An Ursolic Acid Derived Small Molecule Triggers Cancer Cell Death through Hyperstimulation of Macropinocytosis

Article abstract of DOI:10.1021/acs.jmedchem.7b00592

Macropinocytosis is a transient endocytosis that internalizes extracellular fluid and particles into vacuoles. Recent studies suggest that hyperstimulation of macropinocytosis can induce a novel nonapoptotic cell death, methuosis. In this report, we descr

Full text of DOI:10.1021/acs.jmedchem.7b00592

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Article
An Ursolic Acid-derived Small Molecule Triggers Cancer
Cell Death through Hyperstimulation of Macropinocytosis
Lin Sun, Bin Li, Xiaohui Su, Ge Chen, Yaqin Li, Linqian Yu, Li Li, and Wanguo Wei
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00592 • Publication Date (Web): 05 Jul 2017
Downloaded from http://pubs.acs.org on July 5, 2017
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An Ursolic Acidꢀderived Small Molecule Triggers
Cancer Cell Death through Hyperstimulation of
Macropinocytosis
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Lin Sun,† Bin Li,† Xiaohui Su,† Ge Chen,‡ Yaqin Li,† Linqian Yu,† Li Li,*§ and Wanguo Wei*†‡
†Shanghai Advanced Research institute, Chinese Academy of Sciences, University of Chinese
Academy of Sciences, 99 Haike Road, Shanghai, 201210, China
‡School of Life Science and Technology, ShanghaiTech University, 100 Haike Road, Shanghai,
201210, China
§State Key Laboratory of Oncogenes and Related Genes, RenjiꢀMed X Clinical Stem Cell
Research Center, Ren Ji Hospital, School of Biomedical Engineering, Shanghai Jiao Tong
University, Shanghai 200127, China.
KEYWORDS: Ursolic acid (UA)· Cancer · Cell death · Methuosis · Macropinocytosis
ABSTRACT: Macropinocytosis is a transient endocytosis which internalizes extracellular fluid
and particles into vacuoles. Recent studies suggest that hyperstimulation of macropinocytosis can
induce a novel nonꢀapoptotic cell death, methuosis. In this report, we describe the identification
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of an ursolic acidꢀderived small molecule (compound 17), which induces cancer cell death
through hyperstimulation of macropinocytosis. 17 causes the accumulation of vacuoles derived
from macropinosomes based on transmission electron microscopy, timeꢀlapse microscopy and
labeling with extracellular fluid phase tracers. The vacuoles induced by 17 separate from other
cytoplasmic compartments, but acquire some characteristics of late endosomes and lysosomes.
Inhibiting hyperstimulation of macropinocytosis with the specific inhibitor, amiloride, blocks
cell death, implicating that 17 leads to cell death via macropinocytosis, which is coincident with
methuosis. Our results uncovered a novel cell death pathway involved in the activity of 17,
which may provide a basis for further development of naturalꢀproductꢀderived scaffolds for
drugs that trigger cancer cell death by methuosis.
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INTRODUCTION
Resistance of human tumors to cancer chemotherapeutic agents remains a major and unsolved
problem in clinical oncology. In general, cancer cells adapt to the microenvironment changed by
drugs through decreasing intracellular drug accumulation and altering apoptotic pathways.1, 2
Apoptosis is taken as a primary therapeutic focus and target for the treatment of cancer.3
Acquired drug resistance usually occurs in company with genetic and epigenetic changes, such
as p53 mutation, methylation of DNA and acetylation of histone.4, 5 To avoid resistance to proꢀ
apoptotic drugs, strategies have been developed wherein nonꢀapoptotic target is exploited as new
therapeutic strategy for sensitization of malignancies.
Various nonꢀapoptotic forms of cell death have been reported.6, 7 Morphological characteristics
are usually used to distinguish between different forms of cell death. Vacuoles in cytoplasm are
morphological features existing in several nonꢀapoptotic cell death subroutines, such as
autophagic cell death, necroptosis, paraptosis, and oncosis. Methuosis is a newly discovered
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form of nonꢀapoptotic cell death phenotype characterized by accumulation of vacuoles derived
from macropinosomes and endosomes.8, 9 Macropinocytosis was usually recognized as a form of
endocytosis which usually happened in immune responses, while latest evidences reveal that it is
aberrantly activated in methuosis. Presence of large vacuoles in cytoplasm is the most prominent
morphological feature of macropinocytosis. As a novel form of nonꢀapoptotic cell death,
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methuosis has drawn special attention as a new target for cancer therapy, which has been
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confirmed in cells manipulated genetically or pharmacologically.10,
Therefore, the
identification of small molecules that induced methuosis can provide useful probes to study nonꢀ
apoptotic cell death and may lead to the development of new therapeutic interventions. Herein,
we report the identification and preliminary biological characterization of the naturalꢀproductꢀ
derived small molecule 17, which induces cancer cell death through hyperstimulation of
macropinocytosis.
RESULTS AND DISCUSSION
Chemistry. Triterpenoids are a large and diverse class of natural products widely distributed in
the plant kingdom, which exhibit a broad spectrum of pharmacological activities which are under
investigations worldwide for various therapeutic properties.12ꢀ14 For example, ursolic acid (3
βꢀ
hydroxyursꢀ12ꢀenꢀ28ꢀoic acid, UA) is a wellꢀknown pentacyclic triterpenoid carboxylic acid
obtained from various medicinal herbs and fruits. It has been reported to show significant
anticancer activity against different kinds of tumor cell lines.15ꢀ17 However, the limited solubility,
low tumorꢀtargeting specificity and poor bioꢀavailability of UA restricts its further clinical
application.18 Recently, there has been a renewed interest in common dietaries and plantꢀbased
traditional medicines for the prevention and treatment of cancer. Therefore, structural
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modifications of UA have been widely investigated in order to improve its biological activities
and bioavailability.19ꢀ21
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In this regard, a series of novel pyrazoleꢀfused ursolic acid derivatives22ꢀ23 were synthesized as
shown in Scheme 1. First, UA was treated with benzyl bromide in the presence of K2CO3 in
DMF to form benzyl ester (2) to ensure a higher yield and better purification process. Oxidation
of the benzyl ursolate by Jone’s reagent afforded 3ꢀoxoꢀursolic acid (3) in 84% yield. Then,
formylation at C2 position was realized using sodium methoxide and ethyl formate to give
ketoaldehydes 4a (R1 = H). Introduction of methyl carbonyl group was also accomplished using
sodium methoxide and ethyl acetate under the same condition to give 4b (R1 = CH3). While the
installation of trifluoromethyl carbonyl group at C2 position was realized by using Potassium
tertꢀbutoxide and ethyl trifluoroacetate in THF to form 4c (R1 = CF3) in high yield. Subsequently,
the installed carbonyl groups reacted smoothly with various hydrazine hydrochlorides
(R2NHNH2·HCl) under basic conditions, and the pyrazole moieties were then formed
simultaneously to give various pyrazoleꢀfused ursolates with complete regionꢀselectivity. Finally,
the benzyl group was facilely removed by catalytic hydrogenation under 1 atm of H2 to give the
corresponding pyrazoleꢀfused ursolic acid derivatives in high yeilds.
