Acridine Orange And Propidium Iodide Research Articles

Antiproliferative and Apoptosis-Inducing Effects of Ethanolic Extract of Morus Alba Leaves on Human Chronic Myeloid Leukaemia K-562 Cell Lines

The rate of occurrence and prevalence of chronic myeloid leukaemia (CML) are increasing globally, despite it being a rare type of cancer. Morus alba also known as mulberry, has been used in Chinese traditional medicine for a long time to treat various conditions and diseases like hypertension and arthritis. Modern scientific studies have also recognized its medicinal properties, including antioxidant, antidiuretic, antidiabetic, antimicrobial and anticancer. However, its antiproliferative effects on specifically human CML K-562 cell lines remain poorly elucidated. This study aims to determine the antiproliferative effects of M. alba on K-562 cells. In vitro cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Acridine orange and propidium iodide (AO/PI) dual staining were then employed to assess the method of cell death. The ethanolic extract of M. alba leaves exhibited significant antiproliferative activity, with the lowest maximal half inhibitory concentration (IC50) value observed at 72 hours being 23 µg/mL. Notably, AO/PI staining revealed significant morphological changes, such as membrane blebbing, nuclear fragmentation, and indicator of early apoptosis, and late apoptosis. These results demonstrated that ethanolic extract of M. alba leaves induces apoptotic cell death in K-562 cells. Overall, these findings suggested that ethanolic extract of M. alba leaves exerts potent antiproliferative effects on human CML K-562 cells a in time- and dose-dependent manner.

Anticancer activity of cloned Nisin as an alternative therapy for MCF-7 brest cancer cell line.

Despite advancements in treatment and detection, cancer remains one of the most common causes of death worldwide. Conventional chemotherapeutic drugs used to treat cancer have non-specific toxicity toward normal body cells, which leads to several adverse effects. Second, malignancies are known to develop resistance to chemotherapy over time. As a result, the demand for novel anticancer drugs is growing daily. The most frequent type of cancer among women is breast cancer. Utilizing cloned Nisin as an anticancer was the purpose of this study using Gibson cloning and a cell-free peptide synthesis system, then purification of the target protein. The antiproliferative effect of Nisin against a breast cancer MCF-7 cell line was also determined using an MTTassay, and viability in cell lines was measured using acridine orangeand propidium iodide. Our findings demonstrate the successful isolation and cloning of the NisA, gene in addition to inducing of peptide synthesis system and then purification of a target protein. MTTassay results indicate that Nisin exhibits a high and selective cytotoxicity against the MCF-7 cell line with an IC50 value of 11.68 μg/ml. This data suggest that the NisA gene had in vitro antiproliferative effect against breast cancer cell. However, more research, including a combination of the NisA gene with other anticancer therapy in clinical use. In addition, in vivo studies are required.

Anti-Acanthamoeba activity of a semi-synthetic mangostin derivative and its ability in removal of Acanthamoeba triangularis WU19001 on contact lens.

Garcinia mangostana L., also known as the mangosteen tree, is a native medicinal plant in Southeast Asia having a wide variety of pharmacologically active compounds, including xanthonoid mangostin. In this study, we examined the pharmacological activities of the selected semi-synthetic mangostin derivative, namely, amoebicidal activity, encystation inhibition, excystation activity, and removal capacity of adhesive Acanthamoeba from the surface of contact lens (CL). Among the three derivatives, C1 exhibited promising anti-Acanthamoeba activity against Acanthamoeba triangularis WU19001 trophozoites and cysts. SEM images displayed morphological changes in Acanthamoeba trophozoites, including the loss of acanthopodia, pore formation in the cell membrane, and membrane damage. In addition, the treated cyst was shrunken and adopted an irregular flat cyst shape. Under a fluorescence microscope, acridine orange and propidium iodide (AO/PI) staining revealed C1 induced condensation of cytoplasm and chromatin with the loss of cell volume in the treated trophozoites, while calcofluor white staining demonstrated the leakage of cell wall in treated cysts, leading to cell death. Interestingly, at the concentration ranges in which C1 showed the anti-Acanthamoeba effects (IC50 values ranging from 0.035-0.056 mg/mL), they were not toxic to Vero cells. C1 displayed the highest inhibitory effect on A. triangularis encystation at 1/16×MIC value (0.004 mg/mL). While C1 demonstrated the excystation activity at 1/128×MIC value with a high rate of 89.47%. Furthermore, C1 exhibited the removal capacity of adhesive Acanthamoeba from the surface of CL comparable with commercial multipurpose solutions (MPSs). Based on the results obtained, C1 may be a promising lead agent to develop a therapeutic for the treatment of Acanthamoeba infections and disinfectant solutions for CL.

