黃芩素經由泛素-蛋白分解體降解路徑抑制survivin 蛋白的表現

國立交通大學生物科技學系

碩士論文

黃芩素經由泛素-蛋白分解體降解路徑抑制

survivin 蛋白的表現

Inhibition of survivin protein expression by

ubiquitin-proteasome degradation pathway

following baicalein

研究生: 黃厚巽

指導教授: 趙瑞益 教授

黃芩素經由泛素-蛋白分解體降解路徑

抑制

survivin 蛋白的表現

Inhibition of survivin protein expression by

ubiquitin-proteasome degradation pathway following baicalein

研究生: 黃厚巽 Student: Hou-Syun Huang

指導教授: 趙瑞益 Advisor: Jui-I Chao

國立交通大學生物科技學系

碩士論文

A Thesis

Submitted to Department of Biological Science and Technology

College of Biological Science and Technology

National Chiao Tung University

in partial Fulfillment of the Requirements

for the Degree of

Master

In

Biological Science and Technology

July 2010

Hsinchu, Taiwan, Republic of China

致謝

時間過的真快，在趙老師實驗室經歷了兩年的學習與訓練，到如今即將畢 業了。學業上在老師的提攜以及學長姐的帶領下、同學間的教學相長，對於實 驗研究有更進一步的了解與體認，並且增進了自己面對困難、解決問題的能力。 在這段時間以來與實驗室夥伴們的學習相處下，我也學到更多待人處事的經 驗，這是教科書上學不到的東西。在趙老師的指導下，使我學到做研究應有的 邏輯觀念與學術素養，並且讓我有機會嘗試實驗室以往沒有的實驗技術和方 法。實驗室的兩大支柱，惠芳學姊以及光凱學長，在我遭遇問題時挺身而出， 指點迷津讓問題迎刃而解；並且在分生達人的啟蒙下，學習到的分生實驗與知 識上讓我獲益良多。生技公司上班的和欽學長，讓我了解在業界所需具備的能 力以及所應抱持的態度。在剛開始對實驗室不熟悉的階段，阿良跟真宜學姐從 養細胞開始總是不厭其煩地一步一步帶領我走入研究的世界。在實驗室共患 難、同進退的同學們，靜怡、繼慶跟勝壹兩年來的相處下，度過了許多美好的 歡笑時光，也遭遇了許多困難與挫折。遇到困難時大家總是互相討論、幫助我 解決問題以及面對當前的挑戰；遭遇挫折以及心情低落時，他們給我陪伴安慰 以及背後的支持；閒暇之虞，出遊散心以及互相打嘴砲也成為我美好的回憶。 實驗室的美女學妹團，婷婷、淳淳、阿簡和阿蓮，有了她們的加入使得原本枯 燥乏味的實驗生活更顯多采多姿。我們的助理白雲大哥，成為我們實驗室的得 力助手。還有昱耀、旻翰、Tammy 與玉梅有著年輕的活力，他們的認真的學習態 度也是我效訪的對象。還有張家靖老師家與曾慶平老師家無數幫助過我的學長 姐和同學們協助以及指導我解決實驗上許多的問題與困難。天天守候在外的大 溝、耳洞跟皮皮，每天忠實陪伴著我們，在心情低落時，他們就像老朋友一樣 安慰著你，陪你走到車棚為單調的生活帶來一絲樂趣。最後要歸功於我的家人， 辛苦的從小供給我唸書到如今，他們是我最大的後盾，讓我沒有後顧之憂的完 成碩士學位，並且感謝這些日子以來所有幫助過我的每一個人。

Contents

Contents

...

I

中文摘要

...

VI

Abstract

...

VII

1. Introduction

...

1

1.1. Flavonoids and anticancer activities... 1

1.2. Apoptosis pathways ... 2

1.3. Baicalein and apoptosis ... 3

1.4. Baicalein and cell cycle progression... 3

1.5. Baicalein and tumorigenesis inhibition ... 4

1.6. Survivin and cancer ... 5

1.7. Survivin and cell division ... 6

1.8. Survivin and apoptosis... 7

1.9. Survivin and cancer therapy ... 7

1.10. Ubiquitin-proteasome system ... 8

1.11. Survivin and bladder cancer ... 9

1.12. Oxaliplatin and cancer therapy ... 10

2. Materials and methods

...

12

2.1. Reagents and Antibodies ... 12

2.2. Cell culture... 12

2.3. Cell viability assay... 13

2.4. Time-lapse of living cell image analysis ... 13

2.5. Annexin V/PI apoptosis assay ... 14

2.6. Western blot... 14

2.7. Reverse transcription-polymerase chain reaction (RT-PCR) ... 15

2.8. Quantitative real-time PCR... 16

2.9. Immunoprecipitation... 17

2.10. Immunofluorescence staining and confocal microscopy... 18

2.11. Statistical analysis... 19

3. Results

...

