Necrostatins regulate apoptosis, necroptosis, and inflammation in cisplatin-induced nephrotoxicity in LLC-PK1 cells

Article abstract of DOI:10.1016/j.bmcl.2021.128256

Acute kidney injury (AKI) is a common clinical problem that is associated with high mortality due to multiple complex mechanisms. Cisplatin is the most important and highly effective chemotherapeutic agent used for the treatment of various solid tumors; h

Full text of DOI:10.1016/j.bmcl.2021.128256

Bioorg. Med. Chem. Lett. 48 (2021) 128256
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Necrostatins regulate apoptosis, necroptosis, and inflammation in
cisplatin-induced nephrotoxicity in LLC-PK1 cells
,
,
Dahae Leea h, Noriko Yamabea h, Heesu Leeb, Hye Lim Leec, Dong-Wook Kimd,
Jae Wook Leee *, Ki Sung Kanga
,
f
,
g
,
,*
a College of Korean Medicine, Gachon University, Seonngman 13120, Republic of Korea
b Department of Oral Anatomy, College of Dentistry, Gangneung Wonju National University, Gangneung, Republic of Korea
c Department of Pediatrics, College of Korean Medicine, Daejeon University, Daejeon, Republic of Korea
d Department of Pharmaceutical Engineering, Cheongju University, Cheongju 28530, Republic of Korea
e Natural Product Research Center, Korea Institute of Science and Technology, Gangneung 25451, Republic of Korea
f Convergent Research Center for Dementia, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea
g Department of Biological Chemistry, Korea University of Science and Technology, Daejeon 34113, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Acute kidney injury (AKI) is a common clinical problem that is associated with high mortality due to multiple
complex mechanisms. Cisplatin is the most important and highly effective chemotherapeutic agent used for the
treatment of various solid tumors; however, it is associated with dose-dependent adverse effects, particularly in
the kidney where it can cause severe nephrotoxicity. The pathophysiological basis of cisplatin-induced neph-
rotoxicity has been investigated over the last few decades, and the key pathological occurrences in cisplatin
nephrotoxicity include renal tubular cell injury and death. Necrostatin-1 (Nec-1) has been confirmed to act as a
specific and potent small-molecule inhibitor of necroptosis. However, the effects of three structurally distinct
necrostatins on cisplatin-induced nephrotoxicity remain ambiguous. The aim of this study was to determine if
three types of necrostatins (Nec-1, Nec-3-A, and/or Nec-3-B) can exert protective effects in regard to the AKI
induced by cisplatin. Our results indicated that necrostatins can prevent cisplatin induced nephrotoxicity via
modulating apoptotic pathways through the suppression of cleaved caspase-3 and also by influencing the
function of mitogen-activated protein kinase pathway members, including extracellular signal-regulated kinases,
c-Jun N-terminal kinases, and p38, in the renal tubular epithelial cell line LLC-PK1. These findings suggest that
necrostatins exert beneficial anti-apoptotic effects in the context of AKI induced by cisplatin.
Necrostatins
Cisplatin
Acute kidney injury
Apoptosis
Caspase
Mitogen-activated protein kinases
Cisplatin (cis-diamminedichloroplatinum [II]) is a highly effective
chemotherapeutic agent and is widely used in the treatment of
numerous types of solid organ malignancies such as testicular, ovarian,
lung, prostate, and breast cancers and also for head and neck cancer.1–3
Conversely, cisplatin can also induce various toxicities, including gas-
trotoxicity, myelosuppression, ototoxicity, and allergic reactions.4 In
particular, nephrotoxicity is the most serious and one of the most
common adverse effects caused by cisplatin, as this drug accumulates in
renal proximal tubular cells where it can cause proximal tubular injury,
oxidative stress, inflammation, vascular injury, and cell death.5–9
Indeed, approximately 25–30% of cisplatin-recipient patients exhibit
severe renal dysfunction after only a single dose of this drug, and this has
become a principal limitation to determining optimal dosing regi-
mens.10 Therefore, an approach to improve kidney injury caused by
cisplatin has become a crucial problem that must be solved.
The pathophysiology of acute kidney injury (AKI) induced by
cisplatin is complex, and the mechanisms of proximal tubular injury
primarily consist of apoptosis, necrosis, activation of the mitogen-
activated protein (MAP) kinase signaling pathways, and mitochondrial
dysfunction.11–14 Although there are many possible therapeutic targets,
intervention at any single arm typically provides only incomplete pro-
tection. Thus, combinatorial strategies that target multiple mechanisms
of the AKI system, including inhibition of tubular apoptosis or oxidative
stress, stimulation of autophagy and immune suppressive cell
* Corresponding authors at: Convergent Research Center for Dementia, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea (J. Wook Lee);
College of Korean Medicine, Gachon University, Seonngman 13120, Republic of Korea (K. Sung Kang).
E-mail addresses: jwlee5@kist.re.kr (J. Wook Lee), kkang@gachon.ac.kr (K. Sung Kang).
h
These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2021.128256
Received 2 June 2021; Received in revised form 30 June 2021; Accepted 5 July 2021
Available online 10 July 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

