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A simple agarose-trap method for detection of DNA fragmentation in apoptotic cells

Norazizah S, AbuBakar S

JUMMEC, 2001;6:40-41.

Matched MeSH terms: DNA Fragmentation
Comparative analysis of tests used to assess sperm chromatin integrity and DNA fragmentation

Dutta S, Henkel R, Agarwal A

Andrologia, 2021 Mar;53(2):e13718.
PMID: 32628294 DOI: 10.1111/and.13718

Male infertility has a complex etiology, and many times, the cause is unknown. While routine semen analysis provides an overview of basic semen parameters, such as sperm concentration, motility, viability and morphology, a significant overlap of these parameters has been reported in fertile and infertile men. Moreover, conventional semen parameters do not reveal the cellular or molecular mechanisms of sperm dysfunctions leading to infertility. Therefore, sperm functional parameters, including sperm chromatin integrity, are evaluated to provide information on subtle sperm defects that are not routinely identified. Incomplete or defective sperm chromatin condensation increases the susceptibility of the sperm DNA to oxidative damage or other factors. To evaluate sperm chromatin integrity, different methods with varying degrees of diagnostic and prognostic capabilities are available. Among these assays, SCSA, TUNEL and SCD assays are most commonly used. While these assays rather evaluate the DNA directly for damages, the aniline blue and chromomycin A3 stains test for the quality of chromatin condensation. Thus, this review discusses and compares different methods used to evaluate sperm chromatin integrity and condensation, and their inclusion in the routine evaluation of the male infertility.

Matched MeSH terms: DNA Fragmentation
How to select healthy sperm for intracytoplasmic sperm injection in samples with high sperm DNA fragmentation?

Garrido N, Gül M, Jindal S, Vogiatzi P, Saleh R, Durairajanayagam D, et al.

Panminerva Med, 2023 Jun;65(2):148-158.
PMID: 37194246 DOI: 10.23736/S0031-0808.23.04870-X

The body of evidence supports the negative impact of increased sperm DNA fragmentation (SDF) on natural fertility as well as assisted reproduction conditions. High SDF has been correlated with low pregnancy and delivery rates following intrauterine insemination. Also, high SDF is accused of reducing the rates of fertilization, implantation, pregnancy, and live birth following in-vitro fertilization (IVF). Despite no impact of high SDF on fertilization or pregnancy rates following intracytoplasmic sperm injection (ICSI), it has been correlated with poor embryo quality and a higher risk of miscarriage. Several methods have been introduced to help select sperm with the best DNA quality to be used in assisted reproductive technology procedures. These include magnetic-activated cell sorting, intracytoplasmic morphologically selected sperm injection, physiologic ICSI, and microfluidic sperm sorters, among others. This article aimed to discuss the impact of high SDF in infertile men on the reproductive outcome of couples undergoing IVF/ICSI. Additionally, this review highlights the principles, advantages, and limitations of different techniques that are currently used for the selection of sperm with intact DNA to be utilized for ICSI.

Matched MeSH terms: DNA Fragmentation
Fulltext Antioxidant, anticancer, apoptosis properties and chemical composition of black truffle Terfezia claveryi

Dahham SS, Al-Rawi SS, Ibrahim AH, Abdul Majid AS, Abdul Majid AMS

Saudi J Biol Sci, 2018 Dec;25(8):1524-1534.
PMID: 30591773 DOI: 10.1016/j.sjbs.2016.01.031

Desert truffles are seasonal and important edible fungi that grow wild in many countries around the world. Truffles are natural food sources that have significant compositions. In this work, the antioxidant, chemical composition, anticancer, and antiangiogenesis properties of the Terfezia claveryi truffle were investigated. Solvent extractions of the T. claveryi were evaluated for antioxidant activities using (DPPH, FRAP and ABTS methods). The extracts cytotoxicity on the cancer cell lines (HT29, MCF-7, PC3 and U-87 MG) was determined by MTT assay, while the anti-angiogenic efficacy was tested using ex-vivo assay. All extracts showed moderate anticancer activities against all cancer cells (p DNA fragmentation p

Matched MeSH terms: DNA Fragmentation
Fulltext Modified gel preparation for distinct DNA fragment analysis in agarose gel electrophoresis

Lee SV, Bahaman AR

Trop Biomed, 2010 Aug;27(2):351-4.
PMID: 20962737

Agarose gel electrophoresis is the standard method that is used to separate, identify, and purify DNA fragments. However, this method is time-consuming and capable of separating limited range of fragments. A new technique of gel preparation was developed to improve the DNA fragment analysis via electrophoresis.

