The Mechanisms of Carnosol in Chemoprevention of Ultraviolet B-Light-Induced Non-Melanoma Skin Cancer Formation

Carnosol is a natural compound extracted from rosemary and sage, which has been demonstrated to have anti-inflammatory, anti-oxidant, and anti-cancer properties. In this report, we evaluated the therapeutic potential and elucidated the potential mechanism of action of carnosol in chemoprevention of ultraviolet B-light (UVB) induced non-melanoma skin cancer formation. Our data indicated that carnosol could partially reduce UVB-induced reactive oxygen species (ROS) elevation and thus reduce DNA damage. It could also reduce UVB-induced formation of cyclobutane pyrimidine dimers (CDP) in keratinocytes possibly through its ability in absorbing UVB radiation. In addition, carnosol could inhibit the UVB-induced activation of NF-κB and also reduce UVB-induced transformation of keratinocytes. Taken together, the results indicate the role of carnosol as a potential chemopreventive agent upon UVB radiation.

After selecting the dose of carnosol, we next determined the effect of carnosol on UVB-induced ROS elevation in a time dependent manner. We measured the ROS level at 20 minutes intervals for 12 h post UVB radiation. Our data indicated that carnosol treatment did not statistically significantly affect the ROS level in non-radiated cells, but it continuously reduced the ROS level in the irradiated cells from 20 minutes to 10 h after UVB radiation (Fig. 2B). These results indicated that carnosol might selectively inhibit the induction of some ROS induced by UVB radiation.  Carnosol protects DNA from UVB-induced breakage. As increased levels of intracellular ROS causes DNA damage 19 , we next determined whether the reduced ROS by carnosol is correlated to DNA damage upon UVB radiation. We used phosphorylated H2AX (γH2AX) and Chk1 (p-Chk1) as two DNA breakage and damage markers 20,21 . In HaCaT cells, at early phase (15 and 60 minutes) post UVB radiation, the phosphorylation levels of both H2AX and Chk1 was significantly reduced by carnosol treatment (Fig. 3A). We further confirmed the DNA damage in cells using comet and immunofluoresent assays. In comet assay, the intensity of the comet tail was reduced by almost 50% comparing the cells treated with carnosol (20 μM) and non-treated ones (Fig. 3B). The immunofluorescent assay showed decreased level of p-Chk1 in the nucleus when treated with carnosol (20 μM) in UVB irradiated HaCaT cells (Fig. 3C). These results indicated that carnosol could at least partially protect the DNA breakage from UVB radiation.
Carnosol reduces UVB-induced DNA lesions formation. UVB can also damage DNA by inducing the formation of DNA lesions 22 . The two predominant forms of UVB-induced DNA lesions are cyclobutane pyrimidine dimers (CPD) and 6-4 pyrimidone photoproducts (6-4 PP) 23 . To determine whether the formation of DNA lesions is also protected by carnosol, we quantitatively analyzed the UVB-induced formation of CPD and 6-4 PP for cells treated with or without carnosol. Our data demonstrated that UVB radiation increased CPD and 6-4 PP formations by 21.5-fold and 3.3-fold, respectively. Carnosol treatment could decrease UVB-induced CPD formation by approximately 50% to 11.8-fold (Fig. 4A); while the treatment has no significant effect on UVB-induced 6-4 PP formation (Fig. 4B). These results indicated that carnosol might be able to compete with DNA in absorption of UVB radiation. HaCaT cells were exposed to 50 mJ/cm 2 UVB radiation in the presence or absence of carnosol (20 μM). (A) Cells were lysed at indicated time point and protein levels of phosphorylated H2AX (γH2AX) and Chk1 (p-chk1) were determined by western blot. The data represents three sets of independent experiments. (B) Comet assay for HaCaT exposed to UVB radiation. After cell treatment, cells were trypsinized and collected for comet assay. The tail intensity was semi-quantitatively analyzed using Image J. *p < 0.05, **p < 0.01. (C) Cells were immunostained with p-Chk1 upon UVB radiation with or without carnosol (20 μM) treatment. p-Chk1 was stained with green fluorescent dye, while nucleus was stained with DAPI. Scale indicates 20 μm.
