Sulindac sulfone inhibits the mTORC1 pathway in colon cancer cells by directly targeting voltage-dependent anion channel 1 and 2.

Sulindac sulfone is a metabolite of sulindac, a non-steroidal anti-inflammatory drug (NSAID), without anti-inflammatory ability. However, sulindac sulfone has been reported to significantly reduce polyps in patients with colorectal adenomatous polyposis in clinical trials. Thus, sulindac sulfone is expected to be useful for the chemoprevention of neoplasia with few side effects related to anti-inflammatory ability. To date, the molecular targets of sulindac sulfone have not yet fully investigated. Therefore, in order to newly identify sulindac sulfone-binding proteins, we generated sulindac sulfone-fixed FG beads and purified sulindac sulfone-binding proteins from human colon cancer HT-29 cells. we identified mitochondrial outer membrane proteins voltage-dependent anion channel (VDAC) 1 and VDAC2 as novel molecular targets of sulindac sulfone, and sulindac sulfone directly bound to both VDAC1 and VDAC2. Double knockdown of VDAC1 and VDAC2 by siRNA inhibited growth and arrested the cell cycle at G1 phase in HT-29 cells. Depletion of VDAC1 and VDAC2 also inhibited the mTORC1 pathway with a reduction in cyclin D1. Interestingly, these effects were consistent with those of sulindac sulfone against human colon cancer cells, suggesting that sulindac sulfone negatively regulates the function of VDAC1 and VDAC2. In the present study, our data suggested that VDAC1 and VDAC2 are direct targets of sulindac sulfone which suppresses the mTORC1 pathway and induces G1 arrest.

Design, synthesis and evaluation of γ-turn mimetics as LSD1-selective inhibitors.

Lysine-specific demethylase 1 (LSD1) is an attractive molecular target for cancer therapy. We have previously reported potent LSD1-selective inhibitors (i.e., NCD18, NCD38, and their analogs) consisting of trans-2-phenylcyclopropylamine (PCPA) or trans-2-arylcyclopropylamine (ACPA) and a lysine moiety that could form a γ-turn structure in the active site of LSD1. Herein we report the design, synthesis and evaluation of γ-turn mimetic compounds for further improvement of LSD1 inhibitory activity and anticancer activity. Among a series of γ-turn mimetic compounds synthesized by a Mitsunobu-reaction-based amination strategy, we identified 1n as a potent and selective LSD1 inhibitor. Compound 1n induced cell cycle arrest and apoptosis through histone methylation in human lung cancer cells. The γ-turn mimetics approach should offer new insights into drug design for LSD1-selective inhibitors.

Three Combined Treatments, a Novel HDAC Inhibitor OBP-801/YM753, 5-Fluorouracil, and Paclitaxel, Induce G2 Phase Arrest Through the p38 Pathway in Human Ovarian Cancer Cells.

Ovarian cancer is the most lethal disease among gynecological malignancies. More effective therapy is required to counter high recurrence rates and chemotherapy resistance. We investigated the efficacy and molecular mechanisms of three combined treatments (TCTs)-a novel histone deacetylase (HDAC) inhibitor OBP-801/YM753, 5-fluorouracil (5-FU), and paclitaxel (PTX)-in human ovarian cancer SKOV-3 and OVCAR-3 cells. The inhibition of cell growth was stronger with TCTs than with each single agent and with two combined treatments. The TCTs significantly induce G2 phase arrest in both cell lines. We then analyzed the molecular mechanisms and found that the TCTs increased the phosphorylation of p38 (Thr180/Tyr182), decreased the expression of CDC25C, and increased the phosphorylation of CDC2 (Tyr15), an inactive form of CDC2. To examine the responsibilities of the p38 pathway for G2 phase arrest induced by the TCTs, we employed the p38 inhibitor SB203580. SB203580 inhibited G2 phase arrest, suppression of CDC25C, and phosphorylation of CDC2 (Tyr15) induced by the TCTs. These results suggest that the TCTs can induce G2 phase arrest through activation of the p38 signaling pathway. We therefore believe that this combination is promising as a novel therapeutic strategy against ovarian cancer.

