1. Introduction
Senescent cells secrete a senescence-associated secretory phenotype (SASP) that
leads to chronic inflammation, playing a crucial role in age-related functional
decline and senile diseases [1]. Thus, the development of senotherapeutic
interventions to remove senescent cells (senolytic drugs) or to modulate the SASP
(senostatic drugs) could extend a person’s healthspan or treat various diseases
[2]. Several senolytics target the prosurvival pathway, such as kinase inhibitors
(e.g., dasatinib), flavonoids (e.g., quercetin and fisetin), BCL-2/BCL-xL
inhibitors (e.g., navitoclax), and BCL-xL inhibitors (e.g., A1331852 and
A1155463) [3].
Cancer is a representative geriatric disease and is closely related to senescent
cells constituting tissues. Cellular senescence itself suppresses cancer
development despite the accumulation of various genetic mutations, but the SASP
secreted from senescent cells promotes the development of surrounding cancer
cells [4]. Therefore, to develop effective therapeutic agents to remove cancer,
it is crucial to identify candidate substances that can selectively act only on
cancer cells. Additionally, SASP secreted from senescent cells that surround
cancer tissues reduce the effects of various cancer treatments and induce cancer
recurrence through SASP secretion [5, 6, 7]. Therefore, increasing attention is
being paid to the development of cancer therapeutics using senotherapies that
remove senescent cells or suppress SASP secretion. Senolytics, such as quercetin,
navitoclax, and fisetin, are being studied as potential cancer treatments in
nonclinical or initial clinical trials [8, 9, 10]. However, for good therapeutic
effect, candidate drugs should be selective for both cancer and senescent cells.
Piperine is a bioactive phenolic component that has been isolated from plants of
the Piper species, such as black pepper (Piper nigrum) and long
pepper (Piper longum) [11], and has attracted attention as a dietary
phytochemical and medicine [12]. Various pharmacological properties of piperine
have been suggested, including antioxidant activity [13], anti-inflammatory
activity [14] and biological enhancement [15]. Piperine also exerts a
chemopreventive effect [16] and causes cellular toxicity in cancer cells by
inducing various effector proteins involved in apoptosis [17, 18, 19]. Piperine
was reported to suppress tumor development and metastasis in mouse models [17].
In cancer cells, piperine triggers both cell cycle arrest by activating p21 and
apoptosis by activating caspase [20]. Interestingly, a combination therapeutic
model of piperine with curcumin, a yellow pigment in the Indian spice turmeric
(Curcuma longa), demonstrated neurotrophic and neuroprotective effects
in a D-galactose-induced brain aging model [21], preventing the progress of aging
induced by D-galactose as well as reversing hippocampal memory function due to
antioxidant activity [22]. Curcumin is a well-known, promising antiaging
intervention that is easy to add to one’s diet. Curcumin was reported to induce
an extended lifespan in various models, including fruit flies, nematodes, and
mice [23, 24, 25, 26]. While curcumin has demonstrated a direct antiaging effect,
the antiaging effect of piperine has only been attributed to its antioxidant
effect on brain aging in combination with curcumin. Like piperine, piperlongumine
is a natural product found in various Piper species, and its analogs
have also been suggested as senolytic agents through activation of the caspase
pathway in senescent cells [27].
We previously screened single natural compounds that acted differently on cancer
cells compared with premature senescent cells. Most substances showed similar
effects on cancer and senescent cells, but piperine induced toxicity in cancer
cells only. This study investigates the selective effect of piperine as a
potential senostatic agent as well as an anticancer drug.
