Morusin 抗人類大腸直腸癌之分子訊息作用機制(1/2)

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行政院國家科學委員會專題研究計畫期中進度報告

計畫類別：個別型計畫

計畫編號： NSC93-2314-B-006-106-

執行期間： 93 年 08 月 01 日至 94 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：精簡報告

處理方式：本計畫可公開查詢

中華民國 94 年 5 月 6 日

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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(1/2) ※

※※※※※※※※※※※※※※※※※※※※※※※※※※※

計畫類別：■個別型計畫 □整合型計畫計畫編號：NSC 93-2314-B-006-106

執行期間：93 年 08 月 01 日至 94 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：國立成功大學醫學系外科

中華民國 94 年 05 月 1 日

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Morusin 抗人類大腸直腸癌之分子訊息作用機制(1/2)

Antitumor molecular signaling mechanism of Morusin in Human colorectal cancer cells

計畫編號：NSC 92-2314-B-006-106

執行期限：93 年 08 月 01 日至 94 年 07 月 31 日主持人：李政昌國立成功大學醫學系外科

一、中文摘要

在台灣南部，由傳統草藥 M. australis (Moraceae)根部萃取物已被廣泛用在治療腫 瘤疾病，而且普遍施用在人類大腸直腸癌的

治療。不過其確實的機制卻不甚明暸。Morusin

是由 M. australis (Moraceae)的根部純化出的 成份，具有引發癌細胞進行細胞凋亡的效

果。然而morusin 抑制癌細胞生長及促進細胞

死亡的機制卻不甚明暸。大腸直腸癌不論在男女都是引起死亡的主要原因之一，在西方國家和在台灣，已經成為主要的公眾健康問題。目前的研究的具體的目標是去探究 morusin 其抗大腸直腸癌的抗癌機制，進一步

了解 morusin 是否能被用作癌症化學治療藥

劑。目前的研究結果顯示，在以morusin 處理

HT-29 細胞 6 天後，其 50%抑制濃度(IC50)為 5.95 µM，而以 morusin 處理 PBMC 細胞，其 IC50為 29.75 µM。而以軟洋菜膠分析 morusin

抑制HT-29 細胞的細胞群落形成，14 天後其

IC50為 2.76 µM。在以 morusin 處理 HT-29 細胞後，可以發現細胞膜內側的磷脂絲胺酸翻轉至細胞膜外側、DNA 片段化現象、細胞核

型態的改變，以及細胞的 sub-G1 期 DNA 增

加的情形，由以上結果證實morusin 經誘發細

胞凋亡而抑制HT-29 細胞生長。morusin 誘發 HT-29 細胞進行細胞凋亡的分子機制與活化 caspase-8、caspase-9 和 caspase-3 有關，並會造成粒線體膜電位喪失。進一步的研究發現

在morusin 的處理後，會使 HT-29 細胞細胞質

中的Ku70 減少。因此第二年的具體目標將更

深入探討morusin 誘發 HT-29 細胞進行細胞凋亡的分子機制，並進一步以rhodamine 123 染

色佐以共軛焦顯微鏡觀察來確認 morusin 誘

發 HT-29 細胞走向細胞凋亡與粒線體有關。

而細胞凋亡的相關蛋白質，包括 caspase-9、

caspase-3、Bid、Bcl-XL、Bax、Bak、cytochrome c、Smac/DIABLO、nuclear cleaved PARP、AIF 和 DFF40 都將探討其與 morusin 誘發 HT-29 細胞走向細胞凋亡的關聯性。另外也將探討

是否有 cofilin 從細胞質轉位到粒線體的情

形。最後，將以HT-29 細胞植入 SCID 老鼠後，

投予morusin 進行動物實驗，以確知其療效。

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

The crude extracts of M. australis (Moraceae) have been used for cancer therapy and are common remedy to treat human colorectal cancer in southern Taiwan. One pure compound, morusin, was isolated from the crude extract of M. australis. However, the action mechanism of morusin against cancer cell growth and cell death are not well understood. Colorectal cancer, one of the leading causes of death in both men and women, has become a major public health

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problem in western countries as well as in Taiwan. The specific goal of the present study is to explore the anti-tumor mechanism of morusin in human colorectal cancer, and to better understand if it can be used as a chemotherapeutic agent for cancer. The present study showed that the IC50 of morusin on the growth of HT-29 cells was 5.95 µM as compared to peripheral blood mononuclear cells was 29.75 µM at day 6 of post-treatment. The inhibitory efficacy of morusin on colony formation of HT-29 in soft agar was 2.76 µM at day 14 of post-treatment. The susceptibility of cells to morusin is 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, and the loss of mitochondrial membrane potential in HT-29 cells. Moreover, the level of Ku70 in the cytoplasm was decreased after treatment of morusin. Therefore, the specific aim of second year will be concentrated on the molecular mechanisms leading to apoptosis induced by morusin in HT-29 cells. To confirm mitochondria involvement in morusin-induced apoptosis, cells will be stained with rhodamine 123 and measured by confocal microscopy.