C-H activation enables a rapid structure-activity relationship study of arylcyclopropyl amines for potent and selective LSD1 inhibitors.

We describe the structure-activity relationship of various arylcyclopropylamines (ACPAs), which are potent LSD1 inhibitors. More than 45 ACPAs were synthesized rapidly by an unconventional method that we have recently developed, consisting of a C-H borylation and cross-coupling sequence starting from cyclopropylamine. We also generated NCD38 derivatives, which are known as LSD1 selective inhibitors, and discovered a more effective inhibitor compared to the original NCD38.

A Histone Deacetylase Inhibitor, OBP-801, and Celecoxib Synergistically Inhibit the Cell Growth with Apoptosis via a DR5-Dependent Pathway in Bladder Cancer Cells.

The prognosis of muscle-invasive bladder cancer with metastasis is poor. There have been no therapeutic improvements for many years, and an innovative therapy for muscle-invasive bladder cancer has been awaited to replace the conventional cytotoxic chemotherapy. Here, we show a candidate method for the treatment of bladder cancer. The combined treatment with a novel histone deacetylase (HDAC) inhibitor, OBP-801, and celecoxib synergistically inhibited cell growth and markedly induced apoptosis through the caspase-dependent pathway in high-grade bladder cancer cells. Furthermore, the combined treatment induced expression of death receptor 5 (DR5). We identified that knockdown of DR5 by small interfering RNA (siRNA) significantly suppressed apoptosis by the combined treatment. Therefore, we conjectured that the apoptosis induced by OBP-801 and celecoxib is at least partially dependent on DR5. However, it was interesting that the combined treatment drastically suppressed expression of DR5 ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). These data suggest that there is no involvement of TRAIL in the induction of apoptosis by the combination, regardless of the dependence of DR5. Moreover, xenograft studies using human bladder cancer cells showed that the combined therapy suppressed tumor growth by upregulating expressions of DR5 and Bim. The inhibition of tumor growth was significantly more potent than that of each agent alone, without significant weight loss. This combination therapy provided a greater benefit than monotherapy in vitro and in vivo These data show that the combination therapy with OBP-801 and celecoxib is a potential novel therapeutic strategy for patients with muscle-invasive bladder cancer. Mol Cancer Ther; 15(9); 2066-75. ©2016 AACR.

The flavonoid apigenin downregulates CDK1 by directly targeting ribosomal protein S9.

Flavonoids have been reported to inhibit tumor growth by causing cell cycle arrest. However, little is known about the direct targets of flavonoids in tumor growth inhibition. In the present study, we developed a novel method using magnetic FG beads to purify flavonoid-binding proteins, and identified ribosomal protein S9 (RPS9) as a binding partner of the flavonoid apigenin. Similar to treatment with apigenin, knockdown of RPS9 inhibited the growth of human colon cancer cells at the G2/M phase by downregulating cyclin-dependent kinase 1 (CDK1) expression at the promoter level. Furthermore, knockdown of RPS9 suppressed G2/M arrest caused by apigenin. These results suggest that apigenin induces G2/M arrest at least partially by directly binding and inhibiting RPS9 which enhances CDK1 expression. We therefore raise the possibility that identification of the direct targets of flavonoids may contribute to the discovery of novel molecular mechanisms governing tumor growth.

Apigenin sensitizes prostate cancer cells to Apo2L/TRAIL by targeting adenine nucleotide translocase-2.

Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent. Recombinant human Apo2L/TRAIL has been under clinical trials, whereas various kinds of malignant tumors have resistance to Apo2L/TRAIL. We and others have shown that several anticancer agents and flavonoids overcome resistance to Apo2L/TRAIL by upregulating death receptor 5 (DR5) in malignant tumor cells. However, the mechanisms by which these compounds induce DR5 expression remain unknown. Here we show that the dietary flavonoid apigenin binds and inhibits adenine nucleotide translocase-2 (ANT2), resulting in enhancement of Apo2L/TRAIL-induced apoptosis by upregulation of DR5. Apigenin and genistein, which are major flavonoids, enhanced Apo2L/TRAIL-induced apoptosis in cancer cells. Apigenin induced DR5 expression, but genistein did not. Using our method identifying the direct targets of flavonoids, we compared the binding proteins of apigenin with those of genistein. We discovered that ANT2 was a target of apigenin, but not genistein. Similarly to apigenin, knockdown of ANT2 enhanced Apo2L/TRAIL-induced apoptosis by upregulating DR5 expression at the post-transcriptional level. Moreover, silencing of ANT2 attenuated the enhancement of Apo2L/TRAIL-induced apoptosis by apigenin. These results suggest that apigenin upregulates DR5 and enhances Apo2L/TRAIL-induced apoptosis by binding and inhibiting ANT2. We propose that ANT2 inhibitors may contribute to Apo2L/TRAIL therapy.

Perillyl alcohol causes G1 arrest through p15(INK4b) and p21(WAF1/Cip1) induction.

The monoterpene perillyl alcohol (POH) is a naturally occurring compound derived from citrus fruits, mint and herbs. It exhibited chemotherapeutic potential against various malignant tumors in preclinical models and is currently being tested in clinical trials in patients with refractory advanced cancers. POH inhibits cellular proliferation at the G1 phase of the cell cycle in vitro. However, the molecular mechanisms responsible for this effect have not been sufficiently elucidated. Here we showed that 1.0 mM POH upregulates p15(INK4b) and p21(WAF1/Cip1), resulting in hypophosphorylation of the retinoblastoma (RB) protein and subsequent G1 arrest in human immortalized keratinocyte HaCaT cells. The induction of p15(INK4b) was mediated through its promoter, but that of p21(WAF1/Cip1) was not. The small interfering RNA (siRNA) of either p15(INK4b) or p21(WAF1/Cip1) significantly attenuated the increase in the G1 cell population caused by POH. The induction of p15(INK4b) and p21(WAF1/Cip1) and sub-sequent G1 arrest by POH was also observed in other cancer cell lines. These results suggest that the induction of p15(INK4b) as well as p21(WAF1/Cip1) is associated with the antiproliferative effect of POH.

Two-domain arginine kinases from the clams Solen strictus and Corbicula japonica: exceptional amino acid replacement of the functionally important D(62) by G.

Arginine kinases (AKs) isolated from the adductor muscle of the clams Solen strictus and Corbicula japonica have relative molecular masses of 80 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in contrast to the 40 kDa AKs found in Mollusca and Arthropoda. The cDNAs encoding Solen and Corbicula AKs have open reading frames of 2175 nucleotides (724 amino acid protein) and 2172 nucleotides (723 amino acid protein), respectively. The amino acid sequence clearly indicates that Solen and Corbicula AKs have a two-domain structure: the first-domain includes residues 1-363 and the second-domain includes residue 364 to the end. There is approximately 60% inter-domain amino acid identity. It is clear that gene-duplication and subsequent fusion occurred in the immediate ancestor of the clams Solen, Corbicula, and Pseudocardium. During substrate binding, it is proposed that AK undergoes a substrate-induced conformational change and that the hydrogen bond between D(62) and R(193) stabilizes the substrate-bound structure. However, in Solen and Corbicula two-domain AKs, D(62) is replaced by a G, and R(193) by A, S, or D. Consequently, the two-domain AKs can not form the stabilizing hydrogen bond. Nevertheless, the enzyme activity of Corbicula AK is comparable to those of other molluscan 40 kDa AKs. We assumed that the substrate-bound structure of the two-domain AK is stabilized not by the hydrogen bond between D(62) and R(193) but by the bond between H(60) and D(197), characteristic of the unusual two-domain AKs. This explains why D(62) and R(193), which remain highly conserved in other AKs, have undergone amino acid replacements in Solen and Corbicula AKs.