SCientifiC RepoRTS | (2018) 8:3574 | DOI:10.1038/s41598-018-22029-x Carnosol inhibits keratinocyte transformation upon UVB radiation. UVB-induced DNA breakages and lesions are known to be one of the major risk factors in cancer development 24 . To evaluate the potential chemopreventive effect of carnosol on UVB-induced skin cancer formation, we determined the transformation rate of keratinocytes in the presence of carnosol after repeated UVB radiation using soft agar assay. Our data demonstrated that the treatment of carnosol reduced the UVB-induced transformation of keratinocytes from 3.5-fold to 2.2-fold, which is an approximately 50% reduction. This result indicated that carnosol could protect skin cells from transforming to cancerous cell upon UVB radiation (Fig. 5).

Carnosol inhibits UVB-induced NF-κB activity.
Since the protective effect of carnosol on UVB-induced cell death is not as significant as we predicted, we further determined if carnosol could suppress UVB-induced activation of the pro-survival NF-κB pathway as we previously reported 25 . Our data showed that carnosol could partially protect IκB, the inhibitor of NF-κB, from UVB-induced reduction in a dose-dependent manner (1, 10, 20 μM) (Fig. 7A Lanes 6-8 vs. 5; Lanes 10-12 vs. 9). The protection lasted at least 6 hours post-UVB radiation (Fig. 7B). The increased levels of IκB were correlated to a decreased phosphorylation of NF-κB at S276 at 20 μM . These results indicated that carnosol could inhibit UVB-induced activation of NF-κB, which is often considered as a pro-survival and pro-oncogenic factor 26 . HaCaT cells were exposed to 10 mJ/cm 2 UVB radiation every 48 hours for 14 days. Cells were then collected and equal number of cells was re-seeded into 96-well plate with soft agar. After 10 days of incubation, cells were stained and the fluorescent intensity was determined. **p < 0.01.

Discussion
Overexposure to UVB leads to an increased chance of developing various forms of skin cancers, including basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and cutaneous malignant melanoma 15,16 . Identification and characterization of natural compounds as chemopreventive agents for UVB-induced skin cancer formation is very appealing because these compounds are often safer and more environmentally friendly. In this study, we determined the efficacy of carnosol, a natural compound extracted from sage or rosemary, in protecting keratinocytes from UVB-induced DNA damage and transformation. Our results indicated that carnosol reduced skin cell transformation (Fig. 5) possibly through the following mechanisms. First, carnosol protects DNA from UVB-induced breakage by reducing intracellular ROS level in the irradiated cells (Figs 2 and 3). Second, carnosol reduces DNA lesion by reducing the formation of CPD (Fig. 4A), but not 6-4 PP (Fig. 4B) 27 . The selective reduction could be due to that the absorbance wavelength for forming cis-syn CPD is at 280 nm 28 , which overlaps with the absorbance peak of carnosol in 284 nm 14 ; on the other hand, the absorbance wavelength to form 6-4 PP and its Dewar valence isomers is at 313 nm 28 which has no overlap with the absorbance of carnosol. This makes carnosol a good candidate for chemoprevention of UVB-induced skin cancer because (1) CPD is the predominant form of the dTpT dimers induced by UVB radiation (Fig. 4, Panel A vs. B) 30 and (2) the lasting UVB-induced mutation is caused by CPD but not 6-4 PP or oxidative DNA damage 29 . Moreover, carnosol inhibits UVB-induced NF-κB activation (Fig. 7), which is a pro-oncogenic factor 26 elevated in skin cancer cells 31 . Thus, the inhibition of NF-κB activity could potentially reduce the risk of cancer development after overexposure to UVB radiation. Carnosol, a polyphenol, may inhibit UVB-induced NF-κB activation through NADPH oxidase (NOX) 32 , which is known to be activated by UVB and products superoxide (O 2 •− ) 33 . We previously reported that the UVB-induced early activation of NF-κB is dependent on constitutive nitric oxide synthases (cNOS) activation 25 . The nitric oxide (NO • ) produced from cNOS can quickly react with O 2 •− to form peroxynitrite (ONOO − ), which leads to PERK activation, eIF2α phosphorylation, down regulation of IκB synthesis and sequentially NF-κB activation 25,34-36 . By inhibiting NOX and reducing the production superoxide (O 2 •− ), carnosol could reduce the formation of peroxynitrite (ONOO − ), thus down regulate NF-κB activation. Based on these results, carnosol shows the characteristics to be a chemopreventive agent upon UVB-induced skin cancer.