Ruscogenin Ameliorated Sjögren's Syndrome by Inhibiting NLRP3 Inflammasome Activation.

This article investigated the role and the specific mechanism of Ruscogenin in Sjögren's syndrome (SS). NOD/ShiLtJ mice were treated with Ruscogenin, and acinar cells isolated from submandibular glands were treated with TNF-α, Ruscogenin and transfected with NLRP3 overexpression plasmid. Salivary flow rate (SFR) was measured at weeks 11, 13, 15, 17, and 20. Histological analysis of the submandibular glands was conducted by hematoxylin-eosin staining assay. IL-6, IL-17, TNF-α, and IL-1β mRNA expression was detected through qRT-PCR. AQP 5, AQP 4, P2X7R, NLRP3, caspase 1, IL-1β, Bax, and Bcl-2 protein levels were tested by western blot. Cell apoptosis was assessed through acridine orange and propidium iodide (AO/PI) staining assay and flow cytometry assay. Ruscogenin ameliorated the SFR and submandibular gland inflammation of NOD/ShiLtJ mice. Ruscogenin promoted the preservation of acinar cells and suppressed inflammation-related factors (P2X7R, NLRP3, caspase 1, and IL-1β) in submandibular gland tissues of NOD/ShiLtJ mice. Ruscogenin inhibited acinar cell apoptosis in NOD/ShiLtJ mice and reversed TNF-α-induced apoptosis and inflammation of acinar cells. NLRP3 overexpression reversed the repressive effect of Ruscogenin on TNF-α-induced inflammation and apoptosis of acinar cells. Ruscogenin ameliorated SS by inhibiting NLRP3 inflammasome activation.

PEGylated Lecithin-Chitosan Nanoparticle-Encapsulated Alphα-Terpineol for In Vitro Anticancer Effects.

The aim of this study was to fabrication PEGylated lecithin-chitosan nanoparticles (PLC-NPs) as alphα-Terpineol's (αT-PLC-NPs) delivery system and examine its anti-cancer effects. αT-PLC-NPs were synthesized by self-assembling method; after characterization, entrapment efficiency of α-T was measured by HPLC procedure. MTT test was conducted for cytotoxicity evaluation. Chick chorioallantoic membrane (CAM) and quantitative polymerase chain reaction (qPCR) analysis were used to determine the angiogenesis properties, and qPCR, flow cytometry, and acridine orange and propidium iodide (AO/PI) staining were used to evaluate the pro-apoptotic effects of αT-PLC-NPs. Finally, the anti-inflammatory and antibacterial activity of the αT-PLC-NPs was also evaluated. αT-PLC-NPs with a size of 220.8 nm, polydispersity index (PDI) of 0.3, zeta potential of +29.03 mV, and encapsulation efficiency of 82% showed higher inhibitory effect on MCF7 cells (IC50: 750 μg/mL) compared to HFF cells (above 1000 μg/mL). Decreased angiogenesis indices and embryonic growth factors in CAM assay, decreased expression of VEGF and VEGF-R genes, and decreased cell migration showed the inhibitory effect of αT-PLC-NPs on angiogenesis. Increased expression of P53, P21, and caspase9 genes, as well as the results of AO/PI staining along with increasing the number of SubG1 phase cells in flow cytometry, confirmed the pro-apoptotic effects of αT-PLC-NPs. Also, its anti-inflammatory effects were demonstrated by inhibiting the expression of pro-inflammatory cytokines (TNF-α and IL-6). The inhibitory power of αT-PLC-NPs in suppressing gram-positive and negative bacterial strains was demonstrated by disk diffusion (DD), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) methods. PLC-NPs are a promising carrier for α-T transfer for preclinical studies.

