S3). In comparison, inhibition of NF-κB by the same dose of QNZ significantly prevented the induction of p21 by ANE, confirming the validity of the above experiments (Fig. 3 C, S4A). Because the induction is independent of reactive oxygen species-mediated DNA damage, ANE may upregulate NF-κB signaling to directly increase p21 (Fig. S4B). NF-κB inhibition also obviously increased ANE cytotoxicity but not PARP cleavage, an indicator of apoptosis (Fig. 3D, S4 C). Although all these results suggested ANE indeed activated NF-κB to modulate cell functions, NF-κB is not directly involved in the upregulation of IL8 transcription. ANE might also induce a few inflammatory cytokines

via a mechanism in addition to NF-κB. Like Akt, phosphorylation of STAT3 (Y705) was also decreased by ANE under lower serum condition (Fig. 4A, S5). Despite that ANE treatment significantly reduced the phosphorylation of total Ku-0059436 nmr STAT3 (Y705), the

ratio of nuclear to cytoplasmic localization of unphosphorylated STAT3 was not altered (Fig. 4B). As a control, ANE enhanced nuclear translocation of Snail proteins. Moreover, inhibition of STAT3 dimerization by NSC74859, which reduces DNA-binding STAT3 with IC50 of 86 μM, reduced the activation of IL8 promoter (Fig. 4 C) [22]. In contrast, inhibition of STAT3 phosphorylation Pifithrin-�� cost by JAK inhibitor I, a pan JAK inhibitor with IC50 value between 1-15 nM, did not detectably downregulate the reporter activity (Fig. 4 C) [23]. This result suggests STAT3 is required for ANE-induced IL8 transcription but JAK-mediated Y705 phosphorylation is dispensable. Similar effects also could be seen in the transcripts level of IL6 although the case of IL8 was inconsistent possibly because the mRNA stability may be independently regulated (data not shown) [24]. These results increase

a possibility that ANE enhances inflammation in oral mucosa at least Montelukast Sodium via facilitating dephosphorylation of nuclear STAT3. Activated STAT3 is associated with inflammation during tumor progress [25]. However, ANE may modulate the transcription of a few inflammatory cytokines by enhancing Y705 dephosphorylation of STAT3 since un- and phosphorylated STAT3 had been reported to differently regulate several downstream targets [26]. In this study, we provided a few examples to prove serum concentration influenced the effects of ANE in cultured cells. The effects of ANE under different serum condition give a rational explanation to the various alterations in betel quid chewers. In serum-starved cells, ANE caused cell ballooning and nuclear pyknosis. Theoretically, the environment that oral epithelial cells reside in is impossible to be stringently serum free. However, epithelial tissue normally is avascular and less accessible to the circulating nutrients in blood stream. A previous research indicated the epidermis in average has lower ratio of interstitial fluid than dermis [27]. Because in our results even 0.