Jonathan C. King

Baicalein, a component of Scutellaria baicalensis, induces apoptosis by Mcl-1 down-regulation in human pancreatic cancer cells ☆

Scutellaria baicalensis (SB) and SB-derived polyphenols possess anti-proliferative activities in several cancers, including pancreatic cancer (PaCa). However, the precise molecular mechanisms have not been fully defined. SB extract and SB-derived polyphenols (wogonin, baicalin, and baicalein) were used to determine their anti-proliferative mechanisms. Baicalein significantly inhibited the proliferation of PaCa cell lines in a dose-dependent manner, whereas wogonin and baicalin exhibited a much less robust effect. Treatment with baicalein induced apoptosis with release of cytochrome c from mitochondria, and activation of caspase-3 and -7 and PARP. The general caspase inhibitor zVAD-fmk reversed baicalein-induced apoptosis, indicating a caspase-dependent mechanism. Baicalein decreased expression of Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, presumably through a transcriptional mechanism. Genetic knockdown of Mcl-1 resulted in marked induction of apoptosis. The effect of baicalein on apoptosis was significantly attenuated by Mcl-1 over-expression, suggesting a critical role of Mcl-1 in this process. Our results provide evidence that baicalein induces apoptosis in pancreatic cancer cells through down-regulation of the anti-apoptotic Mcl-1 protein.

Overexpression of CXCL5 Is Associated With Poor Survival in Patients With Pancreatic Cancer

Epithelial neutrophil-activating peptide-78 (CXCL5), a member of the CXC chemokine family, has been shown to be involved in angiogenesis, tumor growth, and metastasis. The objective of this study was to determine the relationship between CXCL5 expression and tumor progression in human pancreatic cancer and to elucidate the mechanism underlying CXCL5-mediated tumor angiogenesis and cancer growth. We report herein that CXCL5 is overexpressed in human pancreatic cancer compared with paired normal pancreas tissue. Overexpression of CXCL5 is significantly correlated with poorer tumor differentiation, advanced clinical stage, and shorter patient survival. Patients with pancreatic cancer and CXCL5 overexpression who underwent resection of cancer had a mean survival time 25.5 months shorter than that of patients who did not overexpress CXCL5. Blockade of CXCL5 or its receptor CXCR2 by small-interfering RNA knockdown or antibody neutralization attenuated human pancreatic cancer growth in a nude mouse model. Finally, we demonstrated that CXCL5 mediates pancreatic cancer-derived angiogenesis through activation of several signaling pathways, including protein kinase B (Akt), extracellular signal-regulated kinase (ERK), and signal transducer and activator of transcription (STAT) in human endothelial cells. These data suggest that CXCL5 is an important mediator of tumor-derived angiogenesis and that it may serve as a survival factor for pancreatic cancer. Blockade of either CXCL5 or CXCR2 may be a critical adjunct antiangiogenic therapy against pancreatic cancer.

The flavonoid quercetin inhibits pancreatic cancer growth in vitro and in vivo

Objectives The flavonoid quercetin holds promise as an antitumor agent in several preclinical animal models. However, the efficacy of oral administration of quercetin in a pancreatic cancer mouse model is unknown. Methods The antiproliferative effects of quercetin alone or in combination with gemcitabine were tested in 2 human pancreatic cancer cell lines using cell count and MTT assays. Apoptosis was evaluated by flow cytometry. Tumor growth in vivo was investigated in an orthotopic pancreatic cancer animal model using bioluminescence. Quercetin was administered orally in the diet. Results Quercetin inhibited the growth of pancreatic cancer cell lines, which was caused by an induction of apoptosis. In addition, dietary supplementation of quercetin attenuated the growth of orthotopically transplanted pancreatic xenografts. The combination of gemcitabine and quercetin had no additional effect compared with quercetin alone. In vivo quercetin caused significant apoptosis and reduced tumor cell proliferation. Conclusions Our data provide evidence that oral administration of quercetin was capable of inhibiting growth of orthotopic pancreatic tumors in a nude mouse model. These data suggest a possible benefit of quercetin in patients with pancreatic cancer.

