Mara B. Antonoff

Triptolide induces cell death in pancreatic cancer cells by apoptotic and autophagic pathways

BACKGROUND & AIMSnPancreatic adenocarcinoma, among the most lethal human malignancies, is resistant to current chemotherapies. We previously showed that triptolide inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. This study investigates the mechanism by which triptolide kills pancreatic cancer cells.nnnMETHODSnCells were treated with triptolide and viability and caspase-3 activity were measured using colorimetric assays. Annexin V, propidium iodide, and acridine orange staining were measured by flow cytometry. Immunofluorescence was used to monitor the localization of cytochrome c and Light Chain 3 (LC3) proteins. Caspase-3, Atg5, and Beclin1 levels were down-regulated by exposing cells to their respective short interfering RNA.nnnRESULTSnWe show that triptolide induces apoptosis in MiaPaCa-2, Capan-1, and BxPC-3 cells and induces autophagy in S2-013, S2-VP10, and Hs766T cells. Triptolide-induced autophagy has a pro-death effect, requires autophagy-specific genes, atg5 or beclin1, and is associated with the inactivation of the Protein kinase B (Akt)/mammalian target of Rapamycin/p70S6K pathway and the up-regulation of the Extracellular Signal-Related Kinase (ERK)1/2 pathway. Inhibition of autophagy in S2-013 and S2-VP10 cells results in cell death via the apoptotic pathway whereas inhibition of both autophagy and apoptosis rescues cell death.nnnCONCLUSIONSnThis study shows that triptolide kills pancreatic cancer cells by 2 different pathways. It induces caspase-dependent apoptotic death in MiaPaCa-2, Capan-1, and BxPC-3, and induces caspase-independent autophagic death in metastatic cell lines S2-013, S2-VP10, and Hs766T, thereby making it an attractive chemotherapeutic agent against a broad spectrum of pancreatic cancers.

Triptolide therapy for neuroblastoma decreases cell viability in vitro and inhibits tumor growth in vivo

BACKGROUNDnHeat shock protein (Hsp)-70 is overexpressed in several human malignancies, and its inhibition has been shown to kill cancer cells. Our objectives were to assess the effectiveness of triptolide, an Hsp-70 inhibitor, in treating neuroblastoma in vitro and in vivo, and to measure the associated effects on Hsp-70 levels and apoptosis markers.nnnMETHODSnAfter exposing N2a and SKNSH cell lines to triptolide, cell viability was assessed. Caspase-3 and -9 activities were measured and annexin staining performed to determine if cell death occurred via apoptosis. Hsp-70 protein and mRNA levels were determined using Western blot and real-time polymerase chain reaction. In an orthotopic tumor model, mice received daily triptolide injections and were humanely killed at study completion with tumor measurement.nnnRESULTSnTriptolide treatment resulted in dose- and time-dependent N2a cell death and dose-dependent SKNSH killing. Triptolide exposure was associated with dose-dependent increases in caspase activity and annexin staining. Triptolide decreased Hsp-70 protein and mRNA levels in a dose-dependent fashion. Mice receiving triptolide therapy had significantly smaller tumors than controls.nnnCONCLUSIONnTriptolide therapy decreased neuroblastoma cell viability in vitro and inhibited tumor growth in vivo. Our studies suggest that triptolide killed cells via apoptosis and in association with inhibition of Hsp-70 expression. Triptolide may provide a novel therapy for neuroblastoma.

