ANTICANCER RESEARCH 27: 865-872 (2007)

CPT-11 (SN-38) Chemotherapy may be Selectively Applicable to Biliary Tract Cancer with Low hMLH1 Expression

KEN SATO, YOSHIHIKO KITAJIMA, NAOHIKO KOHYA, YASUO KOGA, KAZUMA OHTAKA and KOHJI MIYAZAKI

Department of Surgery, Saga University Faculty of Medicine, Nabeshima 5-1-1, Saga 849-8501, Japan

Abstract. Biliary tract cancer is of highly malignancy with a Biliary tract cancer is of highly malignancy, leading to a poor 5-year survival. However, established chemotherapeutic poor 5-year survival rate (1). Thus effective chemotherapy regimens have not yet been established. Previously, we have is important in order to prolong the survival of cancer reported that hMLH1, a mismatch repair (MMR) gene was patients, especially in non-resectable or non-curable frequently (57%) found to be lacking in surgically resected resection cases. However, the effective chemotherapeutic biliary tract carcinomas and the patients lacking the expression regimens for biliary tract cancer have not been optimally of hMLH1 revealed a poorer prognosis than those patients who established. Recently, single or combination usage of 5- possessed it. The MMR gene has been considered to be FU, cisplatin and gemcitabine has been suggested for associated with sensitivity to various chemotherapeutic agents biliary tract cancer, however the therapeutic effect is that act on DNA. A loss of MMR expression has been reported limited, the response rate ranging from approximately to increase sensitivity to topoisomerase inhibitors such as 15~35% (2-5). etoposide (ETP) or camptothecins (CPT). In the present study, Recently, it has been reported that the mismatch repair whether or not hMLH1 deficiency resulted in a higher sensitivity (MMR) gene is associated with sensitivity to various to irinotecan (CPT-11) active form (SN-38) was investigated chemotherapeutic agents that act on DNA (6-7). In fact, using a short interfering (Si)RNA system. A quantitative reverse MMR deficient cells have shown resistance to monofunctional transcription-polymerase chain reaction (RT-PCR) was alkylating agents such as N-methyl-N-nitrosourea (MNU), conducted to measure the levels of hMLH1 expression in seven dacarbazine (DTIC) and temozolomide (TMZ) or platinum- cancer cell lines, and this was compared with the drug based drugs such as cisplatin (8-12). The MMR components sensitivity (IC50) to SN-38. The hMLH1 expression was have been shown to participate in the recognition of DNA correlated with the IC50 for SN-38, although the relationship adducts brought about by these drugs (9, 11-13). The was not statistically significant (R=0.717, p=0.0715). SiRNA recognition of the damaged bases by MMR initiates a signal double strand RNA (dsRNA) was transiently transfected into transduction pathway that can activate cell-cycle checkpoints KMG-C (gallbladder cancer) cells. hMLH1 mRNA expression and trigger apoptosis (11, 14-16). Using biliary tract cancer was repressed by hMLH1 dsRNA in a dose-dependent manner cells, we previously demonstrated that the expression of a in comparison to the control dsRNA. The cell growth of the deficient DNA repair gene (MGMT) and a proficient hMLH1 dsRNA transfected group was decreased by mismatch repair gene (hMLH1) are related to sensitivity approximately 50% by SN-38 exposure. Flow cytometry was against the alkylating agents MNU and DTIC (8). A loss of also carried out to examine the effect of the SN-38 treatment MMR expression has also been reported to demonstrate on the cell cycle. Following hMLH1 dsRNA transfection, the sensitivity to topoisomerase inhibitors such as etoposide subG1 fraction was increased in comparison with the control (ETP) or camptothecins (CPT ) (17-19). in a dose-dependent manner. In conclusion, a low expression The camptothecins (CPT) are broad-spectrum anticancer of hMLH1 in biliary tract cancer may aid in predicting its drugs that specifically target DNA topoisomerase I (Topo responsiveness to CPT-11(SN38). I) (20). The CPT interfere with the catalytic cycle of topoisomerases by stabilizing the covalent complex formed by the topoisomerase and cleaved DNA, referred to as the cleavage complex (21). Irinotecan (CPT-11) is a Correspondence to: Kohji Miyazaki, Department of Surgery, Saga semisynthetic analog of camptothecin, originally isolated University Faculty of Medicine, Nabeshima 5-1-1, Saga 849-8501, Japan. e-mail: [email protected] from the Chinese/Tibetan ornamental tree Canptotheca acuminata. CPT-11 has been studied for its effects against Key Words: hMLH1, SN38, biliary tract cancer, SiRNA. esophageal, gastric, lung and especially colon carcinomas

