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T1852 Taurolidine in Pancreatic Cancer
lower in groups HS and HSPTX compared with NS group (p<0.05). No differences in pulmonary MPO activity were observed among these three groups. Twelve hours after reperfusion a significant reduction in pulmonary vascular permeability in group HSPTX was observed when compared to groups HS and NS (p<0.05). HS and HSPTX groups showed a reduction in serum IL-6 when compared to NS group (p<0.05). Conclusion: Addition of pentoxifylline to hypertonic saline solution reduces not only the liver damage but also the pulmonary vascular permeability associated to hepatic ischemia reperfusion.
strikingly favorable toxicity profile: It impairs neither liver and kidney function nor hematopoiesis. We investigated the effect of taurolidine in pancreatic cancer, a disease where therapeutic options are scarce. Methods: Two moderately differentiated ductal pancreatic adenocarcinoma cell lines were investigated: DSL-6A (murine) and HPAF-2 (human). Cells were incubated for 72 hours with increasing concentrations of taurolidine (0-2000 μmol/L), gemcitabine (0-100 μmol/L), or a combination of taurolidine (100 μmol/L) and gemcitabine in either high or low concentration. Cell proliferation and viability were assessed using a hemocytometer for direct cell counting and an MTT assay. Results: Both taurolidine and gemcitabine monotherapy inhibited cell growth and viability in a dose-dependent way. The highest concentrations analyzed inhibited cell proliferation by more than 98%. Taurolidine was no less effective than gemcitabine. In combination therapy, taurolidine was able to enhance efficacy of even high dose gemcitabine (Table). Similar results were achieved for cell viability. Conclusion: In pancreatic cancer cells, taurolidine appears to be as effective as gemcitabine, the current gold standard of cytotoxic therapy. The combination of both agents acts synergistically as compared with either monotherapy. Taurolidine has little side effects In Vivo, so its use both in monotherapy and in combination with gemcitabine warrants further investigations. Studies using an orthotopic tumor model of ductal pancreatic adenocarcinoma are necessary to confirm whether this promising new drug is of relevance in a preclinical therapeutic setting. Number of cells (x10^4/mL ± SEM) under therapy as indicated.
T1850 Molecular Identification & Clinical Immune Characterization of Occult HBV Infections Kewal K. Maudar, Arpit Bhargava, Ram P. Punde, Subodh Varshney, Pradyumna K. Mishra Introduction Hepatitis is one of the major diseases of mankind and is a serious global public health problem. The diagnosis of the disease is not only imperative but also complex because of different viral antigens, which bring about varying serological profiles during different stages of the disease. Recently, new forms of infection such as occult infections of HBV are being reported. Occult HBV infections are defined as the presence of low-level of HBV DNA in blood in patients negative for markers of hepatitis B. Objective The aim of the present study was the molecular identification and clinical immune characterization of occult HBV infections in the routine health workers and blood donors at Bhopal Memorial Hospital & Research Centre. Materials & Methods A total of 1000 samples of blood donors and 100 healthy health care workers were undertaken for the study. Qualitative screening for HBV was done through ELISA while the nucleic acid analysis was performed through COBAS Amplicor Analyzer (minimum detection limit 300 copies/ml) and the negative samples were further confirmed with the Light Cycler 2.0 (minimum detection limit 10 copies/ml). In addition, Th1/Th2 cytokine profiling was done for immune characterization of these infections. Results It was observed that all the samples were negative for the HBV in qualitative screening through standard ELISA. Further nucleic acid analysis through COBAS Amplicor Analyzer showed that 22 cases of blood donors were positive for HBV DNA which was previously reported negative in ELISA while no healthcare worker reported positive. All the samples reported negative by COBAS Amplicor were again confirmed by real time PCR and the similar results were obtained. Additionally, a significant increase in the mean levels of Th2 cytokines IL-4, IL-6 and IL-10 in occult cases was observed in comparison to controls. Conclusion In conclusion it is a unique attempt to determine the frequency and importance of this occult phenomenon. Results of such investigations should provide and improve the basic knowledge necessary to specifically understand on the grounds of occult HBV infections. This background provides the scientific basis for the wide range of investigations. It would not only create health awareness, but would also evoke a perpetual social consciousness about the upcoming infectious states of these diseases.