Apoptotic related proteins including caspase-9, caspase-3, Bid, Bcl-XL, Bax, Bak, cytochrome c, Smac/DIABLO, nuclear cleaved PARP, AIF and DFF40 will be evaluated. Moreover, the translocation of cofilin from cytosol to mitochondria will be determined. Finally, the therapy of morusin on HT-29 cell implanted in SCID mice will be examined.

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

Colorectal cancer, one of the leading causes of death in both men and women, has become a major public health problem in western countries (1) as well as in Taiwan.

Chemotherapeutic agents such as 5-fluorouracil and leucovorin have been widely used

postoperatively in colorectal cancer patients with regional lymph node metastasis in order to reduce the risk of distant metastasis (2).

Moreover, oxaliplatin, irinotecan, or a combination of these drugs with 5-fluorouracil have been used to sustain patients with distant metastasis in order to improve their quality of life and prolong their survival (2). However, colorectal cancer death rates remain unacceptably high and the development of complementary strategies to reduce the burden of this disease is important (3). This fact prompted us to search for potential anticancer agents among natural products of Chinese traditional herbs. Herbaceous plants have been broadly used for food and medicine in China, and have played significant roles in treating diseases and improving the quality of life and maintaining the health of Chinese people for many centuries (4).

The crude extracts of M. australis (Moraceae) have been used for cancer therapy in southern Taiwan. One pure compound comes from the crude extract, morusin, was isolated. The action of morusin against cancer cell growth and cell death are not well understood. In our previous study, morusin can induce apoptosis in HT-29, a colorectal adenocarcinoma cell, by increasing sub-G1 content and DNA fragmentation. The mechanism that morusin triggered apoptosis was mediated by the loss of mitochondria membrane potential and the release of cytochrome c and Smac/DIABLO. Moreover, it can decrease XIAP expression, activate caspase-9 and caspase-3, increase nuclear PARP and DFF40 contents.

Flavonoids, naturally plant products, 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 (5, 6). Among these actions, the antitumor effect may be mediated by the different cell cycle arrest (7) and the induction of apoptosis in tumor cells (8). 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 (9). Previous report had shown that

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morusin significantly inhibits arachidonic acid-, PAF-, and collagen-induced platelet aggregation (10). 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 (11-17). The apoptotic body is engulfed by phagocyte and leads little or no inflammation (18). Apoptotic cell death occurs at an execution phase characterized by condensation and fragmentation of nuclear chromatin with internucleosomal cleavage of DNA (19). The induction of apoptosis is a potentially promising approach for cancer therapy (20). Apoptosis can be induced by stimuli including radiation, hyperthermia, and chemotherapeutic agents including etoposide, adriamycin, mitoxanthrone, cisplatin, and 5-fluorouracil (5-FU) (11) (21, 22) (23) (13, 24). Apoptosis is carried out through the activation of intracellular cysteine protease known as caspase (25, 26). Caspases are expressed as precursors activated in a cascade following a pro-apoptotic stimulus (26, 27-Bid).

Notably, Lassus et al. reported that caspase-2 is required for chemotherapeutic agents-induced apoptosis in human cancer cell lines (28, 29), 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)

(28, 29). 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 (30-32).

Activated Caspase-9 can further activate the executioner caspases including caspase-3 and caspase-7 that promote apoptosis by cleaving cellular substrates (33-35). The substrates of caspase-3 include poly (ADP-ribose) polymerase (PARP) (36, 37), DNA-dependent protein kinase (38, 39) and DFF45 (40). 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 (40-42), which functions as a nuclease and degrades nucleosomal DNA (26). In nonapoptotic cells, CAD/CPAN/DFF40 remains inactive because it is bound to its natural inhibitor ICAD (42). 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 (40, 43). Both ICAD-L/DFF45 and ICAD-S/DFF35 can bind and inhibit CAD/CPAN/DFF40 (40) (40, 41, 44).

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 (33, 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, while cytochrome c stimulates downstream caspase cascades (50) (51-55).

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 (26 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

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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).

NF-κB, 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 (73-76). An inactive NF-κB resides in the cytoplasm bound to an inhibitory protein known as IκB (73).

Activation of NF-κB is trigged by stimuli, and then IκBα is phosphorylated and proteolytically processed by proteasomes (77). This proteolytic process allows NF-κB translocate to the nucleus, and bind to the promoter region of the target gene (78).

The first year data demonstrated that the IC50 of morusin on the growth of HT-29 cells was 5.95 µM as compared to peripheral blood mononuclear cells was 29.75 µM at day 6 of post-treatment. The inhibitory efficacy of morusin on colony formation of HT-29 in soft agar was 2.76 µM at day 14 of post-treatment.

Furthermore, we demonstrated that morusin induced the death of HT-29 cells through an apoptotic pathway involving phosphatidylserine exposure, increase of sub-G1 content, DNA fragmentation, and morphologic change. In addition, the molecular events that occurred in cells treated with morusin, activation of caspase-8, -9 and -3, and loss of mitochondria membrane potential were also observed.

