Yen Ta Huang

Ceftriaxone attenuates hypoxic-ischemic brain injury in neonatal rats.

BackgroundPerinatal brain injury is the leading cause of subsequent neurological disability in both term and preterm baby. Glutamate excitotoxicity is one of the major factors involved in perinatal hypoxic-ischemic encephalopathy (HIE). Glutamate transporter GLT1, expressed mainly in mature astrocytes, is the major glutamate transporter in the brain. HIE induced excessive glutamate release which is not reuptaked by immature astrocytes may induce neuronal damage. Compounds, such as ceftriaxone, that enhance the expression of GLT1 may exert neuroprotective effect in HIE.MethodsWe used a neonatal rat model of HIE by unilateral ligation of carotid artery and subsequent exposure to 8% oxygen for 2 hrs on postnatal day 7 (P7) rats. Neonatal rats were administered three dosages of an antibiotic, ceftriaxone, 48 hrs prior to experimental HIE. Neurobehavioral tests of treated rats were assessed. Brain sections from P14 rats were examined with Nissl and immunohistochemical stain, and TUNEL assay. GLT1 protein expression was evaluated by Western blot and immunohistochemistry.ResultsPre-treatment with 200 mg/kg ceftriaxone significantly reduced the brain injury scores and apoptotic cells in the hippocampus, restored myelination in the external capsule of P14 rats, and improved the hypoxia-ischemia induced learning and memory deficit of P23-24 rats. GLT1 expression was observed in the cortical neurons of ceftriaxone treated rats.ConclusionThese results suggest that pre-treatment of infants at risk for HIE with ceftriaxone may reduce subsequent brain injury.

Novel Survivin Inhibitor YM155 elicits Cytotoxicity in Glioblastoma Cell Lines with Normal or Deficiency DNA-Dependent Protein Kinase Activity

BACKGROUND Pediatric glioblastoma is a malignant disease with an extremely poor clinical outcome. Patients usually suffer from resistance to radiation therapy, so targeted drug treatment may be a new possibility for glioblastoma therapy. Survivin is also overexpressed in glioblastoma. YM155, a novel small-molecule survivin inhibitor, has not been examined for its use in glioblastoma therapy. METHODS The human glioblastoma cell line M059K, which expresses normal DNA-dependent protein kinase (DNA-PK) activity and is radiation-resistant, and M059J, which is deficient in DNA-PK activity and radiation-sensitive, were used in the study. Cell viability, DNA fragmentation, and the expression of survivin and securin following YM155 treatment were examined using MTT (methylthiazolyldiphenyl-tetrazolium) assay, ELISA assay, and Western blot analysis, respectively. RESULTS YM155 caused a concentration-dependent cytotoxic effect, inhibiting the cell viability of both M059K and M059J cells by 70% after 48 hours of treatment with 50 nM YM155. The half-maximal inhibitory concentration (IC50) was around 30-35 nM for both cell lines. Apoptosis was determined to have occurred in both cell lines because immunoreactive signals from the DNA fragments in the cytoplasm were increased 24 hours after treatment with 30 nM YM155. The expression of survivin and securin in the M059K cells was greater than that measured in the M059J cells. Treatment with 30 nM YM155, for both 24 and 48 hours, significantly suppressed the expression of survivin and securin in both cell lines. CONCLUSION The novel survivin inhibitor YM155 elicits potent cytotoxicity in glioblastoma cells in vitro via DNA-PK-independent mechanisms. YM155 could be used as a new therapeutic agent for the treatment of human glioblastomas.

