プレス発表資料
平成 30 年 11 月 13 日
秋
秋田大学研究グループの成果が米国の「Journalof Functional

田

大

学

Foods」にオンライン掲載

カレーのスパイス「クルクミン」が熱変性した
GO-Y022 が胃癌を抑制することを解明
秋田大学大学院医学系研究科臨床腫瘍学講座・腫瘍内科の柴田浩行教授と吉田泰一医員の研究グループは東北大学、金沢大学、癌研究会癌研究所との共同研究により、スパイスのクルク
ミンが熱変性によってできる化合物”GO-Y022”がヒトの胃癌細胞株の増殖を抑制し、アポトー
シスによる細胞死を誘導することを発見しました。また、胃癌を起こすモデルマウスに
GO-Y022 を含む餌を経口摂取させると、GO-Y022 を含まない餌を投与した胃癌モデルマウスと比較して胃癌のサイズが約 1/3 に縮小されました。GO-Y022 は市販のカレーにも含まれていました。今後、「GO-Y022 リッチなカレー」のメニューを考案することなどで医食同源を目指すことが期待されます。
本 研 究 成 果 は 10 月 17 日 に Journal of Functional Foods の オ ン ラ イ ン で（https://authors.elsevier.com/a/1Xv9U6FNx9edP9）公表されました。

胃癌による死亡は世界第 2 位を占める悪性腫瘍です。クルクミンは癌化に関連する分子を抑制することが知られています。しかし、クルクミンの活性は低く、改良の余地があります。そこで、我々は様々なクルクミンの誘導体を合成し、抗腫瘍活性が強い誘導体の合成に成功しました。しかし、これらの誘導体はペンタノイドであり、ヘプタノイドであるクルクミンとは化学構造が大きく異なっていました。近年、これらの誘導体の中で GO-Y022 と名付けたペンタ
ノイドがクルクミンの熱変性で生じることが判明しました。この GO-Y022 は 4 種類の胃癌細胞株に対してクルクミンよりも約 5 倍強い抗腫瘍活性を示し、また、GO-Y022 はクルクミン
よりも強いアポトーシス誘導能を有しています。GO-Y022 は胃癌のマウスモデルの腫瘍のサイズを約 1/3 に抑制し、経口摂取された GO-Y022 は胃の粘膜や胃癌で検出されましたが、血液中には見出されませんでした。GO-Y022 は胃粘膜で作用し、全身性の有害事象も認められませんでした。GO-Y022 は市販のカレーにも含まれ、GO-Y022 には胃癌の抑制効果があり、
実はこれまでにもカレーの成分として食べられていました。これによりカレーの GO-Y022 は機能性食品になりうる可能性が示されました。

【論文著者・タイトル】
Taichi Yoshida, Takashi Maruyama, Masatomo Miura, Masahiro Inoue, Koji Fukuda,
Kazuhiro Shimazu, Daiki Taguchi, Hiroaki Kanda, Masanobu Oshima, Yoshiharu Iwabuchi,
Hiroyuki Shibata

Dietary intake of pyrolyzed deketene curcumin inhibits gastric carcinogenesis【お問い合わせ先】
秋田大学大学院医学系研究科
臨床腫瘍学講座 教授 柴田浩行
TEL：018-884-6261
FAX：018-884-6455
E-mail：m-soumu@hos.akita-u.ac.jp

Journal of Functional Foods 50 (2018) 192–200

Contents lists available at ScienceDirect

Journal of Functional Foods
journal homepage: www.elsevier.com/locate/jff

Dietary intake of pyrolyzed deketene curcumin inhibits gastriccarcinogenesis

T

Taichi Yoshidaa, Takashi Maruyamab, Masatomo Miurac, Masahiro Inouea, Koji Fukudaa,
Kazuhiro Shimazua, Daiki Taguchia, Hiroaki Kandad, Masanobu Oshimae, Yoshiharu Iwabuchif,⁎
Hiroyuki Shibataa,aDepartment of Clinical Oncology, Graduate School of Medicine, Akita University, Akita, Japan
Department of Immunology, Graduate School of Medicine, Akita University, Akita, Japanc
Department of Pharmacy, Akita University Hospital, Akita, Japand
Division of Pathology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japane
Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japanf
Department of Organic Chemistry, Graduate School of Pharmaceutical Science, Tohoku University, Sendai, Japanb

A R T I C LE I N FO

A B S T R A C T

Keywords:
CurcuminChemoprevention
Diarylpentanoid analog
Gastric cancer
Pyrolysis
STAT3 inhibition

Gastric cancer is the second leading cause of cancer-related mortality worldwide. Curcumin, a phytochemical,
possesses molecular inhibitory potentials for regulating malignancies. However, the lower potential of curcumin
warrants improvement. Thus, we synthesized diarylpentanoid analogs with higher potency; however, these
diﬀered structurally from curcumin—a heptanoid. Recently, one diarylpentanoid was formed following pyrolysis
of curcumin, which is identical to our GO-Y022. The growth inhibition of gastric cancer cells by GO-Y022 was
ﬁve-fold higher than by curcumin; GO-Y022 displayed superior apoptosis induction ability. Besides, it suppressed the gastric tumor growth to a third in a mouse model. GO-Y022 was moved to the epithelial and gastrictumors but not detected in the bloodstream. Moreover, oral GO-Y022 was eﬀective topically, exhibited no adverse events in mice, and was detected in the commercially available curry paste. Brieﬂy, GO-Y022 can inhibit
gastric carcinogenesis; it is dietary and can be safely used as an oral functional food.

