Targeting X chromosome-linked inhibitor of apoptosis protein in mucoepidermoid carcinoma of the head and neck: A novel therapeutic strategy using nitidine chloride.

Nitidine chloride (NC) was recently reported to exhibit a wide range of pharmacological properties for several diseases, including cancer. Here we report for the first time that NC is a potential therapeutic agent for mucoepidermoid carcinoma (MEC) occurring in the head and neck because it suppresses X chromosome-linked inhibitor of apoptosis protein (XIAP) in human MEC in vitro and in vivo. The antitumor effects of NC were evaluated by trypan blue exclusion assay, western blotting, live/dead assay, 4',6-diamidino-2-phenylindole (DAPI) staining, human apoptosis antibody array, immunofluorescence staining, immunohistochemistry, small interfering RNA assay, transient transfection of XIAP overexpression vector, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and histopathological examination of organs. NC inhibited cell viability and induced caspase-dependent apoptosis in vitro. A human apoptosis antibody array assay showed that XIAP is suppressed by NC treatment. XIAP was overexpressed in oral squamous cell carcinoma (OSCC) tissues that arose from the head and neck, and high XIAP expression was correlated with poor prognosis in OSCC patients. XIAP depletion significantly increased apoptosis, and ectopic XIAP overexpression attenuated the apoptosis induced by NC treatment. NC suppressed tumor growth in vivo at a dosage of 5 mg/kg/day. The number of TUNEL-positive cells increased and the protein expression of XIAP was consistently downregulated in NC-treated tumor tissues. In addition, NC caused no histopathological changes in the liver or kidney. These findings provide new insights into the mechanism of action underlying the anticancer effects of NC and demonstrate that NC is a promising therapeutic agent for the treatment of human MEC of the head and neck. KEY MESSAGES: • Nitidine chloride induces caspase-dependent apoptosis in MEC of the head and neck. • High XIAP expression correlates with poor prognosis of OSCC patients. • Nitidine chloride suppresses tumor growth in vivo without any systemic toxicities. • Targeting XIAP is a novel chemotherapeutic strategy for MEC of the head and neck.

Effect of a side-stream voltage supplied by sludge recirculation to an anaerobic digestion reactor.

This study showed that side stream voltage supplied by sludge recirculation from an auxiliary bio-electrochemical anaerobic digestion (ABEAD) reactor appears to have a similar effect as main stream voltage supply to an anaerobic digestion (AD) reactor. The increased sludge recirculation rate enhanced the operation stability at a high OLR. H2-producing bacterial community was improved in bio-electrochemical anaerobic digestion (BEAD) and ABEAD reactors and was increased with increase in sludge recirculation rate. Despite the dominance of hydrogenotrophic methanogens in all reactors, high operational performances of BEAD and ABEAD reactors supports the results of H2-producing bacteria increase in those reactors. The ABEAD reactors having 1/7 of the capacity of the main AD reactor showed possibility of integration of BEAD technology into new and existing facilities economically. The findings of this study would provide useful information for approaching the commercialization of BEAD and suggest direction of further research for practical applications.

A new insight into the apoptotic effect of nitidine chloride targeting Checkpoint kinase 2 in human cervical cancer in vitro.

Nitidine chloride (NC), a natural, bioactive, phytochemical alkaloid derived from the roots of Zanthoxylum nitidum, has been reported to exhibit anti-tumor activity against various types of cancer. However, the potential therapeutic role of NC in human cervical cancer has not yet been studied. We are the first to report that NC acts as a potential apoptosis-inducing agent for human cervical cancer in vitro. NC treatment of human cervical cancer cell lines induced caspase-mediated apoptosis, thereby reducing cell viability. Phospho-kinase proteome profiling using a human phospho-kinase array revealed that NC treatment phosphorylated Checkpoint kinase 2 (Chk2) at Thr68, which activates Chk2 in both cell lines. We also found that NC significantly affected the p53/Bim signaling axis, which was accompanied by mitochondrial membrane depolarization and cytochrome c release from the mitochondria into the cytosol. In addition, NC profoundly increased phosphorylation of the histone variant H2AX at Ser139, a typical marker of DNA damage. Taken together, these results provide in vitro evidence that NC can increase Chk2 activation, thereby acting as an attractive cell death inducer for treatment of human cervical cancer.

