4 research outputs found
Nanoparticle-Delivered Antisense MicroRNA-21 Enhances the Effects of Temozolomide on Glioblastoma Cells
Glioblastoma
(GBM) generally exhibits high IC<sub>50</sub> values
for its standard drug treatment, temozolomide (TMZ). MicroRNA-21 (miR-21)
is an oncomiR overexpressed in GBM, thus controlling important aspects
of glioma biology. We hypothesized that PLGA nanoparticles carrying
antisense miR-21 to glioblastoma cells might beneficially knock down
endogenous miR-21 prior to TMZ treatment. PLGA nanoparticles encapsulating
antisense miR-21 were effective in intracellular delivery and sustained
silencing (<i>p</i> < 0.01) of miR-21 function in U87
MG, LN229, and T98G cells. Prior antisense miR-21 delivery significantly
reduced the number of viable cells (<i>p</i> < 0.001),
and increased (1.6-fold) cell cycle arrest at G2/M phase upon TMZ
treatment in U87 MG cells. There was overexpression of the miR-21
target genes <i>PTEN</i> (by 67%) and <i>caspase-3</i> (by 15%) upon cotreatment. This promising PLGA nanoparticle-based
platform for antisense miR-21 delivery to GBM is an effective cotherapeutic
strategy in cell culture, warranting the need for further studies
prior to future clinical translation
Molecular Imaging Biosensor Monitors p53 Sumoylation in Cells and Living Mice
Small
molecule mediated stabilization of p53 tumor suppressor protein
through sumoylation is a promising new strategy for improving cancer
chemotherapy. A molecular tool that monitors p53 sumoylation status
and expedites screening for drugs that enhance p53 sumoylation would
be beneficial. We report a molecularly engineered reporter fragment
complementation biosensor based on optical imaging of Firefly luciferase
(FLuc), to quantitatively image p53 sumoylation and desumoylation
in cells and living mice. We initially characterized this biosensor
by successfully imaging sumoylation of several target proteins, achieving
significant FLuc complementation for ERα (<i>p</i> < 0.01), p53 (<i>p</i> < 0.005), FKBP12 (<i>p</i> < 0.03), ID (<i>p</i> < 0.03), and HDAC1
(<i>p</i> < 0.002). We then rigorously tested the sensitivity
and specificity of the biosensor using several variants of p53 and
SUMO1, including deletion mutants, and those with modified sequences
containing the SUMO-acceptor site of target proteins. Next we evaluated
the performance of the biosensor in HepG2 cells by treatment with
ginkgolic acid, a drug that reduces p53 sumoylation, as well as trichostatin
A, a potential inducer of p53 sumoylation by enhancement of its nuclear
export. Lastly, we demonstrated the in vivo utility of this biosensor
in monitoring and quantifying the effects of these drugs on p53 sumoylation
in living mice using bioluminescence imaging. Adoption of this biosensor
in future high throughput drug screening has the important potential
to help identify new and repurposed small molecules that alter p53
sumoylation, and to preclinically evaluate candidate anticancer drugs
in living animals
Polymer Nanoparticles Mediated Codelivery of AntimiR-10b and AntimiR-21 for Achieving Triple Negative Breast Cancer Therapy
The current study shows the therapeutic outcome achieved in triple negative breast cancer (TNBC) by simultaneously antagonizing miR-21-induced antiapoptosis and miR-10b-induced metastasis, using antisense-miR-21-PS and antisense-miR-10b-PS delivered by polymer nanoparticles (NPs). We synthesized the antisense-miR-21 and antisense-miR-10b loaded PLGA-<i>b</i>-PEG polymer NPs and evaluated their cellular uptake, serum stability, release profile, and the subsequent synchronous blocking of endogenous miR-21 and miR-10b function in TNBC cells in culture, and tumor xenografts in living animals using molecular imaging. Results show that multitarget antagonization of endogenous miRNAs could be an efficient strategy for targeting metastasis and antiapoptosis in the treatment of metastatic cancer. Targeted delivery of antisense-miR-21 and antisense-miR-10b coloaded urokinase plasminogen activator receptor (uPAR) targeted polymer NPs treated mice showed substantial reduction in tumor growth at very low dose of 0.15 mg/kg, compared to the control NPs treated mice and 40% reduction in tumor growth compared to scramble peptide conjugated NPs treated mice, thus demonstrating a potential new therapeutic option for TNBC
Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications
Gaussia
luciferase (GLUC) is a bioluminescent reporter protein
of increasing importance. As a secretory protein, it has increased
sensitivity <i>in vitro</i> and <i>in vivo</i> (∼20 000-fold, and ∼1000-fold, respectively)
over its competitor, secreted alkaline phosphatase. Unfortunately,
this same advantageous secretory nature of GLUC limits its usefulness
for many other possible intracellular applications, <i>e.g.</i>, imaging signaling pathways in intact cells, <i>in vivo</i> imaging, and in developing molecular imaging biosensors to study
protein–protein interactions and protein folding. Hence, to
widen the research applications of GLUC, we developed engineered variants
that increase its intracellular retention both by modifying the N-terminal
secretory signal peptide and by tagging additional sequences to its
C-terminal region. We found that when GLUC was expressed in mammalian
cells, its N-terminal secretory signal peptide comprising amino acids
1–16 was essential for GLUC folding and functional activity
in addition to its inherent secretory property. Modification of the
C-terminus of GLUC by tagging a four amino acid (KDEL) endoplasmic
reticulum targeting peptide in multiple repeats significantly improved
its intracellular retention, with little impact on its folding and
enzymatic activity. We used stable cells expressing this engineered
GLUC with KDEL repeats to monitor chemically induced endoplasmic reticulum
stress on cells. Additionally, we engineered an apoptotic sensor using
modified variants of GLUC containing a four amino acid caspase substrate
peptide (DEVD) between the GLUC protein and the KDEL repeats. Its
use in cell culture resulted in increased GLUC secretion in the growth
medium when cells were treated with the chemotherapeutic drugs doxorubicin,
paclitaxel, and carboplatin. We thus successfully engineered a new
variant GLUC protein that is retained inside cells rather than secreted
extracellularly. We validated this novel reporter by incorporating
it in biosensors for detection of cellular endoplasmic reticulum stress
and caspase activation. This new molecularly engineered enzymatic
reporter has the potential for widespread applications in biological
research