行政院國家科學委員會專題研究計畫成果報告

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行政院國家科學委員會專題研究計畫成果報告

研究肺癌細胞對歐洲紫杉醇衍生物(剋癌易)產生抗藥性之機轉(第 3 年)

研究成果報告(完整版)

計畫類別：個別型

計畫編號： NSC 96-2314-B-040-017-MY3

執行期間： 98 年 08 月 01 日至 99 年 07 月 31 日執行單位：中山醫學大學醫學分子毒理學研究所

計畫主持人：許國堂

處理方式：本計畫涉及專利或其他智慧財產權，1 年後可公開查詢

中華民國 99 年 10 月 27 日

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行政院國家科學委員會補助專題研究計畫 _v 成果報告

研究肺癌細胞對歐洲紫杉醇衍生物(剋癌易)產生抗藥性之機轉 (第 3 年)

計畫類別：v 個別型計畫 □ 整合型計畫計畫編號：NSC 96－2314－B－040－017－MY3 執行期間：96 年 08 月 01 日至 99 年 07 月 31 日

計畫主持人：許國堂共同主持人：

計畫參與人員：碩士班研究生-兼任助理人員：廖福田、丘翎燕、黃馨儀、

黃依柔、曾媄慧

成果報告類型(依經費核定清單規定繳交)：□精簡報告 v 完整報告

本成果報告包括以下應繳交之附件：

□赴國外出差或研習心得報告一份

□赴大陸地區出差或研習心得報告一份

□出席國際學術會議心得報告及發表之論文各一份

□國際合作研究計畫國外研究報告書一份

處理方式：除產學合作研究計畫、提升產業技術及人才培育研究計畫、

列管計畫及下列情形者外，得立即公開查詢

□涉及專利或其他智慧財產權，□一年□二年後可公開查詢

執行單位：中山醫學大學醫學分子毒理學研究所中華民國 99 年 10 月 30 日

附件一

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中文摘要及關鍵詞 (keywords)：

Docetaxel (剋癌易 DOC),Vincristine (VCR), MDR-1(P-glycoprotein, ABCB1), 抗藥性 (drug resistance), 肺癌,p53 promoter, reporter gene assay

本計畫之第一年目標為使用具有 Docetaxel (DOC),Vincristine (VCR)之抗藥性的肺癌細胞株做為材料，並且找出 p53、tubulin 和 MDR-1(P-glycoprotein, ABCB1)與 DOC/VCR 之抗藥性的關係。同時，利用 DNA 微探針陣列比對會被 DOC/VCR 轉錄活化的基因，並且逐步地在本計畫期間進一步比對和驗證其與抗藥性的關聯。我們已經建立了具有 DOC/VCR 之抗藥性的 A549 和 H1299 之肺癌細胞株，並且使用全人類基因 DNA 微探針陣列比對會被 DOC/VCR 轉錄活化的基因。我們發現 MDR-1(ABCB1)基因的過度表現是只與 A549 細胞對 DOC 的抗藥性能力相關，卻與 VCR 的抗藥性能力無關。應該還有其他的基因會影響細胞對 DOC 的抗藥性能力。我們也發現到抗 DOC/VCR 藥的肺癌細胞株其中的 p53、tubulin 的基因表現並未受到抗藥篩選的影響。接著使用鈣離子通道抑制劑(Verapamil, Diltiazem and Nifedipine)來降低其抗藥性。結果證明鈣離子通道抑制劑可以有效增加抗藥之肺癌細胞株對 藥物感受性。此研究結果已經發表於 Toxicology Letters Volume 192, Issue 3, pages 408-418 IF:3.249 (15/75) 15 February, 2010。

本計畫之第二年目標為找出 DOC 如何控制 p53 的基因轉錄活性，因而調節藥物感受性。我們已經將 p53 的基因轉錄調控區分離出來，然後使用 reporter gene assay 尋找 DOC 的反應序列。初步結果證明 DOC 處理後會造成 p53 promoter 的基因轉錄活性。此部分之成果已經完成並且著手進行論文書寫預計 2011 年可以投稿。此稿之題目暫定為(Docetaxel induces human p53 tumor suppressor gene transcription via a short sequence at core promoter element) 。我們也發現高度表現 clusterin 除了會降低肺癌細胞對化學治療藥物的感受性外，也會減少細胞移動的能力。此部分之成果已經完成論文書寫和投稿。此稿之題目為 (Regulation of chemosensitivity and migration by clusterin in non-small cell lung cancer cells)。

