Suppression of invasion of a hamster pancreatic cancer cell line by antisense oligonucleotides mutation-matched to K-ras gene.

The anti-invasive activity of antisense oligonucleotides (ASO) specific to the K-ras gene in hamster pancreatic cancer was investigated. HaP-T1, a cell culture derived from BHP-induced hamster pancreatic cancer, was used. After liposome-mediated transfection with mutation-matched and mutation-mismatched ASO in different concentrations, cell proliferation was studied by MTT and MTT-agarose methods. In vitro chemoinvasion assay with the reconstitution of a matrix of a basement membrane onto a filter in a Boyden chamber was performed. Mutation-matched ASO inhibited the tumor growth and invasiveness of HaP-T1 in a dose-dependent manner, while mutation-mismatched ASO were not effective in inhibiting invasion. The present study suggests that antisense oligonucleotides mutation-matched to the K-ras gene may be a new anticancer strategy for pancreatic cancer since they inhibited not only tumor growth but also invasiveness in vitro.

Different pattern of metastasis in liver implanted pancreatic cancer between young and old Syrian golden hamsters.

We have previously reported on the "return trip" metastases from the liver to the pancreas in a hamster experimental pancreatic cancer model. Because the pancreas is the main metastatic site of liver-implanted pancreatic tumors, our aim was to clarify whether the metastatic sites differ in young and old tumor-bearing animals. HaP-T1, a continuous tissue-cultured cell line, derived from BHP-induced pancreatic adenocarcinoma, was implanted into the liver. The animals were divided into two groups: A) younger than 26 weeks and B) older than 26 weeks. Three animals from each group were sacrificed on Days 35, 42, 49, 56, 63, 70, 77, 84, 91 and 98, to study the metastatic sites. Survival was also studied. After death, necropsy was performed. Resected and necropsied specimens were analyzed histopathologically and by PCR/RFLP analysis to confirm the presence of K-ras point mutation. The success rate of implantation was 100%. Survival was 102.3+/-2.5 days in group A and 95.3+/-1.5 days in group B. Animals of group A, sacrificed weekly until Day 70, showed metastases only to the pancreas ("return trip"), while this phenomenon happened only in animals sacrificed on Day 35 in group B. In group A, on Days 77, 84, 91 and 98, metastases were also found in the kidneys, lymph nodes, ovary and testis. In hamsters of group B, metastases were found in multiple sites such as the pancreas, vas deferens, ovary and testis ("multiple journeys"). All intra-hepatically-implanted tumor and metastatic sites showed the K-ras point mutation. This homologous implantation model may be helpful for further research into the process of metastasis and its relationship with the immunological response.

Antisense therapy specific to mutated K-ras gene in hamster pancreatic cancer model. Can it inhibit the growth of 5-FU and MMC-resistant metastatic and remetastatic cell lines?

UNLABELLED: K-ras point mutation at codon 12 has a relationship greater than 90% with pancreatic cancer. Cancer therapy should also include the treatment of metastatic disease because it is known that the properties of metastatic cells may vary considerably from those of the primary tumor. AIM: To clarify if the same drugs, which can inhibit the tumor growth in the parental cell line, can inhibit the pancreatic metastatic and remetastatic cell lines at the same concentrations and to compare the inhibition with antisense oligonucleotides mismatched to K-ras gene, in Syrian golden hamsters. MATERIALS AND METHODS: HaP-T1, a BHP-induced hamster pancreatic cancer cell line, MS-PaS-1 (a metastatic cell line established from "return trip" metastases from the liver to the pancreas) and MS-PaS-2 named as a "remetastatic cell line", i.e., metastases from MS-PaS-1 were used. MTT and MTT-agarose assays were performed, using 5-Fluorouracil (5-FU), Mitomycin C (MMC) and antisense oligonucleotide specific to K-ras oncogene. RESULTS: The inhibitory concentration (IC50) of 5-FU, which inhibited HaP-T1, had to be increased by 50-fold to inhibit MS-PaS-1 and 100-fold to inhibit MS-PaS-2. MMC had to be increased by 10-fold to inhibit MS-PaS-1 and 50-fold to inhibit MS-PaS-2. However, IC50 was the same when antisense oligonucleotide was tried in these 3 cell lines. CONCLUSION: Antisense oligonucleotide-targeted K-ras gene may be a good choice for therapy because it could inhibit the growth in metastatic and remetastatic cells as well as in primary tumor cells.

Comparison of metastatic behavior of parental and metastatic pancreatic cancer cell lines in Syrian golden hamsters.

The major cause of death in patients with pancreatic cancer is metastatic disease. In fact, at the time of diagnosis, patients usually have locally advanced or metastatic disease involving lymph nodes, liver, lungs or peritoneum. Therefore, for a better understanding of the tumoral behavior to design prevention or treatment strategies, in vivo models are important. We report here the results of the metastatic behavior of parental and metastatic cell lines in a hamster pancreatic cancer model when implanted orthotopically (OIH,OIM) or into the liver (LIH,LIM). Metastatic sites and survival were studied. Survival ranged from 72 to 105 days. The OIH group showed spontaneous metastases to lymph nodes. The second target organ was the lung. Liver metastases appeared earlier in the OIM group than OIH. LIH showed only metastases to the pancreas while the LIM group showed metastases to pancreas, vas deferens and testis. This study suggests that the metastatic behavior of parental and metastatic cell lines is different. Thus, this should be considered in the planning of clinical or surgical treatment against pancreatic cancer.

Detection of K-ras point mutation at codon 12 in pancreatic diseases: a study in a Brazilian casuistic.

OBJECTIVE: To clarify the sensitivity and the validity of K-ras point mutational analysis at codon 12 in Brazilian patients with pancreatic diseases, and the possible correlation between the presence of the mutation and the histopathological findings. PATIENTS: Ninety-seven Brazilian patients with pancreatic ductal adenocarcinoma, pancreatic neuroendocrine tumors and chronic pancreatitis were enrolled in this study. Forty-five patients (46%) were female and 52 patients (54%) were male, having an average age of 60.2+/-9.2 years for adenocarcinoma (n=52), 45.1+/-19.4 years for pancreatic neuroendocrine tumors (n=20), and 46.4+/-11.2 years for chronic pancreatitis (n=25). DNA extracted from 11 normal human peripheric lymphocytes was utilized as a control. RESULTS: The sensitivity of K-ras mutational analysis was 83.3% (25/30) in paraffin-embedded samples and 72.7% (16/22) in surgically resected specimens of the malignancy. On the other hand, no mutations were found in pancreatic neuroendocrine tumors or in chronic pancreatitis. Regarding the histopathological grading, the higher positivity rate was found in poorly-differentiated adenocarcinoma (100%), and progressively decreased in moderately-differentiated adenocarcinoma (72.2%), and well-differentiated adenocarcinoma (66.6%). The positivity rate in non-classified adenocarcinoma was 81.8%. CONCLUSION: K-ras point mutation, in our study, is notably prevalent in malignancies and is absent in chronic pancreatitis and pancreatic neuroendocrine tumors. These results encourage us to consider the possibility of treatment strategies for this oncogene in the future.