Novel Anthraquinone Compounds Induce Cancer Cell Death through Paraptosis

Article abstract of DOI:10.1021/acsmedchemlett.8b00624

Novel anthraquinone compounds that induce ER stress and paraptosis-like cell death were designed and synthesized. Compound 4a is the first organic micromolecule to kill tumor cells by only paraptosis, and its mechanism of action has been further explored. Paraptosis does not appear to involve either phosphatidylserine translocation associated with apoptosis or cell cycle arrest. The bisbenzyloxy and N-(2-hydroxyethyl)formamide structures may be two critical pharmacophores for paraptosis. Bisbenzyloxy can induce ER stress, and the N-(2-hydroxyethyl)formamide structure can increase the ratio of LC3II/I and cytoplasmic vacuolization and facilitates paraptosis. Some antitumor drugs fail to eradicate malignant cell lines with impaired apoptotic pathways; paraptosis may be a route to kill such cells and provides a new potential strategy for cancer chemotherapy.

Full text of DOI:10.1021/acsmedchemlett.8b00624

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Letter
Novel Anthraquinone Compounds Induce
Cancer Cell Death Through Paraptosis
Tian Wei, Junying Li, Zhengying Su, Fu Lan, Zhaoquan Li,
Dandan Liang, Chunmiao Wang, Danrong Li, and Huaxin Hou
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019
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Novel Anthraquinone Compounds Induce Cancer Cell Death Through
Paraptosis
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Wei Tian,†,§ Junying Li,†,§ Zhengying Su,† Fu Lan,† Zhaoquan Li,† Dandan Liang,† Chunmiao Wang,†
Danrong Li, ‡,* and Huaxin Hou†,*
†College of Pharmacy, Guangxi Medical University, Nanning 530021, China
‡Life Sciences Institute, Guangxi Medical University, Nanning 530021, China
KEYWORDS: anthraquinone, paraptosis, ER stress, noncaspase-dependent cell death, vacuolation
ABSTRACT: Novel anthraquinone compounds that induce ER stress and paraptosis-like cell death were designed and
synthesized. Compound 4a is the first organic micromolecule to kill tumor cells by only paraptosis, and its mechanism of
action has been further explored. Paraptosis does not appear to involve either phosphatidylserine translocation associated
with apoptosis or cell cycle arrest. The bisbenzyloxy and N-(2-hydroxyethyl)formamide structures may be two critical
pharmacophores for paraptosis. Bisbenzyloxy can induce ER stress, and the N-(2-hydroxyethyl)formamide structure can increase
the ratio of LC3II/I and cytoplasmic vacuolization and facilitates paraptosis. Some antitumor drugs fail to eradicate malignant cell
lines with impaired apoptotic pathways; paraptosis may be a route to kill such cells and provides a new potential strategy for
cancer chemotherapy.
Programmed cell death is divided into two general types:
caspase-dependent and noncaspase-dependent cell death.
Apoptosis is one of the most typical forms of caspase-
dependent programmed cell death. Paraptosis is a new
noncaspase-dependent type of programmed cell death that
appears to differ from apoptosis, necrosis, and autophagy
and is characterized by ER stress, extensive cytoplasmic
vacuolization, and swelling of the endoplasmic reticulum
and/or mitochondria. Little is known at present regarding
the molecular basis of paraptosis 1-3. Some antitumor drugs
fail to eradicate malignant cell lines with impaired apoptotic
pathways; paraptosis may be a route to kill such cells and
provides a new potential strategy for cancer chemotherapy.
Factors that induce paraptosis include organometallic
complexes, viruses4, natural products and their analogues.