本計畫之第三年目標為找尋逆轉抗藥性的方法和機制來讓具有對化學治療藥物產生多重抗藥性的癌細胞可以進行有效的治療。我們發現具有多重抗藥性的癌細胞可以被 endoplasmic reticulum stress (ER stress) inducers 誘導而死亡。此部分之成果已經完成並且著手進行論文書寫預計 2011 年可以投稿。此稿之題目暫定為(The effect of ER stress on

docetaxel and vincristine-induced multidrug resistance in human lung cancer cell lines)。因此預估本項專題研究計畫應該會有四篇論文發表，其中已經發表壹篇，投稿審查中壹篇，草稿中兩篇。

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英文摘要：

The purpose of this study is to analyze the expression of MDR-associated genes with docetaxel (DOC) or vincristine (VCR) selected A549 and H1299 non-small cell lung cancer (NSCLC) sublines that exhibit MDR phenotypes. Although all drug resistant sublines showed cross-resistance to DOC, VCR, and doxorubicin (DXR); the gene of ATP-binding cassette (ABC) transporter B1 (ABCB1, MDR-1) was only found strongly induced in DOC but not VCR resistant A549 sublines by DNA microarry analysis and quantitative reverse transcription real-time

polymerase chain reaction (qRT-PCR). The results showed that MDR is regulated by ABC transporters and by other factors. Furthermore, verapamil (VER), the inhibitor of ABCB1 and an L-type calcium channel blocker, is capable to reverse the resistance in all drug-resistant sublines independent of ABCB1 expression levels. Other L-type calcium channel blockers, Diltiazem (DIL) and Nifedipine(NIF) also sensitizes MDR sublines without interfering ABCB1 activity but with less efficacy. Our data of the first year already reported in Toxicology Letters Volume 192, Issue 3, pages 408-418 IF:3.249 (15/75) 15 February, 2010.

We also characterized the promoter of p53 to define the cis-element responsible for DOC induction by luciferase reporter gene assay. The upstream region of p53 promoter was cloned and sequenced for characterization. We found that a 120-bp sequence upstream of transcription start site is important for p53 promoter basal activity. We also transfected p53 promoter deletion constructs into lung cancer lines with Doc treatment, the results showed that a 21- bp (PE 21) element can up regulate p53 promoter activity by treated with Doc. To identify the response element of Doc, we generated four mutation constructs (M1-M4) on PE 21 sequence, only M4 has no response to Doc stimulation. In comparison with previous studies, we found that 5-FU not only up regulates p53 RNA and protein, but also may act with similar sequences of Doc response element of p53 promoter. In further experiment we demonstrated that all the constructs are regulated by 5-FU except M4. Therefore, we hypothesized that Doc probably has same response element as 5-FU induction. The results will be submitted in year of 2011 as the manuscript titled (Docetaxel induces human p53 tumor suppressor gene transcription via a short sequence at core promoter element). Another manuscript was submitted with the title (Regulation of chemosensitivity and migration by clusterin in non-small cell lung cancer cells) that is reviewing process.

The third year project was to search the re-sensitization agents that are able to induce the death of the MER-associated NSCLC cells. Furthermore, we also proposed to define the mechanism that regulates the reversal of MDR resistance. We found that the endoplasmic reticulum stress inducers can reverse the MDR-associated resistance. The results is going to be submitted in year of 2011 with the title of (The effect of ER stress on docetaxel and vincristine-induced multidrug resistance in human lung cancer cell lines).

We estimate that from this three-year NSC funding of my project, we would be able to publish four related SCI papers. The first paper is already published and the second paper is submitted under reviewing process. There are two additional papers will be prepared and submitted in near future.

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壹、Ling-Yen Chiu, Jiunn-Liang Ko, Yi-Ju Lee, Tsung-Ying Yang, Yi-Torng Tee, Gwo-Tarng Sheu* (2010) L-type calcium channel blockers reverse docetaxel and vincristine-induced multidrug resistance independent of ABCB1 expression in human lung cancer cell lines Toxicology Letters Volume 192, Issue 3, 408-418 IF:3.249 (15/75) 15 February

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Regulation of chemosensitivity and migration by clusterin in non-small cell lung cancer cells Running Title: Regulation of chemosensitivity and migration by clusterin

Ching-Yuan Cheng^a, Shur-Hueih Cherng^b, Wen-Jun Wu^c, Tsung-Ying Yang^c,d, Xin-Yi Huang^c, Fu-Tien Liao^c, Ming-Fang Wu^e, Gwo-Tarng Sheu^c*

aDivision of General Thoracic Surgery, Department of Surgery, Changhua Christian Hospital, Taiwan

bDepartment of Biotechnology, Hung Kuang University, Taichung, Taiwan

cInstitute of Medicine, Chung Shan Medical University, Taichung, Taiwan

dDivision of Chest Medicine, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan

eDepartment of Medical Oncology, Chung Shan Medical University Hospital, Taichung, Taiwan

*To whom correspondence should be addressed:

Gwo-Tarng Sheu, Ph.D.

