EBV-positive gastric cancer cell lines have been identified by several investigators, by detecting EBV nuclear antigens (EBNAs), latent membrane protein 1 (LMP1), or BARF0 [2, 20]. However, the frequency of LMP2A expression is lower in patients with EBV-positive gastric cancer and in EBV-positive gastric cancer cell lines than in B-cell malignant lymphomas [21–23]. It is difficult to treat EBV-positive gastric cancer successfully because significant resistance to cytotoxic anti-cancer drugs, such as paclitaxel, cisplatin, and 5-fluorouracil (5-FU), has been demonstrated in laboratory studies and clinical trials [24, 25]. Only the lytic form of EBV is affected when infected cancer cells are treated with gancyclovir (GCV), and antiviral drugs are ineffective in some gastric cancer cells, such as SNU-719, because EBV lytic genes are expressed at very low levels . Recently, various experimental treatments for EBV-positive gastric cancers have been studied to improve their efficacy. Representative approaches include the combination of 5-aza-2-deoxycytidine or histone deacetylase (HDAC) inhibitors with cytotoxic anti-cancer drugs or with antiviral drugs such as GCV [26, 27]. We consider that epigenetic modification by HDAC is not essential for changing the latent form of EBV into the lytic form, because it does not directly affect cellular signal transduction pathways.
If the MAPK signal pathway or the phosphatidylinositol-3-kinase (PI3K)/AKT signal pathway is dysregulated in epithelial cells or B-cell lymphomas, EBV is likely to be changed into the lytic form [19, 20]. Therefore, we hypothesized that such cellular signal transduction pathways play important roles in the transformation of EBV into the lytic form, and that resistance to cytotoxic anti-cancer drugs can be overcome if these pathways are effectively inhibited. We also assumed that LMP2A has a greater effect on cellular signal transduction pathways, in terms of converting EBV into the lytic form, than do other viral proteins. Our results confirmed that EBV-positive gastric cancer cells are resistant to 5-FU and that this resistance is reduced when the expression of phosphorylated nuclear factor kappaB (p-NFκB) and phosphorylated AKT (p-AKT) is decreased by combining 5-FU with LY294002, a PI3K inhibitor (Figure 3). Furthermore, apoptosis was enhanced not only when the PI3K/AKT signal pathway is inhibited, but also when p-p53 expression is increased and Bcl-2 expression is reduced (Figure 7).
Even at high concentrations of 5-FU, the number of drug-resistant SNU-719 cells was 20-30% higher than that for the AGS EBV-negative gastric cancer cells. In contrast, LY294002 strongly affected both SNU-719 cells and AGS cells, regardless of their EBV status. However, when EBV-positive gastric cancer cells were treated with enzastaurin, which inhibits both PI3K and protein kinase C (PKC), no cytotoxic effects were observed (data not shown). Lee et al.  reported that synergistic or additive effects were observed in diverse gastric cancer cell lines when treated with enzastaurin alone or in combination with cytotoxic anti-cancer agents; however, significant resistance to enzastaurin treatment alone was observed only in SNU-719 cells (IC50 > 250 μM).
Among the various reported mechanisms of resistance to 5-FU, such as multi-drug resistant proteins (MRP, MDR1) and DPD, we focused on cell signaling proteins . However, previous in vitro studies have reported that several chemotherapeutic agents (e.g., 5-FU, paclitaxel, cisplatin, irinotecan, and doxorubicin) can activate NF-κB, which results in notable suppression of the apoptotic potential . AKT is an important regulator of cell survival and apoptosis. Constitutive activation of NF-κB is observed in various malignant cells, which implies that activated NF-κB induced by AKT may play a major role in the chemo-resistance of gastric cancer cells [16–18, 30, 31]. The expression of this protein is altered in various human tumors, and this aberrant expression may contribute to cellular chemo-resistance. It phosphorylates IκB, thus promoting IκB degradation and thereby increasing the activity of the well-known cell survival factor, NF-κB.