Matched MeSH terms: DNA Fragmentation
Sperm DNA fragmentation test: usefulness in assessing male fertility and assisted reproductive technology outcomes

Evgeni E, Sabbaghian M, Saleh R, Gül M, Vogiatzi P, Durairajanayagam D, et al.

Panminerva Med, 2023 Jun;65(2):135-147.
PMID: 37103485 DOI: 10.23736/S0031-0808.23.04836-X

Male infertility is attributed to multiple factors including high levels of sperm DNA fragmentation (SDF). Conventional semen analysis continues to be the gold standard for diagnosis of male factor infertility around the world. However, the limitations of basic semen analysis have prompted the search for complementary assessments of sperm function and integrity. Sperm DNA fragmentation assays (direct or indirect) are emerging as important diagnostic tools in male infertility workups, and have been advocated for use in infertile couples for a variety of reasons. While a controlled degree of DNA nicking is required for appropriate DNA compaction, excessive fragmentation of sperm DNA is linked to impaired male fertility potential, decreased fertilization, poor embryo quality, recurrent pregnancy loss, and failure of assisted reproductive technology procedures. However, there is an ongoing debate regarding whether or not to employ SDF as a routine test for male infertility. This review compiles up-to-date information regarding the pathophysiology of SDF, the currently available SDF tests, and the role of SDF tests in natural and assisted conception conditions.

Matched MeSH terms: DNA Fragmentation
Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

Tee TT, Cheah YH, Meenakshii N, Mohd Sharom MY, Azimahtol Hawariah LP

Biochem Biophys Res Commun, 2012 Apr 20;420(4):834-8.
PMID: 22465013 DOI: 10.1016/j.bbrc.2012.03.083

Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action of xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X(L) expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.

Matched MeSH terms: DNA Fragmentation*
DNA fragmentation, an indicator of apoptosis, in cultured black tiger shrimp Penaeus monodon infected with white spot syndrome virus (WSSV)

Sahtout AH, Hassan MD, Shariff M

Dis Aquat Organ, 2001 Mar 9;44(2):155-9.
PMID: 11324818

Fifty black tiger shrimp Penaeus monodon from commercial cultivation ponds in Malaysia were examined by Tdt-mediated dUTP nick-end labeling (TUNEL) fluorescence assay and agarose gel electrophoresis of DNA extracts for evidence of DNA fragmentation as an indicator of apoptosis. From these specimens, 30 were grossly normal and 20 showed gross signs of white spot syndrome virus (WSSV) infection. Of the 30 grossly normal shrimp, 5 specimens were found to be positive for WSSV infection by normal histology and by nested polymerase chain reaction (PCR) analysis. All of the specimens showing gross signs of WSSV infection were positive for WSSV by normal histology, while 5 were positive by nested PCR only (indicating light infections) and 15 were positive by 1-step PCR (indicating heavy infections). Typical histological signs of WSSV infection included nuclear hypertrophy, chromatin condensation and margination. None of the 25 grossly normal shrimp negative for WSSV by 1-step PCR showed any signs of DNA fragmentation by TUNEL assay or agarose gel electrophoresis of DNA extracts. The 10 specimens that gave PCR-positive results for WSSV by nested PCR only (i.e., 5 grossly normal shrimp and 5 grossly positive for WSSV) gave mean counts of 16 +/- 8% TUNEL-positive cells, while the 25 specimens PCR positive by 1-step PCR gave mean counts of 40 +/- 7% TUNEL-positive cells. Thus, the number of TUNEL positive cells present in tissues increased with increasing severity of infection, as determined by gross signs of white spots on the cuticle, the number of intranuclear inclusions in histological sections, and results from single and nested PCR assays. DNA extracts of PCR-positive specimens tested by agarose gel electrophoresis showed indications of DNA fragmentation either as smears or as 200 bp ladders. Given that DNA fragmentation is generally considered to be a hallmark of apoptosis, the results suggested that apoptosis might be implicated in shrimp death caused by WSSV.

