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Bestatin Cream Impairs Solar Simulated Light‒Driven Skin Inflammation and Skin Carcinogenesis in Mice

  • Author Footnotes
    6 These authors contributed equally to this work.
    Simin Zhao
    Footnotes
    6 These authors contributed equally to this work.
    Affiliations
    Department of Pathology, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, China

    China-US (Henan) Hormel Cancer Institute, Zhengzhou, China

    Henan Provincial Cooperative Innovation Center for Cancer Chemoprevention, Zhengzhou University, Zhengzhou, China
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  • Author Footnotes
    6 These authors contributed equally to this work.
    Ke Yao
    Footnotes
    6 These authors contributed equally to this work.
    Affiliations
    China-US (Henan) Hormel Cancer Institute, Zhengzhou, China

    The Hormel Institute, University of Minnesota, Austin, Minnesota, USA
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  • Author Footnotes
    6 These authors contributed equally to this work.
    Kangdong Liu
    Footnotes
    6 These authors contributed equally to this work.
    Affiliations
    China-US (Henan) Hormel Cancer Institute, Zhengzhou, China

    Henan Provincial Cooperative Innovation Center for Cancer Chemoprevention, Zhengzhou University, Zhengzhou, China

    Department of Pathophysiology, School of Basic Medical Sciences, Academy of Medical Science, College of Medicine, Zhengzhou University, Zhengzhou, China
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  • Limeng Huang
    Affiliations
    China-US (Henan) Hormel Cancer Institute, Zhengzhou, China
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  • Yanan Jiang
    Affiliations
    Henan Provincial Cooperative Innovation Center for Cancer Chemoprevention, Zhengzhou University, Zhengzhou, China

    Department of Pathophysiology, School of Basic Medical Sciences, Academy of Medical Science, College of Medicine, Zhengzhou University, Zhengzhou, China
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  • Jian Li
    Affiliations
    China-US (Henan) Hormel Cancer Institute, Zhengzhou, China

    The Hormel Institute, University of Minnesota, Austin, Minnesota, USA
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  • Ziming Dong
    Affiliations
    Henan Provincial Cooperative Innovation Center for Cancer Chemoprevention, Zhengzhou University, Zhengzhou, China

    Department of Pathophysiology, School of Basic Medical Sciences, Academy of Medical Science, College of Medicine, Zhengzhou University, Zhengzhou, China
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  • Zigang Dong
    Correspondence
    Correspondence: Zigang Dong, Department of Pathophysiology, School of Basic Medical Sciences, Academy of Medical Science, College of Medicine, Zhengzhou University, 100 Kexue Road, Zhengzhou 450001, China.
    Affiliations
    Department of Pathophysiology, School of Basic Medical Sciences, Academy of Medical Science, College of Medicine, Zhengzhou University, Zhengzhou, China
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  • Author Footnotes
    6 These authors contributed equally to this work.
Open ArchivePublished:May 25, 2021DOI:https://doi.org/10.1016/j.jid.2021.03.032
      Leukotriene A4 hydrolase (LTA4H) is an enzyme that catalyzes the production of the inflammatory mediator leukotriene B4, which is involved in inflammatory responses mediated through the leukotriene B4/leukotriene B4 receptor type 1 (BLT1) signaling pathway. In this study, we investigated whether bestatin, an LTA4H inhibitor, could suppress skin acute inflammation and carcinogenesis. In the clinical sample, BLT1 was significantly induced in human skin tissues after acute solar simulated light (SSL) exposure. BLT1 and NF-κB p65 expressions were also increased in acute SSL‒induced mouse skin tissue. Furthermore, LTA4H and BLT1 were highly expressed in skin chronic inflammation and squamous cell carcinomas. More importantly, topical administration of bestatin cream dramatically inhibited BLT1 expression in acute SSL‒induced human skin tissues. BLT1 and NF-κB p65 expressions were also suppressed in acute SSL‒induced Lta4h-knockout and bestatin-treated mice skin tissues. Moreover, we conducted long-term prevention and therapeutic studies, which showed that bestatin significantly attenuated SSL-induced skin carcinogenesis. Mechanistic studies showed that bestatin inhibited skin carcinogenesis by suppressing cell proliferation and inducing cell apoptosis through LTA4H‒BLT1‒protein kinase B‒NF-κB p65 pathway. Overall, our results suggest that topical application of novel cream containing bestatin might open a helpful avenue for SSL-induced skin carcinogenesis.

