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Introduction

Cutaneous cancer is among one of the most common types of cancer as ~1.5 million individuals worldwide are diagnosed with cutaneous cancer annually, accounting for nearly 10% of all new cancer cases (1). Cutaneous squamous cell carcinoma (cSCC) is a common non-melanoma skin cancer that accounts for ~20% of all skin cancers in the United States and UV light is the main causative factor (2,3). The ozone layer absorbs some of the UVB and all of the UVC, so the UV that reaches the Earth's surface is mainly longwave UVA (320-400 nm) and shortwave UVB (280-320 nm) (4). Prolonged exposure to UV radiation generates reactive oxygen species (ROS), which can directly damage DNA, leading to the activation of proto-oncogenes and the inhibition of anti-oncogenes (5). ROS and its modified intermediates can also oxidize proteins, lipids, nucleic acids and carbohydrates, thereby exacerbating metabolic diseases such as obesity, diabetes and dyslipidemia (6,7). In addition, ROS can also activate inflammatory pathways, release inflammatory regulatory factors and further damage DNA (8,9). Genomic instability occurs when DNA damage is not promptly repaired but rather accumulates. If this damage persists, it can eventually lead to the development of cancer (10-12).

Nuclear factor-E2-related factor 2 (Nrf2) is an essential regulator of certain transcription factors involved in oxidative stress. When healthy cells are damaged, a functional defect in the Nrf2 pathway can increase the tumorigenic potential of early tissue damage (13). However, a previous study reported that Nrf2 is continuously abnormally activated during the progression of certain types of carcinoma, including esophageal SCC, cSCC and non-small cell lung cancer (14). He et al (15) reported that Nrf2 is involved in a number of metabolic processes in cancer cells, including the pentose phosphate pathway, the regulation of glycolysis and fatty acid metabolism. Aberrant activation of Nrf2 may be associated with the accumulation of p62/sequestosome-1, a multidomain protein that competes with Nrf2 for binding to Kelch-like ECH associated protein 1 (Keap1), which can lead to aberrant Nrf2 activation (16).

Sirtuin1 (SIRT1) is a deacetylase that serves an instrumental role in the inflammatory response. The elimination of SIRT1 in the liver, pancreas and brain results in increased inflammatory responses and ROS accumulation (17). Upregulation of SIRT1 can repair UV-induced DNA injury and photoaging in human immortalized keratinocytes and mouse embryonic fibroblasts (18,19). The physiological functions of SIRT1 are primarily regulated through its deacetylation of co-activators, histones and transcription factors, such as E2F transcription factor 1, c-Myc, FOXO1 and NF-κB (20). SIRT1 inhibits the function of NF-кB by deacetylation of the RelA/p65 subunit, which results in cells entering the TNF-α-mediated apoptotic process (21). Chrysanthemum indicum Linnén (C. indicum) is a medicinal and food herb, which is readily available in East Asia (22). Previous studies have reported that C. indicum may have antihypertensive, antioxidant, antiseptic and anticancer properties, as well as inhibiting lipogenesis (22-25). Supercritical carbon dioxide fluid extraction has been successfully employed for the extraction process of flowers and buds of C. indicum because it can ensure a high extraction rate and the structural integrity of its volatile compounds, such as monoterpenes, sesquiterpenes and alkenes (26). A previous study reported that the supercritical carbon dioxide fluid extraction of C. indicum (CISCFE) may show potential for the treatment of liver and brain damage induced by D-galactose in an aging mouse model (22). Moreover, CISCFE combined with bleomycin was reported to improve the anticancer ability of tumor-transplanted mice and reduce the toxicity of bleomycin (27). Furthermore, CISCFE inhibited UV-induced photoaging in a mouse model by reducing inflammation and enhancing antioxidant capacity (28). However, the effect of CISCFE in UV-induced skin carcinogenesis is currently unclear.

The present study aimed to investigate whether local application of CISCFE could alleviate UV-induced skin cancer in a mouse model through macroscopic, histological and immunohistochemical evaluations, and determine whether CISCFE could regulate oxidative stress and inflammation related pathways.

