Reactive Oxygen Species-mediated DNA Damage Research Articles

Ovatodiolide inhibits endometrial cancer stemness via reactive oxygen species-mediated DNA damage and cell cycle arrest

Endometrial cancer (EC) is a common gynecological cancer worldwide, often associated with a poor prognosis after recurrence or metastasis. Ovatodiolide (OVA) is a macrocyclic diterpenoid derived from Anisomeles indica that shows anticancer effects in various malignancies. This study aimed to evaluate the cytotoxic effects of OVA on EC cell proliferation and cancer stem cell (CSC) activity and explore its underlying molecular mechanisms. OVA treatment dose-dependently reduced the viability and colony formation of three EC cell lines (AN3CA, HEC-1A, and EMC6). It induced G2/M phase cell cycle arrest, associated with decreased cell division cycle 25C (CDC25C) expression and reduced activation of cyclin-dependent kinases 1 (CDK1) and 2 (CDK2). OVA also increased reactive oxygen species (ROS) production and DNA damage, activating the DNA damage-sensitive cell cycle checkpoint kinases 1 (CHK1) and 2 (CHK2) and upregulating the DNA damage marker γ-H2A.X variant histone (H2AX). It also suppressed the activation of mechanistic target of rapamycin kinase (mTOR) and nuclear factor kappa B (NF-κB) and downregulated glutathione peroxidase 1 (GPX1), an antioxidant enzyme counteracting oxidative stress. Moreover, OVA reduced the self-renewal capacity of CSCs, reducing the expression of key stemness proteins Nanog homeobox (NANOG) and octamer-binding transcription factor 4 (OCT4). The ROS inhibitor N-acetylcysteine attenuated the anti-proliferative and anti-CSC effects of OVA. Our findings suggest that OVA acts via ROS generation, leading to oxidative stress and DNA damage, culminating in cell cycle arrest and the suppression of CSC activity in EC. Therefore, OVA is a promising therapeutic agent for EC, either as a standalone treatment or an adjunct to existing therapies.

Futuristic advancements in phytoremediation of endocrine disruptor Bisphenol A: A step towards sustainable pollutant degradation for rehabilitated environment

Bisphenol A (BPA) accumulates in the environment at lethal concentrations because of its high production rate and utilization. BPA, originating from industrial effluent, plastic production, and consumer products, poses serious risks to both the environment and human health. The widespread aggregation of BPA leads to endocrine disruption, reactive oxygen species-mediated DNA damage, epigenetic modifications and carcinogenicity, which can disturb the normal homeostasis of the body. The living being in a population is subjected to BPA exposure via air, water and food. Globally, urinary analysis reports have shown higher BPA concentrations in all age groups, with children being particularly susceptible due to its occurrence in items such as milk bottles. The conventional methods are costly with a low removal rate. Since there is no proper eco-friendly and cost-effective degradation of BPA reported so far. The phytoremediation, green-biotechnology based method which is a cost-effective and renewable resource can be used to sequestrate BPA. Phytoremediation is observed in numerous plant species with different mechanisms to remove harmful contaminants. Plants normally undergo several improvements in genetic and molecular levels to withstand stress and lower levels of toxicants. But such natural adaptation requires more time and also higher concentration of contaminants may disrupt the normal growth, survival and yield of the plants. Therefore, natural or synthetic amendments and genetic modifications can improve the xenobiotics removal rate by the plants. Also, constructed wetlands technique utilizes the plant’s phytoremediation mechanisms to remove industrial effluents and medical residues. In this review, we have discussed the limitations and futuristic advancement strategies for degrading BPA using phytoremediation-associated mechanisms.

Loss of Prm1 leads to defective chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice.