1. Introduction
Gastric cancer is the second leading cause of cancer-related mortality globally, with a mortality rate of 700,000 in 2012 (Ferlay,
Soerjomataram, & Dikshit, 2015). Typical risk factors for gastric cancer
comprise Helicobacter pylori infection, obesity, smoking, consumption of
red meat and alcohol, and low socioeconomic status (Hundahl, Phillips,& Menck, 2000). However, the precise mechanisms underlying gastriccancer remain unclear, necessitating the determination of eﬀective
treatment for gastric cancer. Surgical resection, the only approach for acure, is not applicable in patients with stage IV gastric cancer, for whom
chemotherapy remains the primary treatment (Japanese Gastric Cancer
Association, 2017). However, the median survival of stage IV patients isapproximately 1 year at best, even with the use of the newer treatment
options (Cunningham, Starling, & Rao, 2008; Van Cutsem, Moiseyenko,& Tjulandin, 2006).
Previously, several dietary elements, such as phytochemicals, havebeen investigated for cancers in these aspects. Curcumin, a dietary
pigment in use for > 3000 years in Asia primarily, is being increasingly
analyzed for its antitumor potential (Kunnumakkara, Bordoloi, &Harsha, 2017). Reportedly, curcumin can suppress the proliferation ofvarious cancer cell types and induce apoptosis (Kunnumakkara et al.,2017). In addition, it comprises antiangiogenic properties and is known
to enhance cancer immunity and inhibit cancer immunotolerance(Mohamed, Jantan, & Haque, 2017). Furthermore, curcumin inhibits
the growth of gastric cancer cell lines (Zhang, Lin, & Zhou, 2015).
However, curcumin is not used in a clinical setting because of its limitedbioavailability and relatively low potential, and the ongoing research is
focused on overcoming these limitations.
Thus, we successfully synthesized a series of new diarylpentanoid
analogs to attain a new curcumin analog with higher antitumor potential (Ohori, Yamakoshi, & Tomizawa, 2006) and demonstrated thatthe new analogs inhibited the proliferation of various cancer cell types,
including colon cancer stem cells (Lin, Liu, & Li, 2011). In addition, we

Abbreviations: C5, diarylpentanoid; STAT3, signal transducers and activators of transcription 3⁎
Corresponding author at: Department of Clinical Oncology, Graduate School of Medicine, Akita University, Hondo 1-1-1, Akita, Japan.
E-mail address: hiroyuki@med.akita-u.ac.jp (H. Shibata).
https://doi.org/10.1016/j.jﬀ.2018.09.033
Received 2 June 2018; Received in revised form 29 September 2018; Accepted 30 September 2018
1756-4646/ © 2018 Elsevier Ltd. All rights reserved.

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medium added by GO-Y022, and the IC50 values were calculated after
72 h incubation.

determined that these analogs exhibited the safety proﬁle of the original compound curcumin in mouse models (Lin et al., 2011; Shibata,
Yamakoshi, & Sato, 2009) and that one diarylpentanoid, 1,5-bis(3,5dimethoxy-4-methoxymethoxyphenyl)pentadien-3-one (GO-Y031) inhibited gastric carcinogenesis in vivo with a good safety proﬁle (Uehara,
Inoue, & Fukuda, 2014). However, a big gap remains between themouse models and the ﬁrst-in-human study. Our diarylpentanoid analogs (C5) are structurally diﬀerent from curcumin, which is a diarylheptanoid (C7). Recently, a breakthrough ﬁnding was reported demonstrating that a deketene form of curcumin, which is a C5 analog,
formed because of curcumin pyrolysis during cooking of curry(Dahmke, Boettcher, & Groh, 2014), this C5 analog is identical to ananalog we synthesized, 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one (GO-Y022). Thus, this study aims to investigate the efﬁcacy and safety of GO-Y022 in human gastric cancer cell lines and amouse gastric cancer model and determines the pharmacokinetic proﬁle of GO-Y022.

2.5. Apoptosis analysis
Cells were cultured in a 10 cm-dish to semiconﬂuency, then they
were treated with GO-Y022 at the indicated concentration. After 24 htreatment, cells were lysed and the lysate was applied to the ELISA
plate. Apoptosis was determined by the M30-Apoptosense enzymelinked immunosorbent assay (ELISA; VLVbio) from Funakoshi (Tokyo,
Japan), per the manufacturer's protocol. We treated the control with 1%
DMSO alone. The relative values were calculated as the ratio to thecontrol (the control value = 1.0). All cellular experiments were conducted in triplicate, unless otherwise speciﬁed.
2.6. ELISA (β-catenin and STAT3)
Cells were cultured in a 10 cm-dish to semiconﬂuency, then they
were treated with GO-Y022 at the indicated concentration. After 24 htreatment, cells were lysed and the lysate was applied to the ELISA
plate. We determined the activation of β-catenin and phosphorylationof signal transducers and activators of transcription 3 (STAT3) using the
Active β-Catenin MKA ELISA (Symansis, New Zealand) and phosphoSTAT3 (pSTAT3) Tyr705 sandwich ELISA (Cell Signaling Technology
Japan, Tokyo, Japan), respectively. The obtained values were correctedby the cell numbers and then represented as the relative values against
the control (the control value = 1.0).

2. Material and methods
2.1. Compounds
Originally, GO-Y022 was synthesized by the Department of Organic
Chemistry, Graduate School of Pharmaceutical Science at Tohoku
University (Sendai, Japan) and was subsequently purchased from
Nippon Carbide Industries Co., Inc. (Tokyo, Japan; Fig. 1). We dissolved
GO-Y022 in dimethyl sulfoxide (DMSO) at 10–50 mmol/L as a stock
solution. Curcumin was purchased from Wako Pure Chemical Industries(Osaka, Japan), and the high-fat diet (HFD) 32 was purchased from
CLEA Japan (Tokyo, Japan).

2.7. Fluorescence-activated cell sorting (FACS) analysis
Cells were seeded in a 6 well plate to grow semi-conﬂuent. Then,they were treated with compounds for 48 h. Cells were scraped andstained with Annexin V apoptosis detection kit APC (Invitrogen,
Thermo Fisher Scientiﬁc K.K. Tokyo, Japan) and propidium iodide(eBioscience, Thermo Fisher Scientiﬁc K.K.) to detect apoptosis/necrosis, according to the manufacturer’s protocol. The stained cells were
subjected to ﬂow cytometric analysis with BD FACS AriaTM III (BDbioscience, Tokyo, Japan). Data were analyzed with FlowJo software(Tree Star, OR).

