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J Nat Med

DOI 10.1007/s11418-016-1061-6

NOTE

Protein tyrosine phosphatase 1B inhibitory properties


of seco-cucurbitane triterpenes obtained from fruiting bodies
of Russula lepida
Wilmar Maarisit1,2 • Hiroyuki Yamazaki1 • Syu-ichi Kanno1 • Ayako Tomizawa1 •

Jong-Soo Lee1,3 • Michio Namikoshi1

Received: 6 October 2016 / Accepted: 8 November 2016


Ó The Japanese Society of Pharmacognosy and Springer Japan 2016

Abstract The known seco-cucurbitane triterpene, (24E)- Introduction


3,4-seco-cucurbita-4,24-diene-3,26,29-trioic acid (1), has
been isolated as a potent protein tyrosine phosphatase Protein tyrosine phosphatase (PTP) 1B has been implicated
(PTP) 1B inhibitor together with a new analogue, (24E)- in the negative regulation of signal transduction via insulin
3,4-seco-cucurbita-4,24-diene-3-hydroxy-26,29-dioic acid and leptin receptors [1]. Therefore, this enzyme has
(2), from the fruiting bodies of Russula lepida. Further potential as a drug target for the treatment and prevention
evaluation of their biological properties against PTPs of type-2 diabetes and obesity [2, 3]. Although a number of
revealed that compound 1 inhibited T-cell PTP activity PTP1B inhibitors have been obtained from natural sources
similarly to PTP1B and exhibited moderate selectivity and through chemical synthesis [4, 5], new types of PTP1B
against PTP1B over vaccinia H-1-related phosphatase. inhibitors exhibiting strong biological properties are still
Moreover, the in vitro growth inhibitory effects of 1 and 2 required because clinically efficient drugs have not yet
against three human cancer cell lines were examined in been developed.
order to evaluate cell-based efficacy. However, neither 1 In our studies on PTP1B inhibitors from terrestrial and
nor 2 enhanced insulin-stimulated p-Akt levels at non-cy- marine organisms, we recently reported the isolation of the
totoxic concentrations. known seco-cucurbitane triterpene, (24E)-3,4-seco-cucur-
bita-4,24-diene-3,26,29-trioic acid (1), together with a new
Keywords (24E)-3,4-Seco-cucurbita-4,24-diene-3,26,29- congener, (24E)-3,4-seco-cucurbita-4,24-diene-3-hydroxy-
trioic acid  Seco-cucurbitane triterpene  Russula lepida  26,29-dioic acid (2), from the fruiting bodies of Russula
Protein tyrosine phosphatase 1B  Inhibitor lepida collected at Sendai, Japan (Fig. 1) [6]. Compound 1
exhibited potent PTP1B inhibitory activity without cyto-
toxicity, while compound 2 exerted weaker inhibitory
effects against PTP1B. In the present study, the selectivi-
ties of PTP1B over other PTPs and the phosphorylation
levels of Akt were evaluated for compounds 1 and 2. We
herein report the results obtained from enzyme and cell-
based experiments.

& Hiroyuki Yamazaki


yamazaki@tohoku-mpu.ac.jp
Results and discussion
1
Faculty of Pharmaceutical Sciences, Tohoku Medical and
Pharmaceutical University, Sendai 981-8558, Japan We previously demonstrated that compounds 1 and 2
2
Faculty of Fisheries and Marine Science, Sam Ratulangi inhibited PTP1B activity with IC50 values of 0.4 and
University, Kampus Bahu, Manado 95115, Indonesia 20.4 lM, respectively. Moreover, these compounds did not
3
College of Marine Sciences, Gyeongsang National exert growth inhibitory effects against two human cancer cell
University, Tongyeong, Gyeongnam 650-160, Korea lines Huh-7 (hepatoma) and EJ-1 (bladder), up to 50 lM [6].

