行政院國家科學委員會專題研究計畫 成果報告
Morusin 抗人類大腸直腸癌之分子訊息作用機制(2/2)
計畫類別： 個別型計畫
計畫編號： NSC94-2314-B-006-016-
執行期間： 94 年 08 月 01 日至 95 年 07 月 31 日 執行單位： 國立成功大學醫學系外科
計畫主持人： 李政昌 共同主持人： 翁舷誌
報告類型： 完整報告
處理方式： 本計畫可公開查詢
中 華 民 國 95 年 11 月 1 日
附件：封面格式
行政院國家科學委員會補助專題研究計畫成果報告
※ ※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※
※Morusin 抗人類大腸直腸癌之分子訊息作用機(2/2) ※
※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※※
計畫類別：■個別型計畫 □整合型計畫 計畫編號：NSC94－2314－B－006－016
執行期間：94 年 08 月 01 日至 95 年 07 月 31 日
計畫主持人：李政昌
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
□出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
執行單位：國立成功大學醫學院外科部
中 華 民 國 95 年 10 月 25 日
Morusin 抗人類大腸直腸癌之分子訊息作用機 (2/2)
Antitumor molecular signaling mechanism of Morusin in Human colorectal cancer cells
計畫編號：NSC 94-2314-B-006-016 執行期限：94 年 08 月 01 日至 95 年 07 月 31 日
主持人：李政昌 國立成功大學醫學院外科部 一、中文摘要
Morusin 是 由 M. australis (Moraceae)的根部中分離出的成份，桑白 皮的粗抽出物在台灣民間常被用於改善 原住民的癌症，我們以前的研究已証明對 人類大腸直腸細胞(HT-29及HCT116)、人 類 肝 癌 細 胞 (Hep 3B)及人 類 乳 癌 細 胞 (MCF-7)之生長具有抑制的作用。抑制大 腸直腸癌細胞生長主要是使細胞膜內側 的磷脂絲胺酸有翻轉至外側細胞膜，細胞 的sub-G1期的DNA有增加，DNA片段化現象 出現，並造成細胞核濃縮及出現凋亡小體
現象使細胞凋亡，並活化caspase-8、
caspase-9、caspase-3。本年度的研究發 現Morusin 可使HT-29的caspase-8活化
tBid由細胞質轉至粒線體而降低Bcl-XL
表 現 並 使 粒 線 體 膜 電 位 喪 失 ， 而 造 成 cytochrome c及Smac/DIABLO由粒線體中 釋放到細胞質中，另外, Morusin抑制 IKK-α、IKK-β及IκB-α之磷酸化作用、
而增加IκB-α的表現並抑制NF-κB轉入
核內及抑制核內DNA的結合能力。抑制 Ku70的調節作用及抑制NF-κB在核內的 調節作用而致影響XIAP之表現而引起細 胞死亡之信息，簡言之，我們的研究顯示 Morusin引起大腸直腸細胞HT-29的細胞 凋亡可能經由caspases和NF-κB亡途徑。
關鍵詞：Morusin, 人類大腸直腸癌, 細 胞 凋 亡 , caspases, 粒 線 體 , cytochrome c, Smac/DIABLO, Bcl-2, XIAP, PKCs,Akt/PI3K, NF-κB
Abstract
Morusin, isolated from root bark of Morus australis, has been used as a traditional folk remedy for treatment of cancers in Taiwan. Our previous studies demonstrated that morusin significantly inhibited the growth of human colorectal cancer cells (HT-29 and HCT 116), human hepatocellular carcinoma cells (Hep 3B), and human breast adenocarcinoma cells (MCF-7). The susceptibility of cells to morusin was caused by apoptosis through phosphatidylserine exposure, increase of DNA fragmentation, nuclear morphologic change, and sub-G1 content. The molecular mechanism of morusin on apoptosis was associated with the activation of caspase-8, caspase-9 and caspase-3. The present study demonstrates that activation of caspase-8 leads to the translocation of tBid from cytosol to mitochondria, and significant decrease in anti-apoptotic Bcl-XL in mitochondrial. The loss of mitochondrial membrane potential results in the release of cytochrome c and Smac/DIABLO from mitochondria to cytosol in HT-29 cells. Moreover, morusin
inhibits the phosphorylation of IKK-α, IKK-β and IκB-α, increases expression of IκB-α, and suppresses nuclear translocation of NF-κB and its DNA binding activity. Downregulation of Ku70 and NF-κB-mediated suppression of XIAP further insures the signs of cell death. In summary, our present results indicate that morusin induces cell death in human colorectal cancer HT-29 cells via caspases and NF-κB pathway.
Keywords: Morusin, human colorectal cancer, apoptosis, caspases, itochondria, cytochrome c, Smac/DIABLO, Bcl-2, XIAP, PKCs, Akt/PI3K, NF-κB
二、緣由與目的：
Human colorectal cancer is one of the most common malignant tumors which causes major public health problems in western countries (1) and in Taiwan. Development of colon cancer is a multiple process (2) and the activation of oncogene K-ras is an early event (3-5). A drug called 5-fluorouracil is the most widely used medicine in chemotherapy of human colon cancer (6, 7). However, the therapeutic response of this medicine is limited at 15% in advanced human colon cancer.
