17 research outputs found
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Evaluation of integrin αvβ6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis.
Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvβ6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvβ6
Formulation of Anti-miR-21 and 4‑Hydroxytamoxifen Co-loaded Biodegradable Polymer Nanoparticles and Their Antiproliferative Effect on Breast Cancer Cells
Breast cancer is the second leading
cause of cancer-related death
in women. The majority of breast tumors are estrogen receptor-positive
(ER+) and hormone-dependent. Neoadjuvant anti-estrogen therapy has
been widely employed to reduce tumor mass prior to surgery. Tamoxifen
is a broadly used anti-estrogen for early and advanced ER+ breast
cancers in women and the most common hormone treatment for male breast
cancer. 4-Hydroxytamoxifen (4-OHT) is an active metabolite of tamoxifen
that functions as an estrogen receptor antagonist and displays higher
affinity for estrogen receptors than that of tamoxifen and its other
metabolites. MicroRNA-21 (miR-21) is a small noncoding RNA of 23 nucleotides
that regulates several apoptotic and tumor suppressor genes and contributes
to chemoresistance in numerous cancers, including breast cancer. The
present study investigated the therapeutic potential of 4-OHT and
anti-miR-21 coadministration in an attempt to combat tamoxifen resistance,
a common problem often encountered in anti-estrogen therapy. A biodegradable
polyÂ(d,l-lactide-<i>co</i>-glycolide)-<i>block</i>-polyÂ(ethylene glycol) (PLGA-<i>b</i>-PEG-COOH)
copolymer was utilized as a carrier to codeliver 4-OHT and anti-miR-21
to ER+ breast cancer cells. 4-OHT and anti-miR-21 co-loaded PLGA-<i>b</i>-PEG nanoparticles (NPs) were developed using emulsion-diffusion
evaporation (EDE) and water-in-oil-in-water (w/o/w) double emulsion
methods. The EDE method was found to be best method for 4-OHT loading,
and the w/o/w method proved to be more effective for coloading NPs
with anti-miR-21 and 4-OHT. The optimal NPs, which were prepared using
the double emulsion method, were evaluated for their antiproliferative
and apoptotic effects against MCF7, ZR-75-1, and BT-474 human breast
cancer cells as well as against 4T1 mouse mammary carcinoma cells.
We demonstrated that PLGA-<i>b</i>-PEG NP encapsulation
significantly extended 4-OHT’s stability and biological activity
compared to that of free 4-OHT. MTT assays indicated that treatment
of MCF7 cells with 4-OHT–anti-miR-21 co-loaded NPs resulted
in dose-dependent antiproliferative effects at 24 h, which was significantly
higher than what was achieved with free 4-OHT at 48 and 72 h post-treatment.
Cell proliferation analysis showed that 4-OHT and anti-miR-21 co-loaded
NPs significantly inhibited MCF-7 cell growth compared to that of
free 4-OHT (1.9-fold) and untreated cells (5.4-fold) at 1 μM
concentration. The growth rate of MCF7 cells treated with control
NPs or NPs loaded with anti-miR-21 showed no significant difference
from that of untreated cells. These findings demonstrate the utility
of the PLGA-<i>b</i>-PEG polymer NPs as an effective nanocarrier
for co-delivery of anti-miR-21 and 4-OHT as well as the potential
of this drug combination for use in the treatment of ER+ breast cancer
Degron Protease Blockade Sensor to Image Epigenetic Histone Protein Methylation in Cells and Living Animals
Lysine
methylation of histone H3 and H4 has been identified as
a promising therapeutic target in treating various cellular diseases.
The availability of an <i>in vivo</i> assay that enables
rapid screening and preclinical evaluation of drugs that potentially
target this cellular process will significantly expedite the pace
of drug development. This study is the first to report the development
of a real-time molecular imaging biosensor (a fusion protein, [FLuc2]-[Suv39h1]-[(G4S)<sub>3</sub>]-[H3-K9]-[cODC]) that can detect and monitor the methylation
status of a specific histone lysine methylation mark (H3-K9) in live
animals. The sensitivity of this sensor was assessed in various cell
lines, in response to down-regulation of methyltransferase EHMT2 by
specific siRNA, and in nude mice with lysine replacement mutants. <i>In vivo</i> imaging in response to a combination of methyltransferase
inhibitors BIX01294 and Chaetocin in mice reveals the potential of
this sensor for preclinical drug evaluation. This biosensor thus has
demonstrated its utility in the detection of H3-K9 methylations <i>in vivo</i> and potential value in preclinical drug development
Gemcitabine and Antisense-microRNA Co-encapsulated PLGA–PEG Polymer Nanoparticles for Hepatocellular Carcinoma Therapy
Hepatocellular carcinoma
(HCC) is highly prevalent, and the third
most common cause of cancer-associated deaths worldwide. HCC tumors
respond poorly to chemotherapeutic anticancer agents due to inherent
and acquired drug resistance, and low drug permeability. Targeted
drug delivery systems with significant improvement in therapeutic
efficiency are needed for successful HCC therapy. Here, we report
the results of a technique optimized for the synthesis and formulation
of antisense-miRNA-21 and gemcitabine (GEM) co-encapsulated PEGylated-PLGA
nanoparticles (NPs) and their <i>in vitro</i> therapeutic
efficacy in human HCC (Hep3B and HepG2) cells. Water-in-oil-in-water
(w/o/w) double emulsion method was used to coload antisense-miRNA-21
and GEM in PEGylated-PLGA-NPs. The cellular uptake of NPs displayed
time dependent increase of NPs concentration inside the cells. Cell
viability analyses in HCC (Hep3B and HepG2) cells treated with antisense-miRNA-21
and GEM co-encapsulated NPs demonstrated a nanoparticle concentration
dependent decrease in cell proliferation, and the maximum therapeutic
efficiency was attained in cells treated with nanoparticles co-encapsulated
with antisense-miRNA-21 and GEM. Flow cytometry analysis showed that
control NPs and antisense-miRNA-21-loaded NPs are not cytotoxic to
both HCC cell lines, whereas treatment with free GEM and GEM-loaded
NPs resulted in ∼9% and ∼15% apoptosis, respectively.
Cell cycle status analysis of both cell lines treated with free GEM
or NPs loaded with GEM or antisense-miRNA-21 displayed a significant
cell cycle arrest at the S-phase. Cellular pathway analysis indicated
that Bcl2 expression was significantly upregulated in GEM treated
cells, and as expected, PTEN expression was noticeably upregulated
in cells treated with antisense-miRNA-21. In summary, we successfully
synthesized PEGylated-PLGA nanoparticles co- encapsulated with antisense-miRNA-21
and GEM. These co-encapsulated nanoparticles revealed increased treatment
efficacy in HCC cells, compared to cells treated with either antisense-miRNA-21-
or GEM-loaded NPs at equal concentration, indicating that down-regulation
of endogenous miRNA-21 function can reduce HCC cell viability and
proliferation in response to GEM treatment
Suzuki–Miyaura Cross-Coupling of Potassium Trifluoro(<i>N</i>‑methylheteroaryl)borates with Aryl and Heteroaryl Halides
The
synthesis of potassium trifluoroÂ(<i>N</i>-methylheteroaryl)Âborates
and their use in cross-coupling reactions with various aryl and heteroaryl
halides to construct <i>N</i>-methyl heteroaryl-substituted
aromatic and heteroaromatic compounds are reported
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
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