Anticancer Properties of Platinum Nanoparticles and Retinoic Acid: Combination Therapy for the Treatment of Human Neuroblastoma Cancer.

Neuroblastoma is the most common extracranial solid tumor in childhood. The different treatments available for neuroblastoma are challenged by high rates of resistance, recurrence, and progression, most notably in advanced cases and highly malignant tumors. Therefore, the development of more targeted therapies, which are biocompatible and without undesired side effects, is highly desirable. The mechanisms of actions of platinum nanoparticles (PtNPs) and retinoic acid (RA) in neuroblastoma have remained unclear. In this study, the anticancer effects of PtNPs and RA on neuroblastoma were assessed. We demonstrated that treatment of SH-SY5Y cells with the combination of PtNPs and RA resulted in improved anticancer effects. The anticancer effects of the two compounds were mediated by cytotoxicity, oxidative stress (OS), mitochondrial dysfunction, endoplasmic reticulum stress (ERS), and apoptosis-associated networks. Cytotoxicity was confirmed by leakage of lactate dehydrogenase (LDH) and intracellular protease, and oxidative stress increased the level of reactive oxygen species (ROS), 4-hydroxynonenal (HNE), malondialdehyde (MDA), and nitric oxide (NO), and protein carbonyl content (PCC). The combination of PtNPs and RA caused mitochondrial dysfunction by decreasing the mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content, number of mitochondria, and expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Endoplasmic reticulum-mediated stress and apoptosis were confirmed by upregulation of protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), activating transcription factor 4 (ATF4), p53, Bax, and caspase-3 and down regulation of B-cell lymphoma 2 (BCl-2). PtNPs and RA induced apoptosis, and oxidative DNA damage was evident by the accumulation of 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG). Finally, PtNPs and RA increased the differentiation and expression of differentiation markers. Differentiated SH-SY5Y cells pre-treated with PtNPs or RA or the combination of both were more sensitive to the cytotoxic effect of cisplatin than undifferentiated cells. To our knowledge, this is the first study to demonstrate the effect of the combination of PtNPs and RA in neuroblastoma cells. PtNPs may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment. The results of this study provide a rationale for clinical evaluation of the combination of PtNPs and RA for the treatment of children suffering from high-risk neuroblastoma.

Melatonin Enhances Palladium-Nanoparticle-Induced Cytotoxicity and Apoptosis in Human Lung Epithelial Adenocarcinoma Cells A549 and H1229.

Palladium nanoparticles (PdNPs) are increasingly being used in medical and biological applications due to their unique physical and chemical properties. Recent evidence suggests that these nanoparticles can act as both a pro-oxidant and as an antioxidant. Melatonin (MLT), which also shows pro- and antioxidant properties, can enhance the efficacy of chemotherapeutic agents when combined with anticancer drugs. Nevertheless, studies regarding the molecular mechanisms underlying the anticancer effects of PdNPs and MLT in cancer cells are still lacking. Therefore, we aimed to investigate the potential toxicological and molecular mechanisms of PdNPs, MLT, and the combination of PdNPs with MLT in A549 lung epithelial adenocarcinoma cells. We evaluated cell viability, cell proliferation, cytotoxicity, oxidative stress, mitochondrial dysfunction, and apoptosis in cells treated with different concentrations of PdNPs and MLT. PdNPs and MLT induced cytotoxicity, which was confirmed by leakage of lactate dehydrogenase, increased intracellular protease, and reduced membrane integrity. Oxidative stress increased the levels of reactive oxygen species (ROS), malondialdehyde (MDA), nitric oxide (NO), protein carbonyl content (PCC), lipid hydroperoxide (LHP), and 8-isoprostane. Combining PdNPs with MLT elevated the levels of mitochondrial dysfunction by decreasing mitochondrial membrane potential (MMP), ATP content, mitochondrial number, and expression levels of the main regulators of mitochondrial biogenesis. Additionally, PdNPs and MLT induced apoptosis and oxidative DNA damage due to accumulation of 4-hydroxynonenal (HNE), 8-oxo-2'-deoxyguanosine (8-OhdG), and 8-hydroxyguanosine (8-OHG). Finally, PdNPs and MLT increased mitochondrially mediated stress and apoptosis, which was confirmed by the increased expression levels of apoptotic genes. To our knowledge, this is the first study demonstrating the effects of combining PdNPs and MLT in human lung cancer cells. These findings provide valuable insights into the molecular mechanisms involved in PdNP- and MLT-induced toxicity, and it may be that this combination therapy could be a potential effective therapeutic approach. This combination effect provides information to support the clinical evaluation of PdNPs and MLT as a suitable agents for lung cancer treatment, and the combined effect provides therapeutic value, as non-toxic concentrations of PdNPs and MLT are more effective, better tolerated, and show less adverse effects. Finally, this study suggests that MLT could be used as a supplement in nano-mediated combination therapies used to treat lung cancer.