Compounds that induce paraptosis are predominantly
1C, 1D), used for the treatment of various cancers in the
clinic, and their antitumor effect is exerted through the
apoptotic pathway18,19
.
organometallic complexes such as copper5-7, ruthenium8,9
,
titanium10, and rhenium11 complexes. Some natural
products can also induce tumor cell death through
paraptosis such as taxol12,13 (Figure 1A), curcumin14, and
celastrol15. Although organometallic complexes can induce
cell death through the paraptotic pathway, it is easy to cause
heavy metal poisoning during treatment. However, few
natural product derivatives or organics have been reported
to induce paraptosis.
Rhein (Figure 1B) is a natural product isolated from
Rheum palmatum that has moderate anticancer activities in
different human tumor cells and is very well tolerated by
the human body16,17. There are several anthraquinone-
based drugs, such as mitoxantrone and doxorubicin (Figure
Figure 1. Taxol, rhein and anthraquinone-based drugs that
have been used clinically to treat tumors.
It has been reported in the literature that bisbenzyloxy may
be an essential group to induce ER stress in cells20. Here, we
used rhein as a lead compound, introduced a bisbenzyloxy
group at its 1,8-OH position to ensure ER stress, and then
modified and structurally optimized it at the C-3 position and
designed and synthesized a series of novel anthraquinone
compounds that can induce cell death by paraptosis. The
general structure of the compound is shown in Figure 2.
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rough ER, and the vacuoles were wrapped in monolayer
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Optimized compound 4a (Figure 2) is the first organic
micromolecule to kill tumor cells by only paraptosis without
other cell death mechanisms and has excellent activity in vitro.
The morphological characteristics of paraptotic cells and the
expression of related proteins were further examined. Along
with analyzing structure-activity relationships (SARs) and
mechanisms of action, the mechanism by which paraptosis is
induced by the compounds was elucidated.
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membranes rather than bilayer membranes, similar to
autophagy (Figures 3C and S6, S8-9). We also observed that
mitochondrial swelling, vacuoles, and ridges disappeared in
some cells (Figures S11-12). These results indicate that 4a
treatment induced the formation of massive cytoplasmic
vacuoles and damaged the integrity of the ER, mitochondrial
and Golgi apparatus structures in human SMMC-7721 cells. 4a
triggered ER vacuolation with the specific characteristics of
paraptosis.
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Figure 2. Examples of bioactive compounds with paraptotic
activity.
The general procedure for anthraquinone compound
synthesis and the total yield are shown in the Supporting
Information (Scheme S1 and Table S1). The effect of all
compounds on cancer cell proliferation was investigated by
MTT and CCK-8 assay, and their IC50 values and cell
morphology are listed in Table S2 and Figure S1.
Compounds 4a
, 4b and 5c inhibited tumor cell
proliferation and induced cell vacuoles (Figure S1). It is
worth noting that 4a has an IC50 value (1.5±0.3 μM) at the
single digit micromolar level and is more sensitivity in
hepatocellular carcinoma SMMC-7721 cells than in the
other cells tested (Figure S2). Therefore, 4a was selected
for further investigation.
Figure 3. (A) Phase contrast images and HE staining after
SMMC-7721 cells were treated with 4a for 48 h. (B) SMMC-
7721 cells were incubated with 4a for 48 h and analyzed using
ER-Tracker Red and Hoechst 33342 assays by laser scanning
confocal microscopy. ER-Tracker Red (red) and Hoechst
33342 (blue) were used to visualize ER structures and nuclei,
respectively. (C) TEM imaging of control and treated (4a, 5
μM) SMMC-7721 cells. The abnormal ER, mitochondria and
Golgi apparatus are indicated by red arrowheads, green
arrowheads and yellow arrowheads, respectively.
Inverted phase contrast microscopy showed that a large
number of vacuoles appeared in the cells after 4a treatment
for 48 h (Figures 3A and S3). As the concentration of 4a
increased, the number of vacuoles in the cells gradually
increased, but the volume of the vacuoles slowly shrank.