2.2. Cell lines
We obtained the gastric cancer cell lines KATO III and GCIY fromthe American Type Culture Collection through Summit Pharmaceutical
International (Tokyo, Japan) and RIKEN BioResource Research Center(Tsukuba, Japan), respectively. In addition, both H-111-TC and SH-10TC were obtained from Cell Resource Center for Biomedical Research
Cell Bank Tohoku University. Notably, all cell lines were cultured per
the manufacturer's protocols.

2.8. Mice experiments

2.3. In vitro growth assay

We obtained K19-Wnt1/C2 mE (Gan) mice by crossing K19-Wnt1
and K19-C2 mE mice (Oshima, Matsunaga, & Fujimura, 2006). Gan
mice are genetically engineered animals where activated oncogene,
Wnt-1 signaling and inﬂammation related genes such as COX-2 andprostaglandin E synthase, are up-regulated simultaneously. Resulting
gastric cancers arise spontaneously. Every day, Gan mice were fed 5 g
High Fat Diet 32 alone (n = 6) or HFD added with 0.5% (weight/weight)
GO-Y022 (n = 7), starting since 9 weeks of age and were sacriﬁced andexamined at 16 weeks of age with minor modiﬁcations, as described
previously (Uehara et al., 2014). As shown in the previous study, gastriccancer could be detected after 12-weeks of age in the HFD-fed Gan mice
(Uehara et al., 2014). HFD was used, because GO-Y022 is hydrophobic,
and it can be mixed well with oily materials, such as HFD. All animal
experiments were performed humanely and complied with the guidelines set by Akita University and were approved by the related ethics
committee (certiﬁcation #a-1-2641). The tumor volumes were evaluated as follows (Euhus, Hudd, & LaRegina, 1986): tumor volume(mm3) = (shortest tumor diameter)2 × (longest tumor diameter)/2.

We assayed the cell viability by quantiﬁcation of the uptake andmetabolism of WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt), per the manufacturer's instructions (Dojindo Laboratories, Kumamoto, Japan).
Then, we determined the half-maximal inhibitory concentration (IC50)
by the MTT assay as described previously (Kudo, Yamakoshi, & Sato,2011).
2.4. Eﬀect on a normal gastric epithelial cells
A human gastric epithelial cells from 58 year Caucasian, HGaEpC
was purchased from CELL APPLICATIONS, INC (San Diego, CA).
HGaEpC cells (lot 3288) were seeded in each well of a 96 well-plate at
5 × 105 cells. Cells were cultured with the manufactures’ supplied

2.9. Immunohistochemistry
We performed immunohistochemistry as previously described(Uehara et al., 2014) using previously described protocols with thefollowing antibodies: anti-mouse β-catenin (1:1500; C2206, rabbit;

Fig. 1. Schematic diagrams of curcumin and GO-Y022. MeO indicates amethoxy group.
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Sigma-Aldrich, Tokyo, Japan) and anti-mouse pSTAT3-Tyr705 (1:100;
D3A7, rabbit; Cell Signaling Technology, Japan) (Uehara et al., 2014).
To Quantitate the positivity of pStat3 expression, we randomly selected
the specimen and over 80 epithelial cells were counted for the positivityof pStat3 in each mouse group (HFD diet mice; n = 7, GO-022 dietmice; n = 6), and the percentage of positivity is indicated.

methanol and water (1.0 mL each). Next, we washed the cartridge with
1.0 mL water and 1.0 mL 60% methanol in water and eluted with
1.0 mL 100% methanol. Then, the eluate was dried by vortex-vacuum
evaporation at 70 °C using a rotary evaporator (AS-ONE CVE-2AS,
Osaka, Japan). After that, we dissolved the resulting residue in 20 μL
methanol and vortexed for 30 s; in addition, 20 μL of the mobile phase
was added to the sample, which was vortexed for another 30 s. Next, we
processed a 20 μL aliquot of the sample by high-performance liquid
chromatography (HPLC), which was conducted using a PU-2080 plus
chromatography pump (JASCO, Tokyo, Japan) equipped with a
CAPCELL PAK C18 MG П (250 mm × 4.6 mm I.D.; Shiseido, Tokyo,
Japan) HPLC column, a UV-2075 light source, and an ultraviolet detector (JASCO). The mobile phase was water–acetonitrile–methanol(55:25:20, v/v/v), which was degassed in an ultrasonic bath before
using. In addition, the ﬂow rate was 0.5 mL/min at ambient temperature, and we performed the sample detection at 250 nm. The amount of
GO-Y022 was corrected by the tissue wet weight, and indicated as ng/g
tissue. Of note, we also measured blood concentrations of GO-Y022 as
bellow. Five milligram of GO-Y022 (mixed with 200 μL of 1% methyl
cellulose (Wako)) was orally administrated by using a ﬂexible sonde(5202, FUCHIGAMI, Kyoto, Japan) to the twelve week-aged B6 malemice, which were anesthetized with intraperitoneal injection of 50 μL of
the mixture of 10 mg of midazolam (SANDOZ, Tokyo, Japan), 0.75 mg
of medetomidine (ZENOAQ, Koriyama, Japan), and 12.5 mg of butorphanol (Wako) dissolved in 25 mL of distilled water after one-day
fasting. Blood samples were collected at 1, 2, 4, 8 and 24 h after administration (n = 3). The same amount of 200 μL of 1% methyl cellulose alone was orally administrated to the control group mice undersame anesthesia, and the blood samples were obtained at the sameintervals (n = 3). The blood concentration of GO-Y022 and their metabolites were determined by following HPLC method. After addition of
200 μL acetonitrile a 50 μL blood sample, the solution was vortexed for
30 s. This mixture was spun for 5 min at 13,000g. The clear supernatant
was ﬁltered through a Millipore ﬁlter (0.45 μm; Millex-LH®, Tokyo,
Japan) and was then injected into the HPLC apparatus.