Therefore, treatment concentrates on chemoprevention in at-risk and chemotherapy for the inhibition of human colorectal cancer is very meaningful. An important aim in development of cancer therapeutic agents is to lower their side effects, thus led us to search natural products from Chinese traditional herbs for potential anticancer agents. Flavonoids, naturally plant prodcts, have referred as mild tender drugs. They are phenolic plant pigments and are considered to have many pharmacological functions
including anti-inflammatory, antioxidants and antitumor activity (8,
9). Among these actions, the antitumor
effect may be mediated by the different cell cycle arrest (10) and the induction of apoptosis in tumor cells (11).
Morusin, a natural product, isolated from the root bark of Morus australis (Moraceae) that has been used as a traditional Taiwanese folk remedy for treatment human cancer. Morusin is a prenylated flavonoids (chemical entities have an isoprenyl) containing a flavonoid backbone structure (12).
Previous report had shown that morusin significantly inhibits arachidonic acid-, PAF-, and collagen-induced platelet aggregation (13). Notably, no study on the antitumor activity of morusin has been reported. Therefore, we will focus on exploring the antitumor activity and molecular mechanism of morusin in human colorectal cancer.
Apoptosis, programmed cell death, is a regulated process involving activation of molecular episode and induction of cell death characterized by morphological changes, DNA fragmentation, phosphatidylserine externalization, and generation of apoptotic bodies (14-19). The apoptotic body is engulfed by phagocyte and leads little or no inflammation (20). Apoptotic cell death occurs at an execution phase characterized by condensation and fragmentation of nuclear chromatin with internucleosomal cleavage of DNA (21).
The induction of apoptosis is a potentially promising approach for cancer therapy (22). Apoptosis can be induced by stimuli including radiation, hyperthermia, and chemotherapeutic agents including etoposide, adriamycin, mitoxanthrone, cisplatin, and 5-fluorouracil (5-FU) (14, 16, 23-26).
Apoptosis is carried out through the activation of intracellular cysteine protease known as caspase (27, 28).
Caspases are expressed as precursors activated in a cascade following a pro-apoptotic stimulus (28). Notably,
Lassus et al. reported that caspase-2 is required for chemotherapeutic agents-induced apoptosis in human cancer cell lines (29, 30), and the activation of this apical caspase-2 is required for translocation of Bax to the mitochondria and for the releases of cytochrome c and Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP binding protein with low pI) (29, 30). Caspase-9 activation requires binding of the precursor to a complex of two proteins, Apaf-1 and cytochrome c, and is dependent on the hydrolysis of dATP or ATP (31-33). Activated Caspase-9 can further activate the executioner caspases including caspase-3 and caspase-7 that promote apoptosis by cleaving cellular substrates (34-36). The substrates of caspase-3 include poly (ADP-ribose) polymerase (PARP) (37, 38), DNA-dependent protein kinase (39, 40) and DFF45 (41). Recent studies have also indicated that apoptotic DNA fragmentation and associated nuclear changes are largely attributable to a Mr 40,000 nuclease termed CAD3 also known as CPAN or DFF40 (41-43), which functions as a nuclease and degrades nucleosomal DNA (28). In nonapoptotic cells, CAD/CPAN/DFF40 remains inactive because it is bound to its natural inhibitor ICAD (43). ICAD can be expressed as two isoforms, a Mr 45,000 isoform (ICAD-L/DFF45) and a Mr 35,000 isoform (ICAD-S/DFF35), caused by alternative splicing (41, 44).
Both ICAD-L/DFF45 and ICAD-S/DFF35 can bind and inhibit CAD/CPAN/DFF40 (22, 41, 42).
The inhibitor of apoptosis IAP gene family encoded proteins appear as a key intrinsic inhibitors of the caspase cascade, and represent decisive regulatory factors in apoptosis signaling (34, 45, 46). X-linked inhibitor of apoptosis (XIAP; also known as MIHA,
ILP) inhibits mature caspase-9 and caspase-3, respectively (47-49). The caspase-inhibiting effects of XIAP are antagonized by another apoptosis promoting mitochondrial protein, called Smac/DIABLO that synthesized in the cytoplasm and is targeted to the intermembrane space of mitochondria (50, 51). Smac/DIABLO competes with the small subunit of caspase-9 for XIAP, and tears XIAP away to relieve their inhibitory effect on both initiator and effector caspases, while cytochrome c stimulates downstream caspase cascades (50-55).
The Bcl-2 family of proteins is a critical regulator of apoptosis that can either inhibit (Bcl-2 and Bcl-XL) or promot (Bax and Bad) apoptosis (56-58).
The mitochondria-mediated apoptosis is initiated by the release of Smac/DIABLO and cytochrome c into the cytosol and this process is control by Bcl-2 family members (28 review ref).
