Citric acid induced cell cycle arrest and apoptosis in human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent
signaling pathways
Tsung-Ho Ying1,2, Chia-Wei Chen1,Yu-Ping Hsiao1,3, Sung-Jen Hung4, Jing-Gung Chung5*, Jen-Hung Yang4,*
1Institute of Medicine, School of Medicine, Chung Shan Medical University, Taichung, Taiwan
2Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, Taichung, Taiwan
3Department of Dermatology, Chung Shan Medical University Hospital, Taichung, Taiwan
4Tzu Chi University School of Medicine; and Department of Dermatology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan
5School of Biological Science and Biotechnology, China Medical University, Taichung, Taiwan
Tsung-Ho Ying and Chia-Wei Chen contributed equally to this work
*Correspondence: Jen-Hung Yang, MD, Ph.D.; Tzu Chi University School of Medicine; and Department of Dermatology, Buddhist Tzu Chi General Hospital No.701, Zhongyang Rd., Sec .3, Hualien, 97004 Taiwan. Tel: 886-3-8565301; E-mail address: [email protected] (J.-H. Yang)
*Correspondence: Jing-Gung Chung, Ph.D. Department of Biological Science and Technology, China Medical University. No 91, Hsueh-Shih Road, Taichung 40402, Taiwan. Phone: +886-4-2205-3366 ext 2501; FAX: +886-4-2205-3764, Email:
Abstract
Cosmetic dermatology and skin care products are hot topics in recent years, and citric acid (CA) is one of alpha-hydroxyacids (AHAs) have been widely used. However, there is concern about its safety for skin. In this study, we investigated the cytotoxic effect of citric acidin human keratinocyte cell line (HaCaT). HaCaT cells were treated with CA at 2.5~12.5 mM for various time periods. The molecular mechanisms of anti-proliferation through cell cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) stain, flow cytometry, Western blot and confocal microscopy. HaCaT cells were treated with CA at 2.5~12.5 mM for various time periods. The molecular mechanisms of anti-proliferation through cell cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) stain, flow cytometry, Western blot and confocal microscopy. Citric acid not only inhibits proliferation of HaCaT cells in a dose-dependent model, but also induces apoptosis and cell cycle arrest at G2/M phase (before 24 hours) and S phase (after 24 hours). Citric acid increased the level of Bax and inhibited the levels of Bcl-2, Bcl-xL and activate caspase 9 and caspase 3, which subsequently induce apoptosis via caspase-dependent and caspase-independent pathway. Citric acid also activated death receptor and increased the levels of caspase 8, activated Bid protein, AIF, and EndoG. Therefore, citric acid could induce apoptosis through mitochondrial pathway in human keratinocyte cell line (HaCaT). The study results suggested that citric acid is cytotoxic to HaCaT cells via the mechanisms of induction of apoptosis and cell cycle arrest in vitro.
Citric acid (CA) is a sort of the α-hydroxy acids (AHAs) which was widely used in cosmetic products for years . AHAs attract customers with the potential re-juvenate ability to reduce wrinkles, spots, and other signs of photo-aging. .Citric acid with concentration of 20 % increased the thickness of the epidermisand increased glycosamino glycans on the sun-damaged skin . Citric acid also would induce collagen I and procollagen II proliferation . Behind these re-juventative effects, the Food and Drug Administration pronounced a particular concern about AHAs because capability of penetrating the skin barrier and skin irritations Being concerned about the safety of AHAs, especially long term application to the skin, we are interested in exploring the anti-proliferative and apoptotic effects of AHAs in human keratinocytes . We had evaluated the cytotoxic effects of glycolic acid and lactic acid in previous studies, but the in vitro effects of citric acid (CA) still remain unclear. In this study, we investigated the effects of CA in the human keratinocyte cell line (HaCaT).
