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Enhancement of photodynamic therapy for pancreatic cancer using ALA with an iron chelator in hamsters
A524 AGA ABSTRACTS
2783 USE OF A POL¥URETHANE COVERED NITINOL STENT FOR MALIGNANT GASTRODUODENAL OBSTRUCTION. Seonja Park, Hosung Son, Mooin Park, Gyosik Jung, Jayoung Koo, Kosin Med Ctr, Pusan, MD, South Korea. Purpose : To investigate the feasibility and clinical effectiveness of a polyurethane-covered nitinol stent in the treatment of malignant gastroduodenal obstructions Methods: Under fluoroscopic guidance, the stent was placed in 37 consecutive patients with malignant gastric outlet obstruction (n=31) and duodenal obstruction (n=6). All patients had severe nausea and recurrent vomiting. 16 patients underwent implantation of a self expanding uncovered stent from Sep 1998 to March to 1999 and 21 patients underwent implantation of coaxial stent (a polyurethane-covered expandable nitinol stent within uncovered stent) from Apr 1999 to Nov 1999. Contrast radiographs were taken before and after insertion in all patients to confirm patency. Success was defined both technically and clinically. Results: Technical success was achieved in 36 patients (97%). Clinical success was achieved in 36 patients (97%). The one failure was caused by another stenosis at proximal jejunum that was overlooked before stent placement.. During the follow up of 2 to 50 weeks (mean 12 weeks) there were no major complications except that the stents occluded in 3 patients(covered : 2 cases, coaxial : I case) and migrated 7 patients (covered - 5 cases 31.3% and coaxial -2 cases, 9.5%). Conclusions : Palliation of malignant gastroduodenal obstructions with polyurethanecovered expandable nitinol stent is a feasible, effective, and safe altemative to operation. Especially, coaxial stent is better than covered stent in reducing migration.
2784 TWO REGIONAL CHEMOTHERAPIES FOR INOPERABLE COLORECTAL LIVER METASTASES: A COMPARISON. Uwe Pohlen, Gert Berger, Marion Binnenhei, H. J. Buhr, Benjamin Franklin Med Ctr, Berlin, Germany. Introduction and Objective: The application of regional therapies to prolong survival in patients with colorectal liver metastases is controversial. This study compared two different treatment regimens of regional chemotherapy. The goal was to evaluate these regimens as regards survival. Material and Methods: Sixty patients (38 female, 22 male), aged 42 to 72 (mean: 62), underwent regional therapy for inoperable colorectal liver metastases between 1990 and 1997. Group I (22 patients) was administered 600 mg/m 2/BSA 5-fluorouracil (FU) and 300 mg/m 2/BSA folinic acid via an implanted catheter system in the gastroduodenal artery for 5 consecutive days at two week intervals. Group II (38 patients) received regional applications of 600 mg/m2/BSA 5-FU, 500 mg/m 2/BSA folinic acid, 5 mill. IV Intron A and 450 mg starch microspheres (Spherex®) at the same intervals. Before therapy onset and again after 5 cycles, portal CT and chest x-ray examinations were performed and the tumor markers CEA and CA 19-9 were determined. Results: During treatmenta leukopenia WHO I was observed in 80% of the patients. Twenty patients developed chills and septic temperatures after administration of Intron A, which were regressive after i.m. injection of pethidine (Dolantin®). After 14 months, 48 patients (80%) developed a catheter complication, 15 of whom had to discontinue therapy. In the remaining 33 patients, a new catheter was inserted into the hepatic artery via the subclavian artery and treatment was continued. Group I had a response rate of 45%; median survival was 16 months. In Group II the response rate was 78%; the median survival time in the observation period was 31 months. The patients were documented by means of CT imaging and the tumor marker course. Conclusion: We attribute the high response rate (Group II) of 78% with a median survival of 31 months to the combination of 5-FU and folinic acid preceded by flow delay with starch microspheres. The concentration increase in tumor tissue is possibly enhanced by the immunomodulatory effects of interferon alpha. The response rate of 45% and the survival time of 16 months in Group I is markedly better than systemic therapy with both drugs. Group II yielded two-fold the response rate and survival time of Group I. Despite the new chemotherapy drugs and their combination in systemic treatment, a markedly longer survival time was seen by regionally administering a modified 5-FU schema.
2785 INTRA·AORTIC ADMINISTRATION OF 5-FU-PEG LIPOSOMES INCREASES TISSUE CONCENTRATION. Uwe Pohlen, Gert Berger, Marion Binnenhei, R. Reszka, H. J. Buhr, Benjamin Franklin Med Ctr, Berlin, Germany; Max Delbrueck Ctr, Berlin, Germany. Introduction and Objective: The application of liposorne-encapsulated cytostatic drugs leads to an increased tumor concentration. This effect can be enhanced by arterial administration as well as by reducing the blood flow and thus prolonging retention in the tumor. The goal of this work was to measure the 5-fluorouracil (FU) concentration in various organs and in the VX-2 liver tumor after different applications (i.v. versus intra-aortic).