Copy number variation of PR-39 cathelicidin, and identification of PR-35, a natural variant of PR-39 with reduced mammalian cytotoxicity.

Proline-arginine-rich (PR)-39 is neutrophil antimicrobial peptide that has potent antimicrobial activity against a broad spectrum of microorganisms, including bacteria, fungi, and some enveloped viruses as a part of the innate immune system. We analyzed the nucleotide sequence variations of PR-39 exon 4, which is the mature peptide region responsible for antimicrobial activity, from 48 pigs of six breeds using sequence-based typing. The analysis identified four alleles including allele PR-35 with a 12-bp deletion near the N-terminus. Interestingly, 16.7% of individuals showed the presence of three alleles per individual, but only in the Berkshire and Duroc breeds. We further analyzed the genetic diversity of PR-39 for the entire genomic region of the gene from PR-39 exon 1 to the 3' untranslated region for different alleles by PCR amplification and cloning. The antimicrobial activity of chemically synthesized PR-35 was similar to that of PR-39, but the level of mammalian cell cytotoxicity was lower than the wild type. Better knowledge of the genetic diversity of PR-39 among different individuals and breeds may contribute to improved immune defense of pigs. PR-35, as a natural antimicrobial peptide variant, could be an interesting candidate for the development of peptide antibiotics.

Antibacterial Efficacy of Silver Nanoparticles on Endometritis Caused by Prevotella melaninogenica and Arcanobacterum pyogenes in Dairy Cattle.

Bovine postpartum diseases remain one of the most significant and highly prevalent illnesses with negative effects on the productivity, survival, and welfare of dairy cows. Antibiotics are generally considered beneficial in the treatment of endometritis; however, frequent usage of each antibiotic drug is reason for the emergence of multidrug resistance (MDR) of the pathogenic microorganisms, representing a major impediment for the successful diagnosis and management of infectious diseases in both humans and animals. We synthesized silver nanoparticles (AgNPs) with an average size of 10 nm using the novel biomolecule apigenin as a reducing and stabilizing agent, and evaluated the efficacy of the AgNPs on the MDR pathogenic bacteria Prevotella melaninogenica and Arcanobacterium pyogenes isolated from uterine secretion samples. AgNPs inhibited cell viability and biofilm formation in a dose- and time-dependent manner. Moreover, the metabolic toxicity of the AgNPs was assessed through various cellular assays. The major toxic effect of cell death was caused by an increase in oxidative stress, as evidenced by the increased generation of reactive oxygen species (ROS), malondialdehyde, protein carbonyl content, and nitric oxide. The formation of ROS is considered to be the primary mechanism of bacterial death. Therefore, the biomolecule-mediated synthesis of AgNPs shows potential as an alternative antimicrobial therapy for bovine metritis and endometritis.

The cytotoxic effects of dimethyl sulfoxide in mouse preimplantation embryos: a mechanistic study.

