Steven J. Skube

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

BACKGROUND Heat 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. METHODS After 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. RESULTS Triptolide 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. CONCLUSION Triptolide 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.

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

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.

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

BACKGROUND Our recent work demonstrated that treatment of neuroblastoma with triptolide causes apoptotic cell death in vitro and decreases tumor size in vivo. Triptolide therapy has been associated with reduced expression of Hsp-70, suggesting a mechanism of cell killing involving Hsp-70 inhibition. The principal objective of this study was to investigate the role of Hsp-70 in triptolide-mediated cell death in neuroblastoma. MATERIALS AND METHODS Neuroblastoma cells were transfected with Hsp-70-specific siRNA. Viability, caspase activity, and phosphatidylserine externalization were subsequently measured. An orthotopic, syngeneic murine tumor model was developed, and randomized mice received daily injections of triptolide or vehicle. At 21 d, mice were sacrificed. Immunohistochemisty was used to characterize Hsp-70 levels in residual tumors, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to identify cells undergoing apoptosis. RESULTS Targeted silencing of Hsp-70 with siRNA significantly decreased cellular viability, augmented caspase-3 activity, and resulted in increased annexin-V staining. These effects parallel those findings obtained following treatment with triptolide. Residual tumors from triptolide-treated mice showed minimal staining with Hsp-70 immunohistochemistry, while control tumors stained prominently. Tumors from treated mice demonstrated marked staining with the TUNEL assay, while control tumors showed no evidence of apoptosis. CONCLUSIONS Use of siRNA to suppress Hsp-70 expression in neuroblastoma resulted in apoptotic cell death, similar to the effects of triptolide. Residual tumors from triptolide-treated mice expressed decreased levels of Hsp-70 and demonstrated significant apoptosis. These findings support the hypothesis that Hsp-70 inhibition plays a significant role in triptolide-mediated neuroblastoma cell death.

Triptolide abrogates growth of colon cancer and induces cell cycle arrest by inhibiting transcriptional activation of E2F.

Despite significant progress in diagnostics and therapeutics, over 50 thousand patients die from colorectal cancer annually. Hence, there is urgent need for new lines of treatment. Triptolide, a natural compound isolated from the Chinese herb Tripterygium wilfordii, is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer. Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of colorectal cancer. For mechanistic studies, colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate dehydrogenase release, and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F-dependent genes, E2F1- retinoblastoma protein (Rb) binding, and proteins levels of regulator of G1–S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay. Triptolide decreased colon cancer cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than doubles the median survival of animals with liver metastases from colon cancer. Mechanistically, we demonstrate that at low concentrations triptolide induces apoptotic cell death but at higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding. Therefore, we conclude that Triptolide and Minnelide are effective against colon cancer in multiple pre-clinical models.

Characterizing surgical site infection signals in clinical notes

Surgical site infections (SSIs) are the most common and costly of hospital acquired infections. An important step in reducing SSIs is accurate SSI detection, which enables measurement and quality improvement, but currently remains expensive through manual chart review. Building off of previous work for automated and semi-automated SSI detection using expert-derived “strong features” from clinical notes, we hypothesized that additional SSI phrases may be contained in clinical notes. We systematically characterized phrases and expressions associated with SSIs. While 83% of expert-derived original terms overlapped with new terms and modifiers, an additional 362 modifiers associated with both positive and negative SSI signals were identified and 62 new base observations and actions were identified. Clinical note queries with the most common base terms revealed another 49 modifiers. Clinical notes contain a wide variety of expressions describing infections occurring among surgical specialties which may provide value in improving the performance of SSI detection algorithms.

Supplementing Resident Research Funding Through a Partnership With Local Industry

OBJECTIVE To develop a model for the supplementation of resident research funding through a resident-hosted clinical immersion with local industry. DESIGN Designated research residents hosted multiple groups of engineers and business professionals from local industry in general surgery-focused clinical immersion weeks. The participants in these week-long programs are educated about general surgery and brought to the operating room to observe a variety of surgeries. SETTING This study was performed at the University of Minnesota, in Minneapolis, Minnesota, at a tertiary medical center. PARTICIPANTS Ten designated research residents hosted general surgery immersion programs. Fifty-seven engineers and business professionals from 5 different local biomedical firms have participated in this program. RESULTS General surgery research residents (in collaboration with the University of Minnesotas Institute for Engineering in Medicine) have hosted 9 clinical immersion programs since starting the collaborative in 2015. Immersion participant response to the experiences was very positive. Two full-time resident research positions can be funded annually through participation in this program. CONCLUSIONS With decreasing funding available for surgical research, particularly resident research, innovative ways to fund resident research are needed. The general surgery clinical immersion program at the University of Minnesota has proven its value as a supplement for resident research funding and may be a sustainable model for the future.