Therefore, the specific aim of the second year study is to obtain new insights of this antitumor activity and signaling pathways involved by morusin on HT-29 cells in vitro and in vivo. We will attempt to continue previous study to test if

morusin-induced apoptosis is through cytochrome c, Smac/DIABLO and XIAP pathway. Cytochrome c and Smac/DIABLO releases from mitochondria to cytosol and XIAP expression will be examined by Western blotting and imaging 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, Bax, Bad, and Bak, and cleavage PARP, AIF and DFF40 in nuclear will be determined.

Furthermore, the effect of morusin on the NF-κB activity in HT-29 cells will be analyzed.

The suppression of NF-κB activity, the nuclear protein will be extracted and determined by Western blotting and EMSA. To investigate the link between Akt and NF-κB, we will determine the effects of morusin in HT-29 cells on Akt phosphorylation at residues of Thr 308 or Ser 473 (prerequisite for catalytic of Akt). 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 and will provide an alternative therapy for this disease.

三、結果與討論

Growth Inhibition by Morusin in human colorectal cancer cells.

The cytotoxic activity of morusin on HT-29 human colorectal cancer cells was examined by MTT assay. Cells were grown in the absence or presence of various concentrations (2.98-23.8 µM) of morusin for 6 days. The 50% inhibitory concentrations (IC50) for HT-29 cells were determined to be 5.95 µM (Fig. 1). A daily evaluation of morusin on growth inhibition of HT-29 cells revealed that morusin inhibited cell growth in a dosage- and time-dependent manner.

At a concentration of 23.8 µM, it inhibited the proliferation of HT-29 cells approximately 23%

at day 1 and 81.8% at day 6 post-treatment (Figure 1).

Induction of apoptosis in colorectal cancer cells by morusin.

To determine whether morusin induced apoptosis, cells were treated with morusin (23.8 µM) and then assayed for signs of apoptosis at 0, 12, 24, 48, and 72 h post-treatment. The treated cells were stained with MC540 and then

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analyzed by flow cytometry. As seen in Figure 2A, the percentage of MC540 positive HT-29 cells was increased with time from 4.9% at time zero to 23.3% at 12 h, and reached 68.7% at 72 h after treatment of morusin. Furthermore, cells were stained with PI, and the relative number of cells with a sub-G1 DNA content was determined by flow cytometry. As seen in Figure 2B, treatment of HT-29 cells with 23.8 µM of morusin increased the population of cells with a sub-G1 DNA content from 1.3% at time zero to 80.5% at the 72 h time point. To further confirm that morusin-treated cells undergo apoptosis, DNA fragmentation and in situ Hoechst 33258 staining in these cells were examined. Treatment with 23.8 µM of morusin for 48 h resulted in DNA fragmentation (Fig. 2C, lane 11). Following 72 h morusin (2.98, 5.95, 11.9, or 23.8 µM) exposure, morusin-treated cells were found to have a certain degree of DNA fragmentation (Fig. 2C, lane 13-16).

Treatment with morusin (23.8 µM) for 24, 48, or 72 h resulted in a gradual increase in the amount of nuclear morphological condensation changes (Fig. 2 D).

Effects on caspases and its target proteins by morusin

The major enzyme involved in apoptosis is caspase-3, activation of caspase-3 in morusin -treated cells was examined. HT-29 cells were treated with 23.8 µM of morusin. At different time points after treatment, cells were assessed for the occurrence of activated caspase-3 by western blotting. The 17-kDa forms of activated caspase-3 were first detected 24 h after morusin treatment in HT-29 cell lines, and more activated caspase-3 was detected with a longer HT-29 cells treatment (Figure 3A). To determine whether morusin-induced apoptosis was mediated by the extrinsic or intrinsic pathway, activation of caspase-8 or caspase-9 was examined. Caspase-9 activation would implicate an intrinsic pathway, whereas caspase-8 activation would suggest the extrinsic pathway (29). The pro-caspase-9 is cleaved to yield two peptides of 35 and 37 kDa when it was activation. Fig. 3A shows that these two activated forms became visible 12 h after treatment in HT-29 cells and sustained the level at the 24-72 h time point. If the caspase-8 is activated, the pro-caspase-8 is cleaved to yield

two subunits 10 and 18 kDa (79). As shown in Fig. 3B, the pro-caspase-8 was significantly decreased during morusin (23.8 µM) treated at 12 h and reached lowest level at 72 h. In contrast, these two activated subunits 10 and 18 kDa became visible within 12 h after treatment in HT-29 cells and reached the highest level at the 72 h time point (Fig. 3B).

Decrease in the level of Ku70 caused by morusin

Ku70 is known to play a crucial role in apoptosis. Down regulation of Ku70 has been shown to enhance Bax-mediated apoptosis (80) (19), whereas overexpression of Ku70 has been shown to inhibit apoptosis (81). To evaluate whether Ku70 was involved in morusin-induced mitochondrial dysfunction in human colorectal cancer cells, the level of the Ku70 protein was determined by immunoblot analysis. As seen in Figure 4, the level of total and cytosolic Ku70 was decreased at 48-72 h in HT-29 cells after treatment with 23.8 µM of 23.8 µM. This result indicated that the Ku70 protein was not involved in early morusin-induced mitochondrial dysfunction in human colorectal cancer cells.

