Association of the respiratory severity score with bronchopulmonary dysplasia-associated pulmonary hypertension in infants born extremely preterm.

OBJECTIVE: To test the hypothesis that elevations in the respiratory severity score (RSS) are associated with increased probability of bronchopulmonary dysplasia-associated pulmonary hypertension (BPD-PH). STUDY DESIGN: Retrospective cohort study of infants born extremely preterm admitted to a BPD center between 2010 and 2018. Echocardiograms obtained ≥ 36 weeks' post-menstrual age (PMA) were independently adjudicated by two blinded cardiologists to determine the presence/absence of BPD-PH. Multivariable logistic regression estimated the association between RSS and BPD-PH. RESULT: BPD-PH was observed in 68/223 (36%) of subjects. The median RSS at time of echocardiography was 3.04 (Range 0-18.3). A one-point increase in the RSS was associated with BPD-PH, aOR 1.3 (95% CI 1.2-1.4), after adjustment for gestational age and PMA at time of echocardiography. CONCLUSION: Elevations in the RSS were associated with a greater probability of BPD-PH. Prospective studies are needed to determine the validity and performance of RSS as a clinical susceptibility/risk biomarker for BPD-PH.

Proactive neonatal treatment at 22 weeks of gestation: a systematic review and meta-analysis.

OBJECTIVE: The objective of this study was to provide a systematic review and meta-analysis to quantify prognosis and identify factors associated with variations in reported mortality estimates among infants who were born at 22 weeks of gestation and provided proactive treatment (resuscitation and intensive care). DATA SOURCES: PubMed, Scopus, and Web of Science databases, with no language restrictions, were searched for articles published from January 2000 to February 2020. STUDY ELIGIBILITY CRITERIA: Reports on live-born infants who were delivered at 22 weeks of gestation and provided proactive care were included. The primary outcome was survival to hospital discharge; secondary outcomes included survival without major morbidity and survival without neurodevelopmental impairment. Because we expected differences across studies in the definitions for various morbidities, multiple definitions for composite outcomes of major morbidities were prespecified. Neurodevelopmental impairment was based on Bayley Scales of Infant Development II or III. Data extractions were performed independently, and outcomes agreed on a priori. STUDY APPRAISAL AND SYNTHESIS METHODS: Methodological quality was assessed using the Quality in Prognostic Studies tool. An adapted version of the Grading of Recommendations Assessment, Development and Evaluation approach for prognostic studies was used to evaluate confidence in overall estimates. Outcomes were assessed as prevalence and 95% confidence intervals. Variabilities across studies attributable to heterogeneity were estimated with the I2 statistic; publication bias was assessed with the Luis Furuya-Kanamori index. Data were pooled using the inverse variance heterogeneity model. RESULTS: Literature searches returned 21,952 articles, with 2034 considered in full; 31 studies of 2226 infants who were delivered at 22 weeks of gestation and provided proactive neonatal treatment were included. No articles were excluded for study design or risk of bias. The pooled prevalence of survival was 29.0% (95% confidence interval, 17.2-41.6; 31 studies, 2226 infants; I2=79.4%; Luis Furuya-Kanamori index=0.04). Survival among infants born to mothers receiving antenatal corticosteroids was twice the survival of infants born to mothers not receiving antenatal corticosteroids (39.0% vs 19.5%; P<.01). The overall prevalence of survival without major morbidity, using a definition that includes any bronchopulmonary dysplasia, was 11.0% (95% confidence interval, 8.0-14.3; 10 studies, 374 infants; I2=0%; Luis Furuya-Kanamori index=3.02). The overall rate of survival without moderate or severe impairment was 37.0% (95% confidence interval, 14.6-61.5; 5 studies, 39 infants; I2=45%; Luis Furuya-Kanamori index=-0.15). Based on the year of publication, survival rates increased between 2000 and 2020 (slope of the regression line=0.09; standard error=0.03; P<.01). Studies were highly diverse with regard to interventions and outcomes reported. CONCLUSION: The reported survival rates varied greatly among studies and were likely influenced by combining observational data from disparate sources, lack of individual patient-level data, and bias in the component studies from which the data were drawn. Therefore, pooled results should be interpreted with caution. To answer fundamental questions beyond the breadth of available data, multicenter, multidisciplinary collaborations, including alignment of important outcomes by stakeholders, are needed.

