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Citation: An, C.; Gao, L.; Xiang, L.; Qi,
J. IGF-1 and Glucocorticoid Receptors
Are Potential Target Proteins for the
NGF-Mimic Effect of β-Cyclocitral
from Lavandula angustifolia Mill. in
PC12 Cells. Int. J. Mol. Sci. 2024,25,
9763. https://doi.org/10.3390/
ijms25189763
Academic Editor: Alessandro
Castorina
Received: 29 July 2024
Revised: 3 September 2024
Accepted: 8 September 2024
Published: 10 September 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Article
IGF-1 and Glucocorticoid Receptors Are Potential Target Proteins
for the NGF-Mimic Effect of β-Cyclocitral from Lavandula
angustifolia Mill. in PC12 Cells
Chenyue An †, Lijuan Gao †, Lan Xiang * and Jianhua Qi *
College of Pharmaceutical Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058, China;
anchenyue@zju.edu.cn (C.A.); k923146@zju.edu.cn (L.G.)
*Correspondence: lxiang@zju.edu.cn (L.X.); qijianhua@zju.edu.cn (J.Q.)
†These authors contributed equally to this work.
Abstract: In the present study, the PC12 cells as a bioassay system were used to screen the small
molecules with nerve growth factor (NGF)- mimic effect from Lavandula angustifolia Mill. The
β
-Cyclocitral (
β
-cyc) as an active compound was discovered, and its chemical structure was also
determined. Furthermore, we focused on the bioactive and action mechanism of this compound
to do an intensive study with specific protein inhibitors and Western blotting analysis. The
β
-
cyc had novel NGF-mimic and NGF-enhancer effects on PC12 cells, while the insulin-like growth
factor-1 receptor (IGF-1R)/phosphatidylinositol 3 kinase, (PI3K)/serine/threonine-protein kinase
(AKT), and glucocorticoid receptor (GR)/phospholipase C (PLC)/protein kinase C (PKC) signaling
pathways were involved in the bioactivity of
β
-cyc. In addition, the important role of the rat
sarcoma (Ras)/protooncogene serine-threonine protein kinase (Raf) signaling pathway was observed,
although it was independent of tyrosine kinase (Trk) receptors. Moreover, the non-label target protein
discovery techniques, such as the cellular thermal shift assay (CETSA) and drug affinity responsive
target stability (DARTS), were utilized to make predictions of its target protein. The stability of IGF-R
and GR, proteins for temperature and protease, was dose-dependently increased after treatment of
β
-cyc compared with control groups, respectively. These findings indicated that
β
-cyc promoted the
neuron differentiation of PC12 cells via targeting IGF-1R and GR and modification of downstream
signaling pathways.
Keywords: nerve growth factor; Lavender;
β
-Cyclocitral; neurogenesis; IGF-1 receptor; glucocorticoid
receptor
1. Introduction
The rapid aging of the worldwide population is projected to lead to a significant
increase in the percentage of individuals aged 65 and above, from 10% in 2022 to 16% by
2050. This demographic shift is expected to increase the costs associated with dementia
from USD1.3 trillion in 2019 to USD2.8 trillion by 2030 [
1
]. Diseases associated with
aging, including Alzheimer’s disease (AD), cancer, and diabetes, present increasingly
complex challenges. AD, a prominent form of senile dementia, requires early detection,
and recent advancements in blood biomarkers such as plasma p-Tau231 and p-Tau217 have
shown potential for correlating with A
β
deposition and cognitive decline [
2
]. However,
our understanding of the molecular mechanisms of AD, including cholinergic effects,
A
β
amyloid deposition, oxidative stress, Tau protein hyperphosphorylation, and NMDA
receptor signaling, is still incomplete. Currently, the primary treatments available drugs for
AD contain tacrine, rivastigmine, galantamine, donepezil, huperzine A, and memantine [
3
],
and investigational drugs such as Lecanemab [
4
] and Donanemab [
5
] also show promise
in clinical trials. However, existing small-molecule drugs do not effectively halt the onset
Int. J. Mol. Sci. 2024,25, 9763. https://doi.org/10.3390/ijms25189763 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2024,25, 9763 2 of 16
or reverse the progression of AD; the high treatment costs of antibody-based drugs limit
their utilization.
Neurite outgrowth is fundamental to establishing neural circuitry throughout de-
velopment and in the process of regeneration [
6
]. The self-renewal potential of central
and peripheral axons is influenced by a variety of factors, including ribosomal location,
proteomic profile, microtubule stability, and signaling pathways. Following nerve injury,
molecular pathways such as MAPK, AKT/mTORC1/p70S6K, PI3K/AKT, BDNF/Trk,
and Ras/ERK are activated and play a crucial role in axonal regeneration. Furthermore,
astrocytic modulation, growth factors, and microRNAs contribute to this regenerative
process [
7
]. In particular, neurotrophins, such as nerve growth factor (NGF) and brain-
derived neurotrophic factor (BDNF), are crucial for neurite outgrowth and survival. It
is imperative to understand the regulation of neurite growth, as the progressive loss of
neuronal connectivity is a hallmark of AD pathology. Consequently, the study of neurite
growth regulation has become a focal point in the field of AD research.
Nerve growth factor (NGF) plays a pivotal role in cognitive function, supporting the
survival, development, and continual maintenance of neurons. Due to its significance in
the development of AD through its interaction with the A
β
production mechanism [
8
]
and cholinergic system [
9
], NGF has emerged as a potential target for drug development.
However, the strong polarity and substantial molecular weight of NGF hinder its ability
to traverse the blood-brain barrier [
10
]. As a result, current research is concentrating on
strategies to deliver NGF into the brain. One highly studied approach involves the use of
adeno-associated virus vectors expressing human NGF, specifically CERE-110. Although
these vectors have been extensively investigated, further validation is required to determine
their efficacy [
11
]. This limitation necessitates the exploration of additional small molecule
modulators of NGF and its receptors, which can mimic or enhance neurotrophic effects.
LM11A-31, a small molecule agonist that targets the p75 receptor of NGF, has been shown
to reduce microglial activation in mouse models of AD and is currently undergoing clinical
trials [
12
]. Furthermore, several other small molecules with NGF-mimic properties have
been discovered by our group using traditional Chinese medicines such as AMA, Lindersin
B, and Cucurbitacin B [
13
–
15
]. These discoveries were made through the utilization of the
pheochromocytoma cells (PC12) bioassay system, a well-established model for studying
neuronal differentiation and the molecular mechanisms of NGF action [16].
To discover the small molecules with NGF-mimic effect from natural products, the
large screening for natural products was first performed in the early research. The extract
of Lavandula angustifolia Mill, which is well known for antioxidant, anti-inflammatory,
antibacterial, hypnotic, and memory-enhancing properties [
17
], was found to have a novel
NGF-mimic effect on PC12 cells. To understand the active components of Lavandula
angustifolia Mill, the isolation and purification of the extract of this plant was performed
under the guide of a PC12 cell as a bioassay system. The
β
-cyclocitral (
β
-cyc), as its main
component, was discovered and determined the chemical structure. This compound has
been indicated that it has antifungal, antibacterial, and pesticidal properties [
18
]. However,
there is currently scarce research on the impact of
β
-cyc on the ability to promote neurite
outgrowth. In the present study,
β
-cyc demonstrated excellent NGF-mimic and NGF-
enhancing properties in a PC12 cell bioassay system. To fully understand the underlying
mechanisms of action and identify target proteins, critical steps must be taken to consider
β-cyc as a potential candidate for further research and development.
Growth factors, such as NGF and insulin-like growth factor-1 (IGF-1), play a posi-
tive role in neurogenesis in the hippocampus [
19
]. IGF-1, in particular, plays a key role
in regulating cognitive function. Upon binding with IGF-1, the IGF-1 receptor (IGF-1R)
triggers the phosphorylation of its tyrosine kinase domain, initiating intracellular signal-
ing that modulates cell growth, differentiation, and the various life activities of higher
organisms [
19
]. Disruption of the PI3K/AKT/mTOR pathway in the brains of AD pa-
tients, which is associated with disease severity [
20
,
21
], underscores the importance of
balancing IGF-1R/PI3K/AKT for AD treatment. Glucocorticoids have also been found to
Int. J. Mol. Sci. 2024,25, 9763 3 of 16
be involved in cellular proliferation, neurotransmitter synthesis, neuronal survival, and
neuronal differentiation. These actions are closely linked to neurite outgrowth in PC12
cells [
22
]. Additionally, glucocorticoid receptors (GR) have been implicated in stress re-
sponse and central nervous system functions, including learning and memory, suggesting
their role in the neurogenic effects of antidepressants [
16
]. This evidence also indicated
that TrkA/RAS/Raf/MERK, Insulin R or IGF-1R/PI3K/AKT/ERK and GR/PLC/PKC
signaling pathways took important roles in neurite growth of PC12 cells.
In this study, we focused on the mechanism of action for neurite growth to elucidate the
mechanism underlying
β
-cyc-induced neurite growth through the use of specific protein
inhibitors and Western blotting. Furthermore, potential targets were predicted using
specific inhibitor experiments, such as the Cellular Thermal Shift Assay (CETSA) and Drug
Affinity Responsive Target Stability (DARTS). These findings implied that the dual-targeted
impacts of
β
-cyc may involve IGF-1R and GR, ultimately leading to the activation of the
PI3K/AKT and PLC/PKC signaling pathways.
