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Published in final edited form as: Photochem Photobiol. 2020 Apr 13;96(4):870–876. doi: 10.1111/php.13228

Bucillamine Inhibits UVB-induced MAPK Activation and Apoptosis in Human HaCaT Keratinocytes and SKH-1 Hairless Mouse Skin

Adil Anwar 1, Hiba Anwar 1, Takeshi Yamauchi 1, Ryan Tseng 1, Rajesh Agarwal 2, Lawrence D Horwitz 3, Zili Zhai 1,*, Mayumi Fujita 1,4,*
PMCID: PMC7387142  NIHMSID: NIHMS1572740  PMID: 32077107

Abstract

Ultraviolet B (UVB) radiation is known as a culprit in skin carcinogenesis. We have previously reported that bucillamine (N-[2-mercapto-2-methylpropionyl]-L-cysteine), a cysteine derivative with anti-oxidant and anti-inflammatory capacity, protects against UVB-induced p53 activation and inflammatory responses in mouse skin. Since MAPK signaling pathways regulate p53 expression and activation, here we determined bucillamine effect on UVB-mediated MAPK activation in vitro using human skin kerotinocyte cell line HaCaT and in vivo using SKH-1 hairless mouse skin. A single low dose of UVB (30 mJ/cm2) resulted in increased JNK/MAPK phosphorylation and caspase-3 cleavage in HaCaT cells. However, JNK activation and casaspe-3 cleavage were inhibited by pretreatment of HaCaT cells with physiological doses of bucillamine (25 and 100 μM). Consistent with these results, bucillamine pretreatment in mice (20 mg/kg) inhibited JNK/MAPK and ERK/MAPK activation in skin epidermal cells at 6-12 h and 24 h, respectively, after UVB exposure. Moreover, bucillamine attenuated UVB-induced Ki-67-positive cells and cleaved caspase-3-positive cells in mouse skin. These findings demonstrate that bucillamine inhibits UVB-induced MAPK signaling, cell proliferation, and apoptosis. Together with our previous report, we provide evidence that bucillamine has a photoprotective effect against UV exposure.

Keywords: Bucillamine, UVB radiation, keratinocyte, MAPK activation, apoptosis

Graphical Abstract

graphic file with name nihms-1572740-f0005.jpg

Graphical abstract

Bucillamine, a cysteine derivative with anti-oxidant and anti-inflammatory capacity, protects against UVB-induced apoptosis via inhibition of MAPK activation in human HaCaT keratinocytes and SKH-1 hairless mouse skin.

INTRODUCTION

Solar ultraviolet (UV) radiation, especially UVB (290-320 nm), represents an important etiologic factor for a plethora of skin damages ranging from sunburn, photoaging to skin cancer (1, 2). UVB penetrates into epidermis and en route damages keratinocytes, leading to DNA damage, p53 activation, and apoptotic cell death (3, 4). Mounting evidence indicates that elevated levels of reactive oxygen species (ROS) and inflammation initiate and exacerbate UVB-induced photodamage (5, 6). Therefore, in order to protect against photodamage, photoaging, and photocarcinogenesis in the skin, it is important to lessen UVB-mediated ROS and inflammation. A myriad of natural and synthetic products have been evaluated for their photoprotective effects (5, 6).

Bucillamine (N-[2-mercapto-2-methylpropionyl]-L-cysteine) is a cysteine derivative that contains two donatable thiol groups, which allow it to act as an antioxidant (7, 8). Due to these two thiol groups, bucillamine is considerably more potent as an thiol antioxidant than N-acetylcysteine (NAC) and N-(2-mercaptopropionyl)-glycine (Tiopronin), two clinically used thiol drugs with a single thiol group each (9). Bucillamine also harbors an anti-inflammatory property, possibly via a mechanism independent of its antioxidant nature, because its fully oxidized metabolite also has a similar anti-inflammatory activity (10). Therefore, bucillamine is used in a number of conditions involving oxidative stress and inflammation (9). Bucillamine has been marketed in part of East Asia for the treatment of rheumatoid arthritis by daily oral administration at 100-600 mg (11, 12). We had previously evaluated the photoprotective effect of bucillamine on UVB-induced damage in an SKH-1 hairless mouse model (13). The results showed that the pretreatment of mice with bucillamine suppresses UVB-induced inflammatory response and p53 activation by phosphorylating at Ser15 and Ser20 residues (13). Due to the beneficial result of bucillamine on UVB-induced skin damage, it is worthwhile to further understand the mechanisms of photoprotective effects of bucillamine.