GASTROENTEROLOGY Vol. 118, No.4
Material and Methods: In 20 chinchilla rabbits with a VX-2 liver tumor, a catheter indwelling system was implanted via the left common iliac artery and vein. Clips were placed on the distal common iliac artery and vein as well as above the celiac artery and the hepatovenous inflow to the cava vena. An arteriovenous shunt and minipump system was introduced and the abdomen was perfused for 20 min under hypoxic conditions with a flow rate of lOOm1lmin. Group I (n= 10) received an intra-arterial dose of 50 mg 5-FU. Group II (n= 10) was administered 50 mg 5-FU-PEG liposomes intra-arterially via the aorta. Group III (n=lO) received a 50 mg i.v. injection via the ear vein. In Group IV (n= 10), 50 mg 5-FU-PEG liposomes were administered via the ear vein. pH level and p02 were measured every 5 minutes. After treatment was terminated (20 min), the animals were killed. Liver, tumor, and para-aortic lymph nodes were removed and the concentration of 5-FU was determined using HPLC. In the intra-aortic groups (I and II), the concentrations of 5-FU in serum and perfusate were determined. In the i.v. groups (III and IV) only the serum concentration of 5-FU could be determined. Results: Compared to the i.v.-applied singleentity drug, the i.a-administered 5-FU-PEG liposomes combined with a flow delay led to concentration increases of 4O-fold in the tumor tissue and even lOO-fold in the para-aortic lymph nodes. The pH and p02 measurements show a sharp decrease during the perfusion, which suggests sufficient unassisted abdominal perfusion. To exclude large shunt volumes the 5-FU concentrations in serum and perfusate were measured. The levels showed only a small shunt volume. Conclusion: The intra-aortic administration of 5-FU-PEG liposomes increased the 5-FU concentration 40-fold in tumor and 100-fold in para-aortic lymph nodes compared to the i.v, application.
2786 EVALUATION OF ULTRASONOGRAPHIC IMAGING IN LASER· INDUCED THERMOTHERAPY (LITT) IN THE LIVER. Uwe Ritzel, Dinko Berkovic, Urs Leonhardt, Giuliano Ramadori, Univ of Goettingen, Goettingen, Germany. INTRODUCTION: Recently, evidence was given that MR-guided laserinduced thermotherapy (Ll'FT) in combination with MR thermometry might be an option in palliative therapy of liver metastases. Thereby, absorption of laser light energy induces necrosis of tumor cells by coagulation and hyperthermia. However, MR-guided UTI requires a high level of techniqual equipment that reduces its advantage as compared to surgical resection of isolated metastases. Therefore, in the present study we evaluated an ultrasonographic imaging technique for guided UTI in the liver. METHODS: Laser coagulation was performed by using a neodymiumyttrium aluminium gamet (Nd:YAG) laser (Domier MediLas 5060) with a scattering-dome light emitter. Laser light wavelength was 1046 nm. The placement of the laser probe and its coagulation effect was on-line monitored by abdominal ultrasonography (ATI) using porcine livers at 37° C. In a first series of experiments the pure laser probe was placed into the liver by ultrasonographic guidance. In a second series the probe was coated by a special application catheter set allowing water cooling of the laser probe. The performance was varied from 5 W to 20 W corresponding to a laser light energy of 500 J to I()()()() 1. RESULTS: Ultrasonographic imaging allowed an exact placement and monitoring of the laser probes. When laser light emission was performed, the ultrasonographic image strongly correlated with the macroscopically visible coagulation zone of the liver. Using only the pure laser probe, the maximum diameter of the coagulation zone did not exceed I cm because of carbonisation around the diffusing applicator. A laser energy over 1000 J induced thermic destruction of the laser probe. The coagulation zone was significantly increased using the water cooled catheter set which increased the coagulation diameter up to 3 em and allowed laser light emission of 5000 J at 20 W. Increasing the laser energy did not increase the coagulation diameter because of carbonisation, CONCLUSIONS: It was shown by ex vivo experiments that ultrasonographic imaging could be a useful and easy to perform technique for application of UTI in the liver. Using a water cooled application catheter set, the coagulation diameter of a single laser probe could be increased up to 3 em. These preliminary ex vivo experiments give promise for further investigations using the ultrasonographic imaging technique for in situ UTT in minimal invasive palliative treatment of liver tumors.
2787 ENHANCEMENT OF PHOTODYNAMIC THERAPY FOR PAN· CREATIC CANCER USING ALA WITH AN IRON CHELATOR IN HAMSTERS, Agnieszka Z. Rogowska, Gio A. Buonaccorsi, Sandy Mosse, James Connelly, Alexander J. MacRobert, Stephen G. Bown, National Med Laser Clr, UCL, London, United Kingdom; Dept of Med Physics, UCH, London, United Kingdom; National Med Laser Ctr, London, United Kingdom. Background. Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)only produces superficial necrosis in humans as the maximum tolerated dose of ALA generates low level of the photoactive derivative protoporphyrin IX (PPIX). Iron chelators slow the final conversion of PPIX to haem so may enhance ALA-PDT by increasing tissue levels of PPIX.