ABSTRACTOne of the key events during spermiogenesis is the hypercondensation of chromatin by substitution of the majority of histones by protamines. In humans and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/Prm2) are expressed in a species-specific ratio. Using CRISPR-Cas9-mediated gene editing, we generated Prm1-deficient mice and demonstrated that Prm1+/− mice were subfertile, whereas Prm1−/− mice were infertile. Prm1−/− and Prm2−/− sperm showed high levels of reactive oxygen species-mediated DNA damage and increased histone retention. In contrast, Prm1+/− sperm displayed only moderate DNA damage. The majority of Prm1+/− sperm were CMA3 positive, indicating protamine-deficient chromatin, although this was not the result of increased histone retention in Prm1+/− sperm. However, sperm from Prm1+/− and Prm1−/− mice contained high levels of incompletely processed PRM2. Furthermore, the PRM1:PRM2 ratio was skewed from 1:2 in wild type to 1:5 in Prm1+/− animals. Our results reveal that PRM1 is required for proper PRM2 processing to produce mature PRM2, which, together with PRM1, is able to hypercondense DNA. Thus, the species-specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility.

Pharmacological inhibition of serine synthesis enhances temozolomide efficacy by decreasing O6-methylguanine DNA methyltransferase (MGMT) expression and reactive oxygen species (ROS)-mediated DNA damage in glioblastoma

AbstractGlioblastoma (GBM) is the most malignant primary tumor in the central nervous system of adults. Temozolomide (TMZ), an alkylating agent, is the first-line chemotherapeutic agent for GBM patients. However, its efficacy is often limited by innate or acquired chemoresistance. Cancer cells can rewire their metabolic programming to support rapid growth and sustain cell survival against chemotherapies. An example is the de novo serine synthesis pathway (SSP), one of the main branches from glycolysis that is highly activated in multiple cancers in promoting cancer progression and inducing chemotherapy resistance. However, the roles of SSP in TMZ therapy for GBM patients remain unexplored. In this study, we employed NCT503, a highly selective inhibitor of phosphoglycerate dehydrogenase (PHGDH, the first rate-limiting enzyme of SSP), to study whether inhibition of SSP may enhance TMZ efficacy in MGMT-positive GBMs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flowcytometry and colony formation assays demonstrated that NCT503 worked synergistically with TMZ in suppressing GBM cell growth and inducing apoptosis in T98G and U118 cells in vitro. U118 and patient-derived GBM subcutaneous xenograft models showed that combined NCT503 and TMZ treatment inhibited GBM growth and promoted apoptosis more significantly than would each treatment alone in vivo. Mechanistically, we found that NCT503 treatment decreased MGMT expression possibly by modulating the Wnt/β-catenin pathway. Moreover, intracellular levels of reactive oxygen species were elevated especially when NCT503 and TMZ treatments were combined, and the synergistic effects could be partially negated by NAC, a classic scavenger of reactive oxygen species. Taken together, these results suggest that NCT503 may be a promising agent for augmenting TMZ efficacy in the treatment of GBM, especially in TMZ-resistant GBMs with high expression of MGMT.Pharmacological inhibition of the serine synthesis pathway with a highly selective inhibitor NCT503 synergistically works with temozolomide in inhibiting O6-methylguanine DNA methyltransferase (MGMT)-positive glioblastoma cell growth and inducing apoptosis both in vitro and in vivo. Mechanistically, NCT503 treatment reduces MGMT expression possibly via Wnt/βcatenin pathway. Reactive oxygen species-mediated DNA damage is at least partially involved. Combinational administration of NCT503 and TMZ may represent a novel and promising treatment strategy to enhance TMZ efficacy in patients with MGMT-high glioblastoma.

PBX1 Attenuates Hair Follicle-Derived Mesenchymal Stem Cell Senescence and Apoptosis by Alleviating Reactive Oxygen Species-Mediated DNA Damage Instead of Enhancing DNA Damage Repair.

