平成29 年

4月19 日公益財団法人富山第一銀行奨学財団
理事長

金岡純二

殿

助成研究成果概要報告書
教育機関名：富山大学

助成金額

研究代表者 ：Suresh Awale
研究題目：１,０００千円

所属 ：和漢医薬学総合研究所

職位 ：准教授：抗膵臓がん活性を有する有望な植物資源としての富山県産檜に関する天然物化学研究

研究概要
富山県が，我が国の伝統薬産業及び伝統医薬研究をリードしていることは周知の事実である。この事実を踏まえ，本課題は，申請者らが確立してきた栄養飢餓戦略に基づき富山県の有する天然物資源から新規抗腫瘍薬の開発に取り組むものである。特に，今日，膵臓がんに焦点をあてた申請者らの研究では，富山県に自生する檜からの抽出物に抗膵臓がん活性を認め，それが新規の治療薬ソースになる可能性を見出している。

成果要約

The chloroform extract of the Japanese cypress Chamaecyparis obtuse also known as“Hinoki” collected from Toyama Prefecture was found to kill PANC-1 human pancreatic cancer cells
preferentially in the nutrient-deprived medium without causing toxicity in the normal nutrient richcondition. Phytochemical investigation on this extract led to the isolation of two new
germacrane-type sesquiterpenes (1, 2), together with the six sesquiterpenes (3-8) and a lignan (9).

HOO
15HO

HO
H

H

O

H

H

HO
H

OH
O

O

102
4

14O

671311 12HOH

OH

H

HOHO
H

5HO

O
OO

HOH

4321HAcOH

6H
H

789OOFigure 1. Structure of compounds isolated from “hinoki”

The isolated compounds were tested for their preferential cytotoxicity against six different
human pancreatic cancer cell lines [PANC-1, MIA PaCa2, KLM-1, NOR-P1, CAPAN-1 and PSN-1]
by utilizing an antiausterity strategy.

Among these, α-cadinol (3) was identified as the most active

constituent having PC50 values of 22.8 M (PANC-1), 19.3 M (MIA PaCa-2), 31.0 M (CAPAN-1),
31.6 M (PSN-1), and 45.1 M (KLM-1), respectively.
The most active compound, α-cadinol was further evaluated for its effect in real-time against
PANC-1 cells using electronic impedance-based cell analyzer. PANC-1 cells cultured in only NDM(control) showed increased cell index value even after 24 h of starvation. However, treatment withα-cadinol was found to inhibit the cellular index in a concentration dependent manner (fig. 2).

Normalized Cell Index

2.5

2.0

1.5

NDM

1.025 M

0.50481216202450 MTime (h)Figure 2. Real-time cell monitoring of PANC-1 cells treated with α-cadinol in NDM. The
normalized cellular index of PANC-1 cells at 24 hours of treatment (T24) with 25 M ofα-cadinol was found to be the same as initial level (T0) suggesting the complete
inhibition of proliferation. α-cadinol at the concentration of 50 M, however lead thequick decrease in the PANC-1 cell index value within 4 h, and total cell death within 8 h.

Figure 3. α-Cadinol induced morphological alteration against on PANC-1 humanpancreatic cancer cells. I) Fluorescent EB/AO staining [Red: Ethidium Bromide (EB)dead cells’ indicator Green: Acridine Orange (AO) live cells’ indicator] and phase
contract overlay image. II) Fluorescent Hoechst 33342/PI staining [Blue: Hoechst is cellpermeable dye, stains the condensed chromatin in apoptotic cell more brightly than innormal cells, Pink: Propidium iodide (PI) only permeant to dead cells] and phase
contract overlay image.

The α-cadinol was further studied for its effect on cell morphology and apoptosis using
ethidium bromide (EB)/acridine orange (AO) and Hoechst 33342/propidium iodide double staining
assays. As shown in figure 3, control cells showed intact cell morphology with bright green AO

fluorescence in EB/AO and flattened rounded blue nuclei in Hoechst 33342/PI staining. Cells
treated with α-cadinol showed the concentration dependent increase in dead cells. However,nuclear fragmentation was not observed in Hoechst 33342/PI staining, suggesting α-cadinol
induced the necrotic cell death against PANC-1 cells in NDM.
Akt is a pro-survival factor that is activated in a majority of tumors and regulates cellularfunctions such as cell cycle progression, cell migration, invasion, and angiogenesis. Increased Akt
phosphorylation is one of the austerity markers that enable tumor cells to survive and proliferate in
hostile hypovascular tumor environment. Therefore, the effect of α-cadinol on Akt activation undernutrient-deprived conditions was investigated by Western blot analysis. As shown in Figure 6,
PANC-1 cells in showed high level of p-Akt. However, treatment with α-cadinol dramatically inhibited
p-Akt expression within 3 h (Fig. 4). α-cadinol was also found to inhibit the major downstreameffector of Akt activation, mTOR (mammalian target of rapamycin).

DMEM
Exposure time (hour)α-cadinol (M)

NDM
3050012.5

2550

p-AKT S473
AKTp-mTOR
mTOR

LC-3A/B-I
LC-3A/B-II
GAPDH

Figure 4. Effect of α-cadinol on Akt, mTOR, LC3A/B-I and LC3A/B-II by Western blot.

Autophagy is a cellular recycling process to maintain the energy metabolism, and is often activatedin response to various stresses, including nutrient deprivation in the tumor microenvironment.
Therefore, we examined the expression of autophagic markers, microtubule associated protein lightchain LC3-I (16 kDa) and LC3-II (14 kDa). As shown in figure 6, no apparent differences wereobserved in the LC3-I and LC3-II expression in the controls. However, treatment with α-cadinol led
to significant enhancement in the expression of both LC3-I and LC3-II in a concentration dependent
manner. Therefore, hyperactivation of autophagy, together the inhibition of p-Akt (S473) and p-mTOR
expression by α-cadinol could have contributed to the preferential cytotoxicity of PANC-1 cellsduring nutrition deprived condition. Human pancreatic cancer cells are inherently resistant toconventional anticancer drugs in clinical use such as gemcitabine and 5-fluorouracil. Therefore,
α-cadinol isolated from the “Hinoki” collected in Toyama should be an attractive candidates
antiausterity drug development.

研究成果【雑誌論文、学会発表、図書、新聞掲載、作成 Web ページ、特許権等の出願・取得状況】

発表状況
学会発表
Dya Fita Dibwe, Sijia Sun, Jun-ya Ueda, Matsumoto K., Suresh Awale :Cadinane-type

sesquiterpenes

Chamaecyparis obtuse and theirfromantiausterity activity 日本薬学会第 137 年会，2017，3/26-29, 仙台.
Suresh Awale : Recent advances in antiausterity strategy guided anticancer
drug discoveries The 5th International Symposium on Bioactivities and Disease
Prevention of Phytochemicals and Natural Products for Health, National Chiayi
University Chiayi, 2016, 11/25-26, 台湾.
雑誌論文
Dya Fita Dibwe, Jun-ya Ueda, Chandrasekar Balachandran, Sijia Sun,MatsumotoK.,

SureshAwale:

Cadinane-type

sesquiterpenes

fromChamaecyparis obtuse and their antiausterity activity, (in preparation, to besubmitted in April, 2017)

経費の区分執行額（円）

執行状況
【物品費】

有機溶媒類

163,539 円

アッセイ用試薬類

490,628 円

機器使用コスト（NMR、MS、CD など）

263,713 円【旅費】

82,120 円

【謝金】

0円

【その他】

0円

合計

1,000,000 円

備考