Associate Professor Institute of Medicine

Chung Shan Medical University, Research Building Room 702

No. 110, Sec. 1, Jianguo N. Road, Taichung City 402, Taiwan TEL: +886-4-24730022 ext. 11692 FAX: +886-4-24751101 E-mail: [email protected]

Abstract

The most significant role of clusterin (CLU) plays in cells is to protect all cells from environmental stimuli and stress; unfortunately, cancer cells usually benefit from this protection upon medical treatment. Therefore, chemosensitivity of human non-small cell lung (NSCLC) cancer cell lines were examined for the association with the level of CLU. We found that H1355 cells with the highest level of CLU have the least sensitivity to adriamycin (ADR), docetaxel (DOC), and gemcitabine (GEM) treatment. Inhibition of CLU expression by short hairpin RNA interference (shRNAi) resulted in higher chemosensitivity. When CLU is stably expressed in A549 and H1299 cells, only the chemosensitivity of ADR is reduced. The DOC, GEM sensitivity were not markedly altered by CLU overexpression. These data indicate that inhibition of CLU expression enhances chemosensitivity, but effect of CLU overexpression is not always associated with chemosensitivity. Moreover, we found when A549 and H1299 cells overexpressing CLU, their migration were reduced significantly. The transcription of matrix metalloproteinase-2 (MMP2) was markedly reduced in CLU-overexpressing H1299 cells that indicate less metastasis potential of cancer cells when CLU level is high. A hypothesis could be proposed from our data that high level of CLU certainly reduces chemosensitivity and increases survival; but under unknown circumstance, high level of CLU may inhibit cancer cells migration. Therefore, cancer cells have to determine whether expressing more CLU is needed to protect them from toxicity of drugs or with less CLU that enables them to migrate to other tissue sites.

Key words: Lung cancer; Clusterin; Chemosensitivity; Migration

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Introduction

Lung cancer is the second most frequently diagnosed cancer in men and women and accounts for one-third of all cancer-related deaths in the United States every year [1]. Eighty percent of lung cancer cases are non-small cell lung cancer (NSCLC) and the remaining 20% are small cell lung cancer (SCLC). The 5-year survival rate for lung cancer (15%) is the second to the lowest of all cancers, just above pancreatic cancer [1].

Clusterin (CLU) in humans a single copy gene located at chromosome 8 [2]. CLU is a secreted glycoprotein that is translated from a single mRNA as a preprotein [3]. This preprotein is an intracellular 449 amino acid polypeptide chain with an apparent MW of 60 kDa cytosolic clusterin (cCLU), where the first 22 amino acids represent the classical hydrophobic secretory signal sequence [4]. Proteolytically cleaved of mature glycosylated c-CLU into anand chain that are linked by five disulfide bridges [5] results in the mature form of the secreted clusterin protein (sCLU). The processed clusterin protein appears as a smear of about 40 kDa as detected by western blot [6]. Nuclear clusterin (nCLU) is translated from the second ATG from the mRNA without leader peptide. The n-CLU is unglycosylated and it is involved in apoptosis induction [6].

Overexpression of a truncated form of CLU lacking the hydrophobic secretion signal sequence that localizing to the nucleus of prostate epithelial cells, caused induction of apoptosis [7; 8].

The expression of CLU has been extensively investigated in prostate cancer [9; 10], cervical cancer [11], bladder cancer [12], renal cell carcinoma [13; 14] for its prognostic significance that including chemotherapeutic sensitivity and metastasis potential. Inhibition of CLU expression in cancer cells resulted in cells more sensitive to the stress and toxic conditions which enhance cell death [10].