AGAA525
April 2000
Aim. To determine if co-administration of the iron chelator-I,2 diethyl-3hydroxypiridin-4-one (CP94) increases the tissue level of PPIX and enhances ALA-PDT induced necrosis in an animal model of pancreatic cancer. Methods. Cancers were produced in normal, female, Syrian Golden hamsters(loo-120g) by injecting PC-I cells into pancreas at laparotomy. For kinetic studies, animals were given 2oomg/kg ALA alone or with 100,200,400 or 800mg/kg CP94 orally. Tissue from normal pancreas and tumour was taken 30 minutes to 6hr later for measurement of the PPIX level by fluorescence microscopy (3-9 animals analysed per point). For PDT, 2hr after 200mg/kg ALA alone or with 100 or 200mg/kg CP94, red light (635nm, IOOJ, loomW) was delivered to the tumour via a bare optical fibre at laparotomy. 3 days later the area of necrosis was measured macroscopically and specimens were taken for histological assessment. Results. Pharmacokinetic study. Maximum PPIX fluorescence was seen at 2 hrs. Adding 200mg/kg CP94 to 2oomg/kg ALA increased the PPIX by 65% in normal pancreas and by 200% in the tumour compared to ALA alone (37% and 100% increase using loomg/kg CP94). Higher doses of CP94 were poorly tolerated and not studied further, though increased PPIX by up 254% in normal pancreas. Animals given CP94 without ALA showed no increase of PPIX. Spectroscopy confirmed that fluorescence was due to PPIX (maxima at 635nm). PDT study. The combination of ALA and CP94 at least doubled the mean volume (l Zl mnr' v 52mm 3)and depth (6.lmm v 3.3mm) of necrosis compared with ALA alone. Histopathology confirmed necrosis in the treated areas identified macroscopically. Conclusion. Adding the iron chelator CP94 significantly increases PPIX fluorescence and enhances ALA-PDT in this model of pancreatic cancer.
2788 THE MECHANISM OF THE INHIBITORY EFFECT OF CYCLIN D1 ANTISENSE (ASIDI) ON EGF STIMULATED PROLIFERATION OF THE HUMAN GASTRIC CANCER CELL LINE, MKN-74 CELL. Yoshiro Saikawa, Jackie Z. Wang, Laura H. Tang, Masaharu Odo, Irvin M. Modlin, Yale Univ Sch of Medicine, New Haven, CT. Cyclin DI, 36kDa protein is a member of the cyclin DI-cyclin dependent kinase system involved in the regulation of cell cycle and is involved in the integration of extracellular signals from mitogens including epidermal growth factor (EGF). EGF has been reported to regulate the expression of cyclin D I and has been correlated with a decreased clinical prognosis in gastrointestinal neoplasia. Since cyclin D I is a major factor in EGF stimulated cell cycle we propose that cyclinD I may represent a therapeutic target for the modulation of EGF stimulated cell proliferation. In this study we explored the utility of a targeting gene therapy (antisense strategy) against cyclin DI to inhibit the stimulated cell proliferation by EGF by evaluating the inhibitory effect of antisense oligonucleotide against cyclin DI (ASIDI) on EGF-stimulated cell proliferation. We utilized an in vitro system comprising the gastric cancer cell line, MKN-74 as a model. The ASID I anti sense was designed to correspond to the cyclin D I specific DNA coding sequence. Cells (l04/well) were incubated for 24hr in a 96 well plate with control medium without fetal bovine serum. ASIDI (IOOnmol) was applied for 24hrs and lor EGF (range 0-50nmol) for 0-48hrs. The inhibitory effect of ASIDI was examined by two separate methodologies MTT assay and 3H methylthimidine incorporation assay. Cyclin DI was quantified by immunocytochemistry and Western blotting. Apoptotic induction was assessed by Tunnel assay morphologically and ELISA quantitatively. Our results were: I) EGF-stimulation resulted in a significant increase (> 120%) in the expression of cyclin D 1. 2) Immunocytochemistry and western blotting of cyclin DI demonstrated that ASIDI caused an obvious inhibition of EGF stimulated expression of cyclin D I on non-stimulated and stimulated cells, 3) ASIDI alone demonstrated> 80% inhibition of DNA synthesis and a significant inhibitory effect (>20%) on EGF stimulated cell proliferation, 4). EGF plus ASIDI increased the apoptotic rate by -50% and ASIDI alone resulted in an ~75 % increase. These studies demonstrate that ASIDI inhibits EGF stimulated cell proliferation by suppression of cyclin DI expression and that this effect involves apoptotic induction through cell cycle arrest by depletion of cyclin D 1. This data provides support for the proposal that the use of a cyclin D I anti sense probe may provide a therapeutic strategy in the management of neoplasms in which EGF plays a regulatory role.
