Transferrin (Tf)-templated luminescent blue copper nanoclusters (Tf-Cu NCs) are synthesized. They are further formulated into spherical Tf-Cu NC-doxorubicin nanoparticles (Tf-Cu NC-Dox NPs) based on electrostatic interaction with doxorubicin (Dox). The as-synthesized Tf-Cu NC-Dox NPs are explored for bioimaging and targeted drug delivery to delineate high therapeutic efficacy. Förster resonance energy transfer (FRET) within the Tf-Cu NC-Dox NPs exhibited striking red luminescence, wherein the blue luminescence of Tf-Cu NCs (donor) is quenched due to absorption by Dox (acceptor). Interestingly, blue luminescence of Tf-Cu NCs is restored in the cytoplasm of cancer cells upon internalization of the NPs through overexpressed transferrin receptor (TfR) present on the cell surface. Finally, gradual release of Dox from the NPs leads to the generation of its red luminescence inside the nucleus. The biocompatible Tf-Cu NC-Dox NPs displayed superior targeting efficiency on TfR overexpressed cells (HeLa and MCF-7) as compared to the cells expressing less TfR (HEK-293 and 3T3-L1). Combination index (CI) revealed synergistic activity of Tf-Cu NCs and Dox in Tf-Cu NC-Dox NPs. In vivo assessment of the NPs on TfR positive Daltons lymphoma ascites (DLA) bearing mice revealed significant inhibition of tumor growth rendering prolonged survival of the mice.
The gap junction (GJ) protein connexin-43 (Cx43) is considered as a tumour suppressor protein for its role in reversing the phenotype of the cancer cells. In this study, we exploited the antitumor property of Cx43 in conjunction with the artesunate (ART), a plant-based active anti-malarial compound. The reactive oxygen species (ROS) generated by ART resulted in DNA damage, which in turn led to DNA damage response by activation of DNA damage repair proteins. GJ deficient MCF-7 cells transfected with Cx43 gene showed an increased sensitivity towards dose-dependent ART treatment and required a significantly lower dose of ART to attain its IC50, as compared to parental cells. This would ultimately result in reduced dose-dependent side effects of ART. The Co-culture experiments involving GJ intercellular communication (GJIC) deficient and GJIC enabled cells, established the transfer of ROS to the neighbouring cancer cells not exposed to ART. The ROS accumulated in the ART-treated cells induced the oxidative damage in neighbouring cells, leading to bystander cell death and inhibition of bystander cell proliferation. Thus, our study revealed that expression of Cx43 helped in reducing the dose-dependent cytotoxicity of ART as well as enhanced the bystander apoptosis of the neighbouring cells.
To understand the burgeoning challenges of metastasis, a microchannel of 35 μm diameter, constricted to 7 μm for a distance of 200 μm in a total length of 3 mm, was designed and fabricated using a mask aligner made of polydimethylsiloxane (PDMS) to mimic in vivo capillaries. A thin glass cover-slide was mounted on top to monitor the motion of single or aggregated malignant HeLa cells (size 17–30 μm) microscopically through the constricted microchannel at a constant flow rate of 30 μl/h. Quantitative deconvolution of high-speed videographs of a single cell of 30 μm revealed cellular deformation while passing through constriction, having elongation index, average transit velocity and entry time of 2.67, 18 mm/s and 5.1 ms, respectively. Morphological analysis of live and apoptotic cells by dual staining with Acridine Orange/Ethidium Bromide demonstrated retention of a significant viable cell population after exit through the constriction and a viability index of 50% was quantified by dye exclusion assay. The cumulative data for microfluidic parameters, morphology and relevant metastatic MMP2 gene expression efficiency measured by real-time polymerase chain reaction revealed retention of virulence potency that could possibly cause metastasis, would be beneficial in developing futuristic MEMS device for cancer theranostics.
BackgroundAberrant DNA methylation profiles are a characteristic feature of almost all types of cancers including hepatocellular carcinoma (HCC) and play an important role in carcinogenesis. In spite of the accumulating evidence that suggests appearance of such aberrations at precancerous stages, very little effort has been invested to investigate such possible methylation events in patients at risk of developing HCC i.e. those suffering from chronic hepatitis C virus (HCV) infection and liver cirrhosis (LC). We reasoned that such an analysis could lead to the identification of novel predictive biomarkers as well as potential drug targets.MethodsPromoter methylation status of two Wnt inhibitors SFRP2 and DKK1 was quantitatively analyzed by bisulfite pyrosequencing in a series of liver biopsy samples. These biopsies were collected from HCV-infected individuals suffering from chronic hepatitis (CH; n = 15), liver cirrhosis (LC; n = 13) and hepatocellular carcinoma (HCC; n = 41). DNA isolated from infection free normal livers (N; n =10) was used as control.ResultsOur analysis revealed that both of the genomic loci were significantly hypermethylated in CH patients’ livers as compared to normal controls (p = 0.0136 & 0.0084 for SFRP2 and DKK1, respectively; Mann–Whitney U test). DNA methylation levels for both loci were also significantly higher in all the diseased cohorts as compared to normal controls (p < 0.0001 and = 0.0011 for SFRP2 and DKK1, respectively; Kruskal-Wallis test). However, a comparison between three disease cohorts (CH, LC & HCC) revealed no significant difference in levels of DNA methylation at DKK1 promoter. In contrast, a progressive increase in DNA methylation levels was observed at the SFRP2 promoter (i.e. N < CH & LC < HCC).ConclusionThis study demonstrated that in HCV infected liver tissues hypermethylation at promoter regions of key cancer related genes SFRP2 and DKK1, appears early at CH and LC stages, long before the appearance of HCC.
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