11 research outputs found
Retention of Anticancer Activity of Curcumin after Conjugation with Fluorescent Gold Quantum Clusters: An in Vitro and in Vivo Xenograft Study
Gold nanoparticles
(Au NPs) have been thoroughly investigated for
anti-cancer therapy. However, their undesired high gold content remains
a problem when injected into the body for drug delivery applications.
In this report, we made an effort to conjugate the curcumin molecules
on the surface of gold quantum clusters (Au QCs) by a novel in situ
synthesis method which provides an alternative route to not only reduce
the metallic content but also increase the water solubility of curcumin
and the loading efficiency. Here, curcumin itself acts as a reducing
and capping agent for the synthesis of Au QCs. The UV–vis absorption,
fluorescence, transmission electron microscopy, and electrospray ionization
mass spectrometry results confirmed the synthesis of fluorescent Au
QCs. Curcumin-conjugated Au NPs (C-Au NPs) and glutathione (GSH)-conjugated
Au QCs (GSH-Au QCs) were also synthesized to visualize the effect
of particle size and the capping agent, respectively, on the cytotoxicity
to normal and cancer cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay showed that the curcumin-conjugated Au QCs (C-Au QCs)
were less cytotoxic to normal cells while almost the same cytotoxic
to cancer cells in comparison to curcumin itself, which indicates
that curcumin preserves its anticancer property even after binding
to the Au QCs. However, C-Au NPs and GSH-Au QCs did not show any cytotoxicity
against the normal and cancer cells at the concentration used. The
western blot assay indicated that C-Au QCs promote apoptosis in cancer
cells. Further, the in vivo study on severe combined immunodeficiency
mice showed that C-Au QCs also inhibited the tumor growth efficiently
without showing significant toxicity to internal organs
A Cysteine-Specific Fluorescent Switch for Monitoring Oxidative Stress and Quantification of Aminoacylase‑1 in Blood Serum
Reagents
that allows detection and monitoring of crucial biomarkers
with luminescence ON response have significance in clinical diagnostics.
A new coumarin derivative is reported here, which could be used for
specific and efficient chemodosimetric detection of cysteine, an important
biomarker. The probe is successfully used for studying the biochemical
transformation of N-acetylcysteine, a commonly prescribed Cys supplement
drug to Cys by aminoacylase-1 (ACY-1), an important and endogenous
mammalian enzyme. The possibility of using this reagent for quantification
of ACY-1 in blood serum samples is also explored. Nontoxic nature
and cell membrane permeability are key features of this probe and
are ideally suited for imaging intracellular Cys in normal and cancerous
cell lines. Our studies have also revealed that this reagent could
be utilized as a redox switch to monitor the hydrogen-peroxide-induced
oxidative stress in living SW480 cell lines. Peroxide-mediated cysteine
oxidation has a special significance for understanding the cellular-signaling
events
FRET-Based Probe for Monitoring pH Changes in Lipid-Dense Region of Hct116 Cells
A rhodamine
conjugate (<b>L</b>) with a pseudo Stokes shift
of 165 nm is used for probing changes in solution pH under physiological
conditions. This reagent is found to be nontoxic, and the luminescence
response could be used for imaging changes in endogenous pH induced
by dexamethanose (DMT) in the endoplasmic reticulum
A Switch-On NIR Probe for Specific Detection of Hg<sup>2+</sup> Ion in Aqueous Medium and in Mitochondria
A new
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-based probe molecule
(<b>L</b>) is synthesized for specific binding to Hg<sup>2+</sup> ion in physiological condition with an associated <i>luminescence
ON</i> response in the near-IR region of the spectrum. Appropriate
functionalization in the 5-position of each of two pyrrole moieties
with styryl functionality in a BODIPY core helped us in achieving
the extended conjugation and a facile intramolecular charge transfer
transition with a narrow energy gap for frontier orbitals. This accounted
for a poor emission quantum yield for the probe molecule <b>L</b>. Binding to Hg<sup>2+</sup> helped in interrupting the facile intramolecular
charge transfer (ICT) process that was initially operational for <b>L</b>. This resulted in a hypsochromic shift of absorption band
and a <i>turn-on</i> luminescence response with λ<sub>Max</sub><sup>Ems</sup> of 650 nm
on specific binding to Hg<sup>2+</sup>. Observed spectral changes
are rationalized based on quantum chemical calculations. Interestingly,
this reagent is found to be localized preferentially in the mitochondria
of the live human colon cancer (Hct116) cells. Mitochondria is one
of the major targets for localization of Hg<sup>2+</sup>, which actually
decreases the mitochondrial membrane potential and modifies various
proteins having sulfudryl functionalityÂ(ies) to cause cell apoptosis.
