Regulation of Dihydropyrimidinase-like 3 Gene Expression by MicroRNAs in PC12 Cells with Induced Ischaemia and Hypothermia.

Although hypothermic treatment has been reported to have some beneficial effects on ischaemia at the clinical level, the mechanism of ischaemia suppression by hypothermia remains unclear due to a lack of mechanism understanding and insufficient data. The aim of this study was to isolate and characterize microRNAs specifically expressed in ischaemia-hypothermia for the dihydropyrimidinase-like 3 (Dpysl3) gene. PC12 cells were induced with CoCl2 for chemical ischaemia and incubated at 32 ℃ for hypothermia. In ischaemia-hypothermia, four types of microRNAs (miR-106b-5p, miR-194-5p, miR-326-5p, and miR-497-5p) were highly related to the Dpysl3 gene based on exosomal microRNA analysis. Dpysl3 gene expression was up-regulated by miR-497-5p but down-regulated by miR-106b-5p, miR-194-5p and miR-326-5p. Our results suggest that these four microRNAs are involved in the regulation of Dpysl3 gene expression. These findings provide valuable clues that exosomal microRNAs could be used as therapeutic targets for effective treatment of ischaemia.

Efficacy of cardiopulmonary resuscitation performance while wearing a powered air-purifying respirator.

BACKGROUND: The use of personal protective equipment for respiratory infection control during cardiopulmonary resuscitation (CPR) is a physical burden to healthcare providers. The duration for which CPR quality according to recommended guidelines can be maintained under these circumstances is important. We investigated whether a 2-min shift was appropriate for chest compression and determined the duration for which chest compression was maintained in accordance with the recommended guidelines while wearing personal protective equipment. METHODS: This prospective crossover simulation study was performed at a single center from September 2020 to October 2020. Five indicators of CPR quality were measured during the first and second sessions of the study period. All participants wore a Level D powered air-purifying respirator (PAPR), and the experiment was conducted using a Resusci Anne manikin, which can measure the quality of chest compressions. Each participant conducted two sessions. In Session 1, the sequence of 2 min of chest compressions, followed by a 2-min rest, was repeated twice; in Session 2, the sequence of 1-min chest compressions followed by a 1-min rest was repeated four times. RESULTS: All 34 participants completed the study. The sufficiently deep compression rate was 65.9 ± 31.1% in the 1-min shift group and 61.5 ± 30.5% in the 2-min shift group. The mean compression depth was 52.8 ± 4.3 mm in the 1-min shift group and 51.0 ± 6.1 mm in the 2-min shift group. These two parameters were significantly different between the two groups. There was no significant difference in the other values related to CPR quality. CONCLUSIONS: Our findings indicated that 1 min of chest compressions with a 1-min rest maintained a better quality of CPR while wearing a PAPR.

Detection of Testicular Torsion With Preserved Intratesticular Flow By Point of Care Ultrasound in the Emergency Department: A Case Report.

BACKGROUND: Ultrasonography is an effective diagnostic tool for testicular torsion (TT), which is typically characterized by the absence of blood flow in the affected testicle on color Doppler mode. However, there are a few reported cases of TT with symmetrical preserved flow. We report a case of TT with the preserved intratesticular flow on color Doppler ultrasound. CASE REPORT: A 14-year-old boy was admitted due to sudden-onset right scrotal pain. Point-of-care ultrasound (POCUS) revealed that the right testicle was larger than the left. The intratesticular flow in both testicles was preserved. Radiology-performed ultrasound confirmed the preserved intratesticular flow observed on POCUS, but also demonstrated a whirlpool sign of the right spermatic cord. TT was confirmed surgically. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Emergency physicians should investigate the presence of intratesticular blood flow and the whirlpool sign of the spermatic cord or other ultrasound features suggestive of TT, even if testicular blood flow is preserved. Suspicion of TT from POCUS findings warrants further evaluation to preserve the patient's fertility.

Regulation of adipocyte differentiation by clusterin-mediated Krüppel-like factor 5 stabilization.