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Scheme 1. Synthesis of pyrazoleꢀfused ursolic acid derivativesa
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aReagents and conditions: (a) BnBr, K2CO3, DMF, 60 oC, 92%. (b) PCC, CH2Cl2, 0 oC ꢀ rt, 84%.
(c) R1CO2Et (R1 = H, Me), NaOMe, THF, rt; R1CO2Et (R1 = CF3), tꢀBuOK, THF, rt, 70%. (d)
R2NHNH2·HCl, EtOH, rt. (e) H2, Pd/C, MeOH, rt.
Biological Evaluation. The pyrazoleꢀfused ursolic acid derivatives were evaluated for their in
vitro anticancer activities against five human cancer cell lines: cervical carcinoma HeLa cells,
hepatocelluar carcinoma HepG2 cells, fibrosarcoma HT1080 cells, mammary adenocarcinoma
MCFꢀ7 cells, and neuroblastoma SKꢀNꢀMC cells. More than 100 compounds (20 µM in DMSO)
were evaluated by CCKꢀ8 assay in vitro, and found that most of the pyrazoleꢀfused ursolic acid
derivatives also exhibited some inhibitory activities against the five cancer cell lines, and some
of them showed much more stronger antiꢀcancer activities than ursolic acid (UA) in certain
cancer cell lines (Table 1). Interestingly, a number of ursolic acid derivatives were found to
induce cellular vacuolization that are readily detected by phaseꢀcontrast microscopy (see the
Supporting Information, Figure S1).
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Table 1. Biological evaluation of ursolic acidꢀderivatives
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Viability (CCKꢀ8 assay, 48 h)
Compd
(20 µM)
R1
R2
Vacuole
HeLa
43.30±2.22
44.86±2.89
HepG2
34.12±0.68
45.97±1.12
HT1080
39.43±0.52
35.78±0.33
MCFꢀ7
57.64±5.75
47.59±0.47
SKꢀNꢀMC
67.21±1.78
67.44±2.59
UA
NO
−
−
5
H
CH3
YES
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8
H
H
47.05±1.07
30.80±0.42
56.96±2.02
44.57±0.12
39.05±0.61
27.88±0.25
40.53±0.42
36.18±2.38
63.30±0.25
31.40±1.12
NO
YES
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H
19.94±1.83
33.85±0.93
23.49±0.40
33.36±2.60
33.89±1.38
NO
12
17
18
19
20
H
H
H
H
H
54.20±2.19
18.63±2.34
44.16±1.27
40.87±1.44
33.32±1.18
56.80±2.00
27.87±2.98
43.67±1.25
60.08±2.24
29.49±1.63
30.92±2.25
26.78±0.07
55.18±1.67
38.70±0.24
33.50±0.37
39.69±1.84
25.25±0.07
28.91±0.85
46.55±1.67
46.65±1.37
79.88±2.36
28.63±1.03
66.56±2.07
57.40±1.08
24.61±1.10
NO
YES
NO
NO
NO
21
22
23
H
CF3
H
24.39±0.46
44.86±2.89
37.33±1.06
58.63±1.92
45.97±1.12
29.26±1.87
27.30±0.27
35.78±0.33
37.51±1.04
34.29±1.18
47.59±0.47
28.14±1.30
39.36±2.36
67.44±2.59
30.96±2.11
YES
YES
YES
CH3
NC
*Results are expressed as percent of controls that received vehicle alone (DMSO). Values are the mean ± SD.
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Of the compounds, 17 was found to significantly suppress cell viability in all tested carcinoma
cells, with a quick and striking accumulation of numerous phaseꢀlucent cytoplasmic vacuoles
within 12 h (Figure 1B). The effects of 17 on normal human dermal fibroblasts (HDFs) and
myoblast cell line (C2C12) were also evaluated, with less vacuoles and reduced cell viability
(~13% for C2C12 and 28% for fibroblasts) were observed (Figure 2A, B). Therefore, compound
17 was chosen for further study as a potential inducer of cell vacuolization and death.
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Figure 1. 17 induces cellular vacuolization. (A) The structure of 17. (B) HeLa cells were treated
with 17 at 20 ꢁM, and phase contrast images of live cells were obtained.
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Figure 2. Assay for cytotoxicity in normal cells treated with 17. (A) Phaseꢀcontrast images
were obtained after treatment of C2C12 cells for 24 h with 20 ꢁM 17 or 0.1% DMSO, and CCK8
assays were performed after treatment for 24 h or 48 h with 20 ꢁM 17 or 0.1% DMSO. (B)
Phaseꢀcontrast images were obtained after treatment of HDF cells for 24 h with 20 ꢁM 17 or
0.1% DMSO, and CCK8 assays were performed after treatment for 24 h or 48 h with 20 ꢁM 17
or 0.1% DMSO.
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Cell viability assay revealed that 17 displayed moderate cytostatic properties against human
cancer cell lines: HeLa, HepG2, HT1080, MCFꢀ7, and SKꢀNꢀMC with IC50 = 15.1, 16.65, 17.18,
17.58, and 17.06 ꢁM, respectively. We also checked the status of cancer cells treated with 17 by
CCKꢀ8 assay at 24 h, 48 h and 72 h (see the Supporting Information, Figure S2). Almost all cells
had vacuoles in the cytosol at 24 h, while cell viability was up to ~74.24% in HeLa cells.
