Apoptotic DNA Fragmentation Research Articles

Mitigating lead acetate-induced histopathologic and physiologic disorders in rats receiving vitamin C and glutathione supplement.

The present work examines the extreme impact of lead acetate and the preventive function of co-supplementation with vitamin C and glutathione. It hypothesizes that these supplements can alleviate the poisonous effects of lead exposure. Eighty male albino rats, weighing 100±15g, were categorized into four groups: the control group, the second group receiving daily supplements of 100mg/kg of body weight glutathione and 1 mg/100g of body weight vitamin C orally, the third group receiving 100mg/kg body weight of lead acetate orally daily, and the fourth group receiving similar oral dosages of lead acetate along with glutathione and vitamin C. Lead exposure significantly decreased body weight and relative testis weight, while relative organ weights for the liver, kidney, and spleen increased significantly. Additionally, lead acetate increased plasma glutamic pyruvic transaminase and glutamic oxaloacetic transaminase activities and plasma creatinine concentration (p<0.05). Lead concentration rose significantly in blood, urine, liver, and kidney (p<0.05). Examinations revealed that lead acetate exposure induced apoptotic DNA fragmentation in hepatocytes, significantly increasing caspase-3 activity (91%) and annexin V indicators. Moreover, lead exposure induced a decrease in sperm count and motility, along with an increase in abnormal sperm morphology. However, vitamin C and glutathione supplementation significantly improved these adverse impacts, suggesting their protective function in counteracting the harmful impacts of lead acetate in different organs.

Differential cytotoxic effects of Garcinia mangostana pericarp extract on leukaemic versus normal human cell lines: insights into selective anticancer activity

IntroductionGarcinia mangostana L. is widely recognised for its traditional medicinal uses and growing relevance in the nutraceutical sector. This research endeavour is designed to scrutinise the phytochemical composition and cytotoxic effects of an alcoholic extract derived from the pericarps of G. mangostana (ME) on leukaemic cell lines. MethodsG. mangostana L. pericarps were subjected to ethanol extraction, and subsequent compound identification was conducted via Capillary Liquid Chromatography-Electrospray Ionisation-Quadrupole Time-of-Flight Tandem Mass Spectrometry (CapLC-ESI-QTOF-MS/MS). In parallel, the extract was fractionated through High-Performance Liquid Chromatography (HPLC). Furthermore, U937, Jurkat and normal human umbilical vein endothelial cell (HUVEC) lines were exposed to varying concentrations of the extract to assess cell proliferation, viability, cytotoxicity, and apoptotic DNA damage. ResultsInvestigations confirmed the presence of various xanthones, including β-mangostin, α-mangostin, and garcinone E. Significantly, ME displayed pronounced cytotoxic effects specifically on leukaemic cells, distinguishing its impact on malignant cells as opposed to non-malignant ones, and facilitated apoptotic DNA fragmentation. Moreover, the whole extract consistently displayed greater cytotoxicity compared to individual fractions. ConclusionAccording to the results obtained ME selective cytotoxicity against cancer cell lines and greater biological activity than the single compounds in the extract. These findings underscore ME's potential in cancer prevention, complementing dietary strategies ahead of pharmaceutical interventions.

Pathogenesis and New Pharmacological Approaches to Noise-Induced Hearing Loss: A Systematic Review.

Noise-induced hearing loss (NIHL) is responsible for significant adverse effects on cognition, quality of life and work, social relationships, motor skills, and other psychological aspects. The severity of NIHL depends on individual patient characteristics, sound intensity, and mainly the duration of sound exposure. NIHL leads to the production of a reactive oxygen (ROS) inflammatory response and the activation of apoptotic pathways, DNA fragmentation, and cell death. In this situation, antioxidants can interact with free radicals as well as anti-apoptotics or anti-inflammatory substances and stop the reaction before vital molecules are damaged. Therefore, the aim of this study was to analyze the effects of different pharmacological treatments, focusing on exogenous antioxidants, anti-inflammatories, and anti-apoptotics to reduce the cellular damage caused by acoustic trauma in the inner ear. Experimental animal studies using these molecules have shown that they protect hair cells and reduce hearing loss due to acoustic trauma. However, there is a need for more conclusive evidence demonstrating the protective effects of antioxidant/anti-inflammatory or anti-apoptotic drugs' administration, the timeline in which they exert their pharmacological action, and the dose in which they should be used in order to consider them as therapeutic drugs. Further studies are needed to fully understand the potential of these drugs as they may be a promising option to prevent and treat noise-induced hearing loss.