2. Results
2.1. β-cyc Exhibits NGF-Mimic and NGF-Enhancing Effects on PC12 Cells
Initially, we observed the neurotrophic activity of
β
-cyc in PC12 cells. The cells were
treated with various concentrations of
β
-cyc (1, 3, or 10
µ
M) for 48 h, with DMSO serving as
the negative control and NGF (40 ng/mL) acting as the positive control. The results showed
that treatment with
β
-cyc significantly promoted neurite outgrowth. The percentages
of PC12 cells with neurite outgrowth induced by
β
-cyc were 6.0%
±
1%,
42.3% ±1.4%
,
56.3% ±1.6%
, and 40.7%
±
1.15% at concentrations of 0, 1, 3, and 10
µ
M (p< 0.001,
Figure 1B), respectively. Additionally, NGF-enhancing experiments demonstrated that
treatment with 3
µ
M
β
-cyc significantly enhanced the percentage of PC12 cells with positive
neurite outgrowth from 56.3%
±
1.6% to 86.3%
±
1.3% (p< 0.001, Figure 1B), when these
cells were co-treated with low-dose NGF (1 ng/mL). The changes in cell morphology treated
with 3
µ
M
β
-cyc alone or its combination with 1 ng/mL NGF were shown in Figure 1C.
These results indicated that
β
-cyc exhibited both NGF-mimic and NGF-enhancing effects
in PC12 cells.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 of 17
Figure 1. Effects of β-cyc on PC12 cells. (A) The structure of β-cyc. (B) Neurite outgrowth percent-
ages were treated with varying doses of β-cyc alone or in combination with 1 ng/mL of NGF. (C)
The morphological alterations after treatment for 48 h were observed by an inverted optical micro-
scope. (D) Cell viability after exposure to different doses of β-cyc or β-cyc co-treated with 1 ng/mL
of NGF. Each experiment was repeated three times. The data were expressed as a mean ± SEM. * p
< 0.05, ** p < 0.01, and *** p < 0.001, compared with the negative control;
#
p < 0.05 and
###
p < 0.001,
compared with the 3 µM β-cyc group.
2.2. Ras/Raf/MEK/ERK Signaling Pathway Involves in NGF-Mimic and NGF–Enhancing Effect
of β-cyc
Various neurotrophic factors, including NGF and BDNF, selectively interacted with
the tyrosine kinase receptors TrkA and TrkB, activating multiple kinases to promote neu-
ronal differentiation and survival [23,24]. Due to the NGF-mimic activity of β-cyc, it was
hypothesized that β-cyc-induced neurite outgrowth may target the common receptor of
NGF. Consequently, TrkA and TrkB inhibitors were initially used to block the effects of
β-cyc. However, treatment with K252a and ANA-12, inhibitors of TrkA and TrkB, respec-
tively, did not markedly alter the induction of neurite outgrowth induced by β-cyc alone.
This suggested that the involvement of Trks in the neurogenic effect of β-cyc was limited
in PC12 cells (Figure 2A,B). However, the NGF-enhancing activity of β-cyc was signifi-
cantly inhibited by K252a and ANA-12, leading to notable differences between the β-cyc
and β-cyc + NGF 1 ng/mL groups following treatment with K252a and ANA-12 (p < 0.01
and p < 0.05, respectively). These findings prompted us to speculate that K252a and ANA-
12 effectively suppressed the NGF-enhancing activity of β-cyc by inhibiting low-dose
NGF.
NGF initially stimulates TrkA and TrkB, which then induce differentiation in PC12
cells by arresting proliferation and promoting neurite outgrowth through the transient
activation of Ras and Rap1-dependent ERK phosphorylation [25]. Specific inhibitors were
further utilized to investigate the regulation of these signaling pathways. As depicted in
Figure 2C–E, the addition of inhibitors of Ras, Raf, and ERK (FTA, AZ628 and U0126)
significantly reduced the NGF-mimic effects induced by β-cyc, decreasing the percentage
of neurite outgrowth from 51.5% ± 1.7% to 23.3% ± 2.5% (p < 0.01), 17.3% ± 1.0% (p < 0.001),
Figure 1. Effects of
β
-cyc on PC12 cells. (A) The structure of
β
-cyc. (B) Neurite outgrowth percentages
were treated with varying doses of
β
-cyc alone or in combination with 1 ng/mL of NGF. (C) The
morphological alterations after treatment for 48 h were observed by an inverted optical microscope.
(D) Cell viability after exposure to different doses of
β
-cyc or
β
-cyc co-treated with 1 ng/mL of NGF.
Each experiment was repeated three times. The data were expressed as a mean
±
SEM.
*p< 0.05
,
** p< 0.01
, and *** p< 0.001, compared with the negative control;
#
p< 0.05 and
###
p< 0.001, compared
with the 3 µMβ-cyc group.
Int. J. Mol. Sci. 2024,25, 9763 4 of 16
To evaluate whether the neuritogenic activities were not associated with the toxicity
of
β
-cyc, cell viability was evaluated in PC12 cells using the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) bioassay. After treating the cells with
β
-cyc at
doses of 0.03, 0.3, 3, and 10
µ
M, the cell viability was determined to be 106.04%
±
3.2%,
102.0%
±
3.4%, 125.3%
±
5.2%, and 120.3%
±
3.9%, respectively (Figure 1D). Additionally,
when low-dose NGF was added to the group treated with 3
µ
M
β
-cyc, the cell viability
increased significantly to 155.9%
±
8.1% (p< 0.001, Figure 1D). These results suggested
that
β
-cyc alone did not exhibit cytotoxic effects, even at concentrations up to 10
µ
M.
Moreover, the combination of 3
µ
M of
β
-cyc with 1 ng/mL of NGF at a low dose enhanced
the viability of PC12 cells, similar to the effects of NGF at a dose of 40 ng/mL. Overall, the
MTT bioassay results indicated that the tested concentrations of
β
-cyc did not demonstrate
significant cytotoxicity.
2.2. Ras/Raf/MEK/ERK Signaling Pathway Involves in NGF-Mimic and NGF–Enhancing Effect
of β-cyc
Various neurotrophic factors, including NGF and BDNF, selectively interacted with the
tyrosine kinase receptors TrkA and TrkB, activating multiple kinases to promote neuronal
differentiation and survival [
23
,
24
]. Due to the NGF-mimic activity of
β
-cyc, it was hy-
pothesized that
β
-cyc-induced neurite outgrowth may target the common receptor of NGF.
Consequently, TrkA and TrkB inhibitors were initially used to block the effects of
β
-cyc.
However, treatment with K252a and ANA-12, inhibitors of TrkA and TrkB, respectively,
did not markedly alter the induction of neurite outgrowth induced by
β
-cyc alone. This
suggested that the involvement of Trks in the neurogenic effect of
β
-cyc was limited in
PC12 cells (Figure 2A,B). However, the NGF-enhancing activity of
β
-cyc was significantly
inhibited by K252a and ANA-12, leading to notable differences between the
β
-cyc and
β
-cyc + NGF 1 ng/mL groups following treatment with K252a and ANA-12 (p< 0.01 and
p< 0.05, respectively). These findings prompted us to speculate that K252a and ANA-12
effectively suppressed the NGF-enhancing activity of β-cyc by inhibiting low-dose NGF.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 5 of 17
and 25.3% ± 1.8% (p < 0.01), respectively. Similarly, these inhibitors decreased the enhanc-
ing effects of β-cyc combined with low-dose NGF, reducing the percentage from 78.7% ±
3.3% to 33.7% ± 1.6% (p < 0.001), 31.3% ± 2.1% (p < 0.001), and 45.3% ± 2.5% (p < 0.01),
respectively. In conclusion, these findings suggested that the Ras/Raf signaling pathway
may contribute to the neurogenic effect of β-cyc in PC12 cells, albeit independently of the
Trk receptors.
Figure 2. β-cyc modulation of the Ras/Raf signaling pathway in PC12 cells. (A–E) Effects of inhibi-
tors of TrkA, TrkB, Ras, Raf, ERK (K252a, ANA-12, FTA, AZ628, U0126, respectively) on the neurite
outgrowth induced by β-cyc, its combination with NGF, and NGF 1 ng/mL. Each experiment was
repeated three times. The data were expressed as a mean ± SEM. *** p < 0.001, compared with the
negative control; ## p < 0.01 and ### p < 0.001, compared with the 3 µM β-cyc group; $$ p < 0.01 and $$$
p < 0.001, compared with the combination group of β-cyc with low-dose NGF; & p < 0.05 and && p <
0.01, ns means no significant difference.
2.3. IGF-1R/PI3K/AKT Signaling Pathway Takes an Important Role in NGF-Mimic Effect of
β-cyc
Notably, NGF and BDNF also activate the PI3K/AKT/mTOR pathway [20]. Numer-
ous studies have shown that phosphorylation of the IGF-1R receptor can initiate down-
stream changes in metabolic pathways, potentially leading to signal transduction cascades
that regulate various cellular functions. These functions include promoting cell survival
and neurite outgrowth via the PI3K/AKT signaling pathway [26]. Therefore, our objective
was to investigate the potential involvement of this signaling pathway in the neuropro-
tective and neurite-promoting effects induced by β-cyc.
To explore the potential mechanisms of action of β-cyc, we employed the IGF-1R in-
hibitor T9576 to disrupt the activity induced by β-cyc. After treatment with T9576, the
results demonstrated a substantial reduction in neurite outgrowth from 46.0% ± 1.0% to
4.6% ± 0.3% (p < 0.001) attributed to β-cyc (Figure 3A). Furthermore, treatment with
LY294002, a PI3K/AKT inhibitor, notably reduced β-cyc-induced neurite growth from
44.3% ± 0.8% to 13.0% ± 1.3% (p < 0.001) (Figure 3B). Notably, both T9576 and LY294002
effectively inhibited neurite outgrowth induced by the combination of β-cyc and low-dose
NGF in PC12 cells.