The location of Bcl-2 on the outer membrane of mitochondria raises the possibility that its function may be related to the function of mitochondria, which have been implicated in apoptosis (59-63). Previous studies accumulating evidences demonstrated that the mitochondria plays an essential role in many forms of apoptosis (64) by releasing apoptotic factors, such as cytochrome c (65, 66), apoptosis-inducing factor (AIF) (67), Smac/DIABLO (50, 51) from the intermembrane space of mitochondria into the cytosol, which activate the downstream executional phase of apoptosis. Mitochondrial membrane depolarization is an early event of apoptosis, and the overexpression of antiapoptotic Bcl-2 family prevents this event (62) (63, 68). Both Bcl-2 and Bcl-XL block all of the changes in mitochondria including mitochondrial membrane potential loss, and the
releases of Smac/DIABLO and cytochrome c (62, 69-71). Recombinant Bax is reported to induce cytochrome c release and mitochondrial membrane potential losses (69, 70). This effect can be inhibited by cyclosporin A (a permeability transition pore inhibitor) that targets mitochondrial cyclophilin D (72). The cyclosporin A-sensitive permeability transition (PT) pore has been implicated in Bax/Bak-mediated cytochrome c releases and mitochondrial membrane potential losses (64).
Nuclear factor-κB (NF-κB), a mammalian transcription factor, is pivotal in regulation of many genes involved in immune system, inflammatory responses, tumour development, as well as in control of cell proliferation and apoptosis (73-78).
An inactive NF-κB resides in the cytoplasm bound to an inhibitory protein known as IκB (73). Activation of NF-κB is trigged by extracellular stimuli, and then IκBα is phosphorylated and proteolytically processed by proteasomes and other proteases (79). This proteolytic process allows NF-κB translocate from cytosol to the nucleus, and therefore bind to the promoter region of the target gene (80).
Recent work provides direct evidence that NF-κB mediates a critical antiapoptotic signal and leads to rescue cells from apoptosis, and therefore contribute to oncogenesis (75, 81).
Other lately evidence showed that the activation of NF-κB is through Ras and phosphatidylinositol-3-kinase (PI3-K) involving activation of Akt and IKK (82). Phosphorylation and degration of IκBα, and the activation of NF-κB result in the translocation of NF-κB from cytosol to the nucleus. These processes induce NF-κB DNA-binding activity(83, 84). Moreover, current reports indicate clearly that
NF-κB-dependent transcriptional activation of cIAP1 and cIAP2 (inhibitor of apoptosis protein) play a pivotal role in regulating apoptosis, and directly inhibit the activity of caspase 3, 7 and 9 (45, 85, 86). There are several evidences indicating that PKC and Akt kinase are crucial factors for cell death or survival in vivo (87, 88) and in vitro (89-91), and these two kinases are able to phosphorylate Bad protein (92-94).
The phospho-Bad can interact with 14-3-3 protein and restrict in inhibition of mitochondrially located Bcl-2 family and inhibits its function (72, 95, 96).
Our preliminary study demonstrated that the concentration of 50% inhibition (IC50) of morusin on human colorectal cancer HT-29 cell growth was 4.76 µM (Table 1). The inhibiting efficacy of colony formation on HT-29 cell was evaluated at 14 days post-treatment of PF-C at various concentrations. As shown in Table 1, the IC50 value for colony formation was 1.9 µM. Moreover, susceptibility of the cells to morusin may be caused by apoptosis, which was demonstrated by increase of Sub-G1 content and DNA fragmentation. Therefore, the specific goal of the present study is to obtain new insights into the antitumor activity and signaling pathways of morusin on human colorectal cancer HT-29 cells in vitro and in vivo. We will continue our preliminary data, morusin-induced apoptosis in human colorectal cancer HT-29 cells will be investigated to conform again. Hypodiploid DNA will be determined by flow cytometry and agarose gel eletrophoresis by ladder pattern of DNA fragments. The phosphatidylserine export to the outer leaflet of plasma membrane of morusin-treated HT-29 cells will also be investigated by flow cytometry. In addition, morusin induced phenotypic changes in HT-29 cell nucleus will be
stained with Hoechst 33258 examining by fluorescence microscopy. To test if morusin-induced apoptosis depends on protein synthesis or gene expression, cells treated with cyclohexamide or cyclosporin A prior addition of morusin will be examined. Furthermore, the cell cycle distribution in morusin treated-HT-29 cells will be analyzed by flow cytometry. The molecular mechanism of morusin induced programmed cell death will be elucidated. In order to investigate if apoptosis is through AIF, cytochrome c, Smac/DIABLO and caspase pathway, AIF, cytochrome c and Smac/DIABLO releases and caspase (such as caspase-2, -8, -9 and –3) activity will be examined by using Western blotting and imaged by laser scan confocal microscopy. To examine the effect of morusin on apoptotic signal pathway, the following apoptotic proteins such as, Bid, Bcl-2, Bcl-XL, Bad, Bak, cleavage PARP and DFF45 will be determined. Since, multiple mechanisms of action are reported to involve in the regulation of cell death for almost all anticancer agents, to determine if the observed antitumor effects involving the suppression of NF-κB activity, PKC and XIAP expression become important.