UVB-induced early damaging events and signaling pathways converge at p53 (14), which has a central role in the regulation of DNA repair, cell cycle, and apoptosis and is related to human skin carcinogenesis (15). Among the signaling pathways activated by solar UVB, mitogen-activated protein kinase (MAPK) signaling pathways play a critical role in the UVB-associated apoptotic effects (16, 14). Although the activating effects of UVB exposure on different types of MAPKs might be cell type-specific, JNK/MAPK is a direct signaling mediator of UVB-mediated p53 phosphorylation at Ser20 in human keratinocytes (17), while both ERK/MAPK and p38/MAPK are responsible for UVB-mediated p53 phosphorylation at Ser15 (18).

In the present study, we demonstrate the effects of bucillamine on UV-induced activation of MAPKs and subsequent apoptosis in vitro using cultured HaCaT cells and in vivo using SKH-1 hairless mouse skin.

MATERIALS AND METHODS

Bucillamine.

Powdered bucillamine (>99% purity) was purchased from Keystone Biomedical and dissolved in ethanol-containing saline as a stock solution of 200 mM. Due to its extreme acidity in solutions (9), bucillamine was neutralized with NaOH and then sterilized using a 0.2 μm filter before its usage in the following experiments.

HaCaT cell culture.

Human skin keratinocyte cell line HaCaT was obtained from the American Type Culture Collection (Manassas, VA) and grown in DMEM high glucose supplemented with 10% fetal bovine serum, 100 I.U./ml penicillin, and 100 μg/ml streptomycin (Mediatech, Manassas, VA). Cells were regularly monitored for mycoplasma contamination using a PCR method (19), and the cell line has been authenticated using short tandem repeat analysis by Barbara Davis Center BioResource Core at the University of Colorado Anschutz Medical Campus.

Cell viability assay.

To evaluate the cytotoxicity of bucillamine, HaCaT cells were seeded into a 96-well transparent plate at a density of 2 x 103 cells per well and allowed to adhere for 2 h before being exposed to different concentrations of bucilamine in quadruplicate for 48 h. Cell growth was measured using the CellTiter 96 Aqueous One Solution Cell Proliferation assay kit (Promega, Madison, WI).

UVB source.

We used the same UVB light source to expose both HaCaT cells and SKH-1 mice to UVB as described previously (20–22). The UVB light was a bank of four FS24T12-UVB-HO sunlamps equipped with a UVB Spectra 305 Dosimeter (Daavlin Co., Bryan, OH), which emitted ~ 80% radiation in the range of 280 - 340 nm with a peak emission at 314 nm as monitored with a SEL 240 photodetector, 103 filter, and 1008 diffuser attached to an IL1400A research radiometer (International Light, Newburyport, MA). The UVB irradiation doses were also calibrated using an IL1400A radiometer (13, 20–22).

HaCaT cell treatment.

Cells at 80% confluence were pretreated with varying doses of bucillamine for 3 h prior to UVB exposure. The cells were washed twice with phosphate-buffered saline (PBS) and then just enough amount of PBS was added to cover cells for exposure to a single low dose of UVB (30 mJ/cm2) (20, 2). Cells were re-cultured in complete medium overnight (16 h) before collection of cell lysates for immunoblotting analysis. Control cultures were identically manipulated but not irradiated.

Preparation of cell lysates.

