Proteomic and metabolomic profiling combined with in vitro studies reveal the antiproliferative mechanism of silver nanoparticles in MDA-MB-231 breast carcinoma cells.

Silver nanoparticles, shaped and stabilized by various means, are known to alter biological systems and promote cytotoxicity. However, the precise mechanism by which they induce toxic outcomes in cancer cells is poorly understood. Using a combination of cellular and biophysical assays and proteomic and metabolomic analyses, we report the cytotoxic mechanism of action of tryptone-stabilized silver nanoparticles (T-AgNPs). After their facile synthesis and characterization using an assortment of spectroscopic techniques and transmission electron microscopy, the mechanism of action of the particles was elucidated using MDA-MB-231 breast cancer cells as the cell model. The nanoparticles inhibited the proliferative (IC50:100 ± 3 µg mL-1) and clonogenic potential of the cells. Flow cytometry analyses revealed an absence of phase-specific cell cycle arrest but extensive cell death in the treated cells. The mechanism of action of the particles consisted of their direct binding to the microtubule-building protein tubulin and the disruption of its helical integrity, as confirmed via fluorometric analysis and far-UV spectropolarimetry, respectively. The binding hampered the assembly of microtubules, as confirmed via polymer mass analysis of in vitro assembled, purified tubulin and immunofluorescence imaging of cellular microtubules. Proteomic and metabolomic analyses revealed the downregulation of lipid metabolism to be a synergistic contributor to cell death. Taken together, we report a novel antiproliferative mechanism of action of T-AgNPs that involves tubulin disruption and the downregulation of lipid metabolism.

Cell death mechanisms in eukaryotes.

Like the organism they constitute, the cells also die in different ways. The death can be predetermined, programmed, and cleanly executed, as in the case of apoptosis, or it can be traumatic, inflammatory, and sudden as many types of necrosis exemplify. Nevertheless, there are a number of cell deaths-some of them bearing a resemblance to apoptosis and/or necrosis, and many, distinct from each-that serve a multitude of roles in either supporting or disrupting the homoeostasis. Apoptosis is coordinated by death ligands, caspases, b-cell lymphoma-2 (Bcl-2) family proteins, and their downstream effectors. Events that can lead to apoptosis include mitotic catastrophe and anoikis. Necrosis, although it has been considered an abrupt and uncoordinated cell death, has many molecular events associated with it. There are cell death mechanisms that share some standard features with necrosis. These include methuosis, necroptosis, NETosis, pyronecrosis, and pyroptosis. Autophagy, generally a catabolic pathway that operates to ensure cell survival, can also kill the cell through mechanisms such as autosis. Other cell-death mechanisms include entosis, ferroptosis, lysosome-dependent cell death, and parthanatos.

Tryptone-stabilized gold nanoparticles induce unipolar clustering of supernumerary centrosomes and G1 arrest in triple-negative breast cancer cells.

Gold nanoparticles of different sizes, shapes, and decorations exert a variety of effects on biological systems. We report a novel mechanism of action of chemically modified, tryptone-stabilized gold nanoparticles (T-GNPs) in the triple-negative breast cancer (TNBC) cell line, MDA-MB-231. The T-GNPs, synthesized using HAuCl4.3H2O and tryptone and characterized by an assortment of spectroscopy techniques combined with high-resolution electron microscopy, demonstrated strong antiproliferative and anti-clonogenic potential against MDA-MB-231 cells, arresting them at the G1 phase of the cell cycle and promoting apoptosis. The molecular mechanism of action of these particles involved induction of unipolar clustering and hyper amplification of the supernumerary centrosomes (a distinctive feature of many tumour cells, including TNBC cells). The clustering was facilitated by microtubules with suppressed dynamicity. Mass spectrometry-assisted proteomic analysis revealed that the T-GNP-induced G1 arrest was facilitated, at least in part, by downregulation of ribosome biogenesis pathways. Due to the presence of supernumerary centrosomes in many types of tumour cells, we propose chemical induction of their unipolar clustering as a potential therapeutic strategy.