A trial comparing continuous positive airway pressure (CPAP) devices in preterm infants.

OBJECTIVE: To test the hypothesis that infants born <30 weeks' gestation supported by Seattle-PAP will have lower rates of continuous positive airway pressure (CPAP) failure than infants supported with conventional, Fisher&Paykel-CPAP (FP-CPAP). STUDY DESIGN: Randomized trial (3/2017-01/2019) at 5 NICUs. The primary outcome was CPAP failure; subgroup analyses (gestational age, receipt antenatal corticosteroids) were performed. RESULTS: A total of 232 infants were randomized. Infants in the Seattle-PAP and FP-CPAP groups had mean gestational ages of 27.0 and 27.2 weeks, respectively. We observed no differences in rates of treatment failure between Seattle-PAP (40/112, 35.7%) and FP-CPAP (38/120, 31.7%; risk difference, 4.1%; 95% CI, -8.1-16.2; P = 0.51). Subgroup analysis indicated no differences in rates of CPAP failure. We observed no differences between the two groups in frequencies of adverse events or duration of respiratory support. CONCLUSIONS: Among infants born <30 weeks' gestation, rates of CPAP failure did not differ between Seattle-PAP and FP-CPAP.

Extracorporeal Membrane Oxygenation Incidence, Characteristics, and Outcomes in Neonatal Down Syndrome Patients.

Practice patterns for extracorporeal membrane oxygenation (ECMO) use in newborns with Trisomy 21 (T21) have not been fully reported. The goals of this study were to 1) determine the incidence of ECMO use in T21 neonates; 2) identify clinical and demographic characteristics associated with ECMO use in this population; 3) describe outcomes of neonates with T21 supported with ECMO. This was a retrospective cohort study using the Pediatric Health Information System database (January 2000 to January 2014). Given the exploratory nature, only descriptive statistics were used. p < 0.05 was considered significant. Within 43 pediatric hospitals, the incidence of ECMO use in neonates with T21 was 2.3% (131/5,737). Neonates with T21 supported with ECMO were more likely to be admitted earlier; have higher birth weight, gestational age, and longer hospitalization; and have congenital diaphragmatic hernia or select cardiac anomalies versus those who did not require ECMO. T21 neonates supported with ECMO also had higher incidence of bronchopulmonary dysplasia, necrotizing enterocolitis, and mortality (unadjusted odds ratio 12.3 [95% confidence interval: 8.6-17.6]) compared with T21 neonates not exposed to ECMO. Compared with T21 neonates not requiring ECMO, those supported with ECMO had increased morbidity and mortality. Additional investigation on timing, indications, and risk/benefit profiles, for ECMO use in T21 neonates is needed.

Quality metrics in cardiac catheterization for congenital heart disease: utility of 30-day mortality.

OBJECTIVES: To characterize the frequency and attributability of death among patients who died within 30 days of their cardiac catheterization (30-day mortality). BACKGROUND: 30-day postprocedure mortality is commonly used as a quality outcome metric in national cardiac catheterization registries. It is unclear if this parameter is sufficiently specific to meaningfully capture mortality attributable to cardiac catheterization in patients with congenital heart disease (CHD). METHODS: Multicenter cohort study with 3 participating centers. Records were retrospectively reviewed for patients who died within 30 days of catheterization (06/2007-06/2012). Attributability of death was assigned to each case. RESULTS: A total of 14,707 cardiac catheterization procedures were performed during the study period. Death occurred within 30 days in 279/14,707 (1.9%) of cases. Among the patients who died, 53% of cases were emergent or urgent cases. The median age was 4 mos (1 day-45 years). Death was attributable to the catheterization procedure in 29/279 (10%) of cases. Death was attributable to cardiac surgery in 14%, precatheterization clinical status in 34%, postcatheterization clinical status in 22%, and noncardiac comorbidity in 19%. In 1%, death attributability could not be established. CONCLUSIONS: While valuable in adult settings, 30-day mortality is inadequate as a quality metric among patients with CHD undergoing cardiac catheterization. To derive the optimal benefit from catheterization registry data, more robust methodologies to capture procedure-related mortality are needed. © 2014 Wiley Periodicals, Inc.

Real-time, in vivo determination of dynamic changes in lung and heart tissue oxygenation using EPR oximetry.

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.