Clusterin (CLU) is a heterodimeric glycoprotein involved in a range of biological processes. We investigated the function of CLU as a novel regulator of adipogenesis. CLU expression increased during 3T3-L1 preadipocyte differentiation. CLU overexpression promoted adipogenic differentiation of preadipocytes and increased the mRNA levels of adipogenic markers including peroxisome proliferator-activated receptor γ (Pparg) and CCAAT enhancer-binding protein α (Cebpa). Conversely, knockdown of CLU attenuated adipogenesis and reduced transcript levels of Pparg and Cebpa. However, the promoter activities of both the Pparg and the Cebpa gene were not affected by alteration of CLU expression on its own. Additionally, the protein level of Krüppel-like factor 5 (KLF5), an upstream transcription factor of Pparg and Cebpa involved in adipogenic differentiation, was upregulated by CLU overexpression, although the mRNA level of Klf5 was not altered by changes in the expression level of CLU. Cycloheximide chase assay showed that the increased level of KLF5 by CLU overexpression was due to decreased degradation of KLF5 protein. Interestingly, CLU increased the stability of KLF5 by decreasing KLF5 ubiquitination. CLU inhibited the interaction between KLF5 and F-box/WD repeat-containing protein 7, which is an E3 ubiquitin ligase that targets KLF5. The adipogenic role of CLU was also addressed in mesenchymal stem cells (MSCs) and Clu-/- mouse embryonic fibroblasts (MEFs). Furthermore, CLU enhanced KLF5-mediated transcriptional activation of both the Cebpa and the Pparg promoter. Taken together, these results suggest that CLU is a novel regulator of adipocyte differentiation by modulating the protein stability of the adipogenic transcription factor KLF5.

Clinical relationship between blood concentration and clinical symptoms in aconitine intoxication.

BACKGROUND: Aconitine is well-known for its potential analgesic, anti-inflammatory, and circulation promoting effects and has been widely used as a folk medicine in South Korea. Owing to its extremely toxic nature and relatively low safety margin, intoxication is sometimes fatal. The toxic compound mainly affects the central nervous system, heart, and muscle, resulting in cardiovascular complications. PURPOSE: To determine the exact relationship between blood concentration of aconitine and clinical manifestation. BASIC PROCEDURES: The National Forensic Service (NFS) was commissioned to assist in a quantitative analysis of highly toxic aconitine and corresponding blood concentrations by analyzing the body fluids of three patients who were suspected of aconitine poisoning. MAIN FINDINGS: Aconitine blood values tested by the NFS showed that patients with a blood concentration below a certain level developed symptoms slowly and showed a high severity of clinical manifestation. There was no correlation between blood concentration and symptoms or ECG results. CONCLUSIONS: In case of suspected aconitine poisoning, an emergency care department should be visited, even with symptomatic improvement, and the patient should be monitored for at least 24 h, depending on the level of recovery and changes in ECG results.

Protective role of endogenous plasmalogens against hepatic steatosis and steatohepatitis in mice.

Free cholesterol (FC) accumulation in the liver is an important pathogenic mechanism of nonalcoholic steatohepatitis (NASH). Plasmalogens, key structural components of the cell membrane, act as endogenous antioxidants and are primarily synthesized in the liver. However, the role of hepatic plasmalogens in metabolic liver disease is unclear. In this study, we found that hepatic levels of docosahexaenoic acid (DHA)-containing plasmalogens, expression of glyceronephosphate O-acyltransferase (Gnpat; the rate-limiting enzyme in plasmalogen biosynthesis), and expression of Pparα were lower in mice with NASH caused by accumulation of FC in the liver. Cyclodextrin-induced depletion of FC transactivated Δ-6 desaturase by increasing sterol regulatory element-binding protein 2 expression in cultured hepatocytes. DHA, the major product of Δ-6 desaturase activation, activated GNPAT, thereby explaining the association between high hepatic FC and decreased Gnpat expression. Gnpat small interfering RNA treatment significantly decreased peroxisome proliferator-activated receptor α (Pparα) expression in cultured hepatocytes. In addition to GNPAT, DHA activated PPARα and increased expression of Pparα and its target genes, suggesting that DHA in the DHA-containing plasmalogens contributed to activation of PPARα. Accordingly, administration of the plasmalogen precursor, alkyl glycerol (AG), prevented hepatic steatosis and NASH through a PPARα-dependent increase in fatty acid oxidation. Gnpat+/- mice were more susceptible to hepatic lipid accumulation and less responsive to the preventive effect of fluvastatin on NASH development, suggesting that endogenous plasmalogens prevent hepatic steatosis and NASH. CONCLUSION: Increased hepatic FC in animals with NASH decreased plasmalogens, thereby sensitizing animals to hepatocyte injury and NASH. Our findings uncover a novel link between hepatic FC and plasmalogen homeostasis through GNPAT regulation. Further study of AG or other agents that increase hepatic plasmalogen levels may identify novel therapeutic strategies against NASH. (Hepatology 2017;66:416-431).