The total, cytosol, cell membrane and nuclear fractions of proteins will be extracted. We will also examine if PKC, Akt/PI3K and NF-κB could underlie the cytotoxic effects of morusin in HT-29 cells. To investigate whether PI3K/Akt inhibition is required for morusin-induced apoptosis in HT-29 cells, we will perform Western blot analysis for PI3K and phospho-Akt expression. To test the possibility that NF-κB will involve in morusin-induced apoptosis in HT-29 cells, we will determine the nuclear extracts from morusin-treated HT-29 cells. The NF-κB nuclear translocation will be
examined by Western blot, EMSA and confocal microscopy image analysis.
Moreover, we will also investigate upstream signaling of NF-κB pathway including NIK, IKK and IkB protein expression. Finally, the therapy of morusin on colorectal cancer cell implanted severe combined immunodeficiency (SCID) mice will be examined. These results of this study will help us to unveil the effects of morusin on human colorectal cancer cells and provide an alternative therapy for this disease.
三、結果與討論：
Morusin changes ∆ψm and affects mitochondria-related protein expression in HT-29 cells. Apoptotic death signal can proceed via caspases and mitochondria, and the polarization of mitochondrial membrane can be regulated by the members of Bcl-2 family (27, 64, 97). To examine whether the Bcl-2 family proteins participate in morusin-induced apoptosis of human colorectal cancer, subcellular fractions of HT-29 cells were subjected to immunoblotting. As shown in Fig. 1, the expression of pro-apoptotic Bid decreased in the cytosol while its activated form of truncated Bid (tBid) increased in the mitochondria. Translocation of another pro-apoptotic protein, Bax, from cytosol to mitochondria was also revealed at 12-72 h (Fig. 1). Significant decrease in anti-apoptotic Bcl-XL was displayed in mitochondria at 48 h and reached the lowest level (60% decrease) at 72 h (Fig. 1).
However, total expression of Bcl-XL was not affected (Fig. 1). Ku70 is another crucial factor for apoptosis (98).
Downregulation of Ku70 has been reported to enhance Bax-mediated apoptosis, whereas overexpression of Ku70 inhibits the process (99, 100). In Fig. 2, decrease in the level of total and cytosolic Ku70 was exhibited at 48 h, and the expression of the cytosolic Ku70 was almost undetected at 72 h. However, the nuclear expression of Ku70 remained the same (Fig. 2).
To ascertain the role of mitochondria in
morusin-treated HT-29 cells, ∆ψm was evaluated by staining with rhodamine 123 and then analyzed by flow cytometry. As shown in Fig. 3, time-related increase in the percentage of cells with damaged mitochondria was observed, ranging from 2.4% at time zero to 77.4% at 72 h after treatment of 28.6 µM morusin. Change of
∆ψm has been reported to increase the permeability of outer mitochondrial membrane which allows efflux of apoptogenic proteins to the cytosol (97). To address this, the release of cytochrome c and Smac/DIABLO from mitochondria to cytosol was determined by immunoblotting.
As shown in Fig. 4, increase of cytochrome c and Smac/DIABLO in cytosol was first observed at 12 h and reached the highest level at 72 h. As expected, the expression of mitochondrial cytochrome c and Smac/DIABLO decreased with time (Fig.
4). Cytosolic Smac/DIABLO has been reported to bind XIAP and neutralizes its anti-apoptotic activity (101). In this study, decrease in total XIAP was first detected at 24 h and reached the lowest at 72 h in morusin-treated HT-29 cells (Fig. 4).
Apoptosis can be regulated by members of caspase in which caspase-8 and -9 are initiator caspases and caspase-3 is classified into an effecter caspase (27, 102). As shown in Fig. 5, morusin significantly increased the cleavage form of activated caspase-8 at 12 h of treatment, and this activation status sustained to 72 h. Increase in the activation form of cleaved caspase-9 and caspase-3 was also observed at 12-72 h (Fig. 5).
Morusin decreases NF-κB activity in HT-29 cells. NF-κB is a transcription factor, and has been shown to promote cell survival by upregulation of XIAP expression (103, 104). In the present study, significantly decrease in total XIAP protein expression was displayed, which implies the involvement of NF-κB in morusin-induced apoptosis. To ascertain the role NF-κB, nuclear translocation of NF-κB in HT-29 cells was determined by western blotting. As shown in Fig. 6, morusin dramatically decreased the nuclear NF-κB p65 subunit at 3 h of treatment and reached
the lowest level at 6-24 h. Consistently, the significantly decrease in DNA-binding activity of nuclear NF-κB was also detected by EMSA at 3 h (Fig. 7). The NF-κB band was confirmed by observing the elimination of NF-κB band in the presence of unlabeled κB (Fig. 7, lane 8). Unlabeled mutant κB had no effect (Fig. 7, lane 7). The involvement of NF-κB subunits were also confirmed by showing the suppression of NF-κB bands in the presence of anti-p50 and/or anti-p65 antibodies (Fig. 7, lane 9-11). To characterize the signaling pathway in the inhibition of NF-κB activity in morusin-treated HT-29 cells, change of its upstream regulators were determined. As shown in Fig. 8, decrease in IκB-α phosphorylation and increase in total IκB-α protein expression was observed.
