Aims
Zinc oxide nanoparticles (ZnONPs) were successfully synthesized using the culture filtrate of the endophytic fungus Alternaria tenuissima as a rapid, eco‐friendly and cost‐effective method.
Methods and Results
The rapid synthesis of ZnONPs was completed after 20 min as confirmed by UV–Vis spectroscopy. The synthesized ZnONPs showed a single‐phase crystalline structure. Dynamic light scattering analysis showed that the synthesized ZnONPs were monodispersed and the recorded polydispersity index value was 0·311. Zeta potential value of –23·92 mV indicated the high stability of ZnONPs. Transmission electron microscope revealed the spherical shape and the mean particle size was 15.45 nm. Functional groups present in the prepared samples of ZnONPs were confirmed by Fourier transform infrared spectroscopy. Additionally, the biological activities of in vitro antimicrobial, anticancer, antioxidant as well as the photocatalytic activities were evaluated. ZnONPs showed broad spectrum of antimicrobial potential against all the tested plant and human pathogens. Based on the MTT assay, ZnONPs inhibited the proliferation of normal human melanocytes, human breast and liver cancer cell lines with IC50 concentrations of 55·76, 18·02 and 16·87 µg ml−1. ZnONPs exhibited promising antioxidant potential with 50% inhibitory concentration of 102·13 µg ml−1. Moreover, ZnONPs showed efficient degradation of methylene blue dye.
Conclusions
The synthesized ZnONPs showed promising activities that can be better explored in the near future for many medical, agricultural and industrial applications.
Significance and Impact of the Study
This study suggests a new and alternate approach with the excellent biotechnological potentiality for the production of ZnONPs which could open up the way for the industrial manufacture of nanoparticles using microbial platforms.
Repeated cycles of antibody-directed enzyme pro-drug therapy (ADEPT) and the use of glucarpidase in the detoxification of cytotoxic methotrexate (MTX) are highly desirable during cancer therapy but are hampered by the induced human antibody response to glucarpidase. Novel variants of glucarpidase (formal name: carboxypeptidase G2, CPG2) with epitopes not recognized by the immune system are likely to allow repeated cycles of ADEPT for effective cancer therapy. Towards this aim, over two thousand soil samples were collected and screened for folate hydrolyzing bacteria using folate as the sole carbon source. The work led to the isolation and the characterization of three new glucarpidase producing strains, which were designated as: Pseudomonas lubricans strain SF168, Stenotrophomonas sp SA and Xenophilus azovorans SN213. The CPG2 genes of Xenophilus azovorans SN213 (named Xen CPG2) and Stenotrophomonas sp SA (named Sten CPG2) were cloned and molecularly characterized. Both Xen CPG2 and Sten CPG2 share very close amino acid sequences (99%); we therefore, focused on the study of Xen CPG2. Finally, we demonstrated that a polyclonal antibody raised against our new CPG2, Xen CPG2, does not react with the CPG2 from Pseudomonas sp. strain RS-16 (Ps CPG2) that are currently in clinical use. The two enzymes, therefore could potentially be used consecutively in the ADEPT protocol to minimize the effect of the human antibody response that hampers current treatment with Ps CPG2. The identified novel CPG2 in this study will, therefore, pave the way for safer antibody directed enzyme pro-drug therapy for cancer treatment.
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Autophagy functions in both selective and non‐selective ways to maintain cellular homeostasis. Endoplasmic reticulum autophagy (ER‐phagy) is a subclass of autophagy responsible for the degradation of the endoplasmic reticulum through selective encapsulation into autophagosomes. ER‐phagy occurs both under physiological conditions and in response to stress cues, and plays a crucial role in maintaining the homeostatic control of the organelle. Although specific receptors that target parts of the ER membrane, as well as, internal proteins for lysosomal degradation have been identified, the molecular regulation of ER‐phagy has been elusive. Recent work has uncovered novel regulators of ER‐phagy that involve post‐translational modifications of ER‐resident proteins and functional cross‐talk with other cellular processes. Herein, we discuss how morphology affects the function of the peripheral ER, and how ER‐phagy modulates the turnover of this organelle. We also address how ER‐phagy is regulated at the molecular level, considering implications relevant to human diseases.
Background
Glufosfamide (β-d-glucosylisophosphoramide mustard, GLU) is an alkylating cytotoxic agent in which ifosforamide mustard (IPM) is glycosidically linked to the β-d-glucose molecule. GLU exerted its cytotoxic effect as a targeted chemotherapy. Although, its cytotoxic efficacy in a number of cell lines, there were no experimental or clinical data available on the oncolytic effect of oxazaphosphorine drugs in hepatocellular carcinoma. Therefore, the main objective of the current study is to assess the cytotoxic potential of GLU for the first time in the hepatocellular carcinoma HepG2 cell line model.
Methods
Cytotoxicity was assayed by the MTT method, and half-maximal inhibitory concentration (IC50) was calculated. Flow cytometric analysis of apoptosis frequencies was measured by using Annexin V/PI double stain, an immunocytochemical assay of caspase-9, visualization of caspase-3, and Bcl2 gene expression were undertaken as apoptotic markers. Mitochondrial membrane potential was measured using the potentiometric dye; JC-1, as a clue for early apoptosis as well as ATP production, was measured by the luciferase-chemiluminescence assay.
Results
Glufosfamide induced cytotoxicity in HepG2 cells in a concentration- and time-dependent manner. The IC50 values for glufosfamide were significantly lower compared to ifosfamide. The frequency of apoptosis was much higher for glufosfamide than that of ifosfamide. The contents of caspase-9 and caspase-3 were elevated following exposure to GLU more than IFO. The anti-apoptotic Bcl2 gene expression, the mitochondrial membrane potential, and the cellular ATP levels were significantly decreased than in case of ifosfamide.
Conclusions
The current study reported for the first time cytotoxicity activity of glufosfamide in HepG2 cells in vitro. The obtained results confirmed the higher oncolytic activity of glufosfamide than its aglycone ifosfamide. The generated data warrants further elucidations by in vivo study.
Two novel copper (II) complexes [Cu(TFP)(Gly)Cl] • 2H 2 O complex (1) and [Cu(TFP)(His)Cl] • 2H 2 O complex (2) are synthesized, where TFP stands for trifluropromazine, Gly. represents glycine, and His. is histidine. Chemical composition, IR, mass spectra, and magnetic susceptibility tests are performed. Complex binding with macromolecules was investigated using UV-vis, viscosity, gel electrophoresis, and fluorescence quenching. Fluorescence spectroscopy revealed that each complex could replace ethidium bromide (EB). These complexes exhibit grooved, non-covalent, and electrostatic interactions with CT-DNA. Spectroscopy analysis of the BSA interaction showed that complexes bind to protein (K b values for (1) is 5.89 × 10 3 M À 1 and for (2) is 9.08 × 10 3 M À 1 ) more strongly than CT-DNA (K b values for (1) is 5.43 × 10 3 M À 1 and for (2) is 7.17 × 10 3 M À 1 ). Molecular docking analysis and spectral absorption measurements showed high agreement. Antimicrobial, antioxidant, and anti-inflammatory properties were tested in vitro. The druggability of complex (2) should be tested in vivo as it is more biologically active.
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