Therefore, HeLa cells were chosen for further analysis due to their better response to 17
treatment. Colony formation assay showed that 17 significantly reduced the colony numbers of
HeLa cells in a concentrationꢀdependent manner (see the Supporting Information, Figure S3),
suggesting that exposure to 17 significantly reduced longꢀterm cell survival.
An annexin V/PI staining was performed to investigate whether its toxicity was related to the
induction of apoptosis in HeLa cells. Significant increase of the number of apoptotic and necrotic
cells was observed in cells exposure to 17 in a timeꢀ and doseꢀdependent manner (see the
Supporting Information, Figure S4). To further determine whether caspase pathway is involved
in 17ꢀinduced cell death, caspaseꢀ3 and PARP cleavage were analyzed by immunoblot analysis.
Treatment of cells with the indicated concentrations of 17 and taxol (as a positive control)
resulted in an increased cleavage of both caspaseꢀ3 and PARP1 (see the Supporting Information,
Figure S5).
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Our followꢀup studies tried to figure out whether the loss of cell viability induced by 17 was
caspaseꢀdependent or not. HeLa cells were preꢀtreated with ZꢀVADꢀFMK, a panꢀcaspase
inhibitor for 1 h prior to 17 treatment and cell viability was assessed after 48 h incubation. It was
observed that increases in cell death induced by taxol could be reversed by ZꢀVADꢀFMK
pretreatment. However, ZꢀVADꢀFMK could not prevent cell death and vacuolization induced by
17 (Figure 3). These findings indicated that activation of caspase pathway might be a
compensatory stress response rather than a cell death mechanism under such condition, which
also indicated that the mechanism of 17ꢀinduced cell death was nonꢀapoptotic and caspaseꢀ
independent. We then began to investigate whether 17 induces cell death through other nonꢀ
apoptotic types of cell death pathways, such as paraptosis, neroptosis, oncosis and autophagic
death. Among them, cleavage of caspase only happens in autophagic death in special cases.6, 24, 25
We therefore examined the activation of LC3, an autophagosome marker.26 Western blot analysis
showed both the LC3ꢀI (18 KDa) form and active, autophagosome membraneꢀbound LC3ꢀII (16
KDa) form (Figure 4A). The effect of 17 on cell death was also observed in the presence of the
autophagy inhibitor, 3ꢀMA, which blocks autophagy by inhibiting the class III PI3K.27 Western
blot analysis showed that 3ꢀMA attenuated 17ꢀinduced LC3ꢀII accumulation (Figure 4B), but 3ꢀ
MA could not rescue 17ꢀinduced cytotoxicity and cell vacuolization in HeLa cells (Figure 4C,
D). Therefore, the autophagosome marker, LC3ꢀII, is not essential for the formation of
vacuolization in HeLa cells induced by 17, and autophagy may be involved in 17ꢀinduced cell
death, but it was not indispensable. Taken together, these data suggest that the tumorꢀsuppressive
effects of 17 were not mediated via the induction of paraptosis and autophagy.
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Figure 3. (A) Cell viability of HeLa cells with or without zꢀVADꢀFMK. HeLa cells were treated
with 50 ꢁM of zꢀVADꢀFMK for 1 h before 17 or taxol treatment. After 48 h, the cell viability
was determined by CCK8 assay. (B) HeLa cells were pretreated for 1 h in the presence or
absence of zꢀVADꢀFMK prior to addition of 20 ꢁM 17 or DMSO. Phaseꢀcontrast images were
taken after 24 h.
Figure 4. (A) Western blot analysis of LC3 in HeLa cells after incubation with 0.1% DMSO
control or indicated concentrations of 17 for 24 h. (B) HeLa cells were pretreated for 1 h in the
presence or absence of 3ꢀMA prior to addition of 17 or DMSO. Western blot analysis of LC3
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was performed after addition of 20 ꢁM 17 for 24 h. (C) HeLa cells were pretreated for 1 h in the
presence or absence of 3ꢀMA prior to addition of 17 or DMSO. CCK8 assays were performed
after treatment for 24 h or 48 h with 20 ꢁM 17 or 0.1% DMSO. (D) HeLa cells were pretreated
for 1 h in the presence or absence of 3ꢀMA prior to addition of 17 or DMSO. Phaseꢀcontrast
images were obtained after treatment of cells for 24 h.
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Notably, HeLa cells exhibit clear cytoplasmic vacuolization during 17ꢀinduced cell death. To
identify the origin of the 17ꢀinduced vacuoles and detect subtle changes in organelle ultra
structure, HeLa cells were treated with 17 for 24 h and studied by transmission electron
microscopy, in which cytosolic vacuolization was clearly visible. 17ꢀtreated HeLa cells did not
display the apoptotic or autophagic ultrastructural characteristics, like pyknotic chromatin or
cytosolic autophagosomes (Figure 5A). Numerous electronꢀlucent vacuoles ranging from 0.5ꢀ2
ꢁm in diameter were generally devoid of cytoplasmic components or organelles. Some larger
vacuoles about 5 ꢁm in diameter were distinct from autophagosomes, which have luminal
cytoplasmic contents surrounded by double layers of membranes. The morphological
characteristics of the dying cells did not fit the descriptions of apoptosis and autophagic cell
death, and were more similar to the morphological characteristics which were previously
observed in cells undergoing macropinocytosis.
The formation of large vacuoles is one of the most important characters of macropinocytosis.
In order to monitor the progress of vacuole formation induced by 17, time lapse phaseꢀcontrast
microscopy was performed.28 Liveꢀcell imaging at 10x magnification covering the period from
directly before to 72 hours after 17 treatment revealed the formation of vacuoles within 4 hours
of 17 treatment in HeLa cells (see the Supporting Information, Movie S1). As has been shown,
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more and more vacuoles formed in the cytoplasm and then merged with each other to form large
vacuoles, finally resulting in membrane rupture and cell death (Figure 5B).
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Figure 5. 17 induced cell death via a macropinocytosisꢀlike vacuolization. (A) TEM examination
of morphology changes of HeLa cells with or without 20 ꢁM 17 for 24 h. (B) Timeꢀlapse phaseꢀ
contrast microscopy of HeLa cells treated with 17. Images were captured at 90 sec intervals after
the addition of 17 at 20 ꢁM.
