Li-Jiuan Shen

Arginine Starvation Impairs Mitochondrial Respiratory Function in ASS1-Deficient Breast Cancer Cells

Tumors that cannot make arginine undergo mitochondrial dysfunction in the absence of extracellular arginine and die by autophagy. Starved to Death by Lack of Arginine Some breast tumors have low abundance of argininosuccinate synthetase 1 (ASS1), an enzyme involved in the synthesis of the amino acid arginine. Although ASS1 deficiency enhances the growth of these tumors, these cells are sensitive to depletion of external arginine. ADI-PEG20 (pegylated arginine deiminase), a modified form of a microbial enzyme, metabolizes arginine. Qiu et al. found that in various breast cancer cell lines that were deficient in ASS1, addition of ADI-PEG20 to the medium induced widespread mitochondrial dysfunction, which in turn triggered autophagy, an adaptive catabolic process that can lead to cell death if unchecked. In mice, ADI-PEG20 treatment slowed the growth of tumors formed from ASS1-deficient breast cancer cells only if these cells could undergo autophagy. Low ASS1 abundance in tumor samples from patients with breast cancer correlated with low survival rates, suggesting that treatments that induce arginine starvation may be beneficial in patients with ASS1-deficient breast cancers. Autophagy is the principal catabolic response to nutrient starvation and is necessary to clear dysfunctional or damaged organelles, but excessive autophagy can be cytotoxic or cytostatic and contributes to cell death. Depending on the abundance of enzymes involved in molecule biosynthesis, cells can be dependent on uptake of exogenous nutrients to provide these molecules. Argininosuccinate synthetase 1 (ASS1) is a key enzyme in arginine biosynthesis, and its abundance is reduced in many solid tumors, making them sensitive to external arginine depletion. We demonstrated that prolonged arginine starvation by exposure to ADI-PEG20 (pegylated arginine deiminase) induced autophagy-dependent death of ASS1-deficient breast cancer cells, because these cells are arginine auxotrophs (dependent on uptake of extracellular arginine). Indeed, these breast cancer cells died in culture when exposed to ADI-PEG20 or cultured in the absence of arginine. Arginine starvation induced mitochondrial oxidative stress, which impaired mitochondrial bioenergetics and integrity. Furthermore, arginine starvation killed breast cancer cells in vivo and in vitro only if they were autophagy-competent. Thus, a key mechanism underlying the lethality induced by prolonged arginine starvation was the cytotoxic autophagy that occurred in response to mitochondrial damage. Last, ASS1 was either low in abundance or absent in more than 60% of 149 random breast cancer biosamples, suggesting that patients with such tumors could be candidates for arginine starvation therapy.

Resistance to the anti-proliferative activity of recombinant arginine deiminase in cell culture correlates with the endogenous enzyme, argininosuccinate synthetase

Recombinant mycoplasma enzyme, arginine deiminase (rADI), has been proposed as a possible cancer treatment via arginine depletion. However, many cell lines are resistant to rADI-treatment, even though most require arginine for proliferation. We compared eight different cell lines for sensitivity in cell proliferation to the effect of either rADI or arginine deprivation. The activity of argininosuccinate synthetase (AS), the rate-limiting enzyme for converting citrulline to arginine, was also measured. Our results indicate that resistance to rADI-treatment may correlate with cellular AS activity, either constitutive or inducible, allowing cell survival by conversion of the product of the rADI reaction, i.e. citrulline to arginine.

Recombinant arginine deiminase as a differential modulator of inducible (iNOS) and endothelial (eNOS) nitric oxide synthetase activity in cultured endothelial cells

Modulation of the extracellular level of arginine, substrate for nitric oxide synthetases, is a promising modality to alleviate certain pathological conditions where excess nitric oxide (NO) is produced. However, complications arise, as only preferential inhibition of the inducible nitric oxide synthetase (iNOS), but not endothelial nitric oxide synthetase (eNOS), is desired for the treatment of NO over-production. We investigated the effect of arginine deprivation mediated by a recombinant arginine deiminase (rADI) on the activity of iNOS and eNOS in an endothelial cell line, TR-BBB. Our results demonstrated that cytokine-induced NO production depends on the extracellular arginine as substrate. However, if sufficient citrulline is present in the medium, A23187-activated NO production by eNOS does not rely on extracellular arginine. Treatment with rADI can markedly inhibit cytokine-induced NO production via iNOS, but not A23187-activated NO production via eNOS. Our results also showed that the decrease of NO production by iNOS could be achieved by depleting arginine from the medium even under the conditions that would up-regulate iNOS expression. Thus, rADI appears to be a novel selective modulator of iNOS activity that may be a used as a tool in the study of pathological disorders where NO over-production plays a key role.