Tissues and organs undergo structural deterioration and functional decline during aging. DNA damage is considered a major cause of stem cell senescence. Although stem cells develop sophisticated DNA repair systems, when the intrinsic and extrinsic insults exceed the DNA repair capacity, cellular senescence, and age-related diseases inevitably occur. Therefore, the prevention and alleviation of DNA damage is an alternative to DNA repair in attenuating stem cell senescence and preventing age-related diseases. Pre-B-cell leukaemia homeobox 1 (PBX1) participates in maintaining the pluripotency of human embryonic and haematopoietic stem cells. Our recent studies showed that PBX1 promotes hair follicle-derived mesenchymal stem cell (HF-MSC) proliferation, decreases cellular senescence and apoptosis, and enhances induced pluripotent stem cell generation. Whether PBX1 attenuates HF-MSC senescence and apoptosis by alleviating DNA damage or by enhancing DNA repair remains unknown. In this study, we aimed to determine the effects of PBX1 on the intrinsic ROS or extrinsic H2O2-induced cellular senescence of HF-MSCs. To this end, we generated HF-MSCs overexpressing either PBX1, or poly (ADP-ribose) polymerase 1, or both. Our results showed that PBX1 overexpression attenuates HF-MSC senescence and apoptosis by alleviating reactive oxygen species (ROS)-mediated DNA damage instead of enhancing DNA repair. This is the first study to report that PBX1 attenuates stem cell senescence and apoptosis by alleviating DNA damage. It provides new insight into the mechanism of stem cell senescence and lays the foundation for the development of strategies for age-related disease prevention and treatment, and in particular, hair follicle repair and regeneration.

Abstract 2583: Effects of garlic compound diallyl trisulfide on head and neck cancer cells and cancer stem cells in vitro and in vivo

Abstract Head and neck cancer (HNC) is the seventh most prevalent cancer worldwide. The five-year survival rate is less than fifty percent despite significant advances in therapeutic approaches. Early diagnosis and treatment can help in prevention and disease management. Studies have reported that dietary intake of Allium vegetables or processed garlic that contains diallyl trisulfide (DATS) lowered the risk of various cancers. However, it has not been investigated for its efficacy and molecular mechanisms against HNC. Herein, we investigated the effect of DATS on HNC UMSCC-22A, UMSCC-22B, and Cal33 cells in vitro. DATS treatment significantly reduced the cell viability of HNC cells. DATS induced significant G2/M phase cell cycle arrest, which resulted in the accumulation of cyclin B1 and induced levels of p21. DATS-induced M phase arrest was attenuated by N-acetyl cysteine (NAC) treatment, which suggested that DATS mediates growth-suppressive effects by reactive oxygen species (ROS) generation. DATS treatment induced mitochondrial dysfunction. DATS induced ROS production led to apoptosis, which was attenuated by NAC treatment. DATS increased proapoptotic protein cleaved caspase-3, cleaved PARP, and Bax/Bcl2 ratio and decreased the levels of antiapoptotic protein XIAP. DATS induced ROS mediated DNA damage which was attenuated by NAC treatment. Further, DATS mediated increase in level of cleaved PARP, accumulation of Cyclin B1 and decrease in XIAP was attenuated by NAC treatment. DATS (1-10 μM) treatment resulted in inhibition of self renewal of HNC cancer stem cells (HNC CSCs) sphere formation. DATS reduced aldehyde dehydrogenase 1 (ALDH1) activity and CD133high/CD44high HNC CSC fraction. DATS-treated SCID mice tumor xenograft also revealed it's in vivo efficacy on HNC CSCs. Further, DATS treatment reduced the tumor weight, volume and reduced the levels of Ki67 cell proliferation marker in UMSCC22B head and neck cancer CD1 nude mice tumor xenograft. DATS treatment decreased tumor incidence and inhibited tumor promotion and progression in twenty two week C57BL6 4-Nitroquinoline 1-oxide (4-NQO)-induced mouse oral carcinogenesis model. DATS treatment reduced the cell proliferation marker Ki67 and increased TUNEL positive apoptotic cells in treatment groups compared to control group. Collectively, DATS inhibited cell proliferation, induced cell cycle arrest, and cell death via apoptosis involving DNA damage, mitochondrial dysfunction, and ROS generation. DATS treatment also reduced the HNC CSC fraction, sphere formation and ALDH1 activity. More importantly, DATS inhibited cancer incidence and progression in 4-NQO oral carcinogenesis study. Further, DATS inhibited HNC tumor growth and HNC CSC fraction in vivo. Thus, DATS could be a potential chemopreventive and chemotherapeutic agent against head and neck cancer. Citation Format: Sivapar V. Mathan, Su-Hyeong Kim, Shivendra V. Singh, Rana P. Singh. Effects of garlic compound diallyl trisulfide on head and neck cancer cells and cancer stem cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2583.