Damage in mitochondria caused by justicidin A treatment

Activation of caspase-8 suggests that morusin triggers apoptosis by the intrinsic pathway which affects mitochondria. Mitochondria play a pivotal role in apoptotic process of the tumor cells induced by chemotherapeutic agents (82-84). Therefore, the change in the membrane potential of mitochondria (∆ψm) in response to morusin treatment was evaluated by flow cytometry after staining the cells with mitochondrial dye Rhodamone123. A time-dependent decrease in the intensity of Rhodamone123 staining was observed in the mitochondria after treatment of 23.8 µM of morusin in HT-29 cells. The decrease in ∆ψm

was first observed at 6 h and reached the lowest level at 72 h after morusin treatment in HT-29 cells (Fig. 5A). Treatment of HT-29 cells with various concentrations of morusin (9.5-38.1 µM) for 24 h resulted in a dose-dependent loss of

∆ψm (Fig. 5A).

In the second year, we will focus on the following studies. Activation of caspase-9 suggests that morusin triggers apoptosis by the

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intrinsic pathway which affects mitochondria.

Therefore, the change in the membrane potential of mitochondria (∆ψm) in response to morusin treatment will be examined by confocal microscopy after staining the cells with mitochondrial dye rhodamine 123. Cytochrome c releases from mitochondria into the cytosol is required for the activation of caspases and led to the fragmentation of DNA (65). The interaction between Bcl-2 proteins and the mitochondrial membrane can regulate the releases of cytochrome c and Smac/DIABLO from mitochondria into the cytosol (35, 71, 85).

Therefore, the release of mitochondiral cytochrome c and Smac/DIABLO into the cytosol will be determined by immunoblot analysis. Moreover, treatment of HT-29 cell lines with Z-VDVAD-fmk or cyclosporin A (a ligand of cyclophilin D) prior to the addition of morusin to block mitochondrial dysfunction will be imaged by confocal microsopy. XIAP, an inhibitor of apoptosis protein, can inhibit cell death by interaction with caspases (33). To determine whether XIAP protein is involved in the action of morusin, cell lysates with vehicle- or morusin will be analyzed by Western blotting.

Other crucial regulators of apoptotic cell death are Bcl-2 family proteins, including Bcl-2 and Bcl-XL, which suppress apoptosis, and Bax and Bak, which promote apoptosis (86-88).

Anti-apoptotic members of the Bcl-2 family can bind to mitochondria and inhibit the release of cytochrome c and Smac/DIABLO (26, 66).To find out whether the levels of death-related proteins in HT-29 cells influenced by morusin-induced apoptosis, we will measure the expression levels of Bcl-2, Bcl-XL, Bax, and Bak following morusin treatment. Multiple mechanisms of action involved in the regulation of cell death demonstrated for almost all anticancer agents. 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 (73-76). 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 (77). This proteolytic process allows NF-κB translocate from cytosol to the nucleus, and therefore bind to the promoter region of the target gene (78). 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, 89). 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.

Phosphorylation and degration of IκBα, and activation of NF-κB result in the translocation of NF-κB from cytosol to the nucleus. These processes induce NF-κB DNA-binding activity (90, 91). 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 (92, 93).

Furthermore, the effect of morusin on the NF-κB activity in HT-29 cells will be analyzed by Western blotting and EMSA. To determine if the observed antitumor effects involving the suppression of NF-κB activity and XIAP expression, the total and nuclear protein will be extracted and determined by Western blotting and EMSA. To investigate the link between Akt and NF-κB, we will determine the effects of morusin in HT-29 cells on Akt phosphorylation at residues of Thr 308 and Ser 473 (prerequisite for catalytic of Akt), Moreover, cleaved PARP and DFF40 in morusin-treated nuclear fraction will be determined. Finally, the therapy of morusin on human colorectal cancer 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, and will provide an alternative therapy for this disease.

References

1. Pfister, D. G., Benson, A. B., 3rd, and Somerfield, M. R. (2004) Clinical practice. Surveillance strategies after curative treatment of colorectal cancer.

N Engl J Med 350, 2375-2382

2. Mayer, R. J. (2004) Two steps forward in the treatment of colorectal cancer. N

(9)

Engl J Med 350, 2406-2408

3. Hawk, E. T., Umar, A., and Viner, J. L.

(2004) Colorectal cancer chemoprevention--an overview of the science. Gastroenterology 126, 1423-1447

4. Chan, T. Y., Chan, J. C., Tomlinson, B., and Critchley, J. A. (1993) Chinese herbal medicines revisited: a Hong Kong perspective. Lancet 342, 1532-1534