Materials and methods

Preparation of CISCFE

The flowers and buds of C. indicum were purchased from Qingping Chinese herbal medicine market of Guangzhou, Guangdong, China, and were identified by Professor Zi-Ren Su of Guangzhou University of Chinese medicine (Guangdong, China). The extraction and identification procedures of CISCFE were based on previously published literature (27). Briefly, the flowers and buds of C. indicum were loaded into the extraction vessel of the 532 Supercritical Fluid Extraction Equipment (Applied Separations). CISCFE was obtained using an extraction time of 4 h, a pressure of 25 MPa, a temperature of 45˚C and a flow rate of 20 l/h. High-performance liquid chromatography-pulsed amperometric detection (HPLC-PAD) and gas chromatography-mass spectrometry (GC-MS) were used to analyze the chemical composition of CISCFE (Table SI and Fig. S1). HPLC analysis was performed on a Shimadzu LC40 HPLC system. The separation was performed on a ACE Excel 5 Super C18 column (4.6x250 mm, 5 µm; cat. no. EXL-1211-2546U; Advanced Chromatography Technologies) with a flow rate of 1.0 ml/min, column temperature at 30˚C, and injection volume of 10 µl. The mobile phase consisting of acetonitrile (solvent A) and 0.1% aqueous formic acid (solvent B) was used to elute the targets with the gradient mode (0-5 min: 5% A→25% A; 5-15 min: 25% A→25% A; 15-25 min: 25% A→45% A; 25-35 min: 45% A→55% A; 35-40 min: 55% A→70% A). Luteolin-7-glucoside (cat. no. B20887), luteolin (cat. no. B20888), linarin (cat. no. B20860) and chlorogenic acid (cat. no. B20782) standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The content of these compounds was quantitatively analyzed with peak areas under the standard curves at 334 nm. GC-MS analysis was performed on an Agilent 6890-5975 GC-MS system (Agilent Technologies, Inc.). The oven temperature was initially set at 60˚C, then ramped up to 100˚C at a gradient of 10˚C/min (held for 1 min), then to 110˚C at a rate of 1˚C/min (held for 1 min); then to 150˚C at a rate of 3˚C/min (held for 1 min) and finally to 260˚C at a rate of 10˚C/min (held for 5 min). Split injection (0.5 µl) was conducted with a split ratio of 60:1 and helium was used as carrier gas of 1.0 ml/min flow rate. The spectrometer was set to electron impact (EI) mode with an ionization energy of 70 eV, a scanning range of 40-400 amu, and a scanning rate of 0.34 sec/scan. The temperatures of the inlet and ionization source were 230 and 250˚C, respectively. Ultimately, CISCFE at a concentration of 0.48 mg/cm2/mouse (low concentration CISCFE) or 1.6 mg/cm2/mouse (high concentration CISCFE) in 10% Tween 80 were used for subsequent animal experiments.

Animal model

A total of 75 specific-pathogen free male Kunming mice (22-24 g, age 8 weeks) were obtained from the Animal Experiment Center, Guangzhou University of Chinese Medicine (Animal Quality Certificate No. 44005800007154). The mice were housed in an environment conforming to the prescribed humidity (50±5%) and temperature (23±2˚C), with food and water ad libitum and were maintained under a 12 h light/dark cycle. The laboratory animal license number was SCXK (Yue) 2018-0085 and the ethics certification number was 20190304024. Under the supervision of authorized researchers, all experiments in the present study were approved by the Animal Care and Use Committee of Guangzhou University of Chinese Medicine (approval no. 20190304024; Guangzhou, China) based on the Guidelines for the ethical review of laboratory animal welfare People's Republic of China National Standard GB/T 35892-2018(29). According to additional markers that may constitute humane endpoints in tumor research, the experimental endpoint for tumor size took into account the fact that UV exposure on the back of mice causes damage to skin tumors. The experiment was halted when the volume of any of the skin lesions >1,000 mm3, the maximum diameter >10 mm or when the diameter ≤10 mm but interfered with animal feeding or hindered animal movement. According to the humane endpoint guidance of the present animal experiments, two mice were euthanized, one from the CISCFE-L group and one from NAA group (30).

Topical CISCFE treatments and UV exposure

The skin area on the back (2.5x3.0 cm2) of mice was depilated with a shaver (FS607; FLYCO) (Fig. 1). In our previous study, the concentrations of 0.48 mg/cm2/mouse and 1.6 mg/cm2/mouse of CISCFE reduced skin damage caused by UV exposure (28). Therefore, the mice were randomly allocated to five treatment groups: Sham (no medication or UV radiation), model (only UV radiation without drug treatment), low concentration CISCFE (0.48 mg/cm2/mouse), high concentration CISCFE (1.6 mg/cm2/mouse) and positive control nicotinamide (NAA; 0.65 mg/cm2/mouse; Sigma-Aldrich; Merck KGaA) groups. CISCFE and NAA were applied daily to the shaved area of the skin on the back of the mouse.

Figure 1 UV irradiation and topical application of CISCFE during UV-induced skin cancer of mice. Red days of the week indicate when mice were irradiated. W, week; MED, minimum erythema dose; CISCFE, supercritical carbon dioxide fluid extraction of Chrysanthemum indicum Linnén; NAA, nicotinamide.

Ultra-Vitalux light bulbs were used as a source of UV light (UVA:UVB=93:7; Osram). Mice were positioned 30 cm away from the UV lamp at 23˚C and were irradiated on Mondays, Wednesdays, Fridays and Sundays for 31 weeks. According to our previous study (31), the minimum erythema dose (MED) for mice was 100 mJ/cm2. UV intensity was 1 MED for the first week and was increased by 1 MED/week until 400 mJ/cm2 in the 4th week, which was maintained until the end of the experiment. The experiment continued for 31 weeks, with daily observations of mice health and behavior. At 31 weeks, isoflurane (induction, 5%; maintenance, 2%) was used for anesthesia and cervical dislocation was performed on the mice after photographic documentation of the dorsal skin status. Finally, skin tissue from the irradiated area was extracted for subsequent experiments.

