Chemotherapeutic and Antiproliferative Effect of Purified Protein from Marine Catfish Tachysurus Dussumieri on Human Colon Cancer Cell Line.

The present study aimed to investigate the purified protein from the epidermal mucus of marine catfish Tachysurus dussumieri on the human colon cancer cell line. The bioactive protein was purified with the Anion exchange chromatography and the collected fractions were then tested to assess cell viability in HT 29 cells through the MTT assay. The most responding active purified protein fraction (PPF III) was characterized with the MALDI-TOF/MS it shared a similar homology and sequence with 90% of antimicrobial peptides from external secretions of amphibians. Typical morphological changes of apoptotic cells, including cell shrinkage and detachment, DNA damage, and nuclear condensation were observed after the treatment of bioactive protein. PPF III triggered ROS, increasing the LDH activity, disruption of mitochondrial membrane potential, and upregulation of Cleaved caspase 3/9, Cytochrome-c, Bax, and downregulation of Bcl-2 protein and gene expression on HT 29 cells.

Design, Synthesis, Anticancer Evaluation and Molecular Modeling Studies of New Thiazolidinone-Benzoate Scaffold as EGFR Inhibitors, Cell Cycle Interruption and Apoptosis Inducers in HepG2.

Synthesis of new anticancer candidates with protein kinases inhibitory potency is a major goal of pharmaceutical science and synthetic research. This current work represents the synthesis of a series of substituted benzoate-thiazolidinones. Most prepared thiazolidinones were evaluated in vitro for their potential anticancer activity against three cell lines by MTT assay, and they found to be more effective against cancer cell lines with no harm toward normal cells. Thiazolidinones 5 c and 5 h were further evaluated to be kinase inhibitors against EGFR showing effective inhibitory impact (with IC50 value; 0.2±0.009 and 0.098±0.004 µM, for 5 c and 5 h, respectively). Furthermore, 5 c and 5 h have effects on cell cycle and apoptosis induction capability in HepG2 cell lines by DNA-flow cytometry analysis and annexin V-FITC apoptosis assay, respectively. The results showed that they have effect of disrupting the cell cycle and causing cell mortality by apoptosis in the treated cells. Moreover, molecular docking studies showed better binding patterns for 5 c and 5 h with the active site of the epidermal growth factor receptor (EGFR) protein kinase (PDB code 1M17). Finally, toxicity risk and physicochemical characterization by Osiris method was performed on most of the compounds, revealing excellent properties as possible drugs.

Eco-friendly postharvest irradiation strategy with 131I isotope for environmental management of populations of migratory locust, Locusta migratoria.

PURPOSE: Irradiation of food is promising for control of pests to minimize postharvest losses of yields and thus improvement of food safety, shelf life of produce. It is a method of choice that induces a series of lethal biochemical and molecular changes culminating into the engagement of a downstream cascade to cause abnormalities in irradiated pests. In this study, the effects of iodine-131 (131I) isotope radiation on the male gonad development of the migratory locust, Locusta migratoria, were evaluated. MATERIALS AND METHODS: Newly emerged adult male locusts, less than one-day-old, were divided into two groups, control and irradiated. Locusts in the control group (n = 20 insects) didn't drink irradiated water and were reared under normal environmental conditions for one week. Locusts in the irradiated group (n = 20 insects) were exposed to irradiated water at a dose of 30 mCi and they were subsequently observed until they drank the whole quantity. RESULTS: At the end of the experiment, scanning and electron microscopic examination of testes obtained from irradiated locusts revealed several major abnormalities, including malformed nuclei of spermatozoa, irregular plasma membranes, shrinkage of testicular follicles, vacuolated cytoplasm, disintegrated nebenkern and agglutinations of spermatids. Flow cytometry analysis revealed that 131I radiation induced both early and late apoptosis, but not necrosis, in testicular tissues. Testes of irradiated insects also exhibited a burst in reactive oxygen species (ROS), as indicated by significant elevation in amounts of malondialdehyde (MDA), a marker for peroxidation of lipids. In contrast, irradiation coincided with significant reductions in activities of enzymatic antioxidant biomarkers. Relative to controls, a three-fold upregulation of expression of mRNA of heat shock protein, Hsp90, was observed in testicular tissue of irradiated locusts. 131I-irradiated insects exhibited genotoxicity, as indicated by significant increases in various indicators of DNA damage by the comet assay, including tail length (7.80 ± 0.80 µm; p < .01), olive tail moment (40.37 ± 8.08; p < .01) and tail DNA intensity % (5.1 ± 0.51; p < .01), in testicular cells compared to the controls. CONCLUSION: This is the first report on elucidation of I131-irradiation-mediated histopathological, biochemical and molecular mechanisms in gonads of male L. migratoria. Herein, the findings underscore the utility of 131I radiation as an eco-friendly postharvest strategy for management of insect pests and in particular for control of populations of L. migratoria.