As we consider possible molecular mechanisms that 17 causes cell death through
macropinocytosis, our attention was drawn to a series of studies that have defined the signaling
pathway related to macropinocytosis.29 One of the typical features of macropinocytosis is the
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incorporation of extracellularꢀphase fluid tracers.30 Lucifer Yellow (LY) is a wellꢀestablished
tracer of fluidꢀphase pinocytosis that is impermeable to cellular membranes.31 Live cell imaging
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showed that LY was accumulated in the phaseꢀlucent vacuoles induced by 17, similar to what
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was observed with macropinocytosis in previous studies (Figure 6A).
The distribution
of the mitochondrial marker MitoTracker red and LysoTracker red were measured in HeLa cells
after treatment with 17 (20 ꢁM) for 24 h to further confirm the origin of the vacuoles. Live Cell
Imaging with fluorescent tracers demonstrated HeLa cells contained lysosomal or mitochondrial
degradative compartments, but most vacuoles induced by 17 in HeLa cells showed little overlap
with these trackers (Figure 6A). To further determine whether the ability of 17 to induce cell
vacuolization is correlated with macropinocytosis, we performed immunofluorescence assay to
examine the colocalization of LY and the early endosomal marker EEA1, the late endosomal
marker Rab7 and the late endosomal and lysosomal marker LAMPꢀ1.28, 33, 34 EEA1 represented a
transition state between nascent and mature macropinosomes. As shown in Figure 6B, LY had
minimal fusion with EEA1. Immunofluorescence analysis demonstrated that LY in HeLa cells
exposed to 17 displayed an increased fusion with Rab7 and LAMPꢀ1 with time, macropinosomes
acquired characteristics of late endosomes and ultimately fused with lysosomes, which was
consistent with these previous studies on macropinosomes.35, 36
These results are consistent with what were observed about macropinocytosis in methuosis and
the concept that the vacuoles are not derived from swollen mitochondria or lysosomes, 8, 37 but
ultimately macropinosomes have fusion with lysosomes, which makes the vacuoles acquire some
characteristics of lysosomes. These results suggest that the vacuolization in HeLa cells induced
by 17 is associated with macropinocytosis.
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Figure 6. 17 induces macropinocytosis with cytoplasmic vacuolization. (A) Confocal
microscopic analysis of LY accumulation, lysosome and mitochondria in HeLa cells. After being
treated with 20 ꢁM 17 for 24 h, cells were incubated with Lucifer yellow CH (1.25 mg/ml in
HBSS) for 20 min at 37 °C, or followed by labeling with 50 nM LysoTracker Red or 200 nM
MitoTracker Red CMXRos. Phase contrast and fluorescent images of the living cells were
obtained using a LSM710 confocal microscope. (B) HeLa cells were treated with 0.1% DMSO
control or 20 ꢁM 17 for the indicated time, and incubated with Lucifer yellow CH followed by
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staining with antiꢀEEA1, antiꢀLAMP1, antiꢀRab7 and DAPI. Images were acquired on a
LSM710 confocal microscope.
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Methuosis is a novel form of cell death depending on the hyperstimulation of
macropinocytosis. The hyperstimulation of macropinocytosis leads to membrane ruffling,
formation of randomly sized vacuoles and rupture of cell.24 Based on these findings, we test if
macropinocytosis is an essential progress in 17ꢀinduced cell death. To explore the possible
contribution of macropinocytosis to 17ꢀinduced extensive cytoplasmic vacuolization and cell
death, we utilized the specific inhibitor of the vacuolarꢀtype H+ꢀATPase, Bafilomycin A1 (Bafꢀ
A), which could block clathrinꢀindependent endocytosis, like macropinocytosis.9 Incubation of
HeLa cells with 100 nM BafꢀA1 for 1 h prior to 17 treatment mitigated the formation of
vacuoles, and significantly inhibited the cytotoxicity of 17 (see the Supporting Information,
Figure S6 and S7). Macropinocytosis is an actinꢀdependent process, which also requires dynamin
to mediate the release of endocytic vesicles.38 Actin mediates membrane ruffling and leads to the
formation of macropinosomes, which is a characteristic of macropinocytosis distinguishing it
from other forms of endocytosis.39 When the dynamin inhibitor and actin inhibitor, Dynasore and
CytochalasinꢀD,40, 41 were added before 17 treatment, there was little diminution of cell
vacuolization, but cell viability was blocked (see the Supporting Information, Figure S6 and S7).
To further explore the macropinocytosis in cell death induced by 17, we preꢀtreated HeLa cells
with amiloride (EIPA), a specific inhibitor of macropinocytosis through inhibiting the Na+/H+
ion exchange to affect the intracellular pH. We examined macropinocytic activity by counting
the number of HeLa cells with macropinososomes. EIPA could significantly inhibit
vacuolization of HeLa cells induced by 17 (Figure 7A), and the cell death induced by 17 was
significantly blocked by EIPA (Figure 7B). These results demonstrate that the cell death induced
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by 17 is macropinocytosisꢀdependent, and macropinocytic activity of HeLa cells under EIPA
preꢀtreatment decrease with cell proliferation.
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These data suggested macropinocytosis played an important role in 17ꢀinduced cell death.
Compound 17 was toxic to cancer cells, while EIPA could attenuate 17ꢀinduced cell death by
inhibiting macropinocytosis. Taken together with the previous studies of these inhibitors, our
results supported the idea that the mechanism of 17ꢀinduced cell death was the perturbations of
endocytic trafficking that led to cellular vacuolization associated with macropinocytosis, as was
the case for methuosis.
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Figure 7. 17 triggers methuosis through hyperstimulation of macropinocytosis. (A) HeLa cells
were pretreated for 30 min in the presence or absence of 50 ꢁM EIPA prior to addition of 20 ꢁM
17 or DMSO at the indicated time. At each time point, digital images of cells with
macropinosomes in a field were scored manually for assessing the rate of macropinocytosis.
Macropinosome quantification was performed by calculating the percentage of cells with
macropinosomes. (B) HeLa cells were pretreated for 30 min in the presence or absence of 50 ꢁM
EIPA prior to addition of 20 ꢁM 17 or DMSO. CCK8 assay was performed at the indicated time.
