Horizontal transfer of miR-383 sensitise cells to cisplatin by targeting VEGFA-Akt signalling loop.

BACKGROUND: Cellular resistance to cisplatin has been one of the major obstacles in the success of combination therapy for many types of cancers. Emerging evidences suggest that exosomes released by drug resistant tumour cells play significant role in conferring resistance to drug sensitive cells by means of horizontal transfer of genetic materials such as miRNAs. Though exosomal miRNAs have been reported to confer drug resistance, the exact underlying mechanisms are still unclear. METHODS AND RESULTS: In the present study, mature miRNAs secreted differentially by cisplatin resistant and cisplatin sensitive HepG2 cells were profiled and the effect of most significantly lowered miRNA in conferring cisplatin resistance when horizontally transferred, was analysed. we report miR-383 to be present at the lowest levels among the differentially abundant miRNAs expressed in exosomes secreted by cisplatin resistant cells compared to that that of cisplatin sensitive cells. We therefore, checked the effect of ectopic expression of miR-383 in altering cisplatin sensitivity of Hela cells. Drug sensitivity assay and apoptotic assays revealed that miR-383 could sensitise cells to cisplatin by targeting VEGF and its downstream Akt mediated pathway. CONCLUSION: Results presented here provide evidence for the important role of miR-383 in regulating cisplatin sensitivity by modulating VEGF signalling loop upon horizontal transfer across different cell types.

Identification and stability analysis of potential ADP-ribose modification sites on vascular endothelial growth factor (VEGF) through molecular dynamics simulation.

Post-translational modifications (PTMs) are crucial covalent processes that alter protein properties, achieved through proteolytic cleavage or addition of modifying groups like acetyl, phosphoryl, glycosyl, or methyl to amino acids. ADP-ribosylation is a reversible post-translational modification, where ADP-ribose units are covalently attached to target protein side chains. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that plays a key role in physiological and pathological conditions. Studies have reported that ADP-ribosylation affects VEGF's ability to bind to VEGF receptors, impacting angiogenesis signalling. However, the specific amino acid undergoing ADP-ribosylation on VEGF remained unknown. To understand the mechanism of ADP-ribose addition to VEGF, an in silico study was designed. The study initially checked for the presence of any conserved motif where ADP-ribosylation could potentially occur and identified the presence of the EIE motif in VEGF, a probable site for ADP-ribosylation for many proteins. Subsequently, the amino acids near this motif were selected and their structural properties were analyzed. Surface-exposed amino acids were chosen, and ADP-ribose was then added to their side chains. The results revealed that the amino acids ASP (67) and GLU (70) underwent glycosidic linkage with ADP-ribose, indicating that they are the most probable modification sites. Subsequently, Molecular dynamic simulation analysis such as RMSD, RMSF, Rg, PCA, and FEL, along with MM-PBSA binding free energy calculations were performed to understand the stability of the VEGF-ADP-ribose complexes. The analysis revealed that amino acid at position 67 (ASP67) is the most probable site for ADP-ribosylation in VEGF.Communicated by Ramaswamy H. Sarma.

Integrin ß4 induced epithelial-to-mesenchymal transition involves miR-383 mediated regulation of GATA6 levels.

BACKGROUND: The process of transdifferentiating epithelial cells to mesenchymal-like cells (EMT) involves cells gradually taking on an invasive and migratory phenotype. Many cell adhesion molecules are crucial for the management of EMT, integrin ß4 (ITGB4) being one among them. Although signaling downstream of ITGB4 has been reported to cause changes in the expression of several miRNAs, little is known about the role of such miRNAs in the process of EMT. METHODS AND RESULTS: The cytoplasmic domain of ITGB4 (ITGB4CD) was ectopically expressed in HeLa cells to induce ITGB4 signaling, and expression analysis of mesenchymal markers indicated the induction of EMT. ß-catenin and AKT signaling pathways were found to be activated downstream of ITGB4 signaling, as evidenced by the TOPFlash assay and the levels of phosphorylated AKT, respectively. Based on in silico and qRT-PCR analysis, miR-383 was selected for functional validation studies. miR-383 and Sponge were ectopically expressed in HeLa, thereafter, western blot and qRT-PCR analysis revealed that miR-383 regulates GATA binding protein 6 (GATA6) post-transcriptionally. The ectopic expression of shRNA targeting GATA6 caused the reversal of EMT and ß catenin activation downstream of ITGB4 signaling. Cell migration assays revealed significantly high cell migration upon ectopic expression ITGB4CD, which was reversed upon ectopic co-expression of miR-383 or GATA6 shRNA. Besides, ITGB4CD promoted EMT in in ovo xenograft model, which was reversed by ectopic expression of miR-383 or GATA6 shRNA. CONCLUSION: The induction of EMT downstream of ITGB4 involves a signaling axis encompassing AKT/miR-383/GATA6/ß-catenin.

