RSS-Feed abonnieren
DOI: 10.1055/s-2003-38888
Apoptosis-Inducing Properties of ent-Kaurene-Type Diterpenoids from the Liverwort Jungermannia truncata
Publikationsverlauf
Received: July 5, 2002
Accepted: January 25, 2003
Publikationsdatum:
23. April 2003 (online)
![](https://www.thieme-connect.de/media/plantamedica/200304/lookinside/thumbnails/10.1055-s-2003-38888-1.jpg)
Antitumor agents induce apoptosis in some cancer cells both in vitro and in vivo, indicating that apoptosis-inducing agents are promising candidates for antitumor drugs [1]. Apoptosis is triggered via a biochemically complex pathway involving the caspase cascade [2]. Caspases are a family of cysteine proteases expressed as pro-enzymes [3], [4]. So far, 13 members of mammalian caspases have been identified [3], [4]. Among them, caspase-3 is an important effector caspase, and is responsible for the cleavage of crucial substrates such as poly(ADP-ribose) polymerase (PARP) and an inhibitor of caspase-dependent DNase in the apoptotic process [5], [6]. Recently, we found that ent-11α-hydroxy-16-kauren-15-one (1), isolated from the Japanese liverwort Jungermannia truncata Nees, induced apoptosis in human leukemia cell lines (HL-60 cells) [7]. However, the apoptosis-inducing properties of ent-kaurenes remain to be clarified. In this study, we investigated the involvement of caspases in ent-kaurene-induced apoptosis in HL-60 cells.
ent-Kaurene-type diterpenoids 1 - 6 (Fig. [1]) have been isolated from the Japanese liverwort J. truncata [7]. Compounds 1 - 4 possess an enone group at C-15/C-16. Compounds 5 and 6 have a hydroxy group at C-15, an exo-methylene at C-16, or a ketone at C-15 and a secondary methyl at C-16, respectively. As shown in Fig. [2], the cytotoxicities of 5 and 6 were significantly weaker than those of compounds 1 - 4. During apoptosis, PARP is cleaved from the 116 kDa intact form into 85 kDa and 25 kDa fragments by caspases including caspase-3 [5]. Therefore, the activation of caspases was estimated by PARP proteolysis. We confirmed the induction of apoptosis and activation of caspases by treatment with 1 and its related compounds at 5 μM (Fig. [3]). Compounds 5 and 6 did not induce DNA fragmentation, a typical feature of apoptosis, and PARP proteolysis, a sign of activation of caspases [5]. On the other hand, compounds 2 and 3 showed the same activity as mentioned above (Figs. 3A and 3B). Induction of apoptosis by 5 and 6 was observed at higher concentrations (data not shown). These results indicate that the enone group at C-15/C-16 plays an important role in induction of apoptosis by compounds 1 - 3. Interestingly compound 4-mediated cell death was not due to the induction of apoptosis, and was not followed by PARP proteolysis, indicating that the cell death feature was necrosis. Z-Asp-CH2-DCB, a broad spectrum inhibitor of caspases [8], significantly attenuated the cytotoxicity (Fig. [4] A) and significantly diminished DNA ladder formation (Fig. [4] B) induced by compounds 1, 2 or 3. These results indicated the involvement of caspases in 1 and its related compounds-induced apoptosis.
In this study, we demonstrated that ent-kaurenes 1 - 3 induced apoptosis via activation of caspases. Moreover, the enone group at C-15/C-16 in the molecule is an essential functional group for the induction of apoptosis and activation of caspases in human leukemia cell lines (HL-60 cells). In fact, Dirsch et al. reported that sesquiterpene lactones such as helenalin with an α,β-unsaturated ketone, which is a similar functional group to that in compounds 1 - 3, induce apoptosis via activation of caspases in leukemia T cells [9]. Although most antitumor agents are known to induce apoptosis in cancer cells, malignant tumors are resistant to antitumor agents via various protective factors [10]. Especially, overexpression of Bcl-2, a potent protective factor against induction of apoptosis via mitochondria, is observed in many malignant tumors [10]. The cytotoxicity of cisplatin was inhibited by Bcl-2, however compound 1-induced apoptosis was not inhibited by Bcl-2 (data not shown). Therefore, compound 1 could be a useful antitumor agent against Bcl-2 overexpressing tumor cells.