Gemcitabine concentration: Low dose: 0.01 μmol/L for DSL, 1 μmol/L for HPAF. High dose: 10 μmol/L for DSL, 100 μmol/L for HPAF. * p<0.05 compared with control. # p<0.05 compared with taurolidine monotherapy. ~ p<0.05 compared with high dose gemcitabine monotherapy. T1853
Background: Diets rich in fruits and vegetables have been linked to reduced rates of several cancers. Pterostilbene, a stilbenoid related to resveratrol, is a potent antioxidant derived from blueberries. We have shown that pterostilbene inhibits pancreatic cancer cell growth In Vitro by inducing apoptosis. Apoptotic pathways may involve disruption of mitochondrial membrane integrity. We investigated the effect of pterostilbene on the mitochondrial membrane integrity of two pancreatic cancer cell lines, MIA PaCa-2 and PANC-1. Methods: JC1 is a cationic dye that exhibits potential-dependent accumulation in cells with healthy mitochondria. This accumulation leads to the formation of red fluorescent aggregates and a fluorescence emission shift from green to red. Mitochondrial depolarization causes JC-1 leakage and decreased mitochondrial dye aggregation. Mitochondrial depolarization is indicated by a decrease in the red / green fluorescence intensity ratio. Pancreatic cancer cells (3 x 105) were cultured using standard techniques. After 24 hours, 1 x 106 cells were loaded with 2 micrograms of JC-1 for 20 minutes. Cells were then treated with pterostilbene or DMSO control for an additional 20 minutes. Cells were trypsinized, resuspended in PBS and evaluated by flow cytometry. Results: MIA PaCa-2 red/green fluorescence intensity ratio changed from 2.45 with DMSO control to 0.18 with 75 micrograms of pterostilbene treatment. Similarly, this ratio decreased in the PANC-1 cell line from 2.03 in DMSO control to 0.41 with the same pterostilbene treatment. Ratios from treated cells were similar to positive CCCP controls, 0.28 and 0.67, in MIA PaCa-2 and PANC-1 respectively. Thus, pancreatic cancer cells show significant mitochondrial depolarization in response to pterostilbene treatment (p<0.02). Conclusions: Herein we have demonstrated for the first time that pterostilbene induces mitochondrial membrane depolarization in pancreatic cancer cells In Vitro. This provides an initial mechanism for the previously observed growth inhibition and increased apoptosis when pancreatic cancer cells are treated with pterostilbene. Further In Vitro mechanistic studies and In Vivo experiments are warranted and planned to determine the potential role for pterostilbene in pancreatic cancer treatment.
T1851 Changes in Liver Mitochondrial Function Induced By a High-Fat Diet Enriched with ω-3 PUFAS Are Associated with Reduction of Hepatic Lesions Secondary to Ischemia/Reperfusion Ana Maria M. Coelho, Rui Curi, Sandra N. Sampietre, Nilza A. Molan, Hilton Takahashi, Telesforo Bacchella, Marcel C. Machado Previous studies have demonstrated that a high-fat diet enriched with polyunsaturated fatty acids (ω-3 PUFA) has a protective effect on the liver. Lipid emulsion increases hepatocyte uncoupling protein 2 (UCP2) production that seems to have a protective effect by inhibiting mitochondrial ROS production. We hypothesized that a high-fat diet enriched with ω-3 PUFA could modify mitochondrial functions and therefore protect the liver from ischemia/ reperfusion injury. Aim: To evaluate the effect of a high-fat diet enriched with ω-3 PUFA (cold fish oil) on hepatic mitochondrial functions and hepatic disturbances associate with hepatic ischemia/reperfusion (I/R) injury. Methods: Wistar male rats were divided in 2 groups: Group I (n=2):rats with fatty liver as induced by high-fat diet enriched with ω-3 PUFAs given for 4 weeks and Group II (n=20) that received standard diet. Ten animals of each group were killed and their livers were collected for determination of oxidation and phosphorylation of liver mitochondria and liver histological analysis. Activities of AST and ALT were also determined. Ten animals of each group were submitted to a partial liver ischemia performed by clamping the pedicle from medium and left anterior lateral liver segments during an hour. After 2 hours of reperfusion, liver mitochondrial oxidation and phosphorylation were analyzed. Serum levels of TNF-α, IL-6, IL-10, and AST, ALT activities were also determined. Results: Mild liver steatosis was observed in the group I in comparison to group II. The group I showed a significant liver mitochondrial uncoupling: respiration state 4 (23.6±1.97 vs 15.5±1.35 nmol O2 mgprot/min) and