Matched MeSH terms: DNA Fragmentation*
Bioactive compounds from Aspergillus niger extract enhance the antioxidant activity and prevent the genotoxicity in aflatoxin B1-treated rats

Abdel-Wahhab MA, El-Nekeety AA, Hathout AS, Salman AS, Abdel-Aziem SH, Sabry BA, et al.

Toxicon, 2020 Jul 15;181:57-68.
PMID: 32353570 DOI: 10.1016/j.toxicon.2020.04.103

This study aimed to identify the bioactive compounds of the ethyl acetate extract of Aspergillus niger SH2-EGY using GC-MS and to evaluate their protective role against aflatoxin B1 (AFB1)-induced oxidative stress, genotoxicity and cytotoxicity in rats. Six groups of male Sprague-Dawley rats were treated orally for 4 weeks included the control group, AFB1-treated group (80 μg/kg b.w); fungal extract (FE)-treated groups at low (140) or high dose (280) mg/kg b.w and the groups treated with AFB1 plus FE at the two tested doses. The GC-MS analysis identified 26 compounds. The major compounds found were 1,2,3,4,6-Penta-trimethylsilyl Glucopyranose, Fmoc-L-3-(2-Naphthyl)-alanine, D-(-)-Fructopyranose, pentakis (trimethylsilyl) ether, bis (2-ethylhexyl) phthalate, trimethylsilyl ether-glucitol, and octadecanamide, N-(2- methylpropyl)-N-nitroso. The in vivo results showed that AFB1 significantly increased serum ALT, AST, creatinine, uric acid, urea, cholesterol, triglycerides, LDL, carcinoembryonic antigen, alpha-fetoprotein, interleukin-6, Malondialdehyde, nitric oxide, Bax, caspase-3 and P53 mRNA expression, chromosomal aberrations and DNA fragmentation. It decreased serum TP, albumin, HDL, Bcl-2 mRNA expression, hepatic and renal TAC, SOD and GPx content and induced histological changes in the liver and kidney. FE prevented these disturbances in a dosage-dependent manner. It could be concluded that A. niger SH2-EGY extract is safe a promising agent for pharmaceutical and food industries.

Matched MeSH terms: DNA Fragmentation/drug effects
Phyla nodiflora L. Extracts Induce Apoptosis and Cell Cycle Arrest in Human Breast Cancer Cell Line, MCF-7

Teoh PL, Liau M, Cheong BE

Nutr Cancer, 2019;71(4):668-675.
PMID: 30663402 DOI: 10.1080/01635581.2018.1559942

Phyla nodiflora L. has been used as medicinal remedies for various ailments due to its antioxidant, anti-inflammatory, anti-bacterial, anti-tumor activity. Previously, we found that the plant extracts induced DNA fragmentation in MCF-7. This study was to investigate the modes of action of P. nodiflora in inhibiting breast cancer cells using leaf ethyl acetate (EA leaf), stem ethyl acetate (EA stem) and stem methanol (Met stem) extracts. The MTT assay showed that the anti-proliferative effects of P. nodiflora extracts were selective towards MCF-7 with a minimal effect on MCF10A. Morphological changes such as cell shrinkage and nuclear condensation were observed in treated cells. We found that induction of apoptosis by EA leaf and EA stem was mitochondrial-dependent while loss of mitochondrial membrane potential was not found in Met stem-treated cells. In addition, the expression levels of AIFM1, CASP9, CFLAR, and IGF1R were altered after treatment. Decreased BCL-2 expression was found in treated cells while BAX and caspases' expression was upregulated or maintained. All extracts caused perturbation of cell cycle at S phase by dysregulating the expression of cell cycle regulators such as CDKs and cyclins. Our findings indicate that P. nodiflora inhibits MCF-7 cells by inducing apoptosis and perturbing cell cycle.