5. Fiala, E. S., Reddy, B. S., and Weisburger, J. H. (1985) Naturally occurring anticarcinogenic substances in foodstuffs. Annual Rev Nutr 5, 295-321 6. Kuhnau, J. (1976) The flavonoids. A

class of semi-essential food components:

their role in human nutrition. World Rev Nut Diet 24, 117-191

7. Sato, F., Matsukawa, Y., Matsumoto, K., Nishino, H., and Sakai, T. (1994) Apigenin induces morphological differentiation and G2-M arrest in rat neuronal cells. Biochem Biophys Res Commun 204, 578-584

8. Wei, Y. Q., Zhao, X., Kariya, Y., Fukata, H., Teshigawara, K., and Uchida, A.

(1994) Induction of apoptosis by quercetin: involvement of heat shock protein. Cancer Res 54, 4952-4957

9. Chi, Y. S., Jong, H. G., Son, K. H., Chang, H. W., Kang, S. S., and Kim, H.

P. (2001) Effects of naturally occurring prenylated flavonoids on enzymes metabolizing arachidonic acid:

cyclooxygenases and lipoxygenases.

Biochem Pharmacol 62, 1185-1191 10. Ko, H. H., Yu, S. M., Ko, F. N., Teng, C.

M., and Lin, C. N. (1997) Bioactive constituents of Morus australis and Broussonetia papyrifera. J Nat Prod 60, 1008-1011

11. Eastman, A. (1990) Activation of programmed cell death by anticancer agents: cisplatin as a model system.

Cancer Cells 2, 275-280

12. Nicoletii, I., Migliorati, G., Pagliacci, M.

C., Grignami, f., and Riccardi, C. (1991) A rapid and simple method for measuring thymocyte apoptosis by propidum iodide staining and flowcytometry. J Immunol Meth 139, 271-279

13. Evans, D. L., and Dive, C. (1993) Effects of cisplatin on the induction of apoptosis in proliferating hepatoma cells an nonproliferating immature thymocytes. Cancer Res 53, 2133-2139

14. Fadok, V. A. (1992) Exposure of phosphatidyserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148, 2207-2216

15. Martin, S. J. (1995) Early redistribution of plasma membrane phosphatidyserine is a general featur of apoptosis regardless of the initiating stimulus:

inhibition by overexpression of Bcl-2 and AB1. J Exp Med 182, 1545-1556, 16. Verhoven, B., Schlegel, R. A., and

Williamson, P. (1995) Mechanisms of phospatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J Exp Med 182, 1597-1601 17. Fadok, V. A., Bratton, D. L., Rose, D.

M., Pearson, A., Ezekewitz, R. A., and Henson, P. M. (2000) A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 405, 85-90

18. Saikumar, P., Dong, Z., Mikhailov, V., Denton, M., Weinberg, J. M., and Venkatachalam, M. A. (1999) Apoptosis:

definition, mechanisms, and relevance to disease. Am J Med 107, 489-506

19. Kaufmann, S. H., and Earnshaw, W. C.

(2000) Induction of apoptosis by cancer chemotherapy. Exp Cell Res 256, 42-49.

20. Green, D. R. (2000) Apoptotic pathways:

paper wraps stone blunts scissors. Cell 102, 1-4.

21. Sellins, K. S., and Cohen, J. J. (1991) Hyperthermia induces apoptosis in thymocytes. Radiat Res 126, 88-95

22. Hickman, J. A. (1992) Apoptosis induced by anticancer drugs. Cancer Metastasis Rev 11, 121-139

23. Warters, R. L. (1992) Radiation-induced apoptosis in a murine T-cell hybridoma.

Cancer Res 52, 883-890

24. Watabe, M., Machida, K., and Osada, H.

(2000) MT-21 is a synthetic apoptosis inducer that directly induces cytochrome c release from mitochondria. Cancer Res 60, 5214-5222

(10)

25. Mancini, M., Nicholson, D. W., Roy, S., Thornberry, N. A., Peterson, E. P., Casciola-Rosen, L. A., and Rosen, A.

(1998) The caspase-3 precursor has a cytosolic and mitochondrial distribution:

implications for apoptotic signaling. J Cell Biol 140, 1485-1495

26. Shi, Y. (2001) A structural view of mitochondria-mediated apoptosis. Nat Struct Biol 8, 394-401

27. Gross, A., Yin, X., Wang, K., Wei, M.

C., Jockel, J., Milliman, C., Erdjument-Bromage, H., Tempst, P., and Korsmeyer, S. J. (1999) Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. J Biol Chem 274, 1156-1163

28. Lassus, P., Opitz-Araya, X., and Lazebnik, Y. (2002) Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization.

Science 297, 1352-1354

29. Kumar, S., and Vaux, D. L. (2002) Apoptosis. A cinderella caspase takes center stage. Science 297, 1290-1291 30. Adair, E. R., Adams, B. W., Kelleher, S.

A., and Streett, J. W. (1997) Thermoregulatory responses of febrile monkeys during microwave exposure.

Annals of the New York Academy of Sciences 813, 497-507

31. Hu, Y., Benedict, M. A., Ding, L., and Nunez, G. (1999) Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase- 9 activation and apoptosis. Embo J 18, 3586-3595 32. Srinivasula, S. M., Ahmad, M.,

Fernandes-Alnemri, T., and Alnemri, E.