Figure 2.
β
-cyc modulation of the Ras/Raf signaling pathway in PC12 cells. (A–E) Effects of
inhibitors of TrkA, TrkB, Ras, Raf, ERK (K252a, ANA-12, FTA, AZ628, U0126, respectively) on the
neurite outgrowth induced by
β
-cyc, its combination with NGF, and NGF 1 ng/mL. Each experiment
was repeated three times. The data were expressed as a mean
±
SEM. *** p< 0.001, compared with
the negative control;
##
p< 0.01 and
###
p< 0.001, compared with the 3
µ
M
β
-cyc group;
$$
p< 0.01
and
$$$
p< 0.001, compared with the combination group of
β
-cyc with low-dose NGF;
&
p< 0.05 and
&& p< 0.01, ns means no significant difference.
Int. J. Mol. Sci. 2024,25, 9763 5 of 16
NGF initially stimulates TrkA and TrkB, which then induce differentiation in PC12
cells by arresting proliferation and promoting neurite outgrowth through the transient
activation of Ras and Rap1-dependent ERK phosphorylation [
25
]. Specific inhibitors were
further utilized to investigate the regulation of these signaling pathways. As depicted
in
Figure 2C–E
, the addition of inhibitors of Ras, Raf, and ERK (FTA, AZ628 and U0126)
significantly reduced the NGF-mimic effects induced by
β
-cyc, decreasing the percentage of
neurite outgrowth from 51.5%
±
1.7% to 23.3%
±
2.5% (p< 0.01), 17.3%
±
1.0% (
p< 0.001
),
and 25.3%
±
1.8% (p< 0.01), respectively. Similarly, these inhibitors decreased the enhancing
effects of
β
-cyc combined with low-dose NGF, reducing the percentage from
78.7% ±3.3%
to 33.7%
±
1.6% (p< 0.001), 31.3%
±
2.1% (p< 0.001), and 45.3%
±
2.5% (p< 0.01),
respectively. In conclusion, these findings suggested that the Ras/Raf signaling pathway
may contribute to the neurogenic effect of
β
-cyc in PC12 cells, albeit independently of the
Trk receptors.
2.3. IGF-1R/PI3K/AKT Signaling Pathway Takes an Important Role in NGF-Mimic Effect of
β
-cyc
Notably, NGF and BDNF also activate the PI3K/AKT/mTOR pathway [
20
]. Numerous
studies have shown that phosphorylation of the IGF-1R receptor can initiate downstream
changes in metabolic pathways, potentially leading to signal transduction cascades that
regulate various cellular functions. These functions include promoting cell survival and
neurite outgrowth via the PI3K/AKT signaling pathway [
26
]. Therefore, our objective was
to investigate the potential involvement of this signaling pathway in the neuroprotective
and neurite-promoting effects induced by β-cyc.
To explore the potential mechanisms of action of
β
-cyc, we employed the IGF-1R
inhibitor T9576 to disrupt the activity induced by
β
-cyc. After treatment with T9576, the
results demonstrated a substantial reduction in neurite outgrowth from 46.0%
±
1.0%
to 4.6%
±
0.3% (p< 0.001) attributed to
β
-cyc (Figure 3A). Furthermore, treatment with
LY294002, a PI3K/AKT inhibitor, notably reduced
β
-cyc-induced neurite growth from
44.3%
±
0.8% to 13.0%
±
1.3% (p< 0.001) (Figure 3B). Notably, both T9576 and LY294002
effectively inhibited neurite outgrowth induced by the combination of
β
-cyc and low-dose
NGF in PC12 cells.
To further confirm the protein expression levels of members of this signaling pathway,
we conducted a Western blot analysis. We examined the dose-dependent induction of
IGF-1R phosphorylation by
β
-cyc and observed the optimal peak phosphorylation at a
concentration of 3
µ
M (Figures 3C and S2). Subsequently, we evaluated the time-dependent
phosphorylation of IGF-1R, PI3K, and AKT after
β
-cyc treatment. The results revealed an
increase in the levels of phosphorylated IGF-1R induced by
β
-cyc after 10 min, reaching a
peak at 1 h. Similarly, phosphorylated PI3K levels escalated by
β
-cyc after 10 min, reaching
a peak at 2 h. Additionally, the phosphorylation of AKT increased at 5 min and peaked
at 4 h after
β
-cyc treatment. (Figures 3D and S2). T9576 significantly reduced the levels of
IGF-1R and the downstream proteins PI3K and AKT phosphorylation induced by
β
-cyc
in the presence or absence of 1 ng/mL NGF (Figures 4A and S3). Moreover, LY294002
diminished the phosphorylation of PI3K and AKT induced by
β
-cyc, both alone and in
combination with low-dose NGF (Figures 4B and S3). These findings indicated a close
association between the IGF-1R/PI3K/AKT signaling pathway and the neurite outgrowth
induced by β-cyc in PC12 cells.
Int. J. Mol. Sci. 2024,25, 9763 6 of 16
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6 of 17
Figure 3. The IGF-1R signaling pathway plays a primary role in the neurogenesis effect of β-cyc.
(A,B) Effects of the IGF-1R inhibitor T9576 and PI3K/AKT inhibitor LY294002 on the neurite out-
growth elicited by β-cyc, its combination with low-dose NGF, and NGF 1 ng/mL. (C) Western blot
analysis and digitalization of IGF-1R and p-IGF-1R levels induced by β-cyc dose-dependently. (D)
Western blot analysis and digitalized results of p-IGF-1R, IGF-1R, p-PI3K, PI3K, p-AKT, and AKT
induced by β-cyc in a time-dependent experiment. Each experiment was repeated three times. The
data were expressed as a mean ± SEM. * p< 0.05, ** p< 0.01, *** p < 0.001, compared with the negative
control; ### p < 0.001, compared with the 3 µM β-cyc group; $$$ p < 0.001, compared with the combi-
nation group of β-cyc with low-dose NGF; && p < 0.01, compared with the low-dose NGF, ns indicates
no significant difference, compared with the low-dose NGF.
To further confirm the protein expression levels of members of this signaling path-
way, we conducted a Western blot analysis. We examined the dose-dependent induction
of IGF-1R phosphorylation by β-cyc and observed the optimal peak phosphorylation at a
concentration of 3 µM (Figures 3C and S2). Subsequently, we evaluated the time-depend-
ent phosphorylation of IGF-1R, PI3K, and AKT after β-cyc treatment. The results revealed
an increase in the levels of phosphorylated IGF-1R induced by β-cyc after 10 min, reaching
a peak at 1 h. Similarly, phosphorylated PI3K levels escalated by β-cyc after 10 min, reach-
ing a peak at 2 h. Additionally, the phosphorylation of AKT increased at 5 min and peaked
at 4 h after β-cyc treatment. (Figures 3D and S2). T9576 significantly reduced the levels of
IGF-1R and the downstream proteins PI3K and AKT phosphorylation induced by β-cyc
in the presence or absence of 1 ng/mL NGF (Figures 4A and S3). Moreover, LY294002
diminished the phosphorylation of PI3K and AKT induced by β-cyc, both alone and in
combination with low-dose NGF (Figures 4B and S3). These findings indicated a close
association between the IGF-1R/PI3K/AKT signaling pathway and the neurite outgrowth
induced by β-cyc in PC12 cells.
Figure 3. The IGF-1R signaling pathway plays a primary role in the neurogenesis effect of
β
-
cyc. (A,B) Effects of the IGF-1R inhibitor T9576 and PI3K/AKT inhibitor LY294002 on the neurite
outgrowth elicited by
β
-cyc, its combination with low-dose NGF, and NGF 1 ng/mL. (C) Western
blot analysis and digitalization of IGF-1R and p-IGF-1R levels induced by
β
-cyc dose-dependently.
(D) Western blot analysis and digitalized results of p-IGF-1R, IGF-1R, p-PI3K, PI3K, p-AKT, and
AKT induced by
β
-cyc in a time-dependent experiment. Each experiment was repeated three times.
The data were expressed as a mean
±
SEM. * p< 0.05, ** p< 0.01, *** p< 0.001, compared with the
negative control;
###
p< 0.001, compared with the 3
µ
M
β
-cyc group;
$$$
p< 0.001, compared with the
combination group of
β
-cyc with low-dose NGF;
&&
p< 0.01, compared with the low-dose NGF, ns
indicates no significant difference, compared with the low-dose NGF.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 of 17
Figure 4. Related inhibitors on the modulation of the IGF-1R/PI3K/AKT pathway induced by β-cyc
in PC12 cells. (A) The phosphorylation of the IGF-1R, PI3K, and AKT induced by β-cyc or its com-
bination with low-dose NGF were downregulated by T9576. (B) The phosphorylation of PI3K and
AKT stimulated by β-cyc or its combination with low-dose NGF was reduced by LY294002. Each
experiment was repeated three times. The data were expressed as a mean ± SEM. * p < 0.05, ** p <
0.01 and *** p < 0.001, compared with the negative control; ### p < 0.001, compared with the 3 µM β-
cyc group; $ p < 0.05 and $$$ p < 0.001, compared with the combination group of β-cyc with low-dose
NGF.