When applied the association of CLU expression to lung cancer research, the investigators facing a dilemma with inconsistence results from in vitro and in vivo clinical data. Target the CLU gene expression in lung cancer cell lines by antisense oligonucleotides (ASO) and small-interfering RNAs (Si-RNA) resulted in sensitization of cancer cells to radiotherapy [15]

and chemotherapy [16]; furthermore, reduced their migration and invasion ability [17]. Whereas, when tumor specimens of NSCLC were examined, patients with cytoplasmic CLU-positive staining had a better overall survival and less recurrence than those with CLU-negative patients [18]. Apparently, CLU expression seems to be a positive and favorable prognostic factor in patients of NSCLC. The contradictory between the promising in vitro cell lines data and clinical findings would jeopardize the development of a better therapy for lung cancer in future.

Therefore, we are trying to further study the expression of CLU in human lung cancer cell lines and compare to their chemotherapeutic sensitivity with various therapeutic drugs, as well as the role of CLU in migration.

Materials and Methods:

Cell lines and culture

Human NSCLC cell lines (A549, H1299, H1355, H460 and Calu-1) and lung cells (BEAS-2B)from theAmerican TypeCultureCollection werecultured on Dulbecco’smodified Eagle’smedium (DMEM)(GIBCO,Rockville,MD).Thehuman bronchialepithelialcellline BEAS-2B was cultured on LHC-9 medium (GIBCO, Rockville, MD). All lung cancer cell lines

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were maintained at 37 ℃ in a 5% CO2-humidified atmosphere on medium containing 10% fetal bovine serum (FBS) and 100ng/ml each of penicillin and streptomycin (Life Technologies, Rockville, MD).

Drugs and chemicals

DOC was obtained from Aventis Pharmaceuticals Inc. (Bridgewater, NJ, USA). ADR was purchased from Sigma Chemical. GEM was provided by Eli Lilly (Indianapolis, IN, USA).

Cell growth and viability for chemosensitivity (MTS assay)

Cell viability was assessed by CellTiter 96 AQeous One Solution Cell Proliferation Assay (Promega, Madison, WI), according to themanufacturer'sinstructions.In brief,20 μlofMTS reagent was added into each well of the 96-well assay plate containing the samples in 100 μlof culture medium. Plates were incubated at 37 ℃ in a humidified, 5% CO2atmosphere for another 40 min. Absorbance at 490 nm was read by means of an ELISA plate reader. Each experiment was repeated at least three times. Final data were normalized and presented as percentage of controls.

Western blot analysis

The cells were washed with PBS and harvested for sonication in the presence of a protease inhibitor cocktail (Sigma-Aldrich). The concentration of protein was determined by Bradford assay and the amounts of cell lysates (15g) were applied to SDS-PAGE gels. After transferring proteins to a polyvinylidene fluoride (PVDF) membrane, proteins were reacted with anti-clusterin (Santa Cruz Biotechnology), or anti-β-actin (Sigma-Aldrich), followed by anti-mouse IgG conjugated with horseradish peroxidase (Calbiochem). A chemiluminescence detection kit (ECL, Amersham Pharmacia Biotech) was applied to determine the levels of protein expression.

Transfection of clusterin-shRNAi

The V2LHS-150636-clusterin lentiviral shRNAmir (CLU-shRNA) and Non-silencing shRNAmir negative control (RHS4346, shRNA control) were purchased from Open biosystems (Thermo Fisher Scientific). The TurboFectTM in vitro transfection reagent (Fermentas life sciences) was used to transfect shRNA control and CLU-shRNA vectors into H1355 cells (4x10⁵) in 6-cm dishes (3 μg of DNA/dish) followed by 48 and 72 h incubation for western blot analysis.

For MTS assay, H1355 cells (7.5x10⁴) seeded in a 24-well plate were transfected (0.4 μg of DNA/well). After 60 h, the medium was collected for western blotting. The fresh medium with individual drug were added into the cells and incubated for 48 h (ADR, DOC) or 72 h (GEM) with indicated concentration of drug.

Clusterin overexpression

Full-length open reading frame of human clusterin cDNA was generated by RT–PCR from a human Huh7 cell line using the forward primers of 5- GTGACATA TGATGAAGACTCTG and reverse primer of 5- AACGCGTCGACATCTCACTC CTCCCT. To express the gene, the obtained DNA fragment was subcloned into p3XFLAG-CMV-10 (Sigma-Aldrich) followed by DNA sequencing. The CLU expression vector was transfected with TransFastTM Transfection Reagent (Promega) into A549 and H1299 followed by G418 selection (1.5 mg/ml, Sigma-Aldrich). We isolated the well separated clone of cells into a 96-well plate and reamplified until the cell grew in 10-cm dishes that could be examined by immunoblot analysis.

