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Biomedical subjects

M P Callery

Publications and source records attributed to M P Callery.

At least 19 recordsLinked to original sources
PubMed2001

Protective effects of ischemic preconditioning on the cold-preserved liver are tyrosine kinase dependent.

BACKGROUND: Little data exist regarding the use of ischemic preconditioning before sustained hepatic cold storage. We hypothesized that ischemic preconditioning protects hepatic grafts via a tyrosine kinase-dependent pathway. METHODS: Six porcine livers underwent routine harvest (control). Five other livers underwent 15 min of in situ ischemia followed by 15 min of reflow before harvest (ischemic preconditioning). Another five livers were pretreated with a tyrosine kinase inhibitor (genistein) before preconditioning. Upon reperfusion and after 2 hours of cold storage, graft function, graft circulatory impairment, and markers of cellular damage were analyzed. Tissue cytoplasmic extracts were analyzed for tyrosine phosphorylation with Western blot. Significance was determined with t tests. RESULTS: Ischemic-preconditioned grafts demonstrated enhanced bile production, augmented responses to a bile acid challenge, and elevated O2 consumption (P<0.05) compared to controls. Also, preconditioned grafts demonstrated improved hepatic tissue blood flow and decreased hepatic vascular resistance (P<0.005) compared to controls. Endothelial cell preservation (factor VIII immunostain) was improved in preconditioned graft biopsies compared to controls. With genistein pretreatment, all observed improvements returned to control levels. Analysis of cytoplasmic extracts demonstrated an increase in tyrosine phosphorylation before cold ischemia in preconditioned grafts only, but not in control or genistein-pretreated grafts. CONCLUSIONS: The data indicate that ischemic preconditioning protects the liver from sustained cold ischemia and that tyrosine kinases are involved in preconditioning responses.

Alanine Transaminase↗
PubMed2001

Protein kinase C inhibition abrogates hepatic ischemic preconditioning responses.

INTRODUCTION: A transient period of warm ischemia prior to a longer ischemic episode (ischemic preconditioning) protects the hepatic graft from cold ischemia. The mechanism for this protection is unknown, as is the role of protein kinase C in ischemic preconditioning responses. METHODS: Livers from 40 kg Yorkshire pigs were harvested and subjected to 2 h of cold ischemia (n = 6) (control). Another group of harvested livers was pretreated with a 15-min ischemic period followed by 15 min of in situ perfusion with (n = 5) or without (n = 5) a protein kinase C inhibitor, chelerythrine. Following cold ischemia, all grafts were reperfused on a perfusion circuit and the following variables evaluated: (1) hepatic graft function, (2) graft circulatory impairment, (3) hepatocellular damage, and (4) endothelial cell damage. Protein kinase C levels were also evaluated by Western blot in the cytoplasm of all grafts. RESULTS AND DISCUSSION: Ischemic preconditioned grafts demonstrate improved graft function, reduced graft circulatory impairment, and reduced endothelial cell damage as compared to cold ischemia controls. When preconditioned grafts were pretreated with chelerythrine, graft function, graft circulatory impairment, and endothelial cell damage were no different than cold ischemia controls. Ischemic preconditioned grafts demonstrated decreased levels of protein kinase C prior to cold ischemia. There was no change in protein kinase C levels in cold ischemia controls or chelerythrine-pretreated grafts prior to cold ischemia. These data indicate that modulation of protein kinase C is essential for ischemic preconditioning responses in the cold preserved hepatic graft.

Alkaloids↗
PubMed2001

FRAP-p70s6K signaling is required for pancreatic cancer cell proliferation.