We investigated how the expression of p-AKT and p-NF-κB changes with the induction of resistance to 5-FU, based on the finding that LMP2A activates the PI3K/AKT signaling pathway . When SNU-719 cells, with the PI3K/AKT signaling pathway activated by LMP2A, were treated with 5-FU, p-AKT expression was increased. LY294002 showed a significant anti-proliferative effect in both SNU-719 and AGS cells, regardless of their EBV status. In particular, it reduced p-AKT expression remarkably in SNU-719 cells and produced excellent cytotoxicity compared with treatment by 5-FU alone (Figure 3). Generally, over-expression of p-NFκB protein is important for drug resistance to 5-FU, but the activation of PI3K/AKT signal transduction induced by LMP2A appears to be another important cause of resistance to 5-FU in EBV-positive gastric cancer cells.
When SNU-719 cells expressing LMP2A were transfected with LMP2A siRNA, the expression of p-AKT was reduced and the anti-proliferative effect of 5-FU was recovered. Despite the high concentration of 5-FU, SNU-719 cells transfected with scrambled siRNA survived, but LMP2A-knockdown SNU-719 cells were significantly reduced (Figure 6). The changes in gene or protein expression that facilitate the development of gastric cancer have yet to be identified. However, dysregulation of the PI3K/AKT signal pathway after EBV infection is a possible mechanism in the carcinogenesis of gastric cancer.
LY294002 was combined with 5-FU for different sequential treatments and exposure times. When LY294002 was followed by 5-FU, the expression of p-AKT was increased and an antagonistic effect was observed as a combination index [CI] value of 1.1 (data not shown). However, the reverse sequential combination (i.e., 5-FU followed by LY294002) significantly reduced p-AKT expression with a highly synergistic effect (CI = 0.36). These results indicate that p-AKT contributes to the resistance of EBV-positive gastric cancer cells to 5-FU.
The expression of p-NFκB decreased in AGS EBV-negative gastric cancer cells, depending on the concentration of 5-FU when used alone. However, it increased when used in combination with LY294002. The CI value obtained for the two drugs indicated additive effects. In contrast, p-AKT expression increased after treatment with 5-FU alone, but decreased when 5-FU was combined with LY294002. In AGS EBV-negative gastric cancer cells, it is considered that apoptosis is induced by inhibition of the NF-κB signal pathway in the case of treatment with 5-FU alone, and predominantly by the inhibition of p-AKT expression and its downstream signaling molecules in the case of treatment with LY294002.
We observed that 5-FU increased the expression of cyclin A, resulting in S-phase arrest of the SNU-719 cell population. Compared with 5-FU alone, the combination of 5-FU with LY294002 downregulated the expression of cyclin D3 and CDK2, and upregulated the expression of cyclin A and CDK4. Sequential treatment with 5-FU followed by LY294002 resulted in a mixed pattern of DNA condensation and large nucleated cells, when compared with cells treated with the individual drugs. When SNU-719 cells are treated with 5-FU or LY294002, alone or in combination, it is postulated that apoptosis is induced by the inhibition of DNA synthesis or G0/G1 arrest via increased p-p53 expression and reduced p-NF-κB expression. It was also confirmed that apoptotic cells were more significantly augmented by increased p-p53 and decreased Bcl-2 expression using combined treatment than using 5-FU treatment alone. Leung et al.  reported that most EBV-positive gastric cancer cells express p53 protein at low to medium levels and that another mechanism exists which induces the over-expression of p53, in addition to the direct mutation of p53 by EBV.
It is considered that the high levels of Bcl-2 expression adopted as protection from apoptosis in EBV-positive gastric cancer cells result in the natural death of fewer cancer cells than in the case of EBV-negative gastric cancer cells. In addition, increased Bcl-2 expression results in chemo-resistance to anti-cancer drugs by inhibiting p53-mediated apoptosis [11, 33]. However, some studies have reported the absence of altered Bcl-2 expression or p53 accumulation in EBV-positive gastric cancers . Additional research is warranted regarding the roles of Bcl-2 and p53 in the development of drug resistance . In EBV-positive SNU-719 cells, LY294002 enhanced the sensitivity to 5-FU by downregulating activated p-AKT and its downstream molecules, and induced apoptosis by arresting the cell population at G0/G1 phase. Furthermore, increased sensitivity to 5-FU, with remarkable inhibition of the activated PI3K/AKT signaling pathway, was observed in SNU-719 cells transfected with LMP2A siRNA.