Uksha Saini

Preconditioning mesenchymal stem cells with caspase inhibition and hyperoxia prior to hypoxia exposure increases cell proliferation

Myocardial infarction is a leading cause of mortality and morbidity worldwide. Occlusion of a coronary artery produces ischemia and myocardial necrosis that leads to left ventricular (LV) remodeling, dysfunction, and heart failure. Stem cell therapy may decrease infarct size and improve LV function; the hypoxic environment, however, following a myocardial infarction may result in apoptosis, which in turn decreases survival of transplanted stem cells. Therefore, the effects of preconditioned mesenchymal stem cells (MSC) with hyperoxia (100% oxygen), Z‐VAD‐FMK pan‐caspase inhibitor (CI), or both in a hypoxic environment in order to mimic conditions seen in cardiac tissue post‐myocardial infarction were studied in vitro. MSCs preconditioned with hyperoxia or CI significantly decreased apoptosis as suggested by TUNEL assay and Annexin V analysis using fluorescence assisted cell sorting. These effects were more profound when both, hyperoxia and CI, were used. Additionally, gene and protein expression of caspases 1, 3, 6, 7, and 9 were down‐regulated significantly in MSCs preconditioned with hyperoxia, CI, or both, while the survival markers Akt1, NF‐κB, and Bcl‐2 were significantly increased in preconditioned MSCs. These changes ultimately resulted in a significant increase in MSC proliferation in hypoxic environment as determined by BrdU assays compared to MSCs without preconditioning. These effects may prove to be of great clinical significance when transplanting stem cells into the hypoxic myocardium of post‐myocardial infarction patients in order to attenuate LV remodeling and improve LV function. J. Cell. Biochem. 114: 2612–2623, 2013.

Elevated STAT3 expression in ovarian cancer ascites promotes invasion and metastasis: A potential therapeutic target

Although activation of the STAT3 pathway has been associated with tumor progression in a wide variety of cancer types (including ovarian cancer), the precise mechanism of invasion and metastasis due to STAT3 are not fully delineated in ovarian cancer. We found that pSTAT3 Tyr705 is constitutively activated in patient ascites and ascites-derived ovarian cancer cells (ADOCCs), and the range of STAT3 expression could be very high to low. In vivo transplantation of ADOCCs with high pSTAT3 expression into the ovarian bursa of mice resulted in a large primary tumor and widespread peritoneal metastases. In contrast, ADOCCs with low STAT3 expression or ADOCCs with STAT3 expression knockdown, led to reduced tumor growth and an absence of metastases in vivo. Cytokines derived from the ADOCC culture medium activate the interleukin (IL)-6/STAT pathway in the STAT3 knockout (KO) cells, compensating for the absence of inherent STAT3 in the cells. Treatment with HO-3867 (a novel STAT3 inhibitor at 100 p.p.m. in an orthotopic murine model) significantly suppressed ovarian tumor growth, angiogenesis and metastasis by targeting STAT3 and its downstream proteins. HO-3867 was found to have cytotoxic effects in ex vivo cultures of freshly collected human ovarian cancers, including those resistant to platinum-based chemotherapy. Our results show that STAT3 is necessary for ovarian tumor progression/metastasis and highlight the potential for targeting STAT3 by HO-3867 as a therapeutic strategy for ovarian cancer.

Doxorubicin synergizes with 34.5ENVE to enhance antitumor efficacy against metastatic ovarian cancer.