HO-3867, a safe STAT3 inhibitor, is selectively cytotoxic to ovarian cancer.

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.

Safe and targeted anticancer therapy for ovarian cancer using a novel class of curcumin analogs.

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.

Dysregulation of PTEN in cardiopulmonary vascular remodeling induced by pulmonary hypertension.

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.

HO-3867, a curcumin analog, sensitizes cisplatin-resistant ovarian carcinoma, leading to therapeutic synergy through STAT3 inhibition.

Cisplatin resistance is a major obstacle in the treatment of ovarian cancer. Drug combinations with synergistic or complementary functions are a promising strategy to overcome this issue. We studied the anticancer efficacy of a novel compound, HO-3867, used in combination with cisplatin against chemotherapy-resistant ovarian cancer. A2780R cells, a cisplatin-resistant human ovarian cancer cell line, were exposed to 1, 5, or 10 uM of HO-3867 alone or in combination with cisplatin (10 ug/ml) for 24 hours. Cell viability (MTT), proliferation (BrdU), cell-cycle analysis (FACS), and protein expression (western blot) were used for in vitro studies. STAT3 overexpression was performed using transfected STAT3 cDNA. In vivo studies used cisplatin-resistant xenograft tumors grown in nude mice and treated with 100-ppm HO-3867 and weekly injections of 4-mg/kg cisplatin. HO-3867/cisplatin combination treatment significantly inhibited cisplatin-resistant cell proliferation in a concentration-dependent manner. The inhibition was associated with increased expression of p53 and p21, and decreased expression of cdk5 and cyclin D1. Apoptosis was induced by activation of Bax, cytochrome c release, and stimulated cleavage of caspase-9, caspase-3, and PARP. Overexpression of STAT3 decreased the HO-3867-induced apoptosis. The combination treatment significantly inhibited the growth of cisplatin-resistant xenograft tumors with significant downregulation of pSTAT3, and without apparent toxicity to healthy tissues. The combination treatment exhibited synergistic anticancer efficacy, which appears largely due to HO-3867-induced downregulation of pSTAT3. The results, combined with the previously-reported safety features of HO-3867, suggest the potential use of this compound as a safe and effective adjuvant for the treatment of ovarian cancer.

Synthesis of N-substituted 3,5-bis(arylidene)-4-piperidones with high antitumor and antioxidant activity.

A series of 3,5-bis(arylidene)-4-piperidone (DAP) compounds are considered as synthetic analogues of curcumin for anticancer properties. We performed structure-activity relationship studies by synthesizing a number of DAPs N-alkylated or acylated with nitroxides or their amine precursors as potent antioxidant moieties. Both subtituents on arylidene rings and on piperidone nitrogen (five- or six-membered, 2- or 3-substituted or 3,4-disubstituted isoindoline nitroxides) were varied. The anticancer efficacy of the new DAP compounds was tested by measuring their cytotoxicity to cancer cell lines A2780 and MCF-7 and to the H9c2 cell line. The results showed that all DAP compounds induced a significant loss of cell viability in the human cancer cell lines tested; however, only pyrroline appended nitroxides (5c (Selvendiran, K.; Tong, L.; Bratasz, A.; Kuppusamy, L. M.; Ahmed, S.; Ravi, Y.; Trigg, N. J.; Rivera, B. K.; Kálai, T.; Hideg, K.; Kuppusamy, P. Mol. Cancer Ther. 2010, 9, 1169-1179), 5e, 7, 9) showed limited toxicity toward noncancerous cell lines. Computer docking simulations support the biological activity tested. These results suggest that antioxidant-conjugated DAPs will be useful as a safe and effective anticancer agent for cancer therapy.

Effect of oxygenation on stem-cell therapy for myocardial infarction.

