Background: The development of microarrays permits us to monitor transcriptomes on a genome-wide scale. To validate microarray measurements, quantitative-real time-reverse transcription PCR (Q-RT-PCR) is one of the most robust and commonly used approaches. The new challenge in gene quantification analysis is how to explicitly incorporate statistical estimation in such studies. In the realm of statistical analysis, the various available methods of the probe level normalization for microarray analysis may result in distinctly different target selections and variation in the scores for the correlation between microarray and Q-RT-PCR. Moreover, it remains a major challenge to identify a proper internal control for Q-RT-PCR when confirming microarray measurements.

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Findings from this study may provide insight into a possible molecular mechanism by which ellagic acid inhibits LOX-1-induced endothelial dysfunction. Our data indicate that ellagic acid exerts its protective effects by inhibiting NADPH oxidase-induced overproduction of superoxide, suppressing the release of NO by down-regulating iNOS, enhancing cellular antioxidant defenses, and attenuating oxLDL-induced LOX-1 up-regulation and eNOS down-regulation.

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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 ...

Summary Cullin-RING ubiquitin ligases (CRL) are critical in ubiquitinating Myc, while COP9 signalosome (CSN) controls neddylation of Cullin in CRL. The mechanistic link between Cullin neddylation and Myc ubiquitination/degradation is unclear. Here we show that Myc is a target of the CSN subunit 6 (CSN6)–Cullin signaling axis and that CSN6 is a positive regulator of Myc. CSN6 enhanced neddylation of Cullin-1 and facilitated auto-ubiquitination/degradation of Fbxw7, a component of CRL involved in Myc ubiquitination, thereby stabilizing Myc. Csn6 haplo-insufficiency decreased Cullin-1 neddylation but increased Fbxw7 stability to compromise Myc stability and activity in an Eµ-Myc mouse model, resulting in decelerated lymphomagenesis. We found that CSN6 overexpression, which leads to aberrant expression of Myc target genes, is frequent in human cancers. Together, these results define a mechanism for the regulation of Myc stability through the CSN-Cullin-Fbxw7 axis and provide insights into the correlation of CSN6 overexpression with Myc stabilization/activation during tumorigenesis.

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