Considering these, ability of the present reagent to specifically
recognize Hg<sup>2+</sup> in the mitochondrial region of the live
Hct116 cells has significance
Specific Reagent for Cr(III): Imaging Cellular Uptake of Cr(III) in Hct116 Cells and Theoretical Rationalization
A new rhodamine-based reagent (<b>L</b><sub><b>1</b></sub>), trapped inside the micellar structure
of biologically benign
Triton-X 100, could be used for specific recognition of CrÂ(III) in
aqueous buffer medium having physiological pH. This visible light
excitable reagent on selective binding to CrÂ(III) resulted in a strong
fluorescence <i>turn-on</i> response with a maximum at ∼583
nm and tail of that luminescence band extended until 650 nm, an optical
response that is desired for avoiding the cellular autofluorescence.
Interference studies confirm that other metal ions do not interfere
with the detection process of CrÂ(III) in aqueous buffer medium having
pH 7.2. To examine the nature of binding of CrÂ(III) to <b>L</b><sub><b>1</b></sub>, various spectroscopic studies are performed
with the model reagent <b>L</b><sub><b>2</b></sub>, which
tend to support CrÂ(III)-η<sup>2</sup>-olefin Ï€-interactions
involving two olefin bonds in molecular probe <b>L</b><sub><b>1</b></sub>. Computational studies are also performed with another
model reagent <b>L</b><sub><b>M</b></sub> to examine the
possibility of such CrÂ(III)-η<sup>2</sup>-olefin Ï€-interactions.
Presumably, polar functional groups of the model reagent <b>L</b><sub><b>M</b></sub> upon coordination to the CrÂ(III) center
effectively reduce the formal charge on the metal ion and this is
further substantiated by results of the theoretical studies. This
assembly is found to be cell membrane permeable and shows insignificant
toxicity toward live colon cancer cells (Hct116). Confocal laser scanning
microscopic studies further revealed that the reagent <b>L</b><sub><b>1</b></sub> could be used as an imaging reagent for
detection of cellular uptake of CrÂ(III) in pure aqueous buffer medium
by Hct116 cells. Examples of a specific reagent for paramagnetic CrÂ(III)
with luminescence <i>ON</i> response are scanty in the contemporary
literature. This ligand design helped us in achieving the turn on
response by utilizing the conversion from spirolactam to an acyclic
xanthene form on coordination to CrÂ(III)
Capsaicin selectively inhibits VEGF secretion to retard Hy-A549 cell-induced HUVEC cell migration.
<p>(A) Representative phase contrast photomicrographs demonstrating HUVEC migration upon incubation with spent media of Brefeldin-A-pretreated, capsaicin (37.5 µM)-treated Hy-A549 cells (<i>left panel</i>). Percent cell migrated in the wound area has been represented graphically (<i>right panel</i>). (B) Immunoblots showing expression profiles of pro-angiogenic factors VEGF, bFGF, EGF, TGF-β, in presence or absence of capsaicin. (C) Secreted VEGF from cell-free supernatant of Hy-A549 was quantified by ELISA assay (<i>left panel</i>). Time-dependent expression profiles of VEGF-mRNA/-protein in capsaicin-treated Hy-A549 cells were determined by Western blot and RT-PCR respectively (<i>middle panel</i>). Capsaicin-treated Hy-A549 cells were examined for time-dependent variation in the expression profiles of VEGF by quantitative real time PCR analysis and represented graphically (<i>right panel</i>). (D) Immuno-fluorescent images (60x magnification) showing time-dependent pattern of VEGF protein (TRITC-fluorescent) in capsaicin-treated Hy-A549 cells were represented along with nuclear staining (DAPI: blue). (E) Representative images of HUVEC migration upon incubation with (i) recombinant VEGF-supplemented control media, or VEGF-supplemented spent media of capsaicin-treated Hy-A549 cells, or with (ii) anti-VEGF-treated Hy-A549 spent media (<i>left panel</i>). Percent cell migrated in the wound area is being represented graphically (<i>right panel</i>). (F) Representative images of capillary-like sprout formation by HUVECs upon incubation with recombinant VEGF-supplemented spent media of capsaicin-treated Hy-A549 cells or with anti-VEGF-treated Hy-A549 spent media. GAPDH/α-Actin was used as internal loading control. Values are mean ±SEM of three independent experiments in each case or representative of typical experiment.</p
Capsaicin inhibits VEGF transcriptional activation by targeting HIF-1α in a p53-dependent manner.