      Graphical abstract

      Abbreviations:

      Akt (protein kinase B), BLT1 (leukotriene B4 receptor type 1), LTA4H (leukotriene A4 hydrolase), Lta4h-KO (Lta4h-knockout), LTB4 (leukotriene B4), SCC (squamous cell carcinoma), SSL (solar simulated light), SUV (solar UV), WT (wild type)

      Introduction

      Squamous cell carcinomas (SCCs) arising from squamous cells in the epidermis are the second most common form of skin cancer and show a high incidence of lymph node metastasis (
      • Gao G.
      • Zhang T.
      • Wang Q.
      • Reddy K.
      • Chen H.
      • Yao K.
      • et al.
      ADA-07 suppresses solar ultraviolet-induced skin carcinogenesis by directly inhibiting TOPK.
      ;
      • Keyal U.
      • Bhatta A.K.
      • Zhang G.
      • Wang X.L.
      Present and future perspectives of photodynamic therapy for cutaneous squamous cell carcinoma.
      ). Solar UV (SUV) irradiation is the most important environmental carcinogen that is involved in skin cancer (
      • Yao K.
      • Chen H.
      • Liu K.
      • Langfald A.
      • Yang G.
      • Zhang Y.
      • et al.
      Kaempferol targets RSK2 and MSK1 to suppress UV radiation-induced skin cancer.
      ). SUV includes UVA and UVB wavelengths and may contribute to the initiation and progression of skin cancer through inflammatory responses, oxidative stress, DNA damage, and immunosuppressive responses (
      • Liu K.
      • Yu D.
      • Cho Y.Y.
      • Bode A.M.
      • Ma W.
      • Yao K.
      • et al.
      Sunlight UV-induced skin cancer relies upon activation of the p38α signaling pathway.
      ;
      • Pfeifer G.P.
      • You Y.H.
      • Besaratinia A.
      Mutations induced by ultraviolet light.
      ). Inflammation is associated with tumor initiation, tumor promotion, malignant conversion, and metastasis (
      • Grivennikov S.I.
      • Karin M.
      Inflammation and oncogenesis: a vicious connection.
      ). Cancer-related inflammation has emerged as one of the hallmarks of cancer (
      • Fan Y.
      • Mao R.
      • Yang J.
      NF-κB and STAT3 signaling pathways collaboratively link inflammation to cancer.
      ;
      • Mantovani A.
      Cancer: inflaming metastasis.
      ). Certain types of skin cells has reportedly secreted diverse inflammatory mediators, which are involved in skin cancer progression (
      • Lund A.W.
      • Medler T.R.
      • Leachman S.A.
      • Coussens L.M.
      Lymphatic vessels, inflammation, and immunity in skin cancer.
      ;
      • Medler T.R.
      • Coussens L.M.
      Duality of the immune response in cancer: lessons learned from skin.
      ). Therefore, inflammatory pathways could be effective targets for skin cancer prevention and therapeutic approaches.
      The main metabolic pathways of arachidonic acid are cyclooxygenase, lipoxygenase, and P450 epoxygenase pathways (
      • Haeggström J.Z.
      Leukotriene biosynthetic enzymes as therapeutic targets.
      ;
      • Yarla N.S.
      • Bishayee A.
      • Sethi G.
      • Reddanna P.
      • Kalle A.M.
      • Dhananjaya B.L.
      • et al.
      Targeting arachidonic acid pathway by natural products for cancer prevention and therapy.
      ). Clinical and epidemiological studies suggest that aberrant arachidonic acid metabolism through the activation of cyclooxygenase and lipoxygenase pathways might contribute to inflammation and carcinogenesis (
      • Wang D.
      • Dubois R.N.
      Eicosanoids and cancer.
      ). Leukotriene A4 hydrolase (LTA4H), a major enzyme in the lipoxygenase pathway, catalyzes the biosynthesis of proinflammatory leukotriene lipid mediator leukotriene B4 (LTB4) from arachidonic acid. LTB4 is involved in inflammation through LTB4 receptor type 1 (BLT1) signaling pathway. BLT1 acts as a receptor for LTB4, and its expression can also be induced by LTB4 stimulation (
      • Qiu H.
      • Johansson A.S.
      • Sjöström M.
      • Wan M.
      • Schröder O.
      • Palmblad J.
      • et al.
      Differential induction of BLT receptor expression on human endothelial cells by lipopolysaccharide, cytokines, and leukotriene B4.
      ). Bestatin has been described as a potent inhibitor of LTA4H (
      • Lkhagvaa B.
      • Tani K.
      • Sato K.
      • Toyoda Y.
      • Suzuka C.
      • Sone S.
      Bestatin, an inhibitor for aminopeptidases, modulates the production of cytokines and chemokines by activated monocytes and macrophages.
      ;
      • Umezawa H.
      • Aoyagi T.
      • Suda H.
      • Hamada M.
      • Takeuchi T.
      Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes.
      ). Bestatin is currently used for acute nonlymphocytic leukemia as a Food and Drug Administration‒approved drug in Japan (
      • Urabe A.
      • Mutoh Y.
      • Mizoguchi H.
      • Takaku F.
      • Ogawa N.
      Ubenimex in the treatment of acute nonlymphocytic leukemia in adults.
      ). The anti-inflammatory property of bestatin is largely dependent on the suppression of LTA4H enzyme activity, which catalyzes LTB4 biosynthesis. However, it is currently unknown whether the anti-inflammatory activity of bestatin could inhibit the initiation and progression of SUV-induced skin carcinogenesis.
      In this study, we showed that BLT1 was increased in acute solar simulated light (SSL)-induced human and mouse skin tissue. BLT1 was also highly expressed in chronic inflammation and SCC development. The LTA4H inhibitor, bestatin, suppressed acute SSL‒induced skin inflammation and chronic SSL‒induced skin carcinogenesis. More importantly, administration of bestatin reversed acute SSL‒induced BLT1 expression in human skin tissue. Overall, targeting LTA4H with bestatin might be a practical approach to preventing and treating SSL-induced skin carcinogenesis.