Cytotoxicity and Apoptotic DNA Fragmentation Evaluation of Ethanolic Extract of Phyllanthus niruri L. in Vero and Primary Feline Testicular Cells

Phyllanthus niruri or Dukung Anak is native to Southeast Asia, particularly in Malaysia and has been used as traditional preparations and as health supplements. However, previous studies have found that P. niruri can impair the male reproductive functions. With booming population of stray animals within the community, a new controlling method that is more affordable and safer must be developed. This study was aimed to explore the safety of P. niruri as a male herbal contraceptive to be used in veterinary medicine as one of the non-surgical sterilisation methods. For the preliminary part of this ongoing research, we have investigated the cytotoxicity activity of P. niruri ethanolic extract on Vero and primary feline testicular cells (FTC) and DNA apoptotic activity in FTC. Phyllanthus niruri extract was prepared using cold maceration method in 50% ethanol. The FTC were prepared from feline testes obtained from post-routine castration from local private veterinary clinics, while Vero cells were obtained from the archived cells of Virology Laboratory, Faculty of Veterinary Medicine Universiti Malaysia Kelantan (FPV UMK). The MTT assay was performed to evaluate the cytotoxicity of these plant extracts on FTC and Vero cells. Apoptosis was assessed using DNA laddering assay on extracted DNA of the treated FTC cells and observed the DNA patterns in gel electrophoresis. The 50% cytotoxic concentration (CC50) of the ethanolic extract of P. niruri was a dose dependent. The DNA laddering assay revealed that the plant extract does not induce apoptosis in FTC. Therefore, the study outcomes indicated that the ethanolic extract of P. niruri is potentially safe for animal use. However, in vivo studies are required to confirm these findings.

Exploiting mechanoregulation via FAK/YAP to overcome platinum resistance in ovarian cancer

Cancer cells mechanically interact with the tumor microenvironment during cancer development. Mechano-reciprocity has emerged as a crucial factor affecting anti-cancer drug resistance during adjuvant therapy. Here, we investigated the focal adhesion kinase (FAK)/Yes-associated protein (YAP) signaling axis as a prospective strategy for circumventing cisplatin resistance in ovarian cancer (OC). The Cancer Genome Atlas (TCGA) data analysis revealed that FAK overexpression significantly correlated with unfavorable clinical outcomes in patients with ovarian cancer. AFM indentation experiments showed that cell elasticity depends on FAK activity. Notably, the combination of FAK inhibition and cisplatin treatment led to a 69 % reduction in the IC50 of cisplatin. This combined treatment also increased apoptosis compared to the individual treatments, along with the upregulation of the pro-apoptotic factor BAX and cleaved PARP. Suppressing FAK expression sequestered YAP in the cytosol, potentially reducing cellular proliferation and promoting apoptosis. Moreover, reduced FAK expression sensitized drug-resistant OC cells to cisplatin treatment owing to a decrease in nuclear tension, allowing the relocation of YAP to the cytosol. In a mouse model, the co-administration of an FAK inhibitor and cisplatin significantly suppressed tumor growth and increased apoptotic events and DNA fragmentation. Our findings suggest that drug resistance can be attributed to the perturbation of mechanosensing signaling pathways, which drive the mechanical reinforcement of cancer cells. OC cells can restore their sensitivity to cisplatin treatment by strategically reducing YAP localization in the nucleus through FAK downregulation.

Green synthesis of copper oxide nanoparticles using genus Inula and evaluation of biological therapeutics and environmental applications