RCR (3.09±0.22 vs 3.73±0.14) in relation to the group II (p<0.05). AST and ALT activities were not changed. After I/R, there was an increase in state 3 (79.1±5.42 vs 30.8±2.97) and state 4 (26.5±2.45 vs 15.5±1.01) respiration, and in the respiration control rate (3.06±0.11 vs 1.98±0.15) in the group I compared to group II(p<0.05). The group I showed also lower activities of AST: 1087±775 vs 6607±806 and ALT: 1333±892 vs 7132±1102 and the histological changes observed were significantly less intense (p<0.05). A significant reduction was observed in the levels of TNF-α, IL-6, and IL-10 in the group I as compared to group II (p<0.05). Conclusion: High-fat diet enriched with ω-3 PUFA protected the liver from ischemia/reperfusion injury probably by a mechanism associated to changes mitochondrial function. FAPESP, CNPq
T1854 TNFα and Soluble MD-2 (sMD2) Increase LPS Response in CaCo2 Cells Nathan Huber, Stephanie R. Bailey, Alex B. Lentsch, Timothy A. Pritts Intestinal mucosa responds to acute inflammation with increased cytokine production and mucosal barrier breakdown. This process may involve the TLR4 pathway. While intestinal mucosa is resistant to the LPS-induced signaling at baseline, it may become sensitive to LPS during acute inflammation. The mechanisms underlying this phenomenon are not fully understood. We hypothesized that treatment of intestinal epithelial cells with the proinflammatory cytokine TNFα would alter LPS mediated TLR4 pathway signaling. Methods: Caco2 cells, a human intestinal epithelial cell line unresponsive to LPS at baseline, were grown to confluence and treated with serum free media for 24h prior to treatment with TNFα (100ng/mL). TLR4 expression was analyzed by Western blot. In LPS response experiments, cells were pretreated for 24h with TNFα, then media was changed to include a second 24h treatment with TNFα alone, TNFα and LPS, or TNFα, LPS, and sMD-2 (0.1 nM), an important TLR4 cofactor. Supernatant IL8 content was measured by ELISA. Results: TLR4 expression was markedly increased after 24h and 48h of TNFα treatment. LPS treatment alone did not induce IL8. After TNFα pretreatment, addition of LPS resulted in increased
T1852 Taurolidine in Pancreatic Cancer Elisabeth Schellhaas, Elisabeth Rust, Birgit Hotz, Heinz J. Buhr, Hubert G. Hotz Background: The aminosulfoacid derivative taurolidine was originally developed as an antibacterial substance. Recently, taurolidine was shown to exhibit antineoplastic effects both In Vitro and in animal models via inhibition of protein synthesis, induction of apoptosis, and prevention of neoangiogenesis. In contrast to other antineoplastic agents, it has a
A-923
SSAT Abstracts
SSAT Abstracts
Pterostilbene Disrupts Pancreatic Cancer Mitochondrial Membrane Potential In Vitro David W. McFadden, Julie A. Alosi, John Schneider
strikingly favorable toxicity profile: It impairs neither liver and kidney function nor hematopoiesis. We investigated the effect of taurolidine in pancreatic cancer, a disease where therapeutic options are scarce. Methods: Two moderately differentiated ductal pancreatic adenocarcinoma cell lines were investigated: DSL-6A (murine) and HPAF-2 (human). Cells were incubated for 72 hours with increasing concentrations of taurolidine (0-2000 μmol/L), gemcitabine (0-100 μmol/L), or a combination of taurolidine (100 μmol/L) and gemcitabine in either high or low concentration. Cell proliferation and viability were assessed using a hemocytometer for direct cell counting and an MTT assay. Results: Both taurolidine and gemcitabine monotherapy inhibited cell growth and viability in a dose-dependent way. The highest concentrations analyzed inhibited cell proliferation by more than 98%. Taurolidine was no less effective than gemcitabine. In combination therapy, taurolidine was able to enhance efficacy of even high dose gemcitabine (Table). Similar results were achieved for cell viability. Conclusion: In pancreatic cancer cells, taurolidine appears to be as effective as gemcitabine, the current gold standard of cytotoxic therapy. The combination of both agents acts synergistically as compared with either monotherapy. Taurolidine has little side effects In Vivo, so its use both in monotherapy and in combination with gemcitabine warrants further investigations. Studies using an orthotopic tumor model of ductal pancreatic adenocarcinoma are necessary to confirm whether this promising new drug is of relevance in a preclinical therapeutic setting. Number of cells (x10^4/mL ± SEM) under therapy as indicated.