Furthermore, morusin significantly decreased the phosphorylation and total protein expression of both IKK-α and IKK-β (Fig. 8). These results suggest that downregulation of NF-κB activity is involved in human colorectal carcinoma HT-29 cells through the dephosphorylation of IKK-α, IKK-β and IκB-α, and augment of IκB-α and reduction of IKK-α and IKK-β protein expression.
The role of NF-κB in drug resistance has been reported (105). NF-κB activity is elevated in colorectal cancer cells (106, 107). Administration of NF-κB inhibitor enhances the tumoricidal response of the cells (105, 108). 5-fluorouracil (5-FU) is the most common and effective chemotherapeutic agents for the treatment of colorectal cancer (109). However, colon tumors are inherently resistant or develop resistance during treatment with 5-FU (110). Recently, a major mechanism for inherent and inducible chemoresistance has been suggested to associate with activation of NF-κB (111, 112). In the present study, morusin not only induces apoptosis but significantly suppresses NF-κB activity in HT-29 cells. The results suggests that given morusin in conjunction with 5-FU may potentiate the effect of 5-FU in patients with colorectal cancer.
In conclusion, moruisn suppresses the
growth of human colorectal cancer HT-29 cells. Inhibition of constitutive NF- B activity and induction of caspase-mediated apoptosis is exhibited. Morusin decreases the activity of p-IKK-α/p-IKK-β, p-IκB-α and then NF-κB in the cells.
Activated caspase-8 causes the increase in mitochondrial tBid and Bax for the release of cytochrome c and Smac/DIABLO, and thus for the activation of caspase-9 and caspase-3 to promote the process programmed cell death.
四、文獻：
1. S. H. Landis, T. Murray, S. Bolden, P. A. Wingo, CA Cancer J Clin 49, 8 (Jan-Feb, 1999).
2. M. E. Martinez et al., Cancer Res 59, 5181 (1999).
3. B. Vogelstein et al., N Engl J Med 319, 525 (1988).
4. S. R. Hamilton, Cancer 70, 1216 (1992).
5. K. Okumura, S. Shirasawa, M.
Nishioka, T. Sasazuki, Cancer Res 59, 2445 (1999).
6. H. M. Pinedo, G. F. Peters, J Clin Oncol 6, 1653 (Oct, 1988).
7. P. J. Loehrer, Sr. et al., J Clin Oncol 6, 642 (Apr, 1988).
8. J. Kuhnau, World Rev Nutr Diet 24, 117 (1976).
9. E. S. Fiala, B. S. Reddy, J. H.
Weisburger, Annu Rev Nutr 5, 295 (1985).
10. F. Sato, Y. Matsukawa, K.
Matsumoto, H. Nishino, T. Sakai, Biochem. Biophys. Res. Commun.
204, 578 (1994).
11. Y. Q. Wei et al., Cancer Res. 54, 4952 (1994).
12. Y. S. Chi et al., Biochem.
Pharmacol. 62, 1185 (2001).
13. H. H. Ko, S. M. Yu, F. N. Ko, C. M.
Teng, C. N. Lin, J. Nat. Prod. 60, 1008 (1997).
14. A. Eastman, Cancer Cells 2, 275 (Aug-Sep, 1990).
15. I. Nicoletti, G. Migliorati, M. C.
Pagliacci, F. Grignani, C. Riccardi, J Immunol Methods 139, 271 (Jun 3, 1991).
16. D. L. Evans, C. Dive, Cancer Res 53, 2133 (May 1, 1993).
17. V. A. Fadok, J. Immunol. 148, 2207 (1992).
18. S. J. Martin et al., J. Exp. Med. 182, 1545 (Nov 1, 1995).
19. V. A. Fadok et al., Nature 405, 85 (May 4, 2000).
20. P. Saikumar et al., Am J Med 107, 489 (Nov, 1999).
21. S. H. Kaufmann, W. C. Earnshaw, Exp Cell Res 256, 42 (2000).
22. N. Ahmad, P. Cheng, H. Mukhtar, Biochemical & Biophysical
Research Communications 275, 328 (2000).
23. K. S. Sellins, J. J. Cohen, Radiat Res 126, 88 (Apr, 1991).
24. J. A. Hickman, Cancer Metastasis Rev 11, 121 (Sep, 1992).
25. R. L. Warters, Cancer Res 52, 883 (Feb 15, 1992).
26. M. Watabe, K. Machida, H. Osada, Cancer Res. 60, 5214 (2000).
27. M. Mancini et al., J. Cell. Biol. 140, 1485 (1998).
28. Y. Shi, Nat. Struct. Biol. 8, 394 (2001).
29. P. Lassus, X. Opitz-Araya, Y.
Lazebnik, Science 297, 1352 (Aug 23, 2002).
30. S. Kumar, D. L. Vaux, Science 297, 1290 (Aug 23, 2002).
31. E. R. Adair, B. W. Adams, S. A.
Kelleher, J. W. Streett, Annals of the New York Academy of Sciences 813, 497 (1997).
32. Y. Hu, M. A. Benedict, L. Ding, G.
Nunez, Embo J 18, 3586 (1999).
33. S. M. Srinivasula, M. Ahmad, T.
Fernandes-Alnemri, E. S. Alnemri, Mol Cell 1, 949 (1998).