<p>(A) Time-dependent expression profiles of HIF-1α mRNA and -protein were determined by Western blot and RT-PCR, respectively, in capsaicin-treated Hy-A549 cells (<i>left panel</i>). Capsaicin-treated Hy-A549 cells were examined for time-dependent variation in the expression profiles of HIF-1α by quantitative real time PCR analysis and represented graphically (<i>right panel</i>). (B) Hy-A549 cells, transiently transfected with a non-targeting control-siRNA or HIF-1α-siRNA, were incubated with/without capsaicin for 24 h; the cells were then analyzed to determine VEGF expression at protein and mRNA levels (<i>left panel</i>). Immunoblot showing transfection efficiency of HIF-1α (<i>right panel</i>) (C) Control-siRNA/HIF-1α-siRNA-transfected Hy-A549 cell-supernatants were used to assess HUVEC migration by wound healing assay after capsaicin-treatment (37.5 µM; 24 h) and represented graphically. (D) Time-dependent expression profile of p53-mRNA and -protein was determined by Western blotting and RT-PCR in capsaicin-treated Hy-A549 cells (<i>left panel</i>). p53 phosphorylation at Serine-15 position was also evaluated (<i>right panel</i>). (E) Hy-A549 cells, transfected with control-shRNA/p53-shRNA were incubated with capsaicin for 24 h and HIF-1α VEGF-mRNA and -protein were determined by Western blot and RT-PCR (<i>left panel</i>). Transfection efficiency was checked by analyzing p53 expression level (<i>right panel</i>). (F) HIF-1α was immunoprecipitated from capsaicin-treated Hy-A549 cell lysates and immunoblotted with anti-Ub antibody to assay HIF-1α ubiquitination. The ladder of bands represented ubiquitinated HIF-1α. In parallel experiment, immunoprecipitates were assayed for HIF-1α levels by Western blot. Comparable protein input was confirmed by direct Western blotting with anti-α-actin using 20% of the cell lysates that were used for immunoprecipitation. (G) Control and MG-132 drug-pretreated Hy-A549 cells were subjected to capsaicin-treatment for 24 h and then were examined for expression of HIF-1α/VEGF by Western blotting. α-Actin/GAPDH was used as internal loading control. Values are mean ±SEM of three independent experiments in each case or representative of typical experiment.</p
Schematic diagram representing molecular mechanisms of capsaicin-mediated down-regulation of pro-angiogenic factor, VEGF.
<p>Schematic diagram representing molecular mechanisms of capsaicin-mediated down-regulation of pro-angiogenic factor, VEGF.</p
Capsaicin suppresses VEGF expression by SMAR1-mediated down regulation of Cox-2.
<p>(A) Control-/SMAR1-shRNA (<i>left panel</i>) or control-vector/SMAR1-cDNA (<i>right panel</i>) transfected Hy-A549 cells were treated with capsaicin were assayed for Cox-2 expression by Western blot and RT-PCR. Transfection efficiency was verified by Western blot (<i>bottom panels</i>). (B) Cox-2 promoter activity was checked in Hy-A549. Schematic representation of the Cox-2 promoter showing the probable SMAR1-binding sites predicted by using the MARWIZ software, PCR run with eight different sets of primers designed for each probable MAR binding site spreading over four regions (−141 bp to −421 bp; −631 bp to −1121 bp; −1262 bp to −1331 bp; −1471 bp to −1891 bp). ChIP assay with anti-SMAR1 was performed on MAR-binding regions of Cox-2 promoter. Input and control IgG was used as internal control and negative control. (C) The relative abundance of SMAR1 on Cox-2 promoter was analyzed in control and capsaicin treated Hy-A549 cells at binding sites 6 & 7 after ChIP of Cox-2 promoter with anti-SMAR1(<i>left panel</i>) and represented graphically (<i>right panel</i>). (D) Control-/SMAR1-shRNA-transfected Hy-A549 cells were treated with capsaicin and analyzed for reporter HIF-1α and VEGF gene expression by RT-PCR. α-Actin/GAPDH were used as internal loading control. Values are mean ±SEM of three independent experiments in each case or representative of typical experiment.</p
Effect of capsaicin on lung cancer cell spent medium-induced endothelial cell migration and network formation.
<p>(A) Migration of HUVECs in presence or absence of spent media of NME, WI-38, A549 and Hy-A549 (CoCl<sub>2</sub>-stimulated to mimic hypoxic condition required for tumor-induced angiogenesis) were subjected to bi-directional wound healing assay for 24 h (<i>left panel</i>). The number of cells migrated in the wound area are represented graphically (<i>right panel</i>). (B) Representative images of HUVEC migration upon incubation with capsaicin-treated (0–50 µM) Hy-A549 cell spent medium (<i>left panel</i>). Percent cell migrated in the wound area has been represented graphically (<i>right panel</i>). (C) Hy-A549 cells were treated with capsaicin in a dose-dependent manner for 24 h and cell viability was scored by trypan blue dye-exclusion assay (<i>left panel</i>). Hy-A549 cells, treated with capsaicin (37.5 µM), were subjected to Annexin-V-FITC/PI binding and analyzed flow cytometrically for the determination of percent early apoptosis (<i>right panel</i>). (D) Cytotoxic effect of different doses of capsaisin on HUVEC cells were measured by trypan blue dye-exclusion assay. (E) Graphical representation of HUVEC migration upon incubation with spent media from capsaicin-treated (37.5 µM) HBL-100, HCT-15, HeLa and A549. (F) Representative images of capillary-like sprout formation by HUVECs in presence of media alone or spent media of WI-38/A549/Hy-A549/capsaicin-treated Hy-A549. Values are mean ± SEM of three independent experiments in each case or representative of typical experiment.</p