Cisplatin and Pemetrexed Have Distinctive Growth-inhibitory Effects in Monotherapy and Combination Therapy onKRAS-dependent A549 Lung Cancer Cells

Cisplatin combined with pemetrexed disodium heptahydrate (pemetrexed) is considered the standard treatment for patients with advanced, non-squamous, non-small-cell lung cancer. However, its growth-inhibitory effects on KRAS-dependent lung cancer as monotherapy and combination therapy are not well understood. The aim of this study was to compare the effects of cisplatin and pemetrexed on A549 cells as mono- and combination therapies and elucidate the underlying mechanisms. For in vitro studies, A549 cells were exposed to cisplatin with/without pemetrexed for 72 h. The results were then evaluated by cell viability, apoptosis, reactive oxygen species, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and western blotting assays. Our results revealed that cisplatin monotherapy was most cytotoxic to A549 cells, while cisplatin plus pemetrexed combination had an intermediate effect, and pemetrexed monotherapy induced a minimal cytotoxic effect on A549 cells. This effect was evidenced by cell viability results, inhibition of KRAS proto-oncogene, GTPase (KRAS)/Raf proto-oncogene, serine/threonine kinase/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways and apoptosis induction triggered by reactive oxygen species-mediated DNA damage. The immunoblotting result of conversion of microtubule-associated protein 1 light chain 3 alpha (LC3)-I to -II indicated that the greatest inducer of autophagy was combined treatment with cisplatin plus pemetrexed, while pemetrexed monotherapy had the lowest effect on autophagy induction, with cisplatin monotherapy having an intermediate effect. We found that the AKT serine/threonine kinase 1/mechanistic target of rapamycin kinase (mTOR) and AMP-activated protein kinase/mTOR signaling pathways were associated with autophagy activation. Interestingly, combination therapy with cisplatin plus pemetrexed was the primary eliminator of cellular senescence; cisplatin monotherapy had an intermediate effect, while pemetrexed monotherapy increased cellular senescence of A549 cells, as assessed by the expression of β-galactosidase protein. Cisplatin monotherapy may be more effective than pemetrexed monotherapy or cisplatin plus pemetrexed combination therapy against KRAS-dependent lung cancer.

In Situ-Grown Cdot-Wrapped Boehmite Nanoparticles for Cr(VI) Sensing in Wastewater and a Theoretical Probe for Chromium-Induced Carcinogen Detection.

In modern society, massive industrialization escalates environmental degradation by liberating various contaminants into the environment. Hexavalent chromium is a heavy metal that is being discharged from tannery and other industries, resulting in various carcinogenic diseases. This study reports a carbon dot (cdot)-based fluorometric probe for detecting hexavalent chromium in water. This is the very first time that cdots are tailored over the boehmite nanoparticle's surface using an in situ approach. Validation of formation of the nanocomposite has been discussed in detail employing the Rietveld refinement-based X-ray crystallography method. Vibrational spectroscopy and electron microscopy of the sample authenticate the nucleation process and the growth mechanism. The Stern-Volmer approach and time-resolved fluorescence measurements justify the sensitivity of the sensor (∼58 nM), and selectivity is analyzed by exposing the material to different ionic environments. Density functional theory (DFT) is applied herein to analyze the origin of fluorescence and the sensing mechanism of the probe, which shows that photoinduced electron transfer is responsible for the turn-off-based sensing of Cr(VI). The molecular docking simulation is carried out to ensure the binding of cdots to the binding pocket of the glutathione enzyme, which is responsible for treating reactive oxygen species-mediated DNA damage due to elements such as hexavalent chromium. Time-dependent density functional calculations show that the fluorometric probe is capable of detecting Cr(VI) in living cells making it an early stage chromium-mediated carcinogen detector.