      Results

      The inhibitory function of bestatin in acute SSL‒induced human skin tissues

      Inflammation is associated with the development and progression of multiple cancers (
      • Maru G.B.
      • Gandhi K.
      • Ramchandani A.
      • Kumar G.
      The role of inflammation in skin cancer.
      ). The acute SSL‒induced assay was performed to test LTA4H and BLT1 expression in human skin. The results showed that BLT1 but not LTA4H expression was substantially increased after acute SSL exposure (Figure 1a and b). To detect the effect of bestatin in acute SSL‒induced human skin tissues, we grafted normal human skin onto nude mice (Figure 1c). The histological analysis of the human skin graft model showed that the epidermis and dermis of human skin are thicker than those of the mouse skin and that the hair follicles and sebaceous glands are scarce. In contrast, nude mice have thinner epidermis and dermis, which are rich in hair follicles and sebaceous glands (Figure 1d). The results of acute SSL‒induced assay showed that BLT1 but not LTA4H was significantly inhibited in human skin after bestatin treatment (Figure 1e and f), suggesting that bestatin could inhibit human skin BLT1 expression induced by acute SSL. The human skin tissue array results showed that BLT1 expression was much higher in chronic inflammation (Figure 1g) and SCC (Figure 1h) than in normal human skin tissue. These results suggest that the LTA4H/BLT1 pathway may be involved in SSL-induced skin carcinogenesis.
      Figure thumbnail gr1
      Figure 1The inhibitory function of bestatin in acute SSL‒induced human skin tissues. (a) LTA4H and (b) BLT1 expressions in clinical human skin tissues were analyzed by IHC, and the sum of IOD values from each sample was determined using the Image-Pro Plus software program. (c) Graphical abstract of human skin‒transplanted mouse model construction and acute SSL induction. (d) Histological analysis of human skin‒transplanted mouse model. (e) LTA4H and (f) BLT1 expression in bestatin-treated clinical human skin tissues were analyzed by IHC, and the sum of IOD values from each sample was determined using the Image-Pro Plus software program. (g, h) A human skin tissue array was analyzed by IHC to assess the expression of BLT1. Representative IHC images are shown. Data are presented as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Black Bar = 100 μm; blue Bar = 50 μm. BL, baseline; BLT1, leukotriene B4 receptor type 1; h, hour; IHC, immunohistochemistry; IOD, integrated optical density; LTA4H, leukotriene A4 hydrolase; min, minute; SCC, squamous cell carcinoma; SSL, solar simulated light; W/O, without.