D. Lee et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128256
Fig. 1. Chemical structures of Nec-1, Nec-3-A, and Nec-3-B.
Fig. 3. Effects of necrostatins on cisplatin-induced apoptosis in LLC-PK1 cells.
LLC-PK1 cells were exposed to 25 μM cisplatin for 24 h in the presence of Nec-1,
Nec-3-A, or Nec-3-B at concentrations of 50 μM. (A) The representative images
(40 × magnification) are based on Tali-image-based cytometric analyses that
were performed to assess apoptotic cell death. (B) Bars indicate the percentage
of apoptotic cells (mean ± S.E.M., * p < 0.05 compared to cisplatin treated
cells). (C) Western blot analyses were performed to assess cleaved caspase-3.
(D) A bar graph represents densitometric quantification of western blot bands
(mean ± S.E.M., * p < 0.05 compared to cisplatin treated cells).
determined thatnectostatin-1 (Nec-1), a specific and potent small-
molecule inhibitor of necroptosis, functions to block a critical step in
necroptosis. Specifically, Nec-1 inhibits a specific type of non-apoptotic
cell death that occurs in a caspase-independent manner.17 Recently, the
correlation of necroptosis in AKI has also been demonstrated, and the
extensive efficacy of Nec-1 in necroptosis-associated diseases has been
characterized in vitro and in vivo.18 Moreover, necrostatins-3 (Nec-3), a
structurally distinct necrostatin, was demonstrated to exert an inhibitory
effect on necroptosis through mechanisms that are distinct from that of
Nec-1.19 Here, we studied the efficacies of three structurally distinct
necrostatins, Nec-1, Nec-3-A, and Nec-3-B (Fig. 1), in regard to inhib-
iting a critical step in apoptosis in an AKI model induced by cisplatin.
We obtained Nec-1 from chemical vendor. Nec-3-A and Nec-3-B were
synthesized using published procedure.20 The synthesized compounds
were characterized using 1H NMR and mass spectrometry. To evaluate
the renoprotective effects of necrostatins, LLC-PK1 cells were exposed to
Fig. 2. Protective effects of necrostatins on cell viability in the context of
cisplatin-induced nephrotoxicity. LLC-PK1 cells were exposed to 25
μM
cisplatin for 24 h in the presence of Nec-1 (A), Nec-3-A (B), Nec-3-B (C), or NAC
(D) at the indicated respective concentrations. The cell viability was then
determined (mean ± S.E.M., * p < 0.05 compared to cisplatin treated cells).
populations or cytokines, inhibition of MAP kinase pathway, or inter-
vention of mitochondrial activity, result in amelioration of cisplatin-
induced AKI.
25 μM cisplatin for 24 h in the presence of Nec-1, Nec-3-A, or Nec-3-B. As
presented in Fig. 2, cell viability was decreased to approximately 60%
compared to that of the control and cisplatin-treated groups. However,
Nec-1 and Nec-3-B ameliorated cell viability in a dose-dependent
It is well established that various types of cell death, including
apoptosis, necrosis, and necroptosis, occur in response to AKI caused by
nephrotoxic injury.15,16 Over the past few decades, necroptosis that is
characterized by necrotic cell death morphology and activation of
autophagy has been investigated and characterized. Degterev et al.
manner (Fig. 2A and C), where 50 μM Nec-1 and Nec-3-B treatments
resulted in viabilities of 90.5 ± 4.5% and 87.5% ± 2.0%, respectively. In
contrast, a significant increase was observed in the Nec-3-A group at a
2

