海洋天然物做為抗癌藥物之研究

i

國 立 交 通 大 學

分子醫學與生物工程研究所

碩

士

論

文

海洋天然物做為抗癌藥物之研究

Characterization of marine natural products

as anticancer agents

研 究 生：黃俊嵐

指導教授：梁美智 教授

ii

海洋天然物做為抗癌藥物之研究

Characterization of marine natural products as anticancer agents

研 究 生：黃俊嵐 Student：Jin-Lan Huang

指導教授：梁美智 Advisor：Mei-Chih Liang

國 立 交 通 大 學

分子醫學與生物工程研究所

碩 士 論 文

A Thesis

Submitted to Institute of Molecular Medicine and Bioengineering National Chiao Tung University

in partial Fulfillment of the Requirements for the Degree of

Master in

Molecular Medicine and Bioengineering August 2012

Hsinchu, Taiwan, Republic of China

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摘要

做為治療疾病藥物之來源，天然物扮演了非常重要的角色，其獨特的結構與活 性，對於新藥的發現與發展都十分具有幫助。由於知識與技術上的原因，在過去 發現許多的海洋天然物並未就其分子作用機制做深入的研究與探討 。在本研究中，我們將探討從海洋菌 Zooshikella sp.中所萃取出的紅色色素是否 具有抗癌活性。藉由測定經過不同濃度的萃取物處理之後的癌症細胞株之細胞活 性與增殖能力，我們證實了由 Zooshikella sp.中所萃取出的紅色萃取物，對於癌 細胞的確具有細胞毒性。經過一系列的驗證與純化，我們最後得到兩個具有相似 結構的化合物，分別為 WYT1-70-6 (prodigiosin) 與 WYT1-70-10 (2-methyl-3-hep -tyl prodigiosin)。兩者在抑制細胞增殖與誘發細胞凋亡的能力表現相當接近，此 現象可由多二磷酸腺苷核糖聚合酶(PARP)被切割的情況證明。然而，根據結果發 現即使結構相近，prodigiosin 可以抑制腫瘤壞死因子 α (TNFα) 在多發性骨髓瘤 (multiple myeloma) 所 引 發 的 NF-κB 核 轉 移 與 活 化 ； 而 2-methyl-3-heptyl prodigiosin 則無此現象。最後，我們證實了 prodigiosin 可與 bortezomib 共同作用 而得到更好的細胞毒殺性。總結來說，我們的實驗結果顯示：由於抑制了轉錄因 子 NF-κB 的活性，由 Zooshikella sp.所純化出的天然物 prodigiosin 展現了抗癌活 性，並與 bortezomib 在細胞毒殺能力上具有加成性。

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Abstract

Natural products are important sources of bioactive agents for treating diseases. The unique structures and novel activities of marine natural products represent a promising field for drug discovery. For historical reasons, the molecular mechanisms of many bioactive marine natural products have not been well elucidated. In this study, we aimed to investigate the anticancer activity of natural products isolated from

Zooshikella sp., a marine bacterial species producing red pigments. By assessing the

viability and proliferation of cancer cell lines treated with various concentrations of purified extracts/compounds, we have identified several bioactive extracts/molecules from Zooshikella sp. with cell toxicity. Moreover, two of the pure compounds, prodigiosin (WYT1-70-6) and its analog 2-methyl-3-heptyl prodigiosin (WYT1-70-10), induced apoptosis in cancer cell lines, as judged by the site-specific cleavage of PARP. Additionally, our preliminary data have shown that prodigiosin, but not 2-methyl-3-heptyl prodigiosin, inhibited TNFα induced NF-κB nuclear transloca-tion and activatransloca-tion in the RPMI 8226 multiple myeloma cell line. Finally, we demon-strated that prodigiosin in combination with bortezomib had synergistic cell toxicity. In summary, results from this study suggested that marine natural product prodigiosin isolated from Zooshikella sp. exerts anticancer activity and additive interaction with bortezomib by targeting the transcription factor NF-κB signaling pathway.

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誌謝

時光匆匆。轉眼間，兩年的碩班，已走到了盡頭。朝九晚五的生活，喔，不是。 咳，是”午一晚一”的生活，嗯...還是”午二晚三”，隨便啦！總之，愜意的 實驗室生活已經正式畫下了句點。兩年來的收穫不少，標準 SOP: 發現問題，面 對問題，分析問題，解決問題。反覆的討論與實驗交叉印證，比起實驗結果，這 些更是兩年來獲得足以用上一輩子的智慧。中間，最該感謝的當然是辛苦的梁美 智老師了！從一開始帶著實驗新手又是做實驗，又是養老鼠的，各式各樣的疑難 雜症都要請教她，最後在趕畢業的這段時間尤甚，不辭辛勞的為我修改論文，真 可說是勞苦功高啊！再者也要感謝實驗室的大伙們：一起創室的世宏學長、福轅 還有賤人(?!)，喔，是祖元才對！在實驗中給了我許多建議，也在實驗之余陪我 度過了歡樂的時光；千芳、婷媛還有胖子，喔，是逸翔才對！平常實驗室的雜務 真是辛苦你們了！(記得趕快脫手 XD)也感謝妳們在口試時幫忙準備了好吃的東 西；還有親愛的專題生們，including 牟、SJ、朗賢 and 簡立，幫忙插了一盒 又一盒的 tip，還有提供了我消遣的對象(對！SJ 就是你！)，為大家帶來了許多 歡樂!!還要感謝新碩一的小朋友們，佩樺、大頭、奕君還有佳琪，祝你們以後實 驗順利！ 特別感謝奕君還有佳琪，妳們知道我說的是什麼啦！萬分感謝 XD 就說聲再會吧！有緣再聚！

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CONTENTS

Abstract (Chinese)……….……….i Abstract (English)………..………ii Acknowledgement ...iii Contents………iv List of Figures………...………...vii Appendix………...……… .viii List of Abbreviations………..………...ix

Chapter 1. Introduction

1.1. Natural products ... 1

1.1.1. The sources of natural products ... 1

1.1.2. The value of application in clinical ... 3

1.2. NF-κB signal transduction pathway ... 3

1.2.1. Components and function of NF-κB complex ... 5

1.2.2. The IκB family ... 6

1.2.3. The IκB kinase (IKK) complex ... 7

1.2.4. Activation of NF-κB signaling pathway ... 8

1.2.4.1. Tumor necrosis factor receptor (TNFR) ... 9

v

1.2.4.3. T-cell receptor and B-cell receptor (TCR and BCR)... 10

1.3. Inflammation and cancer ... 10

1.3.1. NF-κB dysregulation and caner ... 11

1.4. Multiple myeloma ... 13

1.4.1. NF-κB and multiple myeloma ... 13

1.4.2. The treatment of multiple myeloma ... 14

Specific aims ... 16

Materials and Methods

2.1. Materials ... 17

2.1.1. Chemicals and reagents ... 17

2.1.2. Equipment and materials ... 20

2.1.3. Buffers ... 21

2.2. Cell culture ... 22

2.2.1. Cell lines ... 22

2.2.2. General culturing of cells ... 22

2.3 Cell viability assay ... 22

2.4. Cell treatment ... 23

2.4.1. NF-κB inhibition treatment ... 23

2.4.2. Cell apoptosis treatment ... 24

2.5. Preparation of whole cell lysates and nuclear extract ... 24

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2.5.2. Preparation of nuclear extracts ... 24

2.6. SDS-polyacrylamide gel electrophoresis ... 25

2.7. Western blot analysis ... 26

2.8. Electrophoretic mobility shift assay (EMSA) ... 27

Chapter 3. Results

3.1. Transcription factor NF-κB inhibitor screening from marine natural products .. 29

3.2. WYT1-33-6 inhibits NF-κB activation by blocking IκBα phosphorylation and NF-κB nuclear translocation in TNFα-induced NIH3T3 cells ... 29

3.3. WYT1-33-6 inhibits cell viability and induces apoptosis in the human cervical cancer HeLa cells ... 30

3.4. Major bioactive components of WYT1-33-6, WYT170-6 (prodigiosin) and WYT1-70-10 (a prodigiosin analog), are cytotoxins in multiple myeloma cells ... 32

3.5. The DNA binding ability of NF-κB is inhibited by WYT1-70-6 through blocking NF-κB translocation in human multiple myeloma RPMI 8226 cells ... 33