2789 ANTI-NEOPLASTIC EFFECTS OF PROTEASOME INHIBITOR MG·132. Jinyi Shao, Hongmiao Sheng, Rania A. Helou, Raymond N. DuBois, Vanderbilt Med Ctr, Nashville, TN. Ubiquitin-proteasome-rnediated proteolysis is involved in the regulation of many biochemical processes. Proteasome inhibitors were found to inhibit cell cycle progression and to induce apoptosis in a variety of cells. Inhibition of proteasome function may potentially suppress tumor growth. However, ubiquitin-proteasorne-dependent proteolysis is widely involved
in normal and tumor cellular biology. The anti-tumor effect of proteasome inhibition is not clearly demonstrated. In this study, we investigated the anti-tumor effect of a synthetic proteasome inhibitor, MG-132 (Carbobenzoxy-L-Ieucyl-L-Ieucyl-L-Ieucinal) in human colon cancer cells. Methods: HCT116 cells were maintained in McCoy's medium containing 10% FBS. Immunoblot analysis was employed to determine the levels of gene expression. For tumorigenicity assay, cells were injected into the dorsal subcutaneous tissue of athymic nude mice. The treatment was started when the tumors were 0.5 em in diameter. MG-132 (10 /LM) was injected into the peritoneal cavities of the mice. Tumor volumes were determined by external measurement. Results: Treatment with 0.3 /LM MG-132 resulted in growth arrest at the G2/M phase in HCT-116 cells followed by cell death. Addition of MG-132 prevented HCT-116 cells anchorage-independent growth in soft agarose. The levels of p53, p21, and p27 were increased following the treatment with MG-132. MG-132-induced growth arrest and apoptosis has been also observed in HCT-15 and SW480 cells in which p53 is mutated and in the p21-knockout HCT-1I6 cells. The mitotic CDK, CDC2, was rapidly phosphorylated at tyrosin-15. Addition of cycloheximide, but not actinomycin D, prevented MG-132-induced growth arrest and apoptosis in HCT-116 cells, indicating protein synthesis is required and gene transcription may not be necessary. Administration of MG-132 inhibited the growth of HCT-I16 xenografts in nude mice and significantly increased the sensitivity of HCT-116 cells to irradiation. Combined treatment with both MG-132 (2 injections) and radiation (3 exposures) immediately stopped the growth of established tumors with continued growth suppression. There was no noticeable toxicity observed in the animals that received combined treatment. Conclusions: Inhibition of proteolysis exerts an anti-neoplastic effect in colon carcinoma cells. Proteasome inhibitors might be useful to enhance the effect of radiation therapy in certain cancers. The toxicity of temporary inhibition of proteolysis was not significant in animals.
2790 ANTIPROLIFERATIVE MECHANISMS OF S-ALLYLMERCAPTOCYSTEINE: POTENTIAL CHEMOPREVENTIVE EFFECTS ON COLON CANCER. Haim Shirin, Yuichi Kawabata, John T. Pinto, Jae W. Soh, Vundavalli Murty, Thomas Delohery, Richard S. Rivlin, Steven F. Moss, Peter R. Holt, Bernard I. Weinstein, Herbert Irving Comp Cancer Ctr, Columbia Univ, New York, NY; Memorial Sloan-Kettering Cancer Ctr, New York, NY; St Luke's-Roosevelt Hosp Ctr, New York, NY. Garlic cosumption is associated with prevention of cancer but the mechanisms involved are not known. To better understand the role of garlic derivatives in colon cancer prevention, we examined the effects of two water soluble derivatives of garlic.Svallylcysteine (SAC) and S-allylmercaptocysteine (SAMC), on cell cycle progression, growth, apoptosis, glutathione induction and jun kinase activation and compare them to the well established colon cancer chemopreventive agent, sulindac sulfide (SS). In addition we examined potential additive interactions between SS and SAC or SS and SAMC. Methods: Exponentially growing SW-480 and HT-29 colon cancer cells were co-cultured wiyh SAC, SAMC or SS for 48 hours. Effects on cell cycle distribution and apoptosis were assessed by flow cytometry. Apoptotic cells were considered to constitute the sub-G I cell population. Concentrations of reduced glutathione were determined by HPLC at 3 and 24 hours and jun kinase activity was evaluated by imrnunocomlex enzyme assay at 30, 60, and 120 minutes post incubation with either SAC, SAMC or SS. Results: Similar to the effects of SS, SAMC but not SAC inhibited growth and induced apoptosis and this was associated with increased jun kinase and caspase 3 activity. These effects were also accompanied by increased cellular levels of reduced glutathione. Unlike SS which inhibits cell cycle progression from GI to S, cells treated with SAMC accumulated in the G2/M phase of the cell cycle. Furthermore the G2/M block induced by SAMC involved the M phase, as revealed by flow cytometry determination of MPM-2 antigen and a delay or lag in anaphase stage as shown by fluorescence in situ hybridization. Addition of SAMC to SS enhanced the growth inhibitory and apoptotic effects of SS. Conclusions: These results suggest that SAMC may be useful for colon cancer chemoprevention. Furthermore, in combination with SS, SAMC may augment the chemopreventive effects of SS and thereby allow lower doses and provide less toxicity.