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Cell migration assay with Boyden chamber

In each well of a 48-well bottom chamber, 32μlDMEM with 10% FBS was added. We used forceps to handle the 8-μmporosity polycarbonate membrane and placed the silicone gasket over the membrane. Then, the top chamber was placed over the gasket, and the Boyden chamber was assembled. Total of 52 μl DMEM solution that containing 2% FBS and cancer cells were loaded into each well of the top chamber, and the whole chamber was incubated at 37°C and 5% CO₂for time as indicated. Thereafter, the chamber was disassembled, and the membrane, through which cells had passed and adhered, was immersed in cooled 95% methanol for 10 min to fix migrated cells. This membrane was then stained in 20% Giemsa stain overnight. The stained cells on the underside of membrane were count under a microscope.

Gelatin zymography

Production of MMPs by cancer cells were analyzed by gelatin zymography. To avoid the MMPs contamination, cells were cultured in serum-free medium for 24 h, and conditioned media were collected. Equal amount of conditioned medium samples were mixed with SDS sample buffercontaining 2% SDS withoutβ-mercaptoethanol and applied to 10 % SDS polyacrylamide gels copolymerized with 0.1% gelatin (Sigma-Aldrich) without boiling. After electrophoresis, gels were washed for 1 h at room temperature with gentle agitation in renaturing buffer (2.7%

Triton X-100 in H2O) to remove SDS. The gels were then equilibrated in developing buffer (50 mM Tris-HCl, pH 7.4, 0.2 M NaCl, 5 mM CaCl₂和 0.2% Brji 35) at 37°C overnight. The gels were then stained with 0.5% Coomassie Brilliant Blue and destained. The MMP activities were visualized as clear bands against the blue background of the stained gels.

Semi-quantitative RT-PCR of MMP2 and MMP9

To analyze the mRNA levels of MMP2 and MMP9, total RNA was extracted from H1299 cells that stably expressing clusterin using the TRIzol reagent (Gibco BRL). The purified RNA (2 µg) were reverse-transcribed to cDNA by oligo-dT primers. For MMP2, the forward primer (5’- TTTTCTCGAATCCATGATGG) and the reverse primer (5’-CTGGTGCAGCTCTCATATTT) were used. For MMP9, the forward primer (5’-AAGATGCTGCTGTTCAGCGGG) and the reverse primer (5’- GTCCTCAGGGCACTGCAGGAT) were used. For GAPDH, the forward primer (5’- GCCAAGGTCATCCATGACAAC) and the reverse primer (5’- CAGTAGAGGCAG GGATGATGTTC) were used as the calibration control. The amplified PCR products were analyzed with 1% agarose gel.

Results

Expression levels of CLU in human NSCLC cells and lung cells.

Adenocarcinoma cell lines of A549, H1355, large cell carcinoma cell lines of H1299, H460 and squamous cell carcinoma cell lines of Calu-1, were examined by immunoblot analysis for the levels of cytosolic and secreted CLU protein expression. The highest level of precursor form of the secreted CLU (pre-sCLU) is present in H1355 cells followed by A549, H460 and Calu-1, respectively (Fig. 1A). The mature forms of secreted CLU (sCLU) were present in cell lysate and culture medium (Fig. 1B). The human bronchial epithelial cell line BEAS-2B and H1299 cells contain only an insignificant level of cytosolic CLU. According to the CLU levels, we chose H1355, A549 and H1299 cells to investigate the correlation of CLU and chemosensitivity.

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Chemosensitivity of NSCLC cells to ADR, GEM, and DOC reversely correlated with the CLU levels.

The sensitivity of H1355, A549 and H1299 cells to ADR (Fig. 2A), GEM (Fig. 2B), and DOC (Fig. 2C) were evaluated by MTS assay. The H1355 cells were the least sensitive cells to ADR, GEM and DOC. These data support the protection role of CLU in H1355 cells.

Inhibition of CLU expression results in sensitization of cells to drug treatment.

To determine the effect of CLU associated with drug sensitivity; H1355 cells were transiently transfected with small hairpin interference RNA construct (CLU- shRNA) that inhibit CLU expression. The expression of CLU was significantly reduced after 72 h of transfection that examined by immunoblotting (Fig. 3A). When CLU expression was reduced, the chemosensitivity of H1355 were increased to ADR (Fig. 3B), GEM (Fig. 3C) and DOC (Fig. 3D) by MTS assay. The levels of sCLU proteins in both medium were showed in the small window by western blot analysis.

Secretary CLU collected from H1355 medium reduces drug sensitivity of H1299 cells.