Point-of-Care Ultrasound-Guided Percutaneous Cannulation of Extracorporeal Membrane Oxygenation: Make it Simple.

BACKGROUND: Cannulation of the great vessels is required for extracorporeal membrane oxygenation (ECMO). Currently, there is no guideline for optimal imaging modalities during percutaneous cannulation of ECMO. OBJECTIVE: The purpose of this study was to describe percutaneous cannulation guided by point-of-care ultrasound (POCUS) for ECMO and compare it with fluoroscopy and landmark guidance. METHODS: Three groups (POCUS-, fluoroscopy-, and landmark-guided) of percutaneous cannulation for ECMO were analyzed retrospectively in a tertiary academic hospital. In the POCUS-guided group, visual confirmation of guidewire and cannula by ultrasound in both the access and return cannula were essential for successful cannulation. Fluoroscopy- and landmark-guided groups were cannulated with the conventional technique. RESULTS: A total of 128 patients were treated by ECMO during the study period, of which 94 (73.4%) cases were venoarterial ECMO. This included 56 cases of extracorporeal cardiopulmonary resuscitation. Also, there were 30 (23.4%) cases of venovenous ECMO and 4 (3.1%) cases of venoarteriovenous ECMO. A total of 71 (55.5%) patients were cannulated under POCUS guidance, and 43 (33.6%) patients were cannulated under fluoroscopy guidance and 14 (10.9%) patients were cannulated by landmark guidance. No surgical cut downs were required. Misplacement of cannula occurred in 3 (2.3%) cases. All three occurred in the landmark-guided group. CONCLUSIONS: POCUS-guided cannulation is comparable to fluoroscopy-guided cannulation in terms of avoiding cannula misplacement. In our experience, POCUS-guided cannulation is a useful strategy over fluoroscopy- and landmark-guided cannulation during peripheral ECMO.

Regulation of Nampt expression by transcriptional coactivator NCOA6 in pancreatic ß-cells.

Nuclear receptor coactivator 6 (NCOA6) is a transcriptional coactivator and crucial for insulin secretion and glucose metabolism in pancreatic ß-cells. However, the regulatory mechanism of ß-cell function by NCOA6 is largely unknown. In this study, we found that the transcript levels of nicotinamide phosphoribosyltransferase (Nampt) were decreased in islets of NCOA6+/- mice compared with NCOA6+/+ mice. Moreover, NCOA6 overexpression increased the levels of Nampt transcripts in the mouse pancreatic ß-cell line NIT-1. Promoter analyses showed that transcriptional activity of the Nampt promoter was stimulated by cooperation of sterol regulatory element binding protein-1c (SREBP-1c) and NCOA6. Additional studies using mutant promoters demonstrated that SREBP-1c activates Nampt promoter through the sterol regulatory element (SRE), but not through the E-box. Using chromatin immunoprecipitation assay, NCOA6 was also shown to be directly recruited to the SRE region of the Nampt promoter. Furthermore, treatment with nicotinamide mononucleotide (NMN), a product of the Nampt reaction and a key NAD+ intermediate, ameliorates glucose-stimulated insulin secretion from NCOA6+/- islets. These results suggest that NCOA6 stimulates insulin secretion, at least partially, by modulating Nampt expression in pancreatic ß-cells.

Angiopoietin-like peptide 4 regulates insulin secretion and islet morphology.

Insulin secretion from pancreatic islet ß-cells is primarily regulated by the blood glucose level, and also modulated by a number of biological factors produced inside the islets or released from remote organs. Previous studies have shown that angiopoietin-like protein 4 (Angptl4) controls glucose and lipid metabolism through its actions in the liver, adipose tissue, and skeletal muscles. In this present study, we investigated the possible role of Angptl4 in the regulation of insulin secretion from pancreatic islets. Angptl4 was found to be highly expressed in the α-cells but not ß-cells of rodent islets. Moreover, treatment of rodent islets with Angptl4 peptide potentiated glucose-stimulated insulin secretion through a protein kinase A-dependent mechanism. Consistently, Angptl4 knockout mice showed impaired glucose tolerance. In the cultured islets from Angptl4 knockout mice, glucose-stimulated insulin secretion was significantly lower than in islets from wild type mice. Angptl4 peptide replacement partially reversed this reduction. Moreover, Angptl4 knockout mice had dysmorphic islets with abnormally distributed α-cells. In contrast, the ß-cell mass and distribution were not significantly altered in these knockout mice. Our current data collectively suggest that Angptl4 may play a critical role in the regulation of insulin secretion and islet morphogenesis.