Cytotoxicity and apoptotic cell death induced by Vitis vinifera peel and seed extracts in A431 skin cancer cells.

Vitis vinifera. L is one of the most widely consumed fruits in the world and are rich in antioxidant abundant polyphenols. The present study was carried out to assess the antiproliferative and apoptotic effects of Vitis vinifera peel and seed extracts in an in vitro model using human epidermoid carcinoma A431 cell lines. Vitis vinifera peel and seed extracts were incubated with A431 cells to evaluate the antiproliferative, apoptotic effects and the morphological apoptotic changes induced by the extracts. Mitochondrial membrane potential was also measured after incubating the cells with extracts. At the inhibitory concentration (IC50), grape seed extract (111.11 µg/mL) and grape peel extract (319.14 µg/mL) were incubated for 24 h with A431 cells. Vitis vinifera peel and seed extracts were able to impart cytotoxic effects, induced apoptosis and apoptotic morphological changes in A431 cells significantly (p < 0.01) and this effect is associated with the interference with mitochondrial membrane potential. This reduction in mitochondrial membrane potential probably initiated the apoptotic cascade in the extracts treated cells. Vitis vinifera peel and seed phytochemicals can selectively target cancer cells and the phytochemicals that are occluded can serve as potential anticancer agents providing better efficacy in killing cancer cells.

Vitis vinifera peel and seed gold nanoparticles exhibit chemopreventive potential, antioxidant activity and induce apoptosis through mutant p53, Bcl-2 and pan cytokeratin down-regulation in experimental animals.

Several studies suggest surface modifications of gold nanoparticles (AuNPs) by capping agents or surface coatings could play an important role in biological systems, and site directed delivery. The present study was carried out to assess the antioxidant and apoptotic activities of the Vitis vinifera peel and seed gold nanoparticles in experimentally induced cancer in Swiss albino mice. 12-dimethylbenz [a] anthracene (DMBA) (single application) and 12-O-tetradecanoylphorbol 13-acetate (TPA) (thrice a week) were applied on the dorsal area of the skin to induce skin papillomagenesis in Swiss albino mice for 16 weeks. Gold nanoparticles were synthesized using Vitis vinifera peel and seed aqueous extracts and characterized by Transmission electron microscopic (TEM) analyses. On topical application, peel and seed gold nanoparticles demonstrated chemopreventive potential by significantly (p<0.05) reducing the cumulative number of tumors while increasing the antioxidant enzyme activities in the gold nanoparticles treated mice. The down-regulated expression of mutant p53, Bcl-2 and the levels of pan-cytokeratins might have facilitated the process of apoptosis in the chemical carcinogenesis process. The results were supported by the histopathological evaluation which exhibited mild dysplasia and acanthosis in the skin tissues of Vitis vinifera peel and seed AuNPs treated mice. Based on the present study, the chemopreventive action of Vitis vinifera peel and seed AuNPs is probably due to its ability to stimulate the antioxidant enzymes within the cells and suppressed abnormal skin cell proliferation that occurred during DMBA-induced skin papillomagenesis.

Synthesis and Characterization of Sygyzium cumini Nanoparticles for Its Protective Potential in High Glucose-Induced Cardiac Stress: a Green Approach.

