| Furanonaphthoquinone compounds belong to a big natural product family which exists in many higher plants and actinomycetes, and studies found that they have multiple biological activities, such as anti-viral, anti-tumor and anti-bacterial activities as well as autoimmune diseases regulation.In this work, we studied the anti-tumor activity of FNQ I which was produced by Streptomyces sp. HTZ 27 isolated in our previous work. We measured the cytotoxicity of FNQ I in nine solid tumor cell lines, two myeloma leukemia cell lines and two normal cell lines. The results showed that the IC50 values of FNQ I on tumor cells is about 4-10 fold lower than that against normal cell lines, indicating that the compound have a preferential cytotoxicity.Flow cytometry assays demonstrated that FNQ I could induce apoptosis in H460 cells. The Caspase-3/7 and Caspase-9 activity assays showed that the relative activity of Caspase-3/7 was increased by 29.8%(p <0.05, 50μM) and 79.2%(p <0.01, 100μM) after FNQ I treated for 6h respectively. While the relative activity of Caspase-9 only increased by 37.6%(p <0.05, 50μM) and 25.7%(p <0.05, 100μM) after FNQ I treated for 24 h respectively. Further activity assays of Caspase-8 showed that the relative activity of Caspase-8 increased by 16%(p <0.05, 100μM) after FNQ I treated H460 cells for 6h. The fact that Caspase-3/7 and Caspase-8 was activated much earlier than Caspase-9 suggested that Caspase-3/7 must be activated by Caspase-8 which located in extrinsic pathway rather than Caspase-9. These results implied that the apoptosis-inducing effect of FNQ I to H460 cells first activate extrinsic pathway. However,the relative activity of Caspase-8 decreased while Caspase-9 increased markedly after FNQ I treated H460 cells for 24 h. This result showed that Caspase-9 which located in intrinsic pathway was activated later, and replaced Caspase-8 to activate Caspase-3, inducing apoptosis in tumor cells. This finding, for the first time, revealed the time corse of extrinsic pathway and intrinsic pathway in the process of apoptosis that FNQ compounds induced,and it is in-consistant with conclusion that FNQ analogs induced apoptosis via intrinsic(mitochondrial) pathway in several published papers so far.To further explore the molecular mechanism of apoptosis induced by FNQ I, expression changes of twenty five genes relating to apoptosis, differentiation or cell survival were investigated by realtime quantitative PCR(RT-q PCR) in H460 cells treated with 40 ?M FNQ I for 1-24 h. In H460 cells treated with FNQ I, expression of 7 genes were markedly up-regulated, 2 genes were markedly down-regulated and 3 genes were markedly fluctuated, meanwhile expression of 13 genes showed no significant expression changes. The expression of genes in intrinsic pathway, Bcl-2, BIRC3, PPP3 R and XIAP were significantly changed after H460 cells treated with FNQ I for at least 12 hrs, while the expression of genes encoding interleukins and their receptors(located in extrinsic pathway) was modulated markedly after H460 cells treated with FNQ I for 3hrs(i.e. IL-1A and IL7). Moreover, TNFSF10B(death receptor 5(DR5)) and CD5 was significantly down-regulated after H460 cells treated with FNQ I for 1hr. Overall, these results supported the viewpoint that FNQ I induced apoptosis first via the extrinsic pathway then via the intrinsic pathway.And, according to the fast(1 h) and marked down-regulation of CD5 expression and its function, we hypothesize that CD5 may play a key role in the initiation of apoptosis in FNQ I-treated H460 cells through Fas L and Caspase-8 pathway. |
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