2.4. GR/PLC/PKC Signaling Pathway Participates in the NGF-Mimic Effect of β-cyc
The participation of GR in the neurogenic activity of PC12 cells and its regulation of
a suite of genes crucial for neuronal structural control has been demonstrated [27]. There-
fore, the potential mechanism of action of β-cyc was further elucidated by using the GR
inhibitor RU486. Following pretreatment with RU486, the cell proportion with neurite
outgrowth decreased significantly after 48 h of drug exposure, with a decrease from 50.0%
± 1.5% to 19.3% ± 2.0% (p < 0.001) (Figure 5A).
Considering the pivotal role of PLC/PKC pathway, which is downstream of GR pro-
tein, plays in cell survival and differentiation as documented in reference [28]. This inves-
tigation employed the inhibitors of PLC and PKC (U73343 and Go6983) to explore the
neurite-promoting effects of β-cyc. The results demonstrated that neurite outgrowth in-
duced by β-cyc significantly decreased following the addition of U73343 or Go6983, re-
sulting in percentages of 20.0% ± 1.0% and 30.0% ± 1.0% (p < 0.001), respectively, compared
to the initial 50.0% ± 1.5% (Figure 5B,C). This finding suggested a close association be-
tween the activation of the PLC/PKC pathway and the neurogenic response of PC12 cells
upon stimulation by β-cyc.
The phosphorylation levels of GR, PLC, and PKC were measured and analyzed at
the protein level via Western blotting. The results indicated that the highest ratio of phos-
phorylated GR to total GR protein was achieved following treatment with 1 µM β-cyc
(Figures 5D and S4). Subsequently, the time-dependent phosphorylation of GR induced
by β-cyc at 1 µM was investigated, revealing that GR phosphorylation peaked at 30 min
(Figures 5E and S4). Following pretreatment with RU486 and subsequent β-cyc treatment,
a significant reduction in the phosphorylation of GR and its downstream proteins PLC
and PKC was observed (Figures 5F and S4). Moreover, the phosphorylation of PLC and
Figure 4. Related inhibitors on the modulation of the IGF-1R/PI3K/AKT pathway induced by
β
-cyc in PC12 cells. (A) The phosphorylation of the IGF-1R, PI3K, and AKT induced by
β
-cyc or
its combination with low-dose NGF were downregulated by T9576. (B) The phosphorylation of
PI3K and AKT stimulated by
β
-cyc or its combination with low-dose NGF was reduced by LY294002.
Each experiment was repeated three times. The data were expressed as a mean
±
SEM. * p< 0.05,
** p< 0.01 and *** p< 0.001, compared with the negative control;
###
p< 0.001, compared with the
3
µ
M
β
-cyc group;
$
p< 0.05 and
$$$
p< 0.001, compared with the combination group of
β
-cyc with
low-dose NGF.
Int. J. Mol. Sci. 2024,25, 9763 7 of 16
2.4. GR/PLC/PKC Signaling Pathway Participates in the NGF-Mimic Effect of β-cyc
The participation of GR in the neurogenic activity of PC12 cells and its regulation of a
suite of genes crucial for neuronal structural control has been demonstrated [
27
]. Therefore,
the potential mechanism of action of
β
-cyc was further elucidated by using the GR inhibitor
RU486. Following pretreatment with RU486, the cell proportion with neurite outgrowth
decreased significantly after 48 h of drug exposure, with a decrease from 50.0%
±
1.5% to
19.3% ±2.0% (p< 0.001) (Figure 5A).
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 of 17
PKC, triggered by β-cyc, was attenuated by U73343. Concurrently, Go6983 led to a reduc-
tion in PKC phosphorylation induced by β-cyc (Figures 5G,H and S4). Overall, these re-
sults robustly implied the participation of the GR/PLC/PKC pathway in the facilitation of
neurite outgrowth within PC12 cells mediated by β-cyc.
Figure 5. Modulation of the GR/PLC/PKC pathway by
β
-cyc in PC12 cells. (A–C) Effects of inhibitors
of GR, PLC, and PKC (RU486, U73343, and Go6983) on the neurite outgrowth induced by
β
-cyc,
its combination with low-dose NGF, and NGF 1 ng/mL. (D,E) Western blotting and digitalized
result of p-GR, GR in dose-dependent (D) and time-dependent (E) experiments induced by
β
-cyc.
(F) The induction of phosphorylation in GR, PLC, and PKC by
β
-cyc and RU486. (G) The induction
of phosphorylation in PLC and PKC by
β
-cyc and U73343. (H) Phosphorylation of PKC by
β
-cyc
and Go6983. Each experiment was repeated three times. The data were expressed as a mean
±
SEM.
*p< 0.05
, ** p< 0.01 and *** p< 0.001, compared with the negative control;
#
p< 0.05,
##
p< 0.01 and
###
p< 0.001, compared with the 3
µ
M
β
-cyc group;
$
p< 0.05 and
$$
p< 0.01, compared with the
combination group of
β
-cyc with low-dose NGF;
&
p< 0.05, compared with the low-dose NGF, ns
indicates no significant difference, compared with the low-dose NGF.
Int. J. Mol. Sci. 2024,25, 9763 8 of 16
Considering the pivotal role of PLC/PKC pathway, which is downstream of GR
protein, plays in cell survival and differentiation as documented in reference [
28
]. This
investigation employed the inhibitors of PLC and PKC (U73343 and Go6983) to explore
the neurite-promoting effects of
β
-cyc. The results demonstrated that neurite outgrowth
induced by
β
-cyc significantly decreased following the addition of U73343 or Go6983,
resulting in percentages of 20.0%
±
1.0% and 30.0%
±
1.0% (p< 0.001), respectively, com-
pared to the initial 50.0%
±
1.5% (Figure 5B,C). This finding suggested a close association
between the activation of the PLC/PKC pathway and the neurogenic response of PC12
cells upon stimulation by β-cyc.
The phosphorylation levels of GR, PLC, and PKC were measured and analyzed at
the protein level via Western blotting. The results indicated that the highest ratio of
phosphorylated GR to total GR protein was achieved following treatment with 1
µ
M
β
-cyc
(Figures 5D and S4). Subsequently, the time-dependent phosphorylation of GR induced
by
β
-cyc at 1
µ
M was investigated, revealing that GR phosphorylation peaked at 30 min
(Figures 5E and S4). Following pretreatment with RU486 and subsequent
β
-cyc treatment,
a significant reduction in the phosphorylation of GR and its downstream proteins PLC and
PKC was observed (Figures 5F and S4). Moreover, the phosphorylation of PLC and PKC,
triggered by
β
-cyc, was attenuated by U73343. Concurrently, Go6983 led to a reduction
in PKC phosphorylation induced by
β
-cyc (Figures 5G,H and S4). Overall, these results
robustly implied the participation of the GR/PLC/PKC pathway in the facilitation of
neurite outgrowth within PC12 cells mediated by β-cyc.
2.5. β-cyc Increases the Thermal Stable Ability of IGF-1R in CETSA
Previous inhibitor experiments revealed that ANA-12 did not significantly inhibit
β
-cyc, suggesting that TrkB is not a priority for targeting
β
-cyc activity. In contrast, T9576
and RU486, particularly T9576, exhibited noticeable inhibitory effects on the neurogenic
action of
β
-cyc. This led us to hypothesize that the IGF-1R or GR proteins may serve as
potential targets for β-cyc.
Consequently, the binding between IGF-1R and
β
-cyc was further confirmed to the
potential targets of
β
-cyc using a CETSA. Briefly, cells were treated with either DMSO or
β
-cyc, heated from 37
◦
C to 80
◦
C, and then analyzed through immunoblotting using a
specific antibody against IGF-1R. The results showed that the IGF-1R protein exhibited a
significant shift in thermal stability following
β
-cyc treatment, which was characterized by a
substantial increase in thermal stability. However, in the control group, protein degradation
occurred as the temperature increased (Figures 6A,C and S5).
Consistent with our hypothesis, GR proteins also experienced a shift in thermal
stability after
β
-cyc treatment according to the CETSA (Figures 6B,C and S5). These findings
partially validate that IGF-1R and GR may indeed be potential dual target proteins of
β
-cyc.
Based on these findings, a modified CETSA approach was employed, involving keeping the
temperature fixed while varying the concentrations of the drug to assess the stability of the
drug-protein interactions. For the IGF-1R protein, the concentrations of
β
-cyc-treated cells
ranged from 0.1
µ
M to 10
µ
M at the optimal temperature of 60
◦
C. Western blot analysis
indicated an increase in the stability of the interaction between
β
-cyc and the IGF-1R
protein with increasing concentrations of
β
-cyc, demonstrating a clear dose dependency
(Figures 6D,F and S5). Similarly, the stability of the GR proteins increased with various
concentrations of
β
-cyc from 0.01
µ
M to 10
µ
M, indicating a marked dose dependency
(Figures 6E,F and S5). These findings collectively further confirmed the dual-target effects
of IGF-1R and GR as potential target proteins of β-cyc.
Int. J. Mol. Sci. 2024,25, 9763 9 of 16
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 10 of 17
Figure 6. Target prediction of β-cyc by CETSA. (A,B) The interaction between β-cyc and IGF-1R and
GR protein was studied using CETSA. β-cyc at 3 and 1 µM were assessed to investigate the expres-
sion changes in IGF-1R and GR protein at varying temperatures by Western blotting. (C) Quantifi-
cation of the data for panels (A,B). (D,E) The modified CETSA method was utilized to explore the
interaction between β-cyc and the IGF-1R protein/GR protein following alterations in the concentra-
tion of β-cyc. (F) Quantitative analysis for panels D and E. Each experiment was repeated three
times. The data were expressed as a mean ± SEM. *** p < 0.001, compared with the negative control.
