Background : Histone deacetylase (HDAC) is well known to be associated with
tumorigenesis through epigenetic regulation, and its inhibitors (HDACIs) induce differentiation
and apoptosis of tumor cells. We examined the therapeutic effects of valproic
acid (VPA, a HDACI) with a combination of 5-fluorouracil (5-FU) in vitro. Methods : A human
pancreas cancer cell line (SUIT-2) and a cholangiocarcinoma cell line (HuCCT1) were
used. Cell viabilities were evaluated by a cell proliferation assay. We determined the anticancer
effects of VPA combined with 5-FU in these cell lines. Results : Pancreas cancer
(SUIT-2) : No effect of 5-FU (1.0 μM) was observed, but 17% and 30% of proliferationinhibitory
effects were recognized in a dose of 2.5 or 5.0 μM, respectively. Cell viability
was only weakly reduced by VPA (0.5 mM). However, in combination of 5-FU (1.0 μM)
with VPA (0.5 mM), 19% of inhibitory effect was observed. Cholangiocarcinoma (HuCCT1) :
5-FU (1.0 μM) did not suppress the cell viability, but 5-FU (2.5 μM) suppressed by 23%.
VPA (0.5 mM) did not suppress the cell viability, while VPA (1.0 mM) weakly decreased it
by 11%. Combination of 5-FU (1.0 μM) and VPA (0.5 mM) markedly reduced the cell viability
by 30%. Conclusion : VPA augmented the anti-tumor effects of 5-FU in cancer cell
lines. Therefore, a combination therapy of 5-FU plus VPA may be a promising therapeutic
option for patients with pancreas cancer and cholangiocarcinoma