Purpose: Novel therapeutic regimens are needed to improve dismal outcomes associated with late-stage ovarian cancer. Oncolytic viruses are currently being tested in patients with ovarian cancer. Here, we tested the therapeutic efficacy of combining doxorubicin with 34.5ENVE, an oncolytic herpes simplex virus transcriptionally driven by a modified stem cell–specific nestin promoter, and encoding for antiangiogenic Vasculostatin-120 (VStat120) for use against progressive ovarian cancer. Experimental Design: Antitumor efficacy of 34.5ENVE was assessed in ovarian cancer cell lines, mouse ascites–derived tumor cells, and primary patient ascites–derived tumor cells by standard MTT assay. The ability of conditioned medium derived from 34.5ENVE-infected ovarian cancer cells to inhibit endothelial cell migration was measured by a Transwell chamber assay. Scope of cytotoxic interactions between 34.5ENVE and doxorubicin were evaluated using Chou–Talalay synergy analysis. Viral replication, herpes simplex virus receptor expression, and apoptosis were evaluated. Efficacy of oncolytic viral therapy in combination with doxorubicin was evaluated in vivo in the murine xenograft model of human ovarian cancer. Results: Treatment with 34.5ENVE reduced cell viability of ovarian cancer cell lines, and mouse ascites–derived and patient ascites–derived ovarian tumor cells. Conditioned media from tumor cells infected with 34.5ENVE reduced endothelial cell migration. When combined with doxorubicin, 34.5ENVE killed synergistically with a significant increase in caspase-3/7 activation, and an increase in sub-G1 population of cells. The combination of doxorubicin and 34.5ENVE significantly prolonged survival in nude mice bearing intraperitoneal ovarian cancer tumors. Conclusions: This study indicates significant antitumor efficacy of 34.5ENVE alone, and in combination with doxorubicin against disseminated peritoneal ovarian cancer. Clin Cancer Res; 20(24); 6479–94. ©2014 AACR.

Comparison of Impella and intra-aortic balloon pump in high-risk percutaneous coronary intervention: Vascular complications and incidence of bleeding

Objective: Compare vascular complications and incidence of bleeding of Impella 2.5 and intra-aortic balloon pump (IABP) in high-risk percutaneous coronary interventions (PCI). Background: Large arterial sheath size for device insertion is associated with vascular and/or bleeding complications; gastrointestinal bleeding may also occur with anti-coagulation use. Methods: Patients with an acute coronary syndrome receiving Impella 2.5 or IABP during high-risk PCI were studied (13 Impella; 62 IABP). Vascular complications and incidence of bleeding were compared. Results: Post-procedure hematocrit was similar between groups. Blood transfusion occurred in 38.4% and 32.2% of patients in the Impella and IABP groups, respectively (P = NS); 65.3%, 30.7% and 3.8% of bleeding were due to vascular access site/procedure related, gastrointestinal and genitourinary, respectively. There was no statistical significant difference in vascular complications between the Impella and IABP groups (15.3% and 6.4% of patients, respectively); mesenteric ischemia (n = 1) and aortic rupture (n = 1) were only in the IABP group. In-hospital and one-year mortality were not statistically significant between groups. Conclusion: Impella can be used as safely as IABP during high-risk PCI with similar vascular and bleeding complications. Importantly, approximately one third of bleeding was from the gastrointestinal system warranting careful prophylactic measures and monitoring.

Osmotin-expressing transgenic tea plants have improved stress tolerance and are of higher quality

Drought is a major stress that affects the yield and quality of tea, a widely consumed beverage crop grown in more than 20 countries of the world. Therefore, osmotin gene-expressing transgenic tea plants produced using earlier optimized conditions were evaluated for their tolerance of drought stress and their quality. Improved tolerance of polyethylene glycol-induced water stress and faster recovery from stress were evident in transgenic lines compared with the normal phenotype. Significant improvements in growth under in-vitro conditions were also observed. Besides enhanced reactive oxygen species-scavenging enzyme activity, the transgenic lines contained significantly higher levels of flavan-3-ols and caffeine, key compounds that govern quality and commercial yield of the beverage. The selected transgenic lines have the potential to meet the demands of the tea industry for stress-tolerant plants with higher yield and quality. These traits of the transgenic lines can be effectively maintained for generations because tea is commercially cultivated through vegetative propagation only.