Stem-cell transplantation to treat acute myocardial infarction (MI) is gaining importance as a minimally invasive and potent therapy to replace akinetic scar tissue by viable myocardium. Our recent studies have shown that stem-cell transplantation marginally improves myocardial oxygenation in the infarct tissue leading to improvement in cardiac function. The aim of the present study was to determine the effect of hyperbaric oxygen (HBO) treatment on myocardial oxygenation and recovery of function in MI hearts. Fisher-344 rats were subjected to MI by permanently ligating the left-anterior-descending (LAD) coronary artery. The rats were then exposed to 100% O(2) at a pressure of 2 atmospheres for 90 minutes, and the exposure was repeated for 5 days a week for 2 weeks. Adult bone-marrow-derived rat mesenchymal stem cells (MSC, 5x105 cells) were mixed with OxySpin (LiNc- BuO, oxygen sensor) and implanted in the infarct and peri-infarct regions of the heart. M-mode ultrasound echocardiography was performed at baseline and at 2 weeks post-transplantation. The myocardial pO(2) in the MSC+HBO group (16.2±2.2 mmHg) was significantly higher when compared to untreated MI (3.8±1.9 mmHg) or MSC (9.8±2.3 mmHg) groups. In addition, there was a significant improvement in cardiac function, increased vessel density, and VEGF expression in MSC+HBO group compared to MSC group (p < 0.05). In conclusion, the results suggested a beneficial effect of HBO administration on stem-cell therapy for MI.

Cellular uptake, retention and bioabsorption of HO-3867, a fluorinated curcumin analog with potential antitumor properties.

Curcumin, a naturally-occurring compound found in the rhizome of Curcuma longa plant, is known for its antitumor activities. However, its clinical efficacy is limited due to poor bioabsorption. A new class of synthetic analogs of curcumin, namely diarylidenylpiperidone (DAP), has been developed with substantially higher anticancer activity than curcumin. However, its cellular uptake and bioabsorption have not been evaluated. In this study we have determined the absorption of a representative DAP compound, HO-3867, using optical and electron paramagnetic resonance spectrometry. The cellular uptake of HO-3867 was measured in a variety of cancer cell lines. HO-3867 was taken in cells within 15 minutes of exposure and its uptake was more than 100-fold higher than curcumin. HO-3867 was also retained in cells in an active form for 72 hours and possibly longer. HO-3867 was substantially cytotoxic to all the cancer cells tested. However, there was no direct correlation between cellular uptake and cytotoxicity suggesting that the cytotoxic mechanisms could be cell-type specific. When administered to rats by intraperitoneal injection, significantly high levels of HO-3867 were found in the liver, kidney, stomach, and blood after 3 hours. Also, significant accumulation of HO-3867 was found in murine tumor xenografts with a dose-dependent inhibition of tumor growth. The results suggest that the curcumin analog has substantially higher bioabsorption when compared to curcumin.

HO-3867, a synthetic compound, inhibits the migration and invasion of ovarian carcinoma cells through downregulation of fatty acid synthase and focal adhesion kinase.

Fatty acid synthase (FAS) and focal adhesion kinase (FAK), which are overexpressed in a variety of human epithelial tumors, play a key role in the migration and invasion of cancer cells. Hence, strategies targeted at inhibiting the FAS/FAK proteins may have therapeutic potential for cancer treatment. The goal of the present study was to determine the effect of HO-3867, a synthetic compound, on the migratory ability of ovarian cancer cells and to understand the mechanistic pathways including the involvement of FAS, FAK, and associated signaling proteins. The study was done using two established human ovarian cancer cell lines, A2780 and SKOV3. Incubation with 10 µmol/L HO-3867 for 24 hours significantly inhibited the native as well as the vascular endothelial growth factor (VEGF)-mediated migration and invasion of the cells. HO-3867 significantly attenuated FAS and FAK protein levels apparently through accelerated ubiquitin-dependent degradation, as shown by a clear downregulation of isopeptidase USP2a. Exposure of cells to HO-3867 also significantly inhibited FAS activity and mRNA levels and a number of downstream proteins, including phospho-extracellular signal-regulated kinase 1/2, phospho-human epidermal growth factor receptor 1, sterol regulatory element binding protein 1, VEGF, and matrix metalloproteinase 2. Western blot and immunohistochemical analyses of A2780 xenograft tumors in mice treated with HO-3867 showed significant reduction in FAS, FAK, VEGF, and downstream protein levels when compared with the untreated control. Collectively, the results showed that HO-3867 suppressed the migration and invasion of ovarian cancer cells by inhibiting the expression or activity of FAS and FAK proteins. The study suggests that molecular targeting of FAS and FAK by HO-3867 may be a potential strategy for ovarian cancer therapy.

Oxygen and oxygenation in stem-cell therapy for myocardial infarction.