BACKGROUND: FRAP-p70s6K signaling regulates mitogenic responses to growth factors in eukaryotic cells. Constitutive p70s6K activation occurs in some human malignancies and may contribute to dysregulated cell growth. We examined whether inhibition of this pathway affects mitogen-induced proliferation and cell cycle progression of human pancreatic cancer cells in vitro. METHODS: Quiescent BxPC3 and Panc-1 human pancreatic cancer cells treated with or without 20 ng/mL rapamycin (FRAP inhibitor) were repleted with 10% FCS to induce cell cycle entry. Proliferation was measured with MTT assay. Cell cycle and apoptosis were determined by FACS analysis. Phosphorylation of p70s6K, Akt, and cdc2 was evaluated by Western blot. Statistical analysis was by two-tailed t test (P < 0.05). RESULTS: Rapamycin (Rapa) inhibited the phosphorylation of p70s6K while inducing G(1) cell cycle arrest (P < 0.005). In both cell lines, Rapa inhibited serum-induced proliferation (P < 0.05) without affecting apoptosis. Cdc2 phosphorylation was inhibited by 15 min with Rapa (not shown), consistent with cell cycle arrest. Akt phosphorylation was not affected, indicating FRAP specificity of Rapa. CONCLUSIONS: FRAP-p70s6K signaling appears to be necessary for G(1)-to-S phase progression and proliferation in pancreatic cancer cells. This supports earlier work demonstrating a similar regulatory role for PI-3' kinase, an upstream activator of FRAP-p70s6K.

Adenocarcinoma↗
PubMed2001

Endotoxin (LPS) stimulates 4E-BP1/PHAS-I phosphorylation in macrophages.

INTRODUCTION: Translational control of cytokine production in endotoxin (LPS)-stimulated macrophages is poorly characterized but likely important. An early step in protein translation is engagement of mRNA by eukaryotic initiation factor 4E (eIF-4E). Translation initiation can be prevented by small 4E-binding proteins (4E-BP1 or PHAS-I) which must be phosphorylated in order to disengage eIF-4E. We examined whether LPS alters 4E-BP1 phosphorylation in macrophages. MATERIALS AND METHODS: Elicited rat peritoneal macrophages and Raw 264.7 macrophages were treated with signal transduction inhibitors and then LPS. Cells were harvested and equal protein amounts were electrophoresed (SDS-PAGE). Western blots (WB) were developed with 4E-BP1 antibody. Alternatively cell lysates were exposed to 7-methyl GTP Sepharose beads in order to isolate the cap-binding protein eIF-4E. The relative amounts of 4E-BP1 associated with eIF-4E were then determined by WB. RESULTS: Macrophage 4E-BP1 is phosphorylated upon stimulation by LPS as evidenced by the appearance of a more slowly migrating gamma (hyperphosphorylated) band on gel electrophoresis. Inhibition of both the p42/p44 MAPK pathway (PD 98059) and the p38 MAPK pathway (SB 203580) failed to alter LPS-induced 4E-BP1 phosphorylation. Rapamycin (FRAP/mTOR inhibitor) blocked 4E-BP1 phosphorylation causing a predominance of the alpha (hypophosphorylated) band. This was confirmed further by 7-methyl-GTP Sepharose isolation of eIF-4E with which 4E-BP1 coprecipitates. CONCLUSION: LPS stimulates 4E-BP1 phosphorylation in macrophages through FRAP/mTOR signaling. This pathway may contribute to the translational control of cytokine gene expression in macrophages.

Animals↗
PubMed2001

Elevated intrahepatic pressures and decreased hepatic tissue blood flow prevent gas embolus during limited laparoscopic liver resections.

BACKGROUND: As new techniques are emerging for laparoscopic liver resections, concerns have been raised about the development of gas embolus related to the CO(2) pneumoperitoneum. We hypothesized that elevated intrahepatic vascular pressures and decreased hepatic tissue blood flow (LQB) would prevent gas embolus during laparoscopic liver resections under conventional pneumoperitoneum. METHODS: Intrahepatic vascular pressures and LQB were measured in nine pigs with varying CO(2) pneumoperitoneum. Gas embolus was determined after hepatic incision by monitoring pulmonary arterial pressure (PAP), hepatic venous PCO(2), systemic blood pressure (SBP), and suprahepatic vena cava ultrasound. RESULTS: As the pneumoperitoneum was increased from 0 to 15 mmHg, intrahepatic vascular pressures increased significantly (p < 0.05), while LQB decreased significantly (p < 0.05). A 2.0-cm hepatic incision at 4, 8, 15, and 20mmHg produced no ultrasound evidence of gas embolus and no changes in PAP, SBP, or hepatic venous PCO(2) (p = NS). CONCLUSION: These data suggest that the risk of significant embolus under conventional pneumoperitoneum is minimal during laparoscopic liver resections.