20

3.1. Baicalein induces cytotoxicity and proliferation inhibition in bladder cancer cells ... 20

3.2. Baicalein inhibits survivin protein expression but not altered gene expression in bladder cancer cells ... 20

3.3. Baicalein reduces the protein stability of survivin in bladder cancer cells.. 21

degradation in bladder cancer cells... 22

3.5. Baicalein treatment increases ubiquitination of survivin... 22

3.6. Co-treatment of baicalein and oxaliplatin enhances the cytotoxicity and survivin protein inhibition in bladder cancer cells ... 23

4. Discussion ...24

5. Conclusion ...27

List of figures

Fig. 1. Effect of baicalein on the cell viability in BFTC905 bladder cancer cells... 49

Fig. 2. Time-lapse observation of baicalein-induced cancer cell death... 50

Fig. 3. Effect of baicalein on the protein levels of survivin in bladder cancer cells... 51

Fig. 4. Effect of baicalein on the survivin mRNA level in bladder cancer cells by

RT-PCR analysis. ... 52

Fig. 5. Effect of baicalein on the survivin mRNA level in bladder cancer cells by

real-time PCR ... 53

Fig. 6. Stability of survivin protein in the baicalein-treated in bladder cancer cells .. 54

Fig. 7. Effect of MG132 (a proteasome inhibitor) on the baicalein-inhibited survivin

... 55

Fig. 8. Effect of MG132 on the apoptosis level in the baicalein-treated BFTC905 cells

... 56

Fig. 9. Ubiquitination of survivin in baicalein-treated BFTC905 cells. ... 57

Fig. 10. Effects of baicalein on the co-expression of survivin and ubiquitin proteins in

bladder cancer cells... 58

Fig. 11. Effect of co-treatment with baicalein and oxaliplatin on the cell viability in

bladder cancer cells... 59

cancer cells... 60

Fig. 13. Proposed model of survivin down-regulation by baicalein in human bladder

cancer cells... 61

Appendix 1. Function of survivin in mitosis ... 62

中文摘要

Survivin 是一種抗細胞凋亡的蛋白，具有促進癌細胞存活與增生的功能。黃 芩素會誘發癌細胞生長停止及促進細胞凋亡的作用，然而黃芩素調控survivin 蛋 白表現的機制仍然不清楚。在本研究中，我們探討BFTC905 人類膀胱癌細胞處 理黃芩素後，survivin 的角色與調控機制。處理黃芩素明顯地抑制 BFTC905 細 胞中survivin 蛋白的表現。雖然如此，利用反轉錄聚合酶鏈反應與即時聚合酶鏈 反應分析，發現黃芩素並不會影響survivin mRNA 的表現量。利用蛋白質合成抑 制劑cycloheximide 處理後，會促進黃芩素減少 survivin 蛋白的表現量。相反地， 處理一種蛋白分解體的抑制劑 MG132，能部分回復黃芩素所抑制的 survivin 蛋 白表現，並且MG132 會降低黃芩素所誘發的細胞凋亡。有趣地，利用免疫沉澱 分析發現黃芩素會誘發survivin 蛋白進行泛素化作用，泛素化的 survivin 蛋白被 黃芩素大量誘發。此外，共同處理黃芩素與oxaliplatin 會加強對 BFTC905 細胞 的毒殺作用及抑制survivin 蛋白的表現。綜合以上結果，我們推測黃芩素抑制人 類膀胱癌細胞中survivin 蛋白表現，是經由泛素-蛋白分解體的降解路徑。

Abstract

Survivn is an anti-apoptosis protein that plays the roles in promoting cancer cell

survival and proliferation. Baicalein has been shown to induce growth inhibition and

apoptosis in cancer cells; however, the regulation of survivin protein expression by

baicalein remains unclear. In this study, we investigated the role and regulation of

survivin following baicalein treatment in the BFTC905 human bladder cancer cells.

Treatment with baicalein markedly inhibited survivin protein expression in BFTC905

cells. Nevertheless, the survivin mRNA level did not alter with baicalein treatment

using reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR.

Treatment with a protein synthesis inhibitor (cycloheximide) increased the decrease

of survivin protein level in the baicalein-treated cells. In contrast, a specific

proteasome inhibitor (MG132) partially restored baicalein-inhibited survivin protein

expression. Moreover, MG132 can reduce apoptosis induction by baicalein.

Interestingly, baicalein induced the protein ubiquitination of survivin using

immunoprecipitation assays. The ubiquitinated survivin proteins were increased by

baicalein. Besides, co-treatment of baicalein and oxaliplatin enhanced the cytotoxicity

and survivin protein inhibition in BFTC905 cells. As a consequence, we suggest that

1. Introduction

1.1. Flavonoids and anticancer activities

Flavonoids are a group of polyphenolic compounds that exist in plants,

vegetables, and fruits (Havsteen, 1983). Flavonoids include flavonols, flavones,

isoflavones, flavanones, anthocyanidins, and flavanols (Manach et al., 2004; Scalbert

and Williamson, 2000). Intake of flavonoids has been shown to reduce risk of cancer,

inflammation and heart diseases (Havsteen, 2002; Middleton et al., 2000). Various

types of flavonoids, such as luteolin, quercetin, kaempferol and catechin, display

anticancer effects on growth inhibition and apoptosis (Brusselmans et al., 2005; Lee

et al., 2005a; Psahoulia et al., 2007; Spencer et al., 2003; Yin et al., 2001). It has been

shown that flavonoids can induce release of cytochrome c with activation of

caspase-3 and caspase-9 to promote apoptosis (Michels et al., 2005; Wang et al.,

1999). For example, epigallocatechin gallate (EGCG), the major catechin in tea, has

been shown to induce apoptosis in various cancers, including leukemia (Hibasami et al., 1996), prostatic cancer (Brusselmans et al., 2003; Chung et al., 2001), gastric cancer (Horie et al., 2005), colon cancer (Chen et al., 2003), and lung cancer (Yang et