Tumour generated exosomal miRNAs: A major player in tumour angiogenesis.

One of the major reasons for the non-effectiveness of anticancer therapeutics designed to target tumour angiogenesis is the involvement of multiple layers of cellular communication that exists within the tumour microenvironment. Any attempt to block one signaling pathway by targeting one of its components led to the activation of alternate pathway that overcome this block rendering the therapy ineffective. During the previous decades most of the research was focused on manipulating signaling pathways initiated by soluble factors and their intermediates. More recently the work from our lab and many advanced labs all over the globe have brought into light, the significance of signaling events mediated by exosomes in regulation of tumour progression and angiogenesis. In this mini review we intend to collate the information available from several groups including ours related to the role of exosomal cargo especially exosomal miRNAs in modulating tumour angiogenesis.

Horizontal Transfer of miR-643 from Cisplatin-Resistant Cells Confers Chemoresistance to Recipient Drug-Sensitive Cells by Targeting APOL6.

Acquisition of resistance to cisplatin is a major impediment to the success of cisplatin-based combination therapies for cancer. Recent studies indicate that exosomal miRNAs derived from drug-resistant tumour cells can confer resistance properties to recipient cells by a horizontal transfer mechanism. Although the role of horizontal transfer of a few miRNAs has been described, little is known about the concerted action of horizontal transfer of miRNAs in conferring cisplatin resistance. The present study was designed to identify the role of miR-643, which is one of the most significantly increased miRNA in exosomes released from cisplatin-resistant Heptocarcinoma cells, in altering the cisplatin resistance properties of recipient cells. Drug-sensitivity assays involving miR-643 revealed that ectopic expression of miR-643 can desensitise the cells towards cisplatin. Furthermore, we identified APOL6 as a major target of miR-643. Further mechanistic studies showed that miR-643 can modulate APOL6 mRNA and protein levels, leading to a reversal of APOL6-mediated apoptosis. Altogether, our results suggest an APOL6-dependent mechanism for miR-643 mediated cisplatin resistance upon the horizontal transfer across cell types.

Fabrication of a Greener TiO2@Gum Arabic-Carbon Paste Electrode for the Electrochemical Detection of Pb2+ Ions in Plastic Toys.

A novel greener methodology is reported for the synthesis of titanium dioxide (TiO2) nanoparticles (NPs) using gum Arabic (Acacia senegal) and the characterization of the ensuing TiO2 NPs by various techniques such as X-ray diffraction (XRD), Fourier transform infrared, Raman spectroscopy, scanning electron microscopy-energy dispersive X-ray, transmission electron microscopy (TEM), high resolution-TEM, and UV-visible spectroscopy. The XRD analysis confirmed the formation of TiO2 NPs in the anatase phase with high crystal purity, while TEM confirmed the size to be 8.9 ± 1.5 nm with a spherical morphology. The electrode for the electrochemical detection of Pb2+ ions was modified by a carbon paste fabricated using the synthesized TiO2 NPs. Compared to the bare electrode, the fabricated electrode exhibited improved electro-catalytic activity toward the reduction of Pb2+ ions. The detection limit, quantification limit, and the sensitivity of the developed electrode were observed by using differential pulse voltammetry to be 506 ppb, 1.68 ppm, and 0.52 ± 0.01 µA µM-1, respectively. The constructed electrode was tested for the detection of lead content in plastic toys.

Regulation of poly ADP-ribosylation of VEGF by an interplay between PARP-16 and TNKS-2.

The biological activity of vascular endothelial growth factor (VEGF), the major cytokine regulating the process of angiogenesis is tightly controlled at multiple levels including processes involving post-translational modification such as ADP-ribosylation and glycosylation. ADP-ribosylation is a reversible NAD+-dependent modification, catalyzed by poly ADP-ribose polymerase (PARP) or ADP-ribosyl transferase (ADPRTs) and has been reported by us and others as a modification that reduces the biological activity of VEGF. The factors responsible for any such modification should occur in the secretory pathway, i.e., in the endoplasmic reticulum and Golgi. Our investigation carried out in this direction revealed that ADP-ribosylation of VEGF requires the interplay between members of poly ADP-ribose polymerase (PARP) family in the secretory pathway, viz., ER associated PARP-16 and Golgi associated Tankyrase-2 (TNKS-2). The data presented in this manuscript suggest that PARP-16 catalysis the priming mono ADP-ribosylation of VEGF which is a prerequisite for poly ADP-ribosylation of VEGF by TNKS-2.