S. (1998) Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization.

Mol Cell 1, 949-957

33. Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. (1997) X-linked IAP is a direct inhibitor of cell-death proteases. Nature 388, 300-304

34. Deveraux, Q. L., Roy, N., Stennicke, H.

R., Van Arsdale, T., Zhou, Q., Srinivasula, S. M., Alnemri, E. S., Salvesen, G. S., and Reed, J. C. (1998)

IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. Embo J 17, 2215-2223

35. Deng, Y., Lin, Y., and Wu, X. (2002) TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO. Genes Dev 16, 33-45 36. Darmon, A. J., Nicholson, D. W., and

Bleackley, R. C. (1995) Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B.

Nature 377, 446-448

37. Chinnaiyan, A. M., O'Rourke, K., Tewari, M., and Dixit, V. M. (1995) FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81, 505-512

38. Casciola-Rosen, L., Nicholson, D. W., Chong, T., Rowan, K. R., Thornberry, N.

A., Miller, D. K., and Rosen, A. (1996) Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death.

Journal of Experimental Medicine 183, 1957-1964

39. Song, Z., McCall, K., and Steller, H.

(1997) DCP-1, a Drosophila cell death protease essential for development.

Science 275, 536-540

40. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S.

(1998) A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43-50

41. Halenbeck, R., MacDonald, H., Roulston, A., Chen, T. T., Conroy, L., and Williams, L. T. (1998) CPAN, a human nuclease regulated by the caspase-sensitive inhibitor DFF45. Curr Biol 8, 537-540

42. Chang, M. Y., Won, S. J., and Liu, H. S.

(1997) A ribozyme specifically suppresses transformation and

tumorigenicity of Ha-ras-oncogene-transforme4d

NIH/3T3 cell lines. J. Cancer Res. Clin Oncol 123, 91-99

43. Gu, J., Dong, R. P., Zhang, C., McLaughlin, D. F., Wu, M. X., and Schlossman, S. F. (1999) Functional

(11)

interaction of DFF35 and DFF45 with caspase-activated DNA fragmentation nuclease DFF40. J Biol Chem 274, 20759-20762.

44. Ahmad, N., Cheng, P., and Mukhtar, H.

(2000) Cell cycle dysregulation by green

tea polyphenol epigallocatechin-3-gallate. Biochl

Biophy Res Comm 275, 328-334

45. Deveraux, Q. L., Leo, E., Stennicke, H.

R., Welsh, K., Salvesen, G. S., and Reed, J. C. (1999) Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases. Embo J 18, 5242-5251.

46. Miller, L. K. (1999) An exegesis of IAPs:

salvation and surprises from BIR motifs.

Trends Cell Biol 9, 323-328.

47. Takahashi, R., Deveraux, Q., Tamm, I., Welsh, K., Assa-Munt, N., Salvesen, G.

S., and Reed, J. C. (1998) A single BIR domain of XIAP sufficient for inhibiting caspases. J Biol Chem 273, 7787-7790 48. Sun, C., Cai, M., Gunasekera, A. H.,

Meadows, R. P., Wang, H., Chen, J., Zhang, H., Wu, W., Xu, N., Ng, S. C., and Fesik, S. W. (1999) NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP.

Nature 401, 818-822

49. Chen, C. C., Sun, Y. T., Chen, J. J., and Chiu, K. T. (2000) TNF-alpha-induced cyclooxygenase-2 expression in human lung epithelial cells: involvement of the phospholipase C-gamma 2, protein kinase C-alpha, tyrosine kinase, NF-kappa B-inducing kinase, and I-kappa B kinase 1/2 pathway. J Immunol 165, 2719-2728

50. Adams, D. S., Nathans, R., Pero, S. C., Sen, A., and Wakshull, E. (2000) Activation of a rel-A/CEBP-beta-related transcription factor heteromer by PGG-glucan in a murine monocytic cell line. J Cellu Bioch 77, 221-233

51. Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G.

E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102,

43-53

52. Srinivasula, S. M., Datta, P., Fan, X. J., Fernandes-Alnemri, T., Huang, Z., and Alnemri, E. S. (2000) Molecular Determinants of the caspase-promoting activity of Smac/DIABLO and its role in the death receptor pathway. J Biol Chem 275, 36152-36157.

53. Chai, J., Du, C., Wu, J. W., Kyin, S., Wang, X., and Shi, Y. (2000) Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature 406, 855-862.

54. Liu, Z., Sun, C., Olejniczak, E. T., Meadows, R. P., Betz, S. F., Oost, T., Herrmann, J., Wu, J. C., and Fesik, S. W.

(2000) Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature 408, 1004-1008,

55. Wu, G., Chai, J., Suber, T. L., Wu, J. W., Du, C., Wang, X., and Shi, Y. (2000) Structural basis of IAP recognition by Smac/DIABLO. Nature 408, 1008-1012.

56. Misko, T. P., Moore, W. M., Kasten, T.

P., Nickols, G. A., Corbett, J. A., Tilton, R. G., McDaniel, M. L., Williamson, J.