At the same time, morphological changes such as cell
shrinkage, cytoplasmic loss and nuclear chromatin
pyknosis were observed (Figures 3A and S4). Since
vacuoles were found in the cytosol, to determine if the
vacuoles were derived from the ER, SMMC-7721 cells
were stained with ER-Tracker Red and Hoechst 33342. It
was clearly seen under a laser scanning confocal
microscope that the nuclei shrank after 4a treatment. In
addition, vacuoles were observed and were surrounded by
positive ER-specific markers, indicating that the vacuoles
originated from the ER. With an increase in drug
concentration, the number of vacuoles gradually increased,
the entire endoplasmic reticulum cytoplasm was occupied
by vacuoles, and cell contents began to be lost in large
quantities (Figures 3B and S5).
Next, we further examined the effect of vacuoles on cell
viability. A CCK-8 assay was performed after the treatment of
SMMC-7721 cells with 3, 5, or 10 μM (which can induce cell
vacuole formation) 4a for 48 h. With an increase in the 4a
concentration, the cell viability was highly decreased (Figures
4C and S25). To investigate whether growth inhibition induced
by 4a was associated with regulation of the cell cycle and the
induction of apoptosis, the DNA content of the cellular nuclei
and the percentages of apoptotic cells were detected by flow
cytometry. Given that early changes in apoptosis occur at the
cell membrane surface and that PS is a negatively charged
phospholipid normally present inside the cell membrane, as the
cell undergoes apoptosis, and the distribution of this
asymmetrical phospholipid is disrupted, PS leaks outside the
cell membrane21. Flow cytometry revealed that apoptotic cells
were not detected during 4a-induced cytoplasmic vacuolation-
mediated cell death (Figures 4A, 4D and S13-18). Except in the
10 μM treatment group, in which the cell viability was
22.42±1.42%, the cell membranes remained normal and did not
Changes in cell organelles were observed by transmission
electron microscopy (TEM). After treatment with 4a at 5 μM
for 48 h, several large vacuoles and many small vacuoles were
present in the cytoplasm, the cellular membrane structure
remained intact, the nucleus was concentrated and smaller, and
autophagosomes and apoptotic bodies were not found (Figures
3C and S6, S8, S11). Further observation showed that the
vacuoles originated from the expansion and swelling of the
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appear to indicate PS eversion in early apoptosis (Figures 4A,
The generation of ER vacuoles is usually associated with
persistent ER stress. Therefore, we reasoned that 4a
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4D and S17). Interestingly, the cell cycle was not altered by
treatment with 4a at any concentration (Figures 4B, 4E and S19-
24), which may be a new biological characteristic of paraptosis.
However, we found that treatment with 5c not only induced
paraptosis-like cell death but also induced apoptosis. Vacuolar
cells and a few apoptotic cells were clearly observed (Figure
S26), and flow cytometry also detected apoptotic cells with PS
translocation (Figures S27-31).
-
induced vacuolation might result in the induction of
persistent ER stress. The vacuolization of cells gradually
increased (Figures 6B and S32), the expression of GRP78,
a key hallmark of the ER stress response24, significantly
increased, and this process was time-dependent after
treatment with 4a (Figures 6A and S33). Vacuolation is
also a feature of autophagy, and these results prompted us
to explore the role of autophagy in 4a-induced vacuole
formation and cell death. Therefore, we examined whether
4a activated autophagy in SMMC-7721 cells. The essential
proteins LC3 and p62 in the autophagic flow pathway were
detected by Western blotting. When autophagosomes are
formed, LC3 is synthesized as proLC3 in the cytosol and
cleaved by Atg4 to produce LC3-I. LC3-I is activated by
Atg7, transferred to Atg3 and conjugated to PE. The LC3-
PE conjugate is LC3-II, a membrane-bound form of LC3.