To examine whether the chemosensitivity is regulated by sCLU, the medium from H1355 cells after 24 h incubation was harvested as the source of sCLU. The conditioned medium used for cell treatment was a mixture of one part of fresh and one part of collected medium to avoid metabolic toxicity from the collected medium. Because the H1299 cells contain lower CLU level, therefore, chemosensitivity of H1299 cells were compared in the presence of conditioned medium and fresh medium. The chemosensitivity of H1299 cells were decreased to ADR (Fig.

4A), GEM (Fig. 4B) and DOC (Fig. 4C) treatment with addition of conditioned medium by MTS assay. Apparently, sCLU present in the conditioned medium has protective effect for H1299 cells to against these drugs.

Overexpression of CLU reduced ADR but not GEM and DOC chemosensitivity in A549 and H1299 cells

Since the sCLU is produced from pre-sCLU with modification, we have constructed a full-length gene of CLU that contains the leader signaling sequence for expression. Stably expression of pre-sCLU in A549 and H1299 individual clones were selected by G418 treatment and examined by western blotting (Fig. 5A). Two clones of A549 (A-1, A-2) and H1299 (H-1, H-2) cells that overexpressing CLU were applied to further chemosensitivity assay. The chemosensitivity to ADR was reduced in all A-1, A-2 (Fig. 5B), H-1 and H-2 (Fig. 5C) CLU-overexpressing cells. Interestingly, the chemosensitivity to GEM (Fig. 5D, E) and DOC were not significantly decreased in these cells (Fig. 5F, G).

Migration was reduced in CLU-overexpressing H1299 and A549 cells

The alteration of cell migration by CLU was further investigated in vitro with modified Boyden chamber inserted with polyethylene terephthalate filter. When compared with parental H1299 cells, both clones of H1299 cells that overexpressing CLU (H-1 and H-2) significantly reduced their migration ability (Fig. 6A). Only one clone of A549 cells that overexpressing CLU (A-2) showed reduced migration ability (Fig. 6B).

The transcription of MMP2 but not MMP9 was inhibited in H1299 cells that overexpssing clusterin

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To further explore the factor that may be inhibited by clusterin, we performed gelatin zymography to detecte whether MMP2 and MMP9 activities was altered. Interestingly, the MMP2 activity was lost in H-1 and H-2 clustering overexpressing cells (Fig. 7A). The lost of MMP2 activity was further confirmed by RT-PCR analysis of MMP2 and MMP9 mRNA transcripts. Only the transcription of MMP2 was inhibited in CLU-overexpressing cells (Fig. 7B).

Discussion

Clusterin (CLU), a protein with many identities, present not only inside of cell, it also exist as part of circulating proteins. The extracellular form of CLU is a highly glycosylated α-β-heterodimer linked by five disulphide bonds [19]. Although clusterin expression level associated with the chemo- and radiosensitivity have been investigated extensively and reviewed [20; 21], those results were sometimes contradictory that needed further research. Especially, CLU-positive expression in human lung cancer patients showed better overall and disease-free survival than those CLU-negative patients [18]. Whereas, it was found that in a lung cancer cell line, CLU silenced by siRNA reduced migration and invasion [17]. From the results of these reports it missed a link between the effects of CLU inhibition and CLU overexpression. Therefore, clinical data did not correlate well to cell line data and whether CLU is a favorable prognostic marker can not be concluded.

We started with the examination of CLU level in several human lung cancer cell lines and identified H1355 as a CLU-rich cell line. Inhibition of CLU expression by transient shRNA transfection reduced the chemosensitivity of H1355 cells to ADR, GEM, and DOC. Addition of CLU-rich conditioned medium to H1299 cells resulted in reduced chemosensitivity. Our data is similar to the data reported from July et al that targeted A549 CLU by siRNA or antisense oligonucleotides sensitized paclitaxel or GEM sensitivity in vitro and in vivo [16]. To investigate whether high level of CLU could reduce chemosensitivity, we then expressed exogenous CLU in A549 and H1299 cells. Interestingly, the chemosensitivity of CLU overexpressing cells were not altered markedly. The possible explanation for the later is the nCLU in those CLU overexpressing cells may regulate pro-apoptotic activities simultaneously in contrast to sCLU. Cao et. Al. [15]

also reported that when H460 cells treated with an antisense oligonucleotide against CLU (OGX-011) and followed by radiotherapy; induced tumor regression in xenograft model.

Apparently, inhibition of CLU resulted in high chemosensitivity is true for H1355, A549 and H460 cells and that is not related to the level of CLU.

