| Research Abstract |
Background: Chemotherapy plays a key role in the treatment of pancreatic and biliary carcinomas. However the response rates for gemcitabine (alone or combination), the most potent anti-cancer drug, seldom exceed 30%. It is essential to identify chemosensitive and chemoresistant patients prior to commencement of chemotherapy to achieve optimal treatment outcomes. The present study examined the in vitro chemosensitivity of pancreatic and biliary carcinomas to gemcitabine and 5-fluorouracil using the collagen droplet drug sensitivity test (CD-DST). Current in vitro drug-sensitivity tests have limitations and disadvantages. This study investigated the use of gene expression data to predict the sensitivity of pancreatic cancers to gemcitabine. Rational attempts to overcome clinical drug resistance require an understanding of the molecular mechanisms involved. The aim of the present study was to compare gene expression in normal and 5-fluorouracil (5-FU)-resistant pancreatic cancer cells and
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to identify important pathways associated with resistance.
Methods: The study involved 37 pancreatic or biliary carcinoma patients diagnosed at the Jichi Medical University Hospital from 2002 to 2004. Carcinoma cells isolated from the main tumor or metastatic lesions were tested in vitro for gemcitabine and 5-fluorouracil sensitivity using the CD-DST. Cancer cells isolated from 14 pancreatic cancer patients were tested in vitro for gemcitabine sensitivity using the collagen droplet drug sensitivity test (CD-DST). On the basis of this test, 9 of the 14 cancers were identified as either gemcitabine-sensitive or gemcitabine-resistant Total RNA was extracted from each of those nine cancers and used as a template to synthesize Cy3-labeled cDNA. Pancreatic RNA extracted him 6 normal individuals was used as a control. Labeled probes were hybridized to an Atlas Glass Human 1.0 Microarray chip, after which the chips were washed and scanned, and the data analyzed using Microsoft Excel-embedded software. The expression profiles of selected genes were confirmed using real-time PCR analysis. The human pancreatic cell line MIAPaCa-2 was incubated with increasing concentrations of 5-FU in order to develop the 5-FU-resistant derivative cell line MIA-FU-2.4. Gene expression in MIAPaCa-2 and MIA-FU-2.4 cells was compared using gene array analysis. The altered expression of selected genes was confirmed using real-time PCR assays.
Results: The success rate for primary culture was 80.3%. The mean T/C ratios for gemcitabine and 5-fluorouracil were 57.8 and 75.0, respectively (p41031). The median survival times (MST) for gemcitabine-sensitive and -resistant patients were 16.1 and 10.7 months, respectively (p-1.16). Statistical analysis of the microarray data showed that four genes were differentially expressed in gemcitabine-sensitive cancers: microsomal glutathione S-transferase 1 (GSTT1), topoisomerase II alpha (TOP2A), caspase 3 and ATP-binding cassette and sub-family C member 2 (ABCC2). More than 20 other genes were additionally identified as possible candidate genes associated with drug resistance. Array data identified 1075 genes that were differentially expressed between MIAPaCa-2 and MIA-FU-2.4 cells. 5-FU resistance was found to corrrelate with enhanced expression of genes involved in the DNA damage checkpoint pathway RAD1, HUS1, RFC5, RPA3 and CHK2 genes. In addition, TYMS, UP1, PCNA, FEN1, TRAF2, GADD45B, TIA1 and BIRC5 gene expression correlated with 5-FU resistance.
Conclusion: The CD-DST may be a useful method for identifying chemosensitive pancreatic and biliary carcinoma patients prior to chemotherapy. Expression of drug resistance-related genes appeared to predict whether a cancer was gemcitabine-sensitive or -resistant. Finally we picked up RAD1, HUS1, RFC5, RPA3, CHK2 genes, TYMS, UP1, PCNA, FEN1, TRAF2, GADD45B, TIA1, BIRC5, microsomal glutathione S-transferase 1 (GSTT1), topoisomerase II alpha (TOP2A), caspase 3, ATP-binding cassette and sub-family C member 2 (ABCC2) as candidate genes to predict chemo-sensitive or resistant of pancreatic cancer. Less
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