The Atg16 conjugate functions in LC3 lipidation25,26. LC3-
II is transported in an Atg5-dependent manner to the inner
layer of the autophagosome and binds to receptors (e.g.,
p62, etc.) that recognize the substance to be degraded,
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encapsulating the substance to form the autophagosome27,28
P62 trapped by LC3 is selectively transported into the
autophagosome, which results in the downregulation of
p62 expression in the cells. LC3-II located in the outer
membrane of the autophagosome is cleaved by Atg4 to
LC3-I, PE is separated, and LC3-I is recycled for repeated
use29,30 (Figure 7D). The treatment of cells with 4a
increased the ratio of LC3-II/I. The higher the LC3-II/I
ratio was, the more vacuoles were observed in the cells
(Figures 6A, 6B, and S32-34). Because p62 protein levels
also increased, autophagosome formation was inhibited
following 4a treatment. The evidence strongly suggests
that 4a triggers ER vacuolation death, which does not
depend on the typical autophagy pathway but proceeds
through a paraptotic pathway.
.
Figure 4. (A) Flow cytometric analysis of apoptosis and the cell
cycle. Cells were collected and stained with annexin V-fluorescein
isothiocyanate (FITC) and PI. (B) Flow cytometric analysis of cell
cycle populations. (C) CCK-8 assays were performed after the
treatment of SMMC-7721 cells with 4a for 48 h. (D) Frequency
distribution graph of apoptotic cells. (E) Frequency distribution
graph of the cell cycle.
Caspase-3 is activated in the apoptotic cell both by
extrinsic (death ligand) and intrinsic (mitochondrial)
pathways. As an executioner caspase, the caspase-3 zymogen
has virtually no activity until it is cleaved by an initiator
caspase22-23. However, we did not detect cleaved caspase-3
in 4a-treated cells (Figure 5). The above results indicated
that 4a-induced cell death, which does not cause apoptosis
or affect the cell cycle, is a noncaspase-dependent method
of cell death.
Figure 6. (A) Western blotting analysis the expression of
GRP78, p62, LC3 after 4a (3 μM) treatment at different time
points in SMMC-7721 cells. GAPDH is shown as a loading
control, **=P<0.01. (B) Phase contrast images of SMMC-
7721 cells treated with 3 μM 4a at the indicated time points.
All images were acquired at the same magnification.
Figure 5. Immunoblot analysis of caspase-3. SMMC-7721 cells
were treated for 12, 24, and 48 h with 4a (IC60) and cisplatin (IC60),
and cellular extracts were analyzed to detect both the inactive
(procaspase-3) and active (cleaved caspase-3) forms of caspase-3.
β-Actin was shown as a loading control, and cisplatin was used as
a positive control for apoptosis.
To study the structure-activity relationship (SAR)
between 4a and the induction of paraptosis, we tested the
expression levels of related proteins using Western blotting.
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Figure 7. Summary of the SAR and the mechanism of paraptosis. (A) Western blotting analysis of the expression of GRP78, p62,
and LC3 after the treatment of SMMC-7721 cells with the same concentration of rhein,
a loading control, **=P<0.01. (B) Phase contrast images of SMMC-7721 cells treated with 10 μM rhein,
2
,
4a, and cisplatin. GAPDH is shown as
4a and cisplatin for
2
,
48 h; all images were acquired at the same magnification. (C) Summary of the SAR and mechanism of paraptosis. (D) LC3-PE
binding system during mammalian autophagy.
After treating cells with the same concentration of rhein,
4a and cisplatin, rhein failed to activate the expression of
GRP78, and 4a treatment significantly activated GRP78
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,
mechanism of action and SAR, we confirmed that bisbenzyloxy
is an important pharmacophore that produces a sustained ER
stress response. N-(2-hydroxyethyl)formamide may be related
to the upregulation of LC3-II. Activated LC3-II may promote
cytoplasmic vacuolization and accelerate this process in
paraptosis. These findings provide new insights for us to
determine paraptosis and may be useful for designing new
effective induced paraptosis drugs.