1.2. Apoptosis pathways

Apoptotic features include cell membrane blebbing, cell shrinkage, chromatin

condensation and DNA fragmentation, finally ending with the engulfment by

macrophages or neighboring cells, thereby avoiding an inflammatory response in

surrounding tissues (Savill and Fadok, 2000). Apoptosis can be separated two major

pathways incliding extrinsic and intrinsic pathways. External apoptotic pathway is

initiated by death ligands binding to their receptors such as CD-95/fas receptor and

TNFα-receptor, which is followed by activation of initiator caspase-8 to induce the

downstream apoptotic pathway (Walczak and Krammer, 2000). Intrinsic factors or

intracellular stimuli such as DNA damage can mediate mitochondrial apoptotic

pathway to initiate the release of cytochrome c and SMAC/DIABLO for apoptosis

induction (Shi, 2002). Both extrinsic and intrinsic pathways lead to activation

caspase-3 for apoptotic induction (Scaffidi et al., 1998; Shi, 2002). The inhibitor of

caspase-activated deoxyribonuclease (CAD) can be cleaved by activated caspase-3 to

release CAD (Enari et al., 1998; Sakahira et al., 1998). Then CAD enters the nucleus

to degrade the chromosomal DNA and leading to DNA fragmentation and cell death

(Enari et al., 1998). The NF-κB is a transcriptional factor which activates various

antiapoptotic signals or proteins can promote cancer cell survival and tumorigenesis

(Francois et al., 2005; Nakano et al., 2006; Suh and Rabson, 2004).

1.3. Baicalein and apoptosis

Baicalein is a bioactive flavonoid extracted from root of Scutellaria baicalensis

or Scutellaria radix that contains anticancer activities (Bonham et al., 2005; Chao et

al., 2007; Ma et al., 2005). It has been found that baicalein induce apoptosis in a

variety of human cancer cells (Chao et al., 2007; Chen et al., 2000; Kuntz et al., 1999;

Lee et al., 2005b; Ma et al., 2005; Pidgeon et al., 2002; Wang et al., 2009). Baicalein

induces cancer cell death which is associated with regulating CDK1 kinase and

survivin in bladder cancer cells (Chao et al., 2007). Moreover, baicalein induces

apoptosis by decreasing Bcl-2 and increasing p53 and Bax human in lung cancer cells

(Leung et al., 2007). In addition, NF-κB-regulated anti-apoptotic genes including

Bcl-2 and Bcl-XL have been shown that they are suppressed by baicalein (Lee et al.,

2005b; Pidgeon et al., 2002).

1.4. Baicalein and cell cycle progression

The regulation of cell cycle progression is regulated by cyclin dependent kinase

with cyclins and negatively regulated by CDK inhibitors (CDKIs) (Schwartz and

Shah, 2005; Shapiro, 2006). Baicalein has been shown to reduce cyclin D proteins to

mediate cell cycle arrest in breast, lung and prostate cancer cells (Lee et al., 2005b;

Pidgeon et al., 2002). Furthermore, baicalein declines the protein expression of cyclin

B1 and CDK1 protein levels in lung cancer cells (Lee et al., 2005b; Leung et al.,

2007). Down-regulation of CDK1 and Cyclin B1 by baicalein is involved in the

regulation of S phase progression (Leung et al., 2007). Baicalein suppresses CDK 2/4

expression and inhibits of the expression of p21 and p27 in prostate cancer cells

(Pidgeon et al., 2002). Baicalein also involves G1 and G2 arrest in association with

repression of CDK1, CDK2, cyclin D2 and cyclin A proteins, and with up-regulation

of cyclin E, p15, p53 and p21 (Hsu et al., 2001). In addition, baicalein can induce

G2/M arrest in leukemia cells (Roy et al., 2007). The levels of cyclin B1 and CDK1

are reduced for inducing G2/M arrest by baicalein in bladder cancer cells (Chao et al.,

2007). Moreover, baicalein arrested S and G2/M phase in breast cancer cells (Wang

et al., 2009). Accordingly, baicalein displays anticancer ability by mediating the

blockage of cell cycle progression in various cancer cell types.

1.5. Baicalein and tumorigenesis inhibition

(Bonham et al., 2005; Miocinovic et al., 2005). For example, baicalein displays the

ability to attenuate tumor growth in pancreatic tumor mouse model (Tong et al.,

2002). The antitumor effects of baicalein inhibits tumorigenesis in C3H/HeN mice

implanted with murine bladder cancer cells (Ikemoto et al., 2004).

