DNA Ladder Assay Research Articles

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.

Curcumin Mitigates Streptozotocin-Induced Genotoxicity In Vivo by Activating P53 Protein and Inhibiting Oxidative Stress and Chromosomal Aberration.

Genotoxins are chemical constituents that damage DNA or chromosomal architecture, leading to alterations in the genetic level. Streptozotocin, a monofunctional nitrosourea derivative, is often utilized to induce diabetes mellitus in laboratory animals because of its detrimental effects on pancreatic cells. The purpose of this work was to investigate possible protective efficacy against the genotoxic effects of Streptozotocin. This study examines the potentiality of curcumin, a phytoproduct with anti-genotoxic and anti-diabetic qualities, against streptozotocin-induced chromosomal abnormalities and DNA damage. The study evaluates the possible protective efficacy of curcumin, exploring a variety of biochemical and molecular biology techniques in murine models. The experiment involves a control group, the administration of Streptozotocin (Group S), and a curcumin-pretreated STZ group (Group SC) in mice. The effects of curcumin on DNA damage and chromosomal aberrations were investigated by histopathology, immunofluorescence, evaluation of chromosomal aberrations, detection of Reactive oxygen species, cell viability analysis, and DNA laddering assay. The results demonstrated a significant reduction in DNA damage, chromosomal anomalies, and a decrease in the expression of the p53 protein in the curcumin-treated animals compared to that of STZ-treated mice. The overall results show that curcumin can reduce chromosomal aberrations and DNA damage by altering the expression of p53 repair proteins. This suggests that curcumin has a promising future as a therapeutic agent, especially when it comes to drug-induced toxicity and the development of novel therapeutics.

Hydroxytyrosol acetate from olive leaves (Olea Europaea L.) induces apoptosis via mitochondrial pathway in BEL7402 cell line

Hydroxytyrosol acetate is one of the polyphenolic compounds in olive leaves. Hydroxytyrosol acetate has a variety of biological activities, such as antibacterial, antioxidant, anti-inflammatory, cognitive improvement and neuroprotective effects. However, there is no report on the antitumor activity and the antitumor mechanism of hydroxytyrosol acetate. In our study, we studied the antitumor activity of hydroxytyrosol acetate by MTT assay and determined the antitumor mechanism by DNA ladder assay, mitochondrial membrane potential assay and western blot assay. We found that hydroxytyrosol acetate could inhibit cell proliferation, and the inhibition rate was 78.08%. The further researches showed that hydroxytyrosol acetate could downregulate Bcl-2 protein while upregulate Bax protein. It also could induce mitochondrial depolarisation and release of cytochrome C. These results indicated that hydroxytyrosol acetate might induce BEL7402 cells apoptosis via mitochondrial pathway.

The Effect of Silencing MiR-4270 on Apoptosis in HCC Cell Line

Background:: Hepatocellular carcinoma (HCC) is the most common type of cancer. Although HCC treatment has greatly improved over the past few decades, patient survival rates are still very low. Therefore, it is essential to find new treatments for HCC. Apoptosis has been shown to be the most effective in disrupting cancer growth. Improper functioning of proteins in apoptosis can lead to cancer growth. MicroRNAs (miRNAs) are key regulators in the development and progression of HCC. Objective:: Irregular expression of miRNAs involved in apoptosis signaling can lead to tumorigenesis. Therefore, we investigated the effect of the hsa-miR-4270 inhibitor on cell proliferation and apoptosis. Methods:: HepG2 cells were cultured at 37°C and 95% air. Transfection of HepG2 cells was performed by miR-4270 inhibitor and lipofectamine 2000. Cell proliferation of HepG2 cells was determined with MTT assay and different concentrations of miR-4270 specific inhibitors. DNA laddering assay was performed to evaluate the induction of apoptosis. Finally, the transcription level of genes involved in apoptosis, including BAX, BCL2, Caspase3, and p53, was measured by real-time RT-PCR. Results:: The results of MTT and DNA laddering assays showed that the miR-4270 inhibitor declined cell proliferation and induced apoptosis in HepG2 cells. Also, the results of quantitative real-time RT-PCR indicated an upregulation of transcription of BAX, p53 and Caspase3 genes and a decline in expression of BCL2 gene. Conclusion:: Taken together, we found hsa-miR-4270 inhibitor decreased cell proliferation and induced apoptosis in the HepG2 cell line, which can be used as a new therapeutic strategy for HCC patients.

