Up-regulated sirtuin 1 (SIRT1), an NAD+-dependent class III histone deacetylase, deacetylates p53 and inhibits its transcriptional activity, leading to cell survival. SIRT1 overexpression has been reported to predict poor survival in some malignancies, including gastric cancer. However, the antitumor effect of SIRT1 inhibition remains elusive in gastric cancer. Here, we investigated the antitumor mechanisms of a sirtuin inhibitor, tenovin-6, in seven human gastric cancer cell lines (four cell lines with wild-type TP53, two with mutant-type TP53, and one with null TP53). Interestingly, tenovin-6 induced apoptosis in all cell lines, not only those with wild-type TP53, but also mutant-type and null versions, accompanied by up-regulation of death receptor 5 (DR5). In the KatoIII cell line (TP53-null), DR5 silencing markedly attenuated tenovin-6-induced apoptosis, suggesting that the pivotal mechanism behind its antitumor effects is based on activation of the death receptor signal pathway. Although endoplasmic reticulum stress caused by sirtuin inhibitors was reported to induce DR5 up-regulation in other cancer cell lines, we could not find marked activation of its related molecules, such as ATF6, PERK, and CHOP, in gastric cancer cells treated with tenovin-6. Tenovin-6 in combination with docetaxel or SN-38 exerted a slight to moderate synergistic cytotoxicity against gastric cancer cells. In conclusion, tenovin-6 has potent antitumor activity against human gastric cancer cells via DR5 up-regulation. Our results should be helpful for the future clinical development of sirtuin inhibitors.

We have demonstrated the successful silencing of endogenous gene expression in the skin using the intradermal transfection of unmodified siRNA via electroporation. Using this method, we revealed that COX- 1-mediated prostaglandins may act as endogenous inhibitors of scratching behavior.

Treatments with nonsteroidal anti-inflammatory drugs (NSAIDs) have increased the number of patients with gastrointestinal complications. Qing Dai has been traditionally used in Chinese herbal medicine for various inflammatory diseases such as ulcerative colitis. We previously reported that Qing Dai suppressed inflammations by scavenging reactive oxygen species (ROS) in ulcerative colitis patients. Thus, Qing Dai can attenuate the production of ROS, which play an important role in NSAID-induced gastrointestinal injuries. In this study, we aimed to elucidate whether Qing Dai decreased mitochondrial ROS production in NSAID-treated gastrointestinal cells by examining cellular injury, mitochondrial membrane potentials, and ROS production with specific fluorescent indicators. We also performed electron paramagnetic resonance measurement in isolated mitochondria with a spin-trapping reagent (CYPMPO or DMPO). Treatments with indomethacin and aspirin induced cellular injury and mitochondrial impairment in the gastrointestinal cells. Under these conditions, mitochondrial alterations were observed on electron microscopy. Qing Dai prevented these complications by suppressing ROS production in gastrointestinal cells. These results indicate that Qing Dai attenuated the ROS production from the NSAID-induced mitochondrial alteration in the gastrointestinal epithelial cells. Qing Dai treatment may be considered effective for the prevention NSAID-induced gastrointestinal injury.

It has been reported that upregulated SIRT1 (NAD+-dependent class III histone deacetylase) deacetylates the p53 protein, represses its function, and allows for tumor cell growth in various cancers. Here we investigated antitumor effects of tenovin-6, a small-molecule inhibitor of SIRT1 and SIRT2, in various colon cancer cell lines. Tenovin-6 induced apoptosis in all five colon cancer cell lines investigated (two cell lines with wild-type p53 and three with mutant p53) regardless of the p53 mutation status. This effect was accompanied by accumulation of death receptor 5 (DR5) in most cell lines. DR5 silencing in HCT116 cells strongly attenuated tenovin-6-induced apoptosis. We investigated the effect of combining tenovin-6 with conventional anticancer agents 5-fluorouracil (5-FU), SN-38 (an active metabolite of irinotecan), and oxaliplatin. Synergistic antitumor effects of tenovin-6 were observed in combination with either 5-FU or oxaliplatin in vitro. The combination of tenovin-6 and oxaliplatin exhibited potent growth inhibition of HCT116 xenograft tumors in vivo. In conclusion, tenovin-6 induced apoptosis in human colon cancer cells through the activation of the DR5 signaling pathway and enhanced the antitumor properties of 5-FU and oxaliplatin. These results may help develop a novel treatment option for colorectal cancer using a SIRT inhibitor.

MDM2 and MDM4, a structurally related MDM2 homolog, negatively regulates expression and functions of TP53 tumor suppressor gene. To explore the precise expression patterns and function of MDM2 and MDM4 in wild-type (wt) TP53 cancer cells, we analyzed 11 various cancer cell lines with wt TP53. All cell lines exhibited deregulated expression of MDM2 and MDM4, and were divided into two distinct types; the one expressing high levels of MDM4 and another expressing low levels of MDM4. The low MDM4 type expressed higher MDM2 levels than the high MDM4 type. In cells with high MDM4 expression, knockdown of MDM4 or MDM2 reactivated TP53, and simultaneous knockdown of MDM2 and MDM4 synergistically reactivated TP53. In contrast, in cells with low MDM4 expression, knockdown of only MDM2 reactivated TP53. These results suggest that both MDM2 and MDM4 are closely involved in TP53 inactivation in cancer cells with high MDM4 expression, whereas only MDM2, and not MDM4, is a regulator of TP53 in cells with low MDM4 expression. MDM4 expression in wt TP53-tumors is a potential indicator for TP53 reactivation cancer therapy by simultaneous targeting of MDM4 and MDM2. Specific knockdown of MDM2 and MDM4 might be applicable for TP53 restoration therapy.