Animals↗
PubMed2001

V-PYRRO/NO: an hepato-selective nitric oxide donor improves porcine liver hemodynamics and function after ischemia reperfusion.

BACKGROUND: The role of nitric oxide (NO) in ischemia reperfusion (I/R) injury is controversial as both beneficial and harmful effects have been reported. We explored the potential role of a pharmacological agent recently shown to generate NO metabolically in the liver in an animal model of transplantation. METHODS: The effect of a selective hepatic NO donor, O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), on hepatic hemodynamics and biliary function was evaluated in both the in situ and I/R pig liver. RESULTS: V-PYRRO/NO significantly reduced in situ hepatic vascular resistance (HVR) without altering systolic blood pressure. Portal vein flow was essentially unchanged during in situ infusions while hepatic artery flow nearly doubled (P=0.03). After I/R, V-PYRRO/NO infusions significantly reduced both portal vein pressure (PVP) and HVR (P=0.04). Also, serum bile acid clearance increased from 15% when taurocholate (TC) was infused alone to 46% (P=0.007) when infused simultaneously with V-PYRRO/NO. Aqueous bile production tripled with TC and V-PYRRO/NO as compared to TC alone (P=0.04). Analysis of bile outputs revealed a significant increase in biliary cholesterol, biliary phospholipid, and biliary bile acid (P<0.05) with V-PYRRO/NO infusion. CONCLUSIONS: The hepato-selective nitric oxide donor, V-PYRRO/NO, reduced hepatic resistance parameters of the pig liver both before and after I/R and improved the plasma clearance of bile acid and biliary outputs of bile acid-dependent compounds. The augmented function observed after I/R may be due to improvements in hepatic blood flow secondary to altered hepatic hemodynamics.

Animals↗
PubMed2001

Hemodynamic and metabolic variables predict porcine ex vivo liver function.

Early recognition of hepatic function during initial graft reperfusion is important in beginning hepatic support perfusions as well as in liver transplantation. We hypothesized that both hemodynamic and metabolic perfusion variables obtained immediately after reperfusion predict eventual function during liver support or transplantation. Specific hemodynamic variables, i.e., portal vein pressure and hepatic vascular resistance, as well as metabolic variables, i.e., O(2) consumption and P(CO(2)) gradients, were compared with indices of hepatic function and damage, i.e., aqueous bile production, bile lipid outputs, lactate dehydrogenase levels, and histopathology, during an ex vivo support perfusion. O(2) consumption during early reperfusion correlated directly with unstimulated bile flows (P < 0.02) and histopathology scores (P < 0.05). Hepatic venous P(CO(2)) gradients correlated inversely with unstimulated bile flows (P < 0.05). Hemodynamic variables, i.e., portal vein pressure and hepatic vascular resistance, were inversely related with taurocholate-stimulated bile flows (P < 0.05). Hemodynamic and metabolic variables of early reperfusion are useful parameters in predicting eventual effectiveness of the harvested liver for ex vivo hepatic support perfusions.

Animals↗
PubMed2000

Pancreatic cancer cell proliferation is phosphatidylinositol 3-kinase dependent.