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J Nat Med

PTPs are key regulators of various cellular functions and Prior to performing cell-based experiments on 1 and 2,
consist of more than 100 members, including PTP1B [2]. cell viability was tested in three human cancer cell lines,
Therefore, selective activity against PTP1B over other A549 (lung adenocarcinoma), MCF-7 (breast adenocarci-
PTPs is one of the important properties used in the noma) and K562 (erythroleukemia), in addition to Huh-7
development of clinically useful PTP1B inhibitors. The (hepatoma) and EJ-1 (bladder) cells, which were evaluated
inhibitory effects of 1 and 2 on T-cell protein tyrosine previously [6]. Cancer cells were treated with compounds 1
phosphatase (TCPTP), one of the non-transmembrane PTPs and 2 at 10 or 50 lM for 48 h, and cell proliferation was
[7], and vaccinia H-1-related phosphatase (VHR), one of measured using the WST-1 assay [14]. Similar to the
the dual-specificity phosphatases [8], were evaluated. effects observed against Huh-7 and EJ-1 cells, compounds
The IC50 values of compounds 1 and 2 against TCPTP 1 and 2 did not appear to exhibit cytotoxicity against A549,
were similar to those against PTP1B. On the other hand, MCF-7 or K562 cells (Table 1).
compounds 1 and 2 were less active to VHR than PTP1B PTP1B is mainly expressed in the liver and controls the
(Fig. 2; Table 1). Compound 1 showed threefold greater insulin signal transduction pathway [1]. Thus, the phos-
selectivity against PTP1B than VHR. phorylation levels of Akt, a key downstream effector of the
T-cell protein tyrosine phosphatase shares 72% insulin signaling cascade, were evaluated by Western
sequence similarity in its catalytic domain with PTP1B [7] blotting using Huh-7 cells [15]. As shown in Fig. 3, com-
and is also a negative regulator of insulin and leptin sig- pounds 1 and 2 did not enhance insulin-stimulated p-Akt
naling pathways [3]. Although improved insulin resistance levels in Huh-7 cells up to 50 lM, and did not affect cell
and glucose homeostasis have been reported in ptp1B -/- proliferation.
mice [1, 9, 10], tcptp -/- mice exhibited severe inflam- Although the IC50 value of 1 (0.4 lM) against PTP1B
matory phenotypes [11, 12]. However, recent studies have was more potent than that of oleanolic acid as a positive
indicated that ptp1B ?/- and tcptp ?/- mice with deletions control (1.1 lM) in the PTP1B enzyme assay (Fig. 2;
of single copies of PTP1B and TCPTP displayed no Table 1), phosphorylated Akt levels in Huh-7 cells were
abnormalities [13]. Thus, dual inhibitors against PTP1B not enhanced by 1 (Fig. 3). This discrepancy between the
and TCPTP are still expected to be drug candidates without results obtained in enzyme- and cell-based experiments
major side effects. Compound 1 may become a candidate may be due to low selectivity against PTP1B over other
lead compound in the development of drugs for the treat- PTPs and/or the permeability of 1 in intact cell membranes.
ment of type-2 diabetes and obesity. Therefore, structural optimizations are needed in order for
these compounds to become candidate lead compounds in
therapeutic agents for the treatment of type-2 diabetes.
COOH
H Materials and methods
R
Materials
HOOC
1: R = COOH
(24E)-3,4-Seco-cucurbita-4,24-diene-3,26,29-trioic acid
2: R = CH2OH (1) and (24E)-3,4-seco-cucurbita-4,24-diene-3-hydroxy-
26,29-dioic acid (2) were isolated from the fruiting bodies
Fig. 1 Structures of compounds 1 and 2 isolated from fruiting bodies
of Russula lepida
of R. lepida as described previously [6]. Human
Fig. 2 Inhibitory activities of
compounds 1 (a) and 2
(b) against PTP1B, TCPTP and
VHR

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J Nat Med

Table 1 Biological activities of


Compound PTPs Cytotoxicity
compounds 1 and 2 against
(IC50, lM) (IC50, lM)
PTPs and human cancer cell
lines PTP1B [6] TCPTP VHR A549 MCF-7 K562 Huh-7 [6] EJ-1 [6]

