NCTD is a demethylated form of cantharidin with antitumor properties, which is now in use as a routine anticancer drug against hepatoma. However, there is limited information on the effect of NCTD on human cancer cells. In the present study, NCTD inhibited proliferation, caused mitotic arrest, then progressed to apoptosis within 96 hr in 3 human hepatoma cell lines: HepG2, Hep3B and Huh-7. NCTD treatment (5 g/ml) enhanced the expression of Cdc25C and p21Cip1/Waf1 , increasing the phosphorylation of these 2 proteins. In addition, NCTD treatment induced an earlier increase in cyclin B1-associated histone H1 kinase activity within 48 hr, but an approximately 70% reduction of both protein level and kinase activity of cyclin B1 was observed at 72 hr. Treatment with NCTD significantly decreased the expression of p53 protein but did not affect the expression of Cdk1 and p27 Kip1 . Moreover, NCTD treatment also increased the phosphorylation of Bcl-2 and Bcl-X L but did not affect the expression of Bax or Bad. Bcl-2 phosphorylation appears to inhibit its binding to Bax since less Bax was detected in immunocomplex with Bcl-2 in NCTD-treated HepG2 cells. In addition, NCTD treatment caused activation of caspase-9 and caspase-3, preceding DNA fragmentation and morphologic features of apoptosis. Pretreatment with the broad-spectrum caspase inhibitor z-VAD-fmk markedly inhibited NCTD-induced caspase-3 activity and cell death. These results suggest that phosphorylation of p21 Cip1/Waf1 and Cdc25C and biphasic regulation of cyclin B1-associated kinase activity may contribute to NCTD-induced M-phase cell-cycle arrest. Furthermore, the increase of p21 Cip1/Waf1 , phosphorylation of Bcl-2 and Bcl-X L , activation of caspase-9 and caspase-3 may be the molecular mechanism through which NCTD induces apoptosis. © 2002 Wiley-Liss, Inc. Key words: norcantharidin; cyclin B; Cdc25c; apoptosis; caspase; Bcl-2 Hepatocarcinoma is the leading cause of cancer-related deaths in Taiwan. 1 Treatment of this disease has largely been unsuccessful, mean survival after diagnosis being limited to a few months. 2 Obviously, there is an urgent need to identify new therapeutic agents for the treatment of hepatocarcinoma in vivo. Many lines of evidence have shown that Chinese medicine contains many chemical compounds with anticancer effects. 3 Therefore, we tested whether the active ingredients of specific Chinese medicines have a therapeutic effect on human liver cancer. Cantharidin, an active ingredient of the blister beetle (Mylabris), which has long been used in Chinese traditional medicine, had been shown in China to have an effect on primary hepatoma, breast cancer and abdominal cancer; 4 but its use was limited by its severe toxicity to the mucous membrane of the gastrointestinal and urinary tracts. 3 Synthetic cantharidin derivatives were subsequently developed. NCTD is a demethylated form of cantharidin. [5][6][7] It possesses significant antihepatoma activity but at the same time is relatively free from side effects, including bone marrow ...

BackgroundAllergic disease can be characterized as manifestations of an exaggerated inflammatory response to environmental allergens triggers. Mite allergen Der-p2 is one of the major allergens of the house dust mite, which contributes to TLR4 expression and function in B cells in allergic patients. However, the precise mechanisms of Der-p2 on B cells remain obscure.Methodology/Principal FindingsWe investigated the effects of Der-p2 on proinflammatory cytokines responses and Toll-like receptor-4 (TLR4)-related signaling in human B cells activation. We demonstrated that Der-p2 activates pro-inflammatory cytokines, TLR4 and its co-receptor MD2. ERK inhibitor PD98059 significantly enhanced TLR4/MD2 expression in Der-p2-treated B cells. Der-p2 markedly activated mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) and decreased p38 phosphorylation in B cells. MKP-1-siRNA downregulated TLR4/MD2 expression in Der-p2-treated B cells. In addition, Der-p2 significantly up-regulated expression of co-stimulatory molecules and increased B cell proliferation. Neutralizing Der-p2 antibody could effectively abrogate the Der-p2-induced B cell proliferation. Der-p2 could also markedly induce NF-κB activation in B cells, which could be counteracted by dexamethasone.Conclusions/SignificanceThese results strongly suggest that Der-p2 is capable of triggering B cell activation and MKP-1-activated p38/MAPK dephosphorylation-regulated TLR4 induction, which subsequently enhances host immune, defense responses and development of effective allergic disease therapeutics in B cells.