T1850 Molecular Identification & Clinical Immune Characterization of Occult HBV Infections Kewal K. Maudar, Arpit Bhargava, Ram P. Punde, Subodh Varshney, Pradyumna K. Mishra Introduction Hepatitis is one of the major diseases of mankind and is a serious global public health problem. The diagnosis of the disease is not only imperative but also complex because of different viral antigens, which bring about varying serological profiles during different stages of the disease. Recently, new forms of infection such as occult infections of HBV are being reported. Occult HBV infections are defined as the presence of low-level of HBV DNA in blood in patients negative for markers of hepatitis B. Objective The aim of the present study was the molecular identification and clinical immune characterization of occult HBV infections in the routine health workers and blood donors at Bhopal Memorial Hospital & Research Centre. Materials & Methods A total of 1000 samples of blood donors and 100 healthy health care workers were undertaken for the study. Qualitative screening for HBV was done through ELISA while the nucleic acid analysis was performed through COBAS Amplicor Analyzer (minimum detection limit 300 copies/ml) and the negative samples were further confirmed with the Light Cycler 2.0 (minimum detection limit 10 copies/ml). In addition, Th1/Th2 cytokine profiling was done for immune characterization of these infections. Results It was observed that all the samples were negative for the HBV in qualitative screening through standard ELISA. Further nucleic acid analysis through COBAS Amplicor Analyzer showed that 22 cases of blood donors were positive for HBV DNA which was previously reported negative in ELISA while no healthcare worker reported positive. All the samples reported negative by COBAS Amplicor were again confirmed by real time PCR and the similar results were obtained. Additionally, a significant increase in the mean levels of Th2 cytokines IL-4, IL-6 and IL-10 in occult cases was observed in comparison to controls. Conclusion In conclusion it is a unique attempt to determine the frequency and importance of this occult phenomenon. Results of such investigations should provide and improve the basic knowledge necessary to specifically understand on the grounds of occult HBV infections. This background provides the scientific basis for the wide range of investigations. It would not only create health awareness, but would also evoke a perpetual social consciousness about the upcoming infectious states of these diseases.
Gemcitabine concentration: Low dose: 0.01 μmol/L for DSL, 1 μmol/L for HPAF. High dose: 10 μmol/L for DSL, 100 μmol/L for HPAF. * p<0.05 compared with control. # p<0.05 compared with taurolidine monotherapy. ~ p<0.05 compared with high dose gemcitabine monotherapy. T1853
Background: Diets rich in fruits and vegetables have been linked to reduced rates of several cancers. Pterostilbene, a stilbenoid related to resveratrol, is a potent antioxidant derived from blueberries. We have shown that pterostilbene inhibits pancreatic cancer cell growth In Vitro by inducing apoptosis. Apoptotic pathways may involve disruption of mitochondrial membrane integrity. We investigated the effect of pterostilbene on the mitochondrial membrane integrity of two pancreatic cancer cell lines, MIA PaCa-2 and PANC-1. Methods: JC1 is a cationic dye that exhibits potential-dependent accumulation in cells with healthy mitochondria. This accumulation leads to the formation of red fluorescent aggregates and a fluorescence emission shift from green to red. Mitochondrial depolarization causes JC-1 leakage and decreased mitochondrial dye aggregation. Mitochondrial depolarization is indicated by a decrease in the red / green fluorescence intensity ratio. Pancreatic cancer cells (3 x 105) were cultured using standard techniques. After 24 hours, 1 x 106 cells were loaded with 2 micrograms of JC-1 for 20 minutes. Cells were then treated with pterostilbene or DMSO control for an additional 20 minutes. Cells were trypsinized, resuspended in PBS and evaluated by flow cytometry. Results: MIA PaCa-2 red/green fluorescence intensity ratio changed from 2.45 with DMSO control to 0.18 with 75 micrograms of pterostilbene treatment. Similarly, this ratio decreased in the PANC-1 cell line from 2.03 in DMSO control to 0.41 with the same pterostilbene treatment. Ratios from treated cells were similar to positive CCCP controls, 0.28 and 0.67, in MIA PaCa-2 and PANC-1 respectively. Thus, pancreatic cancer cells show significant mitochondrial depolarization in response to pterostilbene treatment (p<0.02). Conclusions: Herein we have demonstrated for the first time that pterostilbene induces mitochondrial membrane depolarization in pancreatic cancer cells In Vitro. This provides an initial mechanism for the previously observed growth inhibition and increased apoptosis when pancreatic cancer cells are treated with pterostilbene. Further In Vitro mechanistic studies and In Vivo experiments are warranted and planned to determine the potential role for pterostilbene in pancreatic cancer treatment.