BACKGROUND: Genetic mutations found in pancreatic cancer (K-ras, p16, p53) lead to inappropriate cellular proliferation. Mitogens stimulate proliferation via the phosphatidylinositol 3-kinase (PI3K)- and/or the p44/42-mitogen-activated protein kinase [p44/42-MAPK or extracellular signal-regulated kinase (ERK)] signaling pathways. We examined whether inhibition of either PI3K or ERK could limit proliferation in human pancreatic cancer. METHODS: Proliferation was stimulated in quiescent human pancreatic cancer cell lines (BxPC3 and Panc-1) by 10% fetal calf serum (FCS). In certain samples, PD98059 (an ERK inhibitor) or LY294002 (a PI3K inhibitor) was also added. AKT phosphorylation (indicating PI3K activity) and ERK phosphorylation (ERK activation) were determined by Western blot. Cell viability was determined by MTT assay. Cell cycle progression and apoptosis were determined by flow cytometry. A two-tailed t test was used for statistical analysis of the data (significance P < 0.05). RESULTS: LY294002 inhibited the PI3K pathway without affecting ERK activation in response to serum. PD98059 inhibited the ERK pathway specifically. In both BxPC-3 and Panc-1 cell lines, LY294002 inhibited serum-induced proliferation. This was associated with G(1) cell cycle arrest and with an increase in the rate of apoptosis. PD98059 inhibited proliferation only in BxPC3 cells, and to a lesser degree than did LY294002. CONCLUSIONS: PI3K signaling appears to be necessary for G(1)-to-S phase progression and proliferation in pancreatic cancer cells. ERK plays a lesser role in mitogen-induced proliferation. Pharmacological inhibition of PI3K may decrease proliferation, increase apoptosis, and potentially confer therapeutic benefit in pancreatic cancer.

Cell Cycle↗
PubMed2000

NFkappaB expression during cold ischemia correlates with postreperfusion graft function.

In liver transplantation, activation of NFkappaB occurs upon reperfusion, yet few data exist regarding NFkappaB activation during cold ischemia. We hypothesized that activation of NFkappaB may initially occur during cold ischemia, prior to reperfusion, and serve as an important determinant of postreperfusion function. To test this hypothesis, serial biopsies during porcine liver harvest were obtained immediately upon laparotomy, upon completion of dissection, after 45 and 120 min of cold ischemia, and 60 and 180 min after reperfusion. Nuclear extracts were isolated for Western blot analysis of NFkappaB. Hepatic function was assessed through bile output and sorbitol dehydrogenase (SDH) activity. NFkappaB expression was maximal at 45 min of cold ischemia and decreased by 120 min. The expression at 120 min of cold ischemia correlated with markers of postreperfusion function, namely bile flow and SDH activity. During reperfusion a second distinct peak occurred at 180 min. Increased expression of NFkappaB at 180 min of reperfusion correlated directly with prior expression at 120 min during cold ischemia and with increased SDH activity. These data indicate that nuclear expression of NFkappaB demonstrate two distinct peaks of activity, one during cold ischemia and one after reperfusion. Enhanced expression of NFkappaB during cold ischemia not only correlates directly with NFkappaB expression during reperfusion, but also correlates inversely with postreperfusion graft function.

Animals↗
PubMed2000

Laparoscopic surgery and Kupffer cell activation.

BACKGROUND: Evidence tends to support a relative preservation of the systemic immune response with laparoscopy as compared with laparotomy. However, the role of hepatic macrophages, or Kupffer cells, in modulating this immune advantage is unknown. This study investigated the functions of Kupffer cells after either laparoscopy or laparotomy in a rat model. METHODS: Rats underwent laparoscopy, laparotomy, or control operations. Kupffer cells were harvested, cultured, and stimulated with lipopolysaccharide. Culture supernatants were analyzed for tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6). Cytoplasmic lysates were analyzed for activation of two mitogen-activated protein kinases (MAPKs). RESULTS: Production of TNF-alpha and IL-6 was similar in laparoscopy, laparotomy, and control groups. Both laparotomy and laparoscopy showed increased activation of p38 MAPK as compared with controls. Activation of ERK1/2 was decreased during laparotomy as compared with laparoscopy. CONCLUSIONS: Although cytokine production was similar in the laparoscopy and laparotomy groups, changes in MAPK activation suggest that intracellular pathways are more affected during laparotomy than during laparoscopy.

Animals↗
PubMed2000

The feasibility and accuracy of diagnostic laparoscopy in the septic ICU patient.