Histological analysis

The dorsal skin was fixed in 10% formalin at 26˚C for 48 h. Tissue dehydration was then performed with the following procedure: Soaking in 10% formalin at 26˚C for 1 h, soaked in 75% ethanol at 26˚C for 1.5 h, soaked in 85% ethanol at 26˚C for 1.5 h, soaking in 95% ethanol at 26˚C for 1.5 h, soaking in anhydrous ethanol at 26˚C for 1 h three times, soaking in TO Bio-permeable agent (cat. no. AYA0150; Shanghai Acmec Biochemical Technology Co., Ltd.) at 26˚C for 1.5 h twice and soaking in paraffin wax at 60˚C for 2 h. Subsequently, the tissue was embedded in melted paraffin at 65˚C and sectioned (4 µm). Paraffin sections were stained using Gomori aldehyde fuchsin at 26˚C for 10 min (GAF; cat. no. G1593; Beijing Solarbio Science & Technology Co., Ltd.), hematoxylin and eosin at 26˚C for 20 min (H&E; cat. no. 0619A19; Beijing Leagene Biotechnology Co., Ltd.) and Sirius red staining at 26˚C for 1 h (cat. no. DC0041-100; Beijing Leagene Biotechnology Co. Ltd.). The histological changes in the skin were examined using a light microscope (BX53; Olympus Corporation).

Immunohistochemical analysis

The procedure for fixation, embedding and sectioning of skin tissue is the same as in the Histological analysis section. Skin sections (5 µm) were subsequently deparaffinized with the following procedure: Placed in xylene at 26˚C for 10 min three times, in anhydrous ethanol at 26˚C for 5 min and three times, in 95% ethanol at 26˚C for 3 min, in 85% ethanol at 26˚C for 3 min and in 75% ethanol at 26˚C for 3 min. The skin sections were heated in EDTA for 15 min at 95˚C and then the sections were soaked in 3% hydrogen peroxide at 26˚C for 20 min. Tissue sections were then sealed with 10% goat serum (cat. no. SL038; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at 37˚C. Subsequently, sections were incubated with Ki-67 (1:300; cat. no. ab15580; Abcam) and CD11b (1:4,000; cat. no. ab133357; Abcam) antibodies diluted in PBS at 4˚C overnight. Samples were incubated with anti-rabbit IgG H&L (HRP; 1:1,000; cat. no. ab6721; Abcam) at 37˚C for 1 h, followed by incubation with 3,3-diaminobenzidine at 26˚C for 1 min (cat. no. ZLI-9017; ZSGB-BIO) and counterstaining with hematoxylin at 26˚C for 3 min. Sections were imaged using a light microscope (Olympus Co.; BX53). Samples were semi-quantified using ImageJ software (version 1.53e; National Institutes of Health).

ROS accumulation assay

At week 31, the UV-irradiated dorsal skin of mice was removed and analyzed using a ROS assay. The mouse skin was encapsulated in optimal cutting temperature encapsulant (cat. no. 4583; Sakura Finetek USA, Inc.) and frozen sections (-80˚C; thickness, 8 µm) were incubated with DCFH-DA (cat. no. BB18081; Bestbio) at 37˚C for 30 min. The samples were measured at a wavelength of 525 nm using a fluorescence microscope (BX53; Olympus Corporation). The average fluorescence intensity of ROS was analyzed using ImageJ software (version 1.53e; National Institutes of Health).

Catalase (CAT) and superoxide dismutase (SOD) assays

Skin tissue was homogenized by adding 9 times the volume of saline (g/ml) and the supernatant was collected after centrifugation at 3,000 x g for 10 min at 4˚C. The protein concentration of the supernatant was measured using a BCA kit (cat. no. P0012; Beyotime Institute of Biotechnology). CAT and SOD levels were measured according to the manufacturer instructions of the CAT (cat. no. A007-1; Nanjing Jiancheng Bioengineering Institute) and SOD (cat. no. A001-3; Nanjing Jiancheng Bioengineering Institute) assay kits.

Measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG), IL-6 and TNF-α

Skin tissues were ground in PBS with a homogenizer (KZ-III-FP; Wuhan Servicebio Technology Co., Ltd.) at 60 Hz for 6 min at 4˚C and subsequently centrifuged at 4˚C and 3,000 x g for 20 min. The supernatant was collected and ELISA kits were used accordingly to the manufacturer's instructions to measure the levels of TNF-α (cat. no. 430904; BioLegend, Inc.), IL-6 (cat. no. 431304; BioLegend, Inc.) and 8-OHdG (cat. no. MM-0221M1; Jiangsu Meimian Industrial Co., Ltd.).

