PDF file - 138K, Image of H-4073 docked with the STAT3 monomer and STAT3 translocation assay.

PDF file - 116K, Viability of SKOV3, hOSE, and H9c2 cells treated with H-4073 or HO-3867.

PDF file - 146K, Methods for HO-3867, a safe STAT3 inhibitor, is selectively cytotoxic to ovarian cancer.

PDF file - 156K, Effect of H-4073 in both cancer and normal cells. Phosphokinase assay array using CHO cells.

PDF file - 220K, Representative Annexin V flow cytomety plots of CHO, H9c2 and A2780 cells.

PDF file - 77K, Immunoblot analysis for pSTAT3, and Bcl-2 using 2 different mice tissue.

PDF file - 95K, Results of Tukey's post-hoc test and Molecular weights of the pure DAP compounds.

PDF file - 94K, Western blots showing inhibition of pSTAT3 Y705 by HO-3867 treatment in primary cancer cell lines.

PDF file - 177K, Effect of DAPs on SKOV3 ovarian cancer and hOSE cells.

PDF file - 122K, Illustration of the GFP-Histone 2B fusion protein (pBOS H2BGFP) vector.

PDF file - 282K, H&E-stained organ tissue samples and TUNEL Stain.

PDF file - 692K, (A) H-4073 Cell uptake in A2780 and CHO cell lines; (B) Ion chromatogram of pure HO-3867 in A2780 cells; (C) Ion chromatogram of pure HO-3867 in CHO cells; (D) Extracted ion chromatograms - H-4073; (E)Extracted ion chromatograms - H-4073.

PDF file - 172K, EPR spectra from organ tissue samples obtained from HO-3867-treated mice.

PDF file - 163K, Representative Annexin V flow cytomety plots of chemotherapy-resistant primary cell lines.

Myocardial infarction (MI), which occurs often due to acute ischemia followed by reflow, is associated with irreversible loss (death) of cardiomyocytes. If left untreated, MI will lead to progressive loss of viable cardiomyocytes, deterioration of cardiac function, and congestive heart failure. While supplemental oxygen therapy has long been in practice to treat acute MI, there has not been a clear scientific basis for the observed beneficial effects. Further, there is no rationale for the amount or duration of administration of supplemental oxygenation for effective therapy. The goal of the present study was to determine an optimum oxygenation protocol that can be clinically applicable for treating acute MI. Using EPR oximetry, we studied the effect of exposure to supplemental oxygen cycling (OxCy) administered by inhalation of 21-100% oxygen for brief periods (15-90 min), daily for 5 days, using a rat model of acute MI. Myocardial oxygen tension (pO2), cardiac function and pro-survival/apoptotic signaling molecules were used as markers of treatment outcome. OxCy resulted in a significant reduction of infarct size and improvement of cardiac function. An optimal condition of 30-min OxCy with 95% oxygen + 5% CO2 under normobaric conditions was found to be effective for cardioprotection.

Size exclusion chromatography (SEC) is the most commonly used technique for the determination of protein heterogeneity derived from the presence of multiple biomolecules and their interaction via non‐covalent interactions. On‐line detection of eluting species by native mass spectrometry (MS) provides a clear advantage over the traditionally used UV‐spectroscopic detection as it allows for the direct and unambiguous molecular weight measurement of the eluting species. Here, we demonstrate that the combination of sub 2μm particle SEC separation with high‐resolution mass detection is suitable for the analysis of a wide range of proteins. The possibility to detect as little as 100 ng protein makes it possible to analyze complex mixtures at low samples consumption. Non‐ideal interactions of proteins with the stationary phase as indicated by a non‐linear relationship between the logarithm of the molecular weight and the elution time within the column separation range make it challenging to find standard calibration proteins. We show that a mixture of commercially available proteins can be used to assess and monitor the column performance and reproducibility under MS‐compatible mobile phase conditions. The ability to resolve slight molecular weight changes by high resolution MS makes it possible to detect a variety of biomolecular interactions, which can be further studied by surface induced dissociation (SID) and collision induced dissociation (CID). The suitability of this technique to monitor a protein‐ligand interaction in a protein mixture is demonstrated in the case of the biotin‐streptavidin interaction. Support or Funding Information We thank the National Institutes of Health (NIH) for funding [grant R01 GM113658].