D. Lee et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128256
high concentration (84.5% ± 2.5% at 50
μM) (Fig. 2B). We also
demonstrated that when N-acetyl cysteine (NAC), a well-known scav-
enger of ROS, was used as a positive control, a dose-dependent increase
in cell viability was observed at concentrations of 50 to 1000 μM as
indicated in Fig. 2D (89.4 ± 2.8% at 1000 μM). In the previous study in
same cell lines, it is demonstrated that pretreatment with 50 μM NAC
protects LLC-PK1 cells exposed to 50
μ
M cisplatin.21 This discrepancy
with the results of previous studies may be due to several potential
factors such as different number of cell passages, different culture me-
dium composition, and pretreatment time of NAC. Although the con-
centrations of NAC used in each previous studies are different, cell-based
and animal models in previous studies indicates that NAC ameliorates
cisplatin-induced apoptosis and necrosis.22–25
Cisplatin nephrotoxicity can present as various types of symptoms
such as AKI, hypomagnesemia, Fanconi-like syndrome, distal renal
tubular acidosis, hypocalcemia, renal salt wasting, and hyperuricemia.26
However, the most serious and one of the more common side effects is
AKI, a condition that occurs in approximately 25–30% of patients.10 The
pathophysiology of cisplatin-induced nephrotoxicity is complex and
involves numerous cellular processes, including oxidative stress,
apoptosis, and inflammation.12 The primary mechanism underlying
cisplatin-induced cancer cell death is apoptosis, the process of pro-
grammed cell death. Apoptosis is typically characterized by distinct
morphological and biochemical changes to the cell that include cell
shrinkage, chromatin condensation, plasma membrane potential alter-
ations, exposure of phosphatidyl serine at the cell surface, and caspase
activation.27 The stimulation of caspases is a critical step during the
early stages of apoptosis. In the present study, we used cytometric
analysis to identify apoptotic cells, and we demonstrated that Nec-1,
Nec-3-A, or Nec-3-B treatment significantly suppressed cisplatin-
induced apoptosis.
As presented in Fig. 3A, the number of dead and apoptotic cells that
are indicated with red and green staining was increased after cisplatin
treatment. However, co-treatment with 50 μM concentrations of Nec-1,
Nec-3-A, or Nec-3-B markedly decreased the number of apoptotic cells as
assessed by a reduction in green staining. Simultaneously, red-stained
dead cells were observed after Nec-1 co-treatment. Additionally, quan-
titatively analyzed data were used to assess the percentage of apoptotic
cells induced by cisplatin treatment (Fig. 3B). Cisplatin-treated LLC-PK1
cells exhibited a markedly increased number of apoptotic cells, and a
significant reduction in the number of apoptotic cells was observed after
co-treatment with Nec-1, Nec-3-A, or Nec-3-B.
In addition, the formation of cleaved caspase-3 was dramatically
increased in cisplatin-induced nephrotoxicity. However, Nec-1, Nec-3-A,
and Nec-3-B equally suppressed the elevated level of cleaved caspase-3
in response to cisplatin treatment was suppressed in the presence of Nec-
3-A and Nec-3-B, and strong suppression of these events was observed in
the presence of Nec-1 (Fig. 3C and D).
Caspases are characterized as either initiators or executioners of the
apoptotic cell death mechanism. For example, an extracellular or
intracellular stimulus causes cleavage of an initiator caspase (i.e.,
caspase-8 that is activated by plasma membrane death receptors and
caspase-9 that is associated with mitochondrial collapse). These stimuli
result in the cleavage of an executioner caspase (i.e., caspase-3 and ꢀ 7),
ultimately resulting in DNA fragmentation, nuclear condensation, and
cell death.28 Although several initiator caspases exist, the major
executioner caspase implicated and measured in cisplatin-induced AKI is
caspase-3. Cleaved caspase-3 occurs in response to activation of one or
more of the three apoptotic pathways that include the extrinsic (death
receptors), intrinsic (mitochondrial), or endoplasmic reticulum-
mediated pathways that are all mediated by a tightly coordinated
intracellular proteolytic cascade.29–31 Our previous analysis suggested
that Nec-1 strongly suppressed the level of cleaved caspase-3 induced by
cisplatin, and Nec-3-A and Nec-3-B also facilitated moderate suppression
of cleaved caspase-3. Therefore, the necrostatin Nec-1 directly affects
apoptotic pathways through the suppression of cleaved caspase-3.
Fig. 4. The effect of Necrostatins on MAP kinase in the context of cisplatin-
induced nephrotoxicity. LLC-PK1 cells were exposed to 25
μM cisplatin for
24 h in the presence of Nec-1, Nec-3-A, of Nec-3-B at concentrations of 50
μM.
(A) Western blot analyses were performed to assess phospho-c-Jun N-terminal
kinase (P-JNK), JNK, phospho-extracellular signal-regulated kinases (P-ERK),
ERK, and phospho-p38 (P-p38), and p38. (B) Each bar graph represents
densitometric quantification of western blot bands (mean ± S.E.M., * p < 0.05
compared to cisplatin treated cells).
˜
Tristao et al. previously reported that renoprotection by Nec-1
against cisplatin-induced AKI in mice is related to non-apoptotic effi-
cacy.18,32 Nevertheless, Ning et al. demonstrated that Nec-1 protects
mice from cisplatin-induced nephrotoxicity by suppressing necroptosis,
3

D. Lee et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128256
apoptosis, inflammation, and oxidative stress at different doses and in-
jection times.33 Briefly, their study examining a murine in vivo model of
myocardial ischemia–reperfusion injury revealed that lower concen-
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