Repositioning of simvastatin for diabetic colon cancer: role of CDK4 inhibition and apoptosis.

There is increased risk of colon cancer in both men and women having diabetes. The objective of the study was to evaluate the role of simvastatin in colon cancer associated with type 2 diabetes mellitus. Diabetes was induced by administering high fat diet with low dose streptozotocin model. 1,2 dimethylhydrazine (25 mg/kg, sc) was used for colon cancer induction. MTT assay, scratch assay, clonogenic assay and annexin V-FITC assay using flow cytometry were performed on HCT-15 cell line. Simvastatin controlled diabetes and colon cancer in animal models and reduced mRNA expression of CDK4 in colon tissues. In vitro studies revealed that simvastatin showed a decrease in cell viability and produced dose dependent decrease in clone formation. There was decrease in the rate of migration with increase in concentration of simvastatin in scratch assay. Moreover, simvastatin induced apoptosis as depicted from annexin V-FITC assay using flow cytometry as well as that revealed by tunnel assay. Our data suggest that simvastatin exhibits protective role in colon cancer associated with diabetes mellitus and acts possibly via down regulation of CDK4 and induction of apoptosis and hence can be considered for repositioning in diabetic colon cancer.

Genotoxic damage and apoptosis in rat glioma (F98) cell line following exposure to bromuconazole.

Bromuconazole, a fungicide from the triazole family, is widely used to protect the crop from various fungal contaminations to increase product quality and productivity. Although the massive use of bromuconazole poses a serious risk to human health, the exact mechanism of bromuconazole toxicity, especially on brain support cells, called glia cells, remains unclear so far. This study aimed to determine the mechanism of cytotoxicity and genotoxicity of bromuconazole via inspection of apoptotic death in rat glioma (F98) cells. We observed that bromuconazole treatment caused concentration-dependent cell death with an IC50 of 60 µM, and disruption of the cytoskeleton was observed via immunocytochemical analysis. Further, bromuconazole inhibits cell proliferation, it arrests the cell cycle in the G0/G1 phase and so inhibits DNA synthesis. Genotoxic analysis showed that bromuconazole exposition causes DNA fragmentation (comet assay) and nuclear condensation (DAPI staining). Apoptotic cell death was confirmed through: positive Annexin-V/FITC-PI dyes, p53 and Bax overexpression, Bcl2 repression, an increase in Bax/BCL-2 ratios of the mRNA, mitochondrial membrane depolarization, and an increase of caspase-3 activity. All these results demonstrate that bromuconazole exerts its cytotoxic and genotoxic effects through apoptotic cell death, which could implicate mitochondria.

Bromuconazole fungicide induces cell cycle arrest and apoptotic cell death in cultured human colon carcinoma cells (HCT116) via oxidative stress process.

BACKGROUND: Bromuconazole, a fungicide belonging to the triazole family, is a plant protection product used to control, repel or destroy fungi that may develop on crops. We investigated the pro-apoptotic effect of bromuconazole and the role of oxidative stress in the death mechanism induced by this fungicide in this study. METHODS: The human colon HCT116 cell line was treated with Bromuconazole (IC50/4, IC50/2, and IC50) for 24 h. Cells were collected and analysed for biomarkers of apoptotic cell death and oxidative stress as well as for the assessment of genotoxic damage. RESULTS: Our study showed that bromuconazole caused a concentration-dependent increase in cell mortality with an IC50 of 180 µM. Bromuconazole induced cell cycle arrest in the G0/G1 phase and DNA synthesis inhibition. The Comet assay showed that bromuconazole caused DNA damage in a concentration-dependent manner. Bromuconazole-induced apoptosis was observed by, Annexin-V/FITC-PI and BET/AO staining, by mitochondrial membrane depolarisation, and by increased caspase-3 activity. In addition, bromuconazole induced a significant increase in ROS and lipid peroxidation levels and a disruption in SOD and CAT activities. N-acetylcysteine (NAC) strongly prevents cytotoxic and genotoxic damage caused by bromuconazole. CONCLUSION: Bromuconazole toxicity was through the oxidative stress process, which causes DNA damage and mitochondrial dysfunction, leading to cell cycle arrest and apoptotic death of HCT116 cells.

