will also present current strategies for preventing and treating nonmelanoma skin cancer, including ATR pathway inhibition for prevention and photodynamic therapy for treatment.     

Adaptation of cyanobacteria to UV-B stress correlated with oxidative stress and oxidative damage

Cyanobacteria must cope with the negative effects of ultraviolet B (280-315 nm) (UV-B) stress caused by their obligatory light requirement for photosynthesis. The adaptation of the cyanobacterium Anabaena sp. to moderate UV-B radiation has been observed after 2 weeks of irradiation, as indicated by decreased oxidative stress, decreased damage, recovered photosynthetic efficiency and increased survival. Oxidative stress in the form of UV-B-induced production of reactive oxygen species was measured in vivo with the oxidative stress-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate. Photooxidative damage by UV-B radiation, including lipid peroxidation and DNA strand breakage, was determined by a modified method using thiobarbituric acid reactive substances and fluorometric analysis of DNA unwinding. Photosynthetic quantum yield was determined by pulse amplitude-modulated fluorometry. The results suggest that moderate UV-B radiation results in an evident oxidative stress, enhanced lipid peroxidation, increased DNA strand breaks, elevated chlorophyll bleaching as well as decreased photosynthetic efficiency and survival during the initial exposure. However, DNA strand breaks, photosynthetic parameters and chlorophyll bleaching returned to their unirradiated levels after 4-7 days of irradiation. Oxidative stress and lipid peroxidation appeared to respond later because decreases were observed after 7 days of radiation. The survival curve against irradiation time exhibited a close relationship with the changes in photosynthetic quantum yield and DNA damage, with little mortality after 4 days. Growth inhibition by UV-B radiation was observed during the first 7 days of radiation, whereas normal growth resumed even under UV-B stress thereafter. An efficient defense system was assumed to come into play to repair photosynthetic and DNA damage and induce the de novo synthesis of UV-sensitive proteins and lipids, allowing the organisms to adapt to UV-B stress successfully and survive as well as grow. No induction of mycosporine-like amino acids (MAA) was observed during the adaptation of Anabaena sp. to UV-B stress in our work. The adaptation of the cyanobacterium correlated with and could be caused by the oxidative stress and oxidative damage.     

Comparative Nucleosomal Reactivity of 5-Formyl-Uridine and 5-Formyl-Cytidine

5-Formyl-deoxyuridine (fdU) and 5-formyl-deoxycytidine (fdC) are formyl-containing nucleosides that are created by oxidative stress in differentiated cells. While fdU is almost exclusively an oxidative stress lesion formed from deoxythymidine (T), the situation for fdC is more complex. Next to formation as an oxidative lesion, it is particularly abundant in stem cells, where it is more frequently formed in an epigenetically important oxidation reaction performed by α-ketoglutarate dependent TET enzymes from 5-methyl-deoxycytidine (mdC). Recently, it was shown that genomic fdC and fdU can react with the ϵ-aminogroups of nucleosomal lysines to give Schiff base adducts that covalently link nucleosomes to genomic DNA. Here, we show that fdU features a significantly higher reactivity towards lysine side chains compared with fdC. This result shows that depending on the amounts of fdC and fdU, oxidative stress may have a bigger impact on nucleosome binding than epigenetics.     

DNA repair inhibition and cancer therapy.

The DNA repair process in mammalian cells is a multi-pathway mechanism that protects cells from the plethora of DNA damaging agents that are known to attack nuclear DNA. Moreover, the majority of current anticancer therapies (e.g. ionising radiation and chemotoxic therapies) rely on this ability to create DNA lesions, leading to apoptosis/cell death. A cells natural ability to repair such DNA damage is a major cause of resistance to these existing antitumour agents. It seems logical, therefore, that by modulating these repair mechanisms, greater killing effect to anticancer agents would occur. Experimental data support this, either through knockout studies or by the use of pharmacological inhibitors which target some of the key regulatory proteins involved in the DNA repair process. Several of these key DNA repair proteins which are actively under investigation as novel sites for intervention in cancer biology are discussed.     

Biochemistry of Oxidative Stress

As a normal attribute of aerobic life, structural damage to organic compounds of a wide variety (DNA, proteins, carbohydrates and lipids) may occur as a consequence of oxidative reactions. Oxidative damage inflicted by reactive oxygen species has been called “oxidative stress”. Biological systems contain powerful enzymatic and nonenzymatic antioxidant systems, and oxidative stress denotes a shift in the prooxidant/antioxidant balance in favor of the former. Diverse biological processes such as inflammation, carcinogenesis, ageing, radiation damage and photobiological effects appear to involve reactive oxygen species. This field of research provides new perspectives in biochemical pharmacology, toxicology, radiation biochemistry as well as pathophysiology.     