34. Q. L. Deveraux, R. Takahashi, G. S.
Salvesen, J. C. Reed, Nature 388, 300 (Jul 17, 1997).
35. Q. L. Deveraux et al., Embo J 17, 2215 (1998).
36. Y. Deng, Y. Lin, X. Wu, Genes Dev.
16, 33 (2002).
37. A. J. Darmon, D. W. Nicholson, R.
C. Bleackley, Nature 377, 446 (1995).
38. A. M. Chinnaiyan, K. O'Rourke, M.
Tewari, V. M. Dixit, Cell 81, 505 (1995).
39. L. Casciola-Rosen et al., Journal of Experimental Medicine 183, 1957 (1996).
40. Z. Song, K. McCall, H. Steller, Science 275, 536 (1997).
41. M. Enari et al., Nature 391, 43 (Jan 1, 1998).
42. R. Halenbeck et al., Curr. Biol. 8, 537 (1998).
43. M. Y. Chang, S. J. Won, H. S. Liu, J. Cancer Res. Clin. Oncol. 123, 91 (1997).
44. J. Gu et al., J Biol Chem 274, 20759 (1999).
45. Q. L. Deveraux et al., Embo J 18, 5242 (1999).
46. L. K. Miller, Trends Cell Biol 9, 323 (1999).
47. R. Takahashi et al., J. Biol. Chem.
273:, 7787 (1998).
48. C. Sun et al., Nature 401, 818 (1999).
49. C. C. Chen, Y. T. Sun, J. J. Chen, K.
T. Chiu, Journal of Immunology 165, 2719 (2000).
50. D. S. Adams, R. Nathans, S. C. Pero, A. Sen, E. Wakshull, Journal of Cellular Biochemistry 77, 221 (2000).
51. A. M. Verhagen et al., Cell 102, 43 (Jul 7, 2000).
52. S. M. Srinivasula et al., J Biol Chem 275, 36152 (2000).
53. J. Chai et al., Nature 406, 855 (2000).
54. Z. Liu et al., Nature 408:, 1004 (2000).
55. G. Wu et al., Nature 408, 1008 (2000).
56. T. P. Misko et al., Eur J Pharmacol 233, 119 (1993).
57. G. Kroemer, Nat Med 3, 614 (Jun, 1997).
58. K. F. Ferri, G. Kroemer, Nat. Cell.
Biol. 3, E255 (Nov, 2001).
59. P. Monaghan et al., J Histochem Cytochem 40, 1819 (1992).
60. M. I. Walton et al., Cancer Res. 53, 1853 (1993).
61. D. D. Newmeyer, D. M. Farschon, J.
C. Reed, Cell 79, 353 (1994).
62. P. Marchetti et al., J Exp Med 184, 1155 (1996).
63. P. Marchetti et al., Cancer Res 56, 2033 (1996).
64. G. P. Amarante-Mendes et al., Cell Death Differ 5, 298 (1998).
65. M. S. Chaussee, J. Liu, D. L.
Stevens, J. J. Ferretti, Journal of Infectious Diseases 173, 901 (1996).
66. R. M. Kluck, E. Bossy-Wetzel, D. R.
Green, D. D. Newmeyer, Science 275, 1132 (1997).
67. H. K. Lorenzo, S. A. Susin, J.
Penninger, G. Kroemer, Cell Death Differ 6, 516 (Jun, 1999).
68. S. A. Susin et al., J Exp Med 184, 1331 (1996).
69. J. M. Jurgensmeier et al., Proc Natl Acad Sci U S A 95, 4997 (1998).
70. M. Narita et al., Proc Natl Acad Sci U S A 95, 14681 (1998).
71. W. Roth, J. C. Reed, Nat. Med. 8, 216 (2002).
72. S. Desagher, J. C. Martinou, Trends Cell Biol 10, 369 (Sep, 2000).
73. P. A. Baeuerle, D. Baltimore, Cell 87, 13 (Oct 4, 1996).
74. Anonymous, MMWR - Morbidity &
Mortality Weekly Report 47, 877 (1998).
75. S. Y. Foo, G. P. Nolan, Trends Genet 15, 229 (Jun, 1999).
76. T. S. Finco, A. S. Baldwin, Immunity 3, 263 (Sep, 1995).
77. F. Chen, V. Castranova, X. Shi, L.
M. Demers, Clinical Chemistry 45, 7 (1999).
78. K. M. Ryan, M. K. Ernst, N. R. Rice, K. H. Vousden, Nature 404, 892 (Apr 20, 2000).
79. V. Imbert et al., Cell 86, 787 (Sep 6, 1996).
80. M. el Bour et al., Medecine Tropicale 53, 13 (1993).
81. K. G. Waddick, F. M. Uckun, Biochem. Pharmacol. 57, 9 (Jan 1, 1999).
82. R. C. Bates, N. S. Edwards, G. F.
Burns, D. E. Fisher, Cancer Res. 61, 5275 (2001).
83. O. N. Ozes et al., Nature 401, 82 (Sep 2, 1999).
84. J. A. Romashkova, S. S. Makarov, Nature 401, 86 (1999).
85. E. C. LaCasse, S. Baird, R. G.
Korneluk, A. E. MacKenzie,
Oncogene 17, 3247 (Dec 24, 1998).