Medical Student Perceptions of 24-Hour Call

OBJECTIVE To assess the medical student perception and experience of a 24-hour call requirement, and to learn if improvements can be made to improve the 24-hour call requirement. DESIGN Medical students completing their required surgical clerkship over 1 academic year at our institution were surveyed prior to their clerkship and on the last week of clerkship regarding their perceptions and experience with 24-hour call. SETTING This study was performed at the University of Minnesota, in Minneapolis, Minnesota, a medical school and tertiary medical center. PARTICIPANTS Two hundred one medical students were given the option to complete an anonymous survey before and after their required surgical clerkship. RESULTS Response rate for the preclerkship survey was 70% (n = 140) and 58% (n = 117) for the postclerkship survey. The mean age of respondents was 26 years, and the majority of students were in their third year of medical school. After completing the clerkship, students interested in surgery more often agreed the 24-hour call requirement should remain (51% versus 31%, p = 0.01). Students rotating at a Level I Trauma Center were also more likely to agree the call requirement should remain (59% versus 33%, p = 0.008). Medical students generally had less concerns (mental health, fatigue, mistakes, and grade performance) related to 24-hour call after completion of the clerkship. Concerns about the effect of 24-hour call on study schedule remained high in both pre and postclerkship groups. CONCLUSIONS Medical students have concerns about the experience prior to the clerkship that diminished by its completion. To improve medical student perceptions and overall experience of 24-hour call, frequency of shifts could be limited and the 24-hour call requirement sites could be shifted to Level I Trauma Centers.

Creating a Rural Surgery Track and a Review of Rural Surgery Training Programs.

OBJECTIVE The objective of this study was to present the process of developing a rural surgery training track within an established residency program and review the current rural surgery training programs in the nation. DESIGN This study reviews current rural surgery training opportunities at Accreditation Council for Graduate Medical Education accredited surgical residencies in the United States and presents the process of creating the University of Minnesotas rural surgery training track. SETTING This study was performed at the University of Minnesota, in Minneapolis, Minnesota, and at Essentia Health-Saint Marys Medical Center, in Duluth, Minnesota. PARTICIPANTS Accredited general surgery residencies were reviewed. The creation of a designated rural surgery training track added an additional rural-designated surgical resident during each postgraduation year and created a required postgraduation year 2 rural surgery rotation for all categorical surgical residents. RESULTS Two hundred sixty-eight surgery residency programs were reviewed. Twenty-seven had required rural rotations, 10 offered only elective rural rotations, and 4 had dedicated National Resident Matching Program codes for rural training tracks. After review of national rural surgery training opportunities, the University of Minnesotas process of creating a designated rural surgery training program required attention to 5 main components: needs assessment and review of local opportunities, surgery residency review committee approval, funding, surgical education, and clinical/operative education. CONCLUSIONS Increasing opportunities for surgical residents to train in rural settings may help with recruitment of medical students and retention of surgeons pursuing careers in rural surgery.

Using Implementation Science to Adapt a Training Program to Assist Surgeons with High-Stakes Communication

OBJECTIVE Surgeons often conduct difficult conversations with patients near the end of life, yet surgical education provides little formalized communication training. We developed a communication tool, Best Case/Worst Case, and trained surgeons using a one-on-one resource intensive format that was effective but difficult to scale for widespread dissemination. We aimed to generate an implementation package to teach surgeons using fewer resources without sacrificing fidelity. DESIGN, SETTING, AND PARTICIPANTS We used the Replicating Effectiveness Programs framework to guide our implementation strategy and tested our intervention with 39 surgical residents at 4 institutions from September 2016 to June 2017. The implementation package consisted of: (1) instructional video, (2) checklist to assess competence, (3) learner manual, and (4) instructor manual. We focused on 3 implementation outcomes: feasibility, fidelity, and acceptability to participants. RESULTS Attendance rates ranged from 16% to 75%. Site leaders had little difficulty identifying suitable instructors; however, resident recruitment proved challenging. Sixty-nine percent of residents completed the post-training assessment and the mean score was 12.8 (range 8-15) using the 15-point checklist. Across sites, 69% strongly agreed that Best Case/Worst Case is better than how they usually approach high-stakes conversations and 100% felt prepared to use the tool after training. Instructors reported that the training provided residents with the necessary skills to perform the fundamental elements of Best Case/Worst Case. CONCLUSIONS Using implementation science we demonstrated that a resource intensive communication training intervention can be successfully modified for group-learning and wide-scale dissemination. However, we identified barriers to implementation, including challenges with feasibility and programmatic buy-in that inform not only resident education but also communication skills training more broadly.

Penetrating Colon Trauma Outcomes in Black and White Males

Introduction: Racial disparities have been both published and disputed in trauma patient mortality, outcomes, and rehabilitation. In this study, the objective was to assess racial disparities in patients with penetrating colon trauma. Methods: The National Trauma Data Bank was searched for males aged ≥14 years from 2010 through 2014 who underwent operative intervention for penetrating colon trauma. The primary outcomes for this study were stoma formation and transfer to rehabilitation; secondary outcomes were postoperative morbidity and mortality. Analyses were performed in 2016–2018. Results: There were 7,324 patients identified (4,916 black, 2,408 white). Black and white patients underwent fecal diversion with stoma formation at a similar rate (19.6% vs 18.5%, p=0.28). Black patients were more likely than white patients to be uninsured (self-pay; 37.1% vs 29.9%) and more likely to be injured by firearms (88.3% vs 70.2%, p<0.001), but had a lower overall postoperative morbidity rate (52.6% vs 55.3%, p=0.04). The odds of stoma formation (OR=0.92, 95% CI=0.78, 1.09, p=0.35) and the odds of transfer to rehabilitation (OR=1.03, 95% CI=0.82, 1.30, p=0.78) were similar for black versus white patients. Conclusions: Black patients experienced similar rates of stoma formation and transfer to rehabilitation as white patients with penetrating colon trauma. Multivariate analysis confirmed expected findings that trauma severity increased the odds of receiving an ostomy and rehabilitation placement. The protocol-based management approach to emergency trauma care potentially decreases the risk for the racial biases that could lead to healthcare disparities.