LY294002 may overcome 5-FU resistance via down-regulation of activated p-AKT in Epstein-Barr virus-positive gastric cancer cells.
BACKGROUND
As EBV-associated gastric cancer has unique features that are different from EBV (-) gastric cancer, EBV is considered to have a key role in gastric carcinogenesis. It has been reported that viral latent membrane protein 2A (LMP2A) in EBV-transformed tumor cells activates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which provides a survival signal and chemo-resistance to cytotoxic anti-cancer drugs. This study was to evaluate anti-proliferative effect and cell cycle change when 5-FU and LY294002 (LY), a selective inhibitor of PI3K, were treated separately or combined with different schedules in EBV positive gastric cancer cell line, SNU-719.


METHODS
After single treatment and sequential combination of 5-FU and LY, cytotoxic activity was measured by MTS assay. When 5-FU and LY were treated in single and sequential combinations, the expression of p-AKT, p-NFkB, p-p53 and bcl-2 was observed on different concentrations by Western blot analysis. We also investigated the effect on apoptosis and cell cycle distribution using flow cytometry. The LMP2A siRNA inhibition was done to confirm the reversal of decreased 5-FU activity and p-AKT.


RESULTS
When 5-FU was sequentially combined with LY, the combination index (CI) value indicated synergistic anti-proliferative effect. The expression of p-AKT and p-NF kappaB was upregulated by 5-FU alone but sequential treatment of 5-FU and LY decreased the expression of both p-AKT and p-NF kappaB. When 5-FU was combined with LY, G0/G1 and sub G1 cell population (%) increased. When 5-FU was added to the cells transfected with LMP2A siRNA, its anti-proliferative effect increased and the expression of p-AKT decreased. In sequential combination of 5-FU and LY, the expression of p-p53 was increased and bcl-2 expression was diminished compared to 5-FU alone.


CONCLUSIONS
These data suggest that sequential combination of 5-FU and LY induce synergistic cytotoxicity and overcome intrinsic and acquired resistance of 5-FU via downregulation of activated p-AKT and mitochondria-dependent apoptosis in EBV gastric cancer cell line, SNU-719.
Shin
Jung-Young
JY
Department of Biomedical Sciences, The Catholic University of Korea, Seoul, Korea. bearjy@catholic.ac.kr
Kim
Jeong-Oh
JO
Lee
Suk Kyeong
SK
Chae
Hiun-Suk
HS
Kang
Jin-Hyoung
JH
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D002453:Cell Cycle; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D002867:Chromones / Q000494:pharmacology*; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020031:Epstein-Barr Virus Infections / Q000188:drug therapy / Q000378:metabolism / Q000821:virology; D005472:Fluorouracil / Q000494:pharmacology*; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D008928:Mitochondria; D009025:Morpholines / Q000494:pharmacology*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000821:virology; D014407:Tumor Cells, Cultured; D014763:Viral Matrix Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; D002867:Chromones; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D004791:Enzyme Inhibitors; D009025:Morpholines; D016328:NF-kappa B; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014763:Viral Matrix Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D005472:Fluorouracil