Aberrantly activated pSTAT3-Ser727 in human endometrial cancer is suppressed by HO-3867, a novel STAT3 inhibitor

OBJECTIVE Constitutive activation of STAT3 is a hallmark of various human cancers, however an increased pSTAT3 expression in high grade human endometrial cancer has not been reported. In the present study, we examine the expression of STAT family of proteins in endometrial cancer cell lines and the efficacy of HO-3867, a novel STAT3 inhibitor designed in our lab. METHODS Expression of STAT family proteins was evaluated via Western blot. The cell viability, post-treatment with HO-3867, was assessed using MTT, cell-cycle profile and Annexin assay. In vivo efficacy of HO-3867 was evaluated using xenograft mice. RESULTS Expression of activated STATs was inconsistent among the cell lines and 18 human endometrial cancer specimens tested. While pSTAT3 Tyr705 was not expressed in any of the cell lines, pSTAT3 Ser727 was highly expressed in endometrial cancer cell lines and tumor specimens. HO-3867 decreased the expression of pSTAT3 Ser727 while total STAT3 remained constant; cell viability decreased by 50-80% and induced G2/M arrest in 55% of Ishikawa cells at the G2/M cell cycle checkpoint. There was an increase in p53, a decrease in Bcl2 and Bcl-xL, and cleavage of caspase-3, caspase-7 and PARP. HO-3867 mediated a dosage-dependent inhibition of the growth of xenografted endometrial tumors. CONCLUSIONS HO-3867 treatment decreases the high levels of pSTAT3 Ser727 in endometrial cancer cells by inducing cell cycle arrest and apoptosis. This suggests a specific role of serine-phosphorylated STAT3, independent of tyrosine phosphorylation in the oncogenesis of endometrial cancer. HO-3867 could potentially serve as an adjunctive targeted therapy.

Type of Valvular Heart Disease Requiring Surgery in the 21st Century: Mortality and Length-of-Stay Related to Surgery

Aim: While the incidence of rheumatic heart disease has declined dramatically over the last half-century, the number of valve surgeries has not changed. This study was undertaken to define the most common type of valvular heart disease requiring surgery today, and determine in-hospital surgical mortality and length-of-stay (LOS) for isolated aortic or mitral valve surgery in a United States tertiary-care hospital. Methods: Patients with valve surgery between January 2002 to June 2008 at The Ohio State University Medical Center were studied. Patients only with isolated aortic or mitral valve surgery were analyzed. Results: From 915 patients undergoing at least aortic or mitral valve surgery, the majority had concomitant cardiac proce-dures mostly coronary artery bypass grafting (CABG); only 340 patients had isolated aortic (n=204) or mitral (n=136) valve surgery. In-hospital surgical mortality for mitral regurgitation (n=119), aortic stenosis (n=151), aortic insufficiency (n=53) and mitral stenosis (n=17) was 2.5% (replacement 3.4%; repair 1.6%), 3.9%, 5.6% and 5.8%, respectively (p=NS). Median LOS for aortic insufficiency, aortic stenosis, mitral regurgitation, and mitral stenosis was 7, 8, 9 (replacement 11.5; repair 7) and 11 days, respectively (p<0.05 for group). In-hospital surgical mortality for single valve surgery plus CABG was 10.2% (p<0.005 compared to single valve surgery). Conclusions: Aortic stenosis and mitral regurgitation are the most common valvular lesions requiring surgery today. Surgery for isolated aortic or mitral valve disease has low in-hospital mortality with modest LOS. Concomitant CABG with valve surgery increases mortality substantially. Hospital analysis is needed to monitor quality and stimulate improvement among Institutions.

High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer

Objectives STAT3 is over-expressed in endometrial cancer, and diabetes is a risk factor for the development of type 1 endometrial cancer. We therefore investigated whether glucose concentrations influence STAT3 expression in type 1 endometrial cancer, and whether such STAT3 expression might be inhibited by metformin. Methods In Ishikawa (grade 1) endometrial cancer cells subjected to media with low, normal, or high concentrations of glucose, expression of STAT3 and its target proteins was evaluated by real-time quantitative PCR (qPCR). Ishikawa cells were treated with metformin and assessed with cell proliferation, survival, migration, and ubiquitin assays, as well as Western blot and qPCR. Expression of apoptosis proteins was evaluated with Western blot in Ishikawa cells transfected with a STAT3 overexpression plasmid and treated with metformin. A xenograft tumor model was used for studying the in vivo efficacy of metformin. Results Expression of STAT3 and its target proteins was increased in Ishikawa cells cultured in high glucose media. In vitro, metformin inhibited cell proliferation, survival and migration but induced apoptosis. Metformin reduced expression levels of pSTAT3 ser727, total STAT3, and its associated cell survival and anti-apoptotic proteins. Additionally, metformin treatment was associated with increased degradation of pSTAT3 ser727. No change in apoptotic protein expression was noticed with STAT3 overexpression in Ishikawa cells. In vivo, metformin treatment led to a decrease in tumor weight as well as reductions of STAT3, pSTAT3 ser727, its target proteins. Conclusions These results suggest that STAT3 expression in type 1 endometrial cancer is stimulated by a high glucose environment and inhibited by metformin.