When patients choose: comparison of Nuss, Ravitch, and Leonard procedures for primary repair of pectus excavatum

BACKGROUND/PURPOSEnPectus excavatum is a common chest wall deformity, and several procedures have been developed for its correction. We allow patients to choose among Leonard, Nuss, and Ravitch procedures. This study aimed to determine which procedure most patients select and the resultant outcomes.nnnMETHODSnCharts were reviewed of all pectus excavatum repairs performed for 4 years by a practice covering a university-based childrens hospital. Procedure choice, operative time, length of stay, analgesia, fees, and complications were recorded.nnnRESULTSnThe Ravitch procedure was chosen by 60.9% of our patients, Leonard procedure by 23.9%, and Nuss procedure by 15.2%. Operative times were not significantly different among the groups. The mean length of stay was 2.2 days (Ravitch), 1.5 days (Leonard), and 3.9 days (Nuss) (P < .005). Epidural analgesia/patient-controlled analgesia pump requirements were 50% (Ravitch), 5% (Leonard), and 100% (Nuss). The mean charges were

The Significant Impact of a Competency-Based Preparatory Course for Senior Medical Students Entering Surgical Residency

27,414 (Ravitch),

Protocolized management of infants with congenital diaphragmatic hernia: effect on survival.

18,094 (Leonard), and

Prosurvival role of heat shock factor 1 in the pathogenesis of pancreatobiliary tumors

43,749 (Nuss) (P < .05). The overall complication rate was 16.3%. The complications among each group were as follows: Ravitch, 14.3%; Leonard, 9.1%; and Nuss, 35.7%.nnnCONCLUSIONSnWe allow patients to choose among Leonard, Ravitch, and Nuss procedures for repair of pectus excavatum. Most select the Ravitch procedure. Length of stay, fees, analgesic needs, and complication rate were highest among patients in the Nuss group; all of these variables were lowest in the Leonard group.

Triptolide and TRAIL Combination Enhances Apoptosis in Cholangiocarcinoma

Purpose Previous data suggest that formal, structured preparation might improve knowledge and skills of senior medical students (SMSs) as they transition to surgical residency. However, subsequent impact on clinical performance has not been demonstrated. Method The authors developed a comprehensive course for SMSs entering surgical residencies and studied the impact of the course on the subsequent performance of 2010 graduates (n = 22) compared with matched peers (16 nonparticipant controls at authors home institution and 24 nonparticipant peer controls at outside institutions; total n = 62). Through pre- and postcourse surveys, knowledge tests, and technical examinations, they measured confidence and skill acquisition in 32 specific, job-related tasks. They followed participants and matched peers into internship and collected performance evaluations from supervising senior residents to determine whether course graduates would display performance advantages in these same tasks. The authors used t tests for all comparisons, &agr; = 0.05. Results Participants demonstrated marked improvement in task-specific confidence in all 32 tasks from course beginning to end, with improved scores on written and technical skill examinations. Further, course participants outperformed peers in all 32 tasks in July, with their performance advantage predictably dissipating into the third month of residency. There was a marked correlation between confidence and competence in all tasks. Conclusions Competency-based preparation for surgical internship resulted in objective gains in task-specific confidence and test performance at course conclusion, translating to improved performance and better patient care upon residency matriculation. These data emphasize the significant impact of formally preparing SMSs before graduation.

A Novel Critical Skills Curriculum for Surgical Interns Incorporating Simulation Training Improves Readiness for Acute Inpatient Care

BACKGROUND/PURPOSEnIn 2006, we introduced a new protocol for congenital diaphragmatic hernia (CDH) management featuring nitric oxide in the delivery room, gentle ventilation, lower criteria for extracorporeal membrane oxygenation (ECMO), and appropriately timed operative repair on ECMO. Our goals were to assess outcomes after institution of this protocol and to compare results with historical controls.nnnMETHODSnCharts were reviewed of all newborns admitted to a large metropolitan childrens hospital from 2002 to 2009 with a diagnosis of CDH. Data were recorded regarding delivery, ECMO, operative repair, length of stay, comorbidities/anomalies, complications, and survival. Postprotocol outcomes were compared to those from the preprotocol era and to data from the international CDH Registry.nnnRESULTSnComparison of the protocolized group (n = 43) to the historical group (n = 51) revealed no significant differences in gestational age, birth weight, Apgar scores, or comorbidities. New treatment strategies substantially improved survival to discharge (67% preprotocol, 88% postprotocol; P = .015). Among ECMO patients, survival increased to 82% (20% preprotocol; P = .002).nnnCONCLUSIONSnOur new protocol significantly improved survival to discharge for newborns with CDH. Institution of such a protocol is valuable in improving outcomes for patients with CDH and merits consideration for widespread adoption.