2.6. β-cyc Enhances the Stability of IGF-1R and GR Proteins for Pronase E During DARTS
Analysis
Based on previous findings, initial validation focused on the IGF-1R protein using
DARTS assays. The protein levels of IGF-1R in the group exposed to 3 µM β-cyc for 3 h
were significantly greater than those in the untreated group when the concentration of
pronase E was 0.2% (p < 0.001) (Figures 7A,C and S6). Importantly, the levels of IGF-1R in
the group exposed to various concentrations of β-cyc continued to increase and remained
higher than those in the untreated group under identical conditions (p < 0.001) (Figures
7C,D and S6). These results indicated that the effect was dose-dependent as the drug con-
centration increased.
Furthermore, compared with those of GR, the protein stability of GR increased in a
concentration-dependent manner following β-cyc treatment, although the dose depend-
ency and increase in IGF-1R levels were more pronounced (Figures 7B,E,F and S6). Ulti-
mately, these findings provided further support for the notion that IGF-1R and GR are
potential target proteins of β-cyc.
Figure 6. Target prediction of
β
-cyc by CETSA. (A,B) The interaction between
β
-cyc and IGF-1R and
GR protein was studied using CETSA.
β
-cyc at 3 and 1
µ
M were assessed to investigate the expression
changes in IGF-1R and GR protein at varying temperatures by Western blotting. (C) Quantification of
the data for panels (A,B). (D,E) The modified CETSA method was utilized to explore the interaction
between
β
-cyc and the IGF-1R protein/GR protein following alterations in the concentration of
β
-cyc.
(F) Quantitative analysis for panels D and E. Each experiment was repeated three times. The data
were expressed as a mean ±SEM. *** p< 0.001, compared with the negative control.
2.6. β-cyc Enhances the Stability of IGF-1R and GR Proteins for Pronase E During
DARTS Analysis
Based on previous findings, initial validation focused on the IGF-1R protein using
DARTS assays. The protein levels of IGF-1R in the group exposed to 3
µ
M
β
-cyc for 3 h
were significantly greater than those in the untreated group when the concentration of
pronase E was 0.2% (p< 0.001) (Figures 7A,C and S6). Importantly, the levels of IGF-1R in
the group exposed to various concentrations of β-cyc continued to increase and remained
higher than those in the untreated group under identical conditions (p< 0.001) (Figures
7C,D and S6). These results indicated that the effect was dose-dependent as the drug
concentration increased.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 11 of 17
Figure 7. Target prediction of β-cyc by DARTS assay. (A) Western blot analysis of IGF-1R and (B)
GR protein level after adding different concentrations of pronase E. (C) Quantification of the data
obtained from panels (A,B). (D) Protein level of IGF-1R after adding different concentrations of β-
cyc (0.1, 1, 3, 10 µM) by Western blotting. IGF-1R was normalized with β-actin. (E) Western blot
analysis of GR protein level after adding different concentrations of β-cyc (0.01, 0.1, 1, 10 µM). GR
was normalized with β-actin. (F) Quantification of the data obtained from panels D-E. Each experi-
ment was conducted three times.
$
p < 0.05,
$$
p < 0.01 and
$$$
p < 0.001 in comparison to the untreated
group; *** p < 0.001 compared to the negative control without pronase E;
#
p < 0.05 and
###
p < 0.001
compared with the negative control with pronase E.
3. Discussion
Recent failures in clinical trials investigating amyloid-beta clearance have generated
interest in alternative therapeutic strategies for AD. Research suggests that Aβ plaques
may have a protective role in the early stages of AD, although their correlation with dis-
ease severity appears to be weak. Treatments focused on Aβ plaques, such as the γ-secre-
tase inhibitor semagacestat and the β-secretase inhibitor verubecestat, have shown incon-
clusive efficacy [8]. However, further investigations are needed to determine the effective-
ness of other AD drugs, such as aducanumab and GV-971. Therefore, the development of
clinical drugs for AD requires the exploration of diverse mechanisms and targets. In par-
ticular, neurogenesis has become the new strategy for the treatment of AD.
Lavender essential oil, demonstrating neuroprotective effects and promising cogni-
tive improvement, has the anti-AD potential supported by both dementia model rats and
clinical studies [29–32]. However, the material basis for producing anti-AD effects is still
unclear, and the mechanism of activity still needs to be elucidated. Therefore, we em-
ployed a PC12 cell line, a well-established model for studying neuronal differentiation, to
perform bioactivity-guided isolation and purification of lavender essential oil, leading to
the identification of a small molecule, β-cyc. The morphological alterations observed in
PC12 cells after treatment with β-cyc or β-cyc + low dose of NGF in Figure 1 suggested
that β-cyc not only has NGF-mimic effects but also has NGF-enhancing effects. These re-
sults are consistent with our previous findings [13–15]
To clarify the mechanism of action of β-cyc, we first focused on the signaling path-
ways related to the neurogenic effects to do an investigation with specific protein inhibi-
tors and western blotting analysis. Given that TrkA and TrkB serve as specific transmem-
brane targets for the neurotrophins NGF and BDNF, respectively, we initiated a screening
process utilizing inhibitors targeting TrkA, TrkB, Ras, Raf, ERK, SAPK/JNK, and p38
MAPK. Additionally, inhibitors directed against β-arrestin, PKA, and GSK-3β were also
Figure 7. Target prediction of
β
-cyc by DARTS assay. (A) Western blot analysis of IGF-1R and
(B) GR protein level after adding different concentrations of pronase E. (C) Quantification of the
data obtained from panels (A,B). (D) Protein level of IGF-1R after adding different concentrations of
β
-cyc (0.1, 1, 3, 10
µ
M) by Western blotting. IGF-1R was normalized with
β
-actin. (E) Western blot
analysis of GR protein level after adding different concentrations of
β
-cyc (0.01, 0.1, 1, 10
µ
M). GR was
normalized with
β
-actin. (F) Quantification of the data obtained from panels D-E. Each experiment
was conducted three times.
$
p< 0.05,
$$
p< 0.01 and
$$$
p< 0.001 in comparison to the untreated
group; *** p< 0.001 compared to the negative control without pronase E;
#
p< 0.05 and
###
p< 0.001
compared with the negative control with pronase E.
Int. J. Mol. Sci. 2024,25, 9763 10 of 16
Furthermore, compared with those of GR, the protein stability of GR increased in a
concentration-dependent manner following
β
-cyc treatment, although the dose dependency
and increase in IGF-1R levels were more pronounced (Figures 7B,E,F and S6). Ultimately,
these findings provided further support for the notion that IGF-1R and GR are potential
target proteins of β-cyc.
3. Discussion
Recent failures in clinical trials investigating amyloid-beta clearance have generated
interest in alternative therapeutic strategies for AD. Research suggests that A
β
plaques
may have a protective role in the early stages of AD, although their correlation with disease
severity appears to be weak. Treatments focused on A
β
plaques, such as the
γ
-secretase
inhibitor semagacestat and the
β
-secretase inhibitor verubecestat, have shown inconclusive
efficacy [
8
]. However, further investigations are needed to determine the effectiveness of
other AD drugs, such as aducanumab and GV-971. Therefore, the development of clinical
drugs for AD requires the exploration of diverse mechanisms and targets. In particular,
neurogenesis has become the new strategy for the treatment of AD.
Lavender essential oil, demonstrating neuroprotective effects and promising cognitive
improvement, has the anti-AD potential supported by both dementia model rats and
clinical studies [
29
–
32
]. However, the material basis for producing anti-AD effects is
still unclear, and the mechanism of activity still needs to be elucidated. Therefore, we
employed a PC12 cell line, a well-established model for studying neuronal differentiation,
to perform bioactivity-guided isolation and purification of lavender essential oil, leading to
the identification of a small molecule,
β
-cyc. The morphological alterations observed in
PC12 cells after treatment with
β
-cyc or
β
-cyc + low dose of NGF in Figure 1suggested that
β
-cyc not only has NGF-mimic effects but also has NGF-enhancing effects. These results
are consistent with our previous findings [13–15]
To clarify the mechanism of action of
β
-cyc, we first focused on the signaling pathways
related to the neurogenic effects to do an investigation with specific protein inhibitors
and western blotting analysis. Given that TrkA and TrkB serve as specific transmembrane
targets for the neurotrophins NGF and BDNF, respectively, we initiated a screening process
utilizing inhibitors targeting TrkA, TrkB, Ras, Raf, ERK, SAPK/JNK, and p38 MAPK.
Additionally, inhibitors directed against
β
-arrestin, PKA, and GSK-3
β
were also included
owing to their close association with the promotion of neurite outgrowth. The changes
in NGF mimics or enhancer effects of
β
-cyc on PC12 cells after giving these inhibitors in
Figures 1and S1 indicated that TrkA and TrKB were not involved in the NGF-mimic effects
of β-cyc, whereas Ras, Raf, and ERK may be implicated in the neurogenic effect of β-cyc.