Abstract In this research, we produced copper oxide nanoparticles (CuO NPs) using extracts from the entire above-ground portion of plants of genus Inula (Inula graveolens). The synthesis of CuO NPs was verified through various physicochemical analytical methods, including UV–visible, Fourier transform infrared, and transmission electron microscopy. The CuO NPs were found to be around 20 nm in size and spherical in shape. Subsequently, the synthesized compounds were evaluated for their anticancer properties. After treating A549 cells with CuO NPs at concentrations of 15 and 30 μg, we examined their cytotoxicity, lipid peroxidation activity (malondialdehyde level), and antioxidant activity (catalase, superoxide dismutase, and glutathione levels). Additionally, we analyzed the expression of apoptotic marker genes (p53, caspase-3, and caspase-9), cytokine levels (IL-6 and TNF-α), and DNA fragmentation. Our findings demonstrated that CuO NPs enhanced the expression of apoptotic genes, suggesting that phytochemical-derived NPs from Inula extracts induce apoptosis by upregulating tumor suppressor genes and downregulating oncogenes in A549 cells. Furthermore, CuO NPs exhibited higher susceptibility toward B. subtilis and S. aureus compared to ampicillin. Using the response surface methodology, we determined that CuO NPs are effective adsorbents for removing Pb2+ ions from aqueous solutions, making them promising for environmental applications. Overall, our results indicate that CuO NPs have potential as antimicrobial, antioxidant, and anticancer agents and as efficient adsorbents.

Structure-specific nucleases in genome dynamics and strategies for targeting cancers.

Nucleases are a super family of enzymes that hydrolyze phosphodiester bonds present in genomes. They widely vary in substrates, causing differentiation in cleavage patterns and having a diversified role in maintaining genetic material. Through cellular evolution of prokaryotic to eukaryotic, nucleases become structure-specific in recognizing its own or foreign genomic DNA/RNA configurations as its substrates, including flaps, bubbles, and Holliday junctions. These special structural configurations are commonly found as intermediates in processes like DNA replication, repair, and recombination. The structure-specific nature and diversified functions make them essential to maintaining genome integrity and evolution in normal and cancer cells. In this article, we review their roles in various pathways, including Okazaki fragment maturation during DNA replication, end resection in homology-directed recombination repair of DNA double-strand breaks, DNA excision repair and apoptosis DNA fragmentation in response to exogenous DNA damage, and HIV life cycle. As the nucleases serve as key points for the DNA dynamics, cellular apoptosis, and cancer cell survival pathways, we discuss the efforts in the field in developing the therapeutic regimens, taking advantage of recently available knowledge of their diversified structures and functions.

Photoresponsive 'chemo-free' phytotherapy: formulation development for the treatment of triple-negative breast cancer.

Aim: The present investigation aimed to develop a chemo-free, nanophytosomal system to treat triple-negative breast cancer (TNBC) via a phyto-photo dual treatment strategy. Method: Size, shape, surface analysis, photoprovoked release profile, photothermal stability, (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay, apoptotic assay, DNA fragmentation, in vitro cellular uptake evaluation, mitochondrial membrane potential and caspase-3 assay, andphotodynamic evaluation. Results: Biological experiments using MDA-MB-231 cells displayed dose-dependent synergistic anti-TNBC activity of PhytoS/Houttuynia cordata extract (HCE)/IR780 as compared with Phyto/HCE, PhytoS/IR780 and even more promising under laser treatment. Apoptotic assay and DNA fragmentation analysis also showed enhanced anti-TNBC effects. Investigation found that HCE acts via suppression ofmitochondrial membrane potential and inducing caspase-3 activity in cells. Conclusion: Our findings suggested that photo-empowered phytotherapy can be employed effectively and safely against TNBC.

Comparison of angiogenic potential in vitrified vs. slow frozen human ovarian tissue

Vitrification of ovarian tissue is a promising alternative approach to the traditional slow freezing method. Few empirical investigations have been conducted to determine the angiogenic profiles of these two freezing methods. In this study we aimed to answer the question whether one of the cryopreservation methods should be preferred based on the secretion of angiogenic factors. Tissue culture with reduced oxygen (5%) was conducted for 48 h with samples of fresh, slow frozen/thawed and vitrified/rapid warmed ovarian cortex tissue from 20 patients. From each patient, tissue was used in all three treatment groups. Tissue culture supernatants were determined regarding cytokine expression profiles of angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin binding epidermal growth factor, hepatocyte growth factor, Leptin, Platelet-derived growth factor B, placental growth factor and vascular endothelial growth factor A via fluoroimmunoassay. Apoptotic changes were assessed by TUNEL staining of cryosections and supplemented by hematoxylin and eosin and proliferating cell nuclear antigen staining. Comparing the angiogenic expression profiles of vitrified/rapid warmed tissue with slow frozen/thawed tissue samples, no significant differences were observed. Detection of apoptotic DNA fragmentation via TUNEL indicated minor apoptotic profiles that were not significantly different comparing both cryopreservation methods. Vitrification of ovarian cortical tissue does not appear to impact negatively on the expression profile of angiogenic factors and may be regarded as an effective alternative approach to the traditional slow freezing method.