Western blotting

Skin samples were homogenized in RIPA lysis solution (cat. no. P0013B; Beyotime Institute of Biotechnology). Samples were centrifuged at 4˚C and 14,000 x g for 10 min and the protein content was determined using a BCA kit (cat. no. P0010; Beyotime Institute of Biotechnology). The proteins (40 µg/lane) were electrophoresed using a 10% SDS-polyacrylamide gel and transferred to PVDF membranes. PVDF membranes were blocked with 5% skimmed milk for 1 h at 26˚C and incubated overnight at 4˚C with heme oxygenase 1 (HO-1; 1:1,000; cat. no. ab13248; Abcam), CD11b (1:1,000; cat. no. ab133357; Abcam), VEGF (1:1,000; cat. no. SC7269; Santa Cruz Biotechnology, Inc.), c-Myc (1:1,000; cat. no. SC40; Santa Cruz Biotechnology, Inc.), p65 (1:1,000; cat. no. 8242S; Cell Signaling Technology, Inc.), PTEN (1:1,000; cat. no. ET1606-43; HUABIO), phosphorylated (p)-p62 (1:1,000; cat. no. ab211324; Abcam), NAD-dependent protein deacetylase sirtuin-1 (SIRT1; 1:1,000; cat. no. ab110304; Abcam), p-p65 (1:1,000; cat. no. ab76302; Abcam), p-IκBα (1:1,000; cat. no. ab133462; Abcam), acetyl-p65 (1:250; cat. no. ab19870; Abcam, IκBα (1:1,000; cat. no. ab32518; Abcam), Nrf2 (1:1,000; cat. no. ab137550; Abcam), NAD(P)H dehydrogenase [quinone] 1 (NQO1; 1:50,000; cat. no. ab80588; Abcam), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), lamin B1 (1:2,000; cat. no. AF5161; Affinity Biosciences) and Keap1 (1:2,500; cat. no. ab139729; Abcam) antibodies. Subsequently, membranes were incubated with anti-mouse IgG H&L (1:5,000; cat. no. LK2003; Tianjin Sungene Biotech Co., Ltd.) or anti-rabbit IgG H&L (1:5,000; cat. no. ab6721; Abcam) antibodies for 1 h at 26˚C. Blots were visualized using ECL reagents (cat. no. FD8000; Hangzhou Fude Biotechnology Co., Ltd.) and the blot densities were quantified using ImageJ software (version 1.53e; National Institutes of Health). GAPDH or Lamin B1 were used as loading controls (32).

Statistical analysis

Data were expressed as mean ± standard deviation. Data were analyzed using a one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. Data were visualized and analyzed using GraphPad software (version 8.3.0; Dotmatics).

Results

Chemical Composition Analysis of CISCFE.

GC-MS analysis and HPLC analysis were used to detect the chemical constituents of CISCFE. As shown in Table SI, CISCFE mainly contains d-Camphor, Caryophyllene oxide, Endo-Borneol, α-Curcumene, Cis-verbenol, β-Caryophyllene, Eucalyptol, Thymol as detected by GC-MS. In addition, HPLC analysis detected four compounds in CISCFE (Fig. S1), which were Chlorogenic acid, Luteolin-7-glucoside, Linarin and Luteolin.

CISCFE alleviated cutaneous injury induced by UV

Over the course of the present study, it was demonstrated that the skin of the model group showed shallow wrinkles, erythema and a leathery appearance after 9 weeks of UV exposure compared with the sham group (Fig. 2A). The skin in the low dose and high dose CISCFE groups and the NAA group exhibited no erythema and showed few wrinkles compared with the model group. After 24 weeks of UV irradiation, papular lesions and broken crusts were observed on the skin of model mice. At week 31, ulcerative papules, adhesive scales, recurrent local bleeding and crusting on the skin was observed on model mice. However, after pretreatment of mice with low or high dose CISCFE and NAA, there were no papules and deep wrinkles only appeared on the skin at 31 weeks of UV irradiation. Moreover, the dermal vessels in the model group were expanded and proliferated compared with the sham group after 31 weeks of UV irradiation. After pretreatment with CISCFE, at both doses tested, vasodilation and hyperplasia in the dermis caused by UV were markedly reduced (Fig. 2B).

Figure 2 CISCFE ameliorates UV-induced skin lesions and vascular permeability. (A) Macroscopic images of mouse skin after 9, 24 and 31 weeks of UV irradiation. Highlight, ulcerated erythematous papules with scaling. (B) Dermal blood vessels of mice after 31 weeks of UV irradiation. CISCFE, supercritical carbon dioxide fluid extraction of Chrysanthemum indicum Linnén; NAA, nicotinamide; SC, skin cancer; L, low dose; H, high dose; w, week.

CISCFE reduced histological damage caused by UV exposure

To observe the histopathologic changes of mouse skin after UV irradiation, GAF, Sirius red and H&E staining were used (Fig. 3A). H&E staining showed that the skin of model mice exhibited abnormal proliferation of the epidermis, keratinocytes extending into the dermis and extensive infiltration of inflammatory cells at week 31. Sirius red and GAF staining of the mouse skin showed that UV irradiation damaged elastic and collagen fibers and reduced their density. Nevertheless, compared with the model group, low and high dose CISCFE and NAA treatment significantly reduced inflammatory cell infiltration and the deformation and degradation of elastin fibers and collagen fibers and markedly reduced UV-induced abnormal epidermal hyperplasia (Fig. 3B; P<0.01).

Figure 3 CISCFE reduces histological damage caused by UV exposure. (A) H&E, (scale bar, 50 µm; magnification, x200), Gomori aldehyde fuchsin (GAF) staining (scale bar, 20 µm; magnification, x400) and Sirius red staining (scale bar, 50 µm; magnification, x200) of mouse skin after 31 weeks of UV irradiation. (B) Epidermal thickness of UV-irradiated mouse skin. Data were expressed as mean ± standard deviation (n=5). ##P<0.01 vs. sham group; **P<0.01 vs. model group. H&E, hematoxylin and eosin; GAF, Gomori aldehyde fuchsin; ED, epidermis; DR, dermis; DEJ, dermo-epidermal junction; ST, subcutaneous tissue; HF, hair follicle; EF, elastic fiber; CO, collagen; IFI, inflammatory infiltration; CISCFE, supercritical carbon dioxide fluid extraction of Chrysanthemum indicum Linnén; NAA, nicotinamide; L, low dose; H, high dose.