Cytotoxicity Effect and Morphological Changes of Chrysanthemum Morifolium Methanolic Extract against Chronic Myeloid Leukaemia K-562 Cell Line

Chrysanthemum morifolium, also known as “Bunga kekwa” in Malaysia, has various benefits and widely used in Chinese herbal medicines. The plant extract was reported to have significant biological activities, such as anti-inflammation, anti-tumour, anti-oxidant, and anti-cancer. Nonetheless, its anti-cancer potential on chronic myeloid leukaemia has remained elusive. The main goal of this study is to evaluate the cytotoxic effect of C.morifolium buds and flowers in methanolic extracts on chronic myeloid leukaemia malignancy K-562 cell lines. The bud and flower of C.morifolium were macerated for 72 hours in 100% methanol then were concentrated under reduced pressure using a rotary evaporator and oven-dried to obtain crude extracts. K-562 cells were treated with six different concentrations 400, 200, 100, 50, 25, and 12.5 µg/ml and incubated for 24, 48 and 72 hours. The in vitro cytotoxic activity was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) test and was quantified using a microplate reader at 570 nm. Acridine orange and propidium iodide (AO/PI) staining were used to assess morphological alterations. MTT assays results showed moderate toxicity of both extracts. The lowest maximal half inhibitory concentration (IC50) value were observed at 72 hours of incubation; 182 ± 4.04 ug/ml for BM and 161 ± 7.88 ug/ml for flower extract (FM). However, there was a significantly different IC50 value (p<0.05) between the incubation periods of both treatments where the IC50 value at 24 hours was 301.33 ± 8.51 ug/ml 301 µg/ml in BM, 216 ± 10.79 ug/ml 216 µg/ml in FM and at 48 hours was 227 ± 12.25 ug/ml 227 µg/ml in bud extract (BM), 174 ± 11.92 ug/ml 174 µg/ml in FM. The morphological changes evidence was shown in AO/PI staining by the appearance of a mixed population of cells; early apoptosis, late apoptosis and necrotic cells. These findings suggested that methanolic C.morifolium extracts showed moderate cytotoxic effect on chronic myeloid leukaemia K-562 cells. Further study needed to identify the mode and mechanism of cell death in K-562 cells treated with the C.morifolium extracts.

COMMERCIAL EYE DROPS TRIGGERS NECROTIC EFFECT IN Acanthamoeba sp.

Eye infection due to microbial infection is hard to treat and painful. Two types of eye drops that are commonly used to relieve eye pain offered by pharmacists have been tested in this study namely solution A (containing tetrahydrozoline hydrochloride) and solution B (containing gentamicin and dexamethasone). The efficacy and the activities of these eye drops were tested on Acanthamoeba sp. (a clinical isolate from an Acanthamoeba keratitis patient) to study the cytotoxicity effects of the solutions on the Acanthamoeba. The Acanthamoeba were exposed to solution A and B for 24 hr and cell viability was assessed using MTT assays, morphological changes using the light microscope and through acridine orange and propidium iodide (AO/PI) staining for cytoplasmic biochemical activities. The IC50 value for Acanthamoeba cell viability was 45.1% and 20.3% for solutions A and B respectively. Morphological observation shows the inhibition of acanthapodia formation on the surface of the cells. Solution A and B-treated Acanthamoeba appeared in the red color of the cytoplasm upon staining with AO/PI indicating a necrotic mode of cell death. This is due to loss of membrane integrity of Acanthamoeba cell membrane after exposed to solution A and B at their IC50 value. It is shown that solutions A and B can cause cell death in trophozoite of Acanthamoeba cells at moderate IC50 value. Unfortunately, the necrosis mode of cell death is not a preferable type of cell death for treating Acanthamoeba infection. Therefore, it can be concluded that solutions A and B are not suitable to treat eye infected with Acanthamoeba sp. as it does not promise absolute healing as the solution concentration needed is quite high and the cell death mechanism is necrosis.

Neuroprotective Effects of 7-Geranyloxycinnamic Acid from Melicope lunu ankenda Leaves.