Anticancer potential of diarylidenyl piperidone derivatives, HO-4200 and H-4318, in cisplatin resistant primary ovarian cancer

ABSTRACT We have previously developed a novel class of bi-functional compounds based on a diarylidenyl-piperidone (DAP) backbone conjugated to an N-hydroxypyrroline (-NOH; a nitroxide precursor) group capable of selectively inhibiting STAT3 activation, translocation, and DNA binding activity. HO-4200 and H-4318 are 2 such derivatives capable of inducing apoptosis in ovarian cancer cells through this mechanism and demonstrated efficacy in platinum resistant primary ovarian cancer cell populations and tumor tissues. The improved absorption and cellular uptake of HO-4200 by cancer cells was determined using optical and electron paramagnetic resonance spectrometry. Treatment of ovarian cancer cells with HO-4200 and H-4318 resulted in cleavage of caspase proteins 3, 7, and 9, as well as PARP and inhibition of the pro-survival protein, Bcl-xL, resulting in significantly decreased cell survival and increased apoptosis. HO-4200 and H-4318 significantly inhibit fatty acid synthase (FAS) and pSTAT3 and decreased the expression of STAT3 target proteins: Survivin, c-myc, Bcl-xl, Bcl-2, cyclin D1/D2, and VEGF were suppressed as analyzed using quantitative real time PCR. In addition, HO-4200 and H-4318 significantly inhibited migration/invasion, in primary ovarian cancer cell populations isolated from primary and recurrent ovarian cancer patients. Treatment of freshly collected human ovarian tumor sections with HO-4200 demonstrated significant suppression of pSTAT3 Tyr 705, angiogenesis (VEFG), and markers of proliferation (Ki-67) in ex vivo models. We have shown, for the first time, that the DAP compounds, HO-4200 and H-4318, inhibit cell migration/invasion and induce apoptosis by targeting FAS/STAT3 in human ovarian cancer cells, including primary ovarian cancer cell populations and tumor tissues. Therefore, our results highlight the clinical anti-cancer potential of HO-4200 and H-4318.

Optimising parameters for biolistic gun-mediated genetic transformation of tea (Camellia sinensis (L.) O. Kuntze)

Summary Genetic improvement of tea through breeding is difficult. Therefore, transgenic tea plants expressing the osmotin gene from Nicotiana tabacum were produced using parameters optimised for biolistic-gun mediated transformation. During optimisation, a total of 4,500 somatic embryos were bombarded using nine combinations of variable target distances and burst pressures, while keeping the gap distance (0.6 cm) and macrocarrier flight distance (16 mm) constant.A total of 90 independent, PCR-positive lines were generated. Southern hybridisation confirmed integration of the osmotin gene in 26 out of 27 PCR-positive lines (three independent lines from each of the nine parameter combinations were selected at random). Statistical analysis revealed that the efficiency of transgene integration was significantly affected by target distance. Only those lines derived from somatic embryos bombarded with 1.0 µg plasmid DNA using a 7.58 MPa burst pressure and 9-cm target distance showed osmotin expression. This was evident from strong northern hybridisation and RT-PCR signals. Leaves of 4-year-old transgenic plants growing in a contained polythene tunnel showed improved osmotic adjustment in response to osmotic stress imposed by NaCl. The osmotic potentials of transgenic leaves immersed in 100 mM or 200 mM NaCl solutions were more negative than those of non-transformed control leaves.