86. M. Jaattela, Exp Cell Res 248, 30 (Apr 10, 1999).
87. H. Hara, H. Onodera, M. Yoshidomi, Y. Matsuda, K. Kogure, J Cereb Blood Flow Metab 10, 646 (1990).
88. Y. B. Ouyang et al., J Cereb Blood Flow Metab 19, 1126 (1999).
89. M. M. Behrens, J. L. Martinez, C.
Moratilla, J. Renart, Cell Growth Differ 6, 1375 (1995).
90. R. Yao, G. M. Cooper, Science 267, 2003 (1995).
91. A. Bronisz-Kowalczyk, L.
Buzanska, D. Kowalczyk, K.
Domanska-Janik, Folia Neuropathol 38, 13 (2000).
92. Y. Tan, H. Ruan, M. R. Demeter, M.
J. Comb, J Biol Chem 274, 34859 (1999).
93. J. Zhong, J. Troppmair, U. R. Rapp, Oncogene 20, 4807 (2001).
94. S. R. Datta et al., Cell 91, 231 (1997).
95. E. Yang et al., Cell 80, 285 (Jan 27, 1995).
96. A. Gross, J. M. McDonnell, S. J.
Korsmeyer, Genes Dev. 13, 1899 (1999).
97. J. M. Adams, Genes Dev 17, 2481 (Oct 15, 2003).
98. F. Mancinelli, M. Caraglia, A.
Budillon, A. Abbruzzese, E.
Bismuto, J Cell Biochem (Apr 17, 2006).
99. S. F. Nothwehr, J. C. Martinou, Nat Cell Biol 5, 281 (Apr, 2003).
100. M. Sawada et al., Nat Cell Biol 5, 320 (Apr, 2003).
101. S. Chung, T. L. Gumienny, M. O.
Hengartner, M. Driscoll, Nature Cell Biology 2, 931 (2000).
102. K. G. Adcock, R. L. Akins, E. A.
Farrington, Pharmacotherapy 19, 1315 (1999).
103. C. Belka et al., Int J Radiat Oncol
Biol Phys 58, 542 (Feb 1, 2004).
104. R. P. Brandes, J. Kreuzer, Cardiovasc Res 65, 16 (Jan 1, 2005).
105. J. C. Cusack, Jr., R. Liu, A. S.
Baldwin, Jr., Cancer Res 60, 2323 (May 1, 2000).
106. M. C. Wei et al., Science 292, 727 (2001).
107. J. C. Hardwick, G. R. van den Brink, G. J. Offerhaus, S. J. van Deventer, M. P. Peppelenbosch, Oncogene 20, 819 (Feb 15, 2001).
108. J. C. Cusack, Jr. et al., Cancer Res 61, 3535 (May 1, 2001).
109. N. C. Tebbutt, E. Cattell, R.
Midgley, D. Cunningham, D. Kerr, Eur J Cancer 38, 1000 (May, 2002).
110. N. Baldini, Nat Med 3, 378 (Apr, 1997).
111. X. Jiang et al., Science 299, 223 (Jan 10, 2003).
112. R. Voboril et al., J Surg Res 120, 178 (Aug, 2004).
11
Table 1
The IC50 effect of morusin on cell growth and colony formation on human tumor cells
and human PBMCs
Cell lines Growth inhibition (µM/ml) Clonogenic cell inhibition (µM/ml)
HT- 29 6.1 ± 0.3 2.8±0.1
HCT 116 7.2 ± 0.8 3.6±0.9
Hep 3B 8.5 ± 1.1ab 9.2 ± 1.2
MCF-7 12.7± 2.1 10.3 ± 1.6
HEK293 23.9±1.1 PBMC 29.8±0.1
a Control cells were treated with DMEM. Experiments were repeated three times
and each concentration of samples was conducted in eight replicates.
b The experimental data are expressed as mean ± SEM.