Contribution analysis of methane production from food waste in bulk solution and on bio-electrode in a bio-electrochemical anaerobic digestion reactor.

Quantitative evaluation of methane production either in bulk sludge or biofilm on electrodes was performed in a bio-electrochemical anaerobic digestion (BEAD) reactor with a lower electrode surface area/reactor working volume (A/V) ratio (7.0 m2/m3). Methane production by electrochemical reaction was also evaluated in the BEAD reactor with a biofilm-free electrode under the same conditions as in other experimental sets. The contributions of bulk sludge, biofilms on the electrodes, and electrochemical reactions in the BEAD reactor, on methane production, were 70.2%, 29.8%, and 0%, respectively. The principal methane-producing reactions occurred in the bulk sludge facilitated by H2-dependent methylotrophic and hydrogenotrophic methanogens. Hydrogenotrophic methanogenesis was also the main methane-producing reaction in the biofilms attached to the bio-electrodes. Quantitative analysis of methane production (29.8%) in the biofilm revealed that bio-electrochemical processes involving H2 and direct bio-electrochemical methane production contributed 8.7% and less than 0.1%, respectively. Interestingly, biochemical processes (21.1%) contributed the most to the overall production of methane in the biofilm. Bulk sludge contributed more to methane production than the biofilm, but the methane production per unit mass of volatile solid on the electrodes was about 1.6-times higher than that of bulk sludge. Methane was not produced in the BEAD reactor with biofilm-free electrodes. Therefore, formation and maintenance of biofilms on the electrodes are essential for improved methane production in BEAD reactors.

Effects of a novel auxiliary bio-electrochemical reactor on methane production from highly concentrated food waste in an anaerobic digestion reactor.

In this study, the effects of indirect voltage supply to an anaerobic digestion (AD) reactor on methane production and the removal of chemical oxygen demand (COD) were studied at different organic loading rates (OLRs) of food waste by the circulation from an auxiliary bio-electrochemical reactor (ABER) with stainless steel (STS304) electrodes. The effects of the indirect voltage on microbial communities in the AD reactor were also investigated. In a bio-electrochemical anaerobic digestion (BEAD) reactor with direct voltage, it was possible to achieve stable COD removal and methane production even at a higher OLR of 10.0 kg/(m3·d). However, in the AD reactor, the COD removal efficiency and methane production decreased sharply at an OLR of 6.0 kg/(m3·d) due to the accumulation of volatile fatty acids (VFAs) and decreases in the pH and alkalinity. The supply of indirect voltage through the ABER increased the community of exoelectrogenic bacteria and hydrogenotrophic methanogens in the AD + ABER bulk solution. As a result, rapid oxidation of the accumulated VFAs occurred, and methane production increased in the new AD + ABER system. The results confirm that an indirect voltage supply to the new AD + ABER system can have effects similar to those of a direct voltage supply to the BEAD reactor, and the findings are expected to provide useful information for the development and application of BEAD technology for commercialization.

In vitro and in vivo anti-cancer activity of silymarin on oral cancer.

Silymarin, a standardized extract from milk thistle fruits has been found to exhibit anti-cancer effects against various cancers. Here, we explored the anti-cancer activity of silymarin and its molecular target in human oral cancer in vitro and in vivo. Silymarin dose-dependently inhibited the proliferation of HSC-4 oral cancer cells and promoted caspase-dependent apoptosis. A human apoptosis protein array kit showed that death receptor 5 may be involved in silymarin-induced apoptosis, which was also shown through western blotting, immunocytochemistry, and reverse transcription-polymerase chain reaction. Silymarin increased cleaved caspase-8 and truncated Bid, leading to accumulation of cytochrome c. In addition, silymarin activated death receptor 5/caspase-8 to induce apoptotic cell death in two other oral cancer cell lines (YD15 and Ca9.22). Silymarin also suppressed tumor growth and volume without any hepatic or renal toxicity in vivo. Taken together, these results provide in vitro and in vivo evidence supporting the anti-cancer effect of silymarin and death receptor 5, and caspase-8 may be essential players in silymarin-mediated apoptosis in oral cancer.

Nitidine chloride acts as an apoptosis inducer in human oral cancer cells and a nude mouse xenograft model via inhibition of STAT3.