3.6. Evaluating the cell killing effect of WYT1-70-6 in combination with clinical agents for treating multiple myeloma ... 34

Chapter 4. Discussion

4.1. Marine microorganism Zooshikella sp., a new source of prodiginine family .... 36

4.2. Prodigiosin has gotten attention again as an anticancer agent in recent years .... 36

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4.4. Prodigiosin in combination with bortezomib exert synergistic cell toxicity in hu-man multiple myeloma cells ... 37

Reference ... 39

List of Figures

Figure 1. The classical NF-κB signaling pathway ... 50 Figure 2. The alternative NF-κB signaling pathway ... 51 Figure 3. WYT-33-6 dose-dependently inhibits the degradation of IκBα in NIH3T3

cells ... 52 Figure 4. WYT1-33-6 time-dependently blocks the phosphorylation of IκBα in TNFα

induced NIH3T cells ... 53 Figure 5. WYT1-33-6 inhibits NF-κB subunit p65 nuclear translocation in TNFα

in-duced NIH3T3 cells ... 55 Figure 6. WYT1-33-6 causes cellular morphological changes and reduces cell

viabil-ity in the human cervical cancer HeLa cells ... 56 Figure 7. WYT1-33-6 dose-dependently induces apoptosis which in human cervical

cancer HeLa cells ... 58 Figure 8. Identification of fractions of WYT1-33-6 with NF-B inhibition effects in

TNFα induced NIH3T3 cells ... 60 Figure 9. WYT1-70-6 dose-dependently inhibits the degradation of IκBα in TNFα

induced human multiple myeloma RPMI 8226 multiple cells ... 61 Figure 10. WYT1-70-10, the prodigiosin analog dose not block IκBα degradation in

viii

Figure 11. WYT1-70-6 induces apoptosis related PARP –cleavage in human multiple myeloma RPMI 8226 cells ... 63 Figure 12. WYT1-70-10 induces apoptosis related PARP –cleavage in human

multi-ple myeloma RPMI 8226 cells. ... 64 Figure 13. WYT1-70-6 and its analog WYT1-70-10 exhibit a similar cell growth

in-hibition effect in human multiple myeloma RPMI 82206 cells. ... 65 Figure 14. WYT1-70-6 inhibits NF-κB subunit p65 nuclear translocation in TNFα

in-duced RPMI 8226 cells. ... 66 Figure 15. WYT1-70-6 represses the activity of NF-κB complex in TNFα induced

RPMI 8226 cells ... 67 Figure 16. Synergistic anti-myleloma acticity of WYT1-70-6 in combination with

clinically used drugs ... 68 Figure 17. The NF-κB activation induced by bortezomib is suppressed by

WYT1-70-6 ... 69

Supplementary data ... 70

Appendix

Appendix 1. The AKT/PI3K pathway ... 74 Appendix 2. The processes of purifying the natural products ... 75

ix

LIST OF ABBREVIATIONS

APS ammonium persulfate

CHX cycloheximide

COX cyclooxygenase

DMEM Dulbecco's Modified Eagle's Medium

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

DTT dithiothreitol

EDTA ethylenediamine tetraacetic acid disodium salt

EGTA ethylene glycol tetraacetic acid disodium salt

EGF epidermal growth factor

EMSA electrophoretic mobility shift assay

FBS fetal bovine serum

g gram(s)

h hour(s)

IC50 inhibitory concentrations 50

IκBα inhibitor of kappaB alpha

x IL interleukin IL-1 interleukin-1 IL-6 interleukin-6 LPS lipopolysaccharide M molar mA milliamp (current) MeOH methanol min minute(s) ml milliliter(s) MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2 -(4-sulfophenyl)-2H-tetrazolium MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide μg microgram(s) μl microliter(s) MM multiple myeloma mM millimolar μM micromolar MW molecular weight

NEMO NF-κB essential modulator

xi

NIK NF-κB-inducing kinase

NLS nuclear-localization signal

NP-40 nonidet-P 40

O.D. optical density

PAGE polyacrylamide gel electrophoresis

PARP poly(ADP-ribose) polymerase

PBS phosphate-buffered saline

PBST phosphate-buffered saline with Tween-20

PDGF platelet-derived growth factor

p-IкBα Phosphorylation form of IкBα

PI-3K phosphatidylinositol-3-OH kinase

PKC protein kinase C

PMSF phenylmethylsulfonyl fluoride

rpm revolutions per minute

SDS sodium dodecyl sulfate

s second(s)

Ser serine

TEMED N,N,N’,N’-tetramethylethelenediamine

xii TFIID Transcription Factor II D

TNF-α tumor necrosis factor-alpha

TNFR tumor necrosis factor receptor

TRAF tumor necrosis factor receptor associated factor

Tween-20 polyoxyethylene sorbitan monolaurate

UV ultraviolet

1

Introduction

1.1. Natural products

The natural products are small molecules produced by living organism can be ap-plied to pharmacology. Throughout history, natural products have contributed abounding sources of compounds to application in the fields of medicine, pharmacy and biology. More than half of currently available drugs are natural compounds or are related to them (Newman and Cragg, 2007), which indicates that the discovery of a single natural product does not represent only one drug will be developed. By semisynthesis processes, we can modify the functional groups of lead compounds, the original natural products, to generate structural analogues with greater pharmacologi-cal activity and with fewer side effects (Gordaliza, 2007). There are many reports suggest that these synthetic analogs, in particular, could be important candidates for further studies involving structural modifications to improve the pharmacological profile of native organism metabolites (Schumacher et al., 2011). Additionally, it will get higher efficiency to cooperate with combinatorial chemistry, computational chemistry, bioinformatics and high-throughput screening protocols (Gordaliza, 2007).

1.1.1. The sources of natural products

Natural products can be extracted from eukaryotes to prokaryotes, including ani-mals, plants, and microorganism covering land and ocean. For the natural products from animals, most of them are monoclonal antibodies. A famous example is Bevacizumab (Avestin), a vascular endothelial growth factor (VEGF) inhibitor (Los et al., 2007). It was approved by the U.S. Food and Drug Administration (FDA) for combination use with standard chemotherapy for metastatic colon cancer in 2004. On the other hand, since the herbalism, the origin of contemporary medicine, the

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plants have been studied for a very long time in this field. There are many diverse natural compounds have been discovered and isolated from plant sources (Mukherjee et al., 2001). For example, parthenolide which exists in a traditional herb feverfew (Tanacetum parthenium) is used for anti-inflammatory activity. The recent studies re-vealed that it is able to inhibit the NF-κB signaling pathway both in vtro and in vivo (Hehner et al., 1999; Sheehan et al., 2002). Additionally, the anticancer property of parthemolide has been noticed in many kinds of cancer in recent years (Cheng and Xie, 2011; Li et al., 2012). The scientists not only the identified the new compounds, but modified the functional groups to improve the biological activity. A typical exam-ple is the analog development of curcumin. Polyphenol curcumin, the synthetic ana-log of curcumin, displays a higher anticancer property compared to the parent com-pound. (Yadav et al., 2010).

While isolating the natural products from microorganism, compared to from plants, has more advantages. These advantages include: 1. Extracting the compounds is sim-pler since the cell wall of plants is hard to be lysed. 2. Culturing the microorganism is not only much less time cost, but also can obtain many times of amounts of re-sources for extracting. 3. The species of microorganism is much more than plants, and this fact represents that there are still many of unidentified natural compounds in microorganism. According to above opinions suggests that microorganism is a better resources for discovering new natural products which avail against the cancer. In decade, it becomes popular to test the bioactivities of natural compounds isolated from microorganism, especially the ocean microorganism, in cancer cell lines and find the mechanism how it kills the cancer cells. For example, a research group of Univer-sity of California-San Diego published that they isolated an ester-substituted sesquiterpenoid called cryptosphaerolide from a marine-derived ascomycete related to the genus Cryptosphaeria (Oh et al., 2010). This marine extraction shows cytotoxicity

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(IC50 of 4.5 μM) on the HCT-116 colon carcinoma cell line (Oh et al., 2010). A

pre-vious study revealed that this kind of compounds inhibited myeloid leukemia cell dif-ferentiation protein Mcl-1which plays an important role in life/death decisions of in-dividual cells (Michels et al., 2005). Another example is manzamine A, which has been isolated from various marine sponges, displayed a cytotoxic effect against AsPC-1 pancreatic cancer cells, with an IC50 in a range of 4.2 μM, after 3 days of

treatment (Guzman et al., 2011).