Inflammatory macrophage dependence on NAD+ salvage is a consequence of reactive oxygen species-mediated DNA damage.

The adoption of Warburg metabolism is critical for the activation of macrophages in response to lipopolysaccharide. Macrophages stimulated with lipopolysaccharide increase their expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD+ salvage, and loss of NAMPT activity alters their inflammatory potential. However, the events that lead to the cells' becoming dependent on NAD+ salvage remain poorly defined. We found that depletion of NAD+ and increased expression of NAMPT occurred rapidly after inflammatory activation and coincided with DNA damage caused by reactive oxygen species (ROS). ROS produced by complex III of the mitochondrial electron-transport chain were required for macrophage activation. DNA damage was associated with activation of poly(ADP-ribose) polymerase, which led to consumption of NAD+. In this setting, increased NAMPT expression allowed the maintenance of NAD+ pools sufficient for glyceraldehyde-3-phosphate dehydrogenase activity and Warburg metabolism. Our findings provide an integrated explanation for the dependence of inflammatory macrophages on the NAD+ salvage pathway.

Induction of S-Phase Arrest in Human Glioma Cells by Selenocysteine, a Natural Selenium-Containing Agent Via Triggering Reactive Oxygen Species-Mediated DNA Damage and Modulating MAPKs and AKT Pathways.

Selenocysteine (SeC) a natural available selenoamino acid exhibits novel anticancer activities against human cancer cell lines. However, the growth inhibitory effect and mechanism of SeC in human glioma cells remain unclear. The present study reveals that SeC time- and dose-dependently inhibited U251 and U87 human glioma cells growth by induction of S-phase cell cycle arrest, followed by the marked decrease of cyclin A. SeC-induced S-phase arrest was achieved by inducing DNA damage through triggering generation of reactive oxygen species (ROS) and superoxide anion, with concomitant increase of TUNEL-positive cells and induction of p21waf1/Cip1 and p53. SeC treatment also caused the activation of p38MAPK, JNK and ERK, and inactivation of AKT. Four inhibitors of MAPKs and AKT pathways further confirmed their roles in SeC-induced S-phase arrest in human glioma cells. Our findings advance the understanding on the molecular mechanisms of SeC in human glioma management.

Cyanidin suppresses amyloid beta-induced neurotoxicity by inhibiting reactive oxygen species-mediated DNA damage and apoptosis in PC12 cells.

Amyloid beta (Aβ)-induced oxidative stress is a major pathologic hallmark of Alzheimer's disease. Cyanidin, a natural flavonoid compound, is neuroprotective against oxidative damage-mediated degeneration. However, its molecular mechanism remains unclear. Here, we investigated the effects of cyanidin pretreatment against Aβ-induced neurotoxicity in PC12 cells, and explored the underlying mechanisms. Cyanidin pretreatment significantly attenuated Aβ-induced cell mortality and morphological changes in PC12 cells. Mechanistically, cyanidin effectively blocked apoptosis induced by Aβ, by restoring the mitochondrial membrane potential via upregulation of Bcl-2 protein expression. Moreover, cyanidin markedly protected PC12 cells from Aβ-induced DNA damage by blocking reactive oxide species and superoxide accumulation. These results provide evidence that cyanidin suppresses Aβ-induced cytotoxicity, by preventing oxidative damage mediated by reactive oxide species, which in turn inhibits mitochondrial apoptosis. Our study demonstrates the therapeutic potential of cyanidin in the prevention of oxidative stress-mediated Aβ neurotoxicity.