IGF-1 plays an essential role in CNS development and maturation. Recent preclinical
and clinical evidence indicate that IGF-1 not only regulated growth and development
but also prevented neuronal death mediated by amyloidogenesis, neuroinflammation,
and apoptosis through modulation of PI3/Akt kinase, mTOR, and MAPK/ERK signal-
ing [
33
]. Meanwhile, some evidence indicated that GR regulates a series of important
genes for neuronal structure and plasticity and is involved in the neuritogenic activity in
PC12 cells [
13
,
28
]. Since the PI3K/AKT and PLC/PKC signaling pathways demonstrate
significant involvement in neurogenesis [
34
,
35
]. Therefore, we checked whether these two
signaling pathways were involved in the NGF-mimic effects of
β
-cyc with specific protein
inhibitors and western blotting analysis. As we expected, the significant suppression of
the neurogenic effect of
β
-cyc by IGF-1R and GR inhibitors, as well as additional inhibitors
targeting their downstream proteins PI3K/AKT, PLC, and PKC in Figures 3–5, elucidated
that IGF-1R/PI3K/AKT and GR/PLC/PKC signaling pathways took important roles in the
neurogenic effects of
β
-cyc in PC12 cells. Interestingly, the mechanism of the action of
β
-cyc
for its NGF-mimic effect was different from that of compounds such as AMA, Lindersin B,
and CuB. AMA, a secoiridoid glycoside from Gentiana rigescens, promotes neurogenesis by
activating the INSR/PI3K/AKT and Ras/Raf/MEK signaling pathways in PC12 cells [
14
].
Lindersin B, a cucurbitane triterpenoid from Lindernia crustacea, stimulated neuritogenesis
Int. J. Mol. Sci. 2024,25, 9763 11 of 16
via the activation of the TrkA/PI3K/ERK signaling pathway [
15
]. CuB, a terpene from
Cucumis melo (commonly known as Tiangua Di in Chinese), induced neurite growth by
targeting cofilin and altering the GR and TrkA pathways [13].
Identification of the target proteins of small molecules is crucial to explore their
mechanism of action. Therefore, we screened potential target proteins of
β
-cyc with specific
protein inhibitor experiments and label-free protein target techniques, such as DARTS
and CETSA. The effects of IGF-1R and GR inhibitors on the NGF-mimic effect of
β
-cyc in
Figures 4and 5and the increase of thermal stability and pronase E stability of IGF-1R and
GR of CETSA and DARTS in Figures 6and 7demonstrated that IGF-1R and GR might be
potential target protein of
β
-cyc to produce NGF-mimic effect on PC12 cells. Regrettably,
we failed to get direct evidence of which
β
-cyc bound with IGF-1R or GR by SPR analysis.
Emerging research suggests that combination therapy utilizing molecules with diverse
structures and mechanisms holds great promise as an approach to treating AD. In con-
trast, the use of multiple single-target drugs often leads to increased medication burden
and a greater risk of cumulative toxicity and side effects. Dual-targeted therapies are
considered potential solutions for overcoming drug resistance, as they offer synergistic
benefits with reduced adverse effects and decreased toxicity. Progress in dual-target drug
development has been evident in clinical trials. For instance, Blinatumomab, which targets
CD19 and CD3, obtained FDA approval in 2014 for the treatment of acute B lymphoblastic
leukemia [
36
]. Other notable developments include the PD-L1/CTLA-4 bispecific antibody
KN046, which is effective in the treatment of solid tumors, and AK104, targeting PD-1
and CTLA-4, demonstrated increased efficacy with fewer adverse events [
37
,
38
]. This
movement toward dual-target and multitarget drugs represents a crucial research direction.
These results provided insights into the combination that the use of these molecules may
have in increasing the therapy effect for AD.
4. Materials and Methods
4.1. Antibodies and Reagents
DMSO (CAT No.: D8418), NGF (CAT No.: N2513), TrkA inhibitor (k252a, CAT
No.: 420298-M), MEK/ERK inhibitor (U0126, CAT No.: 19-147), PI3K inhibitor (LY294002,
CAT No.: 440202), PKC inhibitor (Go6983, CAT No.: 365251), and GR inhibitor (RU486,
CAS No.: 84371-65-3) were bought from Sigma-Aldrich Co, Boston, MA, USA. The INSR
inhibitor (CAT No.: sc-221730), IGF-1R inhibitor (T9576, CAS No.: 477-47-4), Raf inhibitor
(AZ628, CAT No.: sc-364418), and PLC inhibitor (U-73343, CAT No.: sc-201422) were
obtained from Santa Cruz Biotechnology, Dallas, TX, USA. The Ras inhibitor (FTA, Item
No.: 17474) was obtained from Cayman Chemical, Ann Arbor, MI, USA. The pronase E
(CAT No.: HY-114158) was obtained from MedChemExpress, Shanghai, China, while the
TrkB inhibitor (ANA-12, CAT No.: S7745) was obtained from Selleck, Shanghai, China.
The antibodies against IGF-1R (CAT No.: 3027S), phospho-IGF-1R (Tyr1131, CAT
No.: 3024S), PI3 kinase (CAT No.: 4249S), phospho-PI3 kinase (Tyr458, CAT No.: 4228S),
AKT (CAT No.: 9272S), phospho-AKT (Ser473, CAT No.: 9271S), GR (CAT No.: 12041T),
PLC (CAT No.: 14008S), phospho-PKC (CAT No.: 2261S), and PKC (CAT No.: 38168S) were
obtained from Cell Signaling Technology, Boston, MA, USA, and the antibodies against
phospho-GR (CAT No.: AF2004) and phospho-PLC
γ
(CAT No.: AF4454) were obtained
from Affinity Biosciences, Cincinnati, OH, USA. The
β
-actin antibody (CATNo.: CW0096M),
secondary antibodies horseradish peroxidase-linked anti-mouse (CAT No.: CW0102S) and
anti-rabbit IgGs (CAT No.: CW0103S), and the pico-ECL Western blot chemiluminescence
detection kit (CAT No.: CW0049M) were obtained from Beijing CoWin Biotech Company,
Beijing, China, respectively. In addition, it is with great regret that we cannot replicate the
study in hippocampus-specific cell lines in this revision because there is no cell line in our
laboratory now. We accepted your suggestion and use this cell line as the model in our
study in the future.
Int. J. Mol. Sci. 2024,25, 9763 12 of 16
4.2. Extraction and Isolation
Lavender oil was obtained from Thursday Plantation, Rydalmere, NSW, Australia.
Chromatographic separation was performed on 1.5 g of lavender oil using a silica gel
column, eluting with a gradient of n-hexane/CH
2
Cl
2
(10:0, 7:3, 5:5, 3:7, 0:10), resulting in
six fractions. The most active fraction (206.1 mg) was washed out with CH
2
Cl
2
/MeOH (7:3)
and further purified by HPLC using a Cosmosil 5C18-MS-II packed column (10
×
250 mm)
(Nacalai Tesque, Tokyo, Japan) with a linear gradient of MeOH/H
2
O: 40:60–100:0 over
80 min with a detection wavelength set to 210 nm and using a flow rate set at 3 mL/min.
This process yielded an active compound (13.6 mg, retention time = 49.4 min), which was
identified as
β
-cyc by comparing its
1
H NMR data with the previous literature [
39
].
1
H
NMR (500 MHz, CDCl
3
) data:
δ
10.11 (s, 1H, CO-H), 2.17 (t, J= 6.3 Hz, 2H, CH
2
), 2.08
(s, 3H, CH
2
), 1.63–1.58 (m, 2H, CH
2
), 1.44–1.41 (m, 2H, CH
2
), 1.18 (s, 6H, 2
×
CH
2
). The
structure of β-cyc is shown in Figure 1A.
4.3. Evaluation of the Neuritogenic Activity of PC12 Cells
The evaluation of neuritogenic activity in the PC12 cells was described in our previous
publication [
15
]. Around 5
×
10
4
cells/well were cultured in a 24-well microplate under
suitable conditions. After that, the medium in each well was exchanged with 1 mL of
DMEM containing the test compound dissolved in DMSO or 0.5% DMSO alone. NGF
at a concentration of 40 ng/mL served as the positive control. After 48 h, 100 cells were
randomly selected and counted in triplicate. For inhibitor screening, cells were initially
pretreated with 500
µ
L of DMEM that contained a specific inhibitor for 30 min. After
that, an additional 500
µ
L of DMEM with either the test compound or 0.5% DMSO was
introduced. The alterations in cellular morphology were assessed in each well after 48 h,
and the percentage of positive cells in the selected area was quantified.
4.4. Cell Viability by MTT Bioassay
The cells were incubated with different concentrations of
β
-cyc for 48 h or with a
combination of
β
-cyc (3
µ
M) and low-dose NGF (1 ng/mL). After removing the original
culture medium, 500
µ
L of medium containing 200
µ
g/mL MTT was added to each well
and incubated at 37
◦
C for 2 h. Subsequently, the medium per well was substituted with
200
µ
L of DMSO to dissolve the formazan crystals, which were then detected at 570 nm
using a spectrophotometer.
4.5. Western Blot Analysis
Western blot analysis was conducted in accordance with the methods outlined in
previous studies [
13
]. Briefly, the cells were homogenized in a lysis buffer containing
1% protease and phosphatase inhibitors to extract proteins. Protein concentrations were
measured using the BCA assay, and all samples were denatured at 100
◦
C for 10 min.