Neurodegenerative diseases (NDDs) are chronic conditions that have drawn robust interest from the scientific community. Phytotherapeutic agents are becoming an important source of chemicals for the treatment and management of NDDs. Various secondary metabolites have been isolated from Melicope lunu-ankenda plant leaves, including phenolic acid derivatives. However, their neuroprotective activity remains unclear. Thus, the aim of this study is to elucidate the in vitro neuroprotective activity of 7-geranyloxycinnamic acid isolated from Melicope lunu-ankenda leaves. The neuroprotective activity was evaluated in differentiated human neuroblastoma (SH-SY5Y) cells by monitoring cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Moreover, the potential to impair apoptosis in differentiated cells was investigated employing the Annexin V-FITC assay, acridine orange and propidium iodide (AO/PI) staining, and fluorescence microscopy. Morphological assessment and ultrastructural analysis were performed using scanning and transmission electron microscopy to evaluate the effect of 7-geranyloxycinnamic acid on surface morphology and internal features of the differentiated cells. Pre-treatment of neuronal cells with 7-geranyloxycinnamic acid significantly protected the differentiated SH-SY5Y cells against H2O2-induced apoptosis. Cytoskeleton and cytoplasmic inclusion were similarly protected by the 7-geranyloxycinnamic acid treatment. The present findings demonstrate the neuroprotective potential of 7-geranyloxycinnamic acid against H2O2-induced neurotoxicity in neuronal cells, which is an established hallmark of neuronal disorders.

Vernonia calvoana Shows Promise towards the Treatment of Ovarian Cancer.

The treatment for ovarian cancers includes chemotherapies which use drugs such as cisplatin, paclitaxel, carboplatin, platinum, taxanes, or their combination, and other molecular target therapies. However, these current therapies are often accompanied with side effects. Vernonia calvoana (VC) is a valuable edible medicinal plant that is widespread in West Africa. In vitro data in our lab demonstrated that VC crude extract inhibits human ovarian cancer cells in a dose-dependent manner, suggesting its antitumor activity. From the VC crude extract, we have generated 10 fractions and VC fraction 7 (F7) appears to show the highest antitumor activity towards ovarian cancer cells. However, the mechanisms by which VC F7 exerts its antitumor activity in cancer cells remain largely unknown. We hypothesized that VC F7 inhibits cell proliferation and induces DNA damage and cell cycle arrest in ovarian cells through oxidative stress. To test our hypothesis, we extracted and fractionated VC leaves. The effects of VC F7 were tested in OVCAR-3 cells. Viability was assessed by the means of MTS assay. Cell morphology was analyzed by acridine orange and propidium iodide (AO/PI) dye using a fluorescent microscope. Oxidative stress biomarkers were evaluated by the means of lipid peroxidation, catalase, and glutathione peroxidase assays, respectively. The degree of DNA damage was assessed by comet assay. Cell cycle distribution was assessed by flow cytometry. Data generated from the MTS assay demonstrated that VC F7 inhibits the growth of OVCAR-3 cells in a dose-dependent manner, showing a gradual increase in the loss of viability in VC F7-treated cells. Data obtained from the AO/PI dye assessment revealed morphological alterations and exhibited characteristics such as loss of cellular membrane integrity, cell shrinkage, cell membrane damage, organelle breakdown, and detachment from the culture plate. We observed a significant increase (p < 0.05) in the levels of malondialdhyde (MDA) production in treated cells compared to the control. A gradual decrease in both catalase and glutathione peroxidase activities were observed in the treated cells compared to the control. Data obtained from the comet assay showed a significant increase (p < 0.05) in the percentages of DNA cleavage and comet tail length. The results of the flow cytometry analysis indicated VC F7 treatment caused cell cycle arrest at the S-phase checkpoint. Taken together, our results demonstrate that VC F7 exerts its anticancer activity by inhibiting cell proliferation, inducing DNA damage, and causing cell cycle arrest through oxidative stress in OVAR-3 cells. This finding suggests that VC F7 may be a potential alternative dietary agent for the prevention and/or treatment of ovarian cancer.