Matched MeSH terms: DNA Fragmentation
Fulltext Coenzyme Q10, oxidative stress markers, and sperm DNA damage in men with idiopathic oligoasthenoteratospermia

Alahmar AT, Sengupta P, Dutta S, Calogero AE

Clin Exp Reprod Med, 2021 Jun;48(2):150-155.
PMID: 34078008 DOI: 10.5653/cerm.2020.04084

OBJECTIVE: Oxidative stress (OS) plays a key role in the etiology of unexplained male infertility. Coenzyme Q10 (CoQ10) is a potent antioxidant that may improve semen quality and OS in infertile men with idiopathic oligoasthenoteratospermia (OAT), but the underlying mechanism is unknown. Therefore, the present study was undertaken to investigate the effect of CoQ10 on OS markers and sperm DNA damage in infertile patients with idiopathic OAT.
METHODS: This prospective controlled study included 50 patients with idiopathic OAT and 50 fertile men who served as controls. All patients underwent a comprehensive medical assessment. Patients and controls received 200 mg of oral CoQ10 once daily for 3 months. Semen and blood were collected and analyzed for sperm parameters, seminal CoQ10 levels, reactive oxygen species (ROS) levels, total antioxidant capacity, catalase, sperm DNA fragmentation (SDF), and serum hormonal profile.
RESULTS: The administration of CoQ10 to patients with idiopathic OAT significantly improved sperm quality and seminal antioxidant status and significantly reduced total ROS and SDF levels compared to pretreatment values.
CONCLUSION: CoQ10, at a dose of 200 mg/day for 3 months, may be a potential therapy for infertile patients with idiopathic OAT, as it improved sperm parameters and reduced OS and SDF in these patients.

Matched MeSH terms: DNA Fragmentation
Catharanthus roseus Aqueous Extract is Cytotoxic to Jurkat Leukaemic T-cells but Induces the Proliferation of Normal Peripheral Blood Mononuclear Cells

Ahmad NH, Rahim RA, Mat I

Trop Life Sci Res, 2010 Dec;21(2):101-13.
PMID: 24575203

Research on natural products has been widely used as a strategy to discover new drugs with potential for applications in complementary medicines because they have fewer side effects than conventional drugs. The aim of the present study was to evaluate the in vitro cytotoxic effects of crude aqueous Catharanthus roseus extract on Jurkat cells and normal peripheral blood mononuclear cells (PBMCs). The aqueous extract was standardised to vinblastine by high performance liquid chromatography (HPLC) and was used to determine cytotoxicity by the MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. DNA fragmentation assay was employed to determine if cell death was due to apoptosis. The results showed that the aqueous extract induced cell death of Jurkat cells at 24, 48 and 72 hours post-treatment in a time- and dose-dependent manner. However, cells treated at 48 and 72 hours produced higher cytotoxic effects with half maximal inhibitory concentration (IC50) values of 2.55 μg/ml and 2.38 μg/ml, respectively. In contrast, the extract induced normal PBMC proliferation, especially after 24 hours treatment with 1000 μg/ml. This result indicates that the C. roseus crude aqueous extract showed differential effects of inhibiting the proliferation of the Jurkat cell line and promoting the growth of PBMCs. These data suggest that the extract may be applicable for modulating the normal and transformed immune cells in leukaemia patients.

Matched MeSH terms: DNA Fragmentation
Fulltext Phytochemicals and Cytotoxicity of Quercus infectoria Ethyl Acetate Extracts on Human Cancer Cells

Yusof WNSW, Abdullah H

Trop Life Sci Res, 2020 Apr;31(1):69-84.
PMID: 32963712 DOI: 10.21315/tlsr2020.31.1.5

Conventional and modern cancer treatment were reported to manifest adverse effects to the patients. More researches were conducted to search for selective cytotoxic agent of plant natural product on cancer cells. The presences of wide range phytochemicals in Quercus infectoria (QI) extract have been implicated with the cytotoxic effect against various types of cancer cell which remain undiscovered. This present study aimed to evaluate cytotoxic effect of QI extracts on selected human cancer cells and then, the most potent extract was further analysed for general phytochemical constituents. QI galls were extracted successively with n-hexane, ethyl acetate and methanol yielded three main extracts; n-hexane (QIH), ethyl acetate (QIEA) and methanol (QIM), respectively. The most potent extract was qualitatively analysed for the present of tannin, alkaloids, glycosides, saponins, terpenoids, flavonoids and phenolic compounds. Next, the extracts were tested to determine the cytotoxic activity against cervical cancer cells (HeLa), breast cancer cells (MDA-MB-231) and liver cancer cells (Hep G2) using MTT assay. Cytotoxic activity of QI extracts against normal fibroblast (L929) cell line was also evaluated to determine the cytoselective property. Meanwhile, DMSO-treated cells served as negative control while cisplatin-treated cells served as positive control. The most potent extract then chosen to be further investigated for DNA fragmentation as hallmark of apoptosis using Hoechst staining. Qualitative phytochemical analysis revealed the presence of tannin, alkaloids, glycosides, saponins, terpenoids, flavonoids and phenolic compounds. QIEA extract exhibited the most potent cytotoxic activity against HeLa cells with (IC50 value = 6.33 ± 0.33 μg/mL) and showed cytoselective property against L929 cells. DNA fragmentation revealed QIEA induced apoptosis in the treated cells. The richness of phytochemical constituents in QIEA extract might contribute to the potency of cytotoxic activity towards HeLa cells.