R., and Currie, M. G. (1993) Selective inhibition of the inducible nitric oxide synthase by aminoguanidine. Eur J Pharmacol 233, 119-125

57. Kroemer, G. (1997) The proto-oncogene Bcl-2 and its role in regulating apoptosis.

Nat Med 3, 614-620

58. Ferri, K. F., and Kroemer, G. (2001) Organelle-specific initiation of cell death pathways. Nat Cell Biol 3, E255-263 59. Monaghan, P., Robertson, D., Amos, T.

A., Dyer, M. J., Mason, D. Y., and Greaves, M. F. (1992) Ultrastructural localization of bcl-2 protein. J Histochem Cytochem 40, 1819-1825 60. Walton, M. I., Whysong, D., O'Connor,

P. M., Hockenbery, D., Korsmeyer, S. J., and Kohn, K. W. (1993) Constitutive expression of human Bcl-2 modulates nitrogen mustard and camptothecin induced apoptosis. Cancer Res 53, 1853-1861

61. Newmeyer, D. D., Farschon, D. M., and Reed, J. C. (1994) Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle

(12)

fraction enriched in mitochondria. Cell 79, 353-364

62. Marchetti, P., Castedo, M., Susin, S. A., Zamzami, N., Hirsch, T., Macho, A., Haeffner, A., Hirsch, F., Geuskens, M., and Kroemer, G. (1996) Mitochondrial permeability transition is a central coordinating event of apoptosis. J Exp Med 184, 1155-1160

63. Marchetti, P., Susin, S. A., Decaudin, D., Gamen, S., Castedo, M., Hirsch, T., Zamzami, N., Naval, J., Senik, A., and

Kroemer, G. (1996) Apoptosis-associated derangement of

mitochondrial function in cells lacking mitochondrial DNA. Cancer Res 56, 2033-2038

64. Amarante-Mendes, G. P., Finucane, D.

M., Martin, S. J., Cotter, T. G., Salvesen, G. S., and Green, D. R. (1998) Anti-apoptotic oncogenes prevent caspase-dependent and independent commitment for cell death. Cell Death Differ 5, 298-306

65. Chaussee, M. S., Liu, J., Stevens, D. L., and Ferretti, J. J. (1996) Genetic and phenotypic diversity among isolates of Streptococcus pyogenes from invasive infections. J Inf Dis 173, 901-908

66. Kluck, R. M., Bossy-Wetzel, E., Green, D. R., and Newmeyer, D. D. (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132-1136

67. Lorenzo, H. K., Susin, S. A., Penninger, J., and Kroemer, G. (1999) Apoptosis inducing factor (AIF): a

phylogenetically old, caspase-independent effector of cell

death. Cell Death Differ 6, 516-524 68. Susin, S. A., Zamzami, N., Castedo, M.,

Hirsch, T., Marchetti, P., Macho, A., Daugas, E., Geuskens, M., and Kroemer, G. (1996) Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med 184, 1331-1341.

69. Jurgensmeier, J. M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D., and Reed, J. C. (1998) Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci U S A

95, 4997-5002

70. Narita, M., Shimizu, S., Ito, T., Chittenden, T., Lutz, R. J., Matsuda, H., and Tsujimoto, Y. (1998) Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci U S A 95, 14681-14686

71. Roth, W., and Reed, J. C. (2002) Apoptosis and cancer: when BAX is TRAILing away. Nat. Med. 8, 216-218 72. Desagher, S., and Martinou, J. C. (2000)

Mitochondria as the central control point of apoptosis. Trends Cell Biol 10, 369-377

73. Baeuerle, P. A., and Baltimore, D. (1996) NF-kappa B: ten years after. Cell 87, 13-20

74. Sha, W. C. (1998) Regulation of immune responses by NF-kappa B/Rel transcription factor. J Exp Med 187, 143-146

75. Foo, S. Y., and Nolan, G. P. (1999) NF-kappaB to the rescue: RELs, apoptosis and cellular transformation.

Trends Genet 15, 229-235

76. Ryan, K. M., Ernst, M. K., Rice, N. R., and Vousden, K. H. (2000) Role of NF-kappaB in p53-mediated programmed cell death. Nature 404, 892-897

77. Imbert, V., Rupec, R. A., Livolsi, A., Pahl, H. L., Traenckner, E. B., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P. A., and Peyron, J. F. (1996) Tyrosine phosphorylation of I kappa B-alpha activates NF-kappa B without proteolytic degradation of I kappa B-alpha. Cell 86, 787-798

78. Rice, N. R., and Ernst, M. K. (1993) In vivo control of NF-kappa B activation by I kappa B alpha. Embo J 12, 4685-4695

79. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to

(13)

the CD95 (Fas/APO-1) death--inducing signaling complex. Cell 85, 817-827 80. Nothwehr, S. F., and Martinou, J. C.