Circulating Endothelial Cells As a Marker of Vascular Injury in Pediatric Patients

<h3>Introduction</h3> Thrombotic microangiopathy (TMA), hepatic veno-occlusive disease (VOD) and GVHD are major causes of morbidity and mortality in hematopoietic stem cell transplant (HSCT) recipients. Current strategies to identify these complications involve surveilling markers of immune activation and organ function. Unfortunately, these processes are frequently identified after organ damage has occurred, creating the need for a timelier marker of disease. Circulating endothelial cells (CECs) have been shown to directly reflect vascular injury and are a potential tool for identifying early evidence of endothelial damage. Our study quantifies and characterize CECs in pediatric patients undergoing HSCT and aims to identify the timing of peak vascular injury relative to transplant as well as the relationship between CECs and post-transplant vascular complications. <h3>Methods</h3> CEC analysis was performed prior to and after stem cell infusion. All patients had central venous catheters. CEC identification was done on whole blood using Invitrogen M-450 Tosylactivated Dynabeads coupled to a CD146 antibody. Confirmatory staining after immunomagnetic isolation was done using acridine orange and propidium iodide (AOPI). CEC identification criteria are shown in Figure 1. Two reviewers quantified CECs by morphology and bead number. Negative controls were done using healthy donor blood. <h3>Results</h3> CEC testing was done on 8 HSCT patients (Fanconi Anemia=1, AML=2, ALL=2, ICF-1 syndrome=1, Hemoglobin Hammersmith=1 and macrophage activation syndrome=1). 6 conditioning regimens were myeloablative and 2 were reduced intensity. CEC quantification for individual patients is shown in Figure 2. The vast majority of patients (n=7) had a peak CEC value prior to day 14, including 4 patients whose peak value occurred prior to stem cell infusion. Clinical complications are described in Figure 2. The two highest CEC values recorded were in patients with severe complications, including cardiac arrest (Patient 5, 124 CECs/mL) and death (Patient 7, 84 CECs/mL). The individual difference between lowest recorded CEC value and peak CEC value was also greatest in these patients (Patient 5, 120 CECs/mL; Patient 7, 80 CECs/mL). Mean CEC values for HSCT patients and controls are shown in Figure 3. <h3>Conclusion</h3> CECs reliably peaked within the first 2 weeks of HSCT suggesting marked vascular injury occurs in this timeframe. CEC count peaked prior to stem cell infusion in half of these patients, which implies a large component of vascular injury is driven by the conditioning process. As predicted, the highest CEC values recorded were in patients with grave complications, including one patient who died from multiorgan system failure. Continued data collection will be informative as to whether the nature of these peaks or characteristics of the CECs themselves will predict future vascular complications.

Abstract LB-246: Apoptotic mechanisms of Vernonia amydalina delile in the prevention of prostate cancer

Abstract Prostate cancer is one of the common cancers in males and its incidence keeps increasing globally. Approximately 81% of prostate cancer is diagnosed during the early stage of the disease. The treatment options for prostate care include surgery, radiotherapy, and chemotherapy, but these treatments often have side effects that may result in a poor quality of life such as impotence or decreased bowel function. Our central goal is to test the apoptotic mechanisms of Vernonia amydalina Delile (an edible medicinal plant that is relatively inexpensive, non-toxic, and virtually without side effects) for the prevention of prostate cancer using human adenocarcinoma (PC-3) cells as a test model. To address our specific goal, PC-3 cells were treated with Vernonia amydalina Delile (VAD). Cell viability was determined by the MTT assay and cell morphology was analyzed by acridine orange and propidium iodide (AO/PI) dye using the fluorescent microscope. Cell cycle arrest and cell apoptosis were evaluated by Flow Cytometry assessment. Nucleosomal DNA fragmentation was detected by DNA laddering. Data obtained from the AO/PI dye assessment indicated that VAD significantly reduced the number of live cells in a dose-dependent manner, showing a gradual increase in the loss of viability in VAD-treated cells. A similar result was obtained by the MTT assay. We observed a significant increase in DNA damage in VAD-treated cells compared to the control group. Flow cytometry data demonstrated that VAD induced apoptosis in treated cells compared to the control cells. These results suggest that induction of cell death, cell cycle arrest, and cell apoptosis are involved in the therapeutic efficacy of VAD as anticancer candidate towards the prevention and/or treatment of prostate cancer. Citation Format: Clement G. Yedjou, Solange S. Tchounwou, William K. Johnson, Sylvianne Njiki, Paul B. Tchounwou. Apoptotic mechanisms of Vernonia amydalina delile in the prevention of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-246.