Drug resistance phenotypes decrease the clinical efficacy of anticancer therapies.42 Apoptosis,
taken as a caspaseꢀdependent cell death, is the most common and recognized form of
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programmed cell death. Inhibition of apoptosis is a major type of drug resistance, which is
usually caused by some gene mutations.43 Various forms of nonꢀapoptotic cell death have been
defined in the fields of toxicology and cancer therapy by different cell death phenotype,
including paraptosis, autophagic death, necrosis, methuosis and so on.24,44 Among these cell
death subroutines, methuosis as a novel form of cell death is characterized by an abnormal or
dysregulated macropinocytosis.8,45 In this study, we examined the impact of 17 on
macropinocytosis and the relationship between macropinocytosis and methuosis, a novel nonꢀ
apoptotic cell death induced by 17.
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CONCLUSIONS
In summary, we described compound 17, a naturalꢀproductꢀderived small molecule, was a
novel inducer of macropinocytosis, ultimately leading to cell death. 17 provided an attractive
molecular probe for studying the potential link between macropinocytosis and cell death. Our
results also demonstrated, for the first time, that 17 triggered methuosis, an unusual form of nonꢀ
apoptotic cell death through trafficking macropinosomes that eventually acquired some
characteristics of late endosomes and lysosomes, resulting in catastrophic vacuolization and
rupture of cell membrane in cancer cells. Future research will be focused on elucidating the exact
targets and process of 17 in methuosis. 17 may serve as a prototype for new drugs that could be
used to induce nonꢀapoptotic death in cancer. Activation of macropinocytosis is largely pHꢀ
46
dependent and cancer cells are more acidic than normal cells
,
which may provide a clue as to
why cancer cells are more sensitive to 17 treatment. More insights into this unconventional form
of cell death would offer new and exciting therapeutic opportunities for cancers.
EXPERIMENTAL SECTION
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General Procedures. All reagents were purchased from commercial suppliers and were used
directly without further purification. Flash chromatography was carried out with silica gel (200ꢀ
300 mesh). Analytical TLC was performed with silica gel GF254 plates, and the products were
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visualized by UV detection. H and C NMR (500 MHz and 125 MHz, respectively) spectra
were recorded in CDCl3, CD3OD or DMSOꢀd6 at room temperature. The chemical shifts (δ) are
provided in ppm using TMS as internal standard, and the coupling constants (J) are given in Hz.
The following abbreviations for multiplicities are used: s, singlet; d, doublet; dd, double doublet;
t, triplet; q, quadruplet; m, multiplet; and br, broad. HRMS spectra were recorded on Agilent
Techologies 6230 TOF LC/MS spectrometer. Purify of compounds for biological testing was
confirmed to be >95% as determined by HPLC analysis (for data, see Supporting Information).
HPLC analysis was conducted according to the following method with the retention time
expressed in min at UV detection of 210 nm. For HPLC method, an Agilent 1260 series HPLC
instrument was used, with chromatography performed on a ZORBAX 100 mm × 4.6 mm, 3.5
µM C18 column with mobile phase gradient of 50ꢀ100% CH3CN in H2O with a flow rate of 1.0
mL/min.
Benzyl
(1S,2R,4aS,6aS,6bR,8aR,10S,12aR,12bR,14bS)ꢀ10ꢀhydroxyꢀ1,2,6a,6b,9,9,12aꢀ
heptamethylꢀ1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14bꢀoctadecahydroꢀpiceneꢀ4a(2H)ꢀ
carboxylate (2). A mixture of ursolic acid 1 (460 mg, 1.0 mmol), K2CO3 (280 mg, 2.0 mmol) in
N,Nꢀdimethylformamide (10 mL) was treated with BnBr (300 mg, 1.7 mmol). The reaction
o
mixture was heated at 60 C for 4 h. After cooling, the mixture was poured into 50 mL of water
and white solid was appeared, which was filtered, washed with H2O and dried under vacuum to
give the title compound 2 (0.50 g, 92%) as a white solid. 1H NMR (500 MHz, CDCl3):
δ= 7.36ꢀ
7.29 (m, 5H), 5.24ꢀ5.23 (m, 1H), 5.10 (d, J = 12.5 Hz, 1H), 4.98 (d, J = 12.5 Hz, 1H), 3.20 (dd, J
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= 11.0, 4.5 Hz, 1H), 2.04ꢀ1.98 (m, 1H), 1.89ꢀ1.76 (m, 3H), 1.73ꢀ1.67 (m, 2H), 1.64ꢀ1.55 (m,
4H), 1.53ꢀ1.44 (m, 6H), 1.37ꢀ1.26 (m, 6H), 1.07 (s, 3H), 1.05ꢀ1.02 (m, 2H), 0.98 (s, 3H), 0.93 (d,
J = 6.0 Hz, 3H), 0.89 (s, 3H), 0.85 (d, J = 6.5 Hz, 3H), 0.78 (s, 3H), 0.64 (s, 3H) ppm. 13C NMR
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(125 MHz, CDCl3): δ= 177.3, 138.1, 136.3, 128.4 (2C), 128.1 (2C), 127.9, 125.7, 79.0, 66.0,
55.2, 52.9, 48.1, 47.5, 42.0, 39.5, 39.1, 38.8, 38.7, 38.6, 36.9, 36.6, 33.0, 30.6, 28.1, 27.9, 27.2,
24.2, 23.5, 23.2, 21.2, 18.3, 17.0 (2C), 15.6, 15.4.
Benzyl
(1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS)ꢀ1,2,6a,6b,9,9,12aꢀheptamethylꢀ10ꢀoxoꢀ
1,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14bꢀoctadecaꢀhydropiceneꢀ4a(2H)ꢀcarboxylate (3).