12
Fig. 1
RACK1
Cytosolic Bid 22
Mitochondrial tBid 15
C 12 24 48 72
Time (h) kDa
Morusin (28.6 µM)
23 Cytosolic Bax
RACK1
23
Mitochondrial Bax
32 Total Bcl-XL
RACK1
32 Mitochondrial Bcl-XL
RACK1
RACK1
RACK1
Cytosolic Bid 22
Cytosolic Bid
Cytosolic Bid 22
Mitochondrial tBid 15
Mitochondrial tBid
Mitochondrial tBid 15
C 12 24 48 72
Time (h) kDa
Morusin (28.6 µM)
23
Cytosolic Bax 23
Cytosolic Bax Cytosolic Bax
RACK1 RACK1
23
Mitochondrial Bax 23
Mitochondrial Bax Mitochondrial Bax
32
Total Bcl-XL 32
Total Bcl-XL
RACK1 RACK1 RACK1
32
Mitochondrial Bcl-XL 32
Mitochondrial Bcl-XL
13
Fig. 2
Morusin (28.6 µM)
C 12 24 48 72
Time (h) kDa
70 Total Ku70
Fold of C 1 0.9 0.8 0.5 0.3
70 Cytosolic Ku70
Fold of C 1 0.9 1.2 0.6 0.2
Nuclear Ku70 70
Fold of C 1 1.2 1.1 1.1 1.2 Morusin (28.6 µM)
C 12 24 48 72
Time (h) kDa
70
Total Ku70 70
Total Ku70
Total Ku70
Fold of C 1 0.9 0.8 0.5 0.3
70
Cytosolic Ku70 70
Cytosolic Ku70
Cytosolic Ku70
Fold of C 1 0.9 1.2 0.6 0.2
Nuclear Ku70 70
Nuclear Ku70
Nuclear Ku70 70
Fold of C 1 1.2 1.1 1.1 1.2
14
Fig. 3
Time (h)
C 12 24 48 72
Cell number
Morusin (28.6 µM)
2.4 ± 0.7% 18.6 ± 1.5% 22.6 ± 2.4% 36.0 ± 4.2% 66.2 ± 4.3% 77.4 ± 6.6%
6
24 h
C 9.5 19.1 28.6 38.1
4.1 ± 1.1% 12.8 ± 2.4% 21.4 ± 3.7% 39.9 ± 3.6% 67.1 ± 7.5%
Fluorescence intensity
(µM)
Time (h)
C 12 24 48 72
Cell number
Morusin (28.6 µM)
2.4 ± 0.7% 18.6 ± 1.5% 22.6 ± 2.4% 36.0 ± 4.2% 66.2 ± 4.3% 77.4 ± 6.6%
6
24 h
C 9.5 19.1 28.6 38.1
4.1 ± 1.1% 12.8 ± 2.4% 21.4 ± 3.7% 39.9 ± 3.6% 67.1 ± 7.5%
Fluorescence intensity
(µM)
15
Fig. 4 Morusin (28.6 µM)
C 12 24 48 72
Time (h) kDa
RACK1
15
Cytosolic cytochrome c
15 Mitochondrial cytochrome c
25 Cytosolic Smac/DIABLO
RACK1
25 Mitochondrial Smac/DIABLO
57 Total XIAP
Morusin (28.6 µM)
C 12 24 48 72
Time (h) kDa
RACK1
RACK1
15
Cytosolic cytochrome c 15
Cytosolic cytochrome c Cytosolic cytochrome c
15
Mitochondrial cytochrome c 15
Mitochondrial cytochrome c
25 Cytosolic Smac/DIABLO
RACK1 RACK1 RACK1
25 Mitochondrial Smac/DIABLO
57 Total XIAP
16
Fig. 5
C 12 24 48 72
Time (h)
Morusin (28.6 µM)
kDa
RACK1
46
3735 Cleaved caspase-9
Caspase-9
17
12 RACK1
32
Cleaved caspase-3 Caspase-3
4143
57 Caspase-8
Cleaved caspase-8
RACK1
C 12 24 48 72
Time (h)
Morusin (28.6 µM)
kDa
RACK1 RACK1 RACK1
46
3735 Cleaved caspase-9
Caspase-9
17
12
17
12 RACK1
RACK1
32
Cleaved caspase-3 Caspase-3
4143
57 Caspase-8
Cleaved caspase-8
RACK1 RACK1
17
Fig. 6
0 3 6 12 24
Morusin (28.6 µM)
Time (h) kDa
NF-κB p65 65
Nuclear Ku70
Fold of C 1 0.44 0.13 0.12 0.04
0 3 6 12 24
Morusin (28.6 µM)
Time (h) kDa
NF-κB p65 65
NF-κB p65 65
Nuclear Ku70
Nuclear Ku70
Nuclear Ku70
Fold of C 1 0.44 0.13 0.12 0.04
18
Fig. 7
1 2 3 4 5 6 7 8 9 10
κB
Probe alone 24
12
6 αp50 αp65 αp50+p65 Time(h)
Morusin(28.6 µM) 3
NF-κB
Mutant
11 0
Free probe
Lane
Fold of C 1 0.4 0.1 0.4 0.1 0.9 0 0.7 0.4 0.2
1 2 3 4 5 6 7 8 9 10
κB
Probe alone 24
12
6 αp50 αp65 αp50+p65 Time(h)
Morusin(28.6 µM) Morusin(28.6 µM)
3
NF-κB
Mutant
11 0
Free probe Free probe Lane
Fold of C 1 0.4 0.1 0.4 0.1 0.9 0 0.7 0.4 0.2
19
Fig. 8
kDa
0 1 2 3 6
Morusin (28.6 µM) Time (h)
37 p-IκB-α
RACK1
85 p-IKK-β 87
p-IKK-α
RACK1 IκB-α 37
kDa
0 1 2 3 6
Morusin (28.6 µM) Time (h)
37
p-IκB-α 37
p-IκB-α
RACK1 RACK1 RACK1
85 p-IKK-β 87
p-IKK-α
RACK1
85 p-IKK-β 87
p-IKK-α 85
p-IKK-β 87 p-IKK-β
p-IKK-α
p-IKK-α
RACK1 RACK1 RACK1
IκB-α 3737
IκB-α