Materials and methods
Chemicals and reagents. Citric acid was obtained from Sigma Chemical Co. (St. Louis, MO, USA). propidium iodide (PI), RNase A, and Triton X-100 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco's Modified Eagle's Medium (DMEM), penicillin- streptomycin, trypsin-EDTA, fetal bovine serum (FBS) and L-glutamine were obtained from GIBCO®/Invitrogen (Carlsbad, CA, USA). DCFH-DA, DiOC6 and Fluo-3/AM were obtained from Molecular Probes/Invitrogen (Eugene, OR, USA). Fetal bovine serum, penicillin–streptomycin, trypsin–EDTA and glutamine were obtained from Gibco BRL (Grand Island, NY, USA). Caspase-3 activity assay kit was purchased from Roche Diagnostics (Mannheim, Germany). All of the chemicals used were reagent grade.
Human immortalized keratinocytes (HaCaT) cell line. Cultures of HaCaT cells were kindly provided from Dr. Norbert E. Fusenig (Institute of Biochemistry, German Cancer Research Center) and were grown on Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% L-glutamine, 25mM HEPES, 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA) at 37℃ in a humidified incubator with 5% CO2 atmosphere .
Assessment of cell morphological changes and viability. HaCaT cells at the density of 2×105 cells/well were placed onto 12-well plates and incubated at 37°C for 24 h before being treated with various concentrations of CA for 24 h, 0.5% of DMSO (solvent) was used for the control regimen. At the end of incubation, cells were examined and photographed under contrast phase microscope for morphological changes determination. Then, Cells (1×105 cells per sample) were centrifuged at 1000xg for 5 min, cell pellets were dissolved with 0.5 ml of PBS containing 5 μg/ml PI and viable cells were determined by using a flow cytometer (Becton-Dickinson, San Jose, CA, USA) for determination of viable cells as previously described .
Assessment of cell cycle and apoptosis by flow cytometry. HaCaT cells treated with CA at concentrations were incubated in an incubator for different time periods before the cells were harvested by centrifugation. The cells were fixed gently by 70% ethanol at 4℃ for overnight and were then re-suspended in PBS containing 40 mg/ml PI and 0.1 mg/ml RNase and 0.1% Triton X-100 in a dark room. After incubation at 37℃ for 30 min, the cell cycles and apoptosis were analyzed with a flow cytometer .
Cells at a density of 2×105 cells/well were placed onto 6-well plates and treated with CA (0, 2.5, 5, 7.5, 10 and 12.5 mM) and incubated for 24 h before cells from each treatment were isolated for DAPI staining as described previously . After staining, the cells were examined and photographed using a fluorescence microscope.
Detection of the changes of levels of reactive oxygen species (ROS), Ca2+, and mitochondrial membrane potential (MMP) in HaCaT cells by flow cytometry. The levels of ROS, Ca2+, and MMP of the HaCaT cells were determined by flow cytometry. HaCaT cells were treated with or without CA (12.5 mM) for different time periods (0, 1, 6, 12, 24, and 48 h). The cells were harvested and washed twice, resuspended in 10 µM 2,7-dichlorodihydrofluorescein diacetate (Sigma-Aldrich) and incubated at 37℃ for 30 min and the levels of ROS were analyzed by flow cytometry. We calculated the percentages of ROS by using CellQuest software (Becton- Dickinson) and flow cytometer (Becton-Dickinson). Intracellular ROS was detected by means of an oxidation-sensitive fluorescent probe (DCFH-DA). In the presence of ROS, DCFH-DA was subsequently transferred to DCF and emitted a green fluorescent signal detected by flow cytometry. The closed area was control group, and the open area was experimental group. We used CellQuest and calculated the ratios of the open area / closed area as the percentages of ROS
Cells were treated with CA at 12.5mM for different time (0, 0.5, 1, 6, 12, 24, and 48 h) before detection of the changes of Ca2+ concentrations. The cells were harvested and washed twice, resuspended in a buffer containing Indo-1-AM (3 mg/ml, Calbiochem, La Jolla, California, USA) and incubated at 37°C for 30 min and the level of Ca2+ was analyzed by flow cytometry .
For detecting the changes of levels of MMP in HaCaT cells, HaCaT cells were treated with or without CA (12.5mM) for different time periods (0, 0.5, 1, 6, 12, 24,
and 48 h), the cells were harvested and washed twice, resuspended in 500 µl of DiOC6 (4 mol/L) and incubated at 37°C for 30 min, and subsequently analyzed by flow cytometry .
