12 research outputs found

    Disulfide Cross-Linked Micelles for the Targeted Delivery of Vincristine to B-Cell Lymphoma

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    Vincristine (VCR) is a potent anticancer drug, but its clinical efficacy is limited by neurotoxicity. The field of drug delivery may provide an opportunity to increase the therapeutic index of VCR by delivering the drug specifically to tumor sites while sparing normal tissue. We have recently developed a telodendrimer (PEG<sup>5k</sup>-Cys<sub>4</sub>-L<sub>8</sub>-CA<sub>8</sub>) capable of forming disulfide cross-linked micelles (DCMs) which can encapsulate a variety of chemotherapeutics. In the present study, we encapsulated VCR into these micelles (DCM-VCR) and used them to treat lymphoma bearing mice. DCM-VCR particles have a size of 16 nm, which has been shown to be optimal for their accumulation into tumor via the enhanced permeability and retention (EPR) effect. Compared to our first-generation non-cross-linked micelles (NCMs), DCM-VCR demonstrated greater stability and slower drug release under physiological conditions. In addition, DCM-VCR exhibited a maximum tolerated dose (MTD) of 3.5 mg/kg while the MTD for conventional VCR was only 1.5 mg/kg. Using a near-infrared cyanine dye (DiD) as the surrogate drug, we showed that DCM-VCR accumulated at the tumor site starting 1 h after injection and persisted up to 72 h in lymphoma xenografted nude mice. In an <i>in vivo</i> efficacy study, high dose (2.5 mg/kg) DCM-VCR produced the greatest reduction in tumor volume. High dose DCM-VCR was well tolerated with no significant changes in complete blood count, serum chemistry and histology of the sciatic nerve. Mice treated with an equivalent dose (1 mg/kg) of conventional VCR and DCM-VCR controlled tumor growth equally; however, in combination with on-demand addition of the reducing agent <i>N</i>-acetylcysteine, DCM-VCR exhibited a superior antitumor effect compared to conventional VCR

    CTA095, a Novel Etk and Src Dual Inhibitor, Induces Apoptosis in Prostate Cancer Cells and Overcomes Resistance to Src Inhibitors

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    <div><p>Etk is a non-receptor tyrosine kinase, which provides a strong survival signal in human prostate cancer cells. Src, another tyrosine kinase that cross-activates with Etk, has been shown to play an important role in prostate cancer metastasis. Herein, we discovered a new class of Etk inhibitors. Within those inhibitors, CTA095 was identified as a potent Etk and Src dual inhibitor. CTA095 was found to induce autophagy as well as apoptosis in human prostate cancer cells. In addition, CTA095 inhibited HUVEC cell tube formation and “wound healing” of human prostate cancer cells, implying its role in inhibition of angiogenesis and metastasis of human prostate cancer. More interestingly, CTA095 could overcome Src inhibitor resistance in prostate cancer cells. It induces apoptosis in Src inhibitor resistant prostate cancer cells, likely through a mechanism of down regulation of Myc and BCL2. This finding indicates that simultaneously targeting Etk and Src could be a promising approach to overcome drug resistance in prostate cancer.</p></div

    CTA095 induces apoptosis in Src inhibitor resistant prostate cancer cells through Myc and BCL2 inhibition.

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    <p>PC3-AZD20 (PC3 cell resistant to 20 μM AZD0530) cells were seeded at 10<sup>6</sup> cells/well in 6 well plates overnight. The cells were treated with AZD0530 or CTA095 at 10 μM. Apoptosis was analyzed using Annexin-V FITC apoptosis detection kit (A). The mRNA levels of Myc and BCL2 were measured using real-time PCR (B). pEtk, Etk, pSrc, Src, pStat3, Stat3, Myc and BCL2 levels were measured using the corresponding antibodies through Western blot (C). Columns, mean; bars, standard deviation, n = 3.</p

    Induction of apoptosis of PC3 cells following treatment with CTA095.

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    <p>PC3 cells were seeded at 10<sup>6</sup> cells/ml (2 ml) in a 6-well plate overnight and then treated with CTA095 at the indicated concentrations for 24 h. Cell cycle arrest was analyzed using PI staining (A). Apoptosis was analyzed using Annexin-V FITC apoptosis detection kit (B). Caspase 9 activation was measured using western blot (D). For caspase 3/7 activity, PC3 cells were seeded at 5000 cells/well in 96 well plate overnight and treated with CTA095 at 0–10 μM for 24 h. Caspase-3/7 activities were measured using the Apo-ONE Homogeneous Caspase-3/7 Assay kit (Promega, Madison, WI) according to the manufacturer's instruction. Columns, mean; bars, standard deviation, n = 3. 5 μM and 10 μM are significantly different from 0 μM (*, p<0.05, one-way ANOVA with Tukey test for pair wise comparison).</p

    Molecular Modeling of CTA095-Etk binding.

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    <p>(A) Surface view of Etk docked to CTA095 after 20 ns of MD minimization and relaxation. R<sub>3</sub> and the three-ring core docked deeper between Glycine-rich loop region (cyan) and hinge region (orange), R<sub>1</sub> and R<sub>2</sub> are solvent exposed. Blue: Helix C; Red: Activation Loop; Green: DFG motif (554–556); Cyan: Glycine-rich loop; Orange; Hinge Region. (B) Cartoon representation showing predicted interactions of CTA095 with the gatekeeper Thr489, DFG (554–556) motif, and Cys496. CTA095 binding stabilizes Phe555 in ‘out’ configuration, and affects the active state salt bridge formation between Lys445 and Glu460. Blue: Helix C; Red: Activation Loop; Green: DFG motif (554–556); Cyan: Glycine-rich loop; Orange; Hinge Region. Figures were generated using PyMol.</p

    Induction of autophagy in PC3 cells by CTA095.

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    <p>Cells were grown in 6-well plate to 50% confluence and treated with CTA095. Autophagy was visualized by GFP-LC3 “puncta” (A) and immunoblot of Endogenous LC3 isoforms (B). All experiments were carried out 24 h after treatment.</p

    CTA095 as a chemo sensitizer.

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    <p>Growth Inhibition of CTA095 and in combination with 10 μM chloroquine (CQ), or 2 ng/ml paclitaxel (PTX) to PC3 human prostate cancer cells. Cells were seeded at 5,000 cells/well in 96-well plate overnight and pretreated with the corresponding co-treatments for 1h, then treated with 2.5 μM CTA095. The cell viability was measured using MTT assay after 72 h. Columns, mean; bars, standard deviation, n = 3.</p