Synthesis, Biological Investigation, and Molecular Docking of Novel Benzimidazole‐Hydrazone Hybrids as Potential Anticancer Agent Candidates

AbstractIn this study, six novel Benzimidazole‐hydrazone derivatives starting from the commercially available 1H‐benzo[d]imidazole‐2‐amine were synthesized, and the molecular structure of the obtained compounds were confirmed by NMR, FTIR and MS spectroscopic techniques. The anticancer activities of the synthesized compounds were investigated against HT29 (Human colon adenocarcinoma) and A549 (Human non‐small cell lung cancer) cancer cell lines using xCELLigence real‐time cell analysis. Cell apoptosis was determined by DNA laddering assay and Annexin V/FITC flow cytometer. Compound 4 c exhibited the best anticancer potency against both cancer cell lines (A549 and HT29) with IC50 values of 0.0019 μM and 0.0093 μM, respectively. Compound 4 c reduced the growth of A549 and HT29 cells in vitro and induced early apoptosis of A549 and HT29 cells. Additionaly, all compounds demonstrated favorable pharmacokinetics properties compared to Paclitaxel (reference compound) and the best docking scores were obtained for compounds 4 e and 4 b among the compounds.

The Anticancer Potentials of Substituted Indeno[1,2-b]quinoline Amines against HT29 and SW620: Experimental and In silico Approach

Background: This study aimed the determination of the antiproliferative and cytotoxic activities of recently prepared indeno [1,2-b]quinoline amines against colon carcinoma, HT29 and SW620 cell lines by using cell proliferation and cytotoxicity assays. Methods: In vitro inhibition of cell proliferation of indenoquinoline derivatives was determined with an MTT cell proliferation assay. On the other hand, their cell cytotoxicities and apoptotic potential were investigated by LDH cytotoxicity and DNA laddering assays. Moreover, molecular docking studies were performed between the compounds and PDB ID: 1OLG and 4LVT target proteins using virtual scanning techniques. Results: Most of the compounds (1, 3, and 7-9) exhibit much more potent antiproliferative activity than positive controls against HT29 and SW620 cell lines (IC50 values 1.1 - 4.1 μg/mL) and show slightly toxic properties (percent cytotoxicity 9.8% to 33.5%) to cells compared to positive control. On the other hand, it was determined that effective compounds 1, 2, 3 and 9 stimulated apoptosis on HT29 and SW620. Moreover, the anticancer effect of the recent indeno[1,2-b]quinoline amine derivatives was investigated with the help of molecular docking simulations for their pharmacokinetics. The molecular docking results displayed that mono bromo (1-3) and phenyl (7-9) substituted indeno [1,2-b]quinoline amines interact with mutated p53 and protein Blc-2 residues with hydrogen bonding and polar interactions, respectively. Conclusion: As a result, the preliminary experimental data and in silico studies indicated that the monosubstituted indenoquinoline amine derivatives, especially 1, 3, and 7-9, exhibit effective pharmacological properties.

Rhodamine hydrazide-linked naphthalimide derivative: Selective naked eye detection of Cu2+, S2− and understanding the therapeutic potential of the copper complex as an anti-cervical cancer agent

A naphthalimide-labeled rhodamine hydrazone derivative HL has been synthesized, characterized and examined in metal ion recognition. It shows selective colorimetric detection of Cu2+ over a number of other metal ions with a detection limit of 1.66 × 10−7 M in CH3CN/HEPES buffer (v/v = 2:1, pH = 6.8). The spirolactam ring of rhodamine and the imino-phenol motif of naphthalimide in HL are involved in complexation of Cu2+ as shown by single crystal X-ray. Single crystal of the copper-complex is prepared by utilizing NaSCN and it is characterized as CuL(SCN). The emergence of new absorption at 550 nm in UV–vis and the pink color of the solution reveal the selective interaction toward Cu2+. HL is characterized as a fluorescence resonance energy transfer (FRET) system that remains ‘turned OFF’ while spirolactam ring exists. In the presence of Cu2+, FRET is ‘turned ON’ via the opening of spirolactam ring to give emission at 580 nm which is less intense due to the quenching effect of Cu2+ ion. The complexation is reversible and the ensemble of Cu2+.HL selectively recognizes S2− over a series of different anions involving a color change from pink to colorless via the formation of spirolactam ring. The copper complex CuL(SCN) is further employed to understand its efficacy as a therapeutic agent. The complex is cytotoxic to high-risk HPV positive cervical cancer cell lines like SiHa and HeLa and is efficient in the generation and accumulation of reactive oxygen species (ROS). The complex also initiates nuclear blebbing and shows DNA degradation as understood by DNA laddering assay.