Interestingly, when the extracellular CLU concentration was high; chemosensitivity was also reduced in cancer cells with less endogenous CLU. This result suggested that as long as cancer cells exist in CLU-rich microenvironment, not each cancer cells necessary to express high level of CLU to protect them from medicine treatment. The sCLU from neighboring cells also provide shielding effect for those cells expressing low level CLU.

If high level of CLU benefits cancer cells, why we observed lung cancer cells expressing different levels of CLU? Is there possible disadvantage with high level of CLU to cancer cells?

When the cells overexpressing CLU, we observed that they grew as bunch-like and clustered in petri-dish (data not shown). Therefore, we decided to examine the migration ability of the CLU-overexpressing cells. According to our data, the migration ability was reduced and MMP2

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transcription was inhibited in the cells overexpressing CLU. We have not yet further identified whether the nCLU or sCLU is responsible for the migration inhibition. Obviously, CLU overexpression may reduce the ability of cancer cell to spread out and results in easier detection by immune system.

Human MMPs are a family of over 20 different endopeptidases that are able to degrade various components of the extracellular matrix (ECM). MMP2 is an enzyme that is supposed to have an important role in invasion to the basement membrane. The role of MMP2 on the survival of patients with NSCLC was studied by Qian et al. [22] This study supported the fact that MMP2 could be included in further prospective trials studying prognostic factors in NSCLC.

Our data point to a possible explanation of why better overall and disease-free survival found in human lung cancer patients with CLU -positive expression. In these patients, cancer cells may be less active in metastasis than patients with CLU -negative expression. Of course, the application of ASO against CLU may be required further discussion. Because when CLU is inhibited by ASO, those targeted cancer cells if not killed by medicine or radiation treatment, they might turn into migration active cells which could move to other tissues to rebuild tumor.

Acknowledgements: This work was supported by grants from the National Science Council, Taiwan (NSC-96-2314-B-040-017-MY3) and Chung Shan Medical Universisy, Taiwan (98-CCH-CSMU-02) to G-T Sheu. We also thank the technical support from the Instrument Center of Chung Shan Medical University, which is partly supported by National Science Council, Ministry of Education and Chung Shan Medical University.

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Legends Figure 1

Expression levels of clusterin were examined by western blot analysis. Cells of A549 and H1355 are lung adenocarcinomas; H1299 and H460 are large cell carcinoma and Calu-1 is a squamous carcinoma. BEAS-2B cells were isolated from normal human bronchial epithelium obtained from autopsy of non-cancerous individuals. (A) Total cellular lysate (15 μg) from the cultured cells were analyzed for cytosolic clusterin expression. (B) The medium collected from individual culture (15 μl) were also analyzed.

Figure 2

Chemosensitivity of A549, H1299 and H1355 cells were compared by MTS viability assay.

Triplicate samples of each cancer cells (2x10³cells) were seeded in 96-well plate and (A) treated with 86, 172, 344, 688 and 1032 nM of ADR for 36 (B) 20, 40, 80, 160, and 200 nM of GEM for 48 h (C) 10, 20, 40, 80 and 100 nM of DOC for 48 h followed by MTS assay.

Figure 3

Inhibition of clusterin expression by short hairpin RNA interference (shRNAi) increases chemosensitivity. (A) To measure the effect of CLU inhibited by shRNAi, the H1355 cells were transfected with CLU-shRNA and Non-silencing shRNAmir negative control (control). After 48 and 72 h later, cells were harvested for western blot analysis. (B) For chemosensitivity, cells were treated with (B) 0.344 to 2.58 μM of ADR (C) 0.5 to 2 μM of GEM and (D) 10 to 80 nM of DOC as described in Materials and Methods.

Figure 4

Secretory CLU collected from H1355 medium reduces drug sensitivity of H1299 cells. Triplicate samples of H1299 cells (2x10³ cells) were seeded in 96-well plate and conditioned medium was added together with (A) 86, 172, 344, 688 and 1032 nM of ADR for 36 h (B) 20, 40, 80, 160, and 200 nM of GEM for 48 h (C) 10, 20, 40, 80 and 100 nM of DOC for 48 h followed by MTS assay.

Figure 5

Overexpression of CLU reduced ADR but not GEM and DOC chemosensitivity in A549 and H1299 cells. (A) As described in material and methods, Two CLU-overexpressing H1299 sublines (H1, H2) and A549 sublines (A1, A2) were selected by G418 and verified by western blot analysis. Triplicate samples of cells (2x10³ cells) were seeded in 96-well plate and treated with (B, C) 86 to 1032 nM of ADR for 36 h (D, E) 10 to 80 nM of GEM for 48 h (F, G) 5 to 60 nM of DOC for 48 h followed by MTS assay.

