20 research outputs found
Chemically Modified Peptides Targeting the PDZ Domain of GIPC as a Therapeutic Approach for Cancer
GIPC (GAIP-interacting protein, C terminus) represents
a new target
class for the discovery of chemotherapeutics. While many of the current
generation of anticancer agents function by directly binding to intracellular
kinases or cell surface receptors, the disruption of cytosolic protein–protein
interactions mediated by non-enzymatic domains is an underdeveloped
avenue for inhibiting cancer growth. One such example is the PDZ domain
of GIPC. Previously we developed a molecular probe, the cell-permeable
octapeptide CR1023 (<i>N</i>-myristoyl-PSQSSSEA), which
diminished proliferation of pancreatic cancer cells. We have expanded
upon that discovery using a chemical modification approach and here
report a series of cell-permeable, side chain-modified lipopeptides
that target the GIPC PDZ domain <i>in vitro</i> and <i>in vivo</i>. These peptides exhibit significant activity against
pancreatic and breast cancers, both in cellular and animal models.
CR1166 (<i>N</i>-myristoyl-PSQSKÂ(ε<i>N</i>-4-bromobenzoyl)ÂSKÂ(ε<i>N</i>-4-bromobenzoyl)ÂA), bearing
two halogenated aromatic units on alternate side chains, was found
to be the most active compound, with pronounced down-regulation of
EGFR/1GF-1R expression. We hypothesize that these organic acid-modified
residues extend the productive reach of the peptide beyond the canonical
binding pocket, which defines the limit of accessibility for the native
proteinogenic sequences that the PDZ domain has evolved to recognize.
Cell permeability is achieved with <i>N</i>-terminal lipidation
using myristate, rather than a larger CPP (cell-penetrating peptide)
sequence. This, in conjunction with optimization of targeting through
side chain modification, has yielded an approach that will allow the
discovery and development of next-generation cellular probes for GIPC
PDZ as well as for other PDZ domains
Bmi-1 knockdown perturbs the GSH biosynthesis pathway.
<p>The top panel represents total cellular GSH measured (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017918#s4" target="_blank">Materials and methods</a>) in ovarian
cancer cells transfected with scrambled control or Bmi-1 siRNA treated
with or without cisplatin for 24 h. The bottom panel represents fold
change in gene expression (normalized with beta actin and compared to
scrambled control) as determined by quantitative RT-PCR of ovarian
cancer cells transfected with scrambled control or Bmi-1 siRNA for 48
h.</p
Effect of Bmi-1 knockdown on orthotopic chemoresistant ovarian cancer growth.
<p>To assess the effects of siRNA therapy on tumor growth, treatment was
initiated 1 wk after i.p. injection (1.0×10<sup>6</sup> CP20) of
tumor cells. Mice were divided into four groups
(n = 10 mice per group): (a) control siRNA-DOPC
(150 µg/kg i.p. twice weekly), (b) control siRNA-DOPC +
cisplatin (160 µg/mouse i.p. weekly), (c) Bmi-1 siRNA-DOPC (150
µg/kg i.p. twice weekly), and (d) Bmi-1 siRNA-DOPC +
cisplatin (doses same as individual treatments). Treatment was continued
until 4 weeks after tumor inoculation before sacrifice. (A) Total RNA
was isolated from a portion of the tumor tissues and subjected to RT-PCR
using primers for Bmi-1 and beta actin. The comparative C<sub>t</sub>
method was used to calculate the relative abundance of mRNA compared
with that of beta actin expression. The experiment was performed in
triplicate and significance determined using two-sided Student's t
test, P<0.05 was considered significant. (B) Mouse and tumor weights
and (C) the number of tumor nodules for each group were compared using
Student's t test (for comparisons of two groups). A two-tailed
P≤0.05 was deemed statistically significant.</p
Bmi-1 knockdown augments engagement of the DDR pathway in cisplatin treated ovarian cancer cells.
<p>(A) Ovarian cancer cells transfected with scrambled control or Bmi-1
siRNA were treated with or without cisplatin for 48 h. Western blot was
performed for phospho Chk-2, total Chk-2, phospho-H2AX, total H2AX and
beta actin using respective antibodies. (B) Scrambled control or Bmi-1
siRNA transfected CP-70 cells were subjected to confocal microscopy
using 53BP1 antibody (red) and DAPI (blue nuclear staining) to
demonstrate nuclear foci formation.</p
Bmi-1 knockdown increases cisplatin-mediated ROS production in ovarian cancer cells.
<p>Ovarian cancer cells transfected with scrambled control or Bmi-1 siRNA
were treated with or without cisplatin for 24 h. Subsequently the cells
were incubated with 5 µM carboxy-H2DCFDA in fresh HBSS for 30 min
at 37°C. The cells were harvested with trypsin and fluorescence of
the labeled cells was measured at an excitation wavelength of 485 nm and
emission wavelength of 530 nm by using Fluorolog 3 (Jobin-Yvon Horiba).