Therapeutic potential of sepantronium bromide YM155 in gemcitabine-resistant human urothelial carcinoma cells

Survivin is overexpressed in transitional cell carcinoma (TCC), the most common type of bladder cancer. Previous reports demonstrated that knockdown of survivin by siRNA induced apoptosis of TCC cells. The present study evaluated the therapeutic effects of sepantronium bromide (YM155), a novel small molecule survivin inhibitor under clinical trials, on TCC cells in vitro. BFTC905, a grade III TCC cell line derived from a patient of blackfoot disease in Taiwan, was the most gemcitabine-resistant cell line when compared to BFTC909, TSGH8301 and T24 in cytotoxicity assay, resulting from upregulation of securin and bcl-2 after gemcitabine treatment. YM155 caused potent concentration‑dependent cytotoxicity in 4 TCC cell lines (IC50s ≤20 nM), but exhibited no cytotoxicity in survivin-null primary human urothelial cells. For BFTC905 cells, addition of gemcitabine and/or cisplatin, the standard TCC chemotherapy regimen, to YM155 did not exert additive cytotoxic effects. Molecular analyses indicated that YM155 inhibited the proliferation of BFTC905 cells by increasing p27kip1, suppressing Ki-67, and inducing quiescence. In addition, YM155 elicited apoptosis manifested with DNA fragmentation through suppressing the expression of survivin, securin and bcl-2. Furthermore, YM155 induced autophagy in BFTC905 cells as autophagic inhibitor, 3-methyladenine, attenuated YM155-induced LC3B-II levels and reversed the cytotoxicity of YM155. mTOR inhibitors sirolimus and everolimus did not increase YM155-induced expression of LC3B-II nor augment YM155-induced cytotoxicity. These results indicate that YM155 exerts its lethal effect on BFTC905 cells via apoptotic and autophagic death pathways and suggest that YM155 may be a potential drug for the therapy of gemcitabine-resistant bladder cancer.

Resveratrol alleviates the cytotoxicity induced by the radiocontrast agent, ioxitalamate, by reducing the production of reactive oxygen species in HK-2 human renal proximal tubule epithelial cells in vitro.

Radiocontrast-induced nephropathy (RIN) is one of the leading causes of hospital-acquired acute kidney injury (AKI). The clinical strategies currently available for the prevention of RIN are insufficient. In this study, we aimed to determine whether resveratrol, a polyphenol phytoalexin, can be used to prevent RIN. For this purpose, in vitro experiments were performed using a human renal proximal tubule epithelial cell line (HK-2 cells). Following treatment for 48 h, the highly toxic radiocontrast agent, ioxitalamate, exerted cytotoxic effects on the HK-2 cells in a concentration-dependent manner, as shown by MTT assay. The half maximal inhibitory concentration (IC50) was found to be approximately 30 mg/ml. Flow cytometry also revealed a marked increase in the number of apoptotic cells following exposure to ioxitalamate. In addition, the number of necrotic, but not necroptotic cells was increased. However, treatment with resveratrol (12.5 μM) for 48 h significantly alleviated ioxitalamate (30 mg/ml)-induced cytotoxicity, by reducing cytosolic DNA fragmentation, increasing the expression of the anti-apoptotic protein, Bcl-2 (B-cell lymphoma 2), and survivin, activating caspase-3, preventing autophagic death and suppressing the production of reactive oxygen species (ROS). Resveratrol also suppressed the ioxitalamate-induced formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage. N-acetylcysteine (NAC), a ROS scavenger commonly used to prevent RIN, also reduced ioxitalamate-induced cytotoxicity, but at a high concentration of 1 mM. Sirtuin (SIRT)1 and SIRT3 were not found to play a role in these effects. Overall, our findings suggest that resveratrol may prove to be an effective adjuvant therapy for the prevention of RIN.