Rationale: Dimethyl sulfoxide (DMSO) is commonly used as a solvent for water-insoluble substances, a vehicle for drug therapy, and a cryoprotectant for cultured cells. DMSO induced embryonic defects and its mechanism of action remains unclear. The rationale is based on the assumption that DMSO supplementation should induce long-term negative effects on both pre- and post-implantation embryo development. Methods: DMSO induced oxidative stress, ER stress, autophagy, mitophagy, signaling responsible genes and proteins were determined by RT-qPCR, Western blotting, immunofluorescence, and confocal microscopy. DMSO induced mitochondrial dysfunction was measured by transmission electron microcopy and JC-1 assay. Apoptosis was estimated using TUNEL and comet assay. Post-implantation embryo developmental capability was estimated by implantation site and fetus numbers. Results: Exposure to DMSO induced an early oxidative stress response within 0.5 to 2 h in 1-cell zygotes by disrupting the balance of pro- and anti-oxidants. Notably, DMSO-treated 2-cell embryos showed increased expression of unfolded protein response genes such as Hspa5, Hsp90b1, Ddit3, Atf4, and Xbp1. As a result, the development of many embryos is arrested at the 2-cell, 4-cell, or morula stages in a dose-dependent manner. Further, DMSO-induced endoplasmic reticulum stress increased mitochondrial Ca2+ levels, induced mitochondrial depolarization/dysfunction, and induced apoptotic cell death via the JNK/ATF2-dependent pathway. Consequently, treatment with DMSO increased the expression of autophagy initiation-, phagophore elongation-, and autophagosome formation-related genes, as well as localization of PINK1/Parkin, which are the main mediators of mitophagy, in mitochondria. Interestingly, DMSO causes cytotoxic effects in preimplantation embryos by inducing extensive mitophagy and autophagy. Especially, DMSO treatment decreased the inner cell mass and trophectoderm cell numbers as well as mRNA expression of B3gnt5 and Wnt3a in developed blastocysts, which decreased the implantation and developmental rates of full-term offspring after being transferred into pseudopregnant mice. Conclusion: These results provide a significant contribution to finding effective protective agents to combat DMSO mediated reproductive toxicity for application in human embryos in the near future.

Biochanin A Ameliorates Arsenic-Induced Hepato- and Hematotoxicity in Rats.

Biochanin A (BCA) is a natural organic compound of the phytoestrogenic isoflavone class that has antioxidant and metal chelator properties in the presence of transition metal ions, however, its efficacy in animal models is still obscure. Therefore, the objective of this study was to investigate the protective effects of BCA against arsenic-induced hepatic injury and hematotoxicity in rats. The results suggest that arsenic intoxicated rats showed significantly higher levels of plasma hepatic markers than normal control rats. Furthermore, an increase in lipid peroxidation with depletion of reduced glutathione (GSH) and activities of superoxide dismutase (SOD) and catalase (CAT) occurred in the livers of rats exposed to arsenic. Administration of BCA (20 mg/kg·bw/day) and selenium (3 mg/kg·bw/day) resulted in a significant reversal of hepatic and oxidative stress markers in arsenic-intoxicated rats. A low dose of BCA (10 mg/kg·bw/day) did not show any preventive effect, while a high dose of BCA (40 mg/kg·bw/day) partially prevented all hepatotoxicity events. These biochemical perturbations were supported by histopathological observations of the liver. Our results suggest that administration of BCA (20 mg/kg·bw/day) attenuated the arsenic hepatotoxicity, a property that could contribute to the therapeutic approaches for chronic liver diseases.

Green Chemistry Approach for Synthesis of Effective Anticancer Palladium Nanoparticles.

The purpose of this study was to design and synthesize Palladium nanoparticles (PdNPs) using an environmentally friendly approach and evaluate the in vitro efficacy of PdNPs in human ovarian cancer A2780 cells. Ultraviolet-Visible (UV-Vis) spectroscopy was used to monitor the conversion of Pd(II) ions to Pd(0)NPs. X-ray diffraction (XRD) revealed the crystallinity of the as-synthesized PdNPs and Fourier transform infrared spectroscopy (FTIR) further confirmed the role of the leaf extract of Evolvulus alsinoides as a reducing and stabilizing agent for the synthesis of PdNPs. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) showed that the average size of the NPs was 5 nm. After a 24-h exposure to PdNPs, cell viability and light microscopy assays revealed the dose-dependent toxicity of the PdNPs. Furthermore, the dose-dependent cytotoxicity of the PdNPs was confirmed by lactate dehydrogenase (LDH), increased reactive oxygen species (ROS) generation, activation of PdNPs-induced autophagy, impairment of mitochondrial membrane potential (MMP), enhanced caspase-3 activity, and detection of TUNEL-positive cells. Our study demonstrates a single, simple, dependable and green approach for the synthesis of PdNPs using leaf extracts of Evolvulus alsinoides. Furthermore, the in vitro efficacy of PdNPs in human ovarian cancer cells suggests that it could be an effective therapeutic agent for cancer therapy.

Reduced graphene oxide-silver nanoparticle nanocomposite: a potential anticancer nanotherapy.