To a cooled solution of compound 2 (450 mg, 0.82 mmol) in anhydrous CH2Cl2 (50 mL) was
added PCC (355 g, 1.6 mmol). The reaction mixture was then stirred at room temperature for 12
h. Upon completion, the reaction mixture was filtered through a pad of celite. The filtrate was
evaporated under reduced pressure and the residue was subjected to silica gel column
1
chromatography to afford the title compound 3 (375 mg, 84%) as a white solid. H NMR (500
MHz, CDCl3): δ = 7.37ꢀ7.29 (m, 5H), 5.25 (t, J = 3.5 Hz, 1H), 5.11 (d, J = 12.5 Hz, 1H), 4.99 (d,
J = 12.5 Hz, 1H), 2.57ꢀ2.50 (m, 1H), 2.40ꢀ2.34 (m, 1H), 2.27 (d, J = 11.5 Hz, 1H), 2.04ꢀ1.98 (m,
1H), 1.92ꢀ1.87 (m, 3H), 1.82ꢀ1.68 (m, 3H), 1.64ꢀ1.54 (m, 3H), 1.50ꢀ1.41 (m, 5H), 1.35ꢀ1.28 (m,
4H), 1.10ꢀ1.07 (m, 1H), 1.08 (s, 6H), 1.03 (d, J = 9.0 Hz, 6H), 0.93 (d, J = 6.0 Hz, 3H), 0.85 (d, J
13
= 6.5 Hz, 3H), 0.68 (s, 3H) ppm. C NMR (125 MHz, CDCl3): δ= 177.2, 138.2, 136.3, 128.4
(2C), 128.1 (2C), 127.9, 125.4, 66.0, 55.2, 52.9, 48.1, 47.4, 46.7, 42.1, 39.4, 39.3, 39.1, 38.8,
36.6, 36.5, 34.2, 32.5, 30.6, 27.9, 26.5, 24.2, 23.5, 23.3, 21.4, 21.1, 19.5, 17.0, 16.9, 15.2.
Selected Experimental Procedure. Benzyl (1S,2R,4aS,6aS,6bR,8aR,12aR,12bR,14bS)ꢀ11ꢀ
formylꢀ1,2,6a,6b,9,9, 12aꢀheptamethylꢀ10ꢀoxoꢀ,3,4,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14bꢀ
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octadecahydropiceneꢀ4a(2H)ꢀcarboxylate (4, R1 = H). A mixture of compound 3 (300 mg, 0.55
3
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mmol, 1.0 equiv) in anhydrous THF (20 mL) was treated with sodium methoxide (45 mg, 0.83
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mmol, 1.5 equiv) at 0 C, followed by the addition of ethyl formate (45 mg, 0.60 mmol, 1.1
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equiv). After being stirred at room temperature for 4 h, the reaction was quenched by the
addition of H2O, and the mixture was extracted with EtOAc (3 x 30 mL). The combined organic
layers were washed with brine, dried over Na2SO4, and concentrated to give a crude product,
which was used directly in the next step without further purification. Analytical samples could be
1
obtained by silica gel column chromatography. H NMR (500 MHz, CDCl3): δ = 14.92 (br s,
1H), 8.57 (s, 1H), 7.37ꢀ7.30 (m, 5H), 5.28 (t, J = 3.3 Hz, 1H), 5.12 (d, J = 12.5 Hz, 1H), 4.99 (d,
J = 12.5, 1H), 2.32ꢀ2.28 (m, 2H), 2.05ꢀ1.92 (m, 4H), 1.82ꢀ1.69 (m, 3H), 1.65ꢀ1.56 (m, 2H), 1.50ꢀ
1.43 (m, 3H), 1.39ꢀ1.26 (m, 5H), 1.18 (s, 3H), 1.14ꢀ1.10 (m, 2H), 1.11 (s, 3H), 1.08 (s, 3H), 0.94
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(d, J = 6.5 Hz, 3H), 0.89 (s, 3H), 0.86 (d, J = 6.5 Hz, 3H), 0.68 (s, 3H) ppm. C NMR (125
MHz, CDCl3): δ= 177.2, 138.1, 136.3, 128.4 (2C), 128.2 (2C), 127.9, 125.4, 105.8, 66.0, 53.0,
52.0, 48.1, 45.5, 42.2, 40.1, 39.4, 39.3, 39.1, 38.8, 36.6, 36.2, 32.3, 30.6, 28.4, 27.9, 24.2, 23.4,
23.3, 21.1, 20.9, 19.4, 17.0, 16.9, 14.6. HRMS (ESI): m/z [M+H]+ calcd for C38H52O4: 572.3866;
found: 572.3871.
Selected Experimental Procedure. Benzyl (1S,2R,4aS,6aS,6bR, 8aR,13aR,13bR,15bS)ꢀ10ꢀ(4ꢀ
cyanophenyl)ꢀ1,2,6a,6b,9,9,13aꢀheptamethylꢀ1,2,3,4,5,6,6a,6b,7,8,8a,9,10,13,13a,13b,14,15bꢀ
octadecahydroꢀ4aHꢀchryseno[1,2ꢀf]indazoleꢀ4aꢀcarboxylate (5, R1 = H, R2 = 4ꢀcyanophenyl). A
mixture of 4 (320 mg, 560 mmol, 1.0 equiv), 4ꢀcyanophenylhydrazine hydrochloride (95 mg,
560 mmol, 1.0 equiv) in ethanol (20 mL) was refluxed at 85 °C for 12 h. After cooling the
reaction to room temperature, the solvents were removed and the residue was partitioned
between ethyl acetate and water (20 mL). The organic layer was separated and evaporated. The
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residue was purified by silica gel column chromatography to afford compound 5 (315 mg, 84%)
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as a pale solid. H NMR (500 MHz, CDCl3): δ = 7.78 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 Hz,
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2H), 7.34 (s, 1H), 7.37ꢀ7.31 (m, 5H), 5.32 (t, J = 3.0 Hz, 1H), 5.12 (d, J = 12.5, 1H), 4.98 (d, J =
12.5 Hz, 1H), 4.14ꢀ4.09 (m, 1H), 2.77 (d, J = 15.5 Hz, 1H), 2.31 (d, J = 11.0 Hz, 1H), 2.15 (d, J
= 15.5 Hz, 1H), 2.05ꢀ1.99 (m, 3H), 1.90ꢀ1.84 (m, 1H), 1.82ꢀ1.60 (m, 4H), 1.50ꢀ1.47 (m, 3H),
1.39ꢀ1.24 (m, 6H), 1.12ꢀ1.08 (m, 1H), 1.09 (s, 3H), 1.03 (s, 3H), 1.02 (s, 3H), 0.95 (s, 3H), 0.93
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(s, 3H), 0.88 (d, J = 3.9 Hz, 3H), 0.69 (s, 3H) ppm. C NMR (125 MHz, CDCl3):
δ = 177.3,
146.7, 146.5, 139.5, 138.1, 136.4, 132.6, 130.0, 128.5 (2C), 128.2 (2C), 128.0, 125.7, 118.1,
115.1, 112.9, 66.1, 58.5, 54.6, 53.1, 48.3, 46.4, 42.2, 39.5, 39.2, 38.9, 38.0, 37.1, 36.7, 34.7, 32.6,
30.7, 29.7, 28.0, 24.3, 23.4, 23.3, 22.8, 21.2, 19.2, 18.5, 17.0, 16.9, 15.5. HRMS (ESI): m/z
[M+H]+ calcd for C45H55N3O2: 669.4294; found: 669.4283.