Extracorporeal CPR and intra-aortic balloon pumping in tachycardia-induced cardiomyopathy complicating cardiac arrest.

Although tachycardia-induced cardiomyopathy (TIC) due to atrial fibrillation occurs frequently, it is under-recognized in clinical settings. TIC has a wide range of clinical manifestations, from asymptomatic tachycardia to cardiomyopathy leading to end stage heart failure. We present a case of a 48year-old-woman who presented as cardiogenic shock, and rapidly progressed to cardiac arrest from recently diagnosed but undertreated atrial fibrillation, resulting TIC in the emergency department (ED). She was rescued by extracorporeal cardiopulmonary resuscitation (E-CPR) for refractory cardiac arrest in the ED, and received concomitant intra-aortic balloon counterpulsation (IABP) support for severe left ventricular failure. Cardiogenic shock can present as an initial manifestation of TIC, and E-CPR and subsequent IABP support can be a valuable rescue therapy for severe TIC.

Neurologic outcome of comatose survivors after hanging: a retrospective multicenter study.

PURPOSE: The aim of this study is to identify the neurologic outcome of hanging patients and prognostic factors. MATERIALS AND METHODS: We retrospectively investigated comatose hanging patients who arrived at the emergency departments (EDs) of twelve academic tertiary care centers during a period of seven years (2006-2012). Patients were analyzed separately according to whether out-of-hospital cardiac arrest (OHCA) occurred or not. The neurologic outcome was evaluated using the Cerebral Performance Category (CPC) at the time of hospital discharge. RESULTS: A total of 1118 patients were admitted to the ED after hanging attempts. There were 159 comatose patients who did not experience OHCA. Twelve (7.5%) of 159 patients were discharged from the hospital with a poor neurologic outcome (CPC 3-5). These 12 patients received only conservative management without therapeutic hypothermia. On multivariate logistic regression analysis, mental state upon ED arrival and arterial pH were predicting factors for poor prognosis. One hundred twenty-one patients suffered OHCA and experienced restored spontaneous circulation after cardiopulmonary resuscitation. Among them, only five (4.1%) patients recovered consciousness to the level of CPC 1-2. The initial arterial pH and HCO3(-) were prognostic factors in hanging patients with OHCA. CONCLUSIONS: Even though cardiac arrest did not occur after hanging injuries, 7.5% of patients could not recover consciousness. Therapeutic hypothermia should be considered for such patients. If OHCA occurred after the hanging injury, the proportion of patients with good neurologic outcome was very low at 4.1%.

The Poisoning Information Database Covers a Large Proportion of Real Poisoning Cases in Korea.

The poisoning information database (PIDB) provides clinical toxicological information on commonly encountered toxic substances in Korea. The aim of this study was to estimate the coverage rate of the PIDB by comparing the database with the distribution of toxic substances that real poisoning patients presented to 20 emergency departments. Development of the PIDB started in 2007, and the number of toxic substances increased annually from 50 to 470 substances in 2014. We retrospectively reviewed the medical records of patients with toxic exposure who visited 20 emergency departments in Korea from January to December 2013. Identified toxic substances were classified as prescription drug, agricultural chemical, household product, animal or plant, herbal drug, or other. We calculated the coverage rate of the PIDB for both the number of poisoning cases and the kinds of toxic substances. A total of 10,887 cases of intoxication among 8,145 patients was collected. The 470 substances registered in the PIDB covered 89.3% of 8,891 identified cases related to poisoning, while the same substances only covered 45.3% of the 671 kinds of identified toxic substances. According to category, 211 prescription drugs, 58 agricultural chemicals, 28 household products, and 32 animals or plants were not covered by the PIDB. This study suggested that the PIDB covered a large proportion of real poisoning cases in Korea. However, the database should be continuously extended to provide information for even rare toxic substances.

The E-box-like sterol regulatory element mediates the insulin-stimulated expression of hepatic clusterin.