1.6. Survivin and cancer

The survivin gene is located on the human 17q25 chromosome that expresses a

16.5-kDa protein (Ambrosini et al., 1997). Survivin belongs to the smallest member

of the IAP (inhibitor of apoptosis proteins) family (Deveraux et al., 1998), which

contains a single 76-amino of the characteristic zinc finger baculovirus–inhibitor of

apoptosis repeat (BIR) domain that is essential for the caspase-inhibitory function

(Altieri, 2003; Ambrosini et al., 1997; Li et al., 1998). Survivin, it is homodimeric,

arranged through hydrophobic surface of the BIR domain of each survivin monomer

(Chantalat et al., 2000; Verdecia et al., 2000). Survivin, a unique inhibitor of

apoptosis, expressed in embryonic and fetal organs in the developmental stages but

undetectable in normal adult tissues (Ambrosini et al., 1997). Furthermore, survivin is

selectively expressed in transformed cells and in most human cancers including lung,

breast, pancreatic, and colon carcinomas, soft tissue sarcomas, brain tumors,

Chakravarti et al., 2002; Grossman et al., 1999; Islam et al., 2000; Kappler et al.,

2004; Kawasaki et al., 1998; Monzo et al., 1999; Satoh et al., 2001; Tanaka et al.,

2000). Additionally, three isoforms of survivin have been found in human cells

including survivin‐2β, survivin‐∆Ex3, and survivin‐3β (Mahotka et al., 1999).

1.7. Survivin and cell division

Survivin is one of chromosome passenger proteins that regulates cell division

(Ambrosini et al., 1997). The conserved mitotic complex of aurora‐B, INCENP,

borealin, and survivin is essential for chromosome movements during mitosis, proper

spindle checkpoint surveillance, and execution of cytokinesis (Lens et al., 2006).

Appendix 1 shows that survivin can control motosis progression (Mita et al., 2008).

Chromosome passenger proteins are required to target the complex to kinetochores,

correct misaligned chromosomes, properly form the central spindle and complete

cytokinesis for equal and complete cell division (Gassmann et al., 2004; Honda et al.,

2003; Wheatley et al., 2001). Survivin localizes to kinetochores at metaphase,

transfers to the central spindle midzone at anaphase, and accumulates in midbodies at

telophase (Vagnarelli and Earnshaw, 2004). Moreover, the phosphorylation of

survivin on Thr34 by CDK1-cyclin B1 has been reported with increased survivin

1.8. Survivin and apoptosis

Mammalian IAPs family including XIAP (X-linked IAP), c-IAP1, 2, NAIP

(neuronal apoptosis-inhibiting protein), and survivin specifically inhibit the apoptosis

(Miller, 1999; Salvesen and Duckett, 2002). XIAP, cIAPs, and NAIP have been

shown to inhibit the caspase-3, -7, and -9 proteins by binding specific regions of

caspases (Riedl and Shi, 2004). It has been shown that survivin inhibits caspase-3, -7,

and -9 but not caspase-8 (Shin et al., 2001; Tamm et al., 1998). However, survivin

also can mediate caspase-3-independent pathway (Banks et al., 2000).

Smac/DIABLO is a pro-apoptotic protein and participates in the activation of

caspase-9 (Srinivasula et al., 2000). Survivin has affinity with Smac/DIABLO to

inhibit apoptosis by antagonizing the pro-apoptotic ability of Smac/DIABLO (Altieri,

2003).

1.9. Survivin and cancer therapy

Survivin displays both cell division and anti-apoptosis that can promote

tumorigenesis. Survivin has been shown to promote angiogenesis (Blanc-Brude et al.,

2003; Conway et al., 2003; Kawasaki et al., 2001; O'Connor et al., 2000b; Tran et al.,

inhibit tumorigenesis and angiogenesis providing for cancer therapy. Antisense

oligonucleotides, siRNA and dominant-negative mutants of survivin are successfully

exploited to suppress survivin expression (Koul et al., 2006; Marusawa et al., 2003;

Nakao et al., 2006; Zhang et al., 2001). Small-molecule antagonist suppresses

survivin transcription by inhibiting promoter of survivin providing potential cancer

therapy (Nakahara et al., 2007). Additionally, survivin has been predicted response to

chemotherapy and radiotherapy in patients with bladder cancer (Als et al., 2007),

breast cancer (Hinnis et al., 2007), multiple myeloma (Nakagawa et al., 2006), and

lymphoma (Adida et al., 2000; Schlette et al., 2004; Watanuki-Miyauchi et al., 2005).

1.10. Ubiquitin-proteasome system

Ubiquitin-proteasome system (UPS) is an essential mechanism involved in

cellular process such as degradation, cell cycle regulation, antigen processing, signal

transduction and transcription (Boutillier et al., 1999; Nandi et al., 2006; Orlowski,

1999). One of UPS important function involves in ubiquitin-proteasome-dependent

degradation of proteins (Ciechanover et al., 1980). The target protein is labeled with

multiple ubiquitin moieties and degraded by the 26S proteasome (Ciechanover and

Schwartz, 1998; Laney and Hochstrasser, 1999). The 76-amino-acid ubiquitin

between the ubiquitin and lysine side chains in the target proteins. Free Ub is

recruited by the E1 (Ub-activating enzyme) and transferred to lysine residue of the E2

(Ub-conjugating enzyme) (Haas et al., 1982). E2 and substrate are bound by the E3

(Ub-protein ligases), which is responsible for substrate recognition. E3 involves that

ubiquitin is transferred to a lysine amino groups of the target protein, then E2 and E3

are released (Hershko et al., 1986). The cyclic transfer of more Ub to the first Ub attached to the substrate is by E4 (ubiquitin-chain elongation factor)(Hoppe, 2005).