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(22). However, no study has reported whether or not CPT- MTT assay. Drug sensitivity to SN-38 was analyzed by the MTT 11 is effective against biliary tract cancer. (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay The toxicity of CPT is primarily caused by the conversion using a Cell Titer96 Non-Radioactive Cell Proliferation Assay Kit (Promega, Madison, WI, USA). Each of the cell lines were seeded of a single-strand break (SSB) to a double-strand break 3 in 96-well culture plates at a density ranging from 8~12x10 (DSB) during the S-phase where the replication fork cells/well. After 24 h, the cells were exposed to SN-38 for 72 h, at a collides with the cleavage complexes formed between concentration ranging from 10~20 ÌM. The cells were washed in topoisomerase I-DNA and CPT (23). Some of the MMR phosphate-buffered saline (PBS) and resuspended in fresh medium components participate in various DNA repair processes and then 20 Ìl of MTT was added to the cultures. After 4 h including DSB repair and recombination (24). incubation, the reaction was stopped by the addition of a We recently reported that hMLH1 expression was solubilization/stop solution. Absorbance at 530 nm was measured using a multiwell plate reader (CS9300PC, Shimadzu Co. Kyoto, frequently (57%) found to be lacking in surgically resected Japan). Every experiment was done in triplicate and data are biliary tract cancer and the patients lacking the expression presented as a mean±S.D value of the three individual experiments. of hMLH1 revealed a poorer prognosis than those patients who possessed it (25). The hMLH1 expression might thus Quantitative RT-PCR. For a quantitative estimate of the hMLH1 be a molecular marker in biliary tract cancer possessing high mRNA expression in the six biliary tract cancer cell lines and the SW- malignant potential. 48 cells, real-time PCR was performed using the primer pairs as The aim of the present study was to investigate whether follows: hMLH1 forward, 5'-CCACAAGTATTCAAGTGATT-3', hMLH1 deficiency resulted in a higher sensitivity to CPT- hMLH1 reverse, 5'-GAACCTTTCACATACAATAT3', glyceralde- hyde-3-phosphate dehydrogenase (GAPDH) forward, 5'-TGG TAT 11 (SN-38). In the human body, CPT-11 is converted into CGT GGA AGG ACT CAT GAC-3'; and GAPDH reverse, 5'-ATG its active form, SN-38, by carboxylesterases that are CCA GTG AGC TTC CCG TTC AGC-3'. After reverse abundantly present in the liver (26). SN-38 is widely used transcription, PCR was carried out for each cDNA. PCR amplification for in vitro experiments for assessing the toxicity of CPT-11. was carried out using a Light-Cyclerì system and the Light-Cycler- A quantitative reverse transcription-polymerase chain FastStartì DNA master SYBR green I kit (both Roche, Mannheim, reaction (RT-PCR) was conducted to measure the levels of Germany). The amplifications were performed in a mixture of 1 Ìl of hMLH1 expression in seven cancer cell lines and this was cDNA suspension, 20 pmol of primer, 4 mM of MgCl2 and 1 Ìl of Light-Cycler-FastStartìDNA master SYBR green. After the cDNA compared with drug sensitivity (IC ) to SN-38. 50 was denatured at 95ÆC for 3 min, a PCR of 50 cycles composed of Furthermore, short interfering (Si) RNA of the hMLH1 denaturation for 15 sec at 95ÆC, annealing for 5 sec at 60ÆC, and gene was introduced into the gallbladder cancer cell line extension for 10 sec at 72ÆC was completed. The melting curves were (KMG-C) and the effect of down-regulation of hMLH1 then described according to the protocol with the following conditions: expression on sensitivity to SN-38 was observed. Finally, 0 sec denaturation period at 95ÆC, starting temperature of 65ÆC, flow cytometry was carried out to examine the affect of SN- ending temperature of 95ÆC, and a rate of temperature increase of –1 38 treatment on the cell cycle. 0.1ÆC/sec . The quantitative hMLH1 values for each cell line were estimated by dividing by the levels of GAPDH expression. The experiment for each sample was performed in triplicate and the mean Materials and Methods was then calculated.