Ratio of mean fluorescence intensity (MFI) with respect to the untreated
scrambled control is represented.</p
Effect of Bmi-1 knockdown on apoptotic markers.
<p>Ovarian cancer cells transfected with scrambled control or Bmi-1 siRNA
were treated with or without cisplatin for 48 h. Western blot was
performed for caspase-8, caspase-9 and PARP using respective
antibodies.</p
Glucocorticoid Receptor-Targeted Liposomal Codelivery of Lipophilic Drug and Anti-Hsp90 Gene: Strategy to Induce Drug-Sensitivity, EMT-Reversal, and Reduced Malignancy in Aggressive Tumors
Many
cancers including the late stage ones become drug-resistant and undergo
epithelial-to-mesenchymal transition (EMT). These lead to enhanced
invasion, migration, and metastasis toward manifesting its aggressiveness
and malignancy. One of the key hallmarks of cancer is its overdependence
on glycolysis as its preferred energy metabolism pathway. The strict
avoidance of alternate energy pathway gluconeogenesis by cancer cells
points to a yet-to-be hoisted role of glucocorticoid receptor (GR)
especially in tumor microenvironment, where cells are known to become
drug-sensitive through induction of gluconeogenesis. However, since
GR is involved in metabolism, anti-inflammatory reactions, immunity
besides inducing gluconeogenesis, a greater role of GR in tumor microenvironment
is envisaged. We have shown previously that GR, although ubiquitously
expressed in all cells; afford to be an effective cytoplasmic target
for killing cancer cells selectively. Herein, we report the therapeutic
use of a newly developed GR-targeted liposomal concoction (DXE) coformulating
a lipophilic drug (ESC8) and an anti-Hsp90 anticancer gene against
aggressive tumor models. This induced drug-sensitivity and apoptosis
while reversing EMT in tumor cells toward effective retardation of
aggressive growth in pancreas and skin tumor models. Additionally,
the ESC8-free lipid formulation upon cotreatment with hydrophilic
drugs, gemcitabine and doxorubicin, could effectively sensitize and
kill pancreatic cancer and melanoma cells, respectively. The formulation-triggered
EMT-reversal was GR-dependent. Overall, we found a new strategy for
drug sensitization that led to the advent of new GR-targeted anticancer
therapeutics
The Role of Factor Inhibiting HIF (FIH-1) in Inhibiting HIF-1 Transcriptional Activity in Glioblastoma Multiforme
<div><p>Glioblastoma multiforme (GBM) accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the <u>H</u>ypoxia-<u>I</u>nducible-<u>F</u>actor-1(HIF-1), which is a key regulator mediating the cellular response to hypoxia. It is known that <u>F</u>actor <u>I</u>nhibiting <u>H</u>IF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis.</p></div
Association of HIF-1α with p300 in the presence of FIH-1, PTEN in U87 cells.
<p>A: Western blot of HIF-1α from protein lysates of U87 cells incubated overnight either in hypoxic (3% oxygen) or normoxic (21% oxygen) conditions (top panel). No change of p300 protein levels was observed in the same samples (middle panel). β-actin was used as a loading control (lower panel). B: Association of HIF-1α with p300 in U87 cells: cells were transiently transfected with either FIH-1 or a PTEN expression plasmid, or both of them together for 24 hrs, and then incubated overnight under both normoxic and hypoxic conditions. Nuclear extracts were collected and subjected to immunoprecipitation with anti-p300 antibody followed by Western blot analysis with anti-HIF-1α (top panel) and IgG (middle panel). Nuclear extracts were also subjected to western blot analysis with anti- HIF-1α antibody (bottom panel).</p
The mRNA levels of GLUT-1 were reduced with FIH-1 overexpression.
<p>1A and 1B: U87 cells were transfected with FIH-1-expression plasmid and cultured in both hypoxic (1B) and normoxic (1A) conditions. Total RNA was harvested and RQ-PCR was performed using primers specific for GLUT-1 and 36B4 (internal control). A significant decrease of GLUT-1 mRNA levels was observed in cells transfected with FIH-1 expression plasmid in both normoxia and hypoxia. The data here is the mean from three independent results. * Represents p value less than 0.05. 1C and 1D: The expression levels of GLUT-1 were decreased by overexpression of FIH-1 in U87 cells. U87 cells were transfected with constructs either expressing FIH-1 or with vector alone and incubated for 48 hrs under normoxic condition. Cells were collected and subjected to flow cytometry analysis. A reduction in GLUT-1 levels was detected in cells transfected with FIH-1 plasmid (1D) compared with cells transfected with control vector (1C), as shown in the P3 population.</p