In this paper, we report that (؉)-preussin, a pyrrolidinol alkaloid originally identified as an antifungal agent, has growth-inhibitory and cytotoxic effects on human cancer cells. Preussin was found to be a potent inhibitor of cyclin E kinase (CDK2-cyclin E) in vitro (50% inhibitory concentration; ϳ500 nM) and to inhibit cell cycle progression into S phase. In agreement with these findings, the level of the cyclin-dependent kinase inhibitor p27 KIP-1 is increased in response to preussin treatment while the expression of both cyclin A and the transcription factor E2F-1 is down-regulated. Preussin also induces programmed cell death (apoptosis), which requires caspase activation and involves the release of cytochrome c from mitochondria. This induction of apoptosis is not blocked by high levels of Bcl-2, which usually confers resistance to chemotherapeutic agents. Taken together, our data indicate that preussin could be a promising lead compound for the development of a new class of potent antitumor drugs.
The postgenomic era is characterized by an almost intimidating amount of information regarding the sequences and expression of previously unknown genes. In response, researchers have developed an increasing interest in functional studies. At the start of such a study, one may have little more than sequence information and bioinformatic annotation. The next step is to hypothesize a potential role in the context of a cell. Testing of the hypothesis needs to be fast, cheap, and applicable to a large number of genes. Knockdown methods that rely on binding of antisense oligonucleotides to mRNA combined with a subsequent functional assay in cell culture fulfil these requirements: sequence information is sufficient for synthesis of active inhibitors. Depending on the in vitro model chosen, knockdown of gene expression can be achieved with medium or even high throughput. The two most popular methods of knockdown in cell culture are the use of antisense oligonucleotides that rely on ribonuclease H (RNAse H)-dependent cleavage of mRNA, and RNA interference triggered by small double-stranded RNA molecules. Both methods act in a sequence-specific manner and can give efficient knockdown. In both cases, researchers struggle with nonspecific "off-target" effects and the difficulty of site selection. Studies that compare the methods differ in their judgment as to which method is superior.
The new chemotherapeutic agent, flavopiridol, presently in clinical trials, has been extensively studied yet little is known about its mechanism of action. In this study we show that the induction of apoptosis by flavopiridol is largely independent of Bcl-2. This is indicated by the observation that neither overexpression nor the antisense oligonucleotide-mediated down-regulation of Bcl-2 had any effect on flavopiridol-induced cell killing. Our results suggest that flavopiridol can induce apoptosis through different pathways of caspase activation with caspase 8 playing a pivotal role. In human lung carcinoma cells, which contain high levels of endogenous Bcl-2 and lack procaspase 8, flavopiridol treatment leads to mitochondrial depolarization in the absence of cytochrome c release, followed by the activation of caspase 3 and cell death. These results clearly differ from observations made with other anti-tumor drugs and might explain, at least in part, the unusual anti-tumor properties of flavopiridol.
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