Pancreatic–pleural fistula is best managed by early operative intervention

BACKGROUND Pancreatic-pleural fistula is an uncommon complication of chronic pancreatitis occurring as a result of disruption of the main pancreatic duct and tracking of pancreatic fluid through the retroperitoneum into 1 or both thoracic cavities. The optimal treatment strategy for pancreatic-pleural fistula is unknown; it has traditionally been medical management followed by operative therapy for patients who fail to respond to conservative treatment. Our objective was to compile the case reports of pancreatic-pleural fistula in the literature in order to better define clinical management strategy. METHODS The case management of pancreatic-pleural fistula was reviewed and a structured MEDLINE search for published studies was performed. Descriptive statistical analysis was performed on compiled data. RESULTS Review of the literature revealed 63 adult patients with pancreatic-pleural fistula published in English between 1970 and 2008. The majority of patients were male (71%) and there was a predominance of alcohol-associated chronic pancreatitis (51%). There were 10 complications (16%) and 2 deaths (3%) reported. Most patients were treated initially with medical therapy (87%). Medical therapy was deemed to have failed after an average period of 35+/-5 days. Total duration of therapy for patients in whom operative intervention was required after attempted medical management was 40+/-6 days, which was greater than the surgery alone cohort. In total, operative treatment was successful more often than medical therapy (94% vs 31%). CONCLUSION Analysis from this series indicates that a majority of patients recover from pancreatic-pleural fistula without sequelae (81%). Attempts at prolonged periods of medical therapy tend to delay the resolution of the fistula compared with patients who undergo definitive operative intervention early in the course of treatment.

Evidence for activation of mutated p53 by apigenin in human pancreatic cancer.

Pancreatic cancer is an exceedingly lethal disease with a five-year survival that ranks among the lowest of gastrointestinal malignancies. Part of its lethality is attributable to a generally poor response to existing chemotherapeutic regimens. New therapeutic approaches are urgently needed. We aimed to elucidate the anti-neoplastic mechanisms of apigenin-an abundant, naturally-occurring plant flavonoid-with a particular focus on p53 function. Pancreatic cancer cells (BxPC-3, MiaPaCa-2) experienced dose and time-dependent growth inhibition and increased apoptosis with apigenin treatment. p53 post-translational modification, nuclear translocation, DNA binding, and upregulation of p21 and PUMA were all enhanced by apigenin treatment despite mutated p53 in both cell lines. Transcription-dependent p53 activity was reversed by pifithrin-α, a specific DNA binding inhibitor of p53, but not growth inhibition or apoptosis suggesting transcription-independent p53 activity. This was supported by immunoprecipitation assays which demonstrated disassociation of p53/BclXL and PUMA/BclXL and formation of complexes with Bak followed by cytochrome c release. Treated animals grew smaller tumors with increased cellular apoptosis than those fed control diet. These results suggest that despite deactivating mutation, p53 retains some of its function which is augmented following treatment with apigenin. Cell cycle arrest and apoptosis induction may be mediated by transcription-independent p53 function via interactions with BclXL and PUMA. Further study of flavonoids as chemotherapeutics is warranted.

Safety in Numbers: Progressive Implementation of a Robotics Program in an Academic Surgical Oncology Practice.

Background. Robotic-assisted surgery has potential benefits over laparoscopy yet little has been published on the integration of this platform into complex surgical oncology. We describe the outcomes associated with integration of robotics into a large surgical oncology program, focusing on metrics of safety and efficiency. Methods. A retrospective review of a prospectively maintained database of robotic procedures from July 2009 to October 2014 identifying trends in volume, operative time, complications, conversion to open, and 90-day mortality. Results. Fourteen surgeons performed 1236 cases during the study period: thyroid (246), pancreas/duodenum (458), liver (157), stomach (56), colorectal (129), adrenal (38), cholecystectomy (102), and other (48). There were 38 conversions to open (3.1%), 230 complications (18.6%), and 13 mortalities (1.1%). From 2009 to 2014, operative volume increased (7 cases/month vs 24 cases/month; P < .001) and procedure time decreased (471 ± 166 vs 211 ± 140 minutes; P < .001) with statistically significant decreases for all years except 2014 when volume and time plateaued. Conversion to open decreased (12.1% vs 1.7%; P = .009) and complications decreased (48.5% vs 12.3%; P < .001) despite increasing complexity of cases performed. There were 13 deaths within 90 days (5/13 30-day mortality) and 2 (15.4%) were from palliative surgeries. Conclusions. Implementation of a diverse robotic surgical oncology program utilizing multiple surgeons is safe and feasible. As operative volume increased, operative time, complications, and conversions to open decreased and plateaued at approximately 3 years. No unanticipated adverse events attributable to the introduction of this platform were observed.