Role of Hsp-70 in Triptolide-Mediated Cell Death of Neuroblastoma

Several mechanisms have evolved to ensure the survival of cells under adverse conditions. The heat shock response is one such evolutionarily conserved survival mechanism. Heat shock factor-1 (HSF1) is a transcriptional regulator of the heat shock response. By the very nature of its prosurvival function, HSF1 may contribute to the pathogenesis of cancer. The current study investigates the role of HSF1 in the pathogenesis of pancreatobiliary tumors. HSF1 was downregulated in pancreatic cancer (MIA PaCa-2 and S2-013) and cholangiocarcinoma (KMBC and KMCH) cell lines by HSF1-specific small interfering RNA (siRNA). Nonsilencing siRNA was used as control. The effect of HSF1 downregulation on viability and apoptosis parameters, i.e., annexin V, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), and caspase-3, was measured. To evaluate the cancer-specific effects of HSF1, the effect of HSF1 downregulation on normal human pancreatic ductal cells was also evaluated. HSF1 is abundantly expressed in human pancreatobiliary cancer cell lines, as well as in pancreatic cancer tissue, as demonstrated by Western blot and immunohistochemistry, respectively. Inhibition of HSF1 expression by the HSF1 siRNA sequences leads to time-dependent death in pancreatic and cholangiocarcinoma cell lines. Downregulation of HSF1 expression induces annexin V and TUNEL positivity and caspase-3 activation, suggesting activation of a caspase-dependent apoptotic pathway. Although caspase-3 inhibition protects against cell death induced by HSF1 expression, it does not completely prevent it, suggesting a role for caspase-independent cell death. HSF1 plays a prosurvival role in the pathogenesis of pancreatobiliary tumors. Modulation of HSF1 activity could therefore emerge as a novel therapeutic strategy for cancer treatment.

Personality profiling of the modern surgical trainee: Insights into generation X

BACKGROUNDnCholangiocarcinoma originates from bile duct epithelial cells in the intrahepatic and extrahepatic biliary system. We recently observed that triptolide (a diterpenoid triepoxide) is effective in inducing apoptosis in pancreatic tumors. Death receptors 4 and 5 are overexpressed in several cancer types, and their activation by tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell death. The principal objective of this study was to determine the effects of combination therapy with TRAIL and triptolide in cholangiocarcinoma.nnnMATERIALS AND METHODSnTwo cholangiocarcinoma cell lines were incubated with various doses of triptolide and TRAIL, alone and in combination; cell viability was assessed at 24 and 48 h. Annexin-V staining and caspase-3 activity were measured after 24 h of triptolide, TRAIL or combination treatment. Western blots assessed protein levels of poly(ADP-ribose) polymerase (PARP) and X-linked inhibitor of apoptosis (XIAP).nnnRESULTSnCombination treatment using TRAIL and triptolide decreased cell viability in all cell lines at 48 h, with greater cell killing than that which was observed with either drug alone. This decrease in viability was associated with increases in annexin-V staining and caspase-3 activity. Western blot analysis demonstrated increases in PARP cleavage and decreases in XIAP expression that were dose-dependent.nnnCONCLUSIONSnTRAIL and triptolide in combination decreased cell viability and enhanced apoptosis. Furthermore, Western blot analysis suggests that triptolide sensitizes cells to TRAIL-induced apoptotic cell death by inhibiting expression of XIAP, a protein known to inhibit apoptosis. Our results demonstrate that combination of TRAIL and triptolide enhance apoptosis in cholangiocarcinoma cell lines.