Matched MeSH terms: DNA Fragmentation
Fulltext Decreased Expression of Inhibitor of Caspase-Activated DNase (ICAD) in Renal Cell Carcinoma - Tissue Microarray of Human Samples

Rajandram R, Razack AH, Ng KL, Gobe GC

J Kidney Cancer VHL, 2016;3(1):1-11.
PMID: 28326275 DOI: 10.15586/jkcvhl.2016.47

Although primary localised tumours of renal cell carcinoma (RCC) can be treated relatively successfully with surgery, metastatic RCC has poor prognosis because of late diagnosis and resistance to therapies. In the present study, we were interested in profiling the protein expression of "inhibitor of caspase-activated DNase" (ICAD), an apoptosis inhibitor, in kidney cancer and its paired normal kidney. Immunohistochemistry with automated batch staining and morphometry using digital pathology were used to compare ICAD in 121 RCC specimens with their paired normal kidney tissue. Tissue microarray of formalin-fixed, paraffin-embedded archival tissue was used. Intensity and localisation of ICAD were compared between normal and cancer samples, and against grading within the cancers. The results demonstrated that, in this cohort, ICAD was highly expressed in the proximal tubular epithelium of normal kidney, and significantly decreased in clear cell RCC tissue (p < 0.05) as well as other subtypes of RCC (p < 0.01) compared with normal kidney. There was a tendency towards nuclear localisation of ICAD in clear cell RCC, but not in other subtypes of RCC. No significant association was found between ICAD intensity and grade of RCC. In summary, down-regulation of ICAD occurs in RCC. ICAD normally inhibits DNA fragmentation and apoptosis; thus, its down-regulation was unexpected in a cancer known for its resistance to apoptosis. However, these RCC samples were from primary, not metastatic, RCC sites, and down-regulated ICAD may be part of a progressive pathway that promotes RCC metastasis.

Matched MeSH terms: DNA Fragmentation
Fulltext The effects of vasectomy on testicular tissue of mice: histological changes and DNA fragmentation study

Salman, M.O., Al-Wasiti, E.A., Thamir, K.A., Al-Ani, I.M., Al-Salihi, A.R.

International Medical Journal Malaysia, 2012;11(2):11-17.
MyJurnal

Introduction: We aim to investigate the effect of vasectomy on the histology of the testis as well as to evaluate DNA fragmentation in testicular tissue of male mice. Methods: Bilateral vasectomy was performed on 20 mature male mice; 10 control mice underwent sham-operation. After 6 weeks, the testes were evaluated for histological changes and DNA fragmentation by single cell gel electrophoresis (comet assay). Results: Marked alterations were observed in the testes of vasectomized mice, including degeneration of spermatids, thickened basement membrane, dilatation of the seminiferous tubules, exfoliation of germ cells, reduction in the seminiferous cell population, vacuolated appearance of the epithelium in the tubules and marked interstitial fibrosis. Single cell gel electrophoresis showed a highly significant (P

Matched MeSH terms: DNA Fragmentation
Fulltext Curcumin from turmeric (Curcuma longa) induced apoptosis in human mammary carcinoma cells (MDA-MB-231)

Latifah, S. Y., Faujan, H. A., Sze, L. P., Raha, A. R., Hisyam, A. H., Li, O. C.