CISCFE inhibited the development of UV-induced skin cancer

To examine the effects of CISCFE on proliferation, angiogenesis and the expression levels of cancer-related proteins, immunohistochemical assays and western blotting were used to detect Ki-67, VEGF, c-Myc and PTEN expression levels. After 31 weeks of UV irradiation, the epidermis of the model mice exhibited increased Ki-67 expression compared with the sham group (Fig. 4A and B; P<0.001). The epidermal layer in the CISCFE and NAA groups demonstrated a significant reduction in Ki-67 expression compared with the model group (P<0.001). VEGF and c-Myc protein expression levels were significantly increased and protein expression levels of PTEN were significantly decreased in the model mice compared with the sham group (Fig. 4C-F; P<0.001). After pretreatment with CISCFE or NAA, VEGF and c-Myc protein expression levels were significantly reduced while PTEN protein expression levels were significantly increased compared with the model group (P<0.05).

Figure 4 CISCFE suppresses UV-induced skin carcinogenesis. (A) Immunohistochemical analysis of Ki-67 expression in mice skin at the 31 weeks was conducted on 5 µm sections. Red arrows, expression of Ki-67 in the epidermis; scale bar, 20 µm; magnification, x400. (B) Semi-quantitative analysis of Ki-67 staining. (C) VEGF, c-Myc and PTEN protein expression levels in mouse skin specimens at the 31st week using western blot. Relative changes in protein expression levels intensities of (D) VEGF, (E) c-Myc and (F) PTEN were quantified by densitometric analysis. Data were presented as mean ± standard deviation (n=6). ###P<0.001 vs. sham group; *P<0.05, **P<0.01, ***P<0.001 vs. model group. CISCFE, supercritical carbon dioxide fluid extraction of Chrysanthemum indicum Linnén; NAA, nicotinamide; L, low dose; H, high dose; AOD, average optical density.

CISCFE suppressed UV-induced oxidative stress and inflammation of skin

To investigate whether the inhibition of skin cancer progression by CISCFE is related to its antioxidant effects, the levels of ROS, SOD, CAT and 8-OHdG were assayed. An increase in ROS accumulation was observed in the model group compared with the sham group (Fig. 5A and B). However, compared with the model group, CISCFE and NAA treatment significantly reduced UV-induced ROS overexpression (P<0.05). The activity levels of SOD and CAT were significantly decreased and 8-OHdG was significantly increased in the skin of the model mice at 31 weeks compared with the sham group (Fig. 5C-E; P<0.05). In mice treated with CISCFE and NAA, the levels of SOD and CAT activity were significantly increased (P<0.05), while 8-OHdG levels were significantly decreased in mice treated with low dose CISCFE compared with those in the model group (P<0.01).

Figure 5 CISCFE suppresses UV-induced oxidative stress and inflammation in mice. (A) ROS accumulation in mouse skin was assessed by DCFH-DA at week 31. Scale bar, 100 µm; magnification, x100. Green fluorescence represents the intensity of the generated ROS. Immunohistochemical analysis of CD11b expression in mouse skin at week 31 was conducted on 5 µm skin sections (Red arrows, expression of CD11b in dermis; scale bar, 50 µm; magnification, x200). Semi-quantitative analysis of (B) ROS Data were expressed as mean ± SD (n=6). Activity of antioxidant enzymes (C) SOD and (D) CAT activity levels in mouse skin at the 31 weeks were detected using assay kits. Data were presented as mean ± SD (n=8). (E) Levels of 8-OHdG in skin specimens were measured by ELISA kit at 31 weeks. Data were presented as mean ± SD (n=4). Semi-quantitative analysis of (F) CD11b. Data were expressed as mean ± SD (n=6). (G) Analysis of CD11b protein expression levels in mouse skin specimens at 31 weeks using western blotting. (H) Protein expression levels of CD11b were quantified by densitometric analysis. Data were presented as mean ± SD (n=6). Expression levels of (I) IL-6 and (J) TNF-α in skin specimens were measured using ELISA kits at 31 weeks. #P<0.05, ##P<0.01, ###P<0.001 vs. sham group; *P<0.05, **P<0.01, ***P<0.001 vs. model group. CISCFE, supercritical carbon dioxide fluid extraction of Chrysanthemum indicum Linnén; NAA, nicotinamide; L, low dose; H, high dose; AOD, average optical density; ROS, reactive oxygen species; prot, protein, CAT, catalase, SOD, superoxide dismutase; SD, standard deviation; 8-OHdG, 8-hydroxy-2'-deoxyguanosine.

To explore the level of inflammation in irradiated mouse skin, the expression levels of CD11b, IL-6 and TNF-α were assayed. After 31 weeks of UV irradiation, an increase in CD11b expression was observed in the dermis of model mice compared with the sham group (Fig. 5A and F; P<0.001). The protein expression level of CD11b in the skin of mice treated with CISCFE and NAA was significantly reduced compared with the model group (Fig. 5G and H; P<0.01). The expression levels of IL-6 and TNF-α in the model group were significantly higher compared with the sham group (Fig. 5I and J; P<0.05). Compared with the model group, topical application of CISCFE and NAA significantly reduced the expression levels of IL-6 and TNF-α (P<0.05).