T1851 Changes in Liver Mitochondrial Function Induced By a High-Fat Diet Enriched with ω-3 PUFAS Are Associated with Reduction of Hepatic Lesions Secondary to Ischemia/Reperfusion Ana Maria M. Coelho, Rui Curi, Sandra N. Sampietre, Nilza A. Molan, Hilton Takahashi, Telesforo Bacchella, Marcel C. Machado Previous studies have demonstrated that a high-fat diet enriched with polyunsaturated fatty acids (ω-3 PUFA) has a protective effect on the liver. Lipid emulsion increases hepatocyte uncoupling protein 2 (UCP2) production that seems to have a protective effect by inhibiting mitochondrial ROS production. We hypothesized that a high-fat diet enriched with ω-3 PUFA could modify mitochondrial functions and therefore protect the liver from ischemia/ reperfusion injury. Aim: To evaluate the effect of a high-fat diet enriched with ω-3 PUFA (cold fish oil) on hepatic mitochondrial functions and hepatic disturbances associate with hepatic ischemia/reperfusion (I/R) injury. Methods: Wistar male rats were divided in 2 groups: Group I (n=2):rats with fatty liver as induced by high-fat diet enriched with ω-3 PUFAs given for 4 weeks and Group II (n=20) that received standard diet. Ten animals of each group were killed and their livers were collected for determination of oxidation and phosphorylation of liver mitochondria and liver histological analysis. Activities of AST and ALT were also determined. Ten animals of each group were submitted to a partial liver ischemia performed by clamping the pedicle from medium and left anterior lateral liver segments during an hour. After 2 hours of reperfusion, liver mitochondrial oxidation and phosphorylation were analyzed. Serum levels of TNF-α, IL-6, IL-10, and AST, ALT activities were also determined. Results: Mild liver steatosis was observed in the group I in comparison to group II. The group I showed a significant liver mitochondrial uncoupling: respiration state 4 (23.6±1.97 vs 15.5±1.35 nmol O2 mgprot/min) and RCR (3.09±0.22 vs 3.73±0.14) in relation to the group II (p<0.05). AST and ALT activities were not changed. After I/R, there was an increase in state 3 (79.1±5.42 vs 30.8±2.97) and state 4 (26.5±2.45 vs 15.5±1.01) respiration, and in the respiration control rate (3.06±0.11 vs 1.98±0.15) in the group I compared to group II(p<0.05). The group I showed also lower activities of AST: 1087±775 vs 6607±806 and ALT: 1333±892 vs 7132±1102 and the histological changes observed were significantly less intense (p<0.05). A significant reduction was observed in the levels of TNF-α, IL-6, and IL-10 in the group I as compared to group II (p<0.05). Conclusion: High-fat diet enriched with ω-3 PUFA protected the liver from ischemia/reperfusion injury probably by a mechanism associated to changes mitochondrial function. FAPESP, CNPq
T1854 TNFα and Soluble MD-2 (sMD2) Increase LPS Response in CaCo2 Cells Nathan Huber, Stephanie R. Bailey, Alex B. Lentsch, Timothy A. Pritts Intestinal mucosa responds to acute inflammation with increased cytokine production and mucosal barrier breakdown. This process may involve the TLR4 pathway. While intestinal mucosa is resistant to the LPS-induced signaling at baseline, it may become sensitive to LPS during acute inflammation. The mechanisms underlying this phenomenon are not fully understood. We hypothesized that treatment of intestinal epithelial cells with the proinflammatory cytokine TNFα would alter LPS mediated TLR4 pathway signaling. Methods: Caco2 cells, a human intestinal epithelial cell line unresponsive to LPS at baseline, were grown to confluence and treated with serum free media for 24h prior to treatment with TNFα (100ng/mL). TLR4 expression was analyzed by Western blot. In LPS response experiments, cells were pretreated for 24h with TNFα, then media was changed to include a second 24h treatment with TNFα alone, TNFα and LPS, or TNFα, LPS, and sMD-2 (0.1 nM), an important TLR4 cofactor. Supernatant IL8 content was measured by ELISA. Results: TLR4 expression was markedly increased after 24h and 48h of TNFα treatment. LPS treatment alone did not induce IL8. After TNFα pretreatment, addition of LPS resulted in increased
T1852 Taurolidine in Pancreatic Cancer Elisabeth Schellhaas, Elisabeth Rust, Birgit Hotz, Heinz J. Buhr, Hubert G. Hotz Background: The aminosulfoacid derivative taurolidine was originally developed as an antibacterial substance. Recently, taurolidine was shown to exhibit antineoplastic effects both In Vitro and in animal models via inhibition of protein synthesis, induction of apoptosis, and prevention of neoangiogenesis. In contrast to other antineoplastic agents, it has a
A-923
SSAT Abstracts
SSAT Abstracts
Pterostilbene Disrupts Pancreatic Cancer Mitochondrial Membrane Potential In Vitro David W. McFadden, Julie A. Alosi, John Schneider