Appendix 2 shows that ubiquitin-proteasome system (Donohue and Osna, 2003).

Degradation of survivin occurs by the ubiquitin-proteasome pathway at the G1 phase,

and it is stabilized when heat shock protein 90 (Altieri, 2004; Fortugno et al., 2003;

Zhao et al., 2000). It has been shown that flavonoids such as kaempferol and

quercetin enhance apoptosis by degradation of survivin in glioma cells (Siegelin et al.,

2008; Siegelin et al., 2009). Furthermore, indomethacin reduced half-life of survivin

and increased survivin ubiquitination (Chiou and Mandayam, 2007). In addition,

chlamydocin involved survivin degradation by proteasome in ovarian cancer cells

(De Schepper et al., 2003).

1.11. Survivin and bladder cancer

death in the year 2009 in the United States (Jemal et al., 2009). Survivin expression

improves our prediction of cancer recurrence and survival in bladder cancer patients

(Shariat et al., 2009). Survivin signaling pathways have been evaluated for

survivin-targeted therapy in bladder cancer (Shariat et al., 2007). Blockage of

survivin expression induces apoptosis and suppresses the growth of the tumor in

bladder cancer cells (Fuessel et al., 2006; Ku et al.). Moreover, detection of survivin

and its associated gene may provide an early biomarker of aggressive tumor behavior

in the bladder cancers (Salz et al., 2005).

1.12. Oxaliplatin and cancer therapy

Oxaliplatin, a clinical anticancer drug, is a third-generation platinum compound

that confers a different spectrum of activity compared with cisplatin (Hochster et al.,

2003; Ramanathan et al., 2003). Like cisplatin, oxaliplatin acts as an alkylating agent

on DNA, forming platinated intrastrand cross-linksbetween two adjacent guanine

bases or two adjacent guanine–adenine bases that result in the blockage of replication

and transcription (Fink et al., 1997). The combination has proven efficacy in

5-fluorouracil-resistant advanced disease and in previously untreated colorectal

cancer (Andre et al., 1998; Maindrault-Goebel et al., 1999). Combination of

therapy in recent years. For example, combination of cyclooxygenase-2 inhibitors and

oxaliplatin increases the growth inhibition and death in colon cancer cells (Lin et al.,

2005). Moreover, oxaliplatin can inhibit survivin protein expression in cancer cells

(Lin et al., 2005).

1.13. The purpose of this study

Our laboratory has provided that baicalein inhibited survivn protein expression

in human bladder cancer cells (Chao et al., 2007). However, the regulation and

mechanism of survivin expression after treatment with baicalein remains unclear. In

this study, the regulation of survivin protein expression is investigated following

baicalein treatment in the human bladder cancer cells. We provide that baicalein

reduces survivin protein expression mediated by the ubiquitin-proteasome pathway.

Moreover, the survivin protein expression is additionally inhibited by combination of

baicalein and oxaliplatin. Understanding the mechanism by which survivin regulates

baicalein-induced apoptosis may provide the identification of novel strategies for

2. Materials and methods

2.1. Reagents and Antibodies

Baicalein, oxaliplatin, Hoechst 33258, and 3-(4,5-dimethyl-thiazol-2-yl)

2,5-diphenyl tetrazolium bromide (MTT), and cycloheximide were purchased from

Sigma Chemical Co. (St. Louis, MO) Anti-survivin (FL-142 and D-8), goat

anti-rabbit IgG horseradish peoxidase, goat anti-mouse IgG horseradish peoxidase,

and the FITC (fluorescein isothiocyanate)-labeled goat anti-mouse IgG antibodies

were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Anti-ubiquitin (Apu2) and anti-actin (C4) antibodies were purchased from Millipore

(Bedford, MA). MG132 (Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) were

purchased from Calbiochem (San Diego, CA). Baicalein was dissolved in DMSO,

and the concentration of DMSO was < 1 % in the control and drug-containing

medium.

2.2. Cell culture

The BFTC905 cell line was derived from human bladder papillary transitional

cell carcinoma of a Chinese female patient. The cells were cultured in complete

supplemented with 10 % fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/

ml streptomycin, and L-glutamine (0.03 %, w/v), and cells were incubated at 37 °C

and 5 % CO2.

2.3. Cell viability assay

BFTC905 cells were plated in 96-well plates at a density of 1 × 104 _{cells/well for}

16–20 h and then treated with various concentrations of baicalein for 24 h in

RPMI-1640 medium. After the treatment, the cells were washed with PBS and were

recultured in RPMI-1640 medium for 2 day. The cells were incubated with 0.5 mg/ml

of MTT in RPMI-1640 medium for 4 h. The surviving cells converted MTT to

formazan, which generates a blue-purple color when dissolved in dimethyl sulfoxide.

The intensity of formazan was measured at 565 nm using a plate reader (VERSAmax,

Molecular Dynamics Inc., CA) for enzyme-linked immunosorbent assays. The cell

viability was calculated by dividing the absorbance of treated cells by that of the

control in each experiment.