Assays for Caspase-3, Caspase-8 and Caspase-9 Activities. Cells (total 2×105 cells) in 12-well plates were incubated with 12.5 mM of CA and 0.1% DMSO (vehicle-control) cells for 0, 24, and 48 h. Cells were harvested, and a 50 l of 10 M substrate solution (CaspaLux8-L1D2 and CaspaLux9-M1D2 kits) were then added to the cell pellet. Cells then were incubated at 37℃ for 60 min and were washed once by adding 500 l of fresh PBS. The caspase-3, caspase-8 and caspase-9 activities were analyzed by flow cytometry as previously described .
Apoptotic associated proteins were examined by Western blotting. HaCaT cells at a density of 1×106 cells/ml cells in 6-well plates were treated with 12.5 mM of CA for 0, 12, 24 and 48 h. Cells were harvested from each treatment by centrifugation for the total proteins determination as Bio-Rad kit for western blotting. The protein levels of Bax, Bcl-2, Bcl-xL, AIF, Endo G, cytochrome c, PARP, Fas, Bid, GRP78, and GADD153 were examined by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting as described previously .
Confocal microscopy to detect apoptosis-inducing factor (AIF), endonuclease G (EndoG) and cytochrome c in HaCaT cells exposed to CA. HaCaT cells were gently fixed in fixed buffer (400 mM sucrose, 2 mM EGTA, 4% paraformadehy in PBS) for 15 min, and then, the cells were permeabilized with 0.1% Triton-X in PBS for 5 min. After rinsing cells with PBS, added blocking solution (1% BSA in PBS) for 1 h, and subsequently stained with a mouse monoclonal antibody against EndoG (Santa Cruz
Biotechnology, at 1:200 dilution) or a goat polyclonal antibody against AIF (Santa Cruz Biotechnology, at 1:200 dilution) or a rabbit polyclonal antibody against cytochrome c (Santa Cruz Biotechnology, at 1:200 dilution) at 4°C overnight. After rinsing with PBS, cells were stained with FITC-conjugated fluorescent secondary antibody (Santa Cruz Biotechnology, at 1:200 dilution) for 2 h at room temperature. The cells were rinsed with PBS containing 1 μg/ml PI to act as a nuclear counter stain, and then washed with ice-cold PBS. After mounted with a mounting medium, the cells were examined with Zeiss LSM 510 META microscope fitted with appropriate fluorescence filters .
Statistical analysis. The results are presented as mean ±S.E.M, and the difference between the PEITC-treated andcontrol groups was analyzed by Student’s t-test, a probability of *, P <0.05, *** P <0.001 being considered significant.
Results
CA inhibited cell viability and morphological changes in HaCaT Cells. After HaCaT cells were treated with various concentrations of CA for 24 h, the cell morphological changes also occurred from CA-treated cells and the results are shown in Figure 1A. The cell viability significantly decreased in a dose-dependent manner (Figure 1B). The half maximal inhibitory concentration (IC50) value of CA was close to 12.5mM at a 24-h exposure. Thus, this concentration at 12.5mM of CA was applied for all subsequent experiments.
CA induced cell cycle arrest and apoptosisin HaCaT Cells. The flow cytometry disclosed that treatment of CA at 12.5 mM increased the proportion of cells at G2/M
phases before 24 h, increased the proportion of cells at S phase after 24 h in HaCaT cells (Figure 2A). CA also induced a distinct subG1 peak, which represents the population of apoptotic cells. The increase in the proportions of apoptosis was associated with higher concentrations of CA in Figure 2B.
Apoptotic cells examined by DAPI staining. These CA-treated cells were variable in cell size with less number and fluorescent light than that of control sample(Figure 3). Moreover, cells after treatment with CA displayed chromosomal condensation and formation of apoptotic bodies (Figure 3). These effects are in a dose-dependent manner.