Malaysian Journal of Medicine and Health Sciences, 2006;2(2):71-79.
MyJurnal

Introduction: Curcumin, a natural compound present in turmeric (Curcuma longa) has been known to possess both anti-inflammatory and antioxidant effects. Objectives: The objectives of the study were to evaluate the cytotoxic activities and to determine the mode of cell death induced by curcumin towards the human mammary carcinoma cells (MDAMB-231). Methodology: Cytotoxicity of curcumin and its effect on cell viability were determined by using MTT assay and trypan blue dye exclusion method, respectively. The mode of cell death was detected by viewing under a light microscope and through DNA fragmentation analysis. Results and discussion: Curcumin was cytotoxic to MDA-MB-231 cells with the IC50 of 17.25 ì g/ml. Cell viability treatment using curcumin at concentrations of 30 ì g/ml and 10 ì g/ml was significantly (p

Matched MeSH terms: DNA Fragmentation
Fulltext Cytotoxic Effects of Pinnatane A Extracted from Walsura pinnata (Meliaceae) on Human Liver Cancer Cells

Zakaria N, Mahdzir MA, Yusoff M, Mohd Arshad N, Awang K, Nagoor NH

Molecules, 2018 Oct 23;23(11).
PMID: 30360475 DOI: 10.3390/molecules23112733

BACKGROUND: Pinnatane A from the bark of Walsura pinnata was investigated for its anti-cancer properties by analyzing the cytotoxic activities and cell cycle arrest mechanism induced in two different liver cancer cell lines.
METHODS: A 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to analyze the pinnatane A selectivity in inducing cell death in cancer and normal cells. Various biological assays were carried out to analyze the anti-cancer properties of pinnatane A, such as a live/dead assay for cell death microscopic visualization, cell cycle analysis using propidium iodide (PI) to identify the cell cycle arrest phase, annexin V-fluorescein isothiocyanate (annexin V-FITC)/PI flow cytometry assay to measure percentage of cell populations at different stages of apoptosis and necrosis, and DNA fragmentation assay to verify the late stage of apoptosis.
RESULTS: The MTT assay identified pinnatane A prominent dose- and time-dependent cytotoxicity effects in Hep3B and HepG2 cells, with minimal effect on normal cells. The live/dead assay showed significant cell death, while cell cycle analysis showed arrest at the G₀/G₁ phase in both cell lines. Annexin V-FITC/PI flow cytometry and DNA fragmentation assays identified apoptotic cell death in Hep3B and necrotic cell death in HepG2 cell lines.
CONCLUSIONS: Pinnatane A has the potential for further development as a chemotherapeutic agent prominently against human liver cells.

Matched MeSH terms: DNA Fragmentation
Fulltext Combinatorial Cytotoxic Effects of Gelam Honey and 5-Fluorouracil against Human Adenocarcinoma Colon Cancer HT-29 Cells In Vitro

T-Johari SAT, Hashim F, Ismail WI, Ali AM

Int J Cell Biol, 2019;2019:3059687.
PMID: 30923553 DOI: 10.1155/2019/3059687

Combination of natural products with chemodrugs is becoming a trend in discovering new therapeutics approach for enhancing the cancer treatment process. In the present study, we aimed to investigate the cytotoxic and apoptosis induction of Gelam honey (GH) combined with or without 5-Fluorouracil (5-FU) on HT-29 cells. The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay to assess cytotoxicity. Morphological changes and apoptosis were determined by the inverted microscope, Annexin V-FITC, and DNA fragmentation via flow cytometric analysis, respectively. Our results demonstrate that combined treatment revealed a remarkable and concentration-dependent cytotoxic effect on HT-29 cells in comparison with GH and 5-FU alone. Flow cytometry analysis showed that early apoptosis event was more pronounced in combined treatment. In addition, compared to 5-FU alone, apoptosis of HT-29 cells treated with combinations of GH and 5-FU demonstrated increasing percentages of fragmented DNA. Our results suggest that GH has a synergistic cytotoxic effect with 5-FU in HT-29 cell lines in vitro. Although the actions of the molecular mechanisms are not yet clear, the results reveal that the combination of GH and 5-FU could have the potential as a therapeutic agent.