2. Materials and Methods
2.1 Reagents and Cells
Piperine was kindly provided by Prof. WK Oh (Seoul National University, Korea)
and purchased (Merck, NY, USA). CT26 (mouse colon carcinoma, CRL-2638), T98G
(human glioblastoma, CRL-1690), A431 (human skin carcinoma, CRL-1555), MCF7
(human breast adenocarcinoma, HTB-22), HepG2 (human hepatocellular carcinoma,
HB-8065), and HeLa (human cervix adenocarcinoma, CCL-2) cell lines were purchased
from American Type Culture Collection (Manassas, VA, USA). All cancer cell lines,
except MCF7, were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin
(P/S) at 37 C in a 5% CO incubator. The MCF7 cells were cultured
in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS and 1%
P/S at 37 C in a 5% CO incubator. Human diploid fibroblasts
(HDFs) were cultured as previously described [28]. Briefly, the cells were
maintained in 10-cm cell dishes containing DMEM supplemented with 10% FBS and
1% P/S. The cells were continuously subcultured at a ratio of 1:4. Nonsenescent
HDFs (NS-HDFs) were defined as HDFs resulting from fewer than 30 population
doublings and premature senescent HDFs (S-HDFs) as HDFs resulting from more than
70 population doublings. Premature senescent cells were confirmed by
senescent-associated -galactosidase (SA--gal) staining. All
cell culture reagents were purchased from Gibco-BRL (Grand Island, NY, USA).
2.2 Piperine Treatment
We investigated the selectivity of piperine by observing its effect on the
growth of cancer and normal cells. Cancer and normal cells were cultured in
complete medium at 37 C in a 5% CO incubator and treated once or
twice with piperine (70 M) at 2-day intervals. Thereafter, we analyzed
cytotoxicity using a lactate dehydrogenase (LDH) assay or performed a visual cell
count to analyze cell growth. We also treated S-HDFs in complete medium at 37
C in a 5% CO incubator with piperine (70 M) three times
at 2-day intervals to test the senomorphic effect of piperine on S-HDFs.
2.3 Cytotoxicity Assay
Cytotoxicity was assessed using an LDH assay kit (DG-LDH500; DoGen Bio, Seoul,
Korea) according to the manufacturer’s protocol. Briefly, cancer cells (2
10) were seeded in triplicate in six-well plates and then
treated once or twice with piperine (70 M) at 2-day intervals. Normal
cells, such as NS-HDFs (6 10) and S-HDFs (3
10), were seeded at triplicate in six-well plates and treated two to three
times with piperine (70 M) at 2-day intervals. After the piperine
treatment, the culture supernatants were collected and placed in triplicates of
96-well plates, followed by incubation with LDH solution at room temperature (RT)
in the dark for 30 min. Finally, the optical density was read at 450 nm using a
microplate reader (Molecular Devices Corp., Menlo Park, CA, USA). To identify
nuclear morphology, cells were fixed and stained with the nucleic acid stain
4,6-diamidino-2-phenylindole (DAPI) after once or twice treatment of
piperine. The stained cells were analyzed with the Zeiss LSM 700 confocal
microscope.
2.4 Analysis of Viable Cells
To count the viable cells, we seeded various cancer cell lines (2
10) in six-well plates containing complete medium and treated them with
piperine (70 M) for 24 or 48 h. We also seeded S-HDFs (3
10) in 6-well plates containing complete medium and treated them with
piperine (70 M) or 5 mM nicotinamide (NA, positive control) one to three
times at 2-day intervals. Subsequently, these cells were harvested, resuspended
in medium, and stained with trypan blue solution. The viable cells were counted
using a hemocytometer.
2.5 Cell Proliferation Assay
Cell proliferation assay was assessed using an EZ-Cytox water-soluble
tetrazolium salt (WST) cell proliferation assay kit (EZ-3000; DoGen Bio)
according to the manufacturer’s protocol. Briefly, S-HDFs (8 10)
were seeded in triplicate in 24-well plates containing complete medium, and the
cells were treated one to three times with piperine (70 M) at 2-day
intervals. Subsequently, the cells were incubated with EZ-Cytox solution, which
contains a WST, for 4 h at 37 C in a 5% CO incubator. Cell
proliferation was determined by measuring the absorbance at 450 nm using a
microplate reader.