Clusterin (also known as apolipoprotein J) is a highly conserved glycoprotein involved in various biological processes, including attenuation of complement activity, sperm maturation, apoptosis, and reverse lipid transport. Although clusterin is reportedly associated with metabolic diseases, the metabolic regulation of clusterin expression is largely unknown. We investigated the effect of insulin on hepatic clusterin expression and its underlying mechanisms. Insulin increased the mRNA and protein levels of clusterin in primary hepatocytes and hepatoma cell lines. Serial deletion and mutant analysis of the clusterin promoter demonstrated that insulin-stimulated transactivation is mediated via a non-canonical E-box (NCE-box) motif in the proximal upstream region. Interestingly, sterol regulatory element binding protein-1c (SREBP-1c) co-transfection showed the same transactivation pattern as insulin stimulation in serial deletion and mutant promoter analysis. In contrast, co-transfection with a dominant negative form of SREBP-1c inhibited insulin-stimulated clusterin expression. Furthermore, insulin increased the recruitment of SREBP-1c to the NCE-box of the clusterin promoter region. Taken together, our results suggest that an NCE-box within the clusterin promoter is necessary for insulin-stimulated hepatic expression of clusterin via SREBP-1c.

20(S)-protopanaxatriol inhibits liver X receptor α-mediated expression of lipogenic genes in hepatocytes.

20(S)-protopanaxatriol (PPT) is an aglycone of ginsenosides isolated from Panax ginseng and has several interesting activities, including anti-inflammatory and anti-oxidative stress effects. Herein, PPT was identified as an inhibitor against the ligand-dependent transactivation of liver X receptor α (LXRα) using a Gal4-TK-luciferase reporter system. LXRα is a transcription factor of nuclear hormone receptor family and stimulates the transcription of many metabolic genes, such as lipogenesis- or reverse cholesterol transport (RCT)-related genes. Quantitative RT-PCR analysis showed that PPT inhibited the LXRα-dependent transcription of lipogenic genes, such as sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase, and stearoyl CoA desaturase 1. These inhibitory effects of PPT are, at least in part, a consequence of the reduced recruitment of RNA polymerase II to the LXR response element (LXRE) of the SREBP-1c promoter. Furthermore, LXRα-dependent triglyceride accumulation in primary mouse hepatocytes was significantly reduced by PPT. Interestingly, PPT did not inhibit the LXRα-dependent transcription of ABCA1, a crucial LXRα target gene involved in RCT. Chromatin immunoprecipitation assays revealed that PPT repressed recruitment of the lipogenic coactivator TRAP80 to the SREBP-1c LXRE, but not the ABCA1 LXRE. Overall, these data suggest that PPT has selective inhibitory activity against LXRα-mediated lipogenesis, but not LXRα-stimulated RCT.

Low-dose radiation suppresses Pokemon expression under hypoxic conditions.

Our previous data demonstrated that CoCl2-induced hypoxia controls endoplasmic reticulum (ER) stress-associated and other intracellular factors. One of them, the transcription factor Pokemon, was differentially regulated by low-dose radiation (LDR). There are limited data regarding how this transcription factor is involved in expression of the unfolded protein response (UPR) under hypoxic conditions. The purpose of this study was to obtain clues on how Pokemon is involved in the UPR. Pokemon was selected as a differentially expressed gene under hypoxic conditions; however, its regulation was clearly repressed by LDR. It was also demonstrated that both expression of ER chaperones and ER stress sensors were affected by hypoxic conditions, and the same results were obtained when cells in which Pokemon was up- or down-regulated were used. The current state of UPR and LDR research associated with the Pokemon pathway offers an important opportunity to understand the oncogenesis, senescence, and differentiation of cells, as well as to facilitate introduction of new therapeutic radiopharmaceuticals.

Nuclear receptor coactivator 6 is a critical regulator of NLRP3 inflammasome activation and gouty arthritis.

Transcriptional coactivators regulate the rate of gene expression in the nucleus. Nuclear receptor coactivator 6 (NCOA6), a coactivator, has been implicated in embryonic development, metabolism, and cancer pathogenesis, but its role in innate immunity and inflammatory diseases remains unclear. Here, we demonstrated that NCOA6 was expressed in monocytes and macrophages and that its level was increased under proinflammatory conditions. Unexpectedly, nuclear NCOA6 was found to translocate to the cytoplasm in activated monocytes and then become incorporated into the inflammasome with NLRP3 and ASC, forming cytoplasmic specks. Mechanistically, NCOA6 associated with the ATP hydrolysis motifs in the NACHT domain of NLRP3, promoting the oligomerization of NLRP3 and ASC and thereby instigating the production of IL-1ß and active caspase-1. Of note, Ncoa6 deficiency markedly inhibited NLRP3 hyperactivation caused by the Nlrp3R258W gain-of-function mutation in macrophages. Genetic ablation of Ncoa6 substantially attenuated the severity of two NLRP3-dependent diseases, folic-induced acute tubular necrosis and crystal-induced arthritis, in mice. Consistent with these findings, NCOA6 was highly expressed in macrophages derived from gout patients, and NCOA6-positive macrophages were significantly enriched in gout macrophages according to the transcriptome profiling results. Conclusively, NCOA6 is a critical regulator of NLRP3 inflammasome activation and is therefore a promising target for NLRP3-dependent diseases, including gout.