CISCFE prevented UV-induced tumorigenesis by inhibiting Nrf2 and NF-κB pathways

High expression of Nrf2 in tumor cells promotes tumor development (15). Furthermore, NFκB is an important inflammatory and oncogenic transcription factor, and activation of SIRT1 inhibits the NF-κB pathway and suppresses the inflammatory response (21). To explore the potential mechanism of action of CISCFE against skin cancer in mice, the p62/Keap1-Nrf2 and SIRT1/NF-κB pathway-related proteins were examined. The results showed that the protein expression levels of p62, p-p62, Nrf2, HO-1 and NQO1 were significantly increased, while that of Keap1 significantly decreased in the model group compared with the sham group (Fig. 6; P<0.05). Furthermore, CISCFE treatment significantly decreased the expression levels of p62, p-p62, Nrf2, HO-1 and NQO1 and significantly increased that of Keap1 compared with the model group (P<0.05). However, the p-p62/p62 ratio was not significantly altered in any treatment condition.

Figure 6 CISCFE inhibits the UV-induced p62/Keap1/Nrf2 pathway in UV-irradiated mice. (A) Protein expression levels of p62, p-p62, Keap1, Nrf2, HO-1 and NQO1 were analyzed in skin samples at week 31 using western blotting. (B) Quantification of p-p62/p62 in mouse skin. Relative changes in protein expression levels of (C) p62, (D) p-p62, (E) Keap1, (F) Nrf2, (G) HO-1 and (H) NQO1 were quantified by densitometric analysis. Data were presented as mean ± standard deviation (n=4). #P<0.05, ##P<0.01, ###P<0.001 vs. sham group; *P<0.05, **P<0.01, ***P<0.001 vs. model group. CISCFE, supercritical carbon dioxide fluid extraction of Chrysanthemum indicum Linnén; NAA, nicotinamide; L, low dose; H, high dose; p, phosphorylated; Keap1, Kelch-like ECH associated protein 1; Nrf2, nuclear factor-E2-related factor 2; HO-1, heme oxygenase 1; NQO1, NAD(P)H dehydrogenase [quinone] 1.

Furthermore, there was a significant increase in the protein expression levels of p65, p-p65, acetyl-p65 and p-IκBα in the skin of the model group compared with the sham group (Fig. 7; P<0.05). However, the aforementioned protein expression levels were significantly lower in the CISCFE group compared with the model group (P<0.05). Additionally, the expression levels of IκBα and SIRT1 were significantly lower in the model group after 31 weeks of UV irradiation compared with the sham group (P<0.01). Topical pretreatment of mice with CISCFE significantly increased the protein expression level of IκBα compared with the model group (P<0.05). High dose treatment of mice with CISCFE significantly increased protein expression levels of SIRT1 compared with the model group (P<0.05). Furthermore, p-IκBα/IκBα and p-p65/p65 expression ratios were significantly increased in the model group compared with the sham group (P<0.01). Additionally, p-IκBα/IκBα and p-p65/p65 expression ratios in the skin of CISCFE mice were significantly decreased compared with the model group (P<0.01).

Figure 7 CISCFE affects the SIRT1/NF-κB pathway in UV-irradiated mice. Protein expression levels of (A) IκBα, p-IκBα, p65, p-p65 and (B) acetyl-p65 and SIRT1 were analyzed in mouse skin samples at 31 weeks using western blotting. (C) Quantification of the p-IκBα/IκBα expression level in mouse skin. (D) Protein expression levels of IκBα quantified by densitometric analysis. (E) Quantification of the p-p65/p65 expression level ratio in mouse skin. Protein expression levels of (F) p65, (G) acetyl-p65 and (H) SIRT1 quantified by densitometric analysis. Data were presented as mean ± standard deviation (n=4). ##P<0.01, ###P<0.001 vs. sham group; *P<0.05, **P<0.01, ***P<0.001 vs. model group. CISCFE, supercritical carbon dioxide fluid extraction of Chrysanthemum indicum Linnén; NAA, nicotinamide; L, low dose; H, high dose; p, phosphorylated; SIRT1, NAD-dependent protein deacetylase sirtuin-1.

Discussion

Prolonged exposure to UV radiation can lead to erythema, photoaging, photo immunosuppression and even skin cancer (3). cSCC is a group of skin cancers caused by the malignant growth of epithelial cells, which accounts for 20-50% of skin cancers in the United States (3,33). Although cSCC can be successfully treated surgically, its incidence is still increasing (1). Therefore, it is important to find a drug with low toxicity for prevention and treatment of this condition. It is believed that traditional Chinese herbs have been used for thousands of years to prevent and treat a variety of ailments (34). C. indicum is one of these herbs and is used both as a food source and also for the potential prevention and treatment of skin-related diseases (24,34). In the present study, a mouse model of UV-induced skin cancer was used to investigate the potential chemoprevention effect and mechanism of action of C. indicum on skin cancer. A number of previous studies have reported the effect of NAA in the field of dermatology and its actions in preventing photoaging and skin cancers in humans (35-37). Moreover, studies have reported that NAA prevented UV radiation from reducing ATP levels and inhibiting glycolysis, thus preventing the UV radiation-induced energy crisis in cells (36,37). Therefore, NAA was used as a positive drug control in the present study to evaluate the anti-skin cancer effect of CISCFE.