Selected Experimental Procedure. (1S,2R,4aS,6aS,6bR,8aR,13aR,13bR,15bS)ꢀ10ꢀ(4ꢀ
Cyanophenyl)ꢀ1,2,6a,6b,9,9,13aꢀheptamethylꢀ1,2,3,4,5,6,6a,6b,7,8,8a,9,10,13,13a,3b,14,15bꢀ
octadecahydroꢀ4aHꢀchryseno[1,2ꢀf]indazoleꢀ4aꢀcarboxylic acid (17). A mixture of benzyl ester
5 (67 mg, 0.1 mmol), 10% Pd/C (10 mg) in methanol (20 mL) was stirred under 1 atm H2 at
room temperature and the reaction process was monitored by TLC. After completion of the
reaction, the mixture was filtered through a pad of celite and washed with CH3OH (10 mL). The
filtrate was evaporated to dryness, and the residue was triturated with ethyl ether (2 x 10 mL).
The solid was collected and dried in vacuum to give 17 as a white solid (48 mg, 82%). 1H NMR
(500 MHz, CDCl3): δ = 10.95 (br s, 1H), 7.75 (d, J = 8.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.37
(s, 1H), 5.32 (t, J = 3.5 Hz, 1H), 2.64 (d, J = 15.0 Hz, 1H), 2.23 (d, J = 11.5 Hz, 1H), 2.14 (d, J =
15.5 Hz, 1H), 2.05ꢀ1.99 (m, 3H), 1.90ꢀ1.84 (m, 1H), 1.72ꢀ1.66 (m, 4H), 1.53ꢀ1.49 (m, 3H), 1.42ꢀ
1.26 (m, 6H), 1.15ꢀ1.12 (m, 1H), 1.10 (s, 3H), 1.03 (s, 3H), 0.99 (s, 3H), 0.95 (d, J = 6.5 Hz,
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3H), 0.93 (s, 3H), 0.89 (d, J = 6.0 Hz, 3H), 0.84 (s, 3H) ppm. C NMR (125 MHz, CDCl3):
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= 183.6, 146.6, 146.2, 139.3, 137.8, 132.5 (2C), 129.9 (2C), 125.6, 117.9, 115.0, 112.9, 54.4,
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52.6, 47.9, 46.3, 42.1, 39.4, 39.1, 38.7, 37.9, 36.9, 36.6, 34.6, 32.3, 30.6, 29.5, 27.9, 24.0, 23.4,
23.2, 22.7, 21.1, 19.1, 16.9, 16.7, 15.4. HRMS (ESI): m/z [M+H]+ calcd for C44H63N4O2:
579.3825; found: 579.3816.
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Cell Culture. HeLa, HepG2, MCFꢀ7, SY5Y, A549, HDFs and C2C12 cell lines were obtained
o
from the Shanghai Institute of Cell Biology (Shanghai, China). Cells were grown at 37 C with
5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA) supplemented
with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA) and 100 U/ml penicillin and 100
ꢁg/ml streptomycin (Life Technologies, Grand Island, NY). All cell lines were maintained at 37
°C in 5% CO2 humidified air.
Cell Viability Assay. Cell viability was detected by a Cell Counting Kitꢀ8 (CCKꢀ8; Dojindo,
Japan) according to the manufacturer’s instructions. Briefly, 5000 cells per well were seeded in a
96ꢀwell plate. Preꢀincubate the plate overnight. Then cells were subjected to various conditions.
10 ꢁL of CCKꢀ8 solution was added into the each well. The absorbance was measured at 450 nm
using a microplate reader (SPECTRAFLUOR, TECAN, Sunrise, Austria). The group treated
with DMSO was regarded as control. In each experiment, six paralleled wells were made and all
the experiments were independently repeated at least three times.
For IC50 determination, compound 17 was dissolved at 100 mM, with subsequent serial
dilution in dimethyl sulfoxide (DMSO). Then cells were seeded in 96ꢀwell plate at the density of
1000 cells/well, and cultured with serial 17 (final concentration 0ꢀ50 ꢁM) for 48 h. At the end of
incubation, CCKꢀ8 assay was performed. IC50 values of 17 were calculated with Prism
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(GraphPad Software). Values are presented as an IC50 in ꢁM with s.d. (n = 3 independent
experiments)
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Colony Formation Assay. HeLa cells were treated with various concentrations of 17 (15 ꢁM,
20 ꢁM, 25 ꢁM) for 48 h before collection. Then cell from each group were seeded in sixꢀwell
plates at a density of 500 cells per well. Colony formation was assessed after 15 days by fixing
with methanol and staining with Giemsa. Colonies were photographed and counted.
Apoptosis Assay. In order to evaluate apoptosis, cells were stained using the Alexa Fluor®
488 annexin V/Dead Cell Apoptosis Kit (Invitrogen) as per the manufacturer's instructions. After
incubation under various conditions, each group of cells was harvested and washed twice in cold
PBS. Then 5 ꢁL Alexa Fluor® 488 annexin V and 1 ꢁL 100 ug/mL PI working solution were
added to each 100 ꢁL of cell suspension in 1x annexinꢀbinding buffer. Cells were incubated at
room temperature for 15 minutes followed by adding 400 ꢁL 1x annexinꢀbinding buffer. The
apoptosis was determined using flow cytometry (FACS Calibur, BD Bioscience), and 10000
events from each sample were acquired to ensure adequate data. Unstained cells were used as
gating controls.