1.1.2. The value of application in clinical

The process mentioned above that biochemists cooperate with computational chemistry and bioinformatics to modify the leading compounds which were discov-ered by cancer biologists is very effective for drug development. There are many re-search data can support this fact. Besides, many of new commercial anticancer drugs have gone through this process. An example of these compounds is romidepsin which is a depsipeptide isolated from Chromobacterium violaceum. Romidepsin is a novel agent in a new class of anti-cancer drugs, known as histone deacetylase inhibitors (Glaser, 2007). It has received the Orphan Drug Designation from FDA for the treat-ment of non-Hodgkin T-cell lymphomas (Gordaliza, 2007). Another example is trabectedin (PharmaMar’s Yondelis®) (D'Incalci and Galmarini, 2010). It is the first anticancer drug isolated from the marine source. Although it has not been approved by FDA now, it is still a potential anticancer drug.

1.2. NF-κB signal transduction pathway

NF-κB complex was discovered and defined as nuclear factor in 1986. It binds to the enhancer region of the κB chain of immunoglobulin in B cells (Anand et al., 2008). Therefore, it should have highly connection with immune response. Indeed, the

stud-4

ies of NF-κB have revealed that NF-κB plays a very important role in immune system (Bonizzi and Karin, 2004; Ghosh et al., 1998; Silverman and Maniatis, 2001). Fur-thermore, the inflammation which is mostly controlled by NF-κB has shown high positive connection with cancer. In addition, that NF-κB signal transduction pathway has abnormal expression level, most of cases were over expression or constitutive ex-pression, was reported (Aggarwal and Gehlot, 2009).

The NF-κB signal transduction pathway can be distinguished into two types. One is the classical pathway, the other one is alternative pathway. Either classical pathway or alternative pathway can trigger the NF-κB signaling (Bonizzi and Karin, 2004). For the classical pathway, NF-κB complex exists in cytoplasm and inactivated by binding to IκB family proteins which result in that NF-κB complex cannot be translocated into nucleus. For that reason, NF-κB cannot function as the transcriptional faction through binding to its targeting site. When the cells receive the specific signal, such as proinflammatory cytokine tumor necrosis factor α (TNFα), the IκB protein will be phosphorylated by IκB kinase (IKK) complex and then be degraded through ubiquitination pathway. Consequently, the NF-κB complex will be activated by translocated into nucleus (see Fig. 1). Activated NF-κB can then be down-regulated through multiple mechanisms including the well characterized feedback pathway whereby newly synthesized IκBα protein binds to nuclear NF-κB and exports it out to the cytosol (Hayden and Ghosh, 2004).

As for the alternative pathway, the composition of NF-κB complex is different from the classical. Without binding to IκB proteins, the NF-κB complex maintains unstimulated state itself. While the activator, like CD40L, interacts with receptor and activates the pathway (Nakano et al., 1999), the IKKα homodimers will be phosphor-ylated by NF-κB inducing kinase (NIK) (Senftleben et al., 2001a) and phosphorylate one component of NF-κB complex subsequently. Once the phosphorylation of NF-κB

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complex is happened, the phosphorylated component will be added poly-ubiquitin and processed. Finally, the active form of NF-κB complex will be able to enter the nucleus and turn the specific genes on (see Fig. 2).

1.2.1. Components and function of NF-κB complex

There are five members of the mammalian NF-κB family, including p65 (RelA), RelB, c-Rel, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). They exist in cytoplasm as homo- or heterodimers bound to IκB family proteins at unstimulated state (Hayden and Ghosh, 2004). All of them have around 300- amino acids conserved sequence called Rel homology domain (RHD) which is responsible for dimerization, interaction with IκBs, and binding to DNA. Only RelA, RelB, and c-Rel contain the transactive domain (TAD) which is necessary for driving transcription. Therefore, the research shows that gene expression is repressed when the promoter or enhancer region binds to p50 or p52 homodimers (Zhong et al., 2002).

The p65 (Rel A) usually accompanies p50 to form the NF-κB complex in the classical pathway which is one of the most important pathway regulated immune system. However, the function of p65/p50 heterodimmers is shut down since the binding of IκBα. The crystallographic structures of this binding complex revealed that the IκB proteins mask the nuclear localization sequence (NLS) of p65 (Malek et al., 2001; Malek et al., 2003). Following degradation of IκBα, the released NF-κB is able to bind promoter and enhancer regions containing κB sites with the consensus sequence GGGRNNYYCC (N = any base, R = purine, and Y = pyrimidine). Moreover, the ge-netic studies of p65 through p65−/− mice revealed the crucial role that p65 protects cells or organs from apoptosis induced by TNFα signaling. This model exhibits le-thality caused by liver degeneration at gestational day 15–16 (Beg et al., 1995).

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homodimerize. Furthermore, it is unable to heterodimerize with c-Rel or p65. In fact, RelB forms heterodimers with p100, p52, and p50 (Dobrzanski et al., 1994; Ryseck et al., 1992) and does not interact with any other IκB proteins (Solan et al., 2002). The RelB/p100 is the major complex in alternative pathway. Without binding to IκB pro-teins, the RelB/p100 complex maintains stable state. When the pathway is stimulated, the IKKα is activated and phosphorylates p100, leading to their polyubiquitination and subsequent processing of p100 by the proteasome (Karin and Ben-Neriah, 2000), forming the active RelB/p52 heterodimers. Moreover, both NLS of RelB and p52 cannot be masked by IκBα, resulting in that RelB/p52 heterodimers exhibit constitu-tive nuclear localization (Dobrzanski et al., 1994; Lernbecher et al., 1994; Ryseck et al., 1992). In addition, RelB-deficient mice have decreased baseline NF-κB activity in the thymus and spleen, but increasing inflammatory infiltration in multiple organs and severe deficits in adaptive immunity (Weih et al., 1995).

1.2.2. The IκB family

There are seven members in IκB family, including IκBα, IκBβ, BCL-3, IκBε, IκBγ, and the precursor proteins p100 and p105 which are characterized by the existence of five to seven ankyrin repeats that assemble into elongated cylinders that bind the di-merization domain of NF-κB dimmers (Hatada et al., 1992). However, the role of the IκB family members, exclusive of IκBα, is less well understood.

The activity of p65/p50 is mostly, although not exclusively, regulated by IκBα through inhibiting the translocation of NF-κB complex. This fact was proofed by IκBα-deficient mice which displays increased but not constitutive DNA-binding abil-ity and significantly increased termination time of NF-κB activation in response to TNF-α (Klement et al., 1996). Moreover, IκBα is known to participate in a feedback loop through that newly synthesized IκBα inhibits the activity of NF-κB. Besides,

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IκBα contains nuclear export sequences (NES) resulting in the output of reformed IκBα and NF-κB complex from nucleus to cytosol. This NES characteristic coupled with the unmasked NLS of p50 contributes to constant shuttling of IκBα/NF-κB com-plexes between the nucleus and the cytoplasm, although the steady-state localization is in the cytosol (Huang et al., 2000; Johnson et al., 1999).

Whereas, the researches of the others IκB proteins compared to IκBα have not clearly characterized their functions yet. The IκBβ was identified the ability that it can bind with p65/p50 or p65/c-Rel dimmers, which is similar to IκBα, through crystallo-graphic structure (Malek et al., 2003). This similar complex in cytosol also has the similar regulating function too (Malek et al., 2001). However, hypophosphorylated IκBβ can bind DNA with p65 and cRel, and the DNA-bound NF-κB:IκBβ complexes are resistant to IκBα in vitro, suggesting hypophosphorylated, nuclear IκBβ may pro-long the expression of certain genes (Tran et al., 1997; Weil et al., 1997). Overall, the result of IκBβ−/− mice shows that IκBβ serves to both inhibit and facilitate the in-flammatory response (Rao et al., 2010) .