Therapeutic potential of thalidomide for gemcitabine-resistant bladder cancer

Controversial effects of thalidomide for solid malignancies have been reported. In the present study, we evaluate the effects of thalidomide for transitional cell carcinoma (TCC), the most common type of bladder cancer. Thalidomide precipitates were observed when its DMSO solution was added to the culture medium. No precipitation was found when thalidomide was dissolved in 45% γ-cyclodextrin, and this concentration of γ-cyclodextrin elicited slight cytotoxicity on TCC BFTC905 and primary human urothelial cells. Thalidomide-γ-cyclodextrin complex exerted a concentration-dependent cytotoxicity in TCC cells, but was relatively less cytotoxic (with IC50 of 200 µM) in BFTC905 cells than the other 3 TCC cell lines, possibly due to upregulation of Bcl-xL and HIF-1α mediated carbonic anhydrase IX, and promotion of quiescence. Gemcitabine-resistant BFTC905 cells were chosen for additional experiments. Thalidomide induced apoptosis through downregulation of survivin and securin. The secretion of VEGF and TNF-α was ameliorated by thalidomide, but they did not affect cell proliferation. Immune-modulating lenalidomide and pomalidomide did not elicit cytotoxicity. In addition, cereblon did not play a role in the thalidomide effect. Oxidative DNA damage was triggered by thalidomide, and anti-oxidants reversed the effect. Thalidomide also inhibited TNF-α induced invasion through inhibition of NF-κB, and downregulation of effectors, ICAM-1 and MMP-9. Thalidomide inhibited the growth of BFTC905 xenograft tumors in SCID mice via induction of DNA damage and suppression of angiogenesis. Higher average body weight, indicating less chachexia, was observed in thalidomide treated group. Sedative effect was observed within one-week of treatment. These pre-clinical results suggest therapeutic potential of thalidomide for gemcitabine-resistant bladder cancer.

Overexpression of Securin in Human Transitional Cell Carcinoma Specimens

Abstract Objective Securin, the product of PTTG (pituitary tumor transforming gene), is overexpressed in several tumors, and plays important roles in cancer progression and invasion. In our previous report, securin expression was observed in transitional cell carcinoma (TCC; the most common pathological pattern of bladder cancer) cell lines, including BFTC905, T24, and TSGH8301. However, the existence of securin in human bladder cancer specimens has not been established. Materials and Methods Commercial bladder cancer tissue arrays (BL208 & BLC661) were used. Slides of paraffin-fixed human bladder tissues included all grades of TCC (18, 22 and 29 tissue samples for grades I, II, and III, respectively), 21 superficial and 41 invasive TCC specimens, and 11 normal urothelial tissue samples. The intensities of securin immunostaining were graded as background, mild, and strong (scored as 0, 1, and 2, respectively), and scores from the nucleus and cytosol were analyzed separately. Results We have demonstrated, for the first time, the expression of securin in bladder tissues. Securin was overexpressed in the cytosol and nucleus of all TCC samples compared to normal urothelium, but only cytosolic localization revealed statistical significance. There were no differences in securin immunoreactivity among the different grades of TCC. Significant over-expression of cytosolic but not nuclear securin was found in both superficial and invasive TCC samples compared to normal urothelium. No difference in immunoreactive staining for nuclear and cytosolic securin between superficial and invasive TCC was noted. Conclusion Significant enhanced expression of cytosolic securin protein was found in human bladder cancer specimens, suggesting that the level of tissue securin may be a potential biomarker for the diagnosis of bladder cancer. Studies that investigate the relationship between securin expression and the outcomes of bladder cancer patients could be conducted in the future.

Lestaurtinib is Cytotoxic to Oxaliplatin-resistant Transitional Cell Carcinoma Cell Line T24 In Vitro

Abstract Objective Patients with bladder cancer have responded poorly to oxaliplatin therapy in clinical trials. Blockade of receptor tyrosine kinases is considered a good strategy in cancer therapy. Our previous studies have demonstrated the crucial roles of brain-derived neurotrophic factor and its receptor tropomyosin receptor kinase B (TrkB) in transitional cell carcinoma (TCC). The aim of this study was to examine the cytotoxic effects of lestaurtinib, a new pan-Trk inhibitor, and oxaliplatin on bladder cancer cell lines. Materials and Methods BFTC905 and T24 TCC cell lines were used for investigation in vitro . The effects of oxaliplatin and/or lestaurtinib on cell viability, apoptosis, and expression of survivin and securin were assessed. MTT assay was used for cytotoxic evaluation. DNA fragments were detected in both the culture medium and cytoplasm to differentiate the types of cell death (apoptosis vs . necrosis). Western blots were used to analyze the expression of survivin and securin after oxaliplatin and/or lestaurtinib treatment. Results Oxaliplatin at 3 μg/mL elicited cytotoxicity on BFTC905 but not T24 cells 48 hours after treatment. The addition of 1 or 3 μM lestaurtinib to oxaliplatin did not exert additive cytotoxic effects on BFTC905 cells. Although oxaliplatin at 3 μg/mL had no effect on T24 cells, the addition of 1 or 3 μM lestaurtinib demonstrated concentration-dependent inhibitory effects. Apoptosis of T24 cells was observed after treatment with lestaurtinib alone and lestaurtinib plus oxaliplatin. Furthermore, in T24 cells, the expression of survivin was inhibited by a combination of lestaurtinib and oxaliplatin, while securin expression was inhibited by lestaurtinib alone and lestaurtinib with oxaliplatin. Conclusion Lestaurtinib may be a potential new drug for the targeted therapy of oxaliplatin-resistant TCC. Further in vivo studies are needed.