Growth inhibition of mice mammary carcinoma cell line with green synthesized Magnetic Iron Oxide Nanoparticles

Mammary adenocarcinoma represents one of the most cancers that effect woman in Iraq and worldwide. Finding a definitecure for this type of cancer is a target of considerable research in many laboratories over the globe. In this study, a greensynthesized magnetic iron oxide nanoparticles (MNP) was used to specify their ability to inhibit the growth of mice mammarycarcinoma cell line (AMN3), it comes as a preliminary study of wider ongoing research. After successfully synthesizing magneticiron nanoparticles, it employed in different concentrations to expose AMN3 cells for 24 and 48 hours. The viability of theexposed cells was determined using standard MTT assay. The inhibition concentration 50 was determined afterward and usedto test the possibility of inducing apoptosis in the carcinoma cells using a mixture of fluorescent stains acridine orange andpropidium iodide. Results indicated that green synthesized MNP can inhibit the growth of mammary adenocarcinoma cell lineup to 79% and 89% after 24 and 48 hr of incubation with 1000μg/ml. The IC50 of MNP was capable of inducing apoptosis inthis cell line as shown by fluorescence microscope. The standard hemolysis assay used to test the reaction between the greensynthesized magnetic oxide nanoparticles and normal cells. The IC50 of AMN3 cells growth inhibition only induce 14% ofRBC hemolysis, while the percentage induced by negative control was 10%. In conclusion, green MNP have an effect uponcancer cells greater than normal cells. This material must tested against other types of cancer and normal cells to verify itsaction and study mechanisms of apoptosis induction in detail.Key words: Magnetic iron oxide nanoparticles, AMN3, green synthesis, nanotherapy.

VERNONIA AMYGDALINA DELILE EXHIBITS A POTENTIAL FOR THE TREATMENT OF ACUTE PROMYELOCYTIC LEUKEMIA.

The World Health Organization (WHO) has been on front line to encourage developing countries to identify medicinal plants that are safe and easily available to patients. Traditional medicine represents the first-treatment choice for the healthcare of approximately 80% of people living in developing countries. Also, its use in the United States has increased by 38% during within the last decade of the 20th century alone. Therefore, the aim of the present study was to explore the efficacy of a medicinal plant, Vernonia amygdalina Delile (VAD), as a new targeted therapy for the management of acute promyelocytic leukemia (APL), using HL-60 cells as a test model. To address our specific aim, HL-60 promyelocytic leukemia cells were treated with VAD. Live and dead cells were determined by acridine orange and propidium iodide (AO/PI) dye using the Cellometer Vision. The extent of DNA damage was evaluated by the comet assay. Cell apoptosis was evaluated by flow cytometry assessment. Data obtained from the AO/PI assay indicated that VAD significantly reduced the number of live cells in a dose-dependent manner, showing a gradual increase in the loss of viability in VAD-treated cells. We observed a significant increase in DNA damage in VAD-treated cells compared to the control group. Flow cytometry data demonstrated that VAD induced apoptosis in treated cells compared to the control cells. These results suggest that induction of cell death, DNA damage, and cell apoptosis are involved in the therapeutic efficacy of VAD. Because VAD exerts anticancer activity in vitro, it would be interesting to perform clinical trials to confirm its effectiveness as an anticancer agent towards the treatment of APL patients.

Abstract 838: Natural plant extracts induced DNA damage and apoptosis through oxidative stress in transgenic mouse model