2.10. Evaluation of the safety proﬁle of GO-Y022
As a measure of the overall health status, body weight of mice wasdetermined before sacriﬁce. In addition, the tissue-speciﬁc toxicity
proﬁle of GO-Y022 was assessed by a blood test. Brieﬂy, we collectedblood from the infraorbital venous plexus before sacriﬁce and analyzed
the serum by Oriental Yeast (Tokyo, Japan) to evaluate the total bilirubin (T-Bil), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) for liver toxicity, creatinine (Cre) for renal toxicity,and lactate dehydrogenase (LDH).
2.11. Localization of GO-Y022
We determined tissues localization and blood concentrations of GOY022. Brieﬂy, at the time of sacriﬁce, we collected specimens from theheart, kidney, liver, spleen, and digestive tract, including the esophagus, stomach, and small and large intestines, as well as the gastrictumor. Resected alimentary tracts were incised along their long axis,
and washed in the distilled water with vigorous shaking to remove thefeed of the surface several times. Then, the tracts were expanded withpins on the dork board. The mucosal surface was swabbed by cotton
wetted with the distilled water until the all oily content of High Fat Diet
was disappeared. After measuring the weight of the tissue sample, thenthey were homogenized in the distilled water, and analyzed by HPLC.
The parenchymal organs were directly homogenized in the distilledwater.
The sample of each tissue was homogenized in 200 μL of the distilled water, and it was applied to an Oasis HLB extraction cartridge(Nihon Waters K.K., Tokyo, Japan) that was preactivated with

Fig. 2. The growth inhibition of gastric cancer cell lines treated with GO-Y022. The growth inhibition was calculated as the percentage of the cell numbers against themock (vehicle alone, 1% DMSO). Open circle indicates curcumin, and the closed circle indicates GO-Y022. *P < 0.01; **P < 0.05.
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Fig. 3. Growth properties of gastric cancer cell lines treated with GO-Y022. Control cultures were treated with the vehicle alone (1% DMSO). The starting cellnumber (at 0 hr) is indicated as 1.0, and the cell numbers are indicated as relative values to the start. Open circle indicates the control. Closed circle indicates 5 μM of
curcumin treatment. Open rectangle indicates 5 μM of GO-Y022 treatment. Closed rectangle indicates 10 μM of GO-Y022 treatment. P < 0.01; **P < 0.05.

2.12. Mass spectrometry

2.13. Statistical analysis

Quantiﬁcation of GO-Y022 in curry was outsourced to Japan Food
Research Laboratories (Tokyo, Japan). The procedures were described
brieﬂy. GO-Y022 was dissolved in methanol, and diluted with water/
methanol (1:1) solution to make standard solution ranging
0.0001–0.01 mg/L. Five gram of curry sample was mixed with 20 mL ofhexane, then homogenized with 40 mL of hexane saturated acetonitrile.
After centrifugation (2000g, 5 min), the under (acetonitrile) layer wascollected. Then, remaining hexane layer was again mixed with 40 mL ofacetonitrile, and centrifuged. This step was repeated two times. The
collected acetonitrile layer was mixed. The volume was measured, andone of 200 mL was applied to Sep-Pak Plus C18 column (Waters, Tokyo,
Japan) conditioned by acetonitrile. Then, the sample was eluted with
1 mL of acetonitrile. The eluent was dried, then solved in 2 mL ofwater/methanol (1:1) solution, and ﬁnally it was diluted with water/
methanol (1:1) to 5-fold dilution. Two microliter of sample solution was
analyzed by liquid chromatography-tandem mass spectrometry (LCunit: Agilent 1100 series (Agilent Technologies, Tokyo, Japan), MS unit:
API-4000 (Applied Biosystems, Waltham, MA)). The operation condition was described bellow: column (CAPCELL PAK C18 MG II (SHISEIDO, Tokyo, Japan)), column temperature (40 °C), mobile phase (A;0.1 vol% formic acid-acetonitrile, B; 0.1 vol% formic acid solution)

Data are presented as mean ± standard deviation. The betweengroup diﬀerences were analyzed using the Fisher's exact probability testor unpaired Student's t-test (two-sided) with StatMate III version 3.14
(ATMS, Tokyo, Japan) or BellCurve for Excel version 2.00 (Tokyo,
Japan).

Time (min)Phase A (%)
Phase B (%)

0
55
45

2
95
5

7.5
95
5

7.51
55
45

3. Results
3.1. GO-Y022 suppressed the growth of gastric cancer cell lines
We examined the growth inhibitory potential of GO-Y022 in four
gastric cancer cell lines, including GCIY, KATO III, SH-10-TC, and H111-TC. The IC50 value for GO-Y022 after 96 h treatment for GCIY was
7.32 μM, whereas that for curcumin were 30.4 μM. That of GO-Y022 for
KATO III was 5.98 μM, whereas that of curcumin for KATO III was
24.8 μM. Those of GO-Y022 were 3.28 μM for SH-10-TC, and 3.67 μM
for H-111-TC, respectively, whereas those of curcumin were 31.5 μM
for SH-10-TC, and 25.8 μM for H-111-TC, respectively. The inhibition of
growth was signiﬁcantly higher with GO-Y022 than curcumin in all
four gastric cancer cell lines (Fig. 2). The IC50 value was 4.2-fold higher
in GCIY, and 4.1-fold higher in KATO III. Those were 9.6-, and 7.0-foldhigher in SH-10-TC, and H-111-TC, respectively. In addition, theaverage IC50 of GO-Y022 was 5.06 ± 1.88 μM, which was signiﬁcantly
lower than that of curcumin (27.4 ± 3.55 μM; P < 0.001). These invitro ﬁndings suggested that the growth inhibitory potential of GO-Y022
was 5.4-fold higher than that of curcumin.
Next, we assessed the growth property of each gastric cancer cellline in the presence of 5 μM GO-Y022. The same concentration of curcumin could not inhibit the growth at 24 h following the treatment for
all four cell lines (Fig. 3). Except for GCIY, 5 μM GO-Y022 decreased thegrowth < 50% of the control for three cell lines at 24 h (Fig. 3). For
GCIY, 10 μM GO-Y022 decreased the growth < 50% of the control(Fig. 3). In addition, three of four gastric cancer cell lines could not
grow in the presence of 5 μM GO-Y022, whereas those could grow in the

15
55
45

Flow rate: 0.2 mL/min, Injection volume: 2 μL, Ionization mode: ESI(+), Curtain gas: 40 psi, Nebulizer gas: 60 psi, Drying gas: 70 psi,
Drying gas temperature: 650 °C, Collision gas: nitrogen, Ionization:
5000 V, Declustering potential: 86 V, Multiple reaction monitoringtransition (m/z), collision energy (eV): 327.2 > 89.2 (65),327.2 > 145.1 (39), 327.2 > 177.2 (29).
Pyrolysis was conducted by heating 100 g of curry roux by microwave (250 °C for 20 min).
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T. Yoshida et al.