1.2.3. The IκB kinase (IKK) complex

The degradation of IκB is a highly regulated event that is initiated upon specific phosphorylation by activated IKK. The IKK contained function in cells can be puri-fied as a 700–900 kDa complex, and has been shown to include two kinase subunits, IKKα (IKK1) and IKKβ (IKK2) which both are serine/threonine kinases, and one regulatory subunit, NEMO (NF-κB essential modifier) or IKKγ (Ghosh and Karin, 2002). In vitro studies indicate that the IKKα/IKKβ heterodimers have higher catalyt-ic effcatalyt-iciency than either homodimer (Huynh et al., 2000)

In the classical NF-κB signaling pathway, IKKβ is both necessary and sufficient for phosphorylation of IκBα on Ser 32 and Ser 36, and IκBβ on Ser 19 and Ser 23. The

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role of IKKα in the classical pathway is unclear, although some studies suggest it may regulate gene expression in the nucleus by modifying the phosphorylation status of histones.(Hayden and Ghosh, 2004)

The alternative pathway, however, only depends on the IKKα subunit, which func-tions by phosphorylating p100 and causing its inducible processing to p52 (Hayden and Ghosh, 2004). Additionally, this process of phosphorylation has high selectively, resulting in that processing of p100 releases a subset of transcriptionally active NF-κB dimers, consisting mainly of p52:RelB (Dejardin et al., 2002; Xiao et al., 2004)

The IKKβ-/- phenotype is similar to that observed for the p65 knockout, supporting the central role for IKKβ in regulation of NF-κB signaling via TNFα (Li et al., 1999a; Li et al., 1999b). In addition, compared to TNFR/p65 double knockout mice survive for many months, TNFR/IKKβ double knockouts shows more defect in innate im-mune responses to bacterial infection, only surviving a few days after birth. These da-ta indicate that there is no partial compensation for IKKβ by IKKα or any other kinase (Senftleben et al., 2001b).

1.2.4. Activation of NF-κB signaling pathway

There are various stimuli that can induce NF-κB signaling pathway, including

an-tigen from bacteria or virus, cytokines, oxidation stress, and receptor ligands etc. And these stimuli activate NF-κB correlated to their specific receptors, including tumor necrosis factor receptor (TNFR), toll/interleukin-1 receptor (TIR), B-cell receptor (BCR) and T-cell receptor (TCR). In these activators, the most important one is the cytokines which is quite simpler and affecting all kinds of cells. Therefore, the studies on mechanism of cytokines, such as TNFα, IL-6, is much more integrated and clari-fied.

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1.2.4.1. Tumor necrosis factor receptor (TNFR)

The TNFR superfamily consists of at least 19 ligands and 29 receptors and exhibits a remarkable diversity in tissue distribution and physiology (Aggarwal, 2003). These family receptors lack intrinsic enzyme activity. Instead, signaling is started by re-cruitment of intracellular adapter molecules (Aggarwal, 2003; Wajant et al., 2003). The most famous ligand of this family is TNFα which interestingly stimulates both death and survival. As mentioned before, the p65-/- mice are fatal since TNFα in-duced liver degradation. However, the p65/TAFR double knockout mice are able to grow up. On the other hand, TNFα also up regulates anti-apoptotic proteins, such as Bcl-XL, through activating NF-κB pathway (Hayden and Ghosh, 2004) .

Besides, there is still another thing needed to be mentioned that TNFα signaling via TNFR1 results in the rapid activation of IKK and nearly complete degradation of IκBα around 10 min to 20 min. It is a very quick response compared to other stimuli (Hoffmann et al., 2003; Hoffmann et al., 2002).

1.2.4.2. Toll/interleukin-1 receptor (TIR)

The Toll/IL-1 receptor (TIR) indicates a group of receptors which have highly ho-mologous domain of Toll-like receptors and IL-1 receptor. This intracellular domain mediates interaction with downstream signaling adapters that lead to activation of three key transcription factors, NF-κB, AP-1, and IRF3(Hayden and Ghosh, 2004). The encountering of LPS, bacterial or viral DNA, and flagellin can trigger the path-way through TLR. After activated, TIR domains recruit cytoplasmic adaptor proteins MyD88 which is crucial for normal NF-κB induction in response to IL-1 and IL-18 (Adachi et al., 1998; Kawai et al., 1999). Subsequently, MyD88 will recruit the others adaptor proteins, such as TIRAP, and eventually activates the IKK complex through

10 TRAF6 (Sun et al., 2004; Wu and Arron, 2003).

1.2.4.3. T-cell receptor and B-cell receptor (TCR and BCR)

Signaling from T-cell receptors (TCRs) and B-cell receptors (BCRs) is critical for mounting adaptive immune responses. These signals indeed lead to NF-κB activation (Ruland and Mak, 2003). Subsequently, the activation of NF-κB resulted from TCR and BCR allows antigen-specific proliferation and maturation of lymphocytes into effector cells which are activated and involved in eliminating a pathogen.

The signal from TCR is passed through recruiting the adaptors which contain tyro-sine kinase activity. However, the link between the receptor proximal tyrotyro-sine kinases and NF-κB is poorly defined. One group reported that that PKCθ is essential for acti-vation of NF-κB by TCR through the knockout studies (Sun et al., 2000). And another group reported that the IKK complex is rapidly recruited to the immunological syn-apse through an unknownmechanism and can be colocalized to the TCR by confocal immunofluorescence analysis (Khoshnan et al., 2000). Summarizing these researches, a hypothesis was proposed that PKCθ is capable of directly interacting with the IKK complex and then activates NF-κB in primary T cells.

1.3. Inflammation and cancer

Cancer is now generally believed to be a preventable disease. Only 5% to 10% of all cancers are caused by inheritance of mutated genes and somatic mutations, where-as the remaining 90% to 95% hwhere-as been linked to lifestyle factors and environment (Anand et al., 2008). Interestingly, in all these risk factors that are considered to cause cancers through inflammation, customs of diet have been recognized as major risk factors for the most common types of cancer (Aggarwal et al., 2009). Indeed, these factors of lifestyle and environment, including tobacco, stress, dietary agents, obesity,

11

alcohol, infectious agents, environmental pollutants ,and irradiation, mostly can in-duce inflammatory pathway which totally account for as much as 95% of all cancers (Aggarwal and Gehlot, 2009).

1.3.1. NF-κB dysregulation and caner

As reported by researches, NF-κB signaling pathway is regarded as playing a very important role in inflammation. Therefore, it is reasonable that inflammation pro-motes the progression of cancer through NF-κB pathway. Indeed, the researches re-vealed that NF-κB is not only over expressed but relied in many kinds of cancer cell lines or cancer stem cells (Mimeault et al., 2012; Noma et al., 2012; Rodriguez-Berriguete et al., 2012). Moreover, the study also revealed the characteris-tic that inflammatory cytokines secreted by inflammatory cells which express acti-vated NF-κB contribute to tumor initiation and progression (Coussens and Werb, 2001). Not only the inflammatory cytokines but growth factors, such as epithelium growth factor (EGF) and vascular endothelial growth factor (VEGF), can induce NF-κB pathway although it does not activate the pathway directly. Actually, these growth factors affect NF-κB through the Akt/PI3K pathway and then result in the pro-liferation and antiapoptosis properties in cancer cells (Appendix 1.).

In addition, there is another significant discovery that NF-κB has been shown to be constitutively active in a majority of tumor cell lines, both solid and hematologic tu-mors (Sethi et al., 2008). Furthermore, the presence of constitutively active NF-κB has now been identified in tissue of most cancers including leukemia, lymphoma, and cancers of the prostate, breast, oral cavity, liver, pancreas, colon, and ovary (Aggarwal and Gehlot, 2009). This constitutive NF-κB signaling is utilized by various cancer types to block apoptosis (Basseres and Baldwin, 2006). Besides, constitutive NF-κB activation also has been linked to chemoresistance and radioresistance (Ahn et

12

al., 2007) . Many mechanism of constitutive activated NF-κB has been described, in-cluding overexpression of growth factor receptors, mutation of IκBα such that it can-not bind to NF-κB, constitutive activation of Ras protein, high proteolytic activity di-rected to IκBα, and autocrine secretion of inflammatory cytokines. However, these mechanisms are cases reports but not universe. In the other words, the mechanism of constitutive active NF-κB has not been demonstrated.

Although the mechanism has not been defined, the contributions of constitutive ac-tivated NF-κB to cancer cells are well clarified. These contributions include survival, antiapoptosis, proliferation, invasion and metastasis.