Recombinant arginine deiminase reduces inducible nitric oxide synthase iNOS-mediated neurotoxicity in a coculture of neurons and microglia.

Modulation of nitric oxide (NO) production is considered a promising approach to therapy of diseases involving excessive inducible nitric oxide synthase (iNOS) expression, such as certain neuronal diseases. Recombinant arginine deiminase (rADI, EC3.5.3.6) catalyzes the conversion of L‐arginine (L‐arg), the sole substrate of NOS for NO production, to L‐citrulline (L‐cit) and ammonia. To understand the effect of the depletion of L‐arg by rADI on NO concentration and neuroprotection, a direct coculture of neuron SHSY5Y cells and microglia BV2 cells treated with lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) was used as a model of iNOS induction. The results showed that rADI preserved cell viability (4‐fold higher compared with the cells treated with LPS/IFN‐γ only) by the MTT assay, corresponding with the results of neuronal viability by neuron‐specific immunostaining assay. NO production (mean ± SD) decreased from 67.0 ± 1.3 to 19.5 ± 5.5 μM after a 2‐day treatment of rADI by the Griess assay; meanwhile, induction of iNOS protein expression by rADI was observed. In addition, rADI substantially preserved the neuronal function of dopamine uptake in the coculture. The replenishment of L‐arg in the coculture eliminated the neuroprotective and NO‐suppressive effects of rADI in the coculture, indicating that L‐arg played a crucial role in the effects of rADI. These results highlight the important role of L‐arg in the neuron‐microglia coculture in excessive induction of iNOS. Regulation of L‐arg by ADI demonstrated that rADI has a potentially therapeutic role in iNOS‐related neuronal diseases.

Use of Inhaled Corticosteroids in Patients With COPD and the Risk of TB and Influenza: A Systematic Review and Meta-analysis of Randomized Controlled Trials

Background: The use of inhaled corticosteroids (ICSs) is associated with an increased risk of pneumonia in patients with COPD. However, the risks of other respiratory infections, such as TB and influenza, remain unclear.Methods: Through a comprehensive literature search of MEDLINE, EMBASE, CINAHL, Cochrane Library, and ClinicalTrials.gov from inception to July 2013, we identified randomized controlled trials of ICS therapy lasting at least 6 months. We conducted meta-analyses by the Peto, Mantel-Haenszel, and Bayesian approaches to generate summary estimates comparing ICS with non-ICS treatment on the risk of TB and influenza.Results: Twenty-fi ve trials (22,898 subjects) for TB and 26 trials (23,616 subjects) for influenza were included. Compared with non-ICS treatment, ICS treatment was associated with a significantly higher risk of TB (Peto OR, 2.29; 95% CI, 1.04-5.03) but not influenza (Peto OR, 1.24;95% CI, 0.94-1.63). Results were similar with each meta-analytic approach. Furthermore, the number needed to harm to cause one additional TB event was lower for patients with COPD treated with ICSs in endemic areas than for those in nonendemic areas (909 vs 1,667, respectively).Conclusions: This study raises safety concerns about the risk of TB and influenza associated with ICS use in patients with COPD, which deserve further investigation.

Selective Intracellular Delivery of Recombinant Arginine Deiminase (ADI) Using pH-Sensitive Cell Penetrating Peptides To Overcome ADI Resistance in Hypoxic Breast Cancer Cells.