In vitro anticancer efficacy of Cyperus rotundus (L.) on breast adenocarcinoma cells via the induction of DNA fragmentation and apoptosis

Introduction and Aim: Cyperus rotundus (L.), commonly known as nutgrass, has been in use in Indian medicine traditional system against several ailments. This investigation was performed to assess the in vitro antineoplastic activity of extracts prepared from water and ethanol with C. rotundus rhizomes. Materials and Methods: Toxic effects induced by the C. rotundus extracts on MCF-7 breast adenocarcinoma cells was evaluated using Sulphorhodamine- B (SRB) Assay, Trypan blue assay, Hoechst nuclear staining assay, Acridine Orange-Ethidium bromide (AO-EB) differential fluorescence staining assay and DNA fragmentation assays. Results: The results of SRB assay indicated the IC50 value of aqueous extract as 510.887 µg/mL and that of ethanol extract as 122.98 µg/mL. Trypan blue assay also indicated the similar results, wherein ethanol extract exerted higher cytotoxicity compared to the aqueous extract. MCF-7 cells treated with aqueous and ethanol extracts of C. rotundus rhizomes were exposed to Hoechst staining and AO-EB nuclear staining assays, wherein and ethanol extract induced clear apoptotic bodies in the tested cells. Further, the cells were treated with the ethanol extract and DNA fragmentation was confirmed by DNA ladder assay. MCF-7 cells administered with ethanol extract were subjected to DNA fragmentation assay for assessing the apoptosis induction nature of the extract. Conclusion: Results from our study indicate a promising approach into the possibility of developing C. rotundus ethanol extract into an effective anticancer moiety after appropriate pre-clinical and clinical validations.

Innovative Eco-Friendly Microwave-Assisted Rapid Biosynthesis of Ag/AgCl-NPs Coated with Algae Bloom Extract as Multi-Functional Biomaterials with Non-Toxic Effects on Normal Human Cells.

Harmful algal blooms impact human welfare and are a global concern. Sargassum spp., a type of algae or seaweed that can potentially bloom in certain regions of the sea around Thailand, exhibits a noteworthy electron capacity as the sole reducing and stabilizing agent, which suggests its potential for mediating nanoparticle composites. This study proposes an eco-friendly microwave-assisted biosynthesis (MAS) method to fabricate silver nanoparticles coated with Sargassum aqueous extract (Ag/AgCl-NPs-ME). Ag/AgCl-NPs-ME were successfully synthesized in 1 min using a 20 mM AgNO3 solution without additional hazardous chemicals. UV-visible spectroscopy confirmed their formation through a surface plasmon resonance band at 400-500 nm. XRD and FTIR analyses verified their crystalline nature and involvement of organic molecules. TEM and SEM characterization showed well-dispersed Ag/AgCl-NPs-ME with an average size of 36.43 nm. The EDS results confirmed the presence of metallic Ag+ and Cl- ions. Ag/AgCl-NPs-ME exhibited significant antioxidant activity against free radicals (DPPH, ABTS, and FRAP), suggesting their effectiveness. They also inhibited enzymes (tyrosinase and ACE) linked to diseases, indicating therapeutic potential. Importantly, the Ag/AgCl-NPs-ME displayed remarkable cytotoxicity against cancer cells (A375, A549, and Caco-2) while remaining non-toxic to normal cells. DNA ladder and TUNEL assays confirmed the activation of apoptosis mechanisms in cancer cells after a 48 h treatment. These findings highlight the versatile applications of Ag/AgCl-NPs-ME in food, cosmetics, pharmaceuticals, and nutraceuticals.

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides as an Important Scaffold for Anticancer Drug Discovery-In Vitro and In Silico Evaluation.