Sensitization of cancer cells to radiation by selenadiazole derivatives by regulation of ROS-mediated DNA damage and ERK and AKT pathways

X-ray-based radiotherapy represents one of the most effective ways in treating human cancers. However, radioresistance and side effect remain as the most challenging issue. This study describes the design and application of novel selenadiazole derivatives as radiotherapy sensitizers to enhance X-ray-induced inhibitory effects on A375 human melanoma and Hela human cervical carcinoma cells. The results showed that, pretreatment of the cells with selenadiazole derivatives dramatically enhance X-ray-induced growth inhibition and colony formation. Flow cytometry analysis indicates that the sensitization by selenadiazole derivatives was mainly caused by induction of G2/M cell cycle arrest. Results of Western blotting demonstrated that the combined treatment-induced A375 cells growth inhibition was achieved by triggering reactive oxygen species-mediated DNA damage involving inactivation of AKT and MAPKs. Further investigation revealed that selenadiazole derivative in combination with X-ray could synergistically inhibit the activity of thioredoxin reductase-1 in A375 cells. Taken together, these results suggest that selenadiazole derivatives can act as novel radiosensitizer with potential application in combating human cancers.

Cerium oxide nanoparticles induced toxicity in human lung cells: role of ROS mediated DNA damage and apoptosis.

Cerium oxide nanoparticles (CeO2 NPs) have promising industrial and biomedical applications. In spite of their applications, the toxicity of these NPs in biological/physiological environment is a major concern. Present study aimed to understand the molecular mechanism underlying the toxicity of CeO2 NPs on lung adenocarcinoma (A549) cells. After internalization, CeO2 NPs caused significant cytotoxicity and morphological changes in A549 cells. Further, the cell death was found to be apoptotic as shown by loss in mitochondrial membrane potential and increase in annexin-V positive cells and confirmed by immunoblot analysis of BAX, BCl-2, Cyt C, AIF, caspase-3, and caspase-9. A significant increase in oxidative DNA damage was found which was confirmed by phosphorylation of p53 gene and presence of cleaved poly ADP ribose polymerase (PARP). This damage could be attributed to increased production of reactive oxygen species (ROS) with concomitant decrease in antioxidant “glutathione (GSH)” level. DNA damage and cell death were attenuated by the application of ROS and apoptosis inhibitors N-acetyl-L- cysteine (NAC) and Z-DEVD-fmk, respectively. Our study concludes that ROS mediated DNA damage and cell cycle arrest play a major role in CeO2 NPs induced apoptotic cell death in A549 cells. Apart from beneficial applications, these NPs also impart potential harmful effects which should be properly evaluated prior to their use.

Reactive Oxygen Species-Mediated DNA Damage and Apoptosis in Human Skin Epidermal Cells After Exposure to Nickel Nanoparticles

Nickel nanoparticles (NiNPs) are increasingly used in various applications due to their unique properties. However, there is little information concerning the toxicity of NiNPs in the human skin cell (A431). The present study was designed to investigate the cytotoxicity, apoptosis, and DNA damage due to NiNPs in A431 cells. A cellular proliferative capacity test showed that NiNPs induce significant cytotoxicity in a dose- and time-dependent manner. NiNPs were also found to induce oxidative stress evidenced by the generation of reactive oxygen species (ROS) and depletion of glutathione (GSH). Further, co-treatment with the antioxidant N-acetylcysteine (NAC) mitigated the ROS generation due to NiNPs, suggesting the potential mechanism of oxidative stress. NiNPs also induced significant elevation of lipid peroxidation, catalase, and superoxide dismutase and caspase-3 activity in A431 cells. In addition, NAC suppressed NiNP-induced caspase-3 activity. DNA fragmentation analysis using the comet assay showed that the NiNPs cause genotoxicity in a dose- and time-dependent manner. Therefore, the study points out the capability of the NiNPs to induce oxidative stress resulting in apoptosis and genotoxicity. This study warrants more careful assessment of NiNPs before their industrial applications.