Loss of 15-Hydroxyprostaglandin Dehydrogenase Increases Prostaglandin E2 in Pancreatic Tumors

Objectives: Prostaglandin E2 (PGE2) is a product of cyclooxygenase (COX) and PGE synthase (PGES) and deactivated by 15-hydroxyprostaglandin dehydrogenase (PGDH). Down-regulation of PGDH contributes to PGE2 accumulation in lung and colon cancers but has not been identified in pancreatic cancer. Methods: Normal human pancreatic and tumor-matched tissues, as well as MiaPaCa-2 and BxPC-3 cell lines, were assessed for COX-2, microsomal PGES-1, PGDH, and snail homolog 1 (SNAI1) and SNAI2 expressions by real-time polymerase chain reaction and Western blotting and PGE2 by enzyme-linked immunosorbent assay. Results: Normal tissues exhibited low COX-2 messenger RNA (mRNA) and protein expressions and high PGDH mRNA and protein expressions and PGE2 levels at 13 pg/mg of protein. In contrast, tumor tissues exhibited high COX-2 mRNA and protein expressions and low PGDH mRNA and protein expressions and PGE2 levels at 32 pg/mg of protein. Tumor tissues exhibited significantly elevated expressions of SNAI2 mRNA and protein but not SNAI1 because SNAI1 and SNAI2 reportedly down-regulate PGDH expression. The COX-2-positive BxPC-3 but not the COX-2-negative MiaPaCa-2 treated with 100-nmol/L PGE2 induced phosphorylated extracellular signal-related kinase that was blocked by the mitogen-activated protein kinase kinase inhibitor U0126, demonstrating the ability of PGE2 to activate ERK. Conclusions: These results suggest that enhanced PGE2 production proceeds through the expressions of COX-2 and microsomal PGES-1 and down-regulation of PGDH by SNAI2 in pancreatic tumors.Abbreviations: COX-2 - cyclooxygenase 2, MEK - mitogen-activated protein kinase kinase, MAPK - mitogen-activated protein kinase, ERK - extracellular signal-related kinase, EGF - epidermal growth factor, PKC - protein kinase C, PG - prostaglandin, PGDH - 15-hydroxyprostaglandin dehydrogenase, mPGES - microsomal prostaglandin E synthase

Apigenin inhibits NNK-induced focal adhesion kinase activation in pancreatic cancer cells.

Objectives Tobacco-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) activates &bgr;-adrenergic receptor (&bgr;-AR) signaling through Src/focal adhesion kinases (FAKs)/mitogen-activated protein kinase to modulate proliferation, migration, and survival. Apigenin (4′, 5, 7-trihydroxyflavone) is reported to attenuate proliferation and migration of cancer cells. This study was designed to determine the effects of apigenin on NNK-induced procarcinogenesis using human pancreatic cancer cells BxPC-3 and MIA PaCa-2, which express &bgr;-AR. Methods Proliferation and migration were assessed by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and scratch assays. &bgr;-AR, FAK/mitogen-activated protein kinase and extracellular signal-regulated kinase (ERK) expression and activation were assessed by Western blotting and real-time polymerase chain reaction. Results 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone caused a dose- and time-dependent increase in BxPC-3 and MIA PaCa-2 cell proliferation that was inhibited by propranolol or apigenin. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone also stimulated a time-dependent increase in FAK and ERK activation that was suppressed by propranolol or apigenin. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone–enhanced gap closure at 24 hours was prevented by either propranolol or apigenin. Conclusion Apigenin suppressed the effects of NNK on pancreatic cancer cell proliferation and migration that are mediated through the &bgr;-AR and its downstream signals FAK and ERK activation. These findings suggest a therapeutic role for this natural phytochemical in attenuating the procarcinogenic effects of NNK on pancreatic cancer proliferation and migration.