2783 USE OF A POL¥URETHANE COVERED NITINOL STENT FOR MALIGNANT GASTRODUODENAL OBSTRUCTION. Seonja Park, Hosung Son, Mooin Park, Gyosik Jung, Jayoung Koo, Kosin Med Ctr, Pusan, MD, South Korea. Purpose : To investigate the feasibility and clinical effectiveness of a polyurethane-covered nitinol stent in the treatment of malignant gastroduodenal obstructions Methods: Under fluoroscopic guidance, the stent was placed in 37 consecutive patients with malignant gastric outlet obstruction (n=31) and duodenal obstruction (n=6). All patients had severe nausea and recurrent vomiting. 16 patients underwent implantation of a self expanding uncovered stent from Sep 1998 to March to 1999 and 21 patients underwent implantation of coaxial stent (a polyurethane-covered expandable nitinol stent within uncovered stent) from Apr 1999 to Nov 1999. Contrast radiographs were taken before and after insertion in all patients to confirm patency. Success was defined both technically and clinically. Results: Technical success was achieved in 36 patients (97%). Clinical success was achieved in 36 patients (97%). The one failure was caused by another stenosis at proximal jejunum that was overlooked before stent placement.. During the follow up of 2 to 50 weeks (mean 12 weeks) there were no major complications except that the stents occluded in 3 patients(covered : 2 cases, coaxial : I case) and migrated 7 patients (covered - 5 cases 31.3% and coaxial -2 cases, 9.5%). Conclusions : Palliation of malignant gastroduodenal obstructions with polyurethanecovered expandable nitinol stent is a feasible, effective, and safe altemative to operation. Especially, coaxial stent is better than covered stent in reducing migration.
2784 TWO REGIONAL CHEMOTHERAPIES FOR INOPERABLE COLORECTAL LIVER METASTASES: A COMPARISON. Uwe Pohlen, Gert Berger, Marion Binnenhei, H. J. Buhr, Benjamin Franklin Med Ctr, Berlin, Germany. Introduction and Objective: The application of regional therapies to prolong survival in patients with colorectal liver metastases is controversial. This study compared two different treatment regimens of regional chemotherapy. The goal was to evaluate these regimens as regards survival. Material and Methods: Sixty patients (38 female, 22 male), aged 42 to 72 (mean: 62), underwent regional therapy for inoperable colorectal liver metastases between 1990 and 1997. Group I (22 patients) was administered 600 mg/m 2/BSA 5-fluorouracil (FU) and 300 mg/m 2/BSA folinic acid via an implanted catheter system in the gastroduodenal artery for 5 consecutive days at two week intervals. Group II (38 patients) received regional applications of 600 mg/m2/BSA 5-FU, 500 mg/m 2/BSA folinic acid, 5 mill. IV Intron A and 450 mg starch microspheres (Spherex®) at the same intervals. Before therapy onset and again after 5 cycles, portal CT and chest x-ray examinations were performed and the tumor markers CEA and CA 19-9 were determined. Results: During treatmenta leukopenia WHO I was observed in 80% of the patients. Twenty patients developed chills and septic temperatures after administration of Intron A, which were regressive after i.m. injection of pethidine (Dolantin®). After 14 months, 48 patients (80%) developed a catheter complication, 15 of whom had to discontinue therapy. In the remaining 33 patients, a new catheter was inserted into the hepatic artery via the subclavian artery and treatment was continued. Group I had a response rate of 45%; median survival was 16 months. In Group II the response rate was 78%; the median survival time in the observation period was 31 months. The patients were documented by means of CT imaging and the tumor marker course. Conclusion: We attribute the high response rate (Group II) of 78% with a median survival of 31 months to the combination of 5-FU and folinic acid preceded by flow delay with starch microspheres. The concentration increase in tumor tissue is possibly enhanced by the immunomodulatory effects of interferon alpha. The response rate of 45% and the survival time of 16 months in Group I is markedly better than systemic therapy with both drugs. Group II yielded two-fold the response rate and survival time of Group I. Despite the new chemotherapy drugs and their combination in systemic treatment, a markedly longer survival time was seen by regionally administering a modified 5-FU schema.
2785 INTRA·AORTIC ADMINISTRATION OF 5-FU-PEG LIPOSOMES INCREASES TISSUE CONCENTRATION. Uwe Pohlen, Gert Berger, Marion Binnenhei, R. Reszka, H. J. Buhr, Benjamin Franklin Med Ctr, Berlin, Germany; Max Delbrueck Ctr, Berlin, Germany. Introduction and Objective: The application of liposorne-encapsulated cytostatic drugs leads to an increased tumor concentration. This effect can be enhanced by arterial administration as well as by reducing the blood flow and thus prolonging retention in the tumor. The goal of this work was to measure the 5-fluorouracil (FU) concentration in various organs and in the VX-2 liver tumor after different applications (i.v. versus intra-aortic).