Bromuconazole exposure induced cell cycle arrest in the G0/G1 in HCT116 cells.Bromuconazole caused DNA synthesis inhibition and degradation.Bromuconazole-induced Annexin-V/FITC-PI and BET/AO positive staining, increased caspase-3 activity and MMP.Bromuconazole enhances ROS, MDA levels and disruption of CAT and SOD activities.

Toxicological aspect of bioinsecticide pyrethrum extract and expressions of apoptotic gene levels in human hepotacellular carcinoma HepG2 cells.

Pyrethrum extract (PE), an important natural bioinsecticide, is extensively used across the world to control pest insects in homes and farms. The aim of this study was to evaluate the potential cytotoxic effect of PE using MTT assay and genotoxic effect using micronucleus (MN) assay. The changes in the expressions of the apoptosis genes in mRNA levels were also investigated using Real-Time qPCR analysis as well as the ratio of apoptotic/necrotic cells with AnnexinV-FITC/Propidium iodide (PI) assay in HepG2 cells. PE markedly suppressed the cell proliferation on HepG2 cells. It significantly increased the frequency of micronucleus (MN) at 500 and 1000 µg/mL. PE also induced the percentage of the cell population of late apoptotic/necrotic cells (FITC + PI+) and necrotic cells (FITC- PI+), especially at 4000 µg/mL analyzed by flow cytometry. PE caused significant fold changes in the expression of several apoptotic genes including APAF1, BIK, BAX, BAD, BID, MCL-1, CASP3, CASP1, CASP2, FAS, FADD and TNFRSF1A. In particular, the pro-apoptotic gene Hrk (Harakiri) remarkably and dose-dependently was overexpressed of the mRNA level. As a result, PE may exhibit cyto-genotoxic effects, especially at higher concentrations and lead to significant changes in the expression of mRNA levels in several apoptotic genes.HighlightsNatural bioinsecticide PE exhibited a cytotoxic effect in HepG2 cells.PE significantly induced the micronucleus (MN) frequency at 500 and 1000 µg/mL.This bioinsecticide induced cell death and it lead to significant fold changes in the expression of mRNA levels in several apoptotic genes in HepG2 cells.The highest increase of the expression of mRNA levels was determined in Hrk (Harakiri) at 4000 µg/mL.

Bioengineering bacterial encapsulin nanocompartments as targeted drug delivery system.

The development of Drug Delivery Systems (DDS) has led to increasingly efficient therapies for the treatment and detection of various diseases. DDS use a range of nanoscale delivery platforms produced from polymeric of inorganic materials, such as micelles, and metal and polymeric nanoparticles, but their variant chemical composition make alterations to their size, shape, or structures inherently complex. Genetically encoded protein nanocages are highly promising DDS candidates because of their modular composition, ease of recombinant production in a range of hosts, control over assembly and loading of cargo molecules and biodegradability. One example of naturally occurring nanocompartments are encapsulins, recently discovered bacterial organelles that have been shown to be reprogrammable as nanobioreactors and vaccine candidates. Here we report the design and application of a targeted DDS platform based on the Thermotoga maritima encapsulin reprogrammed to display an antibody mimic protein called Designed Ankyrin repeat protein (DARPin) on the outer surface and to encapsulate a cytotoxic payload. The DARPin9.29 chosen in this study specifically binds to human epidermal growth factor receptor 2 (HER2) on breast cancer cells, as demonstrated in an in vitro cell culture model. The encapsulin-based DDS is assembled in one step in vivo by co-expressing the encapsulin-DARPin9.29 fusion protein with an engineered flavin-binding protein mini-singlet oxygen generator (MiniSOG), from a single plasmid in Escherichia coli. Purified encapsulin-DARPin_miniSOG nanocompartments bind specifically to HER2 positive breast cancer cells and trigger apoptosis, indicating that the system is functional and specific. The DDS is modular and has the potential to form the basis of a multi-receptor targeted system by utilising the DARPin screening libraries, allowing use of new DARPins of known specificities, and through the proven flexibility of the encapsulin cargo loading mechanism, allowing selection of cargo proteins of choice.

