The Study Of Glucosylceramide Synthase In The Multidrug Resistance Of Human Pancreatic Cancer Cells

Pancreatic cancer is one of the most lethal human cancers and is characterized with unresectable stage at diagnosis. Though chemotherapy is one of the most important adjuvant treatments, chemoresistance, especially multidrug resistance, reduces the effect of many chemotherapeutics. Ceramide is produced by cancer cells in response to exposure to radiation and most chemotherapeutics and is an intracellular second messenger that activates enzymes, leading to apoptosis. Glucosylceramide synthase (GCS) is the primary ceramide glycosylase and is found in the Golgi and endoplasmic reticulum. Glucosylceramide is the precursor of the＞200 known gangliosides and protects cells from injury. It is reported that increased GCS activity results in multidrug resistance, whereas decreased GCS expression and / or activity resulted in buildup of ceramide and enhanced chemosenitivity in breast cancer and other cancer cells. It is unclear whether GCS is involved in the chemoresistance of pancreatic cancer. A higher expression of GCS gene was found in drug-resistant human pancreatic cell SW1990/FU, SW1990/ADM and SW1990/GEM than in sensitive cell SW1990, using oligonucleotide microarray. The aim of this study is to detect the expression of GCS and the relationship of GCS and drug resistance in pancreatic cancer cell SW1990 and its drug-resistance sublines SW1990/FU, SW1990/ADM and SW1990/GEM.Methods:SW1990 and its drug-resistance sublines, SW1990/FU, SW1990/ADM and SW1990/GEM were cultured in vitro. CCK-8 (Cell Counting kit 8) assay was used to detect the drug resistance of the sublines. Relative quantitation of GCS mRNA expression was evaluated by real-time PCR and Western blot was adopted to evaluate the expression of GCS protein. GCS gene was cloned into the mammalian expression vector pCI-neo to generate the plasmids pCI-neo-GCS, pCI-neo-GCS was transfected into SW1990 and blank vector pCI-neo was as control. The expression of GCS mRNA was assayed by RT-PCR and the 50% inhibition concentration (IC₅₀) of 5-FU, ADM and GEM on SW1990 was evaluated by CCK-8 assay. Synthetic double strand RNA, StealthTM RNAi, was introduced into SW1990/FU, SW1990/ADM and SW1990/GEM for GCS RNA interference (RNAi). RT-PCR and CCK-8 were used also for the assay of the effect of the RNAi.Results:The results of CCK-8 assay showed that the drug-resistance sublines SW1990/FU, SW1990/ADM and SW1990/GEM were much more chemoresistant than the sensitive cellline SW1990 and their resistance indexes (RI) were 339.7, 11.9 and 56.6 respectively. GCS mRNA and protein were expressed in all SW1990 and its drug-resistance sublines. There was a high expression of GCS mRNA in all sublines and a significant difference of GCS protein expression was detected in SW1990/ADM and SW1990/GEM, 1.22 times and 1.76 times compared with SW1990 respectively. Double enzyme digestion analysis and DNA sequencing confirmed that pCI-neo-GCS was successfully constructed, pCI-neo-GCS was induced into SW1990 by Lipofectamine 2000 and a stable GCS expression cell, SW1990/pCI-neo-GCS, was selected successfully by being cultured with the antibiotic G-418. The result of CCK-8 assay showed that SW1990/pCI-neo-GCS, which overexpresses GCS, was much more resistant to 5-FU, ADM and GEM than SW1990, the RI was 2.63, 1.94 and 1.44 respectively. Forty-eight hours after RNAi, the GCS mRNA expression was significantly lower than the control cells of the drug-resistant sublines. The CCK-8 assay showed that SW1990/ADM and SW1990/GEM became sensitive to ADM and GEM after RNAi, the chemosensitivity of the SW1990/FU did not change.Conclusions:The chemoresistance of SW1990/FU, SW1990/ADM and SW1990/GEM was stable and hereditable. GCS is expressed in SW1990 and its drug-resistance sublines. The expression of GCS mRNA and protein is higher in drug-resistant celllines SW1990/ADM and SW1990/GEM than sensitive cellline SW1990. SW1990/ADM and SW1990/GEM became sensitive to ADM and GEM respectively after decreasing the expression of GCS, which confirms that GCS is one reason of drug-resistance to ADM and GEM in SW1990/ADM and SW1990/GEM respectively.