Arginine depletion strategies, such as pegylated recombinant arginine deiminase (ADI-PEG20), offer a promising anticancer treatment. Many tumor cells have suppressed expression of a key enzyme, argininosuccinate synthetase 1 (ASS1), which converts citrulline to arginine. These tumor cells become arginine auxotrophic, as they can no longer synthesize endogenous arginine intracellularly from citrulline, and are therefore sensitive to arginine depletion therapy. However, since ADI-PEG20 only depletes extracellular arginine due to low internalization, ASS1-expressing cells are not susceptible to treatment since they can synthesize arginine intracellularly. Recent studies have found that several factors influence ASS1 expression. In this study, we evaluated the effect of hypoxia, frequently encountered in many solid tumors, on ASS1 expression and its relationship to ADI-resistance in human MDA-MB-231 breast cancer cells. It was found that MDA-MB-231 cells developed ADI resistance in hypoxic conditions with increased ASS1 expression. To restore ADI sensitivity as well as achieve tumor-selective delivery under hypoxia, we constructed a pH-sensitive cell penetrating peptide (CPP)-based delivery system to carry ADI inside cells to deplete both intra- and extracellular arginine. The delivery system was designed to activate the CPP-mediated internalization only at the mildly acidic pH (6.5-7) associated with the microenvironment of hypoxic tumors, thus achieving better selectivity toward tumor cells. The pH sensitivity of the CPP HBHAc was controlled by recombinant fusion to a histidine-glutamine (HE) oligopeptide, generating HBHAc-HE-ADI. The tumor distribution of HBHAc-HE-ADI was comparable to ADI-PEG20 in a mouse xenograft model of human breast cancer cells in vivo. In addition, HBHAc-HE-ADI showed increased in vitro cellular uptake in cells incubated in a mildly acidic pH (hypoxic conditions) compared to normal pH (normoxic conditions), which correlated with pH-sensitive in vitro cytotoxicity in hypoxic MDA-MB-231 and human prostate cancer PC3 cells. Together, we conclude that the HBHAc-HE-based peptide delivery offers a useful means to overcome hypoxia-induced resistance to ADI in breast cancer cells, and to target the mildly acidic tumor microenvironment.

Impact of ezetimibe coadministered with statins on cardiovascular events following acute coronary syndrome: a 3-year population-based retrospective cohort study in Taiwan.

BACKGROUND Conflicting results using the combination of ezetimibe and statins to reduce the risk of cardiovascular events in patients with acute coronary syndrome (ACS) have been reported. OBJECTIVE The aim of this work was to assess the effectiveness of ezetimibe coadministered with statins in reducing cardiovascular events in patients with ACS. METHODS A retrospective cohort study of patients discharged after hospitalization with ACS was conducted from January 1, 2006, to December 31, 2007, and included those who were prescribed statins alone (n = 37,753) and those who received ezetimibe plus statins (n = 1001) within 365 days after the hospitalization, based on patient data obtained from the National Health Insurance Research Database (NHIRD) in Taiwan. The propensity score method was used to identify a 1:1 matched cohort (n = 2002). Risk of rehospitalization for ACS was analyzed by a multivariable Cox proportional hazards regression model. RESULTS The crude event rate of rehospitalization due to ACS in the original cohort was 13.4 per 100 person-years (268 events) in the combination group and 22.6 per 100 person-years (12,724 events) in the statins-alone group (adjusted hazard ratio [HR] 0.69; 95% CI, 0.62-0.78). The crude event rates of rehospitalization due to ACS in the matched cohort were 13.4 and 20.0 per 100 person-years in the combination group and statins-alone group, respectively (HR 0.62; 95% CI, 0.53-0.73). Compared with statins alone, the adjusted HRs for rehospitalization for percutaneous transluminal coronary angioplasty without stent, with stent, and revascularization for the combination group in the matched cohort were 0.61 (0.50-0.75), 0.62 (0.48-0.81), and 0.62 (0.51-0.76), respectively. CONCLUSIONS Based on the data of Taiwans NHIRD, our findings suggest that patients with ACS on ezetimibe combined with statins had a significantly lower risk of rehospitalization due to ACS, percutaneous transluminal coronary angioplasty, and revascularization than those on statins alone. The generalization of the results is limited because of using claims data of a specific population as the data source.