Cell lines and culture. Six biliary tract cancer cell lines (KMG-C, G- Transient transfection of hMLH1 SiRNA (dsRNA) and protein 415, GB-d1, HBDC, HAG-1, TFK-1) and SW-48 (a colon cancer extraction. 5x105 cells of KMG-C were seeded onto 6-well plates cell line) were used in this study. KMG-C, G-415 and GB-d1 were and incubated overnight. Twenty-one base pairs (bp) of double- obtained as described previously (8). HBDC was established in our strand (ds) RNA for hMLH1, which were constructed by iGene, laboratory (Jiao Wan) (27) and HAG-1 was kindly provided by Dr. Co,Ltd (Tsukuba, Japan), was transiently transfected into KMG-C Nakano (Department of Internal medicine, Kyushu University, cells for 4 h using a OligofectAMINE (Life Technology, Inc, Kyushu, Japan). TFK-1 was obtained from the Cell Resource Japan) at a concentration ranging from 5-40 nM. Randomly Center for Biomedical Research, Tohoku University (Japan). The constructed dsRNA with a 21 bp length was used as a control first five cell lines were cultured in Williams' medium E (W/E, ICN transfection. At 48 h or 72 h after transfection, the protein was Biomedicals INC, Costa Mesa, CA, USA), while TFK-1 and SW-48 extracted from each cell using a lysis buffer (1% Triton X-100, 50 were cultured in RPMI-1640 (Nissui Pharmaceutical, Tokyo, mM Tris, 150 mM Nacl, 0.5 mM phenylmethylsulfonyl fluoride Japan) supplemented with 10% inactivated fetal bovine serum (PMSF)). The lysate was sonicated and the supernatant was (FBS ) (JRH Biosciences, Lenexa, Kansas, USA), 2 mM glutamine, collected by centrifugation at 15000 rpm for 30 min. 100 Ìg/ml kanamycin and 20 Ìg/ml tetracycline hydrochloride. All cell lines were incubated at 37ÆC in a humidified atmosphere of Western blot analysis. Forty Ìg of cell extracts from each cell line were 5% CO2 in air. separated by gel electrophoresis on a 10% Bis-Tris gel (Invitrogen CA, USA). These electrophoretically separated extracts were Drug. SN-38 was supplied from Yakult, Co. Ltd. (Tokyo, Japan) and transferred onto a Hybondì ECLì nitrocellulose membrane 10 mM of the stock solution was prepared in DMSO and diluted in (Amersham Biosciences, NJ, USA). The membrane was blocked with medium at the approximate dose immediately before use. 1 x Tris-buffered saline (TBS) containing 0.1% Tween20 and 1%

866 Sato et al: CPT-11 is Applicable to Biliary Tract Cancer with Low hMLH1 Expression

Figure 1. IC50 for SN-38 in six biliary tract cancer cell lines and SW48 was estimated by MTT assay performed in triplicate. IC50 values were calculated and the mean of three individual experiments was plotted.