Subsequently, 20
µ
g of protein from each sample was uploaded on a sodium dodecyl
sulfate–polyacrylamide gel (SDS–PAGE). The gel electrophoresis was executed at 80 V for
15 min, followed by 120 V for 60 min. The proteins were transferred onto a polyvinylidene
difluoride (PVDF) membrane and then blocked in 5% skim milk for 60 min. The PVDF
membrane underwent overnight incubation at 4
◦
C with primary antibodies, while the
anti-
β
-actin antibody was employed as the normalization control. Following washing,
the membrane underwent incubation with a secondary antibody for 45 min. Finally, the
membrane was washed and exposed using a chemiluminescence detection kit (Beijing
Cowin Biotech Company, Beijing, China). The protein bands were quantified using ImageJ
software (Version 1.42q, National Institutes of Health, Rockville, MD, USA).
4.6. CETSA
CETSA is a widely utilized method for target validation that exploits changes in
protein thermal stability following interaction with ligands to identify target proteins.
Initially, 2
×
10
6
cells were cultured in 60 mm dishes and incubated for 24 h. Subsequently,
Int. J. Mol. Sci. 2024,25, 9763 13 of 16
a control group was treated with 0.5% DMSO, while additional dishes were treated with
3µM
of
β
-cyc. After 24 h of incubation, the proteins were extracted and subjected to heating
using a Veriti 96-well thermal cycler at varying temperatures. Subsequently, changes in the
protein expression of IGF-1R and GR were assessed through Western blotting.
4.7. DARTS
DARTS is a technique utilized to identify interactions between drug molecules and
proteins. Specifically, lysates derived from PC12 cells were incubated with or without
β
-cyc
(3
µ
M) or
β
-cyc (1
µ
M) for 4 h at room temperature. Subsequently, the lysates were divided
into several portions and treated with varying concentrations of pronase E for 25 min at
room temperature. To terminate the digestion process, the samples were promptly boiled
at 100
◦
C for 10 min following the addition of loading buffer. Finally, Western blot analysis
was conducted to identify any alterations in the protein expression of IGF-1R or GR. Upon
determining the optimal concentration of pronase E, different concentrations of
β
-cyc,
lysates, and pronase E were co-incubated, following the incubation steps as described
above. Finally, protein expression was analyzed using Western blotting.
4.8. Quantification and Statistical Analysis
Data from three independent experiments, each performed in triplicate and expressed
as mean
±
SEM, were subjected to one-way ANOVA and subsequent Tukey’s post hoc test
via GraphPad Prism 8.0.2 software (GraphPad Software, San Diego, CA, USA). p< 0.05 was
considered statistically significant.
5. Conclusions
In conclusion,
β
-cyc from Lavender has novel NGF-mimic and enhancer effects on
PC12 cells. This compound existed the NGF-mimic and enhancer effect on PC12 cells via
targeting of the IGF-1R and GR protein and regulation of the PI3K/AKT/Ras/Raf/ERK
and GR/PLC/PKC signaling pathways (Figure 8). This study indicated the potential
applications of
β
-cyc for its neurogenesis effect and provided evidence for the treatment
of neurodegenerative diseases. Furthermore, the leading compound will be determined
via modification of
β
-cyc, and the study of the chemical structure -bioactivity relationship
and the anti-AD effects and the mechanism of actions of the leading compound will be
evaluated and clarified with the AD animal model.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 15 of 17
In conclusion, β-cyc from Lavender has novel NGF-mimic and enhancer effects on
PC12 cells. This compound existed the NGF-mimic and enhancer effect on PC12 cells via
targeting of the IGF-1R and GR protein and regulation of the PI3K/AKT/Ras/Raf/ERK and
GR/PLC/PKC signaling pathways (Figure 8). This study indicated the potential applica-
tions of β-cyc for its neurogenesis effect and provided evidence for the treatment of neu-
rodegenerative diseases. Furthermore, the leading compound will be determined via
modification of β-cyc, and the study of the chemical structure -bioactivity relationship and
the anti-AD effects and the mechanism of actions of the leading compound will be evalu-
ated and clarified with the AD animal model.
Figure 8. Proposed mechanism of action for β-cyc. β-cyc promotes neurogenesis by targeting IGF-
1R and GR to activate downstream PI3K/AKT and PLC/PKC signaling pathways. The up arrow
represents an increase in protein phosphorylation level.
Supplementary Materials: The following supporting information can be downloaded at:
www.mdpi.com/xxx/s1.
Author Contributions: Conceptualization, J.Q., L.X., and L.G.; methodology and experiment, C.A.;
writing—original draft preparation, C.A.; project administration, J.Q., L.X., and L.G.; funding ac-
quisition, J.Q. All authors have read and agreed to the published version of the manuscript.
Funding: This work was financially supported by the National Key R&D Program of China (Grant
Number: 2022YFE0104000) and the National Natural Science Foundation of China (Grant number:
22177102).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data supporting the conclusions of this study can be acquired
from the corresponding author upon a reasonable request.
Conflicts of Interest: The authors declare no conflicts of interest.
Figure 8. Proposed mechanism of action for
β
-cyc.
β
-cyc promotes neurogenesis by targeting IGF-
1R and GR to activate downstream PI3K/AKT and PLC/PKC signaling pathways. The up arrow
represents an increase in protein phosphorylation level.
Int. J. Mol. Sci. 2024,25, 9763 14 of 16
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/ijms25189763/s1.
Author Contributions: Conceptualization, J.Q., L.X., and L.G.; methodology and experiment, C.A.;
writing—original draft preparation, C.A.; project administration, J.Q., L.X., and L.G.; funding acquisi-
tion, J.Q. All authors have read and agreed to the published version of the manuscript.
Funding: This work was financially supported by the National Key R&D Program of China (Grant
Number: 2022YFE0104000) and the National Natural Science Foundation of China (Grant num-
ber: 22177102).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data supporting the conclusions of this study can be acquired from
the corresponding author upon a reasonable request.
Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations
Alzheimer’s disease, AD; amarogentin, AMA; Analysis of variance, ANOVA; beta amyloid,
A
β
bicinchoninic acid, BCA; brain-derived neurotrophic factor, BDNF; cellular thermal shift as-
say CETSA; Cucurbitacin B, CuB; beta-Cyclocitral,
β
-cyc; drug affinity responsive target stability,
DARTS; dulbecco’s modified eagle medium, DMEM; dimethyl sulfoxide, DMSO; extracellular signal-
regulated kinase, ERK; glucocorticoid receptor, GR; insulin-like growth factor-1 receptor, IGF-1R;
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT; N-Methyl-D-Aspartate, Nerve
growth factor, NGF; NMDA; phosphatidylinositol 3 kinase, PI3K; polyvinylidene difluoride, PVDF;
phospholipase C, PLC; rat sarcoma, Ras; raf proto oncogene serine threonine protein kinase, Raf;
Standard error of the mean, SEM; serine/thronin-protein kinase, AKT; sodium dodecyl sulfate–
polyacrylamide gel, SDS–PAGE; tyrosine kinase, Trk; tyrosine kinase A, TrkA; tyrosine kinase B, TrkB.
References
1.
Alzheimer’s Disease International. World Alzheimer Report 2023: Reducing Dementia Risk: Never Too Early, Never Too Late.
2023. Available online: https://www.alzint.org/resource/world-alzheimer-report-2023/ (accessed on 21 September 2023).
2.
Milà-Alomà, M.; Ashton, N.J.; Shekari, M.; Salvadó, G.; Ortiz-Romero, P.; Montoliu-Gaya, L.; Benedet, A.L.; Karikari, T.K.;
Lantero-Rodriguez, J.; Vanmechelen, E.; et al. Plasma p-tau231 and p-tau217 as state markers of amyloid-
β
pathology in preclinical
Alzheimer’s disease. Nat. Med. 2022,28, 1797–1801. [CrossRef] [PubMed]
3.
Schneider, L.S.; Mangialasche, F.; Andreasen, N.; Feldman, H.; Giacobini, E.; Jones, R.; Mantua, V.; Mecocci, P.; Pani, L.; Winblad,
B.; et al. Clinical trials and late-stage drug development for Alzheimer ’s disease: An appraisal from 1984 to 2014. J. Intern. Med.
2014,275, 251–283. [CrossRef] [PubMed]
4.
Tucker, S.; Möller, C.; Tegerstedt, K.; Lord, A.; Laudon, H.; Sjödahl, J.; Söderberg, L.; Spens, E.; Sahlin, C.; Waara, E.R.; et al. The
murine version of BAN2401 (mAb158) selectively reduces amyloid-
β
protofibrils in brain and cerebrospinal fluid of tg-ArcSwe
mice. J. Alzheimers Dis. 2015,43, 575–588. [CrossRef]
5.
Sims, J.R.; Zimmer, J.A.; Evans, C.D.; Lu, M.; Ardayfio, P.; Sparks, J.; Wessels, A.M.; Shcherbinin, S.; Wang, H.; Monkul Nery, E.S.;
et al. Donanemab in Early Symptomatic Alzheimer Disease: The TRAILBLAZER-ALZ 2 Randomized Clinical Trial. JAMA 2023,
330, 512–527. [CrossRef] [PubMed]
6.
Lest’anová, Z.; Bacová, Z.; Havránek, T.; Bakos, J. Mechanizmy zmien d´
lzky axónov a dendritov neurónu Mechanisms of growth
of neuronal axons and dendrites. Cesk Fysiol. 2013,62, 47–53.
7.
Akram, R.; Anwar, H.; Javed, M.S.; Rasul, A.; Imran, A.; Malik, S.A.; Raza, C.; Khan, I.U.; Sajid, F.; Iman, T.; et al. Axonal
Regeneration: Underlying Molecular Mechanisms and Potential Therapeutic Targets. Biomedicines 2022,10, 3186. [CrossRef]
8. Makin, S. The amyloid hypothesis on trial. Nature 2018,559, S4–S7. [CrossRef]
9. De Strooper, B.; Karran, E. The cellular phase of Alzheimer’s disease. Cell 2016,164, 603–615. [CrossRef]
10.
