Shifting of cell cycle arrest from the S-phase to G2/M phase and downregulation of EGFR expression by phytochemical combinations in HeLa cervical cancer cells.

Cervical cancer is a major human papillomavirus-related disease and is the fourth leading cause of death by cancer among women. Plants are an important source of anticancer compounds and many of them are currently used in the treatment of cancer. Several reports suggest the efficacy of plant-derived compounds increases when used in combination. This study was carried out to evaluate the effect of four plant-derived compounds such as curcumin (C), ellagic acid (E), quercetin (Q), and resveratrol (R) when used alone or in combinations using HeLa cervical cancer cells. All four phytocompounds showed effective cytotoxic activities in targeting HeLa cervical cancer cells as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay. The selected phytocompound combinations C + E, C + Q, and Q + R work synergistically while the combination C + R shows additive effects. All four phytocompounds reduce cell migration as determined by in vitro wound-healing assay. The expression level of the epidermal growth factor receptor is significantly downregulated both in individual and combination. The flow cytometry analysis of cell cycle indicates that individual drugs curcumin, ellagic acid, quercetin, and resveratrol, each with 20 µM effectively arrested cell cycle at the S-phase while the combination of drugs (10 + 10 µM) at the G2/M phase.

Leaf Extract of Nerium oleander L. Inhibits Cell Proliferation, Migration and Arrest of Cell Cycle at G2/M Phase in HeLa Cervical Cancer Cell.

BACKGROUND: Cervical cancer is one of the most common gynaecological malignant tumors reported in women. Although a number of early screening and treatment options are available, mortality due to cervical cancer remains high. Nerium oleander L. is a potential medicinal plant that possesses a wide spectrum of pharmacological and physiological activities including anticancer activities. OBJECTIVE: This study aims to evaluate the antiproliferative activity, inhibition of cell migration and cell cycle arrest by the chloroform extract of leaves of Nerium Oleander L. in HeLa cervical cancer cells. The chloroform extract of Catharanthus roseus which contains anti-cancer compounds, Vinblastin and Vincristin, was used as a positive control for this study. METHODS: The chloroform extracts of Nerium oleander L. and Catharanthus roseus were prepared using the standard protocol. The cytotoxic effects were studied by MTT assay. Cell migration was studied by in vitro scratch assay. Analysis of the cell cycle was carried out by Propidium iodide staining and Flow Cytometry. The expression level of various proteins was evaluated by immunocytochemistry. RESULTS: In this study, we showed that the leaf extract of Nerium oleander inhibited the growth of HeLa cervical cancer cells in culture and inhibited cell migration. Besides, it arrested the cell cycle at the G2/M phase. The Epidermal Growth Factor Receptor (EGFR) expression and phosphorylated p-Rb (Ser 780) level were significantly downregulated by leaf extract of Nerium oleander. CONCLUSION: The extract of Nerium oleander L. contains potential bioactive compounds that inhibit HeLa cell proliferation, cell migration and arrest cell cycle at the G2/M phase.

Multifunctional PEG encapsulated Fe3O4@silver hybrid nanoparticles: antibacterial activity, cell imaging and combined photothermo/chemo-therapy.

A class of multifunctional hybrid nanoparticles (NPs) that can integrate a magnetic core, silver (Ag) nanocrystals, and a biocompatible poly(ethylene glycol) (PEG) shell were synthesized and characterized and their applications as antibacterial agents, optical labels for cellular imaging and drug carriers were tested. The synthetic strategy involves a one-step solvothermal synthesis of Fe3O4@PEG template NPs (∼60 nm) with magnetic Fe3O4 nanocrystals in the core and porous PEG as the shell, followed by loading and in situ reduction of Ag+ ions to form Ag nanocrystals in the shell. The size and number of the Ag nanocrystals embedded in the PEG shell can be readily controlled via a simple reaction condition change, resulting in different nanostructures and properties of the hybrid NPs. Such designed Fe3O4@Ag-PEG hybrid NPs can combine the properties and functions from each component. While the Fe3O4 core provides an easy magnetic separation and targeting and magnetic resonance imaging (MRI) contrast ability, the Ag nanocrystals provide stable strong fluorescence and antibacterial activity. The porous PEG shell with excellent stability in water and non-cytotoxicity can be used as a drug carrier for combined photothermo/chemo-therapy. The small hybrid NPs can enter the intracellular region and light up the mouse melanoma B16F10 cells. This class of hybrid NPs with rational integration of functional building blocks should offer broad opportunities for external magnetic manipulation, imaging diagnostics, antibacterial applications and as drug carriers.

Molecular characterization of senescence marker protein-30 gene promoter: identification of repressor elements and functional nuclear factor binding sites.

BACKGROUND: Senescence marker protein-30 (SMP30), whose expression declines during aging in rat liver, has been proposed as an important aging marker. Besides apoptosis, SMP30 also protects cells against various other injuries by enhancement of membrane calcium-pump activity. The mechanism of this differential gene expression mechanism is not known. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed to elucidate the mechanism of transcriptional regulation of SMP30 gene. RESULTS: We have characterized up to -2750 bp of the promoter by DNA-protein interactions studies. Twenty eight transcription factor binding sites have been identified by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Transient transfection of 5' and 3' -deleted promoter-reporter constructs and luciferase assay illustrated the region between -128/+157 bp is sufficient to drive promoter activity. We have mapped an essential regulatory region between -513 to -352 bp which causes a drastic decline of reporter activity. This region contains CdxA, GATA2 and SRY transcription factor binding sites. Individual mutation of these three sites showed increase in reporter activity. Mutation in SRY site (-403/-368) showed maximum increase in reporter activity among these three sites. Therefore, we suggest that SRY like protein may be acting as a strong repressor of SMP30 gene along with CdxA and GATA-2. We also report that mutation of both Sp1 (172/-148 bp) and a C/EBPbeta (-190/-177 bp) transcription binding site located adjacent to each other on SMP30 gene promoter, causes a significant enhancement in reporter activity than individual mutation, thus may be causing the repression of SMP30 promoter activity. CONCLUSION: These studies provide novel insights into the mechanism that regulate SMP30 gene expression.

Cloning, expression, and functional analysis of rat liver cytosolic inorganic pyrophosphatase gene and characterization of its functional promoter.

Inorganic pyrophosphate (PPi) is formed in several metabolic processes and its hydrolysis by the ubiquitously expressed enzyme inorganic pyrophosphatase (iPPase) is essential for the reactions to proceed in the direction of biosynthesis. Recently, we have reported differential expression and activity of cytosolic iPPase in rat liver with aging. In this article we report the cloning of the coding region of rat liver cytosolic iPPase gene in a bacterial expression vector, its expression, purification, and functional analysis by in-gel enzyme assay. SDS-PAGE and Western blot analysis of this expressed protein revealed that its molecular weight (MW) is approximately 33 kDa, while in-gel assay showed that it is functionally active just as the liver cytosolic iPPase. We have determined the genomic organization of this gene by genome blast approach. We have also cloned and characterized its proximal approximate 1 kb functional promoter (-1009 to +82) by transient transfection and luciferase assay of different 5'-deleted iPPase promoter-luciferase constructs and also established its transcription start site by primer extension analysis, along with protein-DNA interaction studies for a few putative transcription factor binding sites.