The clinical manifestations of cSCC may present as small spots and nodules in the early stages of disease, followed by necrosis, ulceration or mycosis, which can present as flat ulcers with raised edges and are accompanied by scaling (38). In the present study, the mice demonstrated clinical manifestations similar to those of cSCC after 31 weeks of UV irradiation. In addition, the diagnosis of skin cancer needs to be combined with histopathological analysis (39). The pathological results of the present study showed abnormal proliferation of the epidermis into the deep dermis in the model mouse group, in addition to increased inflammatory cell infiltration, which is consistent with previously published literature (40,41). The results of the present study suggested that CISCFE treatment may effectively inhibit the development of UV-induced skin cancer in mice.

The anticancer effects of CISCFE in the present study may be closely related to the biological activities of its chemical components. The chemical compositions of CISCFE analyzed using GC-MS (Table SI) showed that the main components were d-camphor, β-caryophyllene and thymol, which exhibit anti-inflammatory and antioxidant capabilities (42-44). In addition, caryophyllene oxide and thymol were reported to have antitumor activities (45,46). Eucalyptol may inhibit skin carcinogenesis in vivo and in vitro by reducing the migration and invasion of cancer cells (43). HPLC analysis was used to quantify four constituents in the present study, which were chlorogenic acid, linarin, luteolin-7-glucoside and luteolin. Chlorogenic acid and linarin inhibit the NF-κB signaling pathway and thereby inhibit cancer cell growth and proliferation (47,48). A previous study reported that lignan-7-glucoside inhibited the migration and invasion of oral cancer cells by regulating matrix metalloproteinase-2 expression and the extracellular signal-regulated kinase pathway (49). Luteolin has also been reported to act as an anti-inflammatory and anticancer agent (50). It could therefore be suggested that the active compounds in CISCFE, particularly the active components with potential anti-inflammatory and anticancer effects, may be key to the prevention and treatment of skin cancer using CISCFE. Therefore, it was investigated whether CISCFE inhibited UV-induced skin cancer in mice by exerting antioxidant and anti-inflammatory effects.

The activation of oncogenes and suppression of anti-oncogenes are important for determining carcinogenesis (51). c-Myc is a recognized oncogenic gene and its variants are observed in >70% of human cancers (52). Pelengaris et al (53) reported that sustained activation of c-Myc can induce abnormal skin hyperplasia, related keratinization insufficiency and angiogenesis. The hyperplastic epidermis was found to be accompanied by the expression of Ki-67, which serves as an attractive prognostic, predictive and potential therapeutic target for malignancies (54). Tumors are often associated with vascular dilation and proliferation, which provides adequate oxygen and nutrients to the cancer cells for proliferation (55). VEGF is crucial in skin angiogenesis and its abnormal activation within tumors causes the blood vessels in and around the tumor to grow exponentially (56). In the present study, the expression levels of c-Myc, VEGF and Ki-67 were increased following UV irradiation, which led to vasodilation in the dermis and hyperplasia in the epidermis. PTEN, a common mutant tumor suppressor gene, has exhibited inactivation or partial loss of function in numerous types of cancer (57). It has been reported that long-term UV irradiation causes genetic alterations in PTEN and reduces its expression level (58). In the present study, it was demonstrated that CISCFE reduced the expression levels of c-Myc, VEGF and Ki-67 and restored that of PTEN, thereby potentially alleviating skin cancer progression.

Long-term UV exposure can cause oxidative imbalance and lead to the accumulation of ROS, which induces a series of signal transduction events contributing to inflammatory immune imbalance, DNA injury and even cancer (59). Excessive accumulation of ROS can be detected through measuring levels of 8-OHdG, a biomarker of oxidative DNA damage (60). Moreover, 8-OHdG has been reported to be elevated in a number of types of cancer, such as colorectal, gastric and melanoma skin cancers (61,62). SOD and CAT serve a crucial role in the cellular antioxidant system, reducing the oxidative induction of proto-oncogenes and structural DNA damage by oxidative carcinogens (63). In the present study, CISCFE reduced the excessive production of ROS and oxidative DNA damage induced by UV and restored the activities of antioxidants including SOD and CAT. Moreover, the inflammatory factors induced by UV can also contribute to the progression of skin cancer and CD11b may be expressed in various types of inflammatory cells, which can be used to determine inflammatory injury (64). Previous studies have reported that photo-carcinogenesis is associated with UV-induced infiltration of CD11b cell populations and CD11b-mediated oxidative damage (65). Reduction of UV-induced infiltration of CD11b+ cells can prevent UV-induced skin aging and skin cancer (66). In the present study, increased CD11b expression was demonstrated in mouse skin tumors, and IL-6 and TNF-α protein expression levels were increased. However, CISCFE reduced the UV-induced infiltration of inflammatory cells, the protein expression levels of IL-6 and TNF-α and the number of CD11b cells. Therefore, CISCFE may potentially prevent skin cancer by inhibiting inflammation and oxidative stress induced by UV.