2.4. Time-lapse of living cell image analysis

BFTC905 bladder cancer cells were plated at a density of 1.5 × 105 cells Petri

or without 60 μM baicalein by time-lapse observation under an optical phase contrast

microscope with an incubator system (Olympus, Tokyo, Japan).

2.5. Annexin V/PI apoptosis assay

BFTC905 bladder cancer cells were plated at a density of 5 × 105 cells Petri

60-mm Petri dish in complete medium for 16 to 20 h. Thereafter, the cells were

treated with or without 5 μM MG132 for 1 h, then were treated with or without 60

μM baicalein for 24 h. Apoptotic cells was performed using an Annexin-V-FITC

Apoptosis Detection Kit (BioVision, Mountain View, CA) according to the

manufacturer’s instructions. Then cells were collected and resuspended in 500 μl of

binding buffer, and added 5 μl of Annexin-V-fluorescein isothiocyanate (FITC) and 5

μl of propidium iodide (PI). Analyses were performed with a FACSCalibur flow

cytometer (Becton Dickinson, Sunnyvale, CA).

2.6. Western blot

At the end of drug treatment, the cells were lysed in the ice-cold whole cell

extract buffer containing the protease inhibitors. The lysate was vibrated for 30 min at

4 °C and centrifuged at 10,000 × rpm for 10 minutes. Protein concentration was

in samples were subjected to electrophoresis of using 12 % sodium dodecyl

sulfate-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride

membranes and the membranes were blocked overnight at 4 °C using blocking buffer

(5 % non-fat dried milk in solution containing 50 mM Tris/HCl (pH 8.0), 2 mM

CaCl2, 80 mM sodium chloride, 0.05 % Tween 20 and 0.02 % sodium azide).

Thereafter, the membrane were incubated for 2 h at 25 °C with specific primary

antibodies followed by anti-rabbit or anti-mouse immunoglobulin G-horseradish

peroxidase conjugate secondary antibodies. The membranes were washed three times

for 10 min with TBS containing 0.05 % Tween 20. The blot was incubated with

enhanced chemiluminescence detection system (PerkinElmer Life Sciences) for 5 min

and then exposed to X-ray film. To verify equal protein loading and transfer, actin

was used as the protein loading control. The software of Un-Scan-It gel (Ver. 5.1,

Silk Scientific, Inc.) was adopted for semi-quantification of the intensity in each

band.

2.7. Reverse transcription-polymerase chain reaction (RT-PCR)

BFTC905 cells were plated at a density of 2 × 106 cells per 60-mm Petri dish in

culture medium. Total cellular RNA was purified by Trizol reagent (Invitrogen,

determined by a spectrophotometer (Eppendorf, Hamburg Germany). cDNAs were

synthesized by SuperScriptTM III reverse transcriptase (Invitrogen) with oligo-dT12-18

primer (Invitrogen). Each reverse transcript was amplified with GAPDH as an

internal control. The following primer pairs were used for amplification: survivin,

forward primer: 5’-GGCATGGGTGCCCCGACGTTG-3’and reverse primer:

5’-CAGAGGCCTCAATCCATGGCA-3’; GAPDH, forward primer:

5’-CGGAGTCAACGGATTTGGTCGTAT-3’ and reverse primer:

5’-AGCCTTCTCCATGGTGGTGAAGAC-3’. RT-PCR was performed by a DNA

thermal cycler, Mastercycler gradient (Eppendorf, Hamburg Germany), 56 °C for 30

s, and 72 °C for 40 s; and 72 °C for 5 min. The PCR products were visualized on 1.2

% agarose gels with ethidium bromide staining under UV transillumination with a

digital camera system (DH27-S3, Medclub, Taoyuan, Taiwan).

2.8. Quantitative real-time PCR

Each real-time PCR was carried out in triplicate in a 25 μl volume using SYBR

Green qPCR Master Mix (Fermentas Life Sciences,St. Leon-Rot, Germany)

according to the manufacturer’s protocol. Primers sequences were as follows:

survivin 5’-ATTCGTCCGGTTGCGCTTTCC-3’ and

5’-GCGAGAAGATGACCCAGATC-3’ and 5’-GGATAGCAACGCCTGGATAG-3’.

The PCR conditions were for 10 min at 95 °C for initial denaturation, followed by 40

cycles of 95 °C for 15 s and 60 °C for 1 min in the ABI Prism 7000 Sequence

Detection System (Applied Biosystems, Foster City, CA). Relative gene expression

quantifications were calculated according to the comparative Ct method using β-actin

as an internal standard. The fold amplification of genes was respectively detected by

calculating the 2-∆∆Ct of the genes.

2.9. Immunoprecipitation

The PureproteomeTM Protein G Magnetic Beads (Millipore, Bedford, MA) were

mixed so that all of the beads are uniformly resuspended. The beads were plased into

a 1.5 ml microcentrifuge tubes, then tubes were removed into the Magna GrIP Rack

(Millipore). Then the storage buffer was removed with a pipette. The beads were

washed by adding 500 μl of PBS containing 0.1 % Tween® 20 surfactant and

vortexing vigorously for 10 seconds. The tubes were returned to the magnetic rack and allow the beads to adhere to the side. The buffer was removed with a pipette. The

washed beads were resuspend in 350 μl of PBS containing 0.1 % Tween 20 surfactant.