Fig. 4. The apoptosis induction with GO-Y022 in gastric cell lines. The amount of an apoptosis indicator, soluble caspase-cleaved keratin 18 was determined by
ELISA. The amount of soluble caspase-cleaved keratin 18 in the mock-treated cells (control) is indicated as 1.0. The relative values to the mock were indicated in eachtreated cells with 5 μM of curcumin or GO-Y022. The statistical signiﬁcance is indicated by asterisks.

presence of 5 μM curcumin. Overall, these ﬁndings suggested that GOY022 exhibited a stronger growth inhibitory potential than curcumin.

treated cells.
After 24 h treatment, the mean relative level of apoptosis in GCIY
cells treated with 5 μM GO-Y022, evaluated per the rate of apoptosis incultures treated with the vehicle DMSO that was set to 1.0, was
3.48 ± 0.64, whereas that with 5 μM curcumin was 1.25 ± 0.42
(Fig. 4). For KATO III, the relative apoptosis level was 10.85 ± 0.21,
whereas that with 5 μM curcumin was 0.70 ± 0.58. For H-111-TC, thatwas 3.24 ± 0.44, whereas that with 5 μM curcumin was 0.65 ± 0.28.
GO-Y022 exhibited 2.78-fold higher potential of the apoptosis

3.2. Apoptosis induction with GO-Y022
We assessed the amount of an apoptosis indicator, soluble caspasecleaved keratin 18 by ELISA at 24 h after treatment. And those amounts
were compared with the mock-treated cells (control) was indicated as
1.0. These relative apoptosis levels to the mock were indicated in eachFig. 5. Eﬀects on the phosphorylation of STAT3 and β-catenin by GO-Y022. A. The levels of phosphorylated STAT3 (pSTAT3) were determined by ELISA methods.
The amounts of pSTAT3 in the mock-treated cells (1% DMSO) treatment as a control are indicated as 1.0 (open rectangle). The relative values of pSTAT3 to the mockin each treated cells with 5 μM of curcumin (rectangle with diagonal line) and GO-Y022 (rectangle with vertical line) were indicated. The statistical signiﬁcance isindicated by asterisks. B. The levels of β-catenin were determined by ELISA methods. The amounts of β-catenin in the mock-treated cells (1% DMSO) treatment as acontrol are indicated as 1.0 (open rectangle). The relative values of β-catenin to the mock in each treated cells with 5 μM of curcumin (rectangle with diagonal line)
and GO-Y022 (rectangle with vertical line) were indicated. The statistical signiﬁcance is indicated by asterisks.
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induction than curcumin in GCIY. GO-Y022 also exhibited 15.6-fold
and 4.98-fold higher apoptosis induction in KATO III and H-111-TC celllines, respectively. However, for SH-10-TC, that was 9.40 ± 0.15,
whereas that with 5 μM curcumin was 8.99 ± 0.23. That was merely
1.05-fold higher than curcumin. So we conﬁrmed the data by FACS
analysis (Fig. S1). Apoptosis/necrosis fraction was 25.5% in 5 μM of
curcumin treated SH-10-TC cells, whereas that was 28.8% in the mocktreated cells. On the other hand, that was 34.9% in 5 μM GO-Y022
treated SH-10-TC cells. Further, that was 45.3% in 10 μM GO-Y022
treated SH-10-TC cells, whereas that was 37.9% in 10 μM curcumin
treated SH-10-TC cells. Similar result was obtained and indicated that
SH-10-TC cells was sensitive to GO-Y022 to induce apoptosis as well as
curcumin.
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.jﬀ.2018.09.033.
The apoptosis induction might be one of the causes of growth inhibition of GO-Y022.

assessed the ability of GO-Y022 to inhibit the STAT3 phosphorylationand to degrade β-catenin in gastric cancer cell lines.
3.3.1. Inhibition of pSTAT3
The relative pSTAT3 level per cell, compared with mock (1%DMSO) was 0.111 ± 0.011 at 5 μM of GO-Y022, in KATO III, whereasthat was 0.621 ± 0.046 in KATO III at 5 μM of curcumin. The relative
pSTAT3 level was 0.850 ± 0.016 in SH-10-TC with 5 μM GO-Y022,
whereas that was 0.661 ± 0.046 in SH-10-TC at 5 μM curcumin. The
relative pSTAT3 level was 0.196 ± 0.097 in H-111-TC, whereas thatwas 0.958 ± 0.060 in H-111-TC at 5 μM curcumin. The relative
pSTAT3 level was 1.063 ± 0.007 in GCIY with 5 μM GO-Y022,
whereas that was 1.002 ± 0.040 in GCIY at 5 μM curcumin (Fig. 5A).
Inhibition of pSTAT3 with 5 μM of GO-Y022 was apparent in KATO III
and H-111-TC, however that was not observed in SH-10-TC and GCIY.
The relationships among the IC50 values, the relative apoptosis induction (at 5 μM) and the relative inhibition of pSTAT3 (at 5 μM) variedamong cell lines. To examine the eﬀect of pSTAT3 inhibition on thegastric cancer cell lines, we used cryptotanshinone (CTS), a known
STAT3 phosphorylation inhibitor (Donmez, Demirezen, & Beksac,
2016). The IC50 value of CTS for KATO III was 4.29 μM. That for GCIY
was 14.02 μM. That for SH-10-TC was 6.91 μM. However, that for H111-TC was > 100.0 μM (Fig. S2).