(2003) A retention factor keeps death at bay. Nat Cell Biol 5, 281-283

81. Sawada, M., Sun, W., Hayes, P., Leskov, K., Boothman, D. A., and Matsuyama, S.

(2003) Ku70 suppresses the apoptotic translocation of Bax to mitochondria.

Nat Cell Biol 5, 320-329

82. Kroemer, G., Zamzami, N., and Susin, S.

A. (1997) Mitochondrial control of apoptosis. Immunol. Today 18, 44-51 83. Brenner, C., and Kroemer, G. (2000)

Apoptosis. Mitochondria--the death signal integrators. Science 289, 1150-1151

84. Joza, N., Susin, S. A., Daugas, E., Stanford, W. L., Cho, S. K., Li, C. Y., Sasaki, T., Elia, A. J., Cheng, H. Y., Ravagnan, L., Ferri, K. F., Zamzami, N., Wakeham, A., Hakem, R., Yoshida, H., Kong, Y. Y., Mak, T. W., Zuniga-Pflucker, J. C., Kroemer, G., and Penninger, J. M. (2001) Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 410, 549-554.

85. Wei, M. C., Lindsten, T., Mootha, V. K., Weiler, S., Gross, A., Ashiya, M., Thompson, C. B., and Korsmeyer, S. J.

(2000) tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev 14, 2060-2071 86. Adams, J. M., and Cory, S. (1998) The

Bcl-2 protein family: arbiters of cell survival. Science 281, 1322-1326

87. Evan, G., and Littlewood, T. (1998) A matter of life and cell death. Science 281, 1317-1322.

88. Thornberry, N. A., and Lazebnik, Y.

(1998) Caspases: enemies within.

Science 281, 1312-1316

89. Waddick, K. G., and Uckun, F. M. (1999) Innovative treatment programs against cancer: II. Nuclear factor-kappaB (NF-kappaB) as a molecular target.

Biochem Pharmacol 57, 9-17

90. Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., and Donner, D. B. (1999) NF-kappaB activation by tumour necrosis factor requires the Akt

serine-threonine kinase. Nature 401, 82-85

91. Romashkova, J. A., and Makarov, S. S.

(1999) NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling. Nature 401, 86-90.

92. LaCasse, E. C., Baird, S., Korneluk, R.

G., and MacKenzie, A. E. (1998) The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 17, 3247-3259

93. Deveraux, Q. L., and Reed, J. C. (1999) IAP family proteins--suppressors of apoptosis. Genes Dev 13, 239-252

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13

0 1 2 3 4 5 6 7

0 20 40 60 80 100

23.8 µM 11.9 µM 5.95 µM 2.98 µM

Percentage of inhibition

Time (Days) IC₅₀= 5.95 µM

0 1 2 3 4 5 6 7

0 20 40 60 80 100

23.8 µM 11.9 µM 5.95 µM 2.98 µM

Percentage of inhibition

Time (Days) IC₅₀= 5.95 µM

Fig. 1

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14

Fig. 2

23.3±1.4% 46.6±2.1% 59.4±2.7% 68.7±3.2%

Morusin (28.6 µM)

12 24 48 72

Fluorescence intensity

Time (h)

Cell number ^4.9±0.1%

C

A

23.3±1.4%

23.3±1.4% 46.6±2.1%46.6±2.1% 59.4±2.7%59.4±2.7% 68.7±3.2%68.7±3.2%

Morusin (28.6 µM)

12 24 48 72

Time (h)

Cell number ^4.9±0.1%Cell number ^4.9±0.1%

C

A

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15

Fig. 2

1.2±0.5% 4.1±1.5% 8.2±1.0% 28.7±1.2% 58.5±0.8%

Morusin (µM)

^Time

(h)

Relative cell number

C 9.5 19.1 28.6 38.1

48 72

2.4±1.0% 17.7±2.2% 40.1±1.3% 61.2±2.5% 80.5±1.0%

1.3±0.4% 2.2±0.7% 3.4±0.6 4.8±1.1% 18.9±1.6%

24

B

1.2±0.5% 4.1±1.5% 8.2±1.0% 28.7±1.2% 58.5±0.8%

Morusin (µM)

^Time

(h)

C 9.5 19.1 28.6 38.1

48 72

2.4±1.0% 17.7±2.2% 40.1±1.3% 61.2±2.5% 80.5±1.0%

1.3±0.4% 2.2±0.7% 3.4±0.6 4.8±1.1% 18.9±1.6%

24

1.2±0.5%1.2±0.5% 4.1±1.5%4.1±1.5% 8.2±1.0%8.2±1.0% 28.7±1.2%28.7±1.2% 58.5±0.8%58.5±0.8%

Morusin (µM)

^Time

(h)

C 9.5 19.1 28.6 38.1

48 72

2.4±1.0%2.4±1.0% 17.7±2.2%17.7±2.2% 40.1±1.3%40.1±1.3% 61.2±2.5%61.2±2.5% 80.5±1.0%80.5±1.0%

1.3±0.4%1.3±0.4% 2.2±0.7%2.2±0.7% 3.4±0.63.4±0.6 4.8±1.1%4.8±1.1% 18.9±1.6%18.9±1.6%