Regulation of hepatic insulin sensitivity by activating signal co-integrator-2.

ASC-2 (activating signal co-integrator-2, also known as AIB3 and NCoA6) is a transcriptional co-activator and regulates insulin secretion and ß-cell survival. The present study was performed to elucidate the role of ASC-2 in the regulation of insulin sensitivity. Although islet cells from 10-week-old ASC-2+/- mice secreted less insulin than wild-type islets, there was no significant difference in glucose tolerance between ASC-2+/- and wild-type mice. However, ASC-2+/- mice did show increased insulin sensitivity compared with wild-type mice in insulin tolerance tests. Consistently, the levels of phosphorylated Akt were higher in ASC-2+/- hepatocytes than in wild-type hepatocytes after insulin treatment. Moreover, decreases in phosphoenol pyruvate carboxykinase mRNA in refed mice were more prominent in ASC-2+/- livers than in wild-type livers. Interestingly, the expression levels of SOCS1 (suppressor of cytokine signalling 1) and SOCS3, well-known insulin signalling inhibitors, were decreased in ASC-2+/- hepatocytes and increased in ASC-2-overexpressing hepatocytes. Furthermore, ASC-2 was recruited to the promoter region of SOCS1 and potentiated the transcription by SREBP-1c (sterol-regulatory-element-binding protein-1c). This transcription-activating function of ASC-2 was diminished by mutations of SREBP-1c-binding sites in the SOCS1 promoter. Taken together, these results suggest that ASC-2 negatively affects hepatic insulin sensitivity, at least in part, through induction of the insulin signalling inhibitors SOCS1 and SOCS3.

Peripheral Microneedle Patch for First-Aid Hemostasis.

Despite the minimized puncture sizes and high efficiency, microneedle (MN) patches have not been used to inject hemostatic drugs into bleeding wounds because they easily destroy capillaries when a tissue is pierced. In this study, a shelf-stable dissolving MN patch is developed to prevent rebleeding during an emergency treatment. A minimally and site-selectively invasive hemostatic drug delivery system is established by using a peripheral MN (p-MN) patch that does not directly intrude the wound site but enables topical drug absorption in the damaged capillaries. The invasiveness of MNs is histologically examined by using a bleeding liver of a Sprague-Dawley (SD) rat as an extreme wound model in vivo. The skin penetration force is quantified to demonstrate that the administration of the p-MN patch is milder than that of the conventional MN patch. Hemostatic performance is systematically studied by analyzing bleeding weight and time and comparing them with that of conventional hemostasis methods. The superior performance of a p-MN for the heparin-pretreated SD rat model is demonstrated by intravenous injection in vivo.

Antitumor agents 294. Novel E-ring-modified camptothecin-4ß-anilino-4'-O-demethyl-epipodophyllotoxin conjugates as DNA topoisomerase I inhibitors and cytotoxic agents.

Two conjugates (1 and 2) of camptothecin (CPT) and 4ß-anilino-4'-O-demethylepipodophyllotoxin were previously shown to exert antitumor activity through inhibition of topoisomerase I (topo I). In this current study, two novel conjugates (1E and 2E) with an open E-ring in the CPT moiety were first synthesized and evaluated for biological activity in comparison with their intact E-ring congeners. This novel class of CPT-derivatives exhibits its antitumor effect against CPT-sensitive and -resistant cells, in part, by inhibiting topo I-linked DNA (TLD) religation. An intact E-ring was not essential for the inhibition of TLD religation, although conjugates with an open E-ring were less potent than the closed ring analogs. This lower religation potency resulted in decreased formation of protein-linked DNA breaks (PLDBs), and hence, less cell growth inhibition. In addition to their impact on topo I, conjugates 1E, 2, and 2E exhibited a minor inhibitory effect on topo II-induced DNA cleavage. The novel structures of 1E and 2E may present scaffolds for further development of dual function topo I and II inhibitors with improved pharmacological profiles and physicochemical properties.