BACKGROUND: Systemic inflammatory response syndrome (SIRS) and sepsis of unknown origin are common complications of critically ill patients in the ICU. These patients frequently have unreliable clinical exams and are candidates for exploratory laparotomy. Although abdominal CT is commonly used because it is less invasive than laparotomy, it is often unreliable or unobtainable. Bedside laparoscopy is an alternative technique that may be more accurate than CT in selected patients and less invasive than laparotomy. METHODS: We performed diagnostic laparoscopy (DL) in a series of ICU patients with SIRS/septic state of unknown origin between May 1997 and June 1998. All patients were unstable and required significant respiratory and hemodynamic support. Laparoscopy was either performed in the ICU at the patient's bedside or in the operating room. CT scan of the abdomen had been performed on most of the patients who were stable enough to transport. Confirmation of diagnosis was obtained either by laparotomy, autopsy, or clinical recovery. RESULTS: Among the 17 eligible patients, 16 underwent successful DL. Insufflation was impossible in one patient because of high intraabdominal pressure. Bedside evaluations were performed in 14 of the 17 patients. There were no complications from the laparoscopy. Six patients were identified as positive (four intestinal ischemia, two cholecystitis); the other 10 had negative explorations. Follow-up on two patients with negative laparoscopy was incomplete due to denied postmortem. Laparoscopic diagnoses were confirmed in the remaining 14 patients by laparotomy (six cases), postmortem (three cases), or recovery (five cases), with an accuracy of 100%. The overall accuracy of abdominal CT obtained in nine of the 14 patients was 33%. CONCLUSIONS: DL in a select group of critical ICU patients is safe and accurate, whereas CT scan tends to be inaccurate and is often unobtainable due to patient instability. Performing the procedure at the bedside can expedite the diagnosis, eliminate the burden for transfer, and save on anesthesia and operating room charges.

Feasibility Studies↗
PubMed2000

A critical appraisal of laparoscopic staging in hepatobiliary and pancreatic malignancy.

Prognosis for patients with hepatobiliary and pancreatic cancers is dismal. Surgery is the best therapeutic option for those with tumors which have not yet metastasized. Standard radiologic tests such as computed tomography (CT) scan and trans-abdominal ultrasound are useful in identifying patients for whom an attempt at resection would be futile. Staging laparoscopy with laparoscopic ultrasound allows greater precision in identifying those for whom resection would be helpful with less morbidity than an open exploration. Metastatic disease can be identified more precisely than with radiologic tests and can be characterized by biopsy techniques. Palliative procedures are now being performed laparoscopically with low morbidity and short hospital stays. The use of laparoscopy prior to open exploration for patients with hepatobiliary and pancreatic tumors is advantageous.

Biliary Tract Neoplasms↗
PubMed2000

Hand-assisted laparoscopic surgery (HALS) with the HandPort system: initial experience with 68 patients.

OBJECTIVE: To evaluate the feasibility and potential benefits of hand-assisted laparoscopic surgery with the HandPort System, a new device. SUMMARY BACKGROUND DATA: In hand-assisted laparoscopic surgery, the surgeon inserts a hand into the abdomen while pneumoperitoneum is maintained. The hand assists laparoscopic instruments and is helpful in complex laparoscopic cases. METHODS: A prospective nonrandomized study was initiated with the participation of 10 laparoscopic surgical centers. Surgeons were free to test the device in any situation where they expected a potential advantage over conventional laparoscopy. RESULTS: Sixty-eight patients were entered in the study. Operations included colorectal procedures (sigmoidectomy, right colectomy, resection rectopexy), splenectomy for splenomegaly, living-related donor nephrectomy, gastric banding for morbid obesity, partial gastrectomy, and various other procedures. Mean incision size for the HandPort was 7.4 cm. Most surgeons (78%) preferred to insert their nondominant hand into the abdomen. Pneumoperitoneum was generally maintained at 14 mmHg, and only one patient required conversion to open surgery as a result of an unmanageable air leak. Hand fatigue during surgery was noted in 20.6%. CONCLUSIONS: The hand-assisted technique appeared to be useful in minimally invasive colorectal surgery, splenectomy for splenomegaly, living-related donor nephrectomy, and procedures considered too complex for a laparoscopic approach. This approach provides excellent means to explore, to retract safely, and to apply immediate hemostasis when needed. Although the data presented here reflect the authors' initial experience, they compare favorably with series of similar procedures performed purely laparoscopically.