2.6 SA--Gal Staining
SA--gal staining was performed as previously described [28]. Briefly,
S-HDFs were treated three times with piperine (70 M) at 2-day intervals.
Thereafter, the cells were fixed with 2% paraformaldehyde containing 0.2%
glutaraldehyde in phosphate-buffered saline (PBS) for 10 min at RT. Then, the
cells were washed twice with 1 PBS (pH 6.0) for 5 min each and
incubated in staining solution (1 mg/mL X-gal, 40 mM citric acid/sodium phosphate
buffer (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM
sodium chloride, and 2 mM magnesium chloride) at 37 C for 16 h. The
next day, we confirmed the presence of stained cells under an inverted
bright-field microscope and captured images of the cells.
2.7 Quantitative Analysis of SA--Gal-Stained Cells
We used MATLAB software (MathWorks Inc., Natick, MA, USA) to quantify the
SA--gal-stained cells. First, the captured images were inverted after
conversion to grayscale. Next, the image noise was removed via cutoff from 8
bit-images using the selected value (155). The sum of the total intensity was
obtained by adding all the pixels with a value greater than 0. The average
intensity per pixel was calculated by dividing the total intensity by the number
of pixels with a value greater than 0. The equation used was as follows: average
intensity = total intensity/number of pixels/number of cells. The
SA--gal-stained cells were quantified using five different image fields.
2.8 Western Blot Analysis
Western blot analysis was performed as previously described [28]. Briefly, total
proteins were extracted from the cells using radioimmunoprecipitation assay
buffer (Biosesang, Seongnam, Korea) containing Protease Inhibitor Cocktail
(Sigma-Aldrich, St Louis, MO, USA) and Phosphatase Inhibitor Cocktail I and II
(Sigma-Aldrich). The protein samples were separated using sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene
fluoride membranes (Millipore, Burlington, MA, USA). The membranes were incubated
with primary antibodies at 4 C overnight. The antibodies used were
anti-p16 (MA5-1742) from Invitrogen (Carlsbad, CA, USA); anti-p21
(sc-397) and anti--actin (sc-47778) from Santa Cruz Biotechnology
(Delaware, CA, USA); and anti-p38 (#8690), anti-phospho-p38 MAPK (Thr180/Tyr182)
(#9211), anti-p44/42 MAPK (Erk1/2), (#9102), anti-phospho-p44/42 (Erk1/2)
(Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb (#4370), anti-SAPK/JNK (#9252), and
anti-phospho-SAPK/JNK (Thr183/Tyr185) (#9251) from Cell Signaling Technology
(Danvers, MA, USA). The following day, the membranes were washed three times and
incubated with peroxidase-conjugated anti-mouse or anti-rabbit secondary
antibodies (Cell Signaling Technology) at RT for 1 h. Protein expression was
visualized using an enhanced chemiluminescence solution (DoGen) and analyzed
using Image J software (V 1.8.0) (National Institutes of Health, Bethesda, MD,
USA).
2.9 Measurement of Mitochondria Membrane Potential (MMP) and
Reactive Oxygen Species (ROS)
We used tetramethylrhodamine (TMRM) (I34361; Invitrogen) to analyze MMP and
dihydroethidium (DHE) (D23107; Molecular Probes, Eugene, OR, USA) to quantify the
levels of cellular ROS. Briefly, S-HDFs were treated with piperine (70 M)
or 5 mM NA (positive control) three times at 2-day intervals. After the piperine
treatment, the cells were stained with TMRM (100 nM) or DHE (5 M) at 37
C in the dark for 30 min. Subsequently, the cells were harvested,
washed with 1 PBS, and analyzed for TMRM or DHE fluorescence by flow
cytometry using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA,
USA). We counted 10,000 events for each sample, and the results were presented as
mean fluorescence intensity using a bar graph.
2.10 Analysis of SASP Production
S-HDFs were treated three times with piperine (70 M) at 2-day intervals.