Myocardial infarction (MI) is caused by deprivation of oxygen and nutrients to the cardiac tissue due to blockade of coronary artery. It is a major contributor to chronic heart disease, a leading cause of mortality in the modern world. Oxygen is required to meet the constant energy demands for heart contractility, and also plays an important role in the regulation of heart function. However, reoxygenation of the ischemic myocardium upon restoration of blood flow may lead to further injury. Controlled oxygen delivery during reperfusion has been advocated to prevent this consequence. Monitoring the myocardial oxygen concentration would play a vital role in understanding the pathological changes in the ischemic heart following myocardial infarction. During the last two decades, several new techniques have become available to monitor myocardial oxygen concentration in vivo. Electron paramagnetic resonance (EPR) oximetry would appear to be the most promising and reliable of these techniques. EPR utilizes crystalline probes which yield a single sharp line, the width of which is highly sensitive to oxygen tension. Decreased oxygen tension results in a sharpening of the EPR spectrum, while an increase results in widening. In our recent studies, we have used EPR oximetry as a valuable tool to monitor myocardial oxygenation for several applications like ischemia-reperfusion injury, stem-cell therapy and hyperbaric oxygen therapy. The results obtained from these studies have demonstrated the importance of tissue oxygen in the application of stem-cell therapy to treat ischemic heart tissues. These results have been summarized in this review article.

Oxygenation inhibits ovarian tumor growth by downregulating STAT3 and cyclin-D1 expressions.

Hypoxia, which is commonly observed in many solid tumors, is a major impediment to chemo- or radiation therapy. Hypoxia is also known to overexpress/activate signal transducer and activator of transcription 3 (STAT3) leading to tumor progression as well as drug resistance. We hypothesized that increased oxygenation of the hypoxic tumor may have an inhibitory effect on STAT3 activation and hence tumor-growth inhibition. Mice containing human ovarian cancer xenograft tumor were exposed to hyperbaric oxygen (HBO; 100% oxygen; 2 atm; 90-min duration) daily, for up to 21 days. Mice exposed to HBO showed a significant reduction in tumor volume, with no effect on body weight. STAT3 (Tyr 705) activation and cyclin-D1 protein/mRNA levels were significantly decreased up on HBO exposure. Interestingly, HBO exposure, in combination with weekly administration of cisplatin, also significantly reduced the tumor volume; however, this group of mice had drastically reduced body weight when compared to other groups. While conventional wisdom might suggest that increased oxygenation of tumors would promote tumor growth, the results of the present study indicated otherwise. Hyperoxia appears to inhibit STAT3 activation, which is a key step in the ovarian tumor progression. The study may have important implications for the treatment of ovarian cancer in the clinic.

Anticancer efficacy of a difluorodiarylidenyl piperidone (HO-3867) in human ovarian cancer cells and tumor xenografts.

The purpose of this study was to evaluate the anticancer potency and mechanism of a novel difluorodiarylidenyl piperidone (H-4073) and its N-hydroxypyrroline modification (HO-3867) in human ovarian cancer. Studies were done using established human ovarian cancer cell lines (A2870, A2780cDDP, OV-4, SKOV3, PA-1, and OVCAR3) as well as in a murine xenograft tumor (A2780) model. Both compounds were comparably and significantly cytotoxic to A2780 cells. However, HO-3867 showed a preferential toxicity toward ovarian cancer cells while sparing healthy cells. HO-3867 induced G(2)-M cell cycle arrest in A2780 cells by modulating cell cycle regulatory molecules p53, p21, p27, cyclin-dependent kinase 2, and cyclin, and promoted apoptosis by caspase-8 and caspase-3 activation. It also caused an increase in the expression of functional Fas/CD95 and decreases in signal transducers and activators of transcription 3 (STAT3; Tyr705) and JAK1 phosphorylation. There was a significant reduction in STAT3 downstream target protein levels including Bcl-xL, Bcl-2, survivin, and vascular endothelial growth factor, suggesting that HO-3867 exposure disrupted the JAK/STAT3 signaling pathway. In addition, HO-3867 significantly inhibited the growth of the ovarian xenografted tumors in a dosage-dependent manner without any apparent toxicity. Western blot analysis of the xenograft tumor tissues showed that HO-3867 inhibited pSTAT3 (Tyr705 and Ser727) and JAK1 and increased apoptotic markers cleaved caspase-3 and poly ADP ribose polymerase. HO-3867 exhibited significant cytotoxicity toward ovarian cancer cells by inhibition of the JAK/STAT3 signaling pathway. The study suggested that HO-3867 may be useful as a safe and effective anticancer agent for ovarian cancer therapy.

