April 2000

2779

THE APOPTOTIC EFFECT OF ISOPRENOIDS LINKED WITHAMINE AND LONG-CHAIN FATTY AMINES ON HUMAN PAN­CREATIC CANCER CELLS.Yusuke Mizukami, Hitoshi Ura, Takeshi Obara, Tsutomu Izawa, NobuyukiYanagawa, Keisuke Kimura, Satoshi Tanno, Yutaka Kohgo, AsahikawaMed Coll, Asahikawa, Japan.

Background/Aim: Isoprenoids have been reported to initiate apoptosis inseveral cancer cell lines, and their analogues with "amine" at the end groupappears to enhance the effect. The aim of this study was to examinewhether the lipids linked with amine induce apoptosis effectively onhuman pancreatic cancer cells. Methods: We assessed the effects of bothisoprenoid-amine and long-chain fatty (alkylj-amine (LFA) on humanpancreatic cancer cells (PK-I, MIAPaCa-2, BxPC-3) and NIH/3T3 cells.Famesylamine (FA), analogue of 15-carbon (CI5) isoprenoid working asfamesyl transferase inhibitor (FTI), and LFAs of various alkyl-chain length(CI2-18) were used. The cytotoxicity was quantified by WST-I assay, andapoptosis was confirmed by DNA laddering and hypoploidity. Immunoblotanalysis of p21-ras and kinase assay for MAPK families were also inves­tigated. Results: Both FA and LFAs induced apoptosis in human pancreaticcancer cells and Ki-ras transformed NIH/3T3 (Ki-ras cells), while not inparental cells. The cytotxic concentration of oleylamine (OA) (IC50; 10-30/LM), most potent apoptotic inducer among LFAs, was similar to that ofFA, but analogues of OA without amino-group were not cytotxic. In­creased kinase activity of INK, stress-induced kinase of MAPK family,was obtained preceding the apoptosis induced by both FA and OA. Block­age of JNK activity by dominant negative mutant completely abrogated theDNA laddering. Although both FA and OA inhibit the ERK activity,specific MEKI/2 inhibitor PD-098059 alone did not induce apoptosis.Caspase inhibitor, z-VAD-frnk, significantly abrogated these compounds­induced apoptosis, but, not affect on JNK activity. FA inhibited p21-rasfamesylation on Ki-ras cells, but such an effect was not observed by LFAs.Conclusions: Certain kind of lipids especially linked with "amine" couldinduce apoptosis on human pancreatic cancer cells and could be a candi­date for novel chemotherapeutic reagent against pancreatic carcinoma.Although distinct mechanism may be involved in the course of apoptosisinduced by FA and LFAs, common signaling pathways including activa­tion of JNK and caspases, and inactivation of ERK, also could mediate theprocess.

2780ROLE OF PREOPERATIVE LOW DOSE RADIOCHEMO­THERAPY IN ADVANCED RECTAL CANCER.Shosaku Nakahara, Makoto Motooka, Makoto Miyoshi, Satoshi Toyo­shima, Kitakyushu Municipal Med Ctr, Kitakyushu, Japan.

Background: Although radiotherapy or radiochemotherapy is accepted asan adjuvant therapy in advanced rectal cancer, its role is still controversial.We have adopted preoperative low dose irradiation and concomitant oralchemotherapy to obtain tumor reduction, improve subjective symptomsand foreknow the efficacy of postoperative adjuvant therapy. The aim ofthe present study was to assess the effect of our treatment on tumorreduction and patients's conditions. Patients and methods: 44pa­tients(61:t 8 years old, 28 male and 16 female) with advanced rectal cancerwere treated for about 2 weeks preoperatively by low dose irradiation(Li­nac X-ray; 2Gy IOFr. 20Gy in total) and oral chemotherapy(Carmofur;600mglday). All patients underwent radical surgery(35 anterior resectionsand 9 abdominoperineal resections)soon after completion of the treatment.Patients were evaluated in the following items before and after the preop­erative treatment. Results: Tumor size(length on Barium enema) decreasedin all but 2 patients, measuring 5.0:t 1.5cm to 4.2:t I.3cm (reduction rate17:t8%(0-57%), p<O.OOI, Wilcoxon test). Serum CEA levels elevated inonly 5 patients and decreased in half of the patients, indicating significantdecrease from 33.7:t95.3ng/ml to 23.0:t51.3ng/ml(p<0.02, Wilcoxontest). In symptons, subileus improved in 8 of 9 patients, pelvic or anal painand massive rectal bleeding disappeared or were reduced in all symptom­atic patients. Histological judgement of the effect showed nothing in1(2.3%), slight in 17(38.6%), mild in 24(54.5%) and moderate in 2 pa­tients(4.5%). Side effects such as diarrhea, easy fatiguability, anorexia andpolakisuria were observed in 16 II % of the patients. WBC decreased inalmost all patients, measuring 7100:t 1800/mm3 to 4600:t 1500/mm3 (bot­tom; 2500/mm3

) . No patient dropped out of the treatment, although oralchemotherapy was discontinued in 2 patients. Anastomotic leak occured in2 of 35 anterior resection patients(5.7%). Recurrence occured in 6 patients;liver in 3, lung in 2 and pelvis in I. Two died from lung metastusis.However, the long term prognosis could not be drawn because of the shortfollow-up period(median 13 months) and the small number. Conclusion:These data suggest that preoperative low dose irradiation with oral che­motherapy is safe and effective adjuvant therapy without prolonging theperiod until surgery. Results were in each patient may give useful infor­mation to the postoperative adjuvant therapy.