Following treatment, the culture supernatants were collected and placed in
triplicates of 96-well plates. Then, the levels of the SASP secretion, including
interleukin (IL)-6, IL-8, and tumor growth factor (TGF)-1, in the
culture medium were measured by enzyme-linked immunosorbent assay (ELISA)
according to the manufacturer’s protocols. The absorbance was read at 450 nm
using a microplate reader.
2.11 Statistical Analysis
Statistical analysis was performed using GraphPad Prism software (V 8.0)
(GraphPad Software, San Diego, CA, USA). Data were presented as the mean
standard error of the mean of at least three independent experiments. The
differences between the experimental groups were analyzed for statistical
significance using the nonparametric Mann–Whitney U test. p values
0.05 were considered significant.
3. Results
3.1 Piperine Selectively Inhibits the Proliferation of Cancer Cells
and Induces Senescent Cell Growth
To confirm the effect of piperine on cancer cells, we treated various cancer
cell lines with piperine (70 M) and analyzed cellular proliferation and
cytotoxicity. The cancer cells showed cytotoxicity and their growth was
significantly inhibited by the piperine treatment (Fig. 1A and
Supplementary Fig. 1). Next, we compared the effect of piperine on HeLa
cells, cervical cancer cells, NS-HDFs, and S-HDFs by treating each cell type
twice with piperine (70 M) at 2-day intervals. Piperine selectively
induced cytotoxicity in HeLa cells but not in NS-HDFs and S-HDFs (Fig. 1B,C and
Supplementary Fig. 2). Interestingly, continuous treatment of S-HDFs
with piperine induced cell growth (Fig. 1D). These results indicate that piperine
affected cancer cells differently than S-HDFs, suggesting the application of
piperidine as both a cancer cell-specific therapeutic agent and a senomorphic
agent to improve senescent cell function.
Fig. 1.
Selective inhibition of cancer cell proliferation by piperine.
Various cancer cells were treated with piperine (70 M) for once. (A)
Cytotoxicity was measured by lactate dehydrogenase (LDH) assay in various cells.
HeLa and normal cells (NS-HDF and S-HDF) were treated twice with piperine (70
M) at 2-day intervals. (B) Morphological changes and (C) Cytotoxicity
between HeLa and normal cells was observed after treating piperine. S-HDFs were
treated three times with piperine (70 M) or 5 mM NA (positive control) at
2-day intervals. (D) Cell proliferation was measured by using a water-soluble
tetrazolium salt (WST) cell proliferation assay, and the number of viable cells
was counted using a hemocytometer. Data are based on three independent
experiments, and statistical significance between the experimental groups was
analyzed using the Mann–Whitney U test. *p 0.05 compared with
untreated and piperine- or NA-treated S-HDF; ***p 0.001 compared
with untreated and piperine-treated cancer cells (A) or compared with
piperine-treated HeLa and piperine-treated NS-HDF or S-HDF (C); ns, not
significant compared with piperine- and NA-treated S-HDF (D). NS-HDF,
nonsenescent human diploid fibroblast; S-HDF, senescent human diploid fibroblast;
CT26, mouse colon carcinoma cells; T98G, human glioblastoma cells; A431, human
skin carcinoma cells; MCF7, human breast adenocarcinoma cells; HepG2, human
hepatocellular carcinoma cells; HeLa, human cervix adenocarcinoma cells; NA,
nicotinamide. Scale bar, 200 m.
3.2 Piperine Induces Senescent Cell Proliferation
We extended the piperine treatment period to three times every 2 days to observe
the senomorphic effects of piperine on senescent cells (Fig. 2A). Cytotoxicity
was not observed in the S-HDFs after the cells had been treated with piperine
three times (Fig. 2B). The effect of piperine treatment was compared with that of
nicotinamide (NA), which induces senescent cell proliferation [29], to confirm
the effect of piperine treatment on S-HDF growth. Then, we performed a WST cell
proliferation assay and counted the number of viable cells. Interestingly,
piperine induced a higher rate of cell proliferation than NA in S-HDFs (Fig. 2C,D). The expression of p16 and p21, markers of senescent cells and cell
cycle checkpoints, respectively, was also significantly reduced in S-HDFs
following piperine treatment (Fig. 2E). These results show a novel effect of
piperine on senescent cells, suggesting that it induces cell division in
senescent cells as opposed to inducing cytotoxicity in cancer cells.