Bilobetin induces apoptosis in human hepatocellular carcinoma cells via ROS level elevation and inhibition of CYP2J2

Bilobetin is a biflavonoid isolated from the leaves of Ginkgo biloba. Bilobetin displays several biological effects, however, the activities of bilobetin against cancer have been solely demonstrated. Thus, the aim of this study is investigating the apoptotic effects of and anticancer mechanism of bilobetin in Huh7 and HepG2 cells, both of human hepatocellular carcinoma (HCC) cell lines. MTT, cell counting, and colony formation assay showed that anti-proliferation effect of bilobetin. Cell cycle analysis revealed that bilobetin induces increase of population of the sub G1 phase. Annexin V/PI staining and TUNEL assay showed that bilobetin treatment promotes apoptotic cell death and DNA fragmentation. Bilobetin also induces ROS elevation and DNA damage in HCC cells. Western blot analysis elucidated that bilobetin displays apoptotic signaling pathway in HCC cells via upregulating cleaved PARP, cleaved caspase3 and Bax and downregulating CYP2J2. Bilobetin inhibited CYP2J2-catalyzed terfenadine and ebastine hydroxylase activities with IC50 values of 0.81 and 2.21 μM, respectively. Transfection of CYP2J2 siRNA increased the anticancer effect of bilobetin in HCC cells through the inhibition of CYP2J2 expression. Taken together, this study suggests that bilobetin induces apoptosis against HCC cells via ROS level elevation and inhibition of CYP2J2.

Biochemical and molecular anticancer approaches for Boerhaavia diffusa root extracts in oral cancer.

Boerhaavia diffusa is a medicinal herb with anti-inflammatory, antiproliferative, anticancer, and immunomodulatory properties, found across India. The present study is designed to investigate the therapeutic potential for B. diffusa root extracts in oral cancer cell line. The aqueous and methanolic extracts of B. diffusa were prepared using Soxhlet apparatus. In order to determine the phytochemical constituents of B. diffusa, the extracts were subjected to gas chromatography-mass spectrometry analysis. The antioxidant potential of B. diffusa extracts was assessed by 2,2-Diphenyl-picrylhydrazyl, ferric ion-reducing antioxidant power, catalase and peroxidase assays. The effective concentration of B. diffusa root on cell viability was analyzed by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The ability of B. diffusa root extracts to modify the cell-cycle phases was performed by FACS analysis. The apoptotic inducing potential of B. diffusa in oral cancer cells was confirmed by acridine orange-ethidium bromide and 4',6-diamidino-2-phenylindole staining. The protein profile of apoptotic processes was validated by the Western blot analysis; docking studies were also performed. We observed that antioxidant activity was higher in B. diffusa methanolic extract compared with aqueous extract. The results showed that the methanolic and aqueous extracts of B. diffusa exhibited significant cytotoxic effect with IC50 value of 36 μg/ml and 30 μg/ml, respectively. The apoptotic DNA fragmentation and the apoptotic inducing potential in KB oral cancer cell line were higher for the methanolic extract compared with the aqueous extract. These results were also confirmed by in-silico analysis. The results indicate that extracts obtained from the roots of B. diffusa inhibit the progression of oral cancer. These compounds of pharmacological importance can be either used alone or in combination with other drugs to treat oral cancer.

Amelioration of mercuric chloride-induced physiologic and histopathologic alterations in rats using vitamin E and zinc chloride supplement