Cell Culture and drug treatment. Human keratinocyte HaCaT cells (kindly provided by Dr. Nihal
Ahmad, University of Wisconsin-Madison) were grown in Dulbecco's Minimal Essential Medium (Cellgro) supplemented with 10% fetal bovine serum and penicillin/streptomycin, at 37 °C with 5% CO 2 . Carnosol (Cayman) was added to cells at indicated concentration at 60 minutes before UVB radiation. After radiation, cells were continuously incubated with or without carnosol in the medium until further analysis. 15-watt UVB tubes (UVP Inc.). The intensity of UVB was calibrated by a UVP model UVX digital radiometer (UVP Inc.) after 5 minutes warming up of the lamps. The dose rate for 10 mJ/cm 2 or 50 mJ/cm 2 of UVB radiation was 0.8 or 3.8 mW/s respectively. Medium was removed before exposing the cells to UVB. After UVB radiation, fresh medium was added to the culture plates with or without drugs, and the cells were kept in incubation at 37 °C with 5% CO 2 until further analysis.

ROS measurement. CM-H 2 DCFDA (Invitrogen) was used to measure the total ROS level in cells.
CM-H 2 DCFDA was dissolved in DMSO to a stock solution of 500 μM and diluted in PBS to final concentration of 5 μM. CM-H 2 DCFDA was added into cells 60 minutes prior to UVB exposure and the reading of fluorescence dye was recorded every 20 minutes using luminometer (Molecular devices Spectra Max M2).
Cell transformation assay. HaCaT cells were radiated by 10 mJ/cm 2 UVB every 48 hours for 14 days with or without carnosol (20 μM) treatment. Transformation assay was performed following the protocol of 96-well cell transformation assay (Cell Biolabs, Inc.). Base agar layers were prepared using 1.2% agar solution with 2X DMEM/20% FBS, and solidified at 4 °C for 30 minutes. Cell agar layers were prepared similarly and 5000 cells/ well were seeded in each well of 96-well plate. 100 μL of cell culture medium with or without carnosol was added into the well, and the plates were incubated at 37 °C and 5% CO 2 for 10 days. For harvest, 50 μL agar solubilization solution was added to each well and incubated for 60 minutes at 37 °C. Then 25 μL lysis buffer was added and the fluorescent was read at Ex/Em 485/520 nm.
Western blot analysis. Cells were lysed with Nonidet P-40 (NP-40) lysis buffer (2% NP-40, 80 mM NaCl, 100 mM Tris-HCl pH 8.0, 0.1% SDS) with proteinase inhibitor mixture (Complete TM , Roche Molecular Biochemicals) at indicated time point. Cell lysates were incubated on ice for 15 minutes and then centrifuged at 13,000 rpm at 4 °C for 15 minutes. Protein concentration was measured by Protein DC Assay kit (Bio-Rad Laboratories). Equal amounts of protein were subjected on SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked in 5% milk in Tris buffered saline plus Tween 20 (TBST) for 45 minutes and probed with anti-γH2AX (Cell Signaling), anti-pCHK1 (Cell Signaling), anti-p276-NF-κB (Santa Cruz), anti-NF-κB p65 (Santa Cruz), anti-IκB (Santa Cruz), or anti-β-actin (Santa Cruz) at 4 °C overnight. After washing with TBST, the membrane was incubated with corresponding HRP-conjugated anti-rabbit or anti-mouse antibody for 45 minutes at room temperature. Membrane was then washed three times in TBST, followed by two times wash in TBS and developed in West Pico Supersignal chemiluminescent substrate (Pierce).