Fig. 2.
Effects of piperine on the proliferation of senescent cells.
(A) The chemical structural of piperine (top panel) and the experimental scheme
of piperine treatment in senescent cells (bottom panel). S-HDFs were treated
three times with piperine (70 M) at 2-day intervals. (B) Cytotoxicity was
measured using an LDH assay. (C) Cell proliferation was measured by treatment of
piperine (70 M) or 5 mM NA (positive control) using a WST cell
proliferation assay and (D) counted using a hemocytometer on the indicated days.
(E) The expression of p16 and p21 in piperine-treated S-HDFs was
analyzed by Western blot using specific antibodies. -actin was used as a
loading control. Data are based on three independent experiments, and statistical
significance between the experimental groups was analyzed using the Mann–Whitney
U test. *p 0.05 compared with untreated and piperine- or NA-treated
S-HDF, compared with piperine- and NA-treated S-HDF (C), and compared with NS-HDF
and S-HDF or compared with untreated and piperine-treated S-HDF (E); ns, not
significant compared with piperine- and NA-treated S-HDF (C) or compared with
NS-HDF and piperine-treated S-HDF (E). p 0.05 and
p 0.01 compared with untreated and piperine-treated S-HDF
(D).
3.3 Piperine Induces Extracellular Signal-Regulated Kinase (Erk1/2)
and c-Jun N-Terminal Kinase (JNK) Phosphorylation in Senescent Cells
Mitogen-activated protein kinases (MAPKs), including Erk1/2, p38, and JNK, have
been implicated in senescence phenotypes such as growth arrest [30, 31],
apoptosis resistance [32], and the SASP secretion [33, 34]. We examined whether
piperine regulates MAPK pathways in S-HDFs in an effort to elucidate the
mechanism of cell division S-HDFs following piperine treatment. Although p38
phosphorylation was unaffected in piperine-treated S-HDFs, Erk1/2 and JNK
phosphorylation were remarkably increased (Fig. 3A). We also investigated the
involvement of various signaling pathways, such as the 5’ adenosine
monophosphate-activated protein kinase pathway, mammalian target of rapamycin
(mTOR) pathway, and autophagy, in piperine-treated S-HDFs. Piperine treatment did
not affect signaling in S-HDFs (Fig. 3A, data not shown). We also investigated
these signaling pathways in HeLa cells. Piperine treatment increased the
phosphorylation of JNK, p38, and mTOR but not Erk1/2 (Fig. 3B). These results
suggest that the signaling mechanism of piperine in senescent cells differs from
that in cancer cells and imply that piperine activates Erk1/2 and JNK signaling
in senescent cells, leading to the reduction of cell cycle inhibitors
p16 and p21, thereby inducing the division of senescent cells.
Fig. 3.
Differential regulation of signaling in senescent cells and
cancer cells following piperine treatment. S-HDFs (A) and HeLa cells (B) were
treated with piperine (70 M) for 16 h. The proteins associated with
various signaling pathways, including the MAPKs and mTOR, were analyzed by
western blot using specific antibodies. Data are based on three independent
experiments, and statistical significance between the experimental groups was
analyzed by the Mann–Whitney U test. *p 0.05 and ns, not
significant compared with untreated and piperine-treated cells. Erk,
extracellular signal-regulated kinase; SAPK/JNK, stress-associated protein
kinase/c-Jun N-terminal kinase; mTOR, mammalian target of rapamycin.