Nitidine chloride (NC) is a natural alkaloid compound derived from the plant Zanthoxylum nitidum and is known for its therapeutic anticancer potential. In this study, we investigated the effects of NC on growth and signaling pathways in human oral cancer cell lines and a tumor xenograft model. The apoptotic effects and related molecular targets of NC on human oral cancer were investigated using trypan blue exclusion assay, DAPI staining, Live/Dead assay, Western blotting, Immunohistochemistry/Immunofluorescence and a nude mouse tumor xenograft. NC decreased cell viability in both HSC3 and HSC4 cell lines; further analysis demonstrated that cell viability was reduced via apoptosis. STAT3 was hyper-phosphorylated in human oral squamous cell carcinoma (OSCC) compared with normal oral mucosa (NOM) and dephosphorylation of STAT3 by the potent STAT3 inhibitor, cryptotanshinone or NC decreased cell viability and induced apoptosis. NC also suppressed cell viability and induced apoptosis accompanied by dephosphorylating STAT3 in four other oral cancer cell lines. In a tumor xenograft model bearing HSC3 cell tumors, NC suppressed tumor growth and induced apoptosis by regulating STAT3 signaling without liver or kidney toxicity. Our findings suggest that NC is a promising chemotherapeutic candidate against human oral cancer.

Apoptosis induced by caffeic acid phenethyl ester in human oral cancer cell lines: Involvement of Puma and Bax activation.

OBJECTIVE: Caffeic acid phenethyl ester (CAPE), a natural honeybee product exhibits a spectrum of biological activities including antimicrobial, anti-inflammatory, antioxidant and antitumor actions. The purpose of this research was to investigate the anticancer potential of CAPE and its molecular mechanism in human oral cancer cell lines (YD15, HSC-4 and HN22 cells). DESIGN: To determine the apoptotic activity of CAPE and identify its molecular targets, trypan blue exclusion assay, soft agar assay, Western blot analysis, DAPI staining, and live/dead assay were performed. RESULTS: CAPE significantly suppressed transformation of neoplastic cells induced by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) without inhibiting growth. CAPE treatment inhibited cell growth, increased the cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and augmented the number of fragmented nuclei in human oral cancer cell lines. CAPE activated Bax protein causing it to undergo a conformational change, translocate to the mitochondrial outer membrane, and oligomere. CAPE also significantly increased Puma expression and interestingly Puma and Bax were co-localized. CONCLUSION: Overall, these results suggest that CAPE is a potent apoptosis-inducing agent in human oral cancer cell lines. Its action is accompanied by up-regulation of Bax and Puma proteins.

Silymarin and its active component silibinin act as novel therapeutic alternatives for salivary gland cancer by targeting the ERK1/2-Bim signaling cascade.

PURPOSE: Approximately 20% of all salivary gland cancer patients who are treated with current treatment modalities will ultimately develop metastases. Its most common form, mucoepidermoid carcinoma (MEC) is a highly aggressive tumor with an overall 5-year survival rate of ~30%. Until now, several chemotherapeutic drugs have been tested for the treatment of salivary gland tumors, but the results have been disappointing and the drugs often cause unwanted side effects. Therefore, several recent studies have focused on the potential of alternative and/or complementary therapeutic options, including the use of silymarin. METHODS: The effects of silymarin and its active component silibinin on salivary gland cancer-derived MC3 and HN22 cells and their underlying molecular mechanisms were examined using trypan blue exclusion, 4'-6-diamidino-2-phenylindole (DAPI) staining, Live/Dead, Annexin V/PI staining, mitochondrial membrane potential (ΔΨm) measurement, quantitative RT-PCR, soft agar colony formation and Western blotting analyses. RESULTS: We found that silymarin and silibinin dramatically increased the expression of the pro-apoptotic protein Bim in a concentration- and time-dependent manner and, concomitantly, induced apoptosis in MC3 and HN22 cells. We also found that ERK1/2 signaling inhibition successfully sensitized these cells to the apoptotic effects of silymarin and silibinin, which indicates that the ERK1/2 signaling pathway may act as an upstream regulator that modulates the silymarin/silibinin-induced Bim signaling pathway. CONCLUSIONS: Taken together, we conclude that ERK1/2 signaling pathway inhibition by silymarin and silibinin increases the expression of the pro-apoptotic Bcl-2 family member Bim which, subsequently, induces mitochondria-mediated apoptosis in salivary gland cancer-derived cells.