The survivin antibody was added to the resuspended beads with incubation at room

then the buffer was removed with a pipette. The beads were washed 3 times with 500

μl of PBS containing 0.1 % Tween 20 surfactant. After the last wash, the tubes were

removed from the rack and the cell lysates were added. According to the relative

protein expression of survivin in control and baicalein-treated samples, the total protein lysates were adjusted in immunoprecipitation analysis for equal survivin

protein expression of control and baicalein-treated sample. Then samples were

immobilized survivin antibody at 2–8 °C with continuous mixing overnight. The

tubes were placed into the magnetic rack, and then removed the sample with a pipette.

The beads were washed 3 times with 500 μl of PBS containing 0.1 % Tween 20

surfactant. After the last wash, the tube were removed from the magnetic rack and

added the sample buffer for western blot analysis.

2.10.

Immunofluorescence staining and confocal microscopy

To view the protein expression of survivin and ubiquitin after baicalein treatment,

the cells were subjected to immunofluorescence staining and confocal microscopy.

After fixation with 4 % paraformaldehyde solution, the cells were washed three times

with PBS, and non-specific binding sites were blocked in PBS containing 10 % FBS

and 0.3 % Triton X-100 for 1 h at 37 °C. Thereafter, the cells were separately

antibody in PBS containing 10 % FBS for 1 h at 37 °C, and washed three times with

0.3 % Triton X-100 in PBS. Then the cells were individually incubated with goat

anti-mouse Cy3 (1:200) and anti-rabbit FITC in PBS containing 10 % FBS for 1 h at

37 °C. The nuclei were stained with Hoechst 33258. The samples were examined

under a confocal microscope Fluoview 300 (Olympus, Tokyo, Japan).

2.11.

Statistical analysis

Data were analyzed using Student’s t test, and a p value of <0.05 was considered

3. Results

3.1. Baicalein induces cytotoxicity and proliferation inhibition in

bladder cancer cells

To examine the cytotoxicity and proliferation following baicalein treatment in

BFTC905 bladder cancer cells, the cells were analyzed by MTT assay. Treatment

with 20–100 μM baicalein for 24 h significantly reduced the cell viability via a

concentration-dependent manner in BFTC905 cells (Fig. 1). The value of IC50 (the

concentration of 50 % inhibition of cell viability) was around 30 μM. Moreover,

baicalein inhibited cell proliferation and induced cell death that can be observed by

time-lapse living cell morphology observation alteration. The arrows show that

baicalein induced the cell death at 24 h observation (Fig. 2). However, the untreated

cells clearly displayed the increase of cell proliferation and cell number at 24 h

observation (Fig. 2).

3.2. Baicalein inhibits survivin protein expression but not altered

gene expression in bladder cancer cells

To study the effect of survivin protein expression by baicalein in BFTC905

protein levels of survivin were decreased by 20–80 μM baicalein for 24 h in

BFTC905 cells (Fig. 3A and 3B). The quantified data also shows that baicalein

significantly reduced survivin protein expression in BFTC905 cells (Fig. 3B).

Moreover, we have further investigated the survivin expression on transcriptional

levels by reverse transcription-PCR and real-time PCR. The qualities of total RNA

extracts were presented by the contents of 28S rRNA and 18S rRNA (Fig. 4A).

However, baicalein did not alter the survivin mRNA expression (Fig. 4B and 4C).

The survivin mRNA expression in baicalein-treated cells was compared to the control

for 24 h by real-time PCR (Fig. 5A). The mRNA level of survvin was not statistically

altered by treatment with baicalein (Fig. 5B).

3.3. Baicalein reduces the protein stability of survivin in bladder

cancer cells

To further determine the effect of baicalein on the protein stability and half-life

of survivin proteins, a protein synthesis inhibitor (cycloheximide, CHX) was

examined on the effect of survivin protein expression. Treatment with 10 μg/ml CHX

for 24 h reduced around the half of total amount of survivin proteins (Fig. 6A).

However, treatment with CHX and baicalein, the survivin protein levels were

and baicalein almost completely blocked the survivin protein expression after 12 h

period (Fig. 6B).

3.4. Proteasome inhibitor decreases baicalein-induced survivin

protein degradation in bladder cancer cells

To investigate the role of proteasome on baicalein-induced down-regulation of

survivin protein expression, MG132 (a proteasome inhibitor) was utilized in this

study. Treatment with 60 μM baicalein for 24 h significantly reduced survivin protein

expression (Fig. 7). Pre-treatment of BFTC905 cells with 5 μM MG132 potentially

reversed survivin protein level in the baicalein-treated cells (Fig. 7). In annevin V/PI

apoptosis assay, baicalein increased higher apoptosis level than untreated sample in

BFTC905 cells (Fig. 8). Moreover, pre-treatment of MG132 potentially inhibited

apoptosis in the baicalein-treated BFTC905 cells (Fig. 8).