Peculiarities of Immune Regulation in Men of Perm Region with Atherosclerosis Related to Polymorphism of Candidate Genes.

The study examined peculiarities of immune regulation and associated polymorphic variants of candidate genes in men with atherosclerosis in Perm region. The revealed deficiency of CD127 lymphocytes and Annexin V-FITC+7AAD- cells, as well as enhanced level of CD3+CD4+ lymphocytes against the background of variant alleles of candidate genes FAS (rs1159120), CPOX (rs1131857) and wild-type alleles SULT1A1 (rs9282861), MMP9 (rs17576) are responsible for peculiar features of hereditary determination and pathogenesis of atherosclerosis in examined sample (p<0.05). The genetically determined degradation of extracellular matrix in vascular wall and implication of regulated Fas/APO1 apoptosis in the development of progressive atherosclerotic lesions indicate important role of immune system in atherogenesis. The revealed immunological and genetic features are recommended as the markers for early diagnosis of atherosclerosis and its prevention in men of Perm region.

Induction of cell death and modulation of Annexin A1 by phytoestrogens in human leukemic cell lines.

BACKGROUND: Phytoestrogens are polyphenolic plant compounds which are structurally similar to the endogenous mammalian estrogen, 17ß-estradiol. Annexin A1 (ANXA1) is an endogenous protein which inhibits cyclo-oxygenase 2 (COX-2) and phospholipase A2, signal transduction, DNA replication, cell transformation, and mediation of apoptosis. OBJECTIVE: This study aimed to determine the effects of selected phytoestrogens on annexin A1 (ANXA1) expression, mode of cell death and cell cycle arrest in different human leukemic cell lines. METHODS: Cells viability were examined by MTT assay and ANXA1 quantification via Enzyme-linked Immunosorbent Assay. Cell cycle and apoptosis were examined by flow cytometer and phagocytosis effect was evaluated using haematoxylin-eosin staining. RESULTS: Coumestrol significantly (p < 0.05) reduced the total level of ANXA1 in both K562 and U937 cells and genistein significantly (p < 0.05) reduced it in K562, Jurkat and U937 cells, meanwhile estradiol and daidzein induced similar reduction in U937 and Jurkat cells. Coumestrol and daidzein induced apoptosis in K562 and Jurkat cells, while genistein and estradiol induced apoptosis in all tested cells. Coumestrol and estradiol induced cell cycle arrest at G2/M phase in K562 and Jurkat cells with an addition of U937 cells for estradiol. Genistein induced cell cycle arrest at S phase for both K562 and Jurkat cells. However, daidzein induced cell cycle arrest at G0/G1 phase in K562, and G2/M phase of Jurkat cells. Coumestrol, genistein and estradiol induced phagocytosis in all tested cells but daidzein induced significant (p < 0.05) phagocytosis in K562 and Jurkat cells only. CONCLUSION: The selected phytoestrogens induced cell cycle arrest, apoptosis and phagocytosis and at the same time they reduced ANXA1 level in the tested cells. The IC50 value of phytoestrogens was undetectable at the concentrations tested, their ability to induce leukemic cells death may be related with their ability to reduce the levels of ANXA1. These findings can be used as a new approach in cancer treatment particularly in leukemia.

In Vitro Anticancer Evaluation of Some Synthesized 2H-Quinolinone and Halogenated 2H-Quinolinone Derivatives as Therapeutic Agents.

BACKGROUND: Searching for new cytotoxic agents with apoptosis induction may represent a viable strategy for cancer treatment to overcome the increased resistance to available anticancer agents. OBJECTIVE: The purpose of the current study was aimed at preparation and anticancer evaluation of two new series of 2H-quinolinone and halogenated 2H-quinolinone derivatives against two cancer cell lines. METHODS: Two new series of 2H-quinolinone and halogenated 2H-quinolinone derivatives were prepared and screened for their cytotoxicity against breast MCF-7 and liver HepG-2 cancer cell lines as well as normal breast MCF-10a. RESULTS: The tested molecules revealed good cytotoxicity and selectivity toward cancer cell lines relative to normal cells. These compounds were analyzed by DNA flow cytometry on MCF-7 cells. They were found to cause G2/M phase arrest and induced apoptosis at the pre-G1 phase. In addition, increased caspase 3/7 activity and decreased osteopontin expression verified the apoptotic activity. CONCLUSION: The potent compounds discovered in this study can be a hit for the discovery of new cytotoxic agents and are worthy of further investigation.