GASTROENTEROLOGY Vol. 118, No.4
Material and Methods: In 20 chinchilla rabbits with a VX-2 liver tumor, a catheter indwelling system was implanted via the left common iliac artery and vein. Clips were placed on the distal common iliac artery and vein as well as above the celiac artery and the hepatovenous inflow to the cava vena. An arteriovenous shunt and minipump system was introduced and the abdomen was perfused for 20 min under hypoxic conditions with a flow rate of lOOm1lmin. Group I (n= 10) received an intra-arterial dose of 50 mg 5-FU. Group II (n= 10) was administered 50 mg 5-FU-PEG liposomes intra-arterially via the aorta. Group III (n=lO) received a 50 mg i.v. injection via the ear vein. In Group IV (n= 10), 50 mg 5-FU-PEG liposomes were administered via the ear vein. pH level and p02 were measured every 5 minutes. After treatment was terminated (20 min), the animals were killed. Liver, tumor, and para-aortic lymph nodes were removed and the concentration of 5-FU was determined using HPLC. In the intra-aortic groups (I and II), the concentrations of 5-FU in serum and perfusate were determined. In the i.v. groups (III and IV) only the serum concentration of 5-FU could be determined. Results: Compared to the i.v.-applied singleentity drug, the i.a-administered 5-FU-PEG liposomes combined with a flow delay led to concentration increases of 4O-fold in the tumor tissue and even lOO-fold in the para-aortic lymph nodes. The pH and p02 measurements show a sharp decrease during the perfusion, which suggests sufficient unassisted abdominal perfusion. To exclude large shunt volumes the 5-FU concentrations in serum and perfusate were measured. The levels showed only a small shunt volume. Conclusion: The intra-aortic administration of 5-FU-PEG liposomes increased the 5-FU concentration 40-fold in tumor and 100-fold in para-aortic lymph nodes compared to the i.v, application.
2786 EVALUATION OF ULTRASONOGRAPHIC IMAGING IN LASER· INDUCED THERMOTHERAPY (LITT) IN THE LIVER. Uwe Ritzel, Dinko Berkovic, Urs Leonhardt, Giuliano Ramadori, Univ of Goettingen, Goettingen, Germany. INTRODUCTION: Recently, evidence was given that MR-guided laserinduced thermotherapy (Ll'FT) in combination with MR thermometry might be an option in palliative therapy of liver metastases. Thereby, absorption of laser light energy induces necrosis of tumor cells by coagulation and hyperthermia. However, MR-guided UTI requires a high level of techniqual equipment that reduces its advantage as compared to surgical resection of isolated metastases. Therefore, in the present study we evaluated an ultrasonographic imaging technique for guided UTI in the liver. METHODS: Laser coagulation was performed by using a neodymiumyttrium aluminium gamet (Nd:YAG) laser (Domier MediLas 5060) with a scattering-dome light emitter. Laser light wavelength was 1046 nm. The placement of the laser probe and its coagulation effect was on-line monitored by abdominal ultrasonography (ATI) using porcine livers at 37° C. In a first series of experiments the pure laser probe was placed into the liver by ultrasonographic guidance. In a second series the probe was coated by a special application catheter set allowing water cooling of the laser probe. The performance was varied from 5 W to 20 W corresponding to a laser light energy of 500 J to I()()()() 1. RESULTS: Ultrasonographic imaging allowed an exact placement and monitoring of the laser probes. When laser light emission was performed, the ultrasonographic image strongly correlated with the macroscopically visible coagulation zone of the liver. Using only the pure laser probe, the maximum diameter of the coagulation zone did not exceed I cm because of carbonisation around the diffusing applicator. A laser energy over 1000 J induced thermic destruction of the laser probe. The coagulation zone was significantly increased using the water cooled catheter set which increased the coagulation diameter up to 3 em and allowed laser light emission of 5000 J at 20 W. Increasing the laser energy did not increase the coagulation diameter because of carbonisation, CONCLUSIONS: It was shown by ex vivo experiments that ultrasonographic imaging could be a useful and easy to perform technique for application of UTI in the liver. Using a water cooled application catheter set, the coagulation diameter of a single laser probe could be increased up to 3 em. These preliminary ex vivo experiments give promise for further investigations using the ultrasonographic imaging technique for in situ UTT in minimal invasive palliative treatment of liver tumors.
2787 ENHANCEMENT OF PHOTODYNAMIC THERAPY FOR PAN· CREATIC CANCER USING ALA WITH AN IRON CHELATOR IN HAMSTERS, Agnieszka Z. Rogowska, Gio A. Buonaccorsi, Sandy Mosse, James Connelly, Alexander J. MacRobert, Stephen G. Bown, National Med Laser Clr, UCL, London, United Kingdom; Dept of Med Physics, UCH, London, United Kingdom; National Med Laser Ctr, London, United Kingdom. Background. Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)only produces superficial necrosis in humans as the maximum tolerated dose of ALA generates low level of the photoactive derivative protoporphyrin IX (PPIX). Iron chelators slow the final conversion of PPIX to haem so may enhance ALA-PDT by increasing tissue levels of PPIX.