Figure 6

Migration ability was reduced in CLU-overexpressing H1299 and A549 cells. To measure the migration ability of CLU-overexpressing cells, modified Boyden chamber was used and the migrated (A) H1299, H-1 and H2 (B) A549, A-1 and A2 were count.

Figure 7

The transcription of MMP2 but not MMP9 was inhibited in H1299 cells that overexpssing clusterin. (A) The activities of MMP2 and 9 from H1299, H-1 and H-2 were characterized by gelatin zymography. Cells (8x10⁵) were seeded in 35-mm dishes and incubated for 24 h with

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serum-free medium. The conditioned medium collected from individual cells (45 μl) was analyzed. (B) Total RNA from H1299, H-1 and H-2 were purified for cDNA synthesis.

Semi-quantitative RT-PCR of MMP2, MMP9 and GAPDH were analyzed.

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無衍生研發成果推廣資料

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96 年度專題研究計畫研究成果彙整表

計畫主持人：許國堂計畫編號：96-2314-B-040-017-MY3 計畫名稱：研究肺癌細胞對歐洲紫杉醇衍生物(剋癌易)產生抗藥性之機轉

量化

成果項目 ^{實際已達成}

數（被接受或已發表）

預期總達成數(含實際已

達成數)

本計畫實際貢獻百

分比

單位

備註（質化說明：如數個計畫共同成果、成果列為該期刊之封面故事 ...

等）

期刊論文 0 0 0%

研究報告/技術報告 0 0 0%

研討會論文 0 0 0%

論文著作篇

專書 0 0 0%

申請中件數 0 0 0%

專利已獲得件數 0 0 0% 件

件數 0 0 0% 件

技術移轉

權利金 0 0 0% 千元

碩士生 3 0 100%

博士生 2 0 100%

博士後研究員 0 0 0%

國內

參與計畫人力

（本國籍）

專任助理 0 0 0%

人次

期刊論文 1 4 100%

研究報告/技術報告 0 0 0%

研討會論文 0 0 0%

論文著作篇

專書 0 0 0% 章/本

申請中件數 0 0 0%

專利已獲得件數 0 0 0% 件

件數 0 0 0% 件

技術移轉

權利金 0 0 0% 千元

碩士生 0 0 0%

博士生 0 0 0%

博士後研究員 0 0 0%

國外

參與計畫人力

（外國籍）

專任助理 0 0 0%

人次

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其他成果

(

無法以量化表達之成

果如辦理學術活動、獲得獎項、重要國際合作、研究成果國際影響力及其他協助產業技術發展之具體效益事項等，請以文字敘述填列。)

無

成果項目量化 名稱或內容性質簡述

測驗工具(含質性與量性) 0

課程/模組 0

電腦及網路系統或工具 0

教材 0

舉辦之活動/競賽 0

研討會/工作坊 0

電子報、網站 0

科教處計畫加填項

目計畫成果推廣之參與（閱聽）人數 0

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國科會補助專題研究計畫成果報告自評表

請就研究內容與原計畫相符程度、達成預期目標情況、研究成果之學術或應用價值（簡要敘述成果所代表之意義、價值、影響或進一步發展之可能性）、是否適合在學術期刊發表或申請專利、主要發現或其他有關價值等，作一綜合評估。

1. 請就研究內容與原計畫相符程度、達成預期目標情況作一綜合評估

■達成目標

□未達成目標（請說明，以 100 字為限）

□實驗失敗

□因故實驗中斷

□其他原因說明：

2. 研究成果在學術期刊發表或申請專利等情形：

論文：■已發表 □未發表之文稿 □撰寫中 □無專利：□已獲得 □申請中 ■無

技轉：□已技轉 □洽談中 ■無其他：（以 100 字為限）

估本項專題研究計畫應該會有四篇論文發表，其中已經發表壹篇，投稿審查中壹篇，草稿中兩篇

3. 請依學術成就、技術創新、社會影響等方面，評估研究成果之學術或應用價值（簡要敘述成果所代表之意義、價值、影響或進一步發展之可能性）（以 500 字為限）

我們希望提供未來化學治療的參考，希望經由這個長期的研究，可以延長病人的存活期，

減少不良作用，達到控制病情的目的，自然而然地會改善肺癌患者的生活品質。

行政院國家科學委員會專題研究計畫 成果報告