Abstract One of the most frightening events that women will ever face is being diagnosed with breast cancer. One in eight women in the United States will be diagnosed with this life-threatening disease during her lifetime. Even with all the advances in pharmaceutical technology, mortality rates for breast cancer have remained stagnant for the past few decades. The development of new drugs from natural products is considered important. The specific aim of the present study was to use transgenic mouse mammary tumors as a test model to explore the therapeutic mechanisms of a novel natural product as anti-cancer agent in the treatment of breast cancer. To achieve our specific aim, we performed both in vitro and in vivo studies. Acridine orange and propidium iodide (AO/PI) staining were used to visualize live and dead cells with the means of Cellometer Vision. Tumor volume and weight were measured. Tumor histology was assessed by immunohistochemistry, and enzymatic activities were determined by spectrophotometry. The extent of DNA damage was evaluated by the Comet assay. Cell/tissue apoptosis was measured by the flow cytometry. Data obtained from the AO/PI dye assessment indicated that the tested natural product significantly reduced the number of live cells in a dose-dependent manner, showing a gradual increase in the loss of viability in treated cells. We observed tumor growth inhibition after 4 weeks of daily intraperitoneal administration of plant extracts to trangenic mice. There was a significant increase in DNA damage in treated mice compared to the control mice. Flow cytometry data showed a strong dose-response relationship between plant extracts treatment and annexin V/PI positive cells. Similarly, a statistically significant and dose-dependent increase (p&amp;lt; 0.05) was recorded with regard to caspase 3 activity. Data from enzyme analysis (alaninine transaminase, aspartate transaminase, and creatinine) revealed that plant extract administration is not toxic to treated mice. These results suggest that induction of cell death, DNA damage, and cell/tissue apoptosis may be involved in the therapeutic mechanisms of plant extracts in breast cancer. Collectively, the findings from this study provide convincing evidence that the tested plant extracts may represent a potential anticancer candidate against breast cancer. Research supported by NIH Grant No. NIMHD-G12MD007581) and Grant No. P20GM103476. Citation Format: Clement G. Yedjou, Paul B. Tchounwou, Lucio Miele, Marinelle Payton. Natural plant extracts induced DNA damage and apoptosis through oxidative stress in transgenic mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 838.

CYTOTOXIC EFFECT OF HEPCIDIN (TH1-5) ON HUMAN BREAST CANCER CELL LINE (MCF7)

Hepcidin (TH1-5) is a cysteine-rich antimicrobial peptide originally isolated from the freshwater fish Oreochromis mossambicus. A synthesized form of the peptide has been reported to exhibit cytotoxic activity against few human cancer cell lines. This study investigated the potential cytotoxicity of the peptide against human breast cancer cell line and normal mouse embryonic fibroblast cell line (NIH/3T3) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Morphological changes by acridine orange and propidium iodide (AO/PI) double staining were also studied to determine apoptotic evidences. Hepcidin (TH1-5) showed cytotoxic activity against MCF7 with an IC50 of 20 mg/mL but no significant effect against NIH/3T3. This outcome indicates hepcidin (TH1-5) to be a promising cytotoxic peptide that warrants further studies as a potential anticancer agent for breast cancer therapy.

Lactoferrin and ovotransferrin contribute toward antioxidative effects of Edible Bird's Nest against hydrogen peroxide-induced oxidative stress in human SH-SY5Y cells.

There are reports of improved redox outcomes due to consumption of Edible Bird's Nest (EBN). Many of the functional effects of EBN can be linked to its high amounts of antioxidants. Interestingly, dietary components with high antioxidants have shown promise in the prevention of aging and its related diseases like Alzheimer's disease. In this study, the antioxidative potentials of EBN and its constituents, lactoferrin (LF) and ovotransferrin (OVF), were determined and protective effects against hydrogen peroxide (H2O2)- induced toxicity on SH-SY5Y cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange and propidium iodide (AO/PI) staining with microscopy were examined. Results showed that EBN and its constituents attenuated H2O2-induced cytotoxicity, and decreased radical oxygen species (ROS) through increased scavenging activity. Furthermore, LF, OVF, and EBN produced transcriptional changes in antioxidant related genes that tended towards neuroprotection as compared to H2O2-treated group. Overall, the results suggest that LF and OVF may produce synergistic or all-or-none antioxidative effects in EBN.

Abstract 5313: The anticancer effect of Vernonia amygdalina in the MMTV-PyVT transgenic mouse model