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides (MM-compounds) are a relatively new class of heterocyclic compounds that exhibit a wide variety of biological actions, including anticancer properties. Here, we used caspase enzyme activity assays, flow cytometry analysis of propidium iodide (PI)-stained cells, and a DNA laddering assay to investigate the mechanisms of cell death triggered by the MM-compounds (MM134, -6, -7, and -9). Due to inconsistent results in caspase activity assays, we have performed a bromodeoxyuridine (BrdU) incorporation assay, colony formation assay, and gene expression profiling. The compounds' cytotoxic and pro-oxidative properties were also assessed. Additionally, computational studies were performed to demonstrate the potential of the scaffold for future drug discovery endeavors. MM-compounds exhibited strong micromolar (0.06-0.35 µM) anti-proliferative and pro-oxidative activity in two cancer cell lines (BxPC-3 and PC-3). Activation of caspase 3/7 was observed following a 24-h treatment of BxPC-3 cells with IC50 concentrations of MM134, -6, and -9 compounds. However, no DNA fragmentation characteristics for apoptosis were observed in the flow cytometry and DNA laddering analysis. Gene expression data indicated up-regulation of BCL10, GADD45A, RIPK2, TNF, TNFRSF10B, and TNFRSF1A (TNF-R1) following treatment of cells with the MM134 compound. Moreover, in silico studies indicated AKT2 kinase as the primary target of compounds. MM-compounds exhibit strong cytotoxic activity with pro-oxidative, pro-apoptotic, and possibly pro-necroptotic properties that could be employed for further drug discovery approaches.

Anticancer Effect of Enterococcus faecium, Isolated from Vaginal Fluid, on Ovarian Cancer Cells

Given the association between cervicovaginal microbiota and OVC, we investigated the effect of E. faecium CM on OVC (Caov-4) cells. CM was obtained from the bacterium E. faecium isolated from the vagina of healthy women. The Caov-4 cells were treated with varying concentrations of CM that comprised co-cultured bacteria with 0.2, 0.5, 1, 1.5, and 2 OD for 12, 24, and 48 h. The apoptosis and growth of cancer cells were evaluated by DAPI staining, flow cytometry, and DNA laddering assay. Moreover, the expression of PTEN, BAX, BCL2, and AKT1 genes were analyzed using real-time PCR. The CM at a concentration of 0.5 OD from the cultured bacteria and incubation time of 48 h showed the highest negative effect on the viability of cancer cells. The CM treatment increased DNA fragmentation and also induced apoptosis in Caov-4 cells. Interestingly, CM could decrease the expression of proapoptotic genes were less, while antiapoptotic genes were more than 5-FU in the presence of CM. CM of human-derived E. faecium could have an anticancer effect on OVC cells in a concentration- and time-dependent manner. This study demonstrated that E. faecium secretes anticancer substances into the CM, which could directly affect the viability and apoptosis of cancer cells.

Anticancer potential of Nymphaea nouchali Brum flowers against Ehrlich ascites carcinoma cell lines.

Nymphaea nouchali Brum is exotic and medicinal plant in India. The main of this study is to evaluate the anticancer properties of Nymphaea nouchali Brum flowers against Ehrlich ascites carcinoma (EAC)-induced Swiss albino mice. The anticancer properties of Nymphaea nouchali Brum dry and fresh methanol extracts was investigated using EAC in Swiss albino mice. After inoculation of EAC cells into mice, treatment with NNDM flower extract (200 and 400 mg/kg) and standard drug 5-Fluorouracil (20 mg/kg) was continued for 9 days. The evaluation of the effect of drug response was made by the study of tumor growth response including increase in lifespan, the study of hematological parameters, biochemical estimations, and antioxidant assay of liver tissue compared to EAC control. The viability of cancer cell lines (such as HeLa, MCF-7, and MDA-MB 231 cells) was evaluated by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Therefore, from the results of the present study, it can be concluded that NNDM exhibited significant antitumor activity against EAC in Swiss albino mice. The effect of NNDM on viability of cancer cell lines (such as HeLa, MCF-7, and MDA-MB 231 cells) was evaluated by MTT assay, apoptosis in HeLa cell lines was evaluated by DNA laddering assay, HeLa cells treated with NNDM exhibited a characteristic "ladder" pattern after separation of the fragments by agarose gel electrophoresis and subsequent visualization, by ethidium bromide staining. NNDM exhibited a significant effect on cell viability. Based on results, it was concluded that NNDM exhibited cytotoxic effect on cancer cells and, from DNA laddering assay, it can be concluded that NNDM-induced apoptosis in EAC cells.

In vitro safety assessment of alkyl lactate esters in human umbilical vein endothelial cells (HUVECs).

