Microscopic Rearrangement of Bound Minor Groove Binders Detected by NMR

Thermodynamic and structural studies are commonly utilized to optimize small molecules for specific DNA interactions, and, thus, a significant amount of binding data is available. However, the dynamic processes that are involved in minor groove complex formation and maintenance are not fully understood. To help define the processes involved, we have conducted 1D and 2D NMR in conjunction with biosensor-SPR experiments with a variety of compounds and symmetric, as well as asymmetric, AT tract DNA sequences. Surprisingly, the NMR data clearly show exchange between equivalent binding sites for strongly binding compounds like netropsin and DB921 (<i>K</i>_a > 10⁸ M^–1) that does not involve dissociation off the DNA. A quantitative analysis of the data revealed that these bound exchange rates are indeed much faster than the macroscopic dissociation rates which were independently determined by biosensor-SPR. Additionally, we could show the existence of at least two 1:1 compound DNA complexes at the same site for the interaction of these compounds with an asymmetric DNA sequence. To explain this behavior we introduced a model in which the ligand is rapidly flipping between two orientations while in close association with the DNA. The ligand reorientation will contribute favorably to the binding entropy. As the potential of minor groove binders to form more than a single complex with asymmetric, as well as symmetric, duplexes is widely unknown, the consequences for binding thermodynamics and compound design are discussed

Gold Nanoclusters Doped with ⁶⁴Cu for CXCR4 Positron Emission Tomography Imaging of Breast Cancer and Metastasis

As an emerging class of nanomaterial, nanoclusters hold great potential for biomedical applications due to their unique sizes and related properties. Herein, we prepared a ⁶⁴Cu doped gold nanocluster (⁶⁴CuAuNC, hydrodynamic size: 4.2 ± 0.5 nm) functionalized with AMD3100 (or Plerixafor) for targeted positron emission tomography (PET) imaging of CXCR4, an up-regulated receptor on primary tumor and lung metastasis in a mouse 4T1 orthotopic breast cancer model. The preparation of targeted ⁶⁴CuAuNCs–AMD3100 (4.5 ± 0.4 nm) was done <i>via</i> one-step reaction with controlled conjugation of AMD3100 and specific activity, as well as improved colloid stability. <i>In vivo</i> pharmacokinetic evaluation showed favorable organ distribution and significant renal and fecal clearance within 48 h post injection. The expression of CXCR4 in tumors and metastasis was characterized by immunohistochemistry, Western blot, and reverse transcription polymerase chain reaction analysis. PET imaging with ⁶⁴CuAuNCs–AMD3100 demonstrated sensitive and accurate detection of CXCR4 in engineered tumors expressing various levels of the receptor, while competitive receptor blocking studies confirmed targeting specificity of the nanoclusters. In contrast to nontargeted ⁶⁴CuAuNCs and ⁶⁴Cu–AMD3100 alone, the targeted ⁶⁴CuAuNCs–AMD3100 detected up-regulated CXCR4 in early stage tumors and premetastatic niche of lung earlier and with greater sensitivity. Taken together, we believe that ⁶⁴CuAuNCs–AMD3100 could serve as a useful platform for early and accurate detection of breast cancer and metastasis providing an essential tool to guide the treatment

BMSCs secrete a soluble factor(s) that protects APL cells from Ara-C induced apoptosis.

<p>(A) Adherence assay: APL cells were cultured alone, co-cultured with M2-BMSCs or with fibronectin-coated plates for 24 hours. Cells were processed and quantification of APL GR1+ cells in the supernatant and adhered fractions were performed by flow cytometry analysis. Adherence was calculated as the percentage of APL cells present in the adhered fraction in relation to the total amount of APL cells in both fractions. For chemosensitivity studies: (B) APL cells were cultured alone, cultured using fibronectin pre-coated wells, or cultured with M2-BMSCs for 4 hours; (C) APL were cultured with or without M2-BMSCs or using a transwell system. Cultures were incubated for 2 hours before treatment with Ara-C (125, 250, 500 ng/ml) for 24 hours or vehicle alone (control). APL cell death was assessed by flow cytometry using the GR-1-APC mouse antibody and the annexin V-PE apoptosis kit. Each bar represents the mean ± SEM of 3 independent experiments. **p<0.01 and ***p<0.001 (all groups versus APL).</p

Primary mouse and human BMSCs supernatant protect leukemia cells from Ara-C induced cytotoxicity.