In normal condition, our body maintains homeostasis through the programmed cell death called apoptosis (Meier et al., 2000). This process usually induced when emer-gence of DNA damage which is repaired invalidly. However, transformed cells re-sulted from inflammatory microenvironment become antiapoptosis and turn out into cancer. The researches indicated that NF-κB regulates the expression of most antiapoptotic gene products (bcl-2, bcl-xl, c-FLIP, XIAP, IAP-1, IAP-2, and survivin) associated with the survival of the tumor (Aggarwal and Gehlot, 2009). All these gene products inhibit the caspase pathway directly. Therefore, cancer cells can keep sur-vival and avoid death.

Besides, cancer cells show not only antiapoptosis but uncontrollable proliferation property. The studies revealed that gene products linked with proliferation of tumors such as c-myc and cyclin D1are tightly regulated through NF-κB (Hwang et al., 2010; Sun et al., 2012). Additionally most growth factors, such as EGF, linked with prolif-eration of tumors either activate NF-kB or are regulated by this transcription factor.

Also, the post-cancerogenesis, including angiogenesis, invasion and metastasis, is promoted through NF-κB which controls the expression of MMP, adhesion molecules, VEGF and TWIST (Aggarwal and Gehlot, 2009).

13

1.4. Multiple myeloma

Multiple myeloma (MM), also known as plasma cell myeloma or Kahler's disease, is a malignant plasma cell proliferation in the bone marrow (BM) associated with monoclonal protein in the serum and/or urine. With a low 5-year survival rate, it has a prevalence of 50,000 patients, occurring in approximately over 16,000 newly diag-nosed cases of MM and over 10,000 deaths within the United States alone in 2005 (Jemal et al., 2007). While in Taiwan in 2006, there were 73,293 patients diagnosed with cancer and 360, around 0.5%, of cases are multiple myeloma and the average survival time is 28 months (statistics from Department of Heath, excusive Yuan, R.O.C). Due to the low 5-years survival rate, drug resistance, and high recurrence, the multiple myeloma is regarded as an incurable cancer.

1.4.1. NF-κB and multiple myeloma

60-70% of MM patients have bone involvement at the time of diagnosis, and 90% of MM patients will develop bone lesions during the course of the disease (Heider et al., 2005; Roodman, 2009). The studies revealed that BM microenvironment plays a crucial role in MM cell pathogenesis since that MM cells adhere to bone marrow stromal cells (BMSCs) and are able to secrete cytokines which induce several path-way, including NF-κB signaling pathpath-way, promoting the proliferation of MM cells (Hideshima et al., 2007).

Furthermore, it has been suggested that activation of the NF-κB pathway is im-portant for the survival of healthy plasma cells and for MM tumors (Moreaux et al., 2009; Tai et al., 2006). And there are many reports show that many of MM cell lines are sensitive to NF-κB inhibitors (Hideshima et al., 2006). Although the mechanism is unclear, the evidence of microarray and quantitative real time polymerase chain reac-tion (qPCR) suggested that MM cells, either from primary or cell lines, contain

con-14

stitutive NF-κB activity (Annunziata et al., 2007). To illuminate the mechanism of constitutive NF-κB signaling in MM, it is reported that both the mutation of NF-κB regulation genes in MM cells and adherence of MM-BMSC which enriches the cyto-kines and growth factors in BM microenvironment contribute to the constitutive NF-κB activation (Keats et al., 2007; Markovina et al., 2010).

1.4.2. The treatment of multiple myeloma

Conventional treatment of multiple myeloma is combined chemotherapy which is often given in cycles over a period of months, followed by a rest period. Commonly used chemodrugs are including melphalan, doxorubicin and dexamethasone. However, the drug resistance occurs frequently (Schmidmaier et al., 2007; Xiang et al., 2011). The radiation therapy is another common therapy for MM patients. Similar to other hematological malignancy, MM patients can receive the bone marrow transplant. Nevertheless, the high rates of tissue rejection associated with donor transplants have made autotransplants the standard for the field. Before accepting autotransplant, pa-tient needs to be treated with high intensity of radiation therapy. After the autotransplant, many of patients still have a relapse since that some residual myeloma cells sometimes survive in the banked stem cells which are given cyclophosphamide and a white blood cell stimulating drug after collection. (Dolgin, 2011).

Although MM remains incurable, novel agents targeting MM cells in the BM mi-croenvironment, such as lenalidomide and bortezomib, when used alone or in combi-nation, can overcome conventional drug resistance and improve patient outcome (Messori et al., 2011; Richardson et al., 2012). Compared to thalidomide which is an old drug for multiple myeloma with serious side effect, bortezomib displays an out-standing therapeutic efficacy for multiple myeloma. Mitsutoshi Satoh et al. demon-strated that bortezomib showed a better effectiveness ratio than thalidomide for

15

drug-resistance multiple myeloma in a rapid effect in the clinical studies. In addition, the overall survival rate inclined toward the bortezomib -treated patients. The results indicated that bortezomib has therapeutic efficacy for patients with thalidomide re-sistance (Satoh et al., 2011).

Since the degradation of IκBα occurs in proteasome, scientists believed that bortezomib causes apoptosis in multiple myeloma through inhibiting NF-κB activity. However, it has not yet been shown that bortezomib induces the inhibition of NF-κB activity in primary tumor cells from multiple myeloma patients. In contrast, the stud-ies demonstrated that both multiple myeloma cell lines and primary cells from pa-tients resisted the bortezomib-induced NF-κB repression (Hideshima et al., 2009; Markovina et al., 2008). Moreover, the previous studies suggested an interesting re-sult that bortezomib up regulate the activity of NF-κB (Hideshima et al., 2009). There are more studies supporting that constitutive NF-κB activity contributed by bone marrow stem cells or cell itself is a key reason of the resistance of bortezomib in mul-tiple myeloma cell lines and stem-like cells of mantle cell lymphoma (Jung et al., 2012; Yang et al., 2008).

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Specific aims

Studies have shown that the transcription factor NF-κB is a promising target for developing novel anticancer therapeutics. In the research collaboration with Dr. Chung-Kuang Lu at National Research Institute of Chinese Medicine, our specific aims are listed below.

Aim 1: to discover potent inhibitors of the NF-κB signaling pathway from extracts prepared from the marine bacterium Zooshikella sp.

Aim 2: to determine whether the purified NF-κB inhibitor(s) from Zooshikella sp. may repress cell growth and induce apoptosis in human cancer cell lines.

Aim 3: to assess whether the purified NF-κB inhibitor(s) from Zooshikella sp. in combination with clinical agents may exert synergistic cell toxicity in human multiple myeloma cells.

17

Materials and Methods

2.1. Materials

2.1.1. Chemicals and reagents

The natural extracts from Zooshikella sp. were kindly provided by Dr. Chung-Kuang Lu’s lab at National Research Institute of Chinese Medicine. The natu-ral extracts were dissolved in methanol to a stock concentration of 10 mg/ml or 10 mM, and stored at -80°C before use. Doxorubicin (Sigma Chemical Co.) was dis-solved in DMSO to 10 mM and stored at -80°C. Bortezomib (Luminescence Tech-nology Corp.) was dissolved in DMSO to 20 mM and stored at -80°C.

Chemicals

2-Mercaptoethanol (2-ME) Sigma Chemical Co.

Acrylamide-Bis solution (29:1), 30% (w/v) SERVA

Ammonium persulfate (APS) J.T. Baker

Bromophenol blue Sigma Chemical Co.

Cycloheximide

Dimethyl sulfoxide (DMSO)

Sigma Chemical Co. Sigma Chemical Co.

Dithiothreitol (DTT) Sigma Chemical Co.

Ethylene glycol tetraacetic acid (EGTA) Sigma Chemical Co. Ethylenediaminetetraacetic acid (EDTA) Sigma Chemical Co.

Glycerol J.T. Baker.

Glycine J.T. Baker

Hepes Sigma Chemical Co.

Igepal ca-630; NonidetP-40 (NP-40) Sigma Chemical Co.

18

Leupeptin Sigma Chemical Co.

Methanol J.T. Baker

N,N,N’,N’-tetramethyl- ethylenediamine (TEMED) J.T. Baker Octyl phenol ethoxylate; Triton X-100 J.T. Baker

Pepstatin Sigma Chemical Co.

phenylmethanesulfonyl fluoride (PMSF) Sigma Chemical Co.

Potassium chloride (KCl ) Sigma Chemical Co.