UVB-exposed HaCaT cells were rinsed twice in ice-cold PBS and lysed in RIPA buffer (Sigma-Aldrich, St. Louis, MO) containing 1% (v/v) Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL). Supernatants were collected after centrifugation at 17,000 g for 10 min at 4°C. Protein concentrations in supernatants were determined using a commercial assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as a standard.

Animal treatment and tissue sample preparation.

We used the tissues collected and prepared from the previous study (13). Briefly, six-week-old female SKH-1 hairless mice were divided into four groups (n = 12 each except for the control group with n = 6): control group without UVB and without treatments, UVB alone group, UVB + saline group, and UVB + bucillamine group. Mice were injected two doses of saline or 20 mg/kg bucillamine subcutaneously, 24 h apart. The dosage and regimen of bucillame were chosen based on their therapeutic efficacy and no adverse effects in previously published data (9) and in our preliminary experiment as described earlier (13). Two hours after saline or bucillamine treatment, mice were exposed to 230 mJ/cm2 of UVB, total two doses with a 24-h interval. Four animals in each UVB-exposed group were sacrificed at 6, 12, and 24 h after the second UVB exposure, and the dorsal skin was surgically removed for preparation of skin lysates and formalin-fixed, paraffin-embedded tissue sections (13). We used the saved skin lysates and tissue sections for immunoblotting and immunohistochemical analysis, respectively, described below.

Immunoblotting.

Protein extracts were separated on 4-15% Mini-PROTEAN TGX precast gels (Bio-Rad), followed by electrotransfer onto Immobilon-P PVDF membranes (Millipore, Burlington, MA). After blocking with 5% nonfat milk, the immunoblots were incubated with primary antibodies and then with a horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich). The primary antibodies included rabbit antibodies against phospho-p44/42 MAPK (p-ERK1/2), p44/42 MAPK (ERK1/2), phospho-p38 MAPK, p38 MAPK, phospho-SAPK/JNK, SAPK/JNK, cleaved caspase-3 and PARP (Cell Signaling Technology, Danvers, MA) as well as mouse anti-β-actin (Sigma-Aldrich). Signals were visualized by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and analyzed using the Odyssey imaging system (LI-COR, Lincoln, NE). The band densities were quantified using the ImageJ software (NIH, MD).

Immunohistochemistry.

Ki-67 and caspase-3 staining was performed following the standard protocols as described previously (21). Briefly, skin tissue sections were deparaffinized, rehydrated, and immersed in 10 mM citrate buffer (pH 6.0, Sigma-Aldrich) in a pressure cooker at 120°C for 20 min for antigen retrieval. Endogenous peroxidase activity was quenched by 3% hydrogen peroxide for 20 min. After blocking with 1% bovine serum albumin, the sections were incubated with rabbit anti-Ki-67 (Abcam, Cambridge, MA) or anti-caspase-3 (Cell Signaling Technology) at 1:200 dilution in a humidity chamber at 4°C overnight, followed by incubation with conjugated HRP secondary antibody. Color development was achieved by incubation with the chromogen DAB (3,3’-diaminobenzidine) for 1-2 min. The sections were counterstained with hematoxylin (Sigma-Aldrich), dehydrated, mounted, and reviewed by two observers.

Statistical analysis.

GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA) was used for the statistical analysis. Numerical data are expressed as mean ± SD. The two-tailed Student’s t-test was used for comparison between two groups only while for comparison among more groups, one-way ANOVA with Bonferroni’s post-test was used. A value p < 0.05 was considered statistically significant.