Safety assessment requires information on both the chronic and acute effects of chemicals. Chemical materials such as food additives have become one of the most critical and compelling issues in the continuing debate on food safety. Calcium lactate (CL) and Sodium lactate (SL) are approved chemicals used in various products. The present study, in vitro study, was designed to evaluate the cyto-genotoxicity effects of CL and SL on human umbilical vein endothelial cells (HUVECs). The cytotoxicity effect of these additives was determined by MTT and Annexin V-FITC apoptosis assays. Besides, genotoxicity parameters such as morphological change of DNA and DNA fragmentation were studied with DAPI staining and DNA ladder assays, respectively. The results showed that the growth of HUVECs decreased upon treatment with CL at higher concentrations, but SL did not significantly alter HUVECs cell number. Annexin V-FITC apoptosis assays revealed that CL could induce cell death based on necrosis rather than apoptosis. In SL, the early and late apoptosis was not considerable in treated cells. Exposure to CL and SL did not lead to morphological change and DNA fragmentation in the HUVECs cell line. Overall, it is concluded from these results that CL and SL have not been cytotoxic and genotoxic effects in low concentrations in human umbilical vein endothelial cells in vitro.

1-Methylcyclopropene (1-MCP) treatment differentially mediated expression of vacuolar processing enzyme (VPE) genes and delayed programmed cell death (PCD) during ripening and senescence of apple fruit

• 1-MCP reduced VPE activity and delayed vacuolar-mediated PCD in apple fruit. • MdVPE s had varying expression patterns in response to 1-MCP during ripening. • MdVPE s were differentially expressed after transient over-expression of MdERF2. The main purpose of this research was to evaluate the impacts of 1-methylcyclopropene (1-MCP) treatment on vacuolar processing enzyme ( VPE ) genes and programmed cell death (PCD) during ripening and senescence of apple fruit ( Malus × domestica Borkh.). The ethylene antagonists 1-MCP (1 μL L −1 ) was applied to apples after harvest. Both treated and control fruit were stored at 20 ± 2°C. The data showed that 1-MCP treatment maintained flesh firmness, delayed climacteric peaks of respiration and ethylene, reduced VPE activity, relative electrical conductivity (REC), malondialdehyde (MDA) content and reactive oxygen species (ROS) accumulation during ripening. Transmission electron microscopy (TEM) and DNA ladder assay revealed that 1-MCP delayed vacuolar-mediated PCD in ripening apple fruit. Generally, expression of MdVPEγ1, MdVPEγ2, MdVPEδ4 and MdVPEδ7 was down-regulated by 1-MCP treatment, while expression of MdVPEα4 and MdVPEβ2 was enhanced by it during ripening compared with controls. 1-MCP treatment differentially reduced expression of MdVPEα1, MdVPEα2 and MdVPEα5 at the early ripening stage but promoted their expression at the late ripening stage, while it had the opposite effects on MdVPEβ1, MdVPEβ3 and MdVPEγ4 expression. After transient overexpression of MdERF2, MdVPEs displayed varying expression patterns, among which MdVPEα2 transcripts increased greater than the other genes. Our results indicated that MdVPEs expression and vacuolar-mediated PCD were mediated by ethylene signal during ripening and senescence of apple fruit.

Naturally Occurring Functional Ingredient from Filamentous Thermophilic Cyanobacterium Leptolyngbya sp. KC45: Phytochemical Characterizations and Their Multiple Bioactivities.

Cyanobacteria are rich in phytochemicals, which have beneficial impacts on the prevention of many diseases. This study aimed to comprehensively characterize phytochemicals and evaluate multifunctional bioactivities in the ethanolic extract of the cyanobacterium Leptolyngbya sp. KC45. Results found that the extract mainly contained chlorophylls, carotenoids, phenolics, and flavonoids. Through LC-ESI-QTOF-MS/MS analysis, 38 phenolic compounds with promising bioactivities were discovered, and a higher diversity of flavonoids was found among the phenolic compounds identified. The extract effectively absorbed the harmful UV rays and showed high antioxidant activity on DPPH, ABTS, and PFRAP. The extract yielded high-efficiency inhibitory effects on enzymes (tyrosinase, collagenase, ACE, and α-glucosidase) related to diseases. Interestingly, the extract showed a strong cytotoxic effect on cancer cells (skin A375, lung A549, and colon Caco-2), but had a much smaller effect on normal cells, indicating a satisfactory level of safety for the extract. More importantly, the combination of the DNA ladder assay and the TUNEL assay proved the appearance of DNA fragmentation in cancer cells after a 48 h treatment with the extract, confirming the apoptosis mechanisms. Our findings suggest that cyanobacterium extract could be potentially used as a functional ingredient for various industrial applications in foods, cosmetics, pharmaceuticals, and nutraceuticals.