Sheltering Effect and Indirect Pathogenesis of Carbapenem-Resistant Acinetobacter baumannii in Polymicrobial Infection

ABSTRACT The role of carbapenem-resistant Acinetobacter baumannii (CRAb) in polymicrobial infection remains elusive. Having observed the ability of CRAb to shelter other susceptible bacteria from carbapenem killing, we sought to determine the factors contributing to this sheltering effect by transforming different recombinant plasmids into recipient A. baumannii cells. The sheltering effects of CRAb were reproduced in recipient A. baumannii cells that highly expressed carbapenem-hydrolyzing class D β-lactamases (CHDLs) through their associated strong promoter. With the use of Western blot analysis and a bioassay, the highly expressed CHDLs were found to be extracellularly released and led to hydrolysis of carbapenem. The level of extracellular CHDLs increased after challenge with a higher concentration of CHDL substrates, such as carbapenem and ticarcillin. This increased CHDL may, in part, be attributed to cell lysis, as indicated by the presence of extracellular gyrase. In the planktonic condition, the sheltering effect for the cocultured susceptible bacteria might represent an indirect and passive effect of the CRAb self-defense mechanism, because coculture with the susceptible pathogen did not augment the amount of the extracellular CHDLs. Polymicrobial infection caused by CRAb and a susceptible counterpart exerted higher pathogenicity than monomicrobial infection caused by either pathogen alone in mice receiving carbapenem therapy. This study demonstrated that CHDL-producing CRAb appears to provide a sheltering effect for carbapenem-susceptible pathogens via the extracellular release of CHDLs and, by this mechanism, can enhance the pathogenesis of polymicrobial infection in the presence of carbapenem therapy.

Statins and the risk of liver injury: a population-based case-control study.

This case‐control study investigated the association between statin use and liver injury using Taiwans National Health Insurance Research Database.

Cardiovascular outcomes associated with concomitant use of clopidogrel and proton pump inhibitors in patients with acute coronary syndrome in Taiwan

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Conflicting results have been reported regarding the increased risk of adverse outcomes in the concomitant use of clopidogrel and proton pump inhibitors (PPIs) compared with the use of clopidogrel alone. WHAT THIS STUDY ADDS Our study indicated no statistically significant increase in the risk of rehospitalization for acute coronary syndrome due to concurrent use of clopidogrel and PPIs in an Asian population with higher prevalence of CYP2C19 intermediate and poor metabolizers. Among all PPIs, only omeprazole was found to be statistically significantly associated with an increased risk of rehospitalization for acute coronary syndrome. AIMS Our study aimed to examine the impact of concomitant use of proton pump inhibitors (PPIs) with clopidogrel on the cardiovascular outcomes of patients with acute coronary syndrome (ACS). Furthermore, we sought to quantify the effects of five individual PPIs when used concomitantly with clopidogrel. METHODS We conducted a retrospective cohort study of patients who were newly hospitalized for ACS between 1 January 2006 and 31 December 2007 retrieved from the Taiwan National Health Insurance Research Database (NHIRD) and who were prescribed clopidogrel (n= 37 099) during the follow-up period. A propensity score technique was used to establish a matched cohort in 1:1 ratio (n= 5173 for each group). The primary clinical outcome was rehospitalization for ACS, while secondary outcomes were rehospitalization for percutaneous transluminal coronary angioplasty (PTCA) with stent, PTCA without stent and revascularization (PTCA or coronary artery bypass graft surgery) after the discharge date for the index ACS event. RESULTS The adjusted hazard ratio of rehospitalization for ACS was 1.052 (95% confidence interval, 0.971-1.139; P= 0.214) in the propensity score matched cohort. Among all PPIs, only omeprazole was found to be statistically significantly associated with an increased risk of rehospitalization for ACS (adjusted hazard ratio, 1.226; 95% confidence interval, 1.066-1.410; P= 0.004). Concomitant use of esomeprazole, pantoprazole, rabeprazole and lansoprazole did not increase the risk. CONCLUSIONS Our study indicated no statistically significant increase in the risk of rehospitalization for ACS due to concurrent use of clopidogrel and PPIs overall. Among individual PPIs, only omeprazole was found to be statistically significantly associated with increased risk of rehospitalization for ACS.