RESULTS

Bucillamine is not toxic to HaCaT cells at physiological doses

Bucillamine is well tolerated by rheumatoid arthritis patients at 100-600 mg/day (11). To achieve a therapeutic effect, intravenous infusion of bucillamine for 3 h at 10 mg/kg/h or total 30 mg/kg is recommended, which could produce a maximal plasma drug level of approximately 200 μM (9). Although the half-life of bucillamine is relatively short, about 25 min, it has a good dose-proportionality for the area under the concentration time curve (9). Therefore, we evaluated the cellular effects of physiological doses and higher doses of bucillamine on the proliferation of HaCaT cells. Figure 1 shows that physiological doses of bucillamine (≤ 200 μM) had no effects on the cell growth rate, and, unexpectedly, we found that higher doses (400 and 1,000 μM) enhanced cell proliferation, suggesting that bucillamine has no toxicity to HaCaT cells at physiological doses.

Figure 1.

Figure 1.

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Effect of bucillamine on HaCaT cell viability. Total 2,000 cells were treated with varying concentrations of bucillamine for 48 h, and cell viability was determined using MTS assay. The data are expressed as the mean ± SD (n = 3-4). **p < 0.01 and ***p < 0.001 vs the untreated control.

Bucillamine inhibits UVB-induced MAPK activation and apoptosis in HaCaT cells

UVB-induced apoptotic cell death is dose- and time-dependent (22). Previous report showed a 10% inhibition of HaCaT cell viability at 48 h post a single dose of UVB exposure (30 mJ/cm2) when compared to the non-irradiated control cells (2). Hence, we investigated the effects of bucillamine on MAPK activation and apoptosis in HaCaT cells at 16 h post exposure to the same single low dose of UVB. We found that UVB induced MAPK activation, measured by phosphorylation of ERK and JNK, as well as increased apoptosis, measured by the presence of PARP and caspase-3 cleavage, in HaCaT cells when compared to the non-irradiated control cells (Figure 2). However, bucillamine at 25 and 100 μM of physiological doses and 400 μM of high dose obviously inhibited the phosphorylation levels of JNK as well as the cleavage of caspase-3; however, bucillamine had no effects on ERK and p38 phosphorylation/activation and a slight effect on PARP cleavage. These results indicate that bucillamine suppresses UVB-induced apoptotic cell death, partially through blocking JNK activation at the observed time point in vitro.

Figure 2.

Figure 2.

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Effect of bucillamine on UVB-induced MAPK activation and apoptosis in HaCaT cells. Cells were treated with varying doses of bucillamine for 3 h. After bucillamine removal, cells were exposed to UVB (30 mJ/cm2) and then incubated without bucillamine overnight (16 h), followed by the collection of cell lysates for Western blotting analysis of MAPK phosphorylation, PARP cleavage (Cl. PARP), and caspase-3 cleavage (Cl. casp-3). The representative imaging is shown with β-actin as loading control. The band densities were quantitated and the ratios of phosphorylated MAPK/total MAPK and Cl. PARP or Cl. casp-3/β-actin were calculated and normalized.

Bucillamine inhibits UVB-induced MAPK activation in SKH-1 hairless mouse skin

Next, we investigated the effect of bucillamine on MAPK activation in vivo. We used the skin lysates harvested in the mouse skin model of UVB damage (13), where SKH-1 mice were given two doses of bucillamine and UVB, and skin samples were collected after the second UVB irradiation. UVB induced MAPK activation even at 6 h post UVB exposure when compared to the non-irradiated mouse skin (Figure 3a–c). Among MAPKs, p38 and JNK phosphorylation was seen as early as 6 h after UVB exposure, but ERK phosphorylation was more obviously increased at 12 and 24 h post UVB exposure. Compared to UVB alone, normal saline had little effects on UVB-mediated MAPK activation. However, bucillamine pretreatment suppressed UVB-mediated MAPK activation compared to saline treatment at the same time points. For examples, bucillamine reduced JNK phosphorylation at 6 and 12 h post UVB irradiation when compared to UVB alone-treated mice, and reduced UVB-induced ERK phosphorylation at 12 and 24 h when compared to saline-treated controls. At 24 h time point, bucillamine also reduced UVB-induced p38 phosphorylation when compared to the saline-treated counterparts (Figure 3a–c). These data suggest that bucillamine at pharmacological doses protects against the activation of MAPK signaling in UVB-exposed mouse skin.