The synergistic antitumour effect of multi-components from Pulsatilla chinensis saponins in NCI-H460 lung cancer cell line through induction of apoptosis.

Context: Pulsatilla chinensis (Bunge) Regel (Ranunculaceae) possess antitumour effects; however, its antitumour potential has not been extensively investigated.Objective: To investigate the synergetic effect of multi-components from P. chinensis induced cell apoptosis and explore the mechanism.Materials and methods: The cytotoxicity was measured against NCI-H460, SMMC-7721, HCT-116 and U251 cell lines treated with eight monomers from P. chinensis. The synergetic effect of a combination of Pulsatilla saponin D (PSD), Raddeanoside R13 (R13), and Pulsatilla saponin A (PSA) was assessed using CalcuSyn3.0. Annexin V-FITC/PI and DAPI staining analyzed apoptosis of NCI-H460 cells treated with PSD, R13 and PSA alone or in combination. Proteins differential expression was analyzed using proteomic, DAVID Bioinformatics Resources, R software environment and KEGG database, and verified by western blotting.Results: PSD, R13, and PSA displayed greater antitumor activity with IC50 values of 5.6, 5.1 and 10.5 µM against NCI-H460 cells compared with other monomers. The combination of PSD, R13, and PSA had a synergistic effect at CI = 0.27 and induced 17.53% cells apoptotic detected by flow cytometric. Bioinformatic analysis showed an overview of the differentially expressed proteins and some signalling pathways. Moreover, some candidate proteins (LDHA, PI3K, NOL3 and cleaved-caspase-3) were validated by western blotting.Discussion and Conclusion: These results show PSD, R13, and PSA are good candidates as natural products for use in the treatment of lung cancer. Potential signalling pathways and protein targets need to be further validated. The application of the drug combination approach also provides a therapeutic strategy for cancer.

Immobilization of Phosphatidylserine by Ethanol and Lysozyme on the Cell Surface for Evaluation of Apoptosis-Like Decay in Activated-Sludge Bacteria.

Accurate determination of microbial viability can be crucial in microbe-dominated biosystems. However, the identification of metabolic decay in bacterial cells can be elaborate and difficult. We sought to identify apoptosis-like bacterial processes by using annexin V-fluorescein isothiocyanate (FITC) (AVF), a probe typically used to stain phosphatidylserine (PS) on exposed cell membranes. The bacterial cell wall provides a barrier that is responsible for low efficiency of direct PS staining of decayed bacterial cells. This can be overcome by pretreatment of the bacteria with 70% ethanol, which fixates the bacteria and preserves the PS status, combined with lysozyme treatment to hydrolyze the cell wall. That treatment improved the efficiency of AVF staining considerably, as shown for pure strains of an Ochrobactrum sp. and a Micrococcus sp. Using this method, decayed bacterial cells (induced by starvation) were more strongly stained, indicating externalization of PS to a greater extent than seen for cells harvested at logarithmic growth. A multispecies microbial sludge was artificially decayed by heat treatment or alternating anoxic-oxic treatment, which also induced increased AVF staining, again presumably via decay-related PS externalization. The method developed proved to be efficient for identification of bacterial decay and has potential for the evaluation of multispecies bacterial samples from sources like soil matrix, bioaerosol, and activated sludge.IMPORTANCE Since the externalization of phosphatidylserine (PS) is considered a crucial characteristic of apoptosis, we sought to identify apoptosis-like decay in bacterial cells by PS staining using AVF. We show that this is possible, provided the bacteria are pretreated with ethanol plus lysozyme to remove a physical staining barrier and preserve the original, decay-related externalization of PS. Our work suggests that PS externalization occurs in starved bacteria and this can be quantified with AVF staining, providing a measure of bacterial decay. Since PS is the common component of the lipid bilayer in bacterial cell membranes, this approach also has potential for evaluation of cell decay of other bacterial species.

Role of HGF/c-Met in the treatment of colorectal cancer with liver metastasis.