Carbon Quantum Dot-Modified Carbon Paste Electrode-Based Sensor for Selective and Sensitive Determination of Adrenaline.

A carbon quantum dot-based carbon paste electrode was fabricated and used for the determination of adrenaline (AD) at the nanomolar level. This fabricated electrode exhibited tremendous electrocatalytic activity for the oxidation of adrenaline in supporting electrolyte (PBS of pH 7.4). Scan rate variation studies with the modified electrode revealed that the overall electrode process was controlled by a diffusion process. A lower detection limit of 6 nM was achieved by chronoamperometry. Interference by biological molecules such as serotonin (5-HT) and ascorbic acid (AA) in the electrochemical oxidation of AD on the fabricated electrode was tested. It was observed that with the modified electrode, the selective determination of AD was possible. Further, with the fabricated electrode, simultaneous analysis of AA, AD, and 5-HT was performed, and it was observed that the overlapped peaks of these analytes on the naked electrode were well resolved into three peaks on the modified electrode. Along with decent sensitivity and selectivity, the electrode also showed higher stability and antifouling nature. The real-time application of the projected scheme was proven by employing the said electrode for adrenaline in adrenaline bitartrate injections.

Role of Sirtuins in Tumor Angiogenesis.

Generally, changes in the metabolic status of cells under conditions like hypoxia and accumulation of lactate can be sensed by various sensing mechanisms, leading to modulation of a number of signal transduction pathways and transcription factors. Several of the proangiogenic cytokines like VEGF, FGF, PDGF, TGF-ß, Ang-2, ILs, etc. are secreted by cancer cells, under hypoxic microenvironment. These cytokines bind to their receptors on the endothelial cells and activates a number of signaling pathways including Akt/PIP3, Src, p38/MAPK, Smad2/3, etc., which ultimately results in the proliferation and migration of endothelial cells. Transcription factors that are activated in response to the metabolic status of tumors include HIFs, NF-κb, p53, El-2, and FOXO. Many of these transcription factors has been reported to be regulated by a class of histone deacetylase called sirtuins. Sirtuins are NAD+ dependent histone deacetylases that play pivotal role in the regulation of tumor cell metabolism, proliferation, migration and angiogenesis. The major function of sirtuins include, deacetylation of histones as well as some non-histone proteins like NF-κB, FOXOs, PPAR⋎, PGC1-α, enzymes like acetyl coenzymeA and structural proteins like α tubulin. In the cell, sirtuins are generally considered as the redox sensors and their activities are dependent on the metabolic status of the cell. Understanding the intricate regulatory mechanisms adopted by sirtuins, is crucial in devising effective therapeutic strategies against angiogenesis, metastasis and tumor progression. Keeping this in mind, the present review focuses on the role of sirtuins in the process of tumor angiogenesis and the regulatory mechanisms employed by them.

Individual and simultaneous electrochemical determination of metanil yellow and curcumin on carbon quantum dots based glassy carbon electrode.

Adulteration of medicinally valuable curcumin (CU) with harmful chemicals as metanil yellow (MY) in recent years have demanded for quick detection techniques of the adulterants. The voltammetric behavior of CU and MY on bare glassy carbon electrode (BGCE) and carbon quantum dots modified glassy carbon electrode (CQDs/GCE) was studied by both cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in phosphate buffer solution of pH 5.4. The MY responded to the CQDs/GCE with two anodic peaks at -0.004 V and 0.136 V and two cathodic peaks at -0.112 and -0.048 V. Under similar conditions CU exhibited two anodic peaks at 0.28 V and 0.55 V and one cathodic peak at 0.25 V. The overlapped voltammogram obtained for CU and MY on BGCE was well separated on the CQDs/GCE. The interference studies revealed that the compounds, demethoxycurcumin and bisdemethoxycurcumin, which are commonly associated with CU, did not interfere with the detection of MY. Real sample was analyzed with fabricated electrode and the recovery values >98% were obtained.

Curcumin and its synthetic analogue dimethoxycurcumin differentially modulates antioxidant status of normal human peripheral blood mononuclear cells.