The drastic effects of mercuric chloride and the protective efficiency of vitamin E and zinc chloride co-supplementation were clearly investigated in this study. Male rats were divided into four groups. The first was the control. The second received vitamin E (100 mg/kg) and zinc chloride (30 mg/kg) daily. In comparison, the third received mercuric chloride (1 mg/kg) daily, and the fourth received the same mercuric chloride dose supplemented with the same vitamin E and zinc chloride doses. Mercury promotes a significant decline in body weight. It causes a considerable reduction in total red blood cells (RBCs) count and hemoglobin concentration; however, white blood cells (WBCs) increased significantly. Significant mercury-induced elevations in hepatic and renal functions were observed. Mercury induced substantial reductions in catalase (CAT) and superoxide dismutase (SOD). Mercury caused apoptotic DNA fragmentation. It induced degeneration and necrosis in the liver and kidney. It induced necrosis, leukocyte infiltration and blood vessel congestion in the cerebral cortex. Shrinkage and deterioration of Purkinje cells of the cerebellum were observed in response to mercuric chloride toxicity. Mercuric chloride enhanced shrinking in seminiferous tubules and Leydig cells. It reduced sperm count, sperm motility, and testosterone concentration; however, it promoted abnormal sperm morphology. Administration of vitamin E and zinc chloride showed marked improvement in different parameters under investigation, however, further research is needed to determine fate of mercury.

The contribution of hyperhomocysteinemia and hypoperfusion to amyloid‐beta induced cerebral endothelial cell dysfunction

AbstractBackgroundCerebrovascular dysfunction has been implicated as a major contributor to Alzheimer’s Disease (AD) pathology, with particularly endothelial cell (EC) stress promoting the focal ischemia, cerebral blood flow impairments, and blood brain barrier (BBB) permeability that are pathologically characteristic in AD. Recent evidence has emerged suggesting a link between cardiovascular (CV) diseases and AD pathology, particularly showing that CV/cerebrovascular risk factors, including hyperhomocysteinemia (Hhcy) and hypoperfusion (oxygen and glucose deprivation (OGD)), contribute to AD pathology and risk. Despite this, the underlying molecular mechanisms for this interaction remain unclear. Previously our lab has demonstrated that amyloid beta (Aβ), particularly Aβ40‐Q22 (the vasculotropic Dutch mutant), promotes TRAIL death receptor (DR)‐mediated apoptosis, barrier permeability, and angiogenic impairment within human cerebral ECs. We tested the hypothesis that Hhcy and hypoperfusion exacerbate Aβ‐induced cerebral EC TRAIL DR‐mediated apoptosis, barrier dysfunction, and angiogenesis defects.MethodHuman cerebral microvascular ECs were challenged with AβQ22 and/or homocysteine (Hcy) in the presence/absence of hypoperfusion. Apoptotic mediator expression, caspase activation, and DNA fragmentation were measured to assess apoptosis. BBB protein expression and trans‐endothelial‐electrical‐resistance (TEER) were measured to assess EC barrier integrity. Angiogenesis inhibition and activation assays were utilized to measure EC angiogenic capability.ResultAβQ22 and Hcy challenge independently upregulated EC expression of TRAIL DR‐related apoptotic mediators and caspase activity, and, at certain time‐points, resulted in an additive upregulation of apoptotic mediators and caspase activity. AβQ22, Hcy, and hypoperfusion individually increased DNA fragmentation within cerebral ECs. Combination treatments of AβQ22 and Hcy created an additive effect on DNA fragmentation. Hypoperfusion and AβQ22 independently decreased BBB protein expression and TEER, while an additive decrease was observed with the combined challenges. AβQ22 and Hcy independently decreased angiogenesis progression, with some timepoints revealing an additive decrease with the combination treatment.ConclusionThe presence of CV risk factors seems to exacerbate Aβ induced‐EC apoptosis, barrier dysfunction, and angiogenic impairment, revealing several of the specific molecular mechanism through which amyloidosis and CV risk factors may additively act to produce EC dysfunction and death in AD pathology.

Detection of apoptotic cells based on in situ hybridization chain reaction using specific hairpins.

There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.

Combination Therapy of Curcumin and Disulfiram Synergistically Inhibits the Growth of B16-F10 Melanoma Cells by Inducing Oxidative Stress