3.3. In vitro eﬀects on the STAT3 phosphorylation and β-catenin activation
with GO-Y022
Several lines of evidence suggest that the suppression of the β-catenin pathway and phosphorylation of STAT3 at Tyr705 contribute to
the inhibition of gastric carcinogenesis (Clements, Wang, & Sarnaik,
2002; Tye, Kennedy, & Najdovska, 2012). As our newly synthesized
curcumin analogs have similar diarylpentanoid structures, they aresupposed to have same targets molecules. For examples, GO-Y030 and
GO-Y031 have suppressive eﬀects on the growth of gastrointestinal
tumors. GO-Y031, another and more potent C5 curcumin analog that is
not found in cooked curry, was previously reported to suppress the βcatenin activation and STAT3 phosphorylation (Uehara et al., 2014).
GO-Y030 was shown to associate with the ATP binding site of STAT3(Lin et al., 2011). Moreover, it was reported that Helicobacter pylori, amajor cause of gastric cancer, activates STAT3 that plays an importantrole in gastric carcinogenesis (Zhao, Dong, & Kang, 2014). Thus, we

3.3.2. Eﬀect on β-catenin
GO-Y030 and GO-Y031 can induce the degradation of β-catenin.
Therefore, we also examined the degradation of β-catenin with GOY022.
At 5 μM, GO-Y022 did not decrease the relative levels of activated βcatenin per cell (Fig. 5B); the relative levels to the control (1% DMSO)
was 1.566 ± 0.163 in GCIY. That was 2.988 ± 0.593 in KATO III.
Those were 1.136 ± 0.099 in SH-10-TC, and 3.057 ± 0.252 in H-111TC cells, respectively. In contrast, that with 5 μM curcumin in GCIY was
1.198 ± 0.060, that in KATO III was 0.638 ± 0.112, that in SH-10-TC
was 1.117 ± 0.056, and that in H-111-TC was 1.009 ± 0.065
Fig. 6. The in vivo inhibition of gastriccancer in a mouse model with the oral administration of GO-Y022. A, Representing
appearance of GO-Y022-treated gastric tumors. Twenty-ﬁve milligram of GO-Y022
was mixed in 5 g of HFD were given daily
and freely to each mouse. Astarisks indicate
the tumors. The average tumor incidence
was two in a mouse. B, HFD alone-treatedgastric tumor. Red asterisk indicates tumor.
C and D, pathological views of A and B, respectively. Bars indicate 500 μm. E, The volume of each tumor in the GO-Y022-treated(n = 7) and the HFD-mice (n = 6). The
average volume with standard deviation ofeach group is indicated (p = 0.0585). (Forinterpretation of the references to colour inthis ﬁgure legend, the reader is referred to
the web version of this article.)197

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T. Yoshida et al.

(Fig. 5B). In the cases of the 5 μM curcumin treatment, the levels ofactivated β-catenin per cell were not decreased and comparable withthe control. Diﬀerent from pSTAT3, β-catenin was activated in thesurviving cells treated with 5 μM GO-Y022. This phenomenon was similar to the observation where β-catenin showed intense positivity inthe non-apoptotic epithelial cells around the apoptotic cells (Ge, Yang,& Chen, 2015).
Degradation of β-catenin by GO-Y022 was not observed. The action
mechanisms of GO-Y022 was diﬀerent from another curcumin analog
such as GO-Y030 and GO-Y031 in this aspect.
3.4. In vivo inhibitory potential of GO-Y022 in a mouse model of gastriccancerWe assessed the in vivo ability of GO-Y022 to inhibit gastric carcinogenesis in Gan mice that were freely administered 0.5% (weight/
weight) GO-Y022 orally for 8 weeks. Histopathologically, all gastric tumors were suggested to be adenocarcinomas. As the tumors grew in a
block, it became diﬃcult to count the multiplicity of tumors; thus, weevaluated the volume of tumors. The average volume of gastric tumors
in mice treated with GO-Y022 for 8 weeks was 39.9 ± 41.8 (range:
0.5–150.0) mm3, whereas that in mock-treated mice was106.7 ± 115.4 (range: 4.0–322.5) mm3 (p = 0.059; Fig. 6), whichsuggested that GO-Y022 could apparently inhibit the gastric cancer
growth in vivo. In addition, we determined the expression of GO-Y022target molecules β-catenin and pSTAT3 by immunohistochemistry (Fig.
S3). The disappearance of the β-catenin signal in the cytosol, which
revealed the accumulation of β-catenin, an indicator of the β-catenin
activation, was not apparent. However, the inhibition of the nuclear
staining for pSTAT3 was apparently detected by immunohistochemistry(IHC). By IHC of Gan mice treated with HFD, it was shown that pStat3
in the nucleus was observed in many cells, however that was not sofrequently observed in the epithelial cells in mice treated with GO-Y022(Fig. S3A and B). Quantitation of the positivity of pStat3 expression
indicated that the percentage of pStat3-positive cells was
64.4 ± 10.1% (range: 54.0–82.0) in Gan mice fed with mock, whereasthat was 20.3 ± 15.2% (range: 2.0–38.0) in Gan mice fed with GOY022 (p = 0.001, Fig. S3C). This data showed that GO-Y022 could inhibit phosphorylation of Stat3 in vivo, and that is a candidate target
molecule of GO-Y022.

Fig. 7. Tissue distribution of GO-Y022. The data shows the tissue distribution of
GO-Y022 in Gan mice (GO-Y022 treated mice (n = 3) and mock-diet mice(n = 3)). H, heart; K, kidney; L, liver; S, spleen; E, esophagus; G, gastric lumen;I, intestine; A, ascending colon; B, blood; T, tumor. Each dot and a star indicate
an individual mouse. The result of HPLC analysis is indicated in the inset, representatively.

judged GO-Y022 and its metabolites were not detected in the blood.
Orally administrated GO-Y022 acted topically in the luminal epithelia of the digestive organs. Finally, the tumor concentrations of GOY022 ranged 80–1500 ng/g tissue.