Nrf2 activation is beneficial to the survival of precancerous or cancer cells because oncogene mutations provide these cells with a higher proliferative capacity and viability by upregulating Nrf2 expression (67). Kim et al (68) reported that the mutation and sustained activation of Nrf2 affected the differentiation of squamous epithelial cells and was ubiquitous in cSCC. Moreover, p62 has been reported to be involved in the activation of Nrf2 in cancer cells. P62 competes with Nrf2 for the Keap1 binding site, especially when p62 is phosphorylated at serine 349 (Ser-349) (69). Keap1 is subsequently degraded and Nrf2 is released, leading to its persistent activation and subsequent transfer to the nucleus to exert its effect (16,70). In the present study, prolonged UV irradiation increased the expression level of Nrf2, p62 phosphorylation at Serine 349 (Ser-349) and increased the expression levels of downstream proteins NQO1 and HO-1. However, CISCFE treatment diminished the UV-induced expression of p-p62 and reduced the continuous expressions of Nrf2 and downstream NQO1 and HO-1, thereby potentially inhibiting the development of skin cancer.

The expression of NF-κB is triggered in tumor cells and cells that constitute the tumor microenvironment, promoting the production of cytokines and ultimately activating genes involved in abnormal growth and malignant tumor expression (71). Nrf2 and activated antioxidant enzymes, such as HO-1, inhibit the NF-κB pathway, thereby reducing inflammatory damage (72). However, CISCFE in the present UV-induced mouse skin cancer model did not inhibit NF-κB expression via the Nrf2/HO-1 pathway, but instead potentially inhibited NF-κB expression via SIRT1. A number of previous studies have reported that NF-κB is present in the cytoplasm as a p50/RelA (p65) dimer or RelB/p52 dimer. SIRT1 can deacetylate lysine 310 of p65 to inhibit the transcription of inflammation-related genes (73-76). The present study indicated that UV irradiation decreased the protein expression levels of SIRT1, which is in accordance with the previous study by Ming et al (76) in which the level of SIRT1 in patients with UV-related skin cancer was reduced. Moreover, the present study found that UV radiation increased the acetylation and phosphorylation of p65 to activate NF-κB. CISCFE increased SIRT1 protein expression levels and inhibited the activation of the NF-κB pathway, thereby potentially reducing the occurrence of skin inflammation, and even cancer.

In summary, CISCFE exhibited potent anti-inflammatory and anti-skin cancer activity (Fig. 8). CISCFE inhibited UV-induced epidermal abnormal proliferation and dermal fiber damage, reducing epidermal cell carcinogenesis. CISCFE also suppressed oxidative stress and the inflammatory response in mouse skin. CISCFE enhanced the protein expression level of SIRT1, which suppressed the abnormal activation of the pro-inflammatory factor NF-κB. Moreover, CISCFE reduced the protein expression level of p62, which reduced the abnormal activation of Nrf2. Therefore, the potential effect of CISCFE on skin cancer may be related to its anti-inflammatory and antioxidant chemical compositions. These findings suggested that CISCFE may potentially be a future prospective drug for the prevention and therapy of UV-induced skin cancer. However, the present study evaluated the anti-UV-induced skin cancer effects of CISCFE in mice and was not evaluated in patients with skin cancer. In future studies, the therapeutic effects of CISCFE should be studied in patients with skin cancer. In addition, the present study evaluated the expression levels of CD11b, which is commonly used as a biomarker for NK cells, monocytes, dendritic cells and neutrophils (77-80). B cells and T cells are also important classes of immune cells that serve an important role in skin inflammation and skin cancer progression (81,82). In future studies, the relationship between T cells and B cells and the progression of skin cancer should be examined.

Figure 8 Summary schema of the protective mechanism of CISCFE against UV-induced skin carcinogenesis. CISCFE, supercritical carbon dioxide fluid extraction of C. indicum; SOD, superoxide dismutase; CAT, catalase; ROS, reactive oxygen species; Keap1, Kelch-like ECH associated protein 1; Nrf2, nuclear factor 2 erythroid 2-related factor 2; SIRT1, NAD-dependent protein deacetylase sirtuin-1; HO-1, heme oxygenase 1; NQO1, NAD(P)H dehydrogenase [quinone] 1; P, phosphorylated; A, acetylated; U, ubiquitinated; C. indicum, Chrysanthemum indicum Linnén; sMaf, small musculoaponeurotic fibrosarcoma; ARE, antioxidant response elements; ED, epidermis; DR, dermis; ST, subcutaneous tissue.

Supplementary Material

High-performance liquid chromatograph of the 75% methyl alcohol layers of the supercritical carbon dioxide fluid extraction of Chrysanthemum indicum Linnén. mAU, m absorbance unit.

Chemical compositions of supercritical carbon dioxide extract from Chrysanthemum indicum Linnén by gas chromatography-mass spectrometry analysis (˃2%) and HPLC analysis.

Acknowledgements

Not applicable.

Funding

Funding: The present study was funded by The Science and Technology Planning Project of Guangzhou (grant no. 202102021263).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

YXZ and BQL developed and designed the study concept. QHL, QYZ, HJC, HEH, YQH, YCL, BL, YQW and SLD conducted the experiments and collected data. QHL, QYZ and HJC interpreted the results and drafted the manuscript. BQL, XHD and YXZ analyzed data and confirm the authenticity of all the raw data. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

The present study was conducted according to the guidelines of the Animal Care and Use Committee of Guangzhou University of Chinese Medicine (approval no. 20190304024; Guangzhou, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References