Abstract STAT3 is well corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (–NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecular modeling studies of this class suggested direct interaction with the STAT3 DNA binding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to be multifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867–mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated from patient ascites, HO-3867 inhibited cell migration/invasion and survival. Our results offer preclinical proof-of-concept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated. Cancer Res; 74(8); 2316–27. ©2014 AACR.

The use of electron paramagnetic resonance (EPR) oximetry for oxygen measurements in deep tissues (>1 cm) is challenging due to the limited penetration depth of the microwave energy. To overcome this limitation, implantable resonators, having a small (0.5 mm diameter) sensory loop containing the oxygen-sensing paramagnetic material connected by a pair of twisted copper wire to a coupling loop (8–10 mm diameter), have been developed, which enable repeated measurements of deep-tissue oxygen levels (pO2, partial pressure of oxygen) in the brain and tumors of rodents. In this study, we have demonstrated the feasibility of measuring dynamic changes in pO2 in the heart and lung of rats using deep-tissue implantable oxygen sensors. The sensory loop of the resonator contained lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) crystals embedded in polydimethylsiloxane (PDMS) polymer and was implanted in the myocardial tissue or lung pleura. The external coupling loop was secured subcutaneously above chest. The rats were exposed to different breathing gas mixtures while undergoing EPR measurements. The results demonstrated that implantable oxygen sensors provide reliable measurements of pO2 in deep tissues such as heart and lung under adverse conditions of cardiac and respiratory motions.

Pulmonary hypertension (PH) is a disorder of lung vasculature characterized by arterial narrowing. Phosphatase-and-tensin homolog on chromosome 10 (PTEN), associated in the progression of multiple cancers, is implicated in arterial remodeling. However, the involvement of PTEN in PH remains unclear. The objective of the present study was to determine the role of PTEN in pulmonary vascular remodeling using established models of PH. The study used rat models of PH, induced by monocrotaline (MCT) administration (60 mg/kg) or continuous hypoxic exposure (10% oxygen) for 3 weeks. Pulmonary artery smooth muscle cells (SMCs) were used for in vitro confirmation. Development of PH was verified by hemodynamic, morphological and histopathology analyses. PTEN and key downstream proteins in pulmonary and cardiac tissues were analyzed by western blotting and RT-PCR. PTEN was significantly decreased (MCT, 53%; Hypoxia, 40%), pAkt was significantly increased (MCT, 42%; Hypoxia, 55%) in tissues of rats with PH. Similar results were observed in SMCs exposed to hypoxia (1% oxygen) for 48 h. Ubiquitination assay showed that PTEN degradation occurs via proteasomal degradation pathway. Western blotting demonstrated a significant downregulation of cell-cycle regulatory proteins p53 and p27, and upregulation of cyclin-D1 in the lungs of both models. The results showed that PTEN-mediated modulation of PI3K pathway was independent of the focal adhesion kinase and fatty acid synthase. The study, for the first time, established that PTEN plays a key role in the progression of pulmonary hypertension. The findings may have potential for the treatment of pulmonary hypertension using PTEN as a target.

A diagnosis of advanced ovarian cancer is the beginning of a long and arduous journey for a patient. Worldwide, approximately half of the individuals undergoing therapy for advanced cancer will succumb to the disease, or consequences of treatment. Well-known and widely-used chemotherapeutic agents such as cisplatin, paclitaxel, 5-fluorouracil, and doxorubicin are toxic to both cancer and non-cancerous cells, and have debilitating side effects Therefore, development of new targeted anticancer therapies that can selectively kill cancer cells while sparing the surrounding healthy tissues is essential to develop more effective therapies. We have developed a new class of synthetic curcumin analogs, diarylidenyl-piperidones (DAPs), which have higher anticancer activity and enhanced bio-absorption than curcumin. The DAP backbone structure exhibits cytotoxic (anticancer) activity, whereas the N-hydroxypyrroline (-NOH) moiety found on some variants functions as a cellular- or tissue-specific modulator (antioxidant) of cytotoxicity. The anticancer activity of the DAPs has been evaluated using a number of ovarian cancer cell lines, and the safety has been evaluated in a number of non-cancerous cell lines. Both variations of the DAP compounds showed similar levels of cell death in ovarian cancer cells, however the compounds with the -NOH modification were less toxic to non-cancerous cells. The selective cytotoxicity of the DAP–NOH compounds suggests that they will be useful as safe and effective anticancer agents. This article reviews some of the key findings of our work with the DAP compounds, and compares this to some of the targeted therapies currently used in ovarian cancer therapy.