Predicting Exocrine Insufficiency Following Pancreatic Resection

We read with interest the article, “Reduced Pancreatic Parenchymal Thickness Indicates Exocrine Pancreatic Insufficiency After Pancreatoduodenectomy” by Dr. Nakamura et al.[1] Their work presents a potentially potent tool by which the pancreatic surgeon may treat their patients following surgery to alleviate the effects of exocrine pancreatic insufficiency (EPI). Importantly the metric they investigated, pancreatic parenchymal thickness on pre- and postoperative computed tomography (CT) scans, is readily available and easily translatable to current clinical practice making their results immediately applicable. Of particular interest was their finding that the preoperative pancreatic duct to parenchyma ratio was correlated to postoperative EPI with a cutoff of 14.5 mm being 67.6% sensitive and 72.2% specific.[1] While it is intuitive that EPI, clinically defined as the onset of steatorrhea, is directly related to the extent of resection, the volume of residual pancreas needed to prevent postoperative EPI is unknown and probably highly variable; the extent of the underlying disease process and preoperative pancreatic function may play a role as exemplified by parenchymal fibrosis seen in chronic pancreatitis. Generally, 90–95% of pancreatic enzyme excretion must be lost before clinical signs of EPI develop.[2] Additionally, the concurrent resection of the duodenum or stomach affects the neurohormonal axis of the foregut with alterations in gastrin, cholecystokinin, and pancreatic polypeptide which exert trophic and secretogogue effects on the pancreas. Loss of these enteric hormones affects the rate of atrophy of the pancreatic remnant as well as its digestive function.[3] Due to these factors, it is difficult to predict the onset of EPI and current clinical practice dictates waiting for clinical signs of steatorrhea or malabsorption to develop before starting patients on oral pancreatic enzyme supplementation. Biochemical tests such as fecal fat content, β-1 Elastase, and N-benzoyl-l-tyrosil-para-aminobenzoic-acid (NBT-PABA) can be cumbersome and/or costly to perform routinely and researchers have failed to show consistent relationships between pre- and postoperative testing that would aid in accurately predicting level of postoperative exocrine pancreatic function.[4] Furthermore, it has been recognized for some time that while most patients who have undergone PD have significantly abnormal fecal fat content, many will not have clinical signs of steatorrhea or require oral pancreatic enzyme supplementation.[5] The use of CT to evaluate the thickness of the remnant pancreas as a marker of pancreatic exocrine insufficiency has been reported by Lemaire et al.[6] They found that all patients undergoing PD with pancreaticogastric anastomosis had atrophy of the pancreatic gland on CT and marked increase in fecal fat and fecal-1 elastase. However, they were unable to correlate the degree of parenchymal atrophy to the degree of exocrine insufficiency by biochemical tests. Tajima et al utilized dynamic MRI to monitor the development of pancreatic fibrosis following PD and found that grading the time-signal intensity curve (MRI-tic) served as a predictor of impending exocrine insufficiency. Patients exhibiting an increase in the pancreatic fibrosis grade by MRI-tic analysis tended to experience significant deterioration of pancreatic exocrine function within approximately one year.[7] Finally, a provocative test utilizing IV secretin administration coupled with magnetic resonance cholangiopancreatography (secretin-MRCP) has been used to evaluate the exocrine function of the pancreas.[8] In a study of postoperative PD patients investigators determined that while there was a significant correlation between pancreatic atrophy and patency of pancreaticogastric anastomosis, secretin-MRCP imaging alone was not sufficiently sensitive to diagnose exocrine insufficiency.[9] Dr. Nakamura and colleagues addressed some of the weaknesses of previous studies by correlating preoperative parenchymal thickness to postoperative EPI with a benchmark value (14.5 mm) below which there was increased risk for EPI. They also established a cutoff for diagnosis of postoperative EPI using postoperative parenchymal thickness (13 mm). It would be interesting to obtain 13CO2 values for preoperative patients as this would establish a baseline in patients with pre-existing pancreatic disease-particularly for the two patients with chronic pancreatitis. Inclusion of patients with preoperative EPI may falsely increase the sensitivity of the test by overestimating the number of patients with %CD-7h of less than 5% that have reduced parenchymal thickness postoperatively (i.e.: falsely elevated true positive). Although the authors comment on the adequacy of a single cross-sectional measurement of pancreatic parenchyma thickness in predicting postoperative EPI, the use of volumetric analysis may increase the specificity of the test by more accurately determining the amount of residual parenchyma. This may be particularly true in cases where there is focal dilation of the pancreatic duct leading to underestimation of the parenchymal thickness while there is relatively normal exocrine pancreatic function thus leading to a false positive test result. The use of abdominal imaging to predict the functional status of the pancreas appears to be a developing modality with significant promise as a clinically relevant and applicable test. Perhaps the greatest potential lies in the use of preoperative imaging to predict the onset of postoperative EPI following pancreatic resection or to be able to plan the extent of resection to minimize the risk of EPI while performing an oncologically sound resection.

Robotic-Assisted Pancreaticoduodenectomy (Whipple)

Pancreaticoduodenectomy (PD; Whipple operation) is widely considered to be among the most technically challenging abdominal operations performed. Minimally invasive PD represents a formidable challenge, which has been mastered by relatively few surgeons. Robotic-assisted PD (RAPD) offers advantages of improved instrumentation and freedom of motion when compared to the laparoscopic approach, which may allow greater adoption in general clinical practice. Here, we outline our approach to RAPD including patient selection and preparation, patient positioning and port placement, instrumentation, and technical aspects of the operation. We review outcomes following RAPD as well.