Matched MeSH terms: DNA Fragmentation
Reversal of oxidative stress, cytokine toxicity and DNA fragmentation by quercetin in dizocilpine-induced animal model of Schizophrenia

Shahzad S, Batool Z, Afzal A, Haider S

Metab Brain Dis, 2022 Dec;37(8):2793-2805.
PMID: 36152087 DOI: 10.1007/s11011-022-01090-6

Quercetin, a polyphenolic compound found in a variety of plant products possesses various biological activities and beneficial effects on human health. Schizophrenia (SZ) is one of the neuropsychiatric disorders in human beings with rapid mortality and intense morbidity which can be treated with antipsychotics, but these commercial drugs exert adverse effects and have less efficacy to treat the full spectrum of SZ. The present study was conducted to evaluate neuroprotective effects of quercetin in the preventive and therapeutic treatment of SZ. Quercetin was administered as pre- and post-regimens at the dose of 50 mg/kg in dizocilpine-induced SZ rat model for two weeks. Rats were then subjected for the assessment of different behaviors followed by biochemical, neurochemical, and inflammatory marker analyses. The present findings revealed that quercetin significantly reverses the effects of dizocilpine-induced psychosis-like symptoms in all behavioral assessments as well as it also combats oxidative stress. This flavonoid also regulates dopaminergic, serotonergic, and glutamatergic neurotransmission. A profound effect on inflammatory cytokines and decreased %DNA fragmentation was also observed following the administration of quercetin. The findings suggest that quercetin can be considered as a preventive as well as therapeutic strategy to attenuate oxidative stress and cytokine toxicity, regulate neurotransmission, and prevent enhanced DNA fragmentation that can lead to the amelioration of psychosis-like symptoms in SZ.

Matched MeSH terms: DNA Fragmentation
Fulltext In Vitro Pro-apoptotic and Anti-migratory Effects of Ficus deltoidea L. Plant Extracts on the Human Prostate Cancer Cell Lines PC3

Hanafi MMM, Afzan A, Yaakob H, Aziz R, Sarmidi MR, Wolfender JL, et al.

Front Pharmacol, 2017;8:895.
PMID: 29326585 DOI: 10.3389/fphar.2017.00895

This study aims to evaluate the in vitro cytotoxic and anti-migratory effects of Ficus deltoidea L. on prostate cancer cells, identify the active compound/s and characterize their mechanism of actions. Two farmed varieties were studied, var. angustifolia (FD1) and var. deltoidea (FD2). Their crude methanolic extracts were partitioned into n-hexane (FD1h, FD2h) chloroform (FD1c, FD2c) and aqueous extracts (FD1a, FD2a). Antiproliferative fractions (IC50 < 30 μg/mL, SRB staining of PC3 cells) were further fractionated. Active compound/s were dereplicated using spectroscopic methods. In vitro mechanistic studies on PC3 and/or LNCaP cells included: annexin V-FITC staining, MMP depolarization measurements, activity of caspases 3 and 7, nuclear DNA fragmentation and cell cycle analysis, modulation of Bax, Bcl-2, Smac/Diablo, and Alox-5 mRNA gene expression by RT-PCR. Effects of cytotoxic fractions on 2D migration and 3D invasion were tested by exclusion assays and modified Boyden chamber, respectively. Their mechanisms of action on these tests were further studied by measuring the expression VEGF-A, CXCR4, and CXCL12 in PC3 cells by RT-PCR. FD1c and FD2c extracts induced cell death (P < 0.05) via apoptosis as evidenced by nuclear DNA fragmentation. This was accompanied by an increase in MMP depolarization (P < 0.05), activation of caspases 3 and 7 (P < 0.05) in both PC3 and LNCaP cell lines. All active plant extracts up-regulated Bax and Smac/DIABLO, down-regulated Bcl-2 (P < 0.05). Both FD1c and FD2c were not cytotoxic against normal human fibroblast cells (HDFa) at the tested concentrations. Both plant extracts inhibited both migration and invasion of PC3 cells (P < 0.05). These effects were accompanied by down-regulation of both VEGF-A and CXCL-12 gene expressions (P < 0.001). LC-MS dereplication using taxonomy filters and molecular networking databases identified isovitexin in FD1c; and oleanolic acid, moretenol, betulin, lupenone, and lupeol in FD2c. In conclusion, FD1c and FD2c were able to overcome three main hallmarks of cancer in PC3 cells: (1) apoptosis by activating of the intrinsic pathway, (2) inhibition of both migration and invasion by modulating the CXCL12-CXCR4 axis, and (3) inhibiting angiogenesis by modulating VEGF-A expression. Moreover, isovitexin is here reported for the first time as an antiproliferative principle (IC50 = 43 μg/mL, SRB staining of PC3 cells).

Matched MeSH terms: DNA Fragmentation

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