1 0.4 0.7 1.2 [10 [10 [50 [50 [50


2 20.4 15.7 [20.5 [10 [10 [50 [50 [50
a
Oleanolic acid 1.1 1.7 5.4
Doxorubicinb 0.043 0.031 0.042 0.36 0.023
a
Positive control for the PTP assay
b
Positive control for cytotoxicity

solution) or VHR (100 ll of a 1.0 lg ml-1 stock solution)


in 50 mM citrate buffer (pH 6.0) containing 0.1 M NaCl,
1 mM dithiothreitol (DTT) and 1 mM EDTA was added to
each well of a 96-well plastic plate. A sample (2.0 ll in
CH3OH) was added to each well to make the final con-
centration and was then incubated at 37 °C for 10 min. The
reaction was initiated by the addition of pNPP in citrate
buffer (100 ll of a 4.0 mM stock solution), incubated at
37 °C for 30 min, and then terminated using 10 ll of a stop
solution (10 M NaOH). The optical density in each well
was measured at 405 nm using a MTP-500 microplate
reader (Corona Electric Co., Ltd., Ibaraki, Japan). PTP1B
inhibitory activity (%) was defined as [1 - (ABSsample
- ABSblank)/(ABScontrol - ABSblank)] 9 100, where
Fig. 3 Enhanced effects of compounds 1 and 2 on insulin-stimulated ABSblank is the absorbance of wells containing only the
Akt phosphorylation levels in Huh-7 cells. Huh-7 cells were buffer and pNPP, ABScontrol is the absorbance of p-nitro-
incubated with the indicated concentration of 1, 2 or sodium phenol liberated by the enzyme in the assay system without
orthovanadate (SOV: positive control) for 2 h, and then stimulated
a test sample, whereas ABSsample is that with a test sample.
with insulin (1 nM) for 5 min. Cells were lysed on ice, and the levels
of p-Akt, t-Akt and GAPDH were detected by Western blotting. The Assays were performed in three duplicate experiments for
p-Akt/t-Akt levels are shown as a ratio of that in the control group all test samples. Oleanolic acid, a known phosphatase
inhibitor [18], was used as a positive control.
recombinant PTP1B, CD45 and VHR were purchased from
Enzo Life Sciences (Farmingdale, NY, USA). Human WST-1 assay
recombinant TCPTP was purchased from SignalChem
(Richmond, BC, Canada). p-Nitrophenyl phosphate (pNPP) Cytotoxicity was assessed using the WST-1 (sodium
was purchased from Sigma-Aldrich (St. Louis, MO, USA). 5-(2,4-disulfophenyl)-2-(4-iodophenyl)-3-(4-nitrophenyl)-
Oleanolic acid was purchased from Tokyo Chemical 2H tetrazolium inner salt) assay, which detects metaboli-
Industry (Tokyo, Japan). Plastic plates (96-well) were cally competent cells with an intact mitochondrial electron
purchased from Corning Inc. (Corning, NY, USA). All transport chain [14]. Briefly, 1 9 104 cells were seeded on
other chemicals including organic solvents were purchased each well of a 96-well plastic plate and cultured overnight.
from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Cells were treated with each test compound and incubated
for 48 h, and this was followed by the addition of medium
Inhibitory activity against PTPs containing WST-1 solution (0.5 mM WST-1 and 0.02 mM
1-methoxy-5-methylphenazinium methylsulfate; 1-PMS)
The effects of compounds 1 and 2 on PTPs were examined to each well. The plate was incubated at 37 °C for 60 min,
by measuring the rate of hydrolysis of the substrate, pNPP, and absorption at 438 nm (reference 620 nm) was mea-
according to a previously described method with slight sured using a SH-1200 microplate reader (Corona Electric).
modifications [16, 17]. Briefly, PTP1B (100 ll of a 0.5 lg Control cells were treated with 0.1% EtOH. Cell viability
ml-1 stock solution), TCPTP (100 ll of a 0.5 lg ml-1 was calculated using the formula: absorbance in the treated
stock solution), CD45 (100 ll of a 0.5 lg ml-1 stock sample/absorbance in the control 9 100 (%).

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Acknowledgements This work was supported in part by a Grant-in- 15. Abdjul DB, Kanno S, Yamazaki H, Ukai K, Namikoshi M (2016)
Aid for Scientific Research (25870660 and 16K21310) from the A dimeric urea of the bisabolene sesquiterpene from the Oki-
Ministry of Education, Culture, Sports, Science, and Technology nawan marine sponge Axinyssa sp. inhibits protein tyrosine
(MEXT) of Japan to H.Y. and the Takeda Science Foundation to H.Y. phosphatase 1B activity in Huh-7 human hepatoma cells. Bioorg
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