AGAA523

2781DEVELOPMENT OF A GENE THERAPY FOR COLORECTALCANCER USING A RECOMBINANT ADENOVIRUS VECTOR EX­PRESSING AN ANTISENSE K-RAS RNA.Masaru Nakano, Toshifumi Hibi, Teruhiko Yoshida, Sch of Med, KeioUniv, Tokyo, Japan; Sch of Medicine, Keio Univ, Keio Cancer Ctr, Tokyo,Japan; Genetics Div, National Cancer Ctr Research Institute, Tokyo, Japan.

Background: The incidence of colorectal cancer has been increasing rap­idly in Japan over the last 20 years. The development of a new modality oftreatment for a colorectal cancer and secondary distant metastases has beeneagerly awaited. To develop a gene therapy for colorectal cancer, weassessed inhibitory effect of a recombinant adenovirus vector expressing anantisense K-ras RNA under the control of the CAG promoter (AxCA-AS­K-ras) in the growth of colorectal cancer cells. Methods: I) K-ras pointmutation on codon 12, 13 and 61 was determined in 7 colorectal cancer celllines. 2) We assessed in vitro inhibitory effect of the AxCA-AS-K-ras anda sense K-ras RNA (AxCA-S-K-ras) in the growth of these cell lines. 3)To assess expression of K-ras p21 protein, western blot analysis wasperformed using a K-ras specific p21 monoclonal antibody. 4) To assess invivo inhibitory effect of the AxCA-AS-K-ras in the growth of these celllines, HCT-15 cells were inoculated subcutaneously into scmmice. Afterthe tumor were established, 1.5 x 108 pfu AxCA-AS-K-ras were injectedinto the tumor 3 times every 24 hours, and diameter of each tumor weremeasured every day for 21 days. Results: I) The growth of all 7 colorectalcancer cell lines expressing with the AxCA-AS-K-ras was decreased, ascompared with those expressing sense K-ras RNA, irrespective of thestatus of K-ras point mutation. At moi=IO, the growth of HCT-15 with aK-ras point mutation on codon 13 were completely suppressed by AxCA­AS-K-ras for a week. 2) There was a different toxicity to the adenovirusvector among 7 colorectal cancer cell lines. 3) Expression of the K-ras p21protein decreased in the HCT-15 cells after expression of antisense K-rasRNA. 4) Intra-tumoral injection of A xCA -A S-K-ras inhibited the growthof tumors developed by subcutaneous transplantation of HCT-15 cells toSCID mice. The growth of subcutaneous tumors were completely sup­pressed by AxCA-AS-K-ras within a week. Conclusion: We demonstratedthe inhibitory effect of a recombinant adenovirus vector expressing anantisense K-ras RNA in in vitro and in vivo growth of colorectal cancercells. Our findings suggest that intra-tumoral injection of this vector may befeasible in the treatment of advanced colorectal cancer.

2782PALLIATIVE THERAPY FOR ADVANCED BILIARY TRACTCARCINOMA WITH OXALIPLATIN, S-FLUOROURACIL (S-FU)AND LEUCOVORIN (LV).Oliver Nehls, Bodo Klump, Michael Gregor, Rainer Porschen, Univ Hosp,Tuebingen, Germany.

Objective: To determine the efficacy and toxicity of oxaliplatin and 5-FUplus LV in patients (pts) with unresectable or metastatic gallbladder car­cinoma (GBC) or cholangiocarcinoma (CCC). Methods: 12 pts (7M, 5F)were included. Median age was 62 yrs (range 39-74). Major eligibility:histologic proven, measurable disease, age oS 75 yrs, ECOG PS oS 2.Metastatic sites: liver (100% of pts), lung (8%), peritoneum (17%), lymphnodes: perihilar (33%), retroperitoneal (58%). 3 pts had surgery prior tochemotherapy. A total number of 101 cycles (median number of cycles perpatient: 7; range 2-16) of combined oxaliplatin (85 mg/rrr', dl) with 5-FUand LV (1500-2000 and 500 mg/nr', dl and d2), was administered bi­monthly for advanced GBC (6 pts), intrahepatic (4 pts) and extrahepaticCCC (2pts). Treatment has been administered on outpatient basis usingdisposable pumps. Pis were assessed for response every 5 cycles. Results:Partial response (PR) was observed in 2 pts (17%); 6 pts (50%) had stabledisease (NC); progressive disease was diagnosed in 4 pts (33%). Sinceinitiation of chemotherapy, median progression-free survival of pts withPR and stable disease was 5.9 months+ (range 3.2-11.9), median overallsurvival was 6.2 months" (range 2-23). No grade 4 toxicities (WHO)occurred; grade 3 toxicities were: thrombocytopenia in 2 pts (3% ofcycles), paresthesia in I pt (4%), decreased taste in 2 pts (5%), dose­limiting peripheral sensory neuropathy in 1 pt (l %; after 12 cycles). Themost common toxicity was grade 1-2 peripheral sensory neuropathy in IIpts (69%). Gastrointestinal toxicities were mild. Conclusions: This phase IIstudy confirms that an outpatient regimen of combined oxaliplatin and5FUILV for advanced biliary tract carcinoma is well tolerated. Our pre­liminary data demonstrating a disease control in 67% of pts with a medianduration of 5.9 months are encouraging, with longer follow-up necessary.