3.6. Safety proﬁle of GO-Y022
First, we examined the eﬀect of GO-Y022 on the normal gastricepithelium using a primary culture of HGaEpC. GO-Y022 has a growth
inhibitory eﬀect on HGaEpC, and the IC50 values was 3.08 μM (Fig. S5).
Spheroids of HGaEpC were observed at 10 μM, but they were absent at
50 μM. The cells were entirely killed at 50 μM.
We assessed the hepatic and renal toxicity proﬁles of GO-Y022 bymeasuring Cre, AST, ALT, LDH, and T-Bil levels (Fig. 8).
While Cre levels were 0.12–0.20 mg/dL in GO-Y022-treated mice,
they were 0.14–0.20 mg/dL in mock-treated mice. The AST levels were
52–108 IU/L in GO-Y022-reated mice and they were 60–186 IU/L in
mock-treated mice. In addition, the ALT levels were 18–70 IU/L in GOY022-treated mice and 18–126 IU/L in mock-treated mice. Those of
LDH were 322–758 IU/L in GO-Y022-treated mice and they were334–674 IU/L in mock-treated mice. Finally, the T-Bil levels were0.04–0.14 mg/dL in GO-Y022-treated mice and 0.06–0.10 mg/dL in
mock-treated mice.
The levels of serum AST and ALT are parameters of liver damage. In
both mice, the levels of AST and ALT were higher than the normal
limits. That was because HFD diet induced a fatty change of liver inboth mice. However, there were no diﬀerences between the mocktreated mice and in GO-Y022-treated mice. We concluded that additional liver damage was not induced in GO-Y022-treated mice.
The serum Cre level is a parameter of renal damage. The serum
creatinine levels were within the normal limit, and there were no differences between both groups. We considered that no renal damage was
induced in GO-Y022-treated mice.
These data suggested that additional damages to the liver or kidney
were not induced in GO-Y022-treated mice. Furthermore, body weightsof GO-Y022-treated mice ranged 23.7–37.6 (average: 32.3 ± 5.88) g,
whereas those of the mock mice ranged 21.7–46.1 (average:
33.9 ± 13.8) g; we observed no signiﬁcant diﬀerence in the bodyweight between the two groups. Overall, these ﬁndings demonstrated
that 0.5% GO-Y022 administrated had a safe proﬁle in mice.

3.5. Localization of GO-Y022 in mice
We assessed localization of GO-Y022 in Gan mice by HPLC. The
tissue GO-Y022 concentrations in the heart, kidney, liver, spleen, as
well as the peripheral blood, were negligible in the treated mice
(Fig. 7). Conversely, GO-Y022 was detected in the digestive tract, including the esophagus, stomach, and small and large intestines of thetreated mice (Fig. 7). In addition, the concentrations of GO-Y022
ranged from 647 ng/g (tissue in the esophagus), 570–2590 ng/g (tissue
in the stomach), to 40–446 ng/g (tissue in the small intestine; Fig. 7).
Detection of GO-Y022 in the tissues varied among samples. We guess
this variation was inﬂuenced by free eating, not gavage. Actually, theleft over of feed was diﬀerent among mice, and the timing of eating
might be varied.
GO-Y022 really could not be absorbed? We added a next experiment
to examine the existence of GO-Y022 including their possible metabolites. In this experiment, we administrated 5 mg of GO-Y022 with gavage mixed with methylcellulose. The blood samples were collected at
6 points of the time, those were just before of administration, 1 h, 2 h,
4 h, 8 h, and 24 h after administration. To identify the metabolites of
GO-Y022, we set control groups administrated with vehicle methylcellulose alone for comparison. When additional peaks are observed by
HPLC, those are considered as GO-Y022 relating metabolites. However,there were no peaks corresponding to GO-Y022 and additional peaks inthe blood samples obtained from 6 time points (Fig. S4). We have198

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Fig. 8. Laboratory parameters in GO-Y022fed mice and HFD-fed mice. A, Data of AST
and ALT levels. B, Data of total bilirubin (TBil) level. C, Data of LDH. D, Data of creatinine (Cre). Black dot indicates GO-Y022fed mouse and white dot indicates HFD-fed
mouse. The numbers surrounded by rectangle indicate the normal limit of eachparameter. The data were obtained fromseven mice in each group.

3.7. GO-Y022, as an ingredient of cooked curry

111-TC was lowest. There might be some resistant mechanisms to
apoptosis occurred in the downstream signaling of pSTAT3. It is
speculated that growth suppression was due to diﬀerent mechanism
from apoptosis in H-111-TC (Table S1).
Inhibition of pSTAT3 with GO-Y022 might depend on cell lines. The
resistance to a STAT3 inhibitor, CTS varied among cell lines. These
diﬀerences might be reﬂected by the molecular backgrounds of thecells. The underlying mechanisms of sensitivity should be elucidated.
In Gan mice, together with the prostaglandin E synthase (PTGES)
overexpression, Wnt signaling and cyclooxygenase-2 (Cox2) expression
contributed to gastric carcinogenesis. Reportedly, a positive feedback
loop between STAT3 and COX2 contributes to gastric tumorigenesis(Xiong, Du, & Sun, 2014). Furthermore, the Wnt/β-catenin pathway
was reported to cross-talk with STAT3 signaling in the retina (Fragoso,
Patel, & Nakamura, 2012). Moreover, PTGES-derived prostaglandin E2
was illustrated to stimulate STAT3 to promote apoptosis of podocytes(Yu, Wu, & Wang, 2017). Notably, STAT3 plays a vital role in the intersection of the Wnt, Cox2, and PTGES signaling pathways and could
aﬀect them. This study suggests pSTAT3 as one of the key molecules
controlling gastric carcinogenesis and that the inhibition of pSTAT3
could suppress the growth of gastric cancer. In GCIY, the inhibition of
pSTAT3 with GC-Y022 was insuﬃcient, and GCIY might be relatively
resistant to GO-Y022.
This study conﬁrmed that the dietary GO-Y022 is present in commercially available curry pastes. Evidently, GO-Y022 can be ingested byhumans. We observed no adverse eﬀects with the administration of
5 mg GO-Y022 to a 20 g mouse per day. A phase I clinical trial reported
the tolerability of a daily curcumin dose of 8 g (Cheng, Hsu, & Lin,
2001). However, whether an equivalent GO-Y022 dose is safe in humans remains unclear.
GO-Y022 is insoluble in water and, thus, cannot be administrated
intravenously. This study demonstrates that the blood GO-Y022 concentration was insigniﬁcant. Nonetheless, orally administrated GOY022 can be directly transferred into the tumors, as well as to the luminal surface of the digestive tract. However, we did not detect apparent toxicity in the epithelial cells of the digestive tract. Moreover,GO-Y022 had somewhat a growth suppressive eﬀect on the primary
gastric epithelial cells less than 10 μM, but a killing eﬀect was apparent
at as high as 50 μM. These aspects of GO-Y022 highlight its beneﬁt incontrolling malignancies of the digestive tract by topical application.
Furthermore, contingent upon the absence of long-term toxic eﬀects,
dietary GO-Y022 should be considered for chemoprevention of gastrointestinal cancers.
In this study, we also assessed whether GO-Y022 could be ingested
as a supplement by determining the amount of GO-Y022 in