bovine serum albumin (BSA), and blots were probed with 1. SW-48 was particularly sensitive to SN-38, whereas TFK-1 monoclonal antibodies specific for hMLH1 (BD Pharmingen, San was the most resistant among the seven cancer cell lines. The Jose, CA, USA). The signals were developed using an ECLì western IC for TFK-1 was 22.5-fold higher than that of SW-48. blotting detection reagents (Amersham Biosciences, NJ, USA). 50 For quantitative analysis, band intensities were evaluated densitometrically using the LAS 1000 plus system (Fujifilm, Tokyo, hMLH1 mRNA expression in the six biliary tract cancer cell lines Japan). and SW-48. The expression of hMLH1 mRNA in each cell Cell growth assay. Ten thousand KMG-C cells were seeded onto 6 line was quantitatively assessed by real-time PCR and well plates. Ds RNA for hMLH1 was transfected in triplicate at a compared with the IC50 for SN-38. SW-48 was used as a final concentration of 20 or 40 nM. At 48 h after transfection, the control because the hMLH1 expression in SW-48 was cells were exposed to SN-38 at a concentration of 50 or 100 nM for deficient by conventional RT-PCR (8). In fact, the hMLH1 48 h. The cells were collected and the mean number for each well mRNA level in SW-48 by real-time PCR was extremely low was calculated. The effects of hMLH1 dsRNA were estimated by and below the threshold level of this experiment. The dividing the cell number of hMLH1 dsRNA transfectants from that relationship between hMLH1 mRNA expression and the IC of control dsRNA transfectants. 50 for SN-38 is shown in Figure 2. A positive correlation was Flow cytometry. The cell preparation was the same as described for found between these two factors, although the relationship the cell growth assay. Cells were harvested using 0.25% tripsin- was not statistically significant (R=0.717, p=0.0715). 0.02% EDTA and were fixed in 70% ethanol at 4ÆC. Later, the ethanol was removed, and the cells were stained with propidium Down-regulation of hMLH1 expression by hMLH1 SiRNA iodide solution (50 Ìg/ml in PBS with 100 U/ml RNaseA) for 30 transfection. In order to down-regulate hMLH1 expression min at room temperature in the dark. All samples were analyzed using FACScalibur (counting 10,000 cells per sample) and Cell in the KMG-C cells, SiRNA (dsRNA) was transiently Quest software Version 2.0 (both Becton Dickinson, NJ, USA). transfected into KMG-C cells. As shown in Figure 3A, 48 h after transfection, hMLH1 mRNA expression was repressed Statistical analysis. The relationships with IC50 for SN-38 with the by hMLH1 dsRNA in a dose-dependent manner in six biliary tract cancer cell lines and SW48 were assessed comparison with the control dsRNA. At 72 h after statistically by Pearson's correlation using the Statview software transfection, hMLH1 mRNA expressions were at lower program (SAS Institute Cary, NC, USA). levels than those at 48 h. The band intensity (%) of each Results group, which is estimated by setting the control as 100% is shown in Figure 3B. In the case of 40 nM of hMLH1 Sensitivity to SN-38 in the six biliary tract cancer cell lines and dsRNA transfection, hMLH1 mRNA expressions were SW-48. Drug sensitivity to SN-38 was assessed by the MTT down-regulated to 34.3% and 24.4% at 48 h and 72 h, assay. The IC50 of SN-38 for each cell line is shown in Figure respectively.

867 ANTICANCER RESEARCH 27: 865-872 (2007)

Figure 2. The relationship hMLH1 expression and IC50 for SN-38 in six biliary tract cancer cell lines and SW48. hMLH1 expression in each cell line was measured by real-time RT-PCR and the quantitative hMLH1 value in each cell line was estimated by dividing them with the GAPDH expression values. A positive correlation between hMLH1 expression and IC50 for SN-38 was observed (R=0.717, p=0.0715).