AGAA525
April 2000
Aim. To determine if co-administration of the iron chelator-I,2 diethyl-3hydroxypiridin-4-one (CP94) increases the tissue level of PPIX and enhances ALA-PDT induced necrosis in an animal model of pancreatic cancer. Methods. Cancers were produced in normal, female, Syrian Golden hamsters(loo-120g) by injecting PC-I cells into pancreas at laparotomy. For kinetic studies, animals were given 2oomg/kg ALA alone or with 100,200,400 or 800mg/kg CP94 orally. Tissue from normal pancreas and tumour was taken 30 minutes to 6hr later for measurement of the PPIX level by fluorescence microscopy (3-9 animals analysed per point). For PDT, 2hr after 200mg/kg ALA alone or with 100 or 200mg/kg CP94, red light (635nm, IOOJ, loomW) was delivered to the tumour via a bare optical fibre at laparotomy. 3 days later the area of necrosis was measured macroscopically and specimens were taken for histological assessment. Results. Pharmacokinetic study. Maximum PPIX fluorescence was seen at 2 hrs. Adding 200mg/kg CP94 to 2oomg/kg ALA increased the PPIX by 65% in normal pancreas and by 200% in the tumour compared to ALA alone (37% and 100% increase using loomg/kg CP94). Higher doses of CP94 were poorly tolerated and not studied further, though increased PPIX by up 254% in normal pancreas. Animals given CP94 without ALA showed no increase of PPIX. Spectroscopy confirmed that fluorescence was due to PPIX (maxima at 635nm). PDT study. The combination of ALA and CP94 at least doubled the mean volume (l Zl mnr' v 52mm 3)and depth (6.lmm v 3.3mm) of necrosis compared with ALA alone. Histopathology confirmed necrosis in the treated areas identified macroscopically. Conclusion. Adding the iron chelator CP94 significantly increases PPIX fluorescence and enhances ALA-PDT in this model of pancreatic cancer.
2788 THE MECHANISM OF THE INHIBITORY EFFECT OF CYCLIN D1 ANTISENSE (ASIDI) ON EGF STIMULATED PROLIFERATION OF THE HUMAN GASTRIC CANCER CELL LINE, MKN-74 CELL. Yoshiro Saikawa, Jackie Z. Wang, Laura H. Tang, Masaharu Odo, Irvin M. Modlin, Yale Univ Sch of Medicine, New Haven, CT. Cyclin DI, 36kDa protein is a member of the cyclin DI-cyclin dependent kinase system involved in the regulation of cell cycle and is involved in the integration of extracellular signals from mitogens including epidermal growth factor (EGF). EGF has been reported to regulate the expression of cyclin D I and has been correlated with a decreased clinical prognosis in gastrointestinal neoplasia. Since cyclin D I is a major factor in EGF stimulated cell cycle we propose that cyclinD I may represent a therapeutic target for the modulation of EGF stimulated cell proliferation. In this study we explored the utility of a targeting gene therapy (antisense strategy) against cyclin DI to inhibit the stimulated cell proliferation by EGF by evaluating the inhibitory effect of antisense oligonucleotide against cyclin DI (ASIDI) on EGF-stimulated cell proliferation. We utilized an in vitro system comprising the gastric cancer cell line, MKN-74 as a model. The ASID I anti sense was designed to correspond to the cyclin D I specific DNA coding sequence. Cells (l04/well) were incubated for 24hr in a 96 well plate with control medium without fetal bovine serum. ASIDI (IOOnmol) was applied for 24hrs and lor EGF (range 0-50nmol) for 0-48hrs. The inhibitory effect of ASIDI was examined by two separate methodologies MTT assay and 3H methylthimidine incorporation assay. Cyclin DI was quantified by immunocytochemistry and Western blotting. Apoptotic induction was assessed by Tunnel assay morphologically and ELISA quantitatively. Our results were: I) EGF-stimulation resulted in a significant increase (> 120%) in the expression of cyclin D 1. 2) Immunocytochemistry and western blotting of cyclin DI demonstrated that ASIDI caused an obvious inhibition of EGF stimulated expression of cyclin D I on non-stimulated and stimulated cells, 3) ASIDI alone demonstrated> 80% inhibition of DNA synthesis and a significant inhibitory effect (>20%) on EGF stimulated cell proliferation, 4). EGF plus ASIDI increased the apoptotic rate by -50% and ASIDI alone resulted in an ~75 % increase. These studies demonstrate that ASIDI inhibits EGF stimulated cell proliferation by suppression of cyclin DI expression and that this effect involves apoptotic induction through cell cycle arrest by depletion of cyclin D 1. This data provides support for the proposal that the use of a cyclin D I anti sense probe may provide a therapeutic strategy in the management of neoplasms in which EGF plays a regulatory role.