Recombinant human TNF-α R&D Systems, Inc.

Sodium chloride (NaCl) J.T. Baker

Sodium dodecyl sulfatea SDS Sigma Chemical Co.

Sodium fluoride (NaF) Sigma Chemical Co.

Sodium orthovanadate (Na3VO4) Sigma Chemical Co.

Tris-base J.T. Baker Tris-HCl J.T. Baker Tween-20 J.T. Baker Reagents 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy- phenyl)-2-(4-sulfoqhenyl)-2H- tetrazolium (MTS) Promega

LightShift® Chemiluminescent EMSA Kit Thermo Protein assay kit

SuperSignal® West Dura Extended Duration Substrate

Bio-Rad Thermo SuperSignal® West Pico Chemiluminescent Substrate Thermo

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Primary antibodies

Target

Dilution

Host

Sources

IкBα 1:500 Rabbit Santa Cruz Biotechnology #sc-371

p65 1:500 Rabbit Santa Cruz Biotechnology #sc-372

PARP-1 1:500 Rabbit Santa Cruz Biotechnology #sc-7150

Phospho - IкBα 1:500 Rabbit Cell Signaling Technology # 2859S

TFIID 1:500 Rabbit Santa Cruz Biotechnology #sc-204

α-Tubulin 1:500 Rabbit Santa Cruz Biotechnology #sc-12462-R

Secondary antibody

Target

Dilution

Host

Sources

Goat anti-Rabbit IgG (H+L),

HRP conjugate

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Cell culture medium

Dulbecco's Modified Eagle's Medium (DMEM) Biowest

Fetal bovine serum (FBS) Biowest

Penicillin streptomycin (P+S) Biowest

Phosphate buffered saline (PBS) Biowest

RPMI 1640 Biowest

Trypsin-EDTA Biowest

2.1.2. Equipment and materials

Centrifuge Eppendorf centrifuge 5702R (for 15 ml or 50 ml) BECKMAN model CS-15R Centrifuge (for 1.5 ml) CO2 incubator

Cross-link

NUAIRE CO2 Incubator NU-5500

UV box of UNIVERSAL HOOD (Bio-Rad) Electrophoresis system

ELISA reader Microscopy

Mini- PROTEAN® Tetra (Bio-Rad) Bio-Rad Model 550

OLYMPUS CKX41

Power supply PowerPacTM Basic (Bio-Rad) Transfer system

PVDF membrane

Mini Trans-Blot® Cell (Bio-Rad)

Immobilon® -P Transfer Membrane, Millipore NC membrane Immobilon-NY+, Millipore

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2.1.3. Buffers

1X SDS-running buffer 25 mM Tris-base, 192 mM glycine, 0.1% (w/v) SDS 1X transfer buffer 20 mM Tris-HCl, 150 mM glycine

5X SDS loading dye 87.5 mM Tris-HCl, pH 6.8, 45% (v/v) glycerol, 5% (w/v) SDS, 0.25 % (w/v) bromophenol blue, 12.5% (v/v) 2-ME

5X TBE 0.5 M AT buffer

Tris-base, 0.45 M boric acid, 10 mM EDTA, pH 8.0 20 mM Hepes, pH 7.9, 1% (w/v) Triton X-100, 20% (w/v) glycerol, 1 mM EDTA, 1 mM EGTA,

Buffer E

20 mM NaF, 1 mM DTT, 1 mM Na3VO4, 1 μg/ml PMSF,

1 μg/ml leupeptin, 1 μg/ml pepstatin

10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, pH 8.0, 0.1 mM EGTA, pH 8.0 supplemented with 1 mM DTT, 1 μg/ml PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin

Buffer F 20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA supplemented with 1 mM DTT, 1 μg/ml PMSF, 1 μg/ml leupeptin and 1 μg/ml pepstatin

Resolving buffer 375 mM Tris-base, pH 8.8, 0.1% (w/v) SDS Stacking buffer 125 mM Tris-HCl, pH 6.8, 0.1% (w/v) SDS

Stripping buffer 62.5 mM Tris-HCl, pH 7.0, 2% SDS, 100 mM 2-ME TBST buffer 20 mM Tris-HCl, 2.5 mM KCl, 150 mM NaCl, 0.05%

22

2.2. Cell culture

2.2.1. Cell lines

The cell lines listed below were used in this study:

NIH3T3 (mouse fibroblast); HeLa (human cervical adenocarcinoma); A293 (human embryonic kidney); RPMI 8226 (human multiple myeloma was purchased from Bioresource Collection and Research Center)

2.2.2. General culturing of cells

NIH3T3 and A293 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1X penicillin streptomycin (P+S). HeLa was cultured in RPMI 1640, 10% FBS and 1X P+S were added as the same. RPMI 8226 was cultured in RPMI 1640 supplemented with 20 % FBS and 1X P+S. All cell lines were incubated at 37°C in humidified 95% air and 5% CO2.

For subculture of adherent cells, cells were washed by pre-warmed phosphate buff-ered saline (PBS) and incubated at 37°C approximately 1 min with 1 ml of tryp-sin-EDTA. Trypsinization was stopped by adding 9 ml of culture medium after cells had released from 10 cm culture dish, and then the cell culture medium was trans-ferred into 50 ml centrifuge tube and centrifuged at 1500 rpm (Eppendorf centrifuge 5702R) for 5 min at room temperature. After removing the supernatant, the cells were resuspended by 10 ml of culture medium and 1 or 2 ml of cell culture medium would be transferred into 10 cm dish containing 10 ml of fresh culture medium. For subcul-ture of suspension cells, cells were directly centrifuged at 1500 rpm. Then, cells were operated as described before.

2.3. Cell viability assay

23

5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfoqhenyl)-2H-

tetrazolium (MTS, Promega) were used to indicate the cell viability. Cells were counted and seeded in 96-well plates (Corning, Inc.) at the amount of 5,000-25,000 cells each well (depends on cell line). After 24 h, cells were treated with different concentration of compounds for another 24 h. For the adherent cells, 20 μl of MTT was added to each well for 3 h. Then, the medium was removed carefully and 200 μl of DMSO was added to each well. Absorbance was measured at 540 nm by ELISA reader (Bio-Rad Model 550). For the suspension cells, on the other hands, 20 μl of MTS was added to each well for 4 h and absorbance was measured directly at 490 nm by ELISA reader. The inhibitory concentrations 50 (IC50) were determined by survival

curves drown by Excel.

2.4. Cell treatment

2.4.1. NF-κB inhibition treatment

Before compounds treatment, adherent cells should be subcultured to 60 mm dish (100 mm dish for nuclear extracts) over night in the cause of cells attachment, while the suspension cells should not. After that, cells were changed to starving environment. For adherent cells, cells were washed by PBS twice and cultured in proper medium described previously with 0.5% FBS and 1X P+S for 24 h. For suspension cells, cells were centrifuged and washed by PBS twice, and then cultured in proper medium with 0.5%FBS and 1X P+S for 24h as the same. Cells were treated with certain concentra-tions of compounds or solvent for 2 h at 37°C. After 2h, cells were stimulated with 5 ng/ml (except where noted) of recombinant human TNF-α (R&D Systems, Inc.) for 20 min. Cells were harvested after 20 min immediately.

24

2.4.2. Cell apoptosis treatment

Before compounds treatment, cells were prepared as previous described but using standard culture medium instead of 0.5% FBS contained medium. And then cells were treated with indicated concentrations of compounds, solvent, or 10 μg/ml of cyclo -heximide (Sigma Chemical Co.) for 24 h before harvest.

2.5. Preparation of whole cell lysates and nuclear extract

2.5.1. Preparation of whole cell lysates

All procedures were done on ice. Cells were washed by ice-cold PBS twice then harvested (adherent cells were scraped off by scraper and suspension cells were cen-trifuged down directly at 4°C, 1500 rpm for 5 min) with 1ml of PBS and transferred to 1.5 ml eppendorf tube. Cells were spun down at 4°C, 10000 rpm for 3 min and added 100 μl of AT buffer [20 mM Hepes, pH 7.9, 1% (w/v) Triton X-100, 20% (w/v) glycerol, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM DTT, 1 mM Na3VO4, 1

μg/ml PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin]. Cells in AT buffer were passed through 26G×1/2” needle around 20 times, and 4 M NaCl was added to a final

con-centration of 150 mM. Finally, cells were centrifuged at 4°C, 13,000rpm for 35 min and the supernatant were collected and stored at -80°C.