Regulation of bcl-2 family in hydrogen peroxide-induced apoptosis in human leukemia HL-60 cells

Numerous types of cells have been shown to undergo apoptosis when exposed to oxidant agent such as hydrogen peroxide. In order to understand the functional relationship between the anti- and pro-apoptotic regulatory proteins in the cells under oxidant stress, we have studied the level of expression of apoptosis regulatory proteins, bcl-2 and bax, in human leukemia HL-60 cells. The exposure of HL-60 cells to different concentrations of H2O2 for 6 h resulted in a typical apoptosis of the cells as characterized by flow cytometry, cell cycle analysis, and DNA fragmantation. There was a block in G1 to S transition and apoptotic cells were mainly derived from S and G2 cells. Kinetic study demonstrated that the levels of both bcl-2-mRNA and -protein expression were decreased with the progression of cellular apoptosis whereas the level of bax-mRNA was unchanged but the expressed bax-protein was not detectable. Cycloheximide, a nonspecific translation inhibitor, did not prevent the hydrogen peroxide-mediated apoptosis in HL-60 cells. These results suggest that the regulation of bcl-2, but not of bax are important factor in the oxidative stress-induced apoptosis in HL-60 cells.     

Age-dependent DNA repair and cell cycle distribution of human skin fibroblasts in response to UVA irradiation

Ageing process in cells is associated with oxidative stress. Ultraviolet A produces reactive oxygen species responsible for accumulation of DNA and cellular damage. After the evaluation of antioxidant enzyme activities and oxidative stress markers at the basal state, we have studied the responses to UVA stress of coetaneous fibroblasts, isolated from different male donors (2-88 years, n=23) in terms of cytotoxicity, genotoxicity and DNA repair capacities. For this purpose, we have determined level of DNA damage using the comet assay (single strand breaks and alkali-labile sites) and the cell cycle distribution after a 5 J/cm2 irradiation. No differences with age were observed for antioxidant enzyme activities and oxidative stress markers. DNA strand breaks after UVA irradiation (5-20 J/cm2), was found to be age-dependent. DNA repair was slow and also significantly affected by ageing. The cell cycle distribution analysis showed that high repair correlated with high proliferative capacities at basal level. Twenty-four hours after the stress, fraction of young fibroblasts blocked in G1 phase was significantly increased whereas significant modifications concerned the G2-M phase for adult and older fibroblasts. These results indicate an age-dependent decline in the DNA repair capacities correlated with modifications of the cell cycle parameters.     

Impact of the Circadian Clock on UV‐Induced DNA Damage Response and Photocarcinogenesis

The skin is in constant exposure to various external environmental stressors, including solar ultraviolet (UV) radiation. Various wavelengths of UV light are absorbed by the DNA and other molecules in the skin to cause DNA damage and induce oxidative stress. The exposure to excessive ultraviolet (UV) radiation and/or accumulation of damage over time can lead to photocarcinogenesis and photoaging. The nucleotide excision repair (NER) system is the sole mechanism for removing UV photoproduct damage from DNA, and genetic disruption of this repair pathway leads to the photosensitive disorder xeroderma pigmentosum (XP). Interestingly, recent work has shown that NER is controlled by the circadian clock, the body's natural time‐keeping mechanism, through regulation of the rate‐limiting repair factor xeroderma pigmentosum group A (XPA). Studies have shown reduced UV‐induced skin cancer after UV exposure in the evening compared to the morning, which corresponds with times of high and low repair capacities, respectively. However, most studies of the circadian clock–NER connection have utilized murine models, and it is therefore important to translate these findings to humans to improve skin cancer prevention and chronotherapy.     

2,4-Dinitrophenol as an Uncoupler Augments the Anthracyclines Toxicity against Prostate Cancer Cells

One of the strategies for the treatment of advanced cancer diseases is targeting the energy metabolism of the cancer cells. The compound 2,4-DNP (2,4-dinitrophenol) disrupts the cell energy metabolism through the ability to decouple oxidative phosphorylation. The aim of the study was to determine the ability of 2,4-DNP to sensitize prostate cancer cells with different metabolic phenotypes to the action of known anthracyclines (doxorubicin and epirubicin). The synergistic effect of the anthracyclines and 2,4-DNP was determined using an MTT assay, apoptosis detection and a cell cycle analysis. The present of oxidative stress in cancer cells was assessed by CellROX, the level of cellular thiols and DNA oxidative damage. The study revealed that the incubation of LNCaP prostate cancer cells (oxidative phenotype) with epirubicin and doxorubicin simultaneously with 2,4-DNP showed the presence of a synergistic effect for both the cytostatics. Moreover, it contributes to the increased induction of oxidative stress, which results in a reduced level of cellular thiols and an increased number of AP sites in the DNA. The synergistic activity may consist of an inhibition of ATP synthesis and the simultaneous production of toxic amounts of ROS, destroying the mitochondria. Additionally, the sensitivity of the LNCaP cell line to the anthracyclines is relatively higher compared to the other two (PC-3, DU-145).