BACKGROUND: Graphene and graphene-based nanocomposites are used in various research areas including sensing, energy storage, and catalysis. The mechanical, thermal, electrical, and biological properties render graphene-based nanocomposites of metallic nanoparticles useful for several biomedical applications. Epithelial ovarian carcinoma is the fifth most deadly cancer in women; most tumors initially respond to chemotherapy, but eventually acquire chemoresistance. Consequently, the development of novel molecules for cancer therapy is essential. This study was designed to develop a simple, non-toxic, environmentally friendly method for the synthesis of reduced graphene oxide-silver (rGO-Ag) nanoparticle nanocomposites using Tilia amurensis plant extracts as reducing and stabilizing agents. The anticancer properties of rGO-Ag were evaluated in ovarian cancer cells. METHODS: The synthesized rGO-Ag nanocomposite was characterized using various analytical techniques. The anticancer properties of the rGO-Ag nanocomposite were evaluated using a series of assays such as cell viability, lactate dehydrogenase leakage, reactive oxygen species generation, cellular levels of malonaldehyde and glutathione, caspase-3 activity, and DNA fragmentation in ovarian cancer cells (A2780). RESULTS: AgNPs with an average size of 20 nm were uniformly dispersed on graphene sheets. The data obtained from the biochemical assays indicate that the rGO-Ag nanocomposite significantly inhibited cell viability in A2780 ovarian cancer cells and increased lactate dehydrogenase leakage, reactive oxygen species generation, caspase-3 activity, and DNA fragmentation compared with other tested nanomaterials such as graphene oxide, rGO, and AgNPs. CONCLUSION: T. amurensis plant extract-mediated rGO-Ag nanocomposites could facilitate the large-scale production of graphene-based nanocomposites; rGO-Ag showed a significant inhibiting effect on cell viability compared to graphene oxide, rGO, and silver nanoparticles. The nanocomposites could be effective non-toxic therapeutic agents for the treatment of both cancer and cancer stem cells.

Comparative assessment of the apoptotic potential of silver nanoparticles synthesized by Bacillus tequilensis and Calocybe indica in MDA-MB-231 human breast cancer cells: targeting p53 for anticancer therapy.

BACKGROUND: Recently, the use of nanotechnology has been expanding very rapidly in diverse areas of research, such as consumer products, energy, materials, and medicine. This is especially true in the area of nanomedicine, due to physicochemical properties, such as mechanical, chemical, magnetic, optical, and electrical properties, compared with bulk materials. The first goal of this study was to produce silver nanoparticles (AgNPs) using two different biological resources as reducing agents, Bacillus tequilensis and Calocybe indica. The second goal was to investigate the apoptotic potential of the as-prepared AgNPs in breast cancer cells. The final goal was to investigate the role of p53 in the cellular response elicited by AgNPs. METHODS: The synthesis and characterization of AgNPs were assessed by various analytical techniques, including ultraviolet-visible (UV-vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The apoptotic efficiency of AgNPs was confirmed using a series of assays, including cell viability, leakage of lactate dehydrogenase (LDH), production of reactive oxygen species (ROS), DNA fragmentation, mitochondrial membrane potential, and Western blot. RESULTS: The absorption spectrum of the yellow AgNPs showed the presence of nanoparticles. XRD and FTIR spectroscopy results confirmed the crystal structure and biomolecules involved in the synthesis of AgNPs. The AgNPs derived from bacteria and fungi showed distinguishable shapes, with an average size of 20 nm. Cell viability assays suggested a dose-dependent toxic effect of AgNPs, which was confirmed by leakage of LDH, activation of ROS, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in MDA-MB-231 breast cancer cells. Western blot analyses revealed that AgNPs induce cellular apoptosis via activation of p53, p-Erk1/2, and caspase-3 signaling, and downregulation of Bcl-2. Cells pretreated with pifithrin-alpha were protected from p53-mediated AgNPs-induced toxicity. CONCLUSION: We have demonstrated a simple approach for the synthesis of AgNPs using the novel strains B. tequilensis and C. indica, as well as their mechanism of cell death in a p53-dependent manner in MDA-MB-231 human breast cancer cells. The present findings could provide insight for the future development of a suitable anticancer drug, which may lead to the development of novel nanotherapeutic molecules for the treatment of cancers.