We determined the GO-Y022 content in cooked curry, which is aregularly consumed food prepared from commercially available retortpouch preparations, by mass spectrometry. Speciﬁcally, we tested nine
diﬀerent curries, revealing that the GO-Y022 concentrations varied
from zero to 98 μg/100 g curry (Table S2). In addition, heating thecurry at 250 °C for 20 min led to an increase in the GO-Y022 content to110 μg/100 g curry. Although the amount of GO-Y022 in the commercially available curries was minimal, we already experienced eating
GO-Y022, a C5 analog of curcumin.

4. Discussion
This study demonstrates that dietary GO-Y022, a C5 curcumin
analog, inhibited the gastric cancer growth both in vitro and in vivo. The
growth suppression potential of GO-Y022 was higher than that of curcumin in four gastric cancer cell lines. Except for GCIY, 5 μM GO-Y022
decreased the growth < 50% of the control. In addition, it was a morepotent inducer of apoptosis compared with curcumin in all cell lines.
Histopathologically, GCIY is a poorly diﬀerentiated adenocarcinoma
and belongs to a diﬀuse type based on the Lauren classiﬁcation (Maki,
Nobutake, & Ken-ichi, 2013). Kato III is a signet-ring cell carcinoma andbelongs to a diﬀuse type. SH-10-TC is a mucinous adenocarcinoma andbelongs to a diﬀuse type. H-111-TC is a well-diﬀerentiated adenocarcinoma and belongs to an intestinal type. We anticipated that GOY022 could be more eﬀective in some gastric cancer cell lines, depending on the pathological subtypes. In this study, we elucidated thatGO-Y022 suppressed pSTAT3 in vitro except GCIY, and in vivo.
Inhibition of pSTAT3 with 5 μM of GO-Y022 was strongest in KATO
III among four cell lines, and KATO III was also most sensitive to CTS.
Apoptosis induction with 5 μM of GO-Y022 in KATO III was also
highest. KATO III is most sensitive to pSTAT3 inhibition including GOY022 (Table S1). Inhibition of pSTAT3 with 5 μM of GO-Y022 was notobserved in GCIY, and GCIY was also rather resistant to CTS. GCIY
seemed to be most resistant to pSTAT3 inhibition, including GO-Y022.
Apoptosis induction with 5 μM of GO-Y022 in GCIY was lower.
Inhibition of pSTAT3 with 5 μM of GO-Y022 was lesser in SH-10-TC,
however SH-10-TC was relatively sensitive to CTS. Apoptosis induction
with 5 μM of GO-Y022 in SH-10-TC was stronger. SH-10-TC is also
sensitive to pSTAT3 inhibition (Table S1). Finally, inhibition of pSTAT3
with 5 μM of GO-Y022 was stronger in H-111-TC, however H-111-TC
was most resistant to CTS growth inhibition. H-111-TC seemed to be
most resistant to pSTAT3 inhibition. Although GO-Y022 suppressed
pSTAT3 expression, apoptosis induction with 5 μM of GO-Y022 in H199

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T. Yoshida et al.

commercially available curry pastes and determined that its concentration ranged from zero to 100 μg in 100 g curry paste, which
varied among the samples. Notably, there remains a room for the development of better recipes for curry paste with higher GO-Y022 concentrations.
Whether C5 analogs other than dietary GO-Y022 can be ingested
safely remains unexplored. Recently, we developed the most potent C5
analog (1E, 4E)-1,5-bis-(3,5(-bismethoxymethoxyphenyl) penta-1,4dien-3-one (GO-Y030) with 10 and 2 times the potency of curcumin and
GO-Y022, respectively, to inhibit the growth of various cancer cells(Ohori et al., 2006). The oral administration of GO-Y030 at the same
dose as GO-Y022 did not correlate with any apparent toxicities inmouse models. Of note, GO-Y030 is insoluble in water and is not absorbed systemically. If the safety proﬁle of the orally administrated GOY030 is found to be similar to that of GO-Y022, GO-Y030 could be abetter alternative in the prevention of malignancies of the digestive
tract by the topical use through oral administration.

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5. Conclusions
A diarylpentanoid analog of curcumin, GO-Y022 is included incurry, and human beings have been eating it for many thousands years.
This compound formed from pyrolysis of curcumin. It has a growth
inhibitory and an apoptosis inducing eﬀects on gastric cancer cells
mainly via pSTAT3 inhibition. GO-Y022 could inhibit the growth ofgastric tumors in mouse model with safe.
Acknowledgments
We thank Mrs. Ikuko Ogasawara for her technical assistance. This
study was partially supported by Akita Industrial/Academic/
Governmental Research Project on Developing Innovations, Joint
Research and Promotion Project on Creating New Technologies andIndustries, and Yamasaki Spice Promotion Foundation. This work wasalso supported by JSPS KAKENHI Grant Number JP 16H06276. The
authors also thank Enago (http://www.enago.jp) for review and revision of the language.
Ethical issues
All animal experiments were performed humanely and compliedwith the guidelines set by Akita University and were approved by the
associated ethics committee.
Disclosure of potential conﬂicts of interest
There are no conﬂicts of interest in this study.
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