Hanaoka, Jun

Ikemoto, Tetsuya

Imura, Satoru

Ishibashi, Hiroki

Iwahashi, Shuichi

Mori, Hiroki

Morine, Yuji

Ochir, Lkhaguva Tovuu

Shimada, Mitsuo

Utsunomiya, Tohru

Tokushima University Institutional Repository

INTRODUCTIONPancreas cancer is one of the most aggressive hu-man cancers. The overall 5-year survival rate amongpatients with pancreatic cancer is5% (1). Cholan-giocarcinoma is a cancer arising from bile duct epi-thelium. This cancer is one of the most difficult dis-eases to treat as pancreas cancer, and no standardchemotherapy has been established (2, 3). There-fore, we have researched about resistance of che-motherapy in pancreatic and biliary tract cancers.5-fluorouracil (5-FU) is a chemotherapeutic drugwhich is widely used mainly for the treatment ofthe digestive system cancer, but the response rateORIGINALEffect of histone deacetylase inhibitor in combinationwith 5-fluorouracil on pancreas cancer and cholan-giocarcinoma cell linesShuichi Iwahashi1, 2, Hiroki Ishibashi1, 2 Tohru Utsunomiya1, Yuji Morine1,Tovuu Lkhaguva Ochir1, Jun Hanaoka1, Hiroki Mori1, Tetsuya Ikemoto1,Satoru Imura1, and Mitsuo Shimada11.Department of Digestive Surgery and Transplantation, Institute of Health Biosciences, the Universityof Tokushima Graduate School, Tokushima, Japan, 2.These authors equally contributed to this study.Abstract : Background : Histone deacetylase (HDAC) is well known to be associated withtumorigenesis through epigenetic regulation, and its inhibitors (HDACIs) induce differ-entiation and apoptosis of tumor cells. We examined the therapeutic effects of valproicacid (VPA, a HDACI) with a combination of 5-fluorouracil (5-FU) in vitro. Methods : A hu-man pancreas cancer cell line (SUIT-2) and a cholangiocarcinoma cell line (HuCCT1) wereused. Cell viabilities were evaluated by a cell proliferation assay. We determined the an-ticancer effects of VPA combined with 5-FU in these cell lines. Results : Pancreas cancer(SUIT-2) : No effect of 5-FU (1.0 μM) was observed, but 17% and 30% of proliferation-inhibitory effects were recognized in a dose of 2.5 or 5.0 μM, respectively. Cell viabilitywas only weakly reduced by VPA (0.5 mM). However, in combination of 5-FU (1.0 μM)with VPA (0.5 mM), 19% of inhibitory effect was observed. Cholangiocarcinoma (HuCCT1) :5-FU (1.0 μM) did not suppress the cell viability, but 5-FU (2.5 μM) suppressed by 23%.VPA (0.5 mM) did not suppress the cell viability, while VPA (1.0 mM) weakly decreased itby 11%. Combination of 5-FU (1.0 μM) and VPA (0.5 mM) markedly reduced the cell vi-ability by 30%. Conclusion : VPA augmented the anti-tumor effects of 5-FU in cancer celllines. Therefore, a combination therapy of 5-FU plus VPA may be a promising therapeu-tic option for patients with pancreas cancer and cholangiocarcinoma. J. Med. Invest.58 : 106-109, February, 2011Keywords : pancreas cancer, cholangiocarcinoma, HDAC inhibitor, valproic acid, epigenetic regulationReceived for publication December 6, 2010 ; accepted Decem-ber 28, 2010.Address correspondence and reprint requests to Mitsuo ShimadaM.D. Professor and Chairman, Department of Digestive Surgeryand Transplantation, Institute of Health Biosciences, the Uni-versity of Tokushima, Kuramoto-cho, Tokushima, 770-8503,Japan and Fax : +81-88-631-9698.The Journal of Medical Investigation Vol. 58 20111061.0 2.5 5.0 1002040608010005FU(䃒M)Cell viability(%)䟼䟼 䟼N.S.A07080901000 0.5 1.0 (mM)VPACell viability(%) 䟼B5FU 1.0 ȣMVPA 0.5mM0708090100Cell viability(%)䟼 䟼N.S. 䟼(-)(-) (-)(+) (-)(+) (+)(+)Cin pancreatic and biliary tract cancers is very low(4, 5). Therefore, new agents and innovative ap-proach to therapy are the important subjects forresearch.Alterations in the epigenetic modulation of geneexpression have been implicated in cancer devel-opment and progression, and histone acetylation,one of the epigenetic regulations, is a posttransla-tional modulation of the nucleosomal histones thataffects chromatin structure and modulates gene ex-pressions. Histone deacetylases (HDACs) comprisean ancient family of enzymes that play crucial rolesin numerous biological processes (6), and HDACsare found to be overexpressed in many tumor types(7, 8). We reported that the survival rate for pan-creas cancer patients with HDAC1-positive was sig-nificantly lower than that for patients with HDAC1-negative, and HDAC1 was considered to be a prom-ising therapeutic target in pancreas cancer (9).HDAC inhibitors induce the differentiation or apop-tosis of cancer cells (10, 11). Therefore, HDAC in-hibitors are promising new agents, in this study, weused Valproic acid (VPA). VPA has the antitumoreffects of a HDAC inhibitor (12), and VPA has beenshown to have anticancer effects in various cancermodels (13).The aim of this study was to investigate the anti-cancer effects of VPA in combination with 5-FU inpancreas cancer and cholangiocarcinoma cell lines.MATERIAL AND METHODCell lines and culture conditionsSUIT-2 cell was purchased from the JapaneseCollection Research Bioresources Cell Bank (Tokyo,Japan). HuCCT-1 was provided by the RIKEN BRCthrough the National Bio-Resource Project of theMEXT, Japan. All cell lines were grown in RPMI1640 supplemented with 10% fetal bovine serum(FBS), 70 μg/mL penicillin and 100 μg/mL strepto-mycin (complete medium) and maintained at 37in a humidiWed incubator with 5% CO2 in air. Thecells were maintained for no longer than 12 weeksafter