Figure 3.

Figure 3.

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Effect of bucillamine on UVB-induced MAPK activation in SKH-1 hairless mouse skin. Mice were treated with two doses of bucillamine (20 mg/kg) or saline (control), 24 h apart. Two hours of each bucillamine treatment, mice were exposed to UVB (230 mJ/cm2). Mice were sacrificed at 6, 12, and 24 h after the second UVB exposure, and the dorsal skin samples were collected for Western blotting analysis of ERK (a), p38 (b), and JNK (c) phosphorylation. The representative imaging is shown with β-actin as loading controls. The band densities were quantitated and normalized.

Bucillamine inhibits UVB-induced cell proliferation and apoptosis in SKH-1 hairless mouse skin

Since we observed the inhibitory effects of bucillamine on UVB-induced apoptosis in vitro and on MPK phosphorylation in vivo, we further evaluated the effects of the drug on UVB-mediated cellular proliferation and apoptosis in vivo by immunohistochemistry. It has been reported that UVB irradiation increases PCNA-positive cells in the epidermis of mouse skin (21). In consistent with this observation, we found increased nuclear immunostaining of Ki-67 in the epidermis of UVB-exposed mice at 6 h post UVB exposure compared to the non-irradiated mice, which was reduced by the pretreatment of mice with bucillamine (Figure 4a). Similar protective effects of bucillamine were observed at 24 h after UVB exposure. In addition, UVB induced apoptosis in mouse skin epidermis, indicated by the presence of cleaved caspase-3-positive cells. However, bucillamine treatment prior to UVB exposure resulted in a significant decrease in cleaved caspased-3-positive cells in the mouse skin epidermis (Figure 4b). These results support the protective role of bucillamine in UVB-induced skin cell apoptosis and damage.

Figure 4.

Figure 4.

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Effect of bucillamine on UVB-induced Ki-67-positive cells and cleaved caspase-3-positive cells in SKH-1 hairless mice skin. Mice were treated with bucillamine and UVB radiation as described in Figure 3. Representative skin sections were stained with Ki-67 (a) as a cell proliferation marker and cleaved caspase-3 (b) as an apoptosis marker. Arrows indicate cleaved caspase-3-positive cells. Bar = 20 μm. Quantitative analysis of Ki-67- and cleaved caspase-3-positive cells was performed with cell counting in several different areas of the skin. Mean ± SD (n = 4). *p < 0.05 and **p < 0.01 vs the non-irradiated control. ##p < 0.01 and ###p < 0.001 vs the untreated control at the same time point.

DISCUSSION

Many studies have implicated the detrimental effects of repeated UVB exposure on human skin, particularly acting as an environmental trigger of melanoma and non-melanoma skin cancers (1, 23). UVB-induced skin damage is associated with DNA lesions, which, when left unrepaired, present their mutagenic potential, leading to the activation of proto-oncogenes or the inactivation of tumor suppression genes (24). Whereas acute, high doses of UVB cause irreparable DNA damage and subsequent apoptosis, chronic, low doses of UVB induce DNA mutation, leading to skin cancer development (22).

In response to DNA damage following UVB exposure, the nuclear transcription factor p53 is induced at protein level and exerts transcriptional regulation of other genes through its phosphorylation and activation (25, 26). As a tumor suppressor, activated p53 induces cell cycle arrest that facilitates DNA repair and/or apoptosis, On the contrary, mutated p53 initiates malignant transformation (26). The p53 protein has multiple phosphorylation sites, including ser15, ser20, ser33, and ser46, and these phosphorylation residues are involved in the cellular responses to UVB exposure and DNA damage (14, 23). MAPKs have been demonstrated to activate p53 activation by direct phosphorylation and thus regulate the transcriptional function of p53 and its downstream target genes, such as PUMA and caspase-3 (27, 14). Activated caspase-3 through cleavage then participates in apoptosis and aggravates skin aging (28). Our findings of bucillamine inhibition of UVB-mediated MAPK activation indicate that this drug regulates the upstream signaling pathways of p53 probably via MAPK regulation, thus reducing p53 expression and its transcriptional function as observed in our previous report (13).