Safe and targeted anticancer efficacy of a novel class of antioxidant-conjugated difluorodiarylidenyl piperidones: differential cytotoxicity in healthy and cancer cells.

The development of smart anticancer drugs that can selectively kill cancer cells while sparing the surrounding healthy tissues/cells is of paramount importance for safe and effective cancer therapy. We report a novel class of bifunctional compounds based on diarylidenyl piperidone (DAP) conjugated to an N-hydroxypyrroline (NOH; a nitroxide precursor) group. We hypothesized that the DAP would have cytotoxic (anticancer) activity, whereas the NOH moiety would function as a tissue-specific modulator (antioxidant) of cytotoxicity. The study used four DAPs, namely H-4073 and H-4318 without NOH and HO-3867 and HO-4200 with NOH substitution. The goal of the study was to evaluate the proof-of-concept anticancer-versus-antioxidant efficacy of the DAPs using a number of cancerous (breast, colon, head and neck, liver, lung, ovarian, and prostate cancer) and noncancerous (smooth muscle, aortic endothelial, and ovarian surface epithelial) human cell lines. Cytotoxicity was determined using an MTT-based cell viability assay. All four compounds induced significant loss of cell viability in cancer cells, whereas HO-3867 and HO-4200 showed significantly less cytotoxicity in noncancerous cells. EPR measurements showed a metabolic conversion of the N-hydroxylamine function to nitroxide with significantly higher levels of the metabolite and superoxide radical-scavenging (antioxidant) activity in noncancerous cells compared to cancer cells. Western blot analysis showed that the DAP-induced growth arrest and apoptosis in cancer cells were mediated by inhibition of STAT3 phosphorylation at the Tyr705 and Ser727 residues and induction of apoptotic markers of cleaved caspase-3 and PARP. The results suggest that the antioxidant-conjugated DAPs will be useful as safe and effective anticancer agents for cancer therapy.

In vivo imaging of free radicals and oxygen.

Free radicals are highly reactive compounds that play an essential role in many biological processes, both beneficial and deleterious. Detection and quantification of these species is critical to develop a better understanding of normal and pathophysiological functions at the cellular and tissue levels. Electron paramagnetic resonance (EPR) spectroscopy is the technique most commonly used for this purpose through the detection of exogenous probes or spin traps that interact with the free radical species of interest. Over the past several years, the spatial and temporal distribution of free radicals within cells and tissues has been of particular interest. This chapter briefly explains the principles and challenges in the use of EPR for biological samples and introduces the concept of EPR for free radical imaging purposes. In addition, specific examples are given for the use of EPR imaging in four principal areas: free radical probes, nitric oxide (NO), redox state, and oxygen (O(2)) concentration.

Hypoxia induces chemoresistance in ovarian cancer cells by activation of signal transducer and activator of transcription 3.

Signal transducer and activator of transcription 3 (STAT3) is activated in a variety of human cancers, including ovarian cancer. The molecular mechanism by which the STAT3 is activated in cancer cells is poorly understood. We observed that human ovarian xenograft tumors (A2780) in mice were severely hypoxic (pO(2) approximately 2 mmHg). We further observed that hypoxic exposure significantly increased the phosphorylation of STAT3 (pSTAT3) at the Tyr705 residue in A2780 cell line. The pSTAT3 (Tyr705) level was highly dependent on cellular oxygenation levels, with a significant increase at <2% O(2), and without any change in the pSTAT3 (Ser727) or total STAT3 levels. The pSTAT3 (Tyr705) elevation following hypoxic exposure could be reversed within 12 hr after returning the cells to normoxia. The increased level of pSTAT3 was partly mediated by increased levels of reactive oxygen species generation in the hypoxic cancer cells. Conventional chemotherapeutic drugs cisplatin and taxol were far less effective in eliminating the hypoxic ovarian cancer cells suggesting a role for pSTAT3 in cellular resistance to chemotherapy. Inhibition of STAT3 by AG490 followed by treatment with cisplatin or taxol resulted in a significant increase in apoptosis suggesting that hypoxia-induced STAT3 activation is responsible for chemoresistance. The results have important clinical implications for the treatment of hypoxic ovarian tumors using STAT3-specific inhibitors.