3.4 Piperine Reverses Senescence Phenotypes with Modulating SASP
Secretion
To further determine whether piperine rescues cellular senescence phenotypes, we
examined the SA--gal activity of S-HDFs following piperine treatment (70
M) under the same experimental conditions. SA--gal activity was
markedly decreased in piperine-treated S-HDFs, but the morphology of S-HDFs
remained unchanged (Fig. 4A). Senescent cells are characterized by a decreased
MMP [35, 36] and increased production of intracellular ROS [37, 38]. Furthermore,
mitochondrial dysfunction and ROS accumulation are associated with age-related
diseases [38, 39, 40]. Thus, we determined the MMP and cytoplasmic ROS levels to
confirm the effect of the piperine on mitochondrial function in senescent cells.
Because NA leads to MMP recovery [41] and reduces ROS levels in senescent cells
[42], we used NA as a control for these experiments. After treating S-HDFs with
piperine (70 M) or 5 mM NA three times at 2-day intervals, the cells were
stained with TMRM to measure MMP or DHE to analyze intracellular ROS levels. We
found that piperine induced MMP (Fig. 4B) while reducing intracellular ROS levels
(Fig. 4C) in S-DHFs, which was similar to the effect of NA treatment. These
results suggest that the piperine not only induces the division of senescent
cells but also restores their functions. SASP secretion includes high levels of
IL-6, IL-8, and TGF-1 [43, 44, 45, 46]. Thus, controlling (modulating)
the secretion of SASP is crucial for the development of senotherapeutic agents.
To investigate the effects of piperine on SASP secretion from senescent cells, we
used ELISAs to determine the secretion levels of IL-6, IL-8, and TGF-1
in cultured senescent cells following piperine treatment. The SASP secretion from
S-HDFs was remarkably reduced by piperine treatment (Fig. 4D). We also examined
whether piperine affected IL-6 secretion in HeLa cells. As expected, piperine
treatment significantly induced IL-6 secretion (Fig. 4E). These findings implied
that piperine treatment restored the functions of senescent cells and suggest
that piperine is a novel senotherapeutic agent capable of suppressing SASP
secretion, which affects surrounding tissues.
Fig. 4.
Rejuvenation effects of piperine on senescent phenotypes.
S-HDFs were treated three times with piperine (70 M) at 2-day intervals.
(A) SA--gal activity was examined in piperine-treated S-HDFs. (B) MMP
function was determined by flow cytometry using the fluorescent dye TMRM. (C) ROS
levels were determined by flow cytometry using the fluorescent dye DHE. The
positive control was 5 mM NA. S-HDFs (D) and HeLa cells (E) were treated with
piperine (70 M) three times at 2-day intervals. Then, the supernatants
were collected and analyzed by ELISA for IL-6, IL-8, and TGF-1
secretion. Data are based on three independent experiments, and statistical
significance between the experimental groups was analyzed using the Mann–Whitney
U test. *p 0.05 compared with untreated and NA-treated S-HDF
(B) or compared with untreated and piperine-treated cells (D); **p
0.01 compared with untreated with untreated and piperine-treated S-HDF;
***p 0.001 compared with untreated and piperine-treated cells (Fig. 2D,E); ns, not significant compared with piperine- and NA-treated cells (Fig. 2B,C). SA--gal, senescent-associated -galactosidase; ROS,
reactive oxygen species; TMRM, tetramethylrhodamine; DHE, dihydroethidium; SASP,
senescence-associated secretory phenotype; IL, interleukin; TGF, tumor growth
factor.
4. Discussion
Cellular senescence is not only closely related to the occurrence and promotion
of cancer but also affects its treatment and recurrence. Although chemotherapy
kills cancer cells, it also provides an environment in which cancer can recur by
inducing senescence of the surrounding cells [47, 48]. When
cyclophosphamide was administered in an animal model with myc-induced lymphoma,
cancer cell senescence was induced by p16 and p53. The induction of
cancer cell senescence has been proposed as an effective cancer treatment method
with low toxicity. However, there are concerns that senescence may induce
resistance to potential cancer treatments [49]. Radiation therapy is also a
cancer treatment that damages DNA in cancer cells, and the subsequent ROS
generation affects cancer cells [49]. Severe DNA damage causes cell death, but
slight DNA damage induces cell senescence, and this phenomenon induces radiation
resistance [50]. In particular, the generation of senescent cells by radiation
therapy is a major cause of recurrence in glioblastoma. Therefore, drugs that
directly destroy senescent cells and inhibit SASP can be effective adjuvants for
cancer treatment.