Shikonin Inhibits Thyroid Cancer Cell Growth and Invasiveness Through Targeting Major Signaling Pathways

Shikonin, which is an active naphthoquinone isolated from traditional Chinese herbal medicine Zi Cao, has been recently developed to use as an antitumor agent in colorectal cancer, melanoma, leukemia, breast cancer, and hepatocellular cancer. However, its antitumor effect in thyroid cancer remains largely unknown. The aim of the study was to test the therapeutic potential of shikonin for thyroid cancer and explore the mechanisms underlying antitumor effects of shikonin. We examined the effects of shikonin on proliferation, cell cycle, apoptosis, migration, invasion, and xenograft tumor growth in thyroid cancer cells and the effect of shikonin on proliferation of primary thyroid cancer cells. Shikonin inhibited thyroid cancer cell proliferation in a dose- and time-dependent manner and induced cell cycle arrest. Moreover, shikonin induced cell apoptosis through reactive oxygen species-mediated DNA damage and activation of the p53 signaling pathway. Our data also showed that shikonin dramatically inhibited thyroid cancer cell migration and invasion by suppressing epithelial-mesenchymal transition and downregulating expression of Slug and MMP-2, -9, and -14. Further elucidation of the mechanisms involved revealed that shikonin markedly repressed the phosphorylation of Erk and Akt and activated the p16/Retinoblastoma protein (Rb) pathway in thyroid cancer cells. Growth of xenograft tumors derived from the thyroid cancer cell line FTC133 in nude mice was significantly inhibited by shikonin. Importantly, we did not find the effect of shikonin on liver function in mice. We for the first time demonstrated that shikonin is a potentially effective antitumor agent for thyroid cancers.

Selenocystine potentiates cancer cell apoptosis induced by 5-fluorouracil by triggering reactive oxygen species-mediated DNA damage and inactivation of the ERK pathway

5-Fluorouracil (5-FU)-based chemotherapy as a first-line treatment is quite limited, because of its inefficiency and clinical resistance to it. The search for chemosensitizers that could augment its efficiency and overcome the drug resistance to 5-FU has kindled great interest among scientists. Selenocystine (SeC), a naturally occurring selenoamino acid, displayed broad-spectrum anticancer activity in our previous studies. This study demonstrates that SeC acts as an effective enhancer of 5-FU-induced apoptosis in A375 human melanoma cells through induction of mitochondria-mediated apoptosis with the involvement of DNA damage-mediated p53 phosphorylation and ERK inactivation. Pretreatment of the cells with SeC significantly enhanced 5-FU-induced loss of mitochondrial membrane potential (∆ψm) by regulating the expression levels of Bcl-2 family proteins. SeC and 5-FU in combination also triggered cell oxidative stress through regulation of the intracellular redox system and led to DNA damage and inactivation of ERK and AKT. Moreover, inhibitors of ERK and AKT effectively enhanced the apoptotic cell death induced by the combined treatment. However, pretreatment of the cells with glutathione reversed the apoptosis induced by SeC and 5-FU and recovered the expression of ERK and AKT inactivation, which revealed the important role of reactive oxygen species in cell apoptosis and regulation of ERK and AKT pathways. Taken together, our results suggest that a strategy of using SeC and 5-FU in combination could be a highly efficient way to achieve anticancer synergism.

A novel study of antibacterial activity of copper iodide nanoparticle mediated by DNA and membrane damage