Curcumin is a polyphenol derived from the herb Curcuma longa, which has been extensively studied in terms of its antitumour, antioxidant, and chemopreventive activity as well as various other effects. In the present work we compared curcumin with its synthetic analogue dimethoxycurcumin (dimc) in terms of its antioxidant enzyme-modulating effects in human peripheral blood mononuclear cells (PBMC). We found that these compounds modulate antioxidant enzymes differentially. Both curcumin and dimethoxycurcumin effected a decrease in lipid peroxidation status in PBMC, however, curcumin had better activity in this regard. An increase in the activity of catalase was seen in the case of curcumin-treated PBMC, whereas dimc increased catalase activity significantly to almost twofold level. Real time-polymerase chain reaction (RT-PCR) analysis revealed significant up-regulation of catalase at mRNA level post treatment with curcumin as well as dimc, however, dimc had better activity in this regard. Glutathione reductase (GR) activity and reduced glutathione levels increased in the case of peripheral blood mononuclear cells (PBMC) treated with curcumin, however, the trend was reversed with dimethoxycurcumin where, both glutathione reductase activity and reduced glutathione levels were significantly reduced. RT-PCR analysis of glutathione reductase mRNA levels showed decrease in mRNA levels post treatment with dimethoxycurcumin (dimc) further corroborating GR enzyme assay results, however, we could not obtain significant result post curcumin treatment. NFkB reporter assay and western blot analysis of nuclear as well as cytosolic fractions of NFkB revealed that curcumin inhibits NFkB activation whereas inhibition was much less with dimc. It has been reported that curcumin and dimc exerts differential cytotoxicity in normal and tumour cells and the reason for this had been attributed to the differential uptake of these compounds by normal cells and tumour cells. Based on our results we propose that differential modulation of antioxidant enzymes via NFkB pathway could be the reason behind differential cytotoxicity of dimc as well as curcumin in normal cells and tumour cells in addition to differential uptake of these compounds as reported previously.

MoS2-ZnO nanocomposites as highly functional agents for anti-angiogenic and anti-cancer theranostics.

Due to its excellent properties, 2D-MoS2 finds potential applications in the fields of electronics, optoelectronics, energy storage and conversion, biomedicine, etc. This work deals with the incorporation of ZnO into 2D-MoS2, its structural, morphological, optical, and magnetic studies and its application as an efficient cancer therapeutic agent. The MoS2-ZnO nanocomposite exhibits remarkable excitation wavelength dependent down-conversion and up-conversion photoluminescence. The observation of wasp-waisted magnetism in the MoS2-ZnO nanocomposite indicates the coupling of ZnO and MoS2 materials inducing multimodal population. The MoS2-ZnO nanocomposite showed cytotoxic properties with a safety index reaching up to ∼2. An in ovo xenograft assay revealed that the MoS2-ZnO nanocomposite retards tumor growth by specifically activating caspase-3 and thereby inducing cellular apoptosis. Moreover, the treatment of xenografts with the MoS2-ZnO nanocomposite down regulated the expression of major pro-angiogenic genes such as VEGF, VEGFR2 etc. thereby curtailing vascularization into the tumor intima. Treatment of tumor xenografts with the MoS2-ZnO nanocomposite caused reduced expression of mesenchymal specific genes and elevated expression of epithelial specific genes, implying a role of the MoS2-ZnO nanocomposite in retarding the process of epithelial to mesenchymal transition (EMT). This study highlights that the introduction of ZnO into MoS2 nanostructures offers a unique idea to design efficient MoS2-based multifunctional nanocomposites that provide opportunities in advanced biomedical and optoelectronic applications.

Horizontal transfer of miR-106a/b from cisplatin resistant hepatocarcinoma cells can alter the sensitivity of cervical cancer cells to cisplatin.

Recent studies indicate that horizontal transfer of genetic material can act as a communication tool between heterogenous populations of tumour cells, thus altering the chemosensitivity of tumour cells. The present study was designed to check whether the horizontal transfer of miRNAs released by cisplatin resistant (Cp-r) Hepatocarcinoma cells can alter the sensitivity of cervical cancer cells. For this exosomes secreted by cisplatin resistant and cisplatin sensitive HepG2 cells (EXres and EXsen) were isolated and characterised. Cytotoxicity analysis showed that EXres can make Hela cells resistant to cisplatin. Analysis of miR-106a/b levels in EXres and EXsen showed that their levels vary. Mechanistic studies showed that miR-106a/b play an important role in EXsen and EXres mediated change in chemosensitivity of Hela cells to cisplatin. Further SIRT1 was identified as a major target of miR-106a/b using in silico tools and this was proved by experimentation. Also the effect of miR-106a/b in chemosensitivity was seen to be dependent on regulation of SIRT1 by miR-106a/b. In brief, this study brings into light, the SIRT1 dependent mechanism of miR-106a/b mediated regulation of chemosensitivity upon the horizontal transfer from one cell type to another.