3.5. Baicalein treatment increases ubiquitination of survivin

We have determined the effect of baicalein on the protein ubiquitination of

survivin. As shown in Fig. 9, baicalein induced ubiquitinated survivin levels in

BFTC905 cells. To further confirm the ubiquitination of survivin proteins, the

microscopy. Baicalein reduced the intensity of red fluorescence (Cy3) of survivin

proteins. However, baicalein induced high intensity of green fluorescence of ubiquitin

proteins. The yellow color indicated that co-locolization of survivin and ubiquitin

(Fig. 9, arrows).

3.6. Co-treatment of baicalein and oxaliplatin enhances the

cytotoxicity and survivin protein inhibition in bladder cancer cells

As shown in Fig. 11, co-treatment with 10–50 μM baicalein and 1 μM

oxaliplatin for 24 h enhanced the cytotoxicity in BFTC905 cells. To study the

combination effect of baicalein and oxaliplatin on the survivin protein inhibition, the

cells were co-treated with baicalein and oxaliplatin followed by Western blot analysis.

Both of baicalein and oxaliplatin significantly reduced survivin protein expression

(Fig. 12A and 12B). Co-treatment with baicalein and oxaliplatin for 24 h enhanced

4. Discussion

Various types of flavonoids display anticancer effects on growth inhibition and

apoptosis (Brusselmans et al., 2005; Lee et al., 2005a; Psahoulia et al., 2007; Spencer

et al., 2003; Yin et al., 2001). In this study, baicalein significantly inhibited cell

viability in the human BFTC905 bladder cancer cells. We also found that baicalein

induced growth inhibition and cell death by time-lapse observation in BFTC905 cells.

Survivin has been demonstrated to inhibit apoptosis and to promote mitotic

progression in cancer cells (Ambrosini et al., 1997; Li et al., 1998). Interestingly,

baicalein significantly reduced survivin protein expression in BFTC905 cells.

However, baicalein did not influence the survivin mRNA expression by RT-PCR and

real-time PCR assays. As a consequence, we suggest that baicalein inhibits the

survivin expression on the alteration of protein level but not gene expression.

Ubiquitin-proteasome pathway is an essential mechanism participating in

cellular process. Proteasome degradation has also been shown to play an important

role in regulation of apoptosis and cell proliferation by indomethacin (Chiou and

Mandayam, 2007). Furthermore, kaempferol and quercetin enhanced apoptosis by

degradation of survivin in glioma cells (Siegelin et al., 2008; Siegelin et al., 2009). It

in a cell cycle-dependent manner (Zhao et al., 2000). We have shown that baicalein

inhibited survivin protein expression (Chao et al., 2007); however, suppression of

survivin on the post-translational level by baicalein has not been shown previously.

We have further determined the half-life of survivin by baicalein. Baicalein enhanced

the survivin protein degradation when the cells were co-treated with CHX. The data

indicates that baicalein induces the survivin protein instability in bladder cancer cells.

Furthermore, MG132 proteasome inhibitor prevented survivin protein degradation in

the baicalein-treated cells. In addition, MG132 can reduce the baicalein-induced

apoptosis. Baicalein also activated ubiquitination of survivin in bladder cancer cells.

Accordingly, our findings suggest that baicalein inhibits survivin protein expression

through the ubiquitin-proteasome pathway in human bladder cancer cells. We provide

a model of baicalein-induced down-regulation of survivin as shown in Fig. 13.

The stability of survivin resulted from the protein phosphorylation at Thr34 by

the mitotic kinase complex CDK1/cyclin B1 (O'Connor et al., 2000a; Wall et al.,

2003). Quercetin increases the survivin protein expression, which correlates with

raising the protein levels of cyclin B1 and phospho-CDK1 (Kuo et al., 2004). The

levels of cyclin B1 and CDK1 were reduced for inducing G2/M arrest by baicalein in

bladder cancer cells (Chao et al., 2007). The role of CDK1/cyclin B1 on the

XAF1-XIAP complex enhanced degradation of survivin protein (Arora et al., 2007).

Therefore, further investigations are required to determine the roles of XAF1-XIAP

and CDK1/cyclinB1 on the regulation of survivin protein expression following

baicalein in bladder cancer cells.

Combination of various anticancer agents may increase the efficiency of cancer

therapy (Hochster et al., 2003; Ramanathan et al., 2003; Rathkopf et al., 2009).

Previously, Oxaliplatin reduced survivin protein expression and induced apoptosis in

cancer cells (Lin et al., 2005). In this study, we have further examined the anticancer

effects of combination of baicalein and oxaliplatin on the cell viability and survivin

expression in human bladder cancer cells. Both baicalein and oxaliplatin significantly

induced cell death in BFTC905 cells. Interestingly, co-treatment of baicalein and

oxaliplatin additively decreased the levels of survivin proteins and increased

cytotoxicity in BFTC905 cells. Accordingly, the combination of baicalein and

oxaliplatin may increase anticancer effects on survivin inhibition and cancer cell

5. Conclusion

We have summerized that the down-regulation of survivin by baicalein is

mediated ubiquitin-proteasome degradation pathway in human bladder cancer cells

(Fig. 13). Our findings indicate that the blockage of survivin by baicalein may

provide the novel strategies for elevating the efficiency of cancer therapy in bladder

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