Oxidative stress plays a central role in the pathophysiology of melanoma. Curcumin (CUR) is a polyphenolic phytochemical that stimulates reactive oxygen species (ROS) production, while disulfiram (DSS) is a US FDA-approved drug for the treatment of alcoholism that can act by inhibiting the intracellular antioxidant system. Therefore, we hypothesized that they act synergistically against melanoma cells. Herein, we aimed to study the antitumor potential of the combination of CUR with DSS in B16-F10 melanoma cells using in vitro and in vivo models. The cytotoxic effects of different combination ratios of CUR and DSS were evaluated using the Alamar Blue method, allowing the production of isobolograms. Apoptosis detection, DNA fragmentation, cell cycle distribution, and mitochondrial superoxide levels were quantified by flow cytometry. Tumor development in vivo was evaluated using C57BL/6 mice bearing B16-F10 cells. The combinations ratios of 1:2, 1:3, and 2:3 showed synergic effects. B16-F10 cells treated with these combinations showed improved apoptotic cell death and DNA fragmentation. Enhanced mitochondrial superoxide levels were observed at combination ratios of 1:2 and 1:3, indicating increased oxidative stress. In vivo tumor growth inhibition for CUR (20 mg/kg), DSS (60 mg/kg), and their combination were 17.0%, 19.8%, and 28.8%, respectively. This study provided data on the potential cytotoxic activity of the combination of CUR with DSS and may provide a useful tool for the development of a therapeutic combination against melanoma.

Relaxin inhibits 177Lu-EDTMP associated cell death in osteosarcoma cells through notch-1 pathway.

177Lu-EDTMP (Ethylenediamine tetramethylene phosphonic acid) is the most used radioactive agent for pain palliation in bone cancer patients. The present study aims to study the impact of relaxin-2 on the 177Lu-EDTMP associated cell toxicity and death in osteosarcoma cells. MG63 and Saos-2 cells were cultured with 177Lu-EDTMP (37 MBq) for 24 h with and without pretreatment of recombinant relaxin 2 (RLXH2) for 12 and 24 h. 177Lu-EDTMP associated cellular deterioration and death was determined by LDH, MTT, and trypan blue dye assays. ELISA-based kit was used to determine apoptotic DNA fragmentation. Western blotting was used to determine expression levels of apoptotic-related signalling pathway proteins like bcl2, poly(ADP-ribose) polymerase (PARP), and MAPK (mitogen-activated protein kinase). Our results found that RLXH2 counters 177Lu-EDTMP associated cellular toxicity. Similarly, RLXH2 was able to counter 177Lu-EDTMP induced cell death in a concentration and time--dependent manner. Furthermore, it was found that RLXH2 treatment prevents apoptosis in 177Lu-EDTMP challenged cells through activation of the notch-1 pathway in a concentration- and time-dependent manner. We reported that RLXH2 significantly declined cellular toxicity and apoptosis associated with 177Lu-EDTMP in MG63 and Saos-2 cells through the notch-1 pathway.

Apple pectin-based Zataria multiflora essential oil (ZEO) nanoemulsion: An approach to enhance ZEO DNA damage induction in breast cancer cells as in vitro and in silico studies reveal.

Zataria multiflora essential oil (ZEO) is a natural complex of compounds with a high apoptotic potential against breast cancer cells and minor toxicity toward normal cells; however, similar to many essential oils, ZEO utilization in pharmaceutical industries has limitations due to its labile and sensitive ingredients. Nanoemulsification based on natural polymers is one approach to overcome this issue. In this study, an apple pectin-ZEO nanoemulsion (AP-ZEONE) was prepared and its morphology, FTIR spectra, and physical properties were characterized. Furthermore, it was shown that AP-ZEONE substantially suppresses the viability of MDA-MB-231, T47D, and MCF-7 breast cancer cells. AP-ZEONE significantly induced apoptotic morphological alterations and DNA fragmentation as confirmed by fluorescent staining and TUNEL assay. Moreover, AP-ZEONE induced apoptosis in MDA-MB-231 cells by loss of mitochondrial membrane potential (ΔΨm) associated with the accumulation of reactive oxygen species (ROS), G2/M cell cycle arrest, and DNA strand breakage as flow cytometry, DNA oxidation, and comet assay analysis revealed, respectively. Spectroscopic and computational studies also confirmed that AP-ZEONE interacts with genomic DNA in a minor groove/partial intercalation binding mode. This study demonstrated the successful inhibitory effect of AP-ZEONE on metastatic breast cancer cells, which may be beneficial in the therapy process.