recovery from frozen stock.ReagentsValproic acid was purchased from Wako PureChemical Industries, Ltd. (Osaka, Japan), and keptat 4and diluted in PBS as necessary at the timeof use. 5-FU was purchased from Kyowa Hakko(Tokyo, Japan) and made fresh in 0.9% NaCl on theday of use.Cell proliferation assayAll of tumor cells (5103) were seeded into 38-mm2 wells of flat-bottomed 96-well plates in quad-ruplicate and allowed to adhere overnight. The spentmedium was then removed, and the cultures wererefed with new medium (negative control) or me-dium containing diVerent concentrations of VPA and5-FU. Incubation was continued for 72 h prior toadding the Cell Counting Kit-8, and after 2 h, theoptical density was measured at 450 nm with a mi-croplate reader (Multiskan JX ; Labsystems).Statistical analysesStatistical comparisons of mean values were con-ducted using oneway ANOVA. All the results arepresented as meanSD. Statistical analysis was per-formed using Stat View 5.0 J software (SAS Institute,Inc., Cary, NC, USA). A P value of less than 0.05was considered to be statistically significant.RESULTSIn pancreas cancer cell line, SUIT-2, no effect of5-FU was observed in dose of 1.0 μM and 17%, 30%and 33% of proliferation-inhibitory effects were ob-served in dose of 2.5, 5.0 and 10 μM (Fig. 1A). VPA(0.5 mM) weakly decreased cell viability by 13%,and VPA (1.0 mM) suppressed by 19% (Fig. 1B). Incombination of 5-FU and VPA, 19% of inhibitory ef-fect was observed in dose of 5-FU 1.0 μM/VPA 0.5mM, the combination effect was significant compareFigure 1 : The effect of 5-FU (A), VPA (B) and combination of5-FU and VPA (C) in inhibiting cell proliferation of human pan-creas cancer cell line, SUIT-2.** : p0.05, * : p0.01.The Journal of Medical Investigation Vol. 58 February 2011 1071.0 2.5 5.0 1002040608010005FU(䃒M)Cell viability(%) 䟼N.S.A07080901000 0.5 1.0 (mM)VPACell viability(%)BN.S. 䟼5FU 1.0 ȣMVPA 0.5mM0708090100Cell viability(%)N.S.䟼 䟼(-)(-) (-)(+) (-)(+) (+)(+)CN.S.to 5-FU alone or VPA alone (P0.01) (Fig. 1C).In cholangiocarcinoma cell line, 5-FU (1.0 μM)did not suppress the cell viability, 5-FU (2.5 μM)suppressed by 23%, and 34% and 39% of proliferation-inhibitory effects were observed in dose of 5.0 and10 μM (Fig. 2A). VPA (0.5 mM) did not suppressthe cell viability, while VPA (1.0 mM) weakly de-creased it by 11% (Fig. 2B). 5-FU (1.0 μM) andVPA (0.5 mM) reduced by 30%, which significantlyaugmented the anticancer effect of 5-FU alone orVPA alone (P0.01) (Fig. 2C).DISCUSSIONIn the present study, we assessed the effect ofHDAC inhibitor (VPA) in combination with 5-FUon pancreatic-biliary carcinoma cell lines. To ourknowledge, this is the first report to show that VPAenhances the effect of 5-FU on both pancreas can-cer and cholangiocarcinoma cell lines.HDAC inhibitors are useful in cancer treatmentwhen used in combination with current chemothera-peutic drugs, especially in combination with 5-FU,HDAC inhibitor (MS275) enhance the effect of 5-FU in colorectal cancer cells (14), and other HDACinhibitor (SAHA) enhance the effect of 5-FU in non-small cell lung cancer (15). The mechanisms of theadditional effects on HDAC inhibitors to the cyto-toxic agent are the enhancement of apoptosis (14)and the up-regulation of p21(waf1/cip1) expression(15). In this study, the mechanisms may be the aug-mentation of apoptosis or the enhancement of p21(waf1/cip1) expression.However, some HDAC inhibitors are of limitedtherapeutic use due to toxic side effects at highdoses (16). VPA is widely used as a therapeuticdrug for epilepsy, its toxicity profile and pharmacoki-netic properties are well established. Furthermore,in our study, the dose of VPA was 0.5 mM, becausethe peak plasma concentration in patients treatedfor epilepsy ranges between 0.5 and 1.2 mM (17).VPA at a dose of 0.5 mM may not cause any seri-ous side effects in clinical setting.Recently, S-1, an oral drug consisting of the 5-FU prodrug tegafur, combined with two modulatorsof 5-FU activity, has been developed (18-20). S-1contains 5-chloro-2,4-dihydroxypyridine (CDHP),CDHP competitively inhibits the 5-FU degradativeenzyme dihydropyrimidine dehydrogenase (DPD),resulting in the retention of a prolonged concentra-tion of 5-FU in blood (18).VPA has been investigated in clinical studies (21,22), we plan the clinical trial of the combination ther-apy, S-1 and VPA. We have expected VPA enhancesthe anti-tumor effect of S-1 in this trial.In conclusion, VPA augmented the inhibitory ef-fects of 5-FU on the proliferation rates of both pan-creas cancer and cholangiocarcinoma cell lines.Therefore, VPA in combination with 5-FU is sug-gested to be a promising therapeutic option for pan-creatic and biliary tract cancers.ACKNOWLEDGEMENTSGrant support was provided by the Grants-in-Aidfor Scientific Researches of the Japan Society for thePromotion of Science (Grant-in-Aid for Young Sci-entists B : No. 22791286). We would like to thankMs. Harada for providing technical assistance.REFERENCES1. 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Effect of histone deacetylase inhibitor in combination with 5-fluorouracil on pancreas cancer and cholangiocarcinoma cell lines

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Effect of histone deacetylase inhibitor in combination with 5-fluorouracil on pancreas cancer and cholangiocarcinoma cell lines

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