Figure 3. A) Western blot analysis of hMLH1 expression after 5, 20 or 40 nM hMLH1 SiRNA(dsRNA) transfection (a: 48 h after transfection), (b: 72 h after transfection). NC: negative control, ds:double strand, transfected with hMLH1 SiRNA(dsRNA). B) The band intensity of each group after hMLH1 SiRNA transfection. Each bar graph demonstrates the relative intensity (%) compared with control (100%).

868 Sato et al: CPT-11 is Applicable to Biliary Tract Cancer with Low hMLH1 Expression

Figure 4. Cell growth assay after hMLH1 SiRNA transfection with or without SN-38 treatment. Gray bars show the cell number of the negative control, while black bars show the cell number after hMLH1 transfection. 4A: hMLH1 dsRNA 20 nM transfected group. 4B: hMLH1 dsRNA 40 nM transfected group.

Increased sensitivity to SN-38 after hMLH1 dsRNA Discussion transfection. The results of the cell growth assay is shown in Figure 4. To examine the influence of transfection, hMLH1 MMR plays an important role in the cellular response to dsRNA at 20 nM (A) or 40 nM (B) was transfected into various DNA damaging agents. The cells that are defective KMG-C cells without SN-38. Forty eight hours later, KMG- in MMR are tolerant to alkylating agents, such as platinum C cells were exposed to SN-38 at 50 nM or 100 nM in cisplatin (9-12, 28), and 5FU (29). In sharp contrast, the concentrations for an additional 48 h. Cell growth was colorectal cancer cells defective in MMR have been simply inhibited by approximately 50% with SN-38 reported to exhibit higher sensitivity to camptothecin (17). exposure. The cell growth of the hMLH1 dsRNA 20 nM In the present study, the hMLH1 expression was correlated transfected group was further inhibited by approximately with the IC50 for SN-38 using six biliary tract cancer cell 70% of that of the negative control (Figure 4A). The cell lines. Moreover, for the first time, evidence that the number of the hMLH1 dsRNA 40 nM transfected group reduction of hMLH1 expression by SiRNA raised sensitivity was repressed to 55.6% and 52.6% of that of the negative to SN-38 has been shown. Furthermore, apoptosis was control by SN-38 at 50nM and 100nM concentration, induced more strongly in the hMLH1 dsRNA transfected respectively. cells in comparison to the control transfected cells under SN-38 exposure. Flow cytometry. The cell fractions of the KMG-C cells which Jacob et al. (17) recently reported that colorectal cancer were transfected by a negative control or by hMLH1 cell lines defective in DNA MMR exhibited high sensitivity dsRNA with or without SN-38 treatment are shown in to topoisomerase inhibitors. Resistance to the drugs was Figure 5. The subG1 fraction slightly increased in achieved by the functional complement of hMLH1 comparison to the KMG-C control in each transfection transfection (17). In contrast, using the SiRNA system, we group. In the negative control dsRNA transfection (NC) have verified the restoration of drug sensitivity to SN-38 in group, the G2-M fraction of the KMG-C cells increased in biliary tract cancer cells. comparison to the no treatment group with the SN-38 SN-38 interacts with the cellular Topo I-DNA complex treatment in a dose-dependent manner. In the hMLH1 (cleavage complex). The response of this drug appears to dsRNA transfection group, the subG1 fraction correspond to the level of the cleavage complexes (30). predominantly increased with treatment by SN-38 in a dose- Stabilization of the cleavage complexes after CPT-11 (SN- dependent manner. 38) treatment is accompanied by G2-M arrest and apoptosis

869 ANTICANCER RESEARCH 27: 865-872 (2007) SN-38 treatment. The top panel shows the fraction . Flowcytometry cell cycle fractions of KMG-C cells transfected by negative control (NC) or hMLH1 dsRNA (hMLH1) with without Figure 5 of KMG-C without transfection or SN-38.