Abdomen↗
PubMed1999

Biliary secretion of extracorporeal porcine livers with single and dual vessel perfusion.

BACKGROUND: Hepatic support systems that provide detoxification without biliary secretion (i.e., isolated hepatocyte systems) are sufficient to improve encephalopathy and bridge patients to transplantation. However, biliary secretion may be critical when hepatic support attempts to restore function and regeneration of the host liver. The purpose of these studies was to optimize the support liver secretory response to bile acid by either single-vessel (portal vein; PV) or dual-vessel (hepatic artery [HA] + PV) perfusions during extracorporeal porcine liver perfusion. METHODS: Extracorporeal porcine liver perfusion of anesthetized pigs was developed using support porcine livers perfused through the PV (n=4) alone and through the HA + PV (n=4) via a venovenous circuit. Support livers were provoked with taurocholate (TC) to enhance bile aqueous and hydrophobic outputs. RESULTS: After cold preservation and reperfusion, both PV and HA + PV livers had initial 1-hr bile aqueous outputs < 15% of in vivo flow, with cholesterol (C) and phospholipid (PC) outputs <25% of in vivo flow. Bile flow was significantly greater for recovered HA + PV livers (3.0+/-0.01 ml/15 min) than PV livers (1.9+/-0.01 ml/15 min). Despite this, PC output was significantly greater for PV than HA + PV livers. The C/PC ratio of PV livers was twice that of HA + PV livers. TC infusion (48 micromol/kg/15 min) of HA + PV livers demonstrated significantly greater increments in bile flow, PC output, and C output than PV livers. CONCLUSION: In the unstimulated state, porcine support livers with dual-vessel perfusion generated greater aqueous and C outputs despite diminished PC output than in those with single-vessel perfusion. TC stimulation increased bile flow, PC output, and C output in dual-perfused livers more than in PV livers. HA + PV perfusion of support livers is the preferred technique for removing hydrophobic compounds that require PC transport for excretion or exist in the aqueous phase.

Animals↗
PubMed1999

Sodium salicylate inhibits macrophage TNF-alpha production and alters MAPK activation.

INTRODUCTION: Transcriptional activation of the TNF-alpha gene in LPS-stimulated macrophages is dependent upon nuclear factor kappa-B (NF-kappaB) activity. Salicylates may interfere with NF-kappaB activity through a MAPK (mitogen-activated protein kinase)-dependent process. These studies investigate the effects of sodium salicylate (NaSal) on TNF-alpha production and MAPK activation in macrophages. METHODS: Rat peritoneal macrophages were pretreated or not with sodium salicylate or ibuprofen for 1 h and then stimulated with 100 ng/ml LPS. Six hours following stimulation, cell viability was assessed by MTT assay. At specified time intervals after LPS stimulation, supernatant TNF-alpha was measured by ELISA. Western blots of cell lysates were performed for analysis of total and activated (phosphorylated) MAPKs. RESULTS: Salicylate and LPS, alone or combined, did not significantly alter macrophage viability. Salicylate, but not ibuprofen, significantly reduced TNF-alpha production in LPS-stimulated macrophages. LPS-stimulated activation of ERK and SAPK/JNK was inhibited by NaSal pretreatment. NaSal treatment of macrophages activated p38 MAPK independent of LPS stimulation. Pretreatment of samples with the specific p38 MAPK inhibitor, SB203580, did not significantly alter TNF-alpha production in either LPS or NaSal and LPS-treated samples. CONCLUSIONS: Salicylates alter MAPK signaling and suppress TNF-alpha production in LPS-stimulated macrophages. Salicylate-induced control of inflammatory mediator production in macrophages may, in part, underlie the clinically significant anti-inflammatory effects of these compounds.