2
expression, and cisplatin treatment strongly inhibited
GRP78 protein expression compared to their expression in
the control group (Figures 7A and S36-37). It has been
shown that bisbenzyloxy is an important group that
produces a sustained ER stress response. Our experimental
data validate speculation in the literature reported by Zheng
H, et al20. In addition, compared with that in the control,
the expression level of p62 did not decrease in any of the
treatment groups, and the LC3-II/I ratio did not change
except in the 4a treatment group (Figures 7A and S36-37).
When SMMC-7721 cells were treated with the same
ASSOCIATED CONTENT
Supporting Information
Experimental details for chemical synthesis, product analysis,
NMR spectroscopy, mass spectrometry, drug screening, protein
expression, cell culture, FACS analysis, cell viability assay, laser
scanning confocal microscopy, and transmission electron
microscopy can be found in the Supporting Information. Figures
S1−S37 are also included (PDF). The Supporting Information is
available free of charge via the Internet at http://pubs.acs.org.
concentration of
2 and 4a for 48 h, the formation of
vacuoles was clearly observed in cells treated with 4a, and
little vacuolation was observed in cells treated with
compound 2 (Figures 7B and S35), although 2 can produce
ER stress. The above results show that an increase in the
ratio of LC3-II/I may be related to the presence of N-(2-
hydroxyethyl)formamide at the C-3 position of the 1,8-
dibenzyloxy-9,10-anthraquinone ring and indicate that the
occurrence of cellular vacuoles may be related to the
upregulation of LC3-II (Figure 7C). LC3-II is the terminal
protein in the autophagy signaling pathway. Activated
LC3-II binds to p62 to form autophagosomes and
participates in autophagy (Figure 7D), and the expression
level of p62 decreases significantly. However, we found
that activated LC3-II did not bind to p62 to form the double
membranes associated with autophagy but may be related
to cytoplasmic vacuolization and the formation of single
membrane vacuoles in paraptosis (Figure 7C, D) after 4a
treatment.
AUTHOR INFORMATION
Corresponding Author
*E-mail: houhuaxin@163.com
*E-mail: danrongli@163.com
ORCID
Huaxin Hou: 0000-0001-8712-6367
Danrong Li: 0000-0002-1654-7807
Author Contributions
§W. T. and J. L. contributed equally.
Funding Sources
Financial support was from the National Natural Science
Foundation of China (81460561).
Notes
The authors declare no competing financial interests.
In summary, the novel anthraquinone compound 4a is the
first organic micromolecule to kill tumor cells by only
paraptosis in vitro. In paraptosis, we found no translocation of
PS and no cell cycle arrest, which is different from the
traditional apoptosis model and is also a new biological
characteristic of paraptosis. Here, with the study of its
ACKNOWLEDGMENT
This work was supported by the National Natural Science
Foundation of China (81460561) and the Key Laboratory of Early
Prevention and Treatment for Regional High-Frequency Tumor,
Guangxi Medical University, Ministry of Education.
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ABBREVIATIONS
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5
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ER, Endoplasmic reticulum; CCK-8, CellCountingKit-8; MTT, 3-
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium
bromide; IC50, 50% inhibitory concentration; GRP78, Glucose-
regulated protein; LC3, Microtubule-associatedprotein light
chain 3; p62, Sequestosome 1; PE, Phosphatidyl ethanolamine;
PS, Phosphatidylserine.
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Stronger proteasomal inhibition and higher CHOP induction are
responsible for more effective induction of paraptosis by
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Paraptosis: Anthraquinone compound 4a induces cancer cell death via paraptosis. Bisbenzyloxy is an essential
group for ER stress, and the N-(2-hydroxyethyl)formamide structure is necessary to upregulate LC3-II. Upregulated
LC3-II can promote cytoplasmic vacuolization and accelerate paraptosis. Treatment with 4a induces cytoplasmic
vacuolation-mediated cell death that does not depend on the typical autophagy pathway, apoptosis and cell cycle arrest
but proceeds through a paraptotic pathway.
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