Novel benzylidene benzofuranone analogues as potential anticancer agents: design, synthesis and in vitro evaluation based on CDK2 inhibition assays

The classical anticancer agents do not have their efficacy on inhibiting the G2 phase of the cell cycle. There are a very few reports available on drugs that work at G2 phase. Flavopiridol is one such drug candidate. In the current study, we sought to make analogues of flavopiridol. Still, the conditions used during their synthesis were unfavourable for the formation of flavopiridol and led to the generation of benzofuranones. In the present work, a new series of benzylidene benzofuranones were designed, synthesized and evaluated for their antioxidant, anti-colorectal cancer activity. Molecular docking, MMGBSA and molecular dynamics studies were conducted to assess their binding affinity at the active site of CDK2. Based on the cytotoxicity exhibited by test compounds, the compound NISOA4 (from isopropyl series) was further selected for mechanistic anticancer studies on HCT 116 cell lines. The compound selected was evaluated by comet assay, DNA fragmentation assay, cell cycle analysis, apoptosis detection by annexin FITC, semi-quantitative RTPCR based gene expression studies and FRET assay on the target CDK2/Cyclin A. Compound NISOA4 exhibited marked olive moments in comet assay studies. The apoptotic DNA fragmentation for compound NISOA4 demonstrated a marked change in the DNA fragmentation. The compound exhibited cell cycle arrest at G2/M phase at both the test concentrations. Apoptosis induction was observed at both the test concentrations and the compound was found to be a potent proapoptotic agent. It exhibited marked inhibition for the CDK2 gene expression and did not show any effect on CyclinA gene expression. However, the compound NISOA4 along with other analogues showed appreciable inhibition for the CDK2/Cyclin A target enzyme.

Hotspots of single-strand DNA "breakome" are enriched at transcriptional start sites of genes.

Single-strand breaks (SSBs) represent one of the most common types of DNA damage, yet not much is known about the genome landscapes of this type of DNA lesions in mammalian cells. Here, we found that SSBs are more likely to occur in certain positions of the human genome—SSB hotspots—in different cells of the same cell type and in different cell types. We hypothesize that the hotspots are likely to represent biologically relevant breaks. Furthermore, we found that the hotspots had a prominent tendency to be enriched in the immediate vicinity of transcriptional start sites (TSSs). We show that these hotspots are not likely to represent technical artifacts or be caused by common mechanisms previously found to cause DNA cleavage at promoters, such as apoptotic DNA fragmentation or topoisomerase type II (TOP2) activity. Therefore, such TSS-associated hotspots could potentially be generated using a novel mechanism that could involve preferential cleavage at cytosines, and their existence is consistent with recent studies suggesting a complex relationship between DNA damage and regulation of gene expression.

Antitumor Effect of Guatteria olivacea R. E. Fr. (Annonaceae) Leaf Essential Oil in Liver Cancer.

Guatteria olivacea R. E. Fries (synonym Guatteria punctata (Aubl.) R.A. Howard) is a tree of 10–27 m tall popularly known as “envira-bobó”, “envira-fofa”, “envireira”, “embira”, “embira-branca”, “embira-preta”, envira-branca”, and “envira-preta”, which can be found in the Brazilian Amazon biome. In this study, we evaluated the cytotoxic and antitumor effects of the essential oil (EO) obtained from the leaves of G. olivacea against liver cancer using HepG2 cells as a model. EO was obtained using a hydrodistillation Clevenger-type apparatus and was qualitatively and quantitatively characterized using GC–MS and GC–FID, respectively. The alamar blue assay was used to assess the cytotoxic potential of EO in a panel of human cancer cell lines and human non-cancerous cells. In HepG2 cells treated with EO, YO-PRO-1/propidium iodide staining, cell cycle distribution, and reactive oxygen species (ROS) were examined. In C.B-17 SCID mice with HepG2 cell xenografts, the efficacy of the EO (20 and 40 mg/kg) was tested in vivo. GC–MS and GC–FID analyses showed germacrene D (17.65%), 1-epi-cubenol (13.21%), caryophyllene oxide (12.03%), spathulenol (11.26%), (E)-caryophyllene (7.26%), bicyclogermacrene (5.87%), and δ-elemene (4.95%) as the major constituents of G. olivacea leaf EO. In vitro cytotoxicity of EO was observed, including anti-liver cancer action with an IC50 value of 30.82 μg/mL for HepG2 cells. In HepG2 cells, EO treatment increased apoptotic cells and DNA fragmentation, without changes in ROS levels. Furthermore, the EO inhibited tumor mass in vivo by 32.8–57.9%. These findings suggest that G. olivacea leaf EO has anti-liver cancer potential.