Procalcitonin levels to predict bacterial infection in Surgical Intensive Care Unit patients

Background: Infection-induced inflammatory response might be aggravated by surgery insults. The clinical presentation of Surgical Intensive Care Unit (SICU) patients might be different from medical critically ill patients. Purpose: To evaluate the diagnostic and prognostic values of procalcitonin (PCT) to predict bacterial infection in SICU patients. Methods: We retrospectively analyzed the 2-year (2013 and 2014) records of 342 adult SICU cases with suspected bacterial infection in SICU of Hualien Tzu Chi Hospital. The past histories, the first infection-related parameters when SICU admission, culture results, infection-related laboratory examinations, and outcomes were collected. Results: Median of PCT level in patients with negative and any positive culture was 0.84 (interquartile range [IQR] 0.18–6.21) and 2.27 (IQR 0.54–9.93) ng/ml, respectively. Infection from blood, urine, and skin/soft tissue elicited significantly higher PCT levels. PCT in receiver operating characteristic (ROC) curve demonstrated the most accurate to predict bacterial infection (area under the ROC curve [AUC]: 0.61; 95% confidence interval [CI]: 0.54–0.63) and bacteremia (AUC: 0.73; 95% CI: 0.66–0.80) compared to white blood cell count, ratio of neutrophils, and neutrophil-to-lymphocyte count ratio (NLCR). Significantly higher PCT levels (4.12 ng/ml, 1.12–19.99; median, IQR) were observed in mortality cases. Higher PCT levels were significantly accompanied with higher NLCR, as well as higher incidence of leukopenia and bandemia. Using Kaplan–Meier analysis, significantly higher intrahospital mortality was observed in cases with above the cutoff PCT levels of 0.5 and 2 ng/ml cases, respectively. Conclusion: PCT is a relatively more useful tool to predict bacterial and particularly bloodstream infection compared to other infection-related parameters in routinely clinical practice. Initial PCT levels may be a prognostic factor of SICU patients with bacterial infection.

Clinical Experiences with Recombinant Activated Factor VII for Managing Uncontrolled Hemorrhage in Non-Hemophilic Patients

Abstract Objective Recombinant activated factor VII (rFVIIa) is a novel hemostatic agent originally developed to treat hemophilia patients who had developed inhibitors with bleeding. Its role in treating uncontrolled bleeding in patients without pre-existing coagulation abnormalities has not been well established. We herein report our experiences with its use in non-hemophilic patients. Patients and Methods Four patients, aged 33 to 94 years, with different underlying diseases were treated with rFVIIa for uncontrolled, life-threatening hemorrhage. rFVIIa was initially administered by intravenous bolus injection at 80–100 mg/kg. Doses were adjusted according to clinical response. Results Clinical response with significant hemostasis was evident in three patients after initial treatment. One patient was unresponsive to rFVIIa treatment and died of uncontrolled bleeding. Of those who achieved initial hemostasis, two died of their underlying diseases. One had recurrent bleeding controlled by subsequent multiple doses of rFVIIa, but she died of acute myocardial infarction, a thromboembolic complication that probably arose from the use of rFVIIa. Conclusion Our results suggest that rFVIIa could play a role in the management of bleeding other than congenital coagulation disorder. However, clinical hemostatic effects that do not translate into a survival benefit require further study, especially with regard to appropriate timing for clinical use. Its potential risk, especially that of thromboembolism when treating bleeding in elderly patients, warrants further investigation.