Immunofluorescence staining of phosphorylated Chk1. Cells were fixed with 3.6% formaldehyde for 10 minutes at room temperature, rinsed with PBS three times and permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. Cells were then blocked with blocking buffer (2 mg/mL BSA in PBS) for 60 minutes before incubating with anti-p-Chk1 antibody (Cell Signaling) for 60 minutes. After three times washing with PBS, cells were incubated with a fluorescein-conjugated horse anti-rabbit antibody (Vector Labs) for 60 minutes, washed with PBS and mounted with ProLong Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). The pictures were taken by NIKON Eclipse E600.
Comet Assay. HaCaT cells (with or without carnosol treatment) were collected 15 minutes after 50 mJ/cm 2 UVB radiation. The alkaline comet assay was performed according to manufacturer's instructions (Trevigen). LMAgarose was added to the cells then pipetted onto CometSlide. After the solidification of the agarose, the slides were immersed into lysis solution for 60 minutes at 4 °C and transferred to alkaline unwinding solution for 60 minutes at 4 °C. The slides were then run in an electrophoresis tank with alkaline electrophoresis solution at 21 volts for 40 minutes, immersed twice in dH 2 O for 5 minutes each, and then in 70% ethanol for 5 minutes. After that, slides were dried at 37 °C for 15 minutes and stained with SYBR gold and detected by fluorescent microscopy at Ex/Em 496/540 nm. CPD and 6-4 PP ELISA assay. HaCaT cells (with or without carnosol treatment) were collected 15 minutes after 50 mJ/cm 2 UVB radiation. CPD and 6-4 PP detection assays were performed following manufacturer's instructions (Cell Biolabs Inc.). DNA was extracted from the cells (Qiagen), heated at 95 °C for 10 minutes and chilled on ice for 10 minutes. Then, 50 μL of 4 μg/mL DNA was added in each well with 50 μL DNA binding solution and incubated overnight. The solution was removed the next day and the wells were washed twice with PBS before adding anti-CPD or anti-6-4 PP antibody. After 60 minutes incubation at room temperature, the wells were washed 5 times with washing buffer followed by 60 minutes incubation with 100 μL secondary antibody. 100 μL substrate solution was then added and incubated for 15 minutes, stopped by adding 100 μL stop solution. Results were read at OD450 nm.
Electrophoretic mobility shift assay. A 22-bp synthetic oligonucleotide (5′-AGTTGAGGGGACTTT CCCAGGC-3′) containing the specific NF-κB-binding site was annealed and labeled with γ-32 P-ATP using T4 polynucleotide kinase. A DNA-binding reaction mixture of total 20 μL containing poly (dI:dC), labeled probe, binding buffer (10 mM pH 8.0 Tris HCl, 150 mM KCl, 0.5 mM EDTA, 0.1% Triton-X 100, 12.5% Glycerol and 0.2 mM DTT) and 10 μg of cell nuclear extract was incubated at room temperature for 30 minutes and loaded onto a 5% non-denaturing polyacrylamide gel for electrophoresis. The gel was run in 0.5 × TBE buffer at 120 V, transferred to a double layer of Whatman paper and dried on a gel dryer for 45-60 minutes at 76 °C. The dried gel was exposed to autoradiography film (Denville) at −80 °C, the NF-κB bound 32 P-labeled DNA was detected and the band intensity was analyzed by Image J.
Fluorescein isothiocyanate (FITC) conjugated-annexin V (ANX5)/propidium iodide (PI) apoptosis detection kit (BD Biosciences) were used to stain the cells through the determination of the loss of membrane phospholipid symmetry and membrane integrity. Cell survival rate (R) was calculated as: R = [1 × 10 5 -number of positive stained cells]/1 × 10 5 . Both floating and attached cells were harvested and washed twice with cold PBS. The cells were then suspended in ANX5 binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl and 2.5 mM CaCl 2 ). The cell suspension was mixed with 5 μL ANX5-FITC and 5 μL PI and incubated for 15 minutes in dark at room temperature. The ANX5/PI double-stained cells were analyzed using a FACSort Flow Cytometer (BD Science) equipped with CellQuest software (BD Science).