CA decreased the levels of mitochondrial membrane potential (MMP), increased the levels of ROS and Ca2+ accumulation in HaCaT cells. To investigate whether CA induces cell death through the dysfunction of mitochondria, ROS, and Ca2+ accumulation, cells were harvested after exposure to CA and were assayed by flow cytometry. Our results, shown in Figure 4A-4C, indicated that CA reduced the level of MMP (Figure 4A) and promoted the production of ROS (Figure 4B)and Ca2+ accumulation(Figure 4C) in a time-dependent. In addition, caspase-3, capase-8 and -9 activities were promoted by CA (Fig. 4D-4F) in HaCaT cells.
Effect of CA on the expressions of apoptosis-related proteins in HaCaT cells. HaCaT cells were treated with CA at 12.5 mM for different time periods (0, 12, 24, and 48 h). In Figure 5A-5D, CA promoted the expression of apoptosis-related protein levels such as Fas, Bax, cytochrome c, PARP, AIF, EndoG, GRP 78, and GADD153 but decreased the levels of Bcl-2, Bcl-xL, and Bid that led to cell apoptosis in HaCaT cells.
Confocal microscopy showing the release of apoptosis-inducing factor (AIF), endonuclease G (EndoG) and cytochrome c in HaCaT cells exposed to CA. To investigate the mechanism underlying apoptosis induced by CA, we tested the effects of CA on AIF and Endo G localizations in HaCaT cells. Thus, our hypothesis was that CA-induced apoptosis in HaCaT may be mediated through the mitochondria -dependent pathways for releasing the AIF and Endo G. The results from confocal laser microscope indicated that CA promoted the release of AIF (Figure 6A), Endo G (Figure 6B) to nuclei in HaCaT cells.
Discussion
Although several reports have declared the safety concern of α-hydroxy acids (AHAs), the exact molecular mechanisms of citric acid (CA) in human epidermal keratinocytes still remain unclear . According to the recommendation of the Department of Health, R.O.C., the concentration of AHAs must be less than 10%, pH 3.5 or more in the nature for consumers . However, chemical peeling agents contained higher concentrations of AHAs (20-70%) were used in the hospitals and local practitioner’s clinics . We worried about the cell toxicity of citric acid to skin, and herein, we investigated CA affecting human epidermal keratinocytes (HaCaT cells) and the results indicated that CA induced cell dead hand morphological changes through the cell cycle arrest and induction of apoptosis and apoptotic characteristics which were observed by DAPI staining and sub-G1 phase by flow cytometric assay. HaCaT cells treated with CA showed apoptotic features such as chromatin condensation and apoptotic bodies, suggesting the morphological apoptotic features of HaCaT cells. To confirm the apoptotic action of CA, cell cycle analysis was
performed. CA also significantly increased sub-G1 peaks in a time-dependent manner in HaCaT cells, indicating the occurrence of apoptosis in HaCaT cells.
Apoptosis can be divided into intrinsic and extrinsic pathways through caspase-dependent and-incaspase-dependent pathways , based on the activation of caspase family members and is a critical component of the apoptotic machinery of caspase-dependent pathways. It was reported that caspase-8, -9, and -10 are the upstream initiator caspases and caspase-3, -6, and -7 are the downstream effectors caspases . Our results from Fig. 4D, 4E and 4F indicated that CA induced caspase-3, -8 and -9 activities, and from Fig. 5C indicated that CA induced Fas protein expressions. Theses data implying both intrinsic and extrinsic pathways are also involved in CA induced apoptosis. We also used FITC-conjugated immuno-fluorescent staining before using confocal laser system microscope to examine apoptosis associated AIF and EndoG proteins translocation from cytoplasm into nuclei and the results indicated that CA promoted the expressions of AIF (Fig. 6A), and EndoG (Fig. 6B) as well as stimulations of intracellular Ca2+ release(Fig. 4C), which also indicated that CA-induced apoptosis in HaCaT cells are part through caspase-independent pathways.