Novel zinc oxide nanoparticles of Teucrium polium suppress the malignant progression of gastric cancer cells through modulating apoptotic signaling pathways and epithelial to mesenchymal transition

Management of gastric cancer is still challenging due to resistance to current chemotherapeutics and recurrent disease. Moreover, green- synthesized zinc oxide nanoparticles (ZnO-NPs) using natural resources are one of the most promising therapeutic agents for anticancer therapy. Here we report the facile green synthesis and characterization of ZnO-NPs from Teucrium polium (TP-ZnO-NP) herb extract and the anticancer activities of these nanoparticles on gastric cancer cells. Facile green synthesis of TP-ZnO-NP was achieved using zinc acetate dihydrate. For the characterization of TP-ZnO-NP, UV–vis spectroscopy, FTIR, SEM, XRD and EDX analyses were performed. Antiproliferative and anticancer activities of TP-ZnO-NP were explored using the HGC-27 gastric cancer cell line model. MTT cell viability and colony formation assays were used for the analysis of cell proliferation and migration. Wound healing assay was used to analyze the migration capacities of cells. Annexin V/PI double staining, DNA ladder assay, and Acridine orange/Ethidium bromide staining were performed to analyze the induction of apoptosis. qPCR was used to determine gene expression levels of apoptotic and epithelial to mesenchymal transition marker genes. The aqueous extract of TP served as both a reducing and capping agent for the successful biosynthesis of zinc oxide nanoparticles. Remarkably, synthesized TP-ZnO-NPs were found to have significant antiproliferative and anticancer activities on HGC-27 gastric cancer cells. Collectively, current data suggest that TP-ZnO-NP is a novel and promising anticancer agent for future therapeutic interventions in gastric cancer.

Apoptotic-like PCD inducing HRC gene when silenced enhances multiple disease resistance in plants

Programmed cell death (PCD) plays an important role in plant environmental stress and has the potential to be manipulated to enhance disease resistance. Plants have innate immunity and, following pathogen perception, the host induces a Hypersensitive Response PCD (HR-PCD), leading to pattern (PTI) or effector triggered immunity (ETI). Here we report a non-HR type or Apoptotic-Like PCD (AL-PCD) in pathogen infected wheat and potato based on apoptotic-like DNA fragmentation. A deletion mutation in the gene encoding histidine rich calcium binding protein (TaHRC) in FHB-resistant wheat (R-NIL) failed to induce AL-PCD. Similarly, the CRISPR-Cas9 based silencing of StHRC gene in Russet Burbank potato failed to induce apoptotic-like DNA fragmentation, proved based on DNA laddering and TUNEL assays. The absence of AL-PCD in wheat R-NIL reduced pathogen biomass and mycotoxins, increasing the accumulation of resistance metabolites and FHB-resistance, and in potato it enhanced resistance to multiple pathogens. In addition, the reduced expressions of metacaspase (StMC7) and Ca2+ dependent endonuclease 2 (StCaN2) genes in potato with Sthrc indicated an involvement of a hierarchy of genes in the induction of AL-PCD. The HRC in commercial varieties of different crops, if functional, can be silenced by genome editing possibly to enhance resistance to multiple pathogens.

Cordycepin Induces Apoptosis through JNK-Mediated Caspase Activation in Human OEC-M1 Oral Cancer Cells.

Cordycepin, a bioactive compound extracted from Cordyceps sinensis, can induce apoptosis in human OEC-M1 oral cancer cells. However, the exact mechanism is still unclear. The present study aimed to investigate the underlying mechanism of cordycepin-induced apoptosis in OEC-M1 cells. Following treatment with cordycepin, apoptosis was examined and quantified using a DNA laddering assay and a cytokeratin 18 fragment enzyme-linked immunosorbent assay, respectively. Expressions of mitogen-activated protein kinases (MAPKs) and apoptosis-related proteins were detected by the western blot analysis. Our results show that a pan-caspase inhibitor, Z-VAD-FMK, could significantly inhibit cordycepin-induced apoptosis in OEC-M1 cells. In addition, treatment with cordycepin not only activated caspase-8, caspase-9, and caspase-3 but also induced Bid and poly ADP-ribose polymerase cleavages. Furthermore, cordycepin also induced the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase, and p38 MAPKs. Among MAPKs, activation of JNK solely contributed to cordycepin-induced apoptosis with the activation of caspase-8, caspase-9, and caspase-3 and cleavage of PARP. Taken together, the present study demonstrated that cordycepin activated JNK and caspase pathways to induce apoptosis in OEC-M1 cells.