PMA has both mitogenic and antiproliferative effects on human hepatoma Hep3B cells. In response to low PMA concentration (10 nM), Hep3B cells displayed an increasing proliferation potentiation. At high PMA concentration (1 microM) Hep3B cells exhibited modest cytostatic effects. Determinations of protein kinase C (PKC) activity in PMA-treated cells revealed that alterations in PKC activity are associated with proliferative capacity. The decrease in PKC activity mediated by a high dose of PMA was accompanied by cell growth inhibition. Increases in PKC activity mediated by a low dose of PMA were consistent with proliferation stimulation. Immunoblot analysis showed that there are at least six PKC isoenzymes: alpha, delta, epsilon, mu, zeta and iota/lambda, constitutively expressed in Hep3B cells. Cellular fractionation and immunocytochemical staining results demonstrated that both 10 nM and 1 microM PMA treatments induced a marked translocation of PKC-alpha from cytosol to membrane or nuclear fraction within 5-30 min. At the same time PKC-delta and epsilon were translocated from the membrane to nuclear fraction. In addition, prolonged treatment with 1 microM PMA, but not with 10 nM PMA, selectively mediated the down-regulation of these three PKC isoenzymes. The distinct effects of different concentrations of PMA on cell proliferation and PKC-alpha, delta and epsilon isoenzyme modulation support the involvement of these three PKC isotypes in the mechanism of action of Hep3B cells in cell growth events.

Over the past few decades, it has been demonstrated that hyperglycemia can promote lung carcinoma growth, potentially through significantly increased glucose metabolism; however, the underlying mechanism remains to be fully elucidated. In the present study, treatment with a high concentration of glucose (HG) significantly promoted the proliferation and migration of A549 cells. Receptor for advanced glycation end‑products (RAGE) has previously been demonstrated to be associated with diabetes mellitus and oxidative stress, and nicotinamide adenine dinucleotide phosphate oxidases (NOXs) are considered to be initiating factors of oxidative stress. Therefore, an MTT assay, wound‑healing assay, quantitative polymerase chain reaction and western blotting assays were used to analyze the RAGE‑NOX‑4 pathway and to determine its potential involvement in glycometabolism‑associated tumorigenesis. The present study demonstrated that HG could increase the protein expression of RAGE and NOX‑4, whereas the inhibitor of RAGE (anti‑RAGE antibody) could suppress this effect. Futhermore, the inhibitor of NOX [diphenyl iodonium chloride (DPI)] could reduce the protein expression of RAGE and NOX‑4. Furthermore, inhibition of RAGE led to the downregulation of vascular endothelial growth factor (VEGF) and hypoxia‑inducible factor‑1α (HIF‑1α), thus suggesting that HG may influence angiogenesis and tumor metabolism via the RAGE‑NOXs pathway. The present study also demonstrated that the RAGE‑blocking antibody downregulated NOX‑4 and subsequently reduced the production of downstream inflammatory factors, whereas DPI did not affect the mRNA expression of RAGE but it did reduce the protein level of RAGE and then attenuate the inflammatory response. These results indicated that inhibition of RAGE or NOXs may promote the reduced expression of VEGF and HIF‑1α, and NOXs may be downstream targets of RAGE, thus indicating a HG‑RAGE‑NOXs‑VEGF/HIF‑1α association. Furthermore, the results indicated that HG may serve a role in the development of lung adenocarcinoma, mediated by the RAGE‑oxidative stress pathway; therefore, the regulation of this glucose‑associated pathway may be a promising novel direction for oncotherapy. However, while certain antidiabetic agents have been verified to exert inhibitory effects on tumor growth, they can also have long‑term adverse effects on the body, which may limit the value of these drugs as anticancer treatments. In conclusion, the present study suggested a novel attempt to suppress glucose‑induced tumor growth using a RAGE inhibitor such as soluble RAGE while avoiding the risk of glucose fluctuation.