In summary, these findings indicate that some ent-kaurene-type compounds induce apoptosis via activation of caspases. Although further investigations for determination of the detailed pathway of apoptosis by the compounds are needed, compound 1 might be a potent lead compound for the development of antitumor agents.
Fig. 1 The structures of ent-kaurene-type diterpenoids.
Fig. 2 Cytotoxicity of 1 and its related compounds in human leukemia HL-60 cells. HL-60 cells (1 × 104 cells/well in 96-well plate) were incubated with the indicated chemicals (open square, 1; open circle, 5; open triangle, 6; closed square, 2; closed triangle, 3; closed circle, 4.) for 24 h, and then the cytotoxicities of the chemicals were determined by the WST-8 assay. Data are presented as mean ± SD (n = 4).
Fig. 3 Structure-activity relationship of compounds 1 - 6. Cells were treated with the indicated chemicals for 18 h at 5 μM, and then DNA ladder formation was examined (A). After 9 h of treatment with the indicated chemicals (5 μM), PARP proteolysis was examined (B).
Fig. 4 Involvement of caspases in 1-, 2- and 3-induced apoptosis. Cells were incubated with or without Z-Asp-CH2-DCB (100 μM), a broad spectrum inhibitor of caspases, for 1 h. Then the cells were treated with 1 (2.5 μM), 2 (5 μM) or 3 (5 μM) derivatives for 6 h, and cytotoxicity (A) and appearance of DNA ladder (B) were evaluated. * p < 0.05, using Student’s t-test.
References
- 1 Kaufmann S H. Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. Cancer Research. 1989; 49 5870-8
- 2 Ferri K F, Kroemer G. Organelle-specific initiation of cell death pathways. Nature Cell Biology. 2001; 3 E255-63
- 3 Nicholson D W, Thornberry N A. Caspases: killer proteases. Trends in Biochemical Sciences. 1997; 22 299-306
- 4 Thornberry N A, Lazebnik Y. Caspases: Enemies within. Science. 1998; 281 1312-6
- 5 Tewari M, Quan L T, O’Rourke K, Desnoyers S, Zeng Z, Beidler D R, Poirier G G, Salvesen G S, Dixit V M. Yama/CPP32beta, a mammalian homolog of ced-3. is a crmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 1995; 81 801-9
- 6 Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 1998; 391 43-50
- 7 Nagashima F, Kondoh M, Uematsu T, Nishiyama A, Saito S, Sato M. Asakawa Y. Cytotoxic and apoptosis-inducing ent-kaurene-type diterpenoids from the Japanese liverwort Jungermannia truncata Nees. Chemical & Pharmaceutical Bulletin. 2002; 50 808-13
- 8 Dolle R E, Hoyer D, Prasad C VC, Schmidt S J, Helaszek C T, Miller R E, Ator M A. P1 aspartate-based peptide alpha-((2,6-dichlorobenzoyl)oxy)methyl ketones as potent time-dependent inhibitors of interleukin-1beta-converting enzyme. Journal of Medicinal Chemistry. 1994; 37 563-4
- 9 Dirsch V M, Stuppner H, Vollmar A M. Helenalin triggers a CD95 death receptor-independent apoptosis that is not affected by overexpressing of Bcl-xL or Bcl-2. Cancer Research. 2001; 61 5817-23
- 10 Johnstone R W, Ruefli A A, Lowe S W. Apoptosis: a link between cancer genetics and chemotherapy. Cell. 2002; 108 153-64
- 11 Watabe M, Machida K, Osada H. MT-21 is a synthetic apoptosis inducer that directly induces cytochrome c release from mitochondria. Cancer Research. 2000; 60 5214-22
Prof. Dr. Y. Asakawa
Faculty of Pharmaceutical Sciences
Tokushima Bunri University
Yamashiro-cho
Tokushima 770-8514
Japan
eMail: asakawa@ph.bunri-u.ac.jp
Fax: +81-88-655-3051