In this article potential activity of nanoparticles (NPs) of copper iodide (CuI) as an antibacterial agent has been presented. The nano particles are synthesized by co-precipitation method with an average size of 8nm as determined by Transmission Electron Microscope (TEM). The average charge of the NPs is −21.5mV at pH 7 as obtained by zeta potential measurement and purity is determined by XRD. These NPs are able to kill both gram positive and gram negative bacteria. Among the bacteria tested, DH5α is more sensitive but Bacillus subtilis is more resistant to NPs of CuI. Consequently, the MIC and MBC values of DH5α is least (0.066mg/ml and 0.083mg/ml respectively) and B. subtilis is highest (0.15mg/ml and 0.18mg/ml respectively) among the tested bacterial strains. From our studies it is inferred that CuI NPs produce reactive oxygen species (ROS) in both gram negative and gram positive bacteria and it also causes ROS mediated DNA damage for the suppression of transcription as revealed by reporter gene assay. Probably ROS is formed on the surface of NPs of CuI in presence of amine functional groups of various biological molecules. Furthermore they induce membrane damage as determined by atomic force microscopy (AFM). Thus production of ROS and membrane damage are major mechanisms of the bactericidal activity of these NPs of CuI.

Neurons Efficiently Repair Glutamate-induced Oxidative DNA Damage by a Process Involving CREB-mediated Up-regulation of Apurinic Endonuclease 1

Glutamate, the major excitatory neurotransmitter in the brain, activates receptors coupled to membrane depolarization and Ca(2+) influx that mediates functional responses of neurons including processes such as learning and memory. Here we show that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using non-toxic physiological levels of glutamate. This DNA damage was prevented by intracellular Ca(2+) chelation, the mitochondrial superoxide dismutase mimetic MnTMPyP (Mn-5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis(methochloride)), and blockade of the permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1). APE1 knockdown induced accumulation of oxidative DNA damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca(2+)/calmodulin-dependent kinase inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca(2+)- and mitochondrial reactive oxygen species-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca(2+)-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors.

Recent Advances in Understanding the Antineoplastic Mechanisms of Farnesyltransferase Inhibitors

Farnesyltransferase (FTase) inhibitors (FTI) have broad antineoplastic actions targeting both cancer cells and mesenchymal cells involved in tumor angiogenesis. The small GTPases H-Ras, Rheb, and RhoB and the centromere proteins CENP-E and CENP-F are relevant targets of farnesylation inhibition; however, their relative importance in the antineoplastic effect of FTIs may vary in different cell types at different stages of the cell cycle and at different stages in oncogenesis. Three recent studies argue that Ras-independent and perhaps even FTase-independent properties are important to the antineoplastic action of this class of drugs. In mice, genetic ablation of FTase does not abolish the oncogenic activity of Ras, limiting the original conception of FTIs as an effective means to target Ras in cancer cells. FTase may not be the sole molecular target of these agents, and one study has suggested that FTIs act by targeting geranylgeranyl transferase II. Lastly, we have obtained evidence that induction of reactive oxygen species and reactive oxygen species-mediated DNA damage by FTIs may be critical for their antineoplastic action as a class. Together, these findings may alter thinking about how to apply FTIs in the clinic.

2′-deoxyguanosine oxidation is associated with decrease in the DNA-binding activity of the transcription factor Sp1 in liver and kidney from diabetic and insulin-resistant rats

Over the years, several lines of evidence have emerged supporting the role of oxidative stress in the development of diabetic complications. This could involve the increase in the production of reactive oxygen species and the decrease in antioxidative defense systems. Modulation of the level of intracellular reactive oxygen species is likely to affect the intracellular redox homeostasis, which is crucial for numerous biological events such as the transcriptional activation of genes. In this work we studied the binding of the redox transcription factors Sp1 and NF-κB extracted from kidney and liver of streptozotocin diabetic (STZ) and fructose-fed rats using electrophoretic mobility shift (EMSA) assay. In addition, the level in 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) was assessed within DNA by high performance liquid chromatography with electrochemical detection (HPLC-EC). A decrease in the affinity of Sp1 to DNA was observed in the kidney of STZ rats and fructose-fed rats (15% ± 8.3 and 54% ± 6.9, respectively, versus control group set to 100%). This was also found to occur to a lower extent, in the liver. Interestingly, higher levels of 8-oxodGuo, a biomarker of DNA oxidation, were measured in the kidney of diabetic rats. Therefore, the modification in the binding efficiency of Sp1 or NF-κB could be related to reactive oxygen species-mediated DNA damage.