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Western Blot Analysis. Primary antibodies against caspaseꢀ3, PARP1, and LC3 were
obtained from (Cell Signaling Technology, Danvers, MA, USA); GAPDH was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies were purchased from
Invitrogen. Cells were lysed in RIPA lysis buffer containing protease inhibitor cocktail (sigma,
USA) on the ice for 30 min. Protein concentration was quantified using the BCA assay. Protein
was separated by SDSꢀPAGE and was transferred onto polyvinylidene difluoride (PVDF)
membrane. After being blocked with 5% nonꢀfat milk for 1 h at room temperature, the
membranes were incubated with primary antibodies overnight at 4 °C. Then the membranes were
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washed with TBST buffer, and incubated with corresponding secondary antibodies at room
temperature for 2 h. The protein bands were washed again with TBST buffer, and visualized by
the Odyssey Infrared imaging system (LiꢀCor Biosciences, Lincoln, NE). GAPDH was used as a
loading control for normalization.
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Time-Lapse Imaging. 50,000 HeLa cells were plated in a 20 mm glass bottom culture dish
(NEST Biotechnology). Preꢀincubate the plate overnight. Then cells were treated with 17 at 20
ꢁM. The dish was placed in a live cell chamber with 5% CO2 and 37 °C. Live imaging was
performed using an Olympus 1 x 80 inverted microscope. Phaseꢀcontrast images were acquired
every 90 sec for 72 hours.
Live Cell Imaging. Lucifer yellow CH, MitoTracker Red CMXRos and LysoTracker Red
were purchased from Invitrogen/Molecular Probes (Carlsbad, CA). Lucifer yellow (LY) was a
favorite tool for monitoring Extracellular fluid uptake. After being treated with compound for 24
h, cells were incubated with LY (1.25 mg/mL in HBSS) for 20 min at 37 °C and washed with
PBS twice. To visualize active lysosome and mitochondria in cells, LysoTracker and
MitoTracker Red CMXRos were used, respectively, according to the directions supplied by the
manufacturers. Briefly, cells were incubated with 50 nM LysoTracker Red in medium for 30 min
to label intracellular acidic compartments. Staining the mitochondria was performed by
incubating live cells with 200 nM MitoTracker Red CMXRos in medium for 1 h. Phase contrast
and fluorescent images of the living cells were obtained using a LSM710 confocal microscope
(Zeiss).
Electron Microscopy. HeLa cells were exposed to 20 ꢁM 17 for 24 h, and then fixed,
dehydrated, embedded in resin. Images were obtained by the Electron Microscopy Facility at
National Center for Protein Science Shanghai, Institute of Biochemistry and Cell Biology
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(Shanghai, China). Ultrathin sections were mounted on copper grids, and then stained with
uranyl acetate and lead citrate. TEM was performed with a FEI Tecnai G2 Spirit Twin electron
microscope (FEI Co., Eindhoven, The Netherlands).
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Immunofluorescence Analysis. Following treatment of HeLa cells on polyꢀLꢀlysineꢀcoated
glass coverslips, cells were fixed with 4% paraformaldehyde (PFA) for 30 min and permeablized
with 0.1% Triton Xꢀ100 for 15 min at room temperature. Then, nonspecific binding sites were
blocked by 5% bovine serum albumin (BSA) for at room temperature. For staining, HeLa cells
were incubated with antiꢀLAMPꢀ1 primary antibody at 4 °C overnight. Subsequently, cells were
incubated for 1 h at room temperature with Alexa Fluor488ꢀconjugated secondary antibody.
Finally, nuclei were stained with 6ꢀdiaminoꢀ2ꢀphenylindole dihydrochloride (DAPI) for 15 min.
Images were acquired on a LSM710 confocal microscope with 488ꢀ and 405ꢀnm laser excitation
and ZEN software (Carl Zeiss, Oberkochen, Germany).
Quantitative Assessment of Macropinocytosis. The macropinocytosis quantification assay
measuring the amount of cell with macropinosomes was performed as described previously.47
HeLa cells were incubated with EIPA (50 ꢁM) for 30 min, and then treated with 17 for 4 h, 8 h,
12 h, 18 h, 24 h, 36 h, 48 h, 60 h and 72 h, respectively. At each time point, digital images of
cells with macropinosomes in a field were scored manually for assessing the rate of
macropinocytosis. Macropinosome quantification was performed by calculating the percentage
of cells with macropinosomes.
ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS Publication website at
http://pubs.acs.org. Additional figures illustration inhibition data; vacuole formation movies;
copies of NMR spectra; analytical HPLC data on tested compounds (PDF); SMILES molecular
strings (CSV)
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AUTHOR INFORMATION
Corresponding Author
*For W. Wei (lead contact): Phone: (+86)21ꢀ20350941; Eꢀmail: weiwg@sari.ac.cn.
*For L. Li: Phone: (+86)21ꢀ62933629; Eꢀmail: lil@sjtu.edu.cn.
ORCID
Wanguo Wei: 0000ꢀ0002ꢀ2429ꢀ8281
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was partially supported by grants from the Chinese Ministry of Science and
Technology (2013CB967402), NSFC (21202100, 81502985), Science and Technology
Commission of Shanghai Municipality (16140902100), and the Strategic Priority Research
Program of the Chinese Academy of Sciences (XDA01040302). We thank Dr. Naitao Wang and
Dr. Minglei Wang for help with TimeꢀLapse Imaging.
ABBREVIATIONS USED
UA, ursolic acid; DMF, N,Nꢀdimethylformide; THF, tetrahydrofuran; DMSO, dimethyl
sulfoxide; CCKꢀ8, Cell Counting Kitꢀ8; PARP, Poly[ADPꢀribose] polymerase; LC3,
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(microtubuleꢀassociated protein 1 light chain 3; 3ꢀMA, 3ꢀMethyladenine; PI3K, phosphoinositide
3ꢀkinase; TEM, Transmission electron microscopy; LY, Lucifer Yellow; LAMPꢀ1, Lysosomalꢀ
associated membrane protein 1; EEA1, Early Endosome Antigen 1; HBSS, Hank's Balanced Salt
Solution; BafꢀA, Bafilomycin A1; EIPA, amiloride; DMEM, Dulbecco’s modified Eagle’s
medium; FBS, fetal bovine serum; PVDF, polyvinylidene difluoride; PFA, paraformaldehyde;
BSA, bovine serum albumin; DAPI, 6ꢀdiaminoꢀ2ꢀphenylindole dihydrochloride
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