Targeting p65 to inhibit Cas3 transcription by Onjisaponin B for radiation damage therapy in p65+/- mice

BackgroundIn response to radiation injury, p65 becomes activated. The formation of p65 is one target of Onjisaponin B (OB), but it has not been studied in radioprotection. In addition, there is a binding site for p65 in the promoter region of Cas3. This study evaluates the use of OB as an intervention to modulate p65/Cas3 following radiation exposure. PurposeThis study aimed to confirm that OB regulated the transcription of Cas3 via p65 to overcome radiation-induced damage. Study design and methodsCells and mice were exposed to X-rays at a dose of 6 Gy. Immunofluorescence was used to locate intracellular p65. For the protein and mRNA analyses, Western blotting and RT-qPCR-based assays were conducted accordingly. HE staining was used to observe pathological changes in tissues. DNA damage was detected by the comet assay and DNA ladder assay. Next, apoptosis was detected by flow cytometry and Hoechst staining. ResultsCompared with the radiation group, the expression levels of p-p65 and c-Cas3 in the drug group were significantly down-regulated by OB 20 µg/ml. When the expression of p65 was suppressed in V79 and TC cells, OB did not significantly inhibit the activation of p65 or Cas3 in response to irradiation, nor did it significantly inhibit the phosphorylation of p65 and subsequent nuclear translocation. Overexpression of p65 in V79 and MTEC-1 cells resulted in OB significantly inhibiting the activation of p65 and Cas3, and the phosphorylation and translocation of p65 into the nucleus. At 3 d for V79 cells and 24 h for MTEC-1 cells after radiation, compared with the Cas3 over plasmid transfection group, the drug transfection group had no significant effect on reducing apoptosis. In p65+/- mice, expression of the p65 gene was knocked down, leading to increased tissue apoptosis and inflammation, and serious tissue pathological changes. The inhibition of p65 activation by OB after radiation exposure was not apparent in the thymus, although it was observed in the lung. ConclusionsOB interfered with radiation injury by targeting and regulating p65/Cas3. Therefore, it has been concluded that p65 is an important target molecule for the treatment of radiation injury.

Investigation of the Effect of Lactobacillus Brevis Bacteria on the Expression of Rel A, IKB, and Casp3 Genes in HT29 Colon Cancer Cells

Aims Studies have shown that probiotic bacteria inhibit the onset and progression of carcinogenesis through different pathways. Our objective in this study was to determine the effect of probiotic bacteria on the expression of growth-related genes Rel A, IKB, and Casp3 in HT29 colon cancer cells Methods & Materials In this study, the Lactobacillus brevis probiotic bacteria were first cultured, and after the supply of media conditioning, they were treated on HT29 cancer cells. The bacterium’s cytotoxic effect (bacterial T cells) was investigated using a microculture tetrazolium test (MTT). DNA was extracted from the treated cells, and a DNA Ladder assay was performed. Also, the 4′,6-diamidino-2-phenylindole (DAPI) test was performed to show cell apoptosis. After ribonucleic acid (RNA) extraction and complementary DNA (cDNA) preparation to determine the mechanism of the effect of this bacterium on cellular signaling, the expression of growth-related genes Rel A, IKB, and Cas3 was measured using a real-time polymerase chain reaction (PCR) method. Findings The microculture tetrazolium (MTT) test showed that L. b bacteria inhibit HT29 cells’ proliferation, induce apoptosis in these cells, and inhibit Rel A gene proliferation by increasing IKB gene expression. Also, 4′,6-diamidino-2-phenylindole (DAPI), and DNA ladder assay following the treatment of HT29 cells regarding the mentioned bacteria showed qualitative changes in cell apoptosis. In addition, real-time polymerase chain reaction (PCR) results showed that L. b increased Casp3 gene expression in HT29 colon cancer cells (P=0.038). Conclusion Our findings indicate that L. b stimulates the apoptotic cell signaling pathway in HT29 colon cancer cells. It can be used as a new treatment strategy or therapy for colon cancer treatment.