Piperine inhibits the proliferation and survival of various cancer cell lines by
regulating the cell cycle and activating apoptosis-related signaling within cells
[51]. This compound directly modifies functions involved in the activity of
various enzymes and transcription factors involved in cancer cell invasion,
metastasis, and angiogenesis.
Similar to previous reports, piperine induced high cytotoxicity in various
cancer cell lines was not toxic to normal and senescent cells (Fig. 1).
Additionally, piperine stimulated cell division, decreased SA-ꞵ-gal activity,
recovered MMP, and reduced ROS generation in senescent cells, similar to the
effect of NA, which has been reported to restore the function of senescent cells
[29] (Figs. 2,4). We found that piperine differently affected cancer cells and
senescent cells due to the different responses of intracellular signaling
pathways. In senescent cells, piperine promoted Erk1/2 phosphorylation, which is
involved in cell growth, whereas in HeLa cells, a cancer cell line, Erk1/2
phosphorylation was decreased and JNK and p38 phosphorylation were increased
(Fig. 3). Interestingly, piperine induced different responses in senescent and
cancer cells, not only in cell signaling but also in SASP secretion. Piperine
increased SASP secretion in cancer cells while significantly decreasing the
secretion of three SASP factors (IL-6, IL-8, and TGF-1) in senescent
cells (Fig. 4). Because SASP might contribute to several side effects after
treatment with a cancer drug, these results provide piperine as the safe and
effective drug for cancer treatment.
From recent studies, the specific removal effects of senolytics on senescent
cancer cells have been demonstrated. The specific inhibitor of the BCL-2 family
(ABT263) successfully remove a range of senescent cancer cells and in
vivo study, ABT263 suppresses cancer recurrence and metastasis by eliminating
chemotherapy-induced senescent cells [52]. However, dasatinib+quercetin, another
senolytic cocktail drug, did not kill senescent hepatocellular carcinoma (HCC)
cells and reduce the growth of HCC [53]. Senostatics is also effective cancer
therapy by synergistic effects. Metformin leads reduction of prostate cancer
cells cultured with media from metformin-treated senescent cells by suppressing
SASP [52].
5. Conclusions
It is difficult to predict the various side effects of cancer treatment
substances because most cell models or animal models show the death of cancer
cells but do not prove the effect on surrounding normal cells. Piperine showed
the effect of inducing cancer cell-specific toxicity that does not affect normal
cells, and further showed the effect of restoring the function of senescent cells
that may exist around cancer cells. Therefore, we propose piperine as an
effective cancer treatment that can simultaneously induce senostatic effects and
the removal of cancer cells, not as an adjuvant to the existing senostatics for
cancer treatment.
Author Contributions
KAC and WKO designed the research study. JSL, DYL and JHL performed the
research. JTP and SCP advised experimental design. KAC and SCP analyzed the data.
KAC and JSL wrote the manuscript. All authors contributed to editorial changes in
the manuscript. All authors read and approved the final manuscript.
Ethics Approval and Consent to Participate
Not applicable.
Acknowledgment
We thank Young Sam Lee at DGIST for the kind discussion of the results.
Funding
This work was supported by the National Research Foundation of Korea (NRF) grant
funded by the Korean government (MSIT) (No. NRF-2017R1E1A2A02081815 and
NRF-2017R1E1A1A01074674; K.A.C.) and by the Basic Science Research Program,
through the NRF funded by the Ministry of Education (grant number
NRF-2018R1D1A1B07051207; J.S.L).
Conflict of Interest
The authors declare no conflict of interest.