There exists a complex and multifactorial relationship between diabetes and cardiovascular disease. Hyperglycemia is an important factor imposing damage (glucose toxicity) on cardiac cell leading to diabetic cardiomyopathy. There are substantial clinical evidences on the adverse effects of conventional therapies in the prevention/treatment of diabetic cardiovascular complications. Currently, green-synthesized nanoparticles have emerged as a safe, efficient, and inexpensive alternative for therapeutic uses. The present study discloses the silver nanoparticle biosynthesizing capability and cardioprotective potential of Syzygium cumini seeds already reported to have antidiabetic properties. Newly generated silver nanoparticles S. cumini MSE silver nanoparticles (SmSNPs) were characterized by UV-visible spectroscopy, scanning electron microscopy (SEM), zeta sizer, X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Using methanolic extract of S. cumini seeds, an average size of 40-100-nm nanoparticles with 43.02 nm and -19.6 mV zeta potential were synthesized. The crystalline nature of SmSNPs was identified by using XRD. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assays revealed the antioxidative potential to be 66.87 (±0.7) % and 86.07 (±0.92) % compared to 60.29 (±0.02) % and 85.67 (±1.27) % for S. cumini MSE. In vitro study on glucose-stressed H9C2 cardiac cells showed restoration in cell size, nuclear morphology, and lipid peroxide formation upon treatment of SmSNPs. Our findings concluded that S. cumini MSE SmSNPs significantly suppress the glucose-induced cardiac stress in vitro by maintaining the cellular integrity and reducing the oxidative damages therefore establishing its therapeutic potential in diabetic cardiomyopathy.

Detection and genotyping of high-risk HPV and evaluation of anti-oxidant status in cervical carcinoma patients in Tamil Nadu State, India--a case control study.

Cervical cancer is the second common type of cancer among women worldwide, with the human papillomavirus (HPV) recognized as the major causative agent. The HPV 16/18 prevalance in cervical cancer patients from the Trichy and Coimbatore districts of Tamil Nadu state, India, was evaluated in addition to an assessment of oxidative stress and antioxidant status. MDA, GSH, GPx, GST, SOD, vitamin C and vitamin E were estimated in the plasma and erythrocytes of the twenty patients and an equal number of age matched normal subjects as controls. 119 paraffin embedded tissue samples were collected to perform DNA extraction and genotyping of HPV 16/18 using specific primers. Plasma and erythrocyte TBARS level was significantly elevated in the cervical cancer patients compared to normal. It was observed that SOD, GPx, GSH levels in the erythrocyte and plasma was significantly lower in cervical cancer patients, as well as GST and Vitamins E and C levels in the plasma and catalase enzyme levels in the erythrocytes. Genotyping showed 57% positive for HPV16 and 18% for HPV18, indicating that vaccination against these two will effectively reduce the burden associated with the disease. These findings suggest possible use of antioxidant supplementation as prophylactic agents for prevention and treatment of cervical cancer.

Allele sequencing of the barley stem rust resistance gene Rpg1 identifies regions relevant to disease resistance.

The stem rust resistance gene Rpg1 has protected North American barley cultivars from significant yield losses for over 65 years. The remarkable durability of this gene warrants further study as to its possible origin and allelic variation. Eight Swiss barley (Hordeum vulgare) landraces and eight wild barley (H. vulgare subsp. spontaneum) accessions from diverse geographic regions were analyzed to uncover new alleles of Rpg1 and learn about its possible origin. The two germplasm groups included accessions that were resistant and susceptible to Puccinia graminis f. sp. tritici pathotype MCCF. Allele-specific primers were utilized to amplify 1 kbp overlapping fragments spanning the Rpg1 gene and sequenced if a polymerase chain reaction (PCR) fragment was generated. Variation among the PCR products revealed significant polymorphisms among these Hordeum accessions. Landraces and wild barley accessions susceptible to pathotype MCCF exhibited the highest degree of Rpg1 polymorphism. One resistant landrace (Hv672) and one resistant wild barley accession (WBDC040) yielded all seven Rpg1-specific PCR fragments, but only landrace Hv672 coded for an apparently functional Rpg1 as determined by comparison to previously characterized resistant and susceptible alleles and also resistance to HKHJ, a stem rust pathotype that can specifically detect Rpg1 in the presence of other resistance genes. Accessions resistant to stem rust pathotype MCCF, but completely lacking Rpg1-specific PCR amplification and hybridization with an Rpg1-specific probe, suggested the presence of stem rust resistant gene(s) different from Rpg1 in the Hordeum germplasm pool. Some Rpg1 alleles that retained the ability to autophosphorylate did not confer resistance to Puccinia graminis f. sp. tritici pathotype MCCF, confirming our previous observations that autophosphorylation is essential, but not sufficient for disease resistance. Thus, the RPG1 protein plays a complex role in the stem rust disease resistance-signaling pathway.