Ginkgo biloba: a natural reducing agent for the synthesis of cytocompatible graphene.

BACKGROUND: Graphene is a novel two-dimensional planar nanocomposite material consisting of rings of carbon atoms with a hexagonal lattice structure. Graphene exhibits unique physical, chemical, mechanical, electrical, elasticity, and cytocompatible properties that lead to many potential biomedical applications. Nevertheless, the water-insoluble property of graphene restricts its application in various aspects of biomedical fields. Therefore, the objective of this work was to find a novel biological approach for an efficient method to synthesize water-soluble and cytocompatible graphene using Ginkgo biloba extract (GbE) as a reducing and stabilizing agent. In addition, we investigated the biocompatibility effects of graphene in MDA-MB-231 human breast cancer cells. MATERIALS AND METHODS: Synthesized graphene oxide (GO) and GbE-reduced GO (Gb-rGO) were characterized using various sequences of techniques: ultraviolet-visible (UV-vis) spectroscopy, Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), scanning electron microscopy (SEM), atomic force microscopy (AFM), and Raman spectroscopy. Biocompatibility of GO and Gb-rGO was assessed in human breast cancer cells using a series of assays, including cell viability, apoptosis, and alkaline phosphatase (ALP) activity. RESULTS: The successful synthesis of graphene was confirmed by UV-vis spectroscopy and FTIR. DLS analysis was performed to determine the average size of GO and Gb-rGO. X-ray diffraction studies confirmed the crystalline nature of graphene. SEM was used to investigate the surface morphologies of GO and Gb-rGO. AFM was employed to investigate the morphologies of prepared graphene and the height profile of GO and Gb-rGO. The formation of defects in Gb-rGO was confirmed by Raman spectroscopy. The biocompatibility of the prepared GO and Gb-rGO was investigated using a water-soluble tetrazolium 8 assay on human breast cancer cells. GO exhibited a dose-dependent toxicity, whereas Gb-rGO-treated cells showed significant biocompatibility and increased ALP activity compared to GO. CONCLUSION: In this work, a nontoxic natural reducing agent of GbE was used to prepare soluble graphene. The as-prepared Gb-rGO showed significant biocompatibility with human cancer cells. This simple, cost-effective, and green procedure offers an alternative route for large-scale production of rGO, and could be used for various biomedical applications, such as tissue engineering, drug delivery, biosensing, and molecular imaging.

Quantitative analysis of tetracycline-inducible expression of the green fluorescent protein gene in transgenic chickens.

The use of transgenic farm animals as "bioreactors" to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side-effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognized limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet. As a proof of principle study, however, quantitative assessment of expression was not possible, as only one G0 and one G1 transgenic chicken was obtained. In the current study, a sufficient number of G2 and G3 transgenic chickens were obtained, and quantification analysis demonstrated up to a 20-fold induction of expression by doxycycline. In addition, stable transmission of the transgene without any apparent genetic modifications was observed through several generations. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene. Importantly, these results also support the use of the retroviral system for generating transgenic animals with minimal risk in terms of biosafety.

Tetracycline-inducible gene expression in nuclear transfer embryos derived from porcine fetal fibroblasts transformed with retrovirus vectors.

A critical problem of transgenic livestock production is uncontrollable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in transgenic animals. One of the best solutions for this problem may be use of controllable gene expression system. In this study, using retrovirus vectors designed to express the enhanced green fluorescent protein (EGFP) gene under the control of the tetracycline-inducible promoter, we examined whether the expression of the transgene could be controllable in fibroblast cells and nuclear transfer (NT) embryos of porcine. Transformed fibroblast cells were cultured in medium supplemented with or without doxycycline (a tetracycline analog) for 48 hr, and the induction efficiency was measured by comparing EGFP gene expression using epifluorescence microscopy and Western and Northern blot analyses. After the addition of doxycycline, EGFP expression increased up to 17-fold. The nuclei of transformed fibroblast cells were transferred into enucleated oocytes. Fluorescence emission data revealed strong EGFP gene expression in embryos cultured with doxycycline, but little or no expression in the absence of the antibiotic. Our results demonstrate the successful regulation of transgene expression in porcine nuclear transfer embryos, and support the application of an inducible expression system in transgenic pig production to solve the inherent problems of side-effects due to constitutive expression of the transgene.