Animals↗
PubMed1999

Salicylates inhibit NF-kappaB activation and enhance TNF-alpha-induced apoptosis in human pancreatic cancer cells.

INTRODUCTION: Tumor necrosis factor (TNF-alpha)-induced apoptosis is limited by its coactivation of nuclear factor kappa B (NF-kappaB)-dependent anti-apoptotic genes. Sodium salicylate (NaSal) inhibits NF-kappaB activation by limiting phosphorylation and degradation of its bound inhibitor protein, IkappaB-alpha. We examined whether NaSal enhances TNF-alpha-induced apoptosis in cultured human pancreatic cancer cell lines. METHODS: Two cultured human pancreatic cancer cell lines were studied. PANC-1 and BxPC-3 cells were serum-starved for 12 h, pretreated or not for 1 h with NaSal (5-20 mM), and then stimulated with recombinant human TNF-alpha (400 units/ml). Western blots of cytoplasmic lysates were performed to demonstrate IkappaB-alpha phosphorylation and degradation. Western blots of nuclear extracts were performed to assess nuclear translocation of NF-kappaB. In separate cultures, apoptosis was measured 4.5 h after TNF-alpha stimulation by both ELISA detection of interhistone DNA fragments and flow cytometry with propidium iodide staining. RESULTS: TNF-alpha induced IkappaB-alpha phosphorylation and degradation, which was inhibited by NaSal in both cell lines. TNF-alpha-induced apoptosis (DNA fragmentation) increased significantly when BxPC-3 cells were pretreated with NaSal. Flow cytometry confirmed this, demonstrating increases in apoptotic cell fractions: 8.5% (untreated), 9.3% (TNF-alpha alone), 14.9% (15 mM NaSal), and 22.9% (NaSal and TNF-alpha). In contrast, no increases in apoptosis were measured in the PANC-1 cell line among the various treatment groups. CONCLUSIONS: NaSal enhances TNF-alpha-induced apoptosis while inhibiting IkappaB-alpha phosphorylation and degradation in BxPC-3 human pancreatic cancer cells.

Adenocarcinoma↗
PubMed1999

Ubiquitin-proteasome inhibition enhances apoptosis of human pancreatic cancer cells.

BACKGROUND: Tumor necrosis factor (TNF-a)-induced apoptosis is limited by coactivation of nuclear factor kappa B (NF-kb)-dependent antiapoptotic genes. Nuclear translocation of NF-kB requires degradation of ubiquitinated phospho-IkB-a by the 26S proteasome. We examined whether inhibition of the ubiquitin-proteasome pathway enhances TNF-a-induced apoptosis in BxPC-3 human pancreatic cancer cells. METHODS: Serum-starved BxPC-3 cells (12 hours) were pretreated or not for 50 minutes with PSI (30 m mol/L), a peptide aldehyde known to inhibit specifically the chymotrypsin-like activity of the 26S proteasome. Cells were subsequently stimulated with recombinant human TNF-a (400 units/mL). Western blots were performed using antibodies to IkB-a and phospho-IkB-a. Level of apoptosis was determined by two methods: enzyme-linked immunosorbent assay detection of interhistone DNA fragments and flow cytometry with propidium iodide staining. RESULTS: TNF-a-induced degradation of IkB-a was inhibited by PSI. Phospho-IkB-a accumulation was observed 20 minutes after TNF-a stimulation. Apoptosis relative to constitutive levels was significantly increased after PSI pretreatment, as measured by DNA fragmentation (P < or = .05 by Student t test). Percent apoptosis by flow cytometry confirmed marked increases in apoptotic cell fractions from 5.9% (untreated) to 6.8% (TNF-a alone), 16.4% (PSI alone), and 18.9% (PSI and TNF-a). CONCLUSIONS: PSI enhances both constitutive and TNF-a-induced apoptosis through inhibition of IkB-a degradation in BxPC-3 human pancreatic cancer cells.

Apoptosis↗