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 ${}^{\circ }$C in a 5% CO${}_2$ incubator. The MCF7 cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS and 1% P/S at 37 ${}^{\circ }$C in a 5% CO${}_2$ 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 $\beta$-galactosidase (SA-$\beta$-gal) staining. All cell culture reagents were purchased from Gibco-BRL (Grand Island, NY, USA).

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 ${}^{\circ }$C in a 5% CO${}_2$ incubator and treated once or twice with piperine (70 $\mu$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 ${}^{\circ }$C in a 5% CO${}_2$ incubator with piperine (70 $\mu$M) three times at 2-day intervals to test the senomorphic effect of piperine on S-HDFs.

Cytotoxicity was assessed using an LDH assay kit (DG-LDH500; DoGen Bio, Seoul, Korea) according to the manufacturer’s protocol. Briefly, cancer cells (2 $\times$ 10${}^5$) were seeded in triplicate in six-well plates and then treated once or twice with piperine (70 $\mu$M) at 2-day intervals. Normal cells, such as NS-HDFs (6 $\times$ 10${}^4$) and S-HDFs (3 $\times$ 10${}^4$), were seeded at triplicate in six-well plates and treated two to three times with piperine (70 $\mu$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${}^{\prime }$,6${}^{\prime }$-diamidino-2-phenylindole (DAPI) after once or twice treatment of piperine. The stained cells were analyzed with the Zeiss LSM 700 confocal microscope.

To count the viable cells, we seeded various cancer cell lines (2 $\times$ 10${}^5$) in six-well plates containing complete medium and treated them with piperine (70 $\mu$M) for 24 or 48 h. We also seeded S-HDFs (3 $\times$ 10${}^4$) in 6-well plates containing complete medium and treated them with piperine (70 $\mu$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.

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 $\times$ 10${}^3$) were seeded in triplicate in 24-well plates containing complete medium, and the cells were treated one to three times with piperine (70 $\mu$M) at 2-day intervals. Subsequently, the cells were incubated with EZ-Cytox solution, which contains a WST, for 4 h at 37 ${}^{\circ }$C in a 5% CO${}_2$ incubator. Cell proliferation was determined by measuring the absorbance at 450 nm using a microplate reader.

SA-$\beta$-gal staining was performed as previously described [28]. Briefly, S-HDFs were treated three times with piperine (70 $\mu$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 $\times$ 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 ${}^{\circ }$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.

We used MATLAB software (MathWorks Inc., Natick, MA, USA) to quantify the SA-$\beta$-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-$\beta$-gal-stained cells were quantified using five different image fields.

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 ${}^{\circ }$C overnight. The antibodies used were anti-p16${}^{\text{INK4a}}$ (MA5-1742) from Invitrogen (Carlsbad, CA, USA); anti-p21 (sc-397) and anti-$\beta$-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).

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 $\mu$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 $\mu$M) at 37 ${}^{\circ }$C in the dark for 30 min. Subsequently, the cells were harvested, washed with 1 $\times$ 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.

S-HDFs were treated three times with piperine (70 $\mu$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)-$\beta$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.

Statistical analysis was performed using GraphPad Prism software (V 8.0) (GraphPad Software, San Diego, CA, USA). Data were presented as the mean $\pm$ 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.

To confirm the effect of piperine on cancer cells, we treated various cancer cell lines with piperine (70 $\mu$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 $\mu$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 $\mu$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 $\mu$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 $\mu$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 $\mu$m.

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${}^{\text{INK4a}}$ 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 $\mu$M) at 2-day intervals. (B) Cytotoxicity was measured using an LDH assay. (C) Cell proliferation was measured by treatment of piperine (70 $\mu$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${}^{\text{INK4a}}$ and p21 in piperine-treated S-HDFs was analyzed by Western blot using specific antibodies. $\beta$-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). ${}^{\mathrm{\#}}$p 0.05 and ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 compared with untreated and piperine-treated S-HDF (D).

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${}^{\text{INK4a}}$ 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 $\mu$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.

To further determine whether piperine rescues cellular senescence phenotypes, we examined the SA-$\beta$-gal activity of S-HDFs following piperine treatment (70 $\mu$M) under the same experimental conditions. SA-$\beta$-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 $\mu$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-$\beta$1 [43, 44, 45, 46]. Thus, controlling (modulating) the secretion of SASP is crucial for the development of senotherapeutic agents. To investigate the effects of piperine on SASP secretion from senescent cells, we used ELISAs to determine the secretion levels of IL-6, IL-8, and TGF-$\beta$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 $\mu$M) at 2-day intervals. (A) SA-$\beta$-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 $\mu$M) three times at 2-day intervals. Then, the supernatants were collected and analyzed by ELISA for IL-6, IL-8, and TGF-$\beta$1 secretion. Data are based on three independent experiments, and statistical significance between the experimental groups was analyzed using the Mann–Whitney U test. *p 0.05 compared with untreated and NA-treated S-HDF (B) or compared with untreated and piperine-treated cells (D); **p 0.01 compared with untreated with untreated and piperine-treated S-HDF; ***p 0.001 compared with untreated and piperine-treated cells (Fig. 2D,E); ns, not significant compared with piperine- and NA-treated cells (Fig. 2B,C). SA-$\beta$-gal, senescent-associated $\beta$-galactosidase; ROS, reactive oxygen species; TMRM, tetramethylrhodamine; DHE, dihydroethidium; SASP, senescence-associated secretory phenotype; IL, interleukin; TGF, tumor growth factor.