2-Deoxy glucose regulate MMP-9 in a SIRT-1 dependent and NFkB independent mechanism.

MMP9 is a member of the family of zinc-containing endopeptidases which degrade various components of the extracellular matrix, thereby regulating matrix remodeling. Since matrix remodeling plays an important role during growth and progression of cancer and considering the fact that, tumor cells switch to aerobic glycolysis as its major energy source, this study was designed to analyze if partial inhibition of glycolysis (the major energy pathway during hypoxia) can be used as a means to control matrix remodeling in terms of MMP9 activity and expression. For this, human epithelial carcinoma cells were treated with glycolytic inhibitor, 2-deoxy glucose (2DG) at sub-lethal concentrations followed by analysis of the expression and activity of MMP2 and MMP9. The experimental findings demonstrate that exposure of cancer cells to glycolytic inhibitor at concentration that does not induce ER stress, downregulates the activity and expression of MMP9 without affecting the expression levels and activity of MMP2. Further mechanistic analysis revealed that the regulation of MMP9 was mediated in a SIRT-1 dependent mechanism and did not alter the NFkB signaling pathway. The overall results presented here, therefore suggest that the use of glycolytic inhibitor, 2DG at concentration that do not affect cell viability or induce ER stress can be an effective strategy to control matrix remodeling.

Angiogenic Profiling of Synthesized Carbon Quantum Dots.

A simple method was employed for the synthesis of green luminescent carbon quantum dots (CQDs) from styrene soot. The CQDs were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared, and Raman spectroscopy. The prepared carbon quantum dots did not show cellular toxicity and could successfully be used for labeling cells. We also evaluated the effects of carbon quantum dots on the process of angiogenesis. Results of a chorioallantoic membrane (CAM) assay revealed the significant decrease in the density of branched vessels after their treatment with CQDs. Further application of CQDs significantly downregulated the expression levels of pro-angiogenic growth factors like VEGF and FGF. Expression of VEGFR2 and levels of hemoglobin were also significantly lower in CAMs treated with CQDs, indicating that the CQDs inhibit angiogenesis. Data presented here also show that CQDs can selectively target cancer cells and therefore hold potential in the field of cancer therapy.

Regulation of vascular endothelial growth factor by metabolic context of the cell.

Expression of vascular endothelial growth factor, major endothelial specific glycoprotein growth factor that promotes angiogenesis is regulated at transcriptional, post transcriptional and posttranslational levels. One of the key posttranslational modifications involved in regulating the angiogenic potential of VEGF is covalent modification involving polyADP ribosylation. Major factors contributing to the regulation of VEGF include factors relating to hypoxia, growth factors and cytokines and hormones. Apart from these, the metabolite status of the cell as sensed by various metabolite regulators can influence the angiogenic potential. Changes in the metabolite status of the cell occur during different conditions associated with excessive or insufficient angiogenesis contributing to pathology. Effect of metabolites, as exemplified by certain metabolites such as lactate, citrate, sarcosine, metabolites of arachidonic acid on angiogenesis through the regulation of expression of VEGF as well as its angiogenic potential through polyADP ribosylation is discussed.

The tumour suppressor LKB1 regulates myelination through mitochondrial metabolism.

A prerequisite to myelination of peripheral axons by Schwann cells (SCs) is SC differentiation, and recent evidence indicates that reprogramming from a glycolytic to oxidative metabolism occurs during cellular differentiation. Whether this reprogramming is essential for SC differentiation, and the genes that regulate this critical metabolic transition are unknown. Here we show that the tumour suppressor Lkb1 is essential for this metabolic transition and myelination of peripheral axons. Hypomyelination in the Lkb1-mutant nerves and muscle atrophy lead to hindlimb dysfunction and peripheral neuropathy. Lkb1-null SCs failed to optimally activate mitochondrial oxidative metabolism during differentiation. This deficit was caused by Lkb1-regulated diminished production of the mitochondrial Krebs cycle substrate citrate, a precursor to cellular lipids. Consequently, myelin lipids were reduced in Lkb1-mutant mice. Restoring citrate partially rescued Lkb1-mutant SC defects. Thus, Lkb1-mediated metabolic shift during SC differentiation increases mitochondrial metabolism and lipogenesis, necessary for normal myelination.