Abstract One of the most frightening events that women will ever face is being diagnosed with breast cancer. One in eight women in the United States will be diagnosed with this life-threatening disease during her lifetime. Even with all the advances in pharmaceutical technology, mortality rates for breast cancer have remained stagnant for the past few decades. The development of new drugs from natural products is considered important. Previous studies from our laboratory show that a novel natural product, Vernonia amygdalina (V. amygdalina) leaf extract, exerts DNA-damaging anticancer activities against breast cancer. Therefore, the specific aim of the present study was to use transgenic mouse mammary tumors as a test model to explore the therapeutic mechanisms of V. amygdalina as anti-cancer agent in the treatment of breast cancer. To achieve our specific aim, we performed both in vitro and in vivo studies. Acridine orange and propidium iodide (AO/PI) staining were used to visualize live and dead cells with the means of Cellometer Vision. Tumor volume and weight were measured. Tumor histology was assessed by immunohistochemistry, and enzymatic activities were determined by spectrophotometry. The extent of DNA damage was evaluated by the Comet assay. Cell/tissue apoptosis was measured by the flow cytometry. Data obtained from the AO/PI dye assessment indicated that V. amygdalina significantly reduced the number of live cells in a dose-dependent manner, showing a gradual increase in the loss of viability in V. amygdalina-treated cells. We observed tumor growth inhibition after 4 weeks of daily intraperitoneal administration of V. amygdalina to trangenic mice. There was a significant increase in DNA damage in V. amygdalina-treated mice compared to the control mice. Flow cytometry data showed a strong dose-response relationship between V. amygdalina treatment and annexin V/PI positive cells. Similarly, a statistically significant and dose-dependent increase (p&amp;lt; 0.05) was recorded with regard to caspase 3 activity. Data from enzyme analysis (alaninine transaminase, aspartate transaminase, and creatinine) revealed that V. amygdalina administration is not toxic to treated mice. These results suggest that induction of cell death, DNA damage, and cell/tissue apoptosis may be involved in the therapeutic mechanisms of V. amygdalina in breast cancer. Collectively, the findings from this study provide convincing evidence that V. amygdalina may represent a potential anticancer candidate against breast cancer. Citation Format: Clement G. Yedjou, Paul B. Tchounwou, Lucio Miele, Ifedayo V. Ogungbe, Robert J. Brown, Marinelle Payton. The anticancer effect of Vernonia amygdalina in the MMTV-PyVT transgenic mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5313. doi:10.1158/1538-7445.AM2015-5313

Anti-amoebic properties of carbonyl thiourea derivatives.

Thiourea derivatives display a broad spectrum of applications in chemistry, various industries, medicines and various other fields. Recently, different thiourea derivatives have been synthesized and explored for their anti-microbial properties. In this study, four carbonyl thiourea derivatives were synthesized and characterized, and then further tested for their anti-amoebic properties on two potential pathogenic species of Acanthamoeba, namely A. castellanii (CCAP 1501/2A) and A. polyphaga (CCAP 1501/3A). The results indicate that these newly-synthesized thiourea derivatives are active against both Acanthamoeba species. The IC50 values obtained were in the range of 2.39–8.77 µg·mL‑1 (9.47–30.46 µM) for A. castellanii and 3.74–9.30 µg·mL‑1 (14.84–31.91 µM) for A. polyphaga. Observations on the amoeba morphology indicated that the compounds caused the reduction of the amoeba size, shortening of their acanthopodia structures, and gave no distinct vacuolar and nuclear structures in the amoeba cells. Meanwhile, fluorescence microscopic observation using acridine orange and propidium iodide (AOPI) staining revealed that the synthesized compounds induced compromised-membrane in the amoeba cells. The results of this study proved that these new carbonyl thiourea derivatives, especially compounds M1 and M2 provide potent cytotoxic properties toward pathogenic Acanthamoeba to suggest that they can be developed as new anti-amoebic agents for the treatment of Acanthamoeba keratitis.

Rapid assessment of islet viability with acridine orange and propidium iodide.

A simple, rapid method for estimating the viability of isolated islets of Langerhans with fluorescent dyes is described. Low concentrations of acridine orange and propidium iodide (AO/PI) were used to visualize living and dead islet cells simultaneously. AO/PI-stained islets can be divided into three distinct groups. Group A islets fluoresce green, contain insulin, and have normal ultrastructure; group C islets fluoresce primarily red, contain little or no insulin, and have cells with disrupted cellular membranes. Group B islets fluoresce red, green, and yellow. The yellow color is due to the addition of two primary colors from the superimposed red and green fluorescing cells. In this assay, the interpretation that red islet cells are dead and green islet cells are alive was confirmed by sequentially staining single islet cells with AO/PI and trypan blue. The observation that red islets are dead was confirmed by heat-killing, enzymatically damaging, treating with ethanol, or depriving islets of nutrients and observing the red fluorescence. This assay should be useful in studies where the assessment of islet viability is essential.