Bucillamine was originally developed in Japan and used as a first-line drug for the treatment of rheumatoid arthritis since late 1980s (12). Although bucillamine is not currently used worldwide, this thiol compound has an enormous potential for developing to be alternative therapy for other conditions characterized by oxidative stress and inflammation. Animal studies showed that bucillamine provides a certain protection against myocardial reperfusion injury (7), ovalbumin sensitization (8), and choroidal neovascularization (29). This drug is being tested in patients with cystinuria (30) and gout flare (31). Bucillamine has been proven to exhibit ROS scavenging, anti-inflammatoty, and immunomodulating activities (7, 8, 10).

In addition to DNA damage, UVB exposure triggers an array of biological responses and molecular events involving ROS generation, inflammation, and activation of signaling transduction pathways (6). Considering the effects of bucillamine to scavenge ROS and inhibit oxidation and inflammation, it is reasonable to understand that many of UVB-mediated biological events are regulated by bucillamine. Among the signaling pathways stimulated by UVB, MAPKs (especially p38 and JNK (23, 27)) are undoubtedly playing an important role by regulating UVB damaging effects. As an effector of cellular stress responses, MAPK signaling determines cell fate by controlling cell cycle progression and elicits either pro- or anti-survival mechanism (23). MAPKs can directly regulate p53 activity or activate it through their other downstream targets, such as transcription factors cAMP-responsive element-binding protein and activator protein-1 (32, 33), which are both involved in inflammation, cell proliferation, and tumorigenesis. Therefore, the inhibitory effect of bucillamine on MAPK signaling may be widespread, not just limited to p53 inactivation.

Effective photoprotective agents are important for prevention of skin aging and skin cancer development. Many synthetic and natural compounds have been reported to possess beneficial effects on cell damage and skin injury post-exposure to UVB. The nonsteroidal anti-inflammatory drug sulindac and its metabolites attenuate UVB-induced inflammatory responses and reactions relevant to carcinogenesis (34, 35). The photoprotective effects of antioxidant phytochemicals have been extensively studied, including flavonoids such as epigallocatechin gallate and silibibin as well as non-flavonoids such as resveratrol and phenolic acids. Similar to bucillamine, these synthetic and natural chemicals exert robust inhibition of MAPKs as one of the mechanisms for the prevention or treatment of UVB-mediated damage (35, 5, 6, 36–38). Therefore, the inhibition of MAPKs by bucillamine and phytochemicals may be resulted, at least partially, from scavenging ROS and free radicals, which drive MAPK activation due to UVB (6). However, the mechanistic basis for the inhibition of MAPKs by sulindac and its metabolites seems to be complex, since they not only scavenge ROS in chemical systems (39), but also enhance the production of ROS in cancer cells (40). In addition to antioxidant and anti-inflammatory activities, many phytochemicals display a wide pharmacological profile, such as immunomodulation and dermal extracellular matrix remodeling (35, 5). The role of bucillamine in photoprotection besides its thiol-dependent antioxidant and anti-inflammatory activities is still unclear and needs further study.

In summary, our data demonstrate that bucillamine attenuates UVB-mediated MAPK activation and apoptosis in both HaCaT cells and mouse skin, indicating that bucillamine may be effective for preventing and relieving sun exposure-related skin damage.

ACKNOWLEDGEMENTS

This work was supported, in whole or in part, by Veterans Affairs Merit Review Awards 5I01BX001228 (MF), NIH/NCI R01 CA197919 (MF), and Cancer League of Colorado (MF). We thank the University of Colorado Cancer Center Support Grant (P30CA046934), the Skin Diseases Research Cores Grant (P30AR057212), and the Gates Summer Internship Program for their support.

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