<p>APL and U-937cells were cultured with or without primary mouse BM stromal cell supernatant (PM-BM SN) or human BM SN (HS5-BM SN) for 2 hours before treatment with Ara-C (0, 250 and 500 ng/ml) (A) or Ara-C (0, 300 and 600 ng/ml) (B) for 24 hours. Leukemia cell viability was assessed by the MTT assay. Each bar represents the mean ± SD of 3 independent experiments. ***<i>p</i><0.001 (leukemia cells versus leukemia cells + mouse or human BM SN).</p

BMSCs supernatant caused no APL cell cycle arrest.

<p>APL cells were cultured with or without M2-BM SN for 24 hours. Cells were harvested, fixed and stained with PI and analyzed by flow cytometry. Upper panels show representative histograms of APL cells in the absence (left upper panel) and presence of M2-BM SN (right upper panel). Lower figure shows mean and SD of three separate experiments.</p

BMSCs mediated chemoprotection prevents the activation of the apoptotic cell death pathway.

<p>APL cells were cultured alone or co-cultured with M2-BMSCs for 2 hours before treatment with Ara-C (125, 250 and 500 ng/ml) or vehicle alone (control) for 24 hours. (A) Caspase-3 activation was assessed by flow cytometry using a PE-active caspase-3 monoclonal antibody and mouse GR-1-FITC antibody. (B) Cell lysates were processed for western blot analysis to detect cleaved PARP using a mouse monoclonal antibody. Each bar represents the mean ± SD of triplicates from individual experiments. *<i>p</i><0.05; and ***<i>p</i><0.001 (APL versus APL + BMSCs).</p

BMSC soluble factor(s) inhibits mENT1 activity and protect APL cells from AraC-induced apoptosis.

<p>(A) Measurement of mENT1 activity. APL cells were cultured alone or with M2-BM SN for 24 hours. Cells were harvested and mENT1 activity was measured by the incorporation of radioactive ³H-adenosine (B) RNA extraction from APL cells cultured alone or in presence of M2-BM SN for a time-course of 0, 3, 6, 9, 15, and 24 hours was used for RT-PCR to detect expression of mENT1. (C) Western blot analysis for mENT1 using APL cells with or without M2-BM SN for 24 hours. Each bar represents the mean ± SD of 3 independent experiments. ***<i>p</i><0.001 (APL versus APL SN).</p

BM microenvironment modulates protection of APL cells to Ara-C induced apoptosis but not to radiation in vitro and in vivo.

<p>(A) APL cells were cultured in absence or presence of M2-BMSCs for 2 hours before treatment with Ara-C (125, 250, 500 ng/ml) or vehicle alone (control). APL cell death was assessed by flow cytometry using a GR-1-APC mouse antibody and the annexin V-FITC apoptosis kit. (B) Cultures (as described 1A) were exposed to various radiation doses (200, 400 and 600 cGy) and allowed to recover for 24 before cell death was assessed as mentioned in (1A). (C) Kaplan Meier plot of overall survival of mice. Syngeneic B6129F1 recipient mice were intravenously injected with 10⁶ APL cells. On day 12 post-APL injection, mice were left untreated (control; n = 6) or treated with AMD3100 alone (n = 7), Ara-C alone (n = 8), or the combination of AMD3100 and Ara-C (n = 8). Mice treated with chemotherapy received a single injection of Ara-C (500 mg/Kg) on days 12 and 13 post-APL injection. Mice treated with AMD3100 (5 mg/Kg) received subcutaneous injections 1 hour before and three hours after Ara-C treatment. (D) Kaplan Meier plot of overall survival of mice. Syngeneic B6129F1 recipient mice were intravenously injected with 10⁶ APL cells. On day 12 post-APL injection, mice were left untreated (control; n = 6), exposed to radiation (350 cGy) (n = 8), or the combination of AMD3100 and radiation (n = 8). Mice treated with AMD3100 (5 mg/Kg) received a single subcutaneous injection 2 hours before radiation treatment. Each bar represents the mean ± SEM of 3 independent experiments. ***<i>p</i><0.001 (APL + BMSCs versus APL). Overall survival of leukemic mice is not significantly prolonged when recipients are treated with the combination of AMD3100 and radiation versus radiation cohorts).</p