870 Sato et al: CPT-11 is Applicable to Biliary Tract Cancer with Low hMLH1 Expression

(31). However, the mechanism of apoptosis induction by the 2 Ducreux M, Rougier P, Fandi A, Clavero-Fabri MC, Villing cleavage complexes is not yet understood (32). AL, Fassone F, Fandi L, Zarba J and Armand JP: Effective MMR participates in the induction of apoptosis in treatment of advanced biliary tract carcinoma using 5- fluorouracil continuous infusion with cisplatin. Ann Oncol 9: 653- response to a variety of DNA lesions (14, 33). MMR also 656, 1998. recognizes and repairs base-base mispairs, as well as 3 Taieb J, Mitry E, Boige V, Artru P, Ezenfis J, Lecomte T, small insertion or deletion loops arising during DNA Clavero-Fabri MC, Vaillant JN, Rougier P and Ducreux M: replication (33-35). In addition to mutation avoidance, Optimization of 5-fluorouracil (5-FU)/cisplatin combination some of the MMR components participate in various chemotherapy with a new schedule of leucovorin, 5-FU and DNA repair processes including DSB repair and DNA cisplatin (LV5FU2-P regimen) in patients with biliary tract recombination (24). carcinoma. Ann Oncol 13: 1192-1196, 2002. In our study, the SN-38 treatment induced a G -M arrest 4 Gebbia V, Giuliani F, Verderame F, Borsellino N, Mauceri G, 2 Tirrito ML and Valdesi M: Treatment of inoperable and/or in the KMG-C cells transfected with a control SiRNA. metastatic biliary tree carcinoma with single-agent gemcitabine Furthermore, the repression of hMLH1 expression by or in combination with levofornic acid and infusional hMLH1 SiRNA increased the apoptotic rather than the G2- fluorouracil: results of a multicenter phase II study. J Clin M fraction with SN-38 treatment. These data may indicate Oncol 19: 4089-4091, 2001. that topo I-DNA complexes are more stabilized in the 5 Kim ST, Park JO, Lee J, Lee KT, Lee JK, Choi SH, Heo JS, down-regulation of hMLH1, which could possibly have led Park YS, Kang WK and Park K: A phase II study of to a higher level of apoptosis with drug treatment. However, gemcitabine and cisplatin in advanced biliary tract cancer. Cancer 106: 1339-1346, 2006. we cannot define the precise mechanism at present. 6 Jirieny J and Nystrom-Lahti M: Mismatch repair defects in Several authors have reported the implication of the p53 cancer. Curr Opin Genet Dev 10: 157-161, 2000. gene and MMR system in the efficacy of CPT-11. Magrini et 7 Aquilina G and Bignami M: Mismatch repair in correction of al. reported the effects of the expression of these two genes replication errors and processing of DNA damage. J Cell on CPT-11 on colon cancer cell lines. They showed that Physiol 187: 145-154, 2001. apoptosis was most induced by CPT-11 exposure in the 8 Sato K, Kitajima Y, Kohya N, Miyoshi A, Koga Y and Miyazaki p53–/– hMLH1–/– group, compared with the p53+/+ K: Deficient MGMT and proficient hMLH1 expression renders gallbladder carcinoma cells sensitive to alkylating agents hMLH1–/– and the p53+/+ hMLH1+/+ group (36). In the through G2-M cell cycle arrest. Int J Oncol 26: 1653-1661, 2005. present study, apoptosis following SN-38 treatment was 9 Fink D, Aebi S and Howell SB: The role of DNA mismatch more strongly induced in the hMLH1 cells down-regulated repair in drug resistance. Clin Cancer Res 4: 1-6, 1998. by hMLH1 SiRNA, in comparison to the control SiRNA 10 D'atri S, Tentori L, Lacal PM, Graziani G, Pagani E, Benincasa transfection. 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