2.5.2. Preparation of nuclear extracts

All procedures were done on ice. As the preparation of whole cell lysates, cells were harvested by 1 ml of ice-cold PBS and transferred to a 1.5 ml eppendorf tube (two 100 mm dishes of cells were collected to one tube). And then, cells were resus-pended in 400 μl of buffer E (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, pH 8.0, 0.1 mM EGTA, pH 8.0) supplemented with 1 mM DTT, 1 μg/ml PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin and incubated at 4°C for 15 min. The following

25

step should be done twice. Cells were added 25 μl of 10% NP-40 and vortexed for 10 s. The mixtures were centrifuged at 10,000 rpm, 4°C for 1 min, and then the pellets were washed by adding 300 μl of PBS and centrifuging down twice. The pellets were then resuspended in 80 μl of buffer F (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA) supplemented with 1 mM DTT, 1 μg/ml PMSF, 1 μg/ml leupeptin and 1 μg /ml pepstatin. The next step should be done twice. The mixtures were vortexed for 30 s and incubated at -80°C for 20 min followed by thawing the mixtures at room temperature. The mixtures were then vortexed for 30 s and centri-fuged at 13,000 rpm, 4°C for 5 min. The supernatant (nuclear extracts) were collected and stored at -80°C.

2.6. SDS-polyacrylamide gel electrophoresis

SDS- polyacrylamide gel was prepared and electrophoresed by Mini- PROTEAN® Tetra (Bio-Rad). The gel comprises a resolving gel and a stacking gel and. For the preparation of resolving gel, 1X resolving buffer [375 mM Tris-base, pH 8.8, 0.1% (w/v) SDS] containing 10% acrylamide which was diluted from 30% acrylamide (SERVA) was polymerized by adding appropriate of 10% ammonium persulfate (APS) and N,N,N’,N’-tetramethyl- ethylenediamine (TEMED). The bubbles were expelled by the addition of 200 μl of isopropanol. The isopropanol was removed until the re-solving gel was already polymerized, and stacking gel would be prepared by the mix-ture of 1X stacking buffer [125 mM Trix-HCl, pH 6.8, 0.1% (w/v) SDS], 5% acryla-mide, and appropriate 10% APS and TEMED. The stacking gel mixture then was poured on the top of resolving gel and covered by comb.

To prepare samples, the 25 μg (depends on what kind of lysates and western blot target) of protein extract were diluted with 5X SDS loading dye [87.5 mM Tris-HCl, pH 6.8, 45% (v/v) glycerol, 5% (w/v)SDS, 0.25% (w/v) bromophenol blue, 12.5%

26

(v/v) 2-mercaptoethanol (2-ME)] to final concentration of 1X SDS loading dye and filled to total 20 μl by ddH2O. The samples were heated at 98°C for 10 min and

cooled down at room temperature before loaded. Then, the gel was electrophoresed in 1X SDS-running buffer [25 mM Tris-base, 192 mM glycine, 0.1% (w/v) SDS] at 80 volts until the loading dye passed through the stacking gel, and then the voltage was changed to 120 volts.

2.7. Western blot analysis

When gel electrophoresis was finish, protein should be transferred to PVDF mem-brane (Immobilon® -P Transfer Memmem-brane, Millipore) in 1X transfer buffer (20 mM Tris-HCl, 150 mM glycine) containing 20% methanol at 250 mA for 1-2 h by using the transfer system (Mini Trans-Blot® Cell, Bio-Rad). It’s important that PVDF membrane needed to activate by methanol before transferring. After transfering, the membrane was blocked in 5% (w/v) not-fat milk dissolved in TBST buffer [20 mM Tris-HCl, 2.5 mM KCl, 150 mM NaCl, 0.05% (v/v) Tween-20, pH 7.5] for 1 h at room temperature or overnight at 4oC. Then, membrane was dipped in primary anti-body diluted with 5% not-fat milk in TBST at room temperature for 1 h; as for the anti phospho-protein antibody, membrane was suggested to incubate at 4°C overnight. Followed by washing 3 times for 10 min by TBST, membrane was transferred to sec-ondary antibody which was conjugated with Horseradish peroxidase (HRP) at room temperature for 1 h. After 3 times washing by TBST, the proteins were detected by SuperSignal® West Dura Extended Duration Substrate or SuperSignal® West Pico Chemiluminescent Substrate (Thermo).

For stripping the previous antibody, the membrane was incubated in stripping buffer (62.5 mM Tris-HCl, pH 7.0, 2% SDS, 100 mM 2-ME) at 50oC for 5 min, and then was washed three times in TBST.

27

The antibodies used in this study were listed previous.

2.8. Electrophoretic mobility shift assay (EMSA)

The native gel for EMSA was prepared and electrophoresed by Mini- PROTEAN® Tetra (Bio-Rad). A native gel was made by that 1X TBE contained 6% of acrylamide and 2.5% of glycerol, followed by adding appropriate 10% APS and TEMED in order to polymerize the gel. The κB site probe (κB site: 5’-GGGAAATTCC-3’ (Liang et al., 2006) labeled with biotin was ordered from Protech Technology Enterprise Co., LTD. And the binding buffer and biotin detection system were from LightShift® Chemiluminescent EMSA Kit (Thermo).

For the binding reaction, 15 μg of nuclear extract and 25 nM probe for final con-centration were mixed in binding buffer in total volume of 20 μl, and the mixtures were incubate at room temperature for 20 min. Then, the sample mixtures were loaded with 5X loading dye contained in the kit and electrophoresed in 0.5X TBE at 80 volts until the loading dye migrated approximately 3/4 down the length of the gel. Before transfer, the NC membrane was soaked in 0.5X TBE for at least 10 min. The transfer was carried out by transfer system (Mini Trans-Blot® Cell, Bio-Rad) in ice-cold 0.5X TBE buffer at 380 mA for 1 h. Subsequently, the transferred DNA was cross-linked to membrane for 20 min with the membrane face down on a transilluminator equipped with 312 nm bulbs (UV box of UNIVERSAL HOOD, Bio-Rad)

Before the detection of biotin-labeled DNA, the blocking buffer and 4X wash buff-er have to pre-warm until all particulate is dissolved. To block the membrane, mem-brane was soaked in blocking buffer for 15 min with gentle shaking, followed by transferring the membrane to anti-biotin antibody which is prepared with blocking buffer in 1:300 dilution and incubating for 15 min. After the incubation of antibody, the membrane was washed 4 times with 1X wash buffer which was diluted by ddH2O

28

each time for 5 min. And then, membrane was removed to appropriate volume of Substrate Equilibration Buffer for 5 min with gentle shaking. Finally, the membrane was transferred to substrate mixture which is prepared before used for 3-5 min, and detected by exposure to film.

29

Results

3.1. Transcription factor NF-κB inhibitor screening from marine natural

prod-ucts

In a collaborative effort to discover potent NF-κB inhibitor, we screened more than 70 extracts (supplementary data 1) from a variety of marine resources provided by Dr. Chung-Kuang Lu’s lab at National Research Institute of Chinese Medicine. Using a previously established NIH3T3 cell system (Liang et al., 2003; Liang et al., 2006), we were able to utilize TNFα as an activator of the NF-κB signaling for detecting inhibi-tors of this pathway.

Among all extracts tested, we found that WYT1-33-6, an extract from the marine bacterium Zooshikella sp., was a potent NF-κB inhibitor. At a concentration of 20 µg/ml, WYT1-33-6 effectively protected IκBα from TNFα-induced degradation in the NIH3T3 cells (supplementary data 1). Furthermore, we treated cells with increasing concentrations (0-20 µg/ml) of WYT-33-6 and found that TNFα induced IκBα degra-dation was inhibited in a dose-dependent manner (Fig. 3A). This result suggested that WYT1-33-6 can against TNFα induced signal and inhibit the degradation of IκBα. However, this inhibition ability seems to reach the top at 10 μg/ml of WYT1-33-6 since the data showed that there was no apparent difference between IκBα ratio in cells treated with either 10 μg/ml or 20 μg/ml of WYT1-33-6 (Fig. 3B).