The system of hepatocyte growth factor (HGF) and its receptor c-Met plays a critical role in tumor invasive growth and metastasis. The mortality rate of colorectal cancer (CRC), one of the most commonly diagnosed malignancies, is increased by it gradual development into metastasis, most frequently in the liver. Overexpression of c-Met, the protein tyrosine kinase receptor for the HCF/scatter factor, has been implicated in the progression and metastasis of human colorectal carcinoma. In this study, we aimed to investigate the role of c-Met in CRC liver metastasis and illustrate the clinical impact of regulating HGF/c-Met signaling in patients with CRC liver metastasis. We found that (I) higher levels of c-Met expression (mRNA and Protein) in CRC liver metastasis than primary CRC by assessing the patient tissue samples; (II) a positive correlation of c-Met expression with tumor stages of CRC liver metastasis, as well as c-Met expression in CRC, live metastasis concurred with regional lymph node metastasis; (III) the clinical impact of downregulation of HGF/c-Met signaling on the reduction of proliferation and invasion in CRC liver metastasis. Therefore, we demonstrate that the regulation of HGF/c-Met pathways may be a promising strategy in the treatment of patients with CRC liver metastasis.

Ex Vivo Pharmacological Profiling in Chronic Lymphocytic Leukemia Cells.

In vitro drug combination studies are commonly used for CLL primary lymphocytes. An advancement in this method is to perform ex vivo drug testing where the first agent is administered to patients and second drug is tested in these patients' cells in vitro. These assays have been effective in identifying novel agents that work additively or synergistically. In this chapter, we provide a step-by-step protocol for ex vivo drug testing that can be used for combination strategies.

Dicranopteris linearis extract inhibits the proliferation of human breast cancer cell line (MDA-MB-231) via induction of S-phase arrest and apoptosis.

CONTEXT: Dicranopteris linearis (Burm.f.) Underw. (Gleicheniaceae) has been scientifically proven to exert various pharmacological activities. Nevertheless, its anti-proliferative potential has not been extensively investigated. OBJECTIVE: To investigate the anti-proliferative potential of D. linearis leaves and determine possible mechanistic pathways. MATERIALS AND METHODS: MTT assay was used to determine the cytotoxic effects of D. linearis methanol (MEDL) and petroleum ether (PEEDL) extracts at concentrations of 100, 50, 25, 12.5, 6.25 and 3.125 µg/mL against a panel of cancer cell lines (breast [MCF-7 and MDA-MB-231], cervical [HeLa], colon [HT-29], hepatocellular [HepG2] and lung [A549]), as compared to negative (untreated) and positive [5-fluorouracil (5-FU)-treated] control groups. Mouse fibroblast cells (3T3) were used as normal cells. The mode of cell death was examined using morphological analysis via acridine orange (AO) and propidium iodide (PI) double staining. Cell cycle arrest was determined using flow cytometer, followed by annexin V-PI apoptosis detection kit. RESULTS: MEDL demonstrated the most significant growth inhibition against MDA-MB-231 cells (IC50 22.4 µg/mL). PEEDL showed no cytotoxic effect. Induction of apoptosis by MEDL was evidenced via morphological analysis and acridine orange propidium iodide staining. MEDL could induce S phase cell cycle arrest after 72 h of incubation. Early apoptosis induction in MDA-MB-231 cells was confirmed by annexin V-FITC and PI staining. Significant increase in apoptotic cells were detected after 24 h of treatment with 15.07% cells underwent apoptosis, and the amount escalated to 18.24% with prolonged 48 h incubation. CONCLUSIONS: MEDL has potential as a potent cytotoxic agent against MDA-MB-231 adenocarcinoma.

Detection of Apoptosis-like Cell Death in Ustilago maydis by Annexin V-FITC Staining.

Programmed cell death (PCD) guides the transition between key developmental stages in many organisms. PCD also remains an important fate for many organisms upon exposure to different stress conditions. Therefore, an insight into the progression of PCD during the execution of a biological phenomenon can yield significant details of the underlying mechanism. Apoptosis, as well as apoptosis-like programmed cell death, constitutes one of the forms of PCD in higher and lower eukaryotes respectively. Flipping of phosphatidylserine (PS) from the inner leaflet of the plasma membrane to the outer leaflet is among the different hallmarks of apoptosis/apoptosis-like PCD that marks the initiation of the said cell death event. This flipping can be detected through staining of the target cells using annexin V-FITC that binds specifically to PS. In Ustilago maydis the staining of the externally exposed PS by annexin V-FITC is difficult due to the presence of cell wall. The key to such staining, therefore, relies on the gentle removal of the cell wall without significantly altering the underlying plasma membrane architecture/topology. This protocol highlights the dependence of the PS staining on the extent of protoplastation of the stressed cells in Ustilago maydis.