2789 ANTI-NEOPLASTIC EFFECTS OF PROTEASOME INHIBITOR MG·132. Jinyi Shao, Hongmiao Sheng, Rania A. Helou, Raymond N. DuBois, Vanderbilt Med Ctr, Nashville, TN. Ubiquitin-proteasome-rnediated proteolysis is involved in the regulation of many biochemical processes. Proteasome inhibitors were found to inhibit cell cycle progression and to induce apoptosis in a variety of cells. Inhibition of proteasome function may potentially suppress tumor growth. However, ubiquitin-proteasorne-dependent proteolysis is widely involved
in normal and tumor cellular biology. The anti-tumor effect of proteasome inhibition is not clearly demonstrated. In this study, we investigated the anti-tumor effect of a synthetic proteasome inhibitor, MG-132 (Carbobenzoxy-L-Ieucyl-L-Ieucyl-L-Ieucinal) in human colon cancer cells. Methods: HCT116 cells were maintained in McCoy's medium containing 10% FBS. Immunoblot analysis was employed to determine the levels of gene expression. For tumorigenicity assay, cells were injected into the dorsal subcutaneous tissue of athymic nude mice. The treatment was started when the tumors were 0.5 em in diameter. MG-132 (10 /LM) was injected into the peritoneal cavities of the mice. Tumor volumes were determined by external measurement. Results: Treatment with 0.3 /LM MG-132 resulted in growth arrest at the G2/M phase in HCT-116 cells followed by cell death. Addition of MG-132 prevented HCT-116 cells anchorage-independent growth in soft agarose. The levels of p53, p21, and p27 were increased following the treatment with MG-132. MG-132-induced growth arrest and apoptosis has been also observed in HCT-15 and SW480 cells in which p53 is mutated and in the p21-knockout HCT-1I6 cells. The mitotic CDK, CDC2, was rapidly phosphorylated at tyrosin-15. Addition of cycloheximide, but not actinomycin D, prevented MG-132-induced growth arrest and apoptosis in HCT-116 cells, indicating protein synthesis is required and gene transcription may not be necessary. Administration of MG-132 inhibited the growth of HCT-I16 xenografts in nude mice and significantly increased the sensitivity of HCT-116 cells to irradiation. Combined treatment with both MG-132 (2 injections) and radiation (3 exposures) immediately stopped the growth of established tumors with continued growth suppression. There was no noticeable toxicity observed in the animals that received combined treatment. Conclusions: Inhibition of proteolysis exerts an anti-neoplastic effect in colon carcinoma cells. Proteasome inhibitors might be useful to enhance the effect of radiation therapy in certain cancers. The toxicity of temporary inhibition of proteolysis was not significant in animals.
2790 ANTIPROLIFERATIVE MECHANISMS OF S-ALLYLMERCAPTOCYSTEINE: POTENTIAL CHEMOPREVENTIVE EFFECTS ON COLON CANCER. Haim Shirin, Yuichi Kawabata, John T. Pinto, Jae W. Soh, Vundavalli Murty, Thomas Delohery, Richard S. Rivlin, Steven F. Moss, Peter R. Holt, Bernard I. Weinstein, Herbert Irving Comp Cancer Ctr, Columbia Univ, New York, NY; Memorial Sloan-Kettering Cancer Ctr, New York, NY; St Luke's-Roosevelt Hosp Ctr, New York, NY. Garlic cosumption is associated with prevention of cancer but the mechanisms involved are not known. To better understand the role of garlic derivatives in colon cancer prevention, we examined the effects of two water soluble derivatives of garlic.Svallylcysteine (SAC) and S-allylmercaptocysteine (SAMC), on cell cycle progression, growth, apoptosis, glutathione induction and jun kinase activation and compare them to the well established colon cancer chemopreventive agent, sulindac sulfide (SS). In addition we examined potential additive interactions between SS and SAC or SS and SAMC. Methods: Exponentially growing SW-480 and HT-29 colon cancer cells were co-cultured wiyh SAC, SAMC or SS for 48 hours. Effects on cell cycle distribution and apoptosis were assessed by flow cytometry. Apoptotic cells were considered to constitute the sub-G I cell population. Concentrations of reduced glutathione were determined by HPLC at 3 and 24 hours and jun kinase activity was evaluated by imrnunocomlex enzyme assay at 30, 60, and 120 minutes post incubation with either SAC, SAMC or SS. Results: Similar to the effects of SS, SAMC but not SAC inhibited growth and induced apoptosis and this was associated with increased jun kinase and caspase 3 activity. These effects were also accompanied by increased cellular levels of reduced glutathione. Unlike SS which inhibits cell cycle progression from GI to S, cells treated with SAMC accumulated in the G2/M phase of the cell cycle. Furthermore the G2/M block induced by SAMC involved the M phase, as revealed by flow cytometry determination of MPM-2 antigen and a delay or lag in anaphase stage as shown by fluorescence in situ hybridization. Addition of SAMC to SS enhanced the growth inhibitory and apoptotic effects of SS. Conclusions: These results suggest that SAMC may be useful for colon cancer chemoprevention. Furthermore, in combination with SS, SAMC may augment the chemopreventive effects of SS and thereby allow lower doses and provide less toxicity.