3.2. WYT1-33-6 inhibits NF-κB activation by blocking IκBα phosphorylation

and NF-κB nuclear translocation in TNFα-induced NIH3T3 cells

Previous studies revealed that phosphorylation of IκBα N-terminal Ser residues is a prerequisite event for proteasome-mediated IκBα degradation (Hayden and Ghosh, 2004). Therefore, we were interested in determining whether WYT1-33-6 could block

30

the phosphorylation of IκBα. Results from Figure 4 showed that the amount of phos-phorylated IκBα and total IκBα in different TNFα induction time from 0 to 20 min. The IκBα protein of the solvent control was phosphorylated within 5 min after TNFα induction (Fig. 4A, Fig. 4B), and then the phosphorylated IκBα protein was degraded by proteasome at 10 and 20 min after TNFα induction (Fig. 4A, Fig. 4C). In contrast, 20 μg/ml WYT1-33-6 treated cells presented low level of phosphorylated IκBα and constant amount of IκBα (Fig 4). However, the 20-min TNFα induction in the solvent control (Figure 4A) showed a rising IκBα expression which is contrary to the previous data (Fig. 3). Indeed, the IκBα upregulation supports the general understanding that IκBα is resynthesized once NF-κB signaling pathway was activated and can be ex-plained by delay of harvesting. Overall, it was proved that WYT1-33-6 blocks the degradation of IκBα through repressing the phosphorylation of IκBα.

NF-κB nuclear translocation is a downstream event following IκBα degradation in the classical TNFα-induced NF-κB activation (Hayden and Ghosh, 2004). Because our data showed that TNFα-induced IκBα phosphorylation and degradation were blocked by WYT1-33-6 (Figures 3 and 4), we then tested whether WYT1-33-6 could inhibit the NF-κB subunit p65 nuclear translocation in the 3T3 cells. The result showed that the amount of the nuclear p65 protein in the 20 μg/ml WYT-33-6 treated cells was significantly less than that of the solvent control in the TNFα-activated NIH3T3 cells (Fig. 5). In summary, this result is consistent with our previous data and suggests that WYT-33-6 is a potent inhibitor of the NF-κB activation by blocking IκBα phosphorylation and degradation as well as NF-κB nuclear translocation.

3.3. WYT1-33-6 inhibits cell viability and induces apoptosis in the human

cer-vical cancer HeLa cells

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Zooshikella sp., is an effective inhibitor of the NF-κB activation in TNFα-induced

mouse fibroblast 3T3 cells. We then examined whether WYT-33-6 could also inhibit NF-κB activation in human cancer cell lines. We first tested the frequently used hu-man cervical cancer cell line HeLa and found that WYT-33-6 did not block TNFα-induced NF-κB activation in this particular cell line (data not shown). This re-sult indicates that the NF--κB inhibition effect of WYT-33-6 is cell line-dependent.

We next studied whether WYT1-33-6 could exert cell toxicity in HeLa cells. Cells were treated with increasing concentrations of WYT1-33-6 for 24 h and the cellular morphological changes were examined using microscopy. Cycloheximide, the protein biosynthesis inhibitor in eukaryotic organisms, was used as a control of cell toxicity. After 24 h incubation, HeLa cells treated with 10 μg/ml of WYT1-33-6, compared with the solvent control, had a dramatic decrease in cell number and showed cell shrinkage in size(Fig. 6A). On the other hand, cycloheximide treated control also had cell shrinkage morphology. Interestingly, WYT1-33-6 treated cells showed a deep red color, the same color of the WYT1-33-6 (6A).

In addition to cellular morphological study, we assessed cell viability using MTT assay in WYT1-33-6 treated HeLa cells. After 24 h of drug incubation, the cell growth inhibition effect of WYT1-33-6 was determined by the MTT assay. The 50% of cell growth inhibition (IC50) of WYT1-33-6 was approximately 15μg/ml in HeLa

cells (Fig. 6B).

Because WYT1-33-6 inhibited cell growth as judged by MTT assay, we wanted to determine whether this agent could also induce apoptosis in HeLa cells. Cycloheximide was used as a positive apoptosis control. Poly (adenosine 5’-diphosphoateribose) polymerase (PARP) cleavage was used as an apoptosis marker. Same amount of HeLa cells were seeded and treated with different concentrations of WYT1-33-6 for 24 h and cell lysate was subjected to Western blots for PARP

cleav-32

age. The results revealed that WYT1-33-6 dose-dependently induced PARP cleav-age.(Fig. 7). In summary, our studies showed that WYT1-33-6 is a potent agent in in-hibiting cell growth and inducing apoptosis in HeLa cells.

3.4. Major bioactive components of WYT1-33-6, WYT170-6 (prodigiosin) and

WYT1-70-10 (a prodigiosin analog), are cytotoxins in multiple myeloma cells

To take a further study in WYT1-33-6, our collaborator Dr. Chung-Kuang Lu and his lab members separated WYT1-33-6 into 11 fractions (WYT1-49-1 to WYT1-49-11). In order to examine which fraction contributed most to the bioactivity found in WYT1-33-6, we took the NF-κB inhibitor screening procedures previously mentioned in the TNFα induced 3T3 cells. The results suggested that two fractions, WYT1-49-6 and WYT1-49-8, could inhibit IκBα degradation (Fig. 8A). Also, results from cell growth inhibition effect assessed by MTT and MTS assay in several cancer cell lines supported the suggestion that these two were the most bioactive fractions in WYT1-33-6 (supplementary data 2). Dr. Lu and his students further purified WYT1-49-8 and obtained 2 compounds, prodigiosin (WYT1-70-6) and its analog 2-methyl-3-heptyl prodigiosin (WYT1-70-10) (Fig. 8B). Moreover, WYT1-49-6 was also separated into 6 fractions (WYT1-96-1 to WYT1-96-6). However, only one frac-tion, WYT1-96-4, had NFκB inhibitory effect in the TNFα induced 3T3 cells (data not shown). Our studies later confirmed that the main effective component of WYT1-96-4 was also prodigiosin (Appendix 2.). For this reason, the following studies were focused on two bioactive compounds WYT1-70-6 (prodigiosin) and WYT1-70-10 (a prodigiosin analog).

Not only the high connection between NF-κB signaling pathway and multiple my-eloma but the incurable property and low 5 years survival rate, we selected the multi-ple myeloma cell line, RPMI 8226, as the target for our research. As precious

proce-33

dure described, we first examined whether WYT1-70-6 and WYT1-70-10 could in-hibit TNFα induced IκBα degradation in 3T3 cells. Our results showed that WYT1-70-6 mediated NF-κB inhibition in TNFα-induced RPMI 8226 cells in a dose-depend manner (Fig. 9), whereas the WYT1-70-10 displayed no NF-κB inhibi-tion (Fig. 10). Interestingly, these two compounds were both effective in inducing apoptosis and suppressing cell growth in the multiple myeloma RPMI 8226 cell line (Fig. 13). Both compounds began to induce PARP cleavage at the concentration of 2.5 μM (Fig. 11, Fig. 12). Moreover, the IC50 value for both compounds were

approxi-mately 0.5 μM as judged by MTS assay (Fig. 13). Although WYT1-70-10 could in-duce apoptosis and inhibiting cell viability in RPMI 8226 cells, we were unable to determine its molecular target yet. Therefore, we decided to focus our studies on the NF-κB inhibitor WYT1-70-6 (prodigiosin).

3.5. The DNA binding ability of NF-κB is inhibited by WYT1-70-6 through

blocking NF-κB translocation in human multiple myeloma RPMI 8226 cells

Our previous study showed that WYT1-70-6 (prodigiosin) blocked the degradation of IκBα in TNFα induced human multiple myeloma RPMI 8226 cells (Fig. 9). We next examined whether NF-κB nuclear translocation could be blocked by WYT1-70-6 as judged by Western blot for the NF-κB subunit p65 using cell nuclear extracts. The result showed that the level of p65 protein in the nuclear extracts of WYT1-70-6 treated RPMI 8226 cells was significantly decreased compared with that of the sol-vent control (Fig. 14). The result suggested that WYT1-70-6 effectively blocks the nuclear translocation of the NF-κB subunit p65.

Although our studies demonstrated that p65 translocation was inhibited by WYT1-70-6 in RPMI 8226 cells, the function of NF-κB which binds to specific se-quence of DNA (κB site) and turns on the downstream genes had not been examined.