Cytotoxic effect of Cyperus rotundus rhizome extract on human cancer cell lines.

The wild weed Cyperus rotundus is commonly used as traditional medicine in different parts of the world. Sequential extraction of C. rotundus rhizome with solvents of different polarity namely hexane, chloroform, ethyl acetate, methanol and water were prepared and the free radical scavenging activity was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Based on high antioxidant activity of methanolic extract of C. rotundus rhizome (MRCr) was further investigated for its cytotoxic effect on different human cancer cell lines-breast (MCF-7), cervical (HeLa), liver (Hep G2), prostate (PC-3), colorectal (HT-29) and normal cell line (MCF-12A) by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay evaluated as 50% inhibition of growth (IC50). Apoptosis cells were analysed by flow cytometry stained with annexin V-Fluorescein isothiocyanate conjugate (AF) and propidium iodide (PI). The cellular and nuclear changes were examined under light and fluorescent microscope using 4', 6' diamino-2-phenylindole (DAPI) stain, dual stains of AF/PI and acridine orange/ethidium bromide (AO/EB). The cytotoxic effects on the tested cancer cell lines ranged from 4.52±0.57 to 9.85±0.68µgml-1. The migration assay was showed the inhibitory effect with MRCr. The MRCr showed significant anticancer activity against all the tested cancer cell lines and also protected the non-cancer cells. The anticancer activity suggests further elucidation for the formulation of natural pharmaceutical products in the treatment of cancer.

2-aryl benzimidazole conjugate induced apoptosis in human breast cancer MCF-7 cells through caspase independent pathway.

Apoptosis is a representative form of programmed cell death, which has been assumed to be critical for cancer prevention. Thus, any agent that can induce apoptosis may be useful for cancer treatment and apoptosis induction is arguably the most potent defense against cancer promotion. In our previous studies, 2-aryl benzimidazole conjugates were synthesized and evaluated for their antiproliferative activity and one of the new molecule (2f) was considered as a potential lead. This lead molecule showed significant antiproliferative activity against human breast cancer cell line, MCF-7. The results of the present study revealed that this compound arrested the cell cycle at G2/M phase. Topoisomerase II inhibition assay and Western blot analysis suggested that this compound effectively inhibits topoisomerase II activity which leads to apoptotic cell death. Apoptosis induction in MCF-7 cells was further confirmed by loss of mitochondrial membrane potential (∆Ψm), release of cytochrome c from mitochondria, an increase in the level of apoptosis inducing factor (AIF), generation of reactive oxygen species (ROS), up regulation of proapoptotic protein Bax and down regulation of anti apoptotic protein Bcl-2. Apoptosis assay using Annexin V-FITC assay also suggested that this compound induced cell death by apoptosis. However, compound 2f induced apoptosis could not be reversed by Z-VAD-FMK (a pan-caspase inhibitor) demonstrated that the 2f induced apoptosis was caspase independent. Further, 2f treatment did not activate caspase-7 and caspase-9 activity, suggesting that this compound induced apoptosis in breast cancer cells via a caspase independent pathway. Most importantly, this compound was less toxic towards non-tumorigenic breast epithelial cells, MCF-10A. Furthermore, docking studies also support the potentiality of this molecule to bind to the DNA topoisomerase II.

Design, synthesis of phenstatin/isocombretastatin-oxindole conjugates as antimitotic agents.

A series of phenstatin/isocombretastatin-oxindole conjugates was synthesized and tested for their cytotoxic activity against five human cancer cells such as prostate (DU-145), lung (A549), colon (HT-29), breast (MCF-7), liver (HepG2) cancer cells with IC50 values ranging from 0.049 to 38.90 µM. Amongst them, two conjugates (5c and 5d) showed broad spectrum of antiproliferative efficacy on lung cancer cells with an IC50 value of 79 nM and 93 nM, respectively, whereas on colon cancer cells with an IC50 values 45 nM and 49 nM, respectively. In addition, cell cycle assay revealed that these conjugates (5c and 5d) arrest at the G2/M phase and leads to apoptotic cell death which was confirmed by Annexin V-FITC and mitochondrial membrane depolarization. Further, the tubulin polymerization assay analysis results suggest that these conjugates particularly 5c and 5d exhibit significant inhibitory effect on the tubulin assembly with an IC50 value of 1.23 µM and 1.01 µM, respectively. Molecular docking studies indicated that these compounds (5c and 5d) occupy the colchicine binding site of the tubulin.