The stem rust resistance gene Rpg5 encodes a protein with nucleotide-binding-site, leucine-rich, and protein kinase domains.

We isolated the barley stem rust resistance genes Rpg5 and rpg4 by map-based cloning. These genes are colocalized on a 70-kb genomic region that was delimited by recombination. The Rpg5 gene consists of an unusual structure encoding three typical plant disease resistance protein domains: nucleotide-binding site, leucine-rich repeat, and serine threonine protein kinase. The predicted RPG5 protein has two putative transmembrane sites possibly involved in membrane binding. The gene is expressed at low but detectable levels. Posttranscriptional gene silencing using VIGS resulted in a compatible reaction with a normally incompatible stem rust pathogen. Allele sequencing also validated the candidate Rpg5 gene. Allele and recombinant sequencing suggested that the probable rpg4 gene encoded an actin depolymerizing factor-like protein. Involvement of actin depolymerizing factor genes in nonhost resistance has been documented, but discovery of their role in gene-for-gene interaction would be novel and needs to be further substantiated.

Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley.

Rpg1 is a stem rust resistance gene that has protected barley from severe losses for over 60 years in the US and Canada. It confers resistance to many, but not all, pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici. A fast neutron induced deletion mutant, showing susceptibility to stem rust pathotype Pgt-MCC, was identified in barley cv. Morex, which carries Rpg1. Genetic and Rpg1 mRNA and protein expression level analyses showed that the mutation was a suppressor of Rpg1 and was designated Rpr1 (Required for P. graminis resistance). Genome-wide expression profiling, using the Affymetrix Barley1 GeneChip containing approximately 22,840 probe sets, was conducted with Morex and the rpr1 mutant. Of the genes represented on the Barley1 microarray, 20 were up-regulated and 33 were down-regulated by greater than twofold in the mutant, while the Rpg1 mRNA level remained constant. Among the highly down-regulated genes (greater than fourfold), genomic PCR, RT-PCR and Southern analyses identified that three genes (Contig4901_s_at, HU03D17U_s_at, and Contig7061_s_at), were deleted in the rpr1 mutant. These three genes mapped to chromosome 4(4H) bin 5 and co-segregated with the rpr1-mediated susceptible phenotype. The loss of resistance was presumed to be due to a mutation in one or more of these genes. However, the possibility exists that there are other genes within the deletions, which are not represented on the Barley1 GeneChip. The Rpr1 gene was not required for Rpg5- and rpg4-mediated stem rust resistance, indicating that it shows specificity to the Rpg1-mediated resistance pathway.

The effect of thiourea on ureide metabolism in Neurospora crassa.

The wild-type strain of Neurospora crassa Em 5297a can utilize allantoin as a sole nitrogen source. The pathway of allantoin utilization is via its conversion into allantoic acid and urea, followed by the breakdown of urea to ammonia. This is shown by the inability of the urease-less mutant, N. crassa 1229, to grow on allantoin as a sole nitrogen source and by the formation of allantoate and urea by pre-formed mycelia of this mutant. In the wild strain (Em 5297a) thiourea is tenfold more toxic on an allantoin medium than on an inorganic nitrogen medium; allantoin as well as urea counteract thiourea toxicity in the allantoin nitrogen medium. This selective toxicity of thiourea for the mould utilizing allantoin nitrogen does not, however, result in an impairment of allantoin uptake, allantoinase activity or the formation of urea from allantoin. The only process affected by thiourea is the synthesis of urease; urea antagonizes this effect of thiourea in N. crassa.