Loy, R.; Taglialatela, G.; Angelucci, L.; Heyer, D.; Perez-Polo, R. Regional CNS uptake of blood-borne nerve growth factor. J.
Neurosci. Res. 1994,39, 339–346. [CrossRef]
11.
Xu, C.J.; Wang, J.L.; Jin, W.L. The emerging therapeutic role of NGF in Alzheimer’s disease. Neurochem. Res. 2016,41, 1211–1218.
[CrossRef]
12.
Mitra, S.; Behbahani, H.; Eriksdotter, M. Innovative therapy for Alzheimer’s disease-with focus on biodelivery of NGF. Front.
Neurosci. 2019,13, 38. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2024,25, 9763 15 of 16
13.
Li, J.; Sun, K.; Muroi, M.; Gao, L.; Chang, Y.; Osada, H.; Xiang, L.; Qi, J. Cucurbitacin B induces neurogenesis in PC12 cells and
protects memory in APP/PS1 mice. J. Cell Mol. Med. 2019,23, 6283–6294. [CrossRef] [PubMed]
14.
Cheng, L.; Osada, H.; Xing, T.; Yoshida, M.; Xiang, L.; Qi, J. The Insulin receptor: A potential target of amarogentin isolated from
Gentiana rigescens Franch that induces neurogenesis in PC12 Cells. Biomedicines 2021,9, 581. [CrossRef] [PubMed]
15.
Cheng, L.; Ye, Y.; Xiang, L.; Osada, H.; Qi, J. Lindersin B from Lindernia crustacea induces neuritogenesis by activation of tyrosine
kinase A/phosphatidylinositol 3 kinase/extracellular signal-regulated kinase signaling pathway. Phytomedicine 2017,24, 31–38.
[CrossRef]
16.
Tsai, S.J.; Liu, W.H.; Yin, M.C. Trans Fatty Acids Enhanced Beta-Amyloid Induced Oxidative Stress in Nerve Growth Factor
Differentiated PC12 Cells. Springer 2012,37, 786–794. [CrossRef]
17.
El Abdali, Y.; Agour, A.; Allali, A.; Bourhia, M.; El Moussaoui, A.; Eloutassi, N.; Salamatullah, A.M.; Alzahrani, A.; Ouahmane, L.;
Aboul-Soud, M.A.M.; et al. Lavandula dentata L.: Phytochemical Analysis, Antioxidant, Antifungal and Insecticidal Activities of
Its Essential Oil. Plants 2022,11, 311. [CrossRef]
18.
Zheng, T.F.; Zhou, M.; Yang, L.; Wang, Y.Y.; Meng, Y.Y.; Liu, J.L.; Zuo, A.J. Effects of high light and temperature on Microcystis
aeruginosa cell growth and β-cyclocitral emission. Ecotoxicol. Environ. Saf. 2020,192, 110313. [CrossRef]
19.
O’Neill, C.; Kiely, A.P.; Coakley, M.F.; Manning, S.; Long-Smith, C.M. Insulin and IGF-1 signaling: Longevity, protein homoeostasis
and Alzheimer’s disease. Biochem. Soc. Trans. 2012,40, 721–727. [CrossRef]
20.
Lejri, I.; Grimm, A.; Eckert, A. Ginkgo biloba extract increases neurite outgrowth and activates the AKT/mTOR pathway. PLoS
ONE 2019,14, e0225761. [CrossRef]
21.
O’ Neill, C. PI3-kinase/AKT/mTOR signaling: Impaired on/off switches in aging, cognitive decline and Alzheimer’s disease.
Exp. Gerontol. 2013,48, 647–653. [CrossRef]
22.
Terada, K.; Kojima, Y.; Watanabe, T.; Izumo, N.; Chiba, K.; Karube, Y. Inhibition of nerve growth factor-induced neurite outgrowth
from PC12 cells by dexamethasone: Signaling pathways through the glucocorticoid receptor and phosphorylated AKT and
ERK1/2. PLoS ONE 2014,9, e93223. [CrossRef] [PubMed]
23.
Ebendal, T. Function and evolution in the NGF family and its receptors. J. Neurosci. Res. 1992,32, 461–470. [CrossRef] [PubMed]
24. Bothwell, M. NGF, BDNF, NT3, and NT4. Handb. Exp. Pharmacol. 2014,220, 3–15. [CrossRef]
25.
Vaudry, D.; Stork, P.J.S.; Lazarovici, P.; Eiden, L.E. Signaling pathways for PC12 cell differentiation: Making the right connections.
Science 2002,296, 1648–1649. [CrossRef]
26.
Li, W.; Miller, W.T. Role of the activation loop tyrosines in regulation of the Insulin-like growth factor I receptor-tyrosine Kinase. J.
Biol. Chem. 2006,281, 23785–23791. [CrossRef]
27.
Polman, J.A.; Welten, J.E.; Bosch, D.S.; de Jonge, R.T.; Balog, J.; van der Maarel, S.M.; de Kloet, E.R.; Datson, N.A. A genome-wide
signature of glucocorticoid receptor binding in neuronal PC12 cells. BMC Neurosci. 2012,13, 118. [CrossRef]
28.
Jozic, I.; Vukelic, S.; Stojadinovic, O.; Liang, L.; Ramirez, H.A.; Pastar, I.; Tomic Canic, M. Stress Signals, Mediated by Membranous
Glucocorticoid Receptor, Activate PLC/PKC/GSK-3
β
/
β
-catenin Pathway to Inhibit Wound Closure. J. Investig. Dermatol. 2017,
137, 1144–1154. [CrossRef]
29.
Kashani, M.S.; Tavirani, M.R.; Talaei, S.A.; Salami, M. Aqueous extract of lavender (Lavandula angustifolia) improves the spatial
performance of a rat model of Alzheimer’s disease. Neurosci. Bull. 2011,27, 99–106. [CrossRef] [PubMed]
30.
Wang, D.; Yuan, X.; Liu, T.; Liu, L.; Hu, Y.; Wang, Z.; Zheng, Q. Neuroprotective activity of lavender oil on transient focal cerebral
ischemia in mice. Molecules 2012,17, 9803–9817. [CrossRef]
31.
Jimbo, D.; Kimura, Y.; Taniguchi, M.; Inoue, M.; Urakami, K. Effect of aromatherapy on patients with Alzheimer’s disease.
Psychogeriatrics 2009,9, 173–179. [CrossRef]
32. Degel, J.; Köster, E.P. Odors: Implicit memory and performance effects. Chem. Senses. 1999,24, 317–325. [CrossRef] [PubMed]
33.
Bhalla, S.; Mehan, S.; Khan, A.; Rehman, M.U. Protective role of IGF-1 and GLP-1 signaling activation in neurological dysfunctions.
Neurosci. Biobehav. Rev. 2022,142, 104896. [CrossRef] [PubMed]
34.
Long, H.Z.; Cheng, Y.; Zhou, Z.W.; Luo, H.Y.; Wen, D.D.; Gao, L.C. PI3K/AKT Signal Pathway: A Target of Natural Products in
the Prevention and Treatment of Alzheimer’s Disease and Parkinson’s Disease. Front. Pharmacol. 2021,12, 648636. [CrossRef]
[PubMed]
35.
Sheng, H.; Xu, Y.; Chen, Y.; Zhang, Y.; Ni, X. Corticotropin-releasing hormone stimulates mitotic kinesin-like protein 1 expression
via a PLC/PKC-dependent signaling pathway in hippocampal neurons. Mol. Cell Endocrinol. 2012,362, 157–164. [CrossRef]
36.
Pulte, E.D.; Vallejo, J.; Przepiorka, D.; Nie, L.; Farrell, A.T.; Goldberg, K.B.; McKee, A.E.; Pazdur, R. FDA Supplemental Approval:
Blinatumomab for Treatment of Relapsed and Refractory Precursor B-Cell Acute Lymphoblastic Leukemia. Oncologist 2018,23,
1366–1371. [CrossRef]
37.
Li, Q.; Liu, J.; Zhang, Q.; Ouyang, Q.; Zhang, Y.; Liu, Q.; Sun, T.; Ye, F.; Zhang, B.; Xia, S.; et al. The anti-PD-L1/CTLA-4
bispecific antibody KN046 in combination with nab-paclitaxel in first-line treatment of metastatic triple-negative breast cancer: A
multicenter phase II trial. Nat. Commun. 2024,15, 1015. [CrossRef]
Int. J. Mol. Sci. 2024,25, 9763 16 of 16
38.
Gao, X.; Xu, N.; Li, Z.; Shen, L.; Ji, K.; Zheng, Z.; Liu, D.; Lou, H.; Bai, L.; Liu, T.; et al. Safety and antitumour activity
of cadonilimab, an anti-PD-1/CTLA-4 bispecific antibody, for patients with advanced solid tumours (COMPASSION-03): A
multicentre, open-label, phase 1b/2 trial. Lancet Oncol. 2023,24, 1134–1146. [CrossRef]
39.
Zha, G.; Fang, W.; Leng, J.; Qin, H. A simple, mild and general oxidation of alcohols to aldehydes or ketones by SO2F2/K2CO3
using DMSO as solvent and oxidant. Adv. Synth. Catal. 2019,361, 2262–2267. [CrossRef]
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