In recent years, numerous studies have demonstrated that AIF-induced caspase-independent apoptosis under various conditions . Furthermore, Ca2+ has been shown to involve AIF cleavage and release from mitochondria . In the present study, our findings also indicated that CA promoted Ca2+ levels (Fig. 4C) and AIF release (Fig. 5B) from mitochondria which indicated that CA induced apoptosis in HaCaT cells via the caspase-independent pathways. Results from Western blotting (Fig. 5C) indicated that CA induced apoptosis might be through Fas receptor to activatecaspase-8 or through mitochondria pathways. Results also showed that CA promoted the expression of pro-apoptotic proteins Bid, Bax, PARP, and decreased the
anti-apoptotic proteins Bcl-2 and Bcl-XL (Fig. 5A). It is well known that the ration of Bax/Bcl-2 are involved with the levels of mitochondrial membrane potential (MMP) . Our results also showed that CA decreased the levels of MMP, leading to cytochrome c release, activations of caspase-9 followed by the activation ofcaspase-3 for causing apoptosis.
In summary, immortalized human epidermal keratinocytes (HaCaT) cells after treatment with the CA process exhibit apoptotic features of DNA damage, apoptotic bodies, and an increase of sub-G1 peaks, resulting from activations of caspase-8, -9, and -3 and the induction of AIF and Endo G release from mitochondria. These findings are summarized in Fig. 7.
Conclusion
In conclusion, we demonstrated the apoptotic and cell toxic effect of CA in a human keratinocytes cell line (HaCaT) in vitro. Our investigations highlight consumers and physicians the potential risks of CA and/or other AHA-containing cosmetic products.
Acknowledgements
This work was supported by the grant NSC97-2314-B-040-026, NSC102-2314-B-303-002 from the National Science Council, Taiwan, Republic of China, the grant TCRD102-42 from Buddhist Tzu Chi General Hospital, and the grant CSH-2011-D-002from Chung Shan Medical University Hospital.
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Legends to Figures
Figure 1. Citric acid treated HaCaT cells morphological changes were examined and
photographed under contrast phase microscope at 24 h (A). The percentage of viable cells was measured by flow cytometric assay at 24 h (B). Data are presented as the mean ±SD of three independent experiments. *, P<0.05, ***, P<0.001 significantly different compared with control treatment.
Figure 2. The effects of CA on cell cycle distribution in HaCaT cells. Cells were
treated with 12.5mM of CA for 0, 12, 24, 48, and 72 h (A). The cell cycle distribution
and sub-G1 group (apoptosis phase) were determined using flow cytometric analysis
(B) and obtained from three independent experiments with similar results. Data
represent mean ± SD of the results from three experiments (n=3). *P<0.05.
Figure 3. HaCaT cells were incubated with CA at various concentrations for 24 h.
There was an increase in the number of higher intensity DAPI-staining cells and
fragmented nuclei at higher concentrations of CA treatment (the size of scale bar was
20 μm, fluorescence microscope, x200).
Figure 4. CA affected the levels of mitochondrial membrane potential (MMP), ROS,
Ca2+, caspase-3, -8 and -9 activities in HaCaT cells. Cells were treated with 12.5 mM
of CA for various time periods. All samples were analyzed by flow cytometric assay
independent experiments. *, P <0.05, ***, P <0.001, significantly different compared
with CA-treatment.
Figure 5. Western blot demonstrated the increase of Fas, Bax, Bid caspase-3, -8, -9,
AIF, Endo G, cytochrome c, PARP, GADD153, GRP78 and decrease of Bcl-2
andBcl-xl in HaCaT cells exposed to CA at 12.5 mM for different incubation periods.
Figure 6. Confocal microscopy showed a marked increase in the intra cytosolic
release of AIF and EndoG in the cytosol; in addition, we found the translocation of
AIF and EndoG from cytoplasm to the nucleus in HaCaT cells treated with CA at 12.5
mM for 24h. AIF, EndoG, and cytochrome c stained by FITC showed green
fluorescence, whereas nucleus stained by DAPI disclosed blue fluorescence (the size
of scale bar was 20 μm).
Figure 7. Molecular pathways involved in effects of CA in HaCaT cells. CA inhibits