      The inhibitory function of bestatin in acute SSL‒induced mouse skin tissues

      In the acute SSL‒exposed mouse model, BLT1 and NF-κB p65 expressions were significantly increased after acute SSL irradiation. In addition, LTB4 concentration was also significantly increased. More importantly, bestatin treatment inhibited BLT1, NF-κB p65 expression, and LTB4 concentration in the acute SSL‒induced mouse model (Figure 2a‒g). To further determine whether bestatin inhibited BLT1 and NF-κB p65 expression through targeting LTA4H, an acute SSL‒induced assay was performed using Lta4h-knockout (Lta4h-KO) mice. After acute SSL induction, BLT1 and NF-κB p65 expressions were significantly increased in wide-type (WT) mice; however, there was no induced elevation in Lta4h-KO mice. When WT mice were treated with bestatin, BLT1 and NF-κB P65 expressions were significantly reduced, but there were still no changes in Lta4h-KO mice (Figures 2h‒m). In addition, LTB4 levels in WT mice were significantly increased after acute SSL induction but not in Lta4h-KO mice. After bestatin treatment, SSL-induced LTB4 in WT mice was suppressed; however, there was no change of LTB4 expression in Lta4h-KO mice (Figure 2n). We also induced nude mice with acute SSL; the results showed that LTB4 levels and BLT1 and NF-κB P65 expression were significantly increased. When the nude mice were treated with bestatin, LTB4, BLT1, and NF-κB P65 levels were significantly inhibited (Supplementary Figure S1). In summary, our results suggest that bestatin might inhibit the acute SSL‒induced inflammation by targeting LTA4H.
      Figure thumbnail gr2
      Figure 2The inhibitory function of bestatin in acute SSL‒induced mouse skin tissues. (a) LTA4H and (b) BLT1 expressions were detected in bestatin-pretreated SKH-1 mouse skin tissues after 24 h and 48 h of SSL irradiation. (c) NF-κB p65 expression was detected in bestatin-pretreated BALB/C mouse skin tissues after 24 h and 48 h of SSL irradiation. The expression of (d) LTA4H, (e) BLT1, (f) NF-κB p65 were statistically analyzed. (g) The mice serum LTB4 was measured by ELISA assay in an acute SSL‒induced mouse model. (h) LTA4H, (i) BLT1, and (j) NF-κB p65 expressions were detected in bestatin-pretreated Lta4h-KO and WT mice skin tissues after 24 h and 48 h of SSL irradiation. The expression of (k) LTA4H, (l) BLT1, (m) NF-κB p65 in Lta4h-KO mice and WT mice were statistically analyzed. (n) The serum LTB4 was measured by ELISA assay in acute SSL‒induced Lta4h-KO mice and WT mice. Data are presented as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Black Bar = 100 μm. BLT1, leukotriene B4 receptor type 1; h, hour; IOD, integrated optical density; LTA4H, leukotriene A4 hydrolase; Lta4h-KO, Lta4h- knockout; LTB4, leukotriene B4; ns, not significant; SSL, solar simulated light; WT, wild type.

      Bestatin blocks epidermal cell transformation and inhibits the proliferation of skin cancer cells

      We next examined LTA4H expression in skin cell lines (Supplementary Figure S2). The results indicated that LTA4H is highly expressed in skin cancer cells. Bestatin treatment inhibited 12-O-tetradecanoylphorbol-13-acetate‒induced JB6 CI41 cell transformation in a dose-dependent manner (Figure 3a). Bestatin also decreased A431 and SCC12 cell proliferation in a dose-dependent manner (Figure 3b and c). To determine the potential mechanism responsible for the growth inhibitory effect of bestatin, we treated LTA4H-knockdown and scramble cells with bestatin in a cell proliferation assay. The results showed that bestatin significantly inhibited cell proliferation in scramble cells, but there was no change in LTA4H-knockdown cells (Supplementary Figure S3), consistent with the results of bestatin-treated Lta4h-KO mice. This suggested that the inhibitory effect of bestatin might attribute to targeting LTA4H. Next, we verified whether bestatin inhibited the expression of BLT1 and phosphorylated protein kinase B (Akt), mTOR, and p70S6K induced by SSL (60 kJ/m2 UVA and 2.9 kJ/m2 UVB) in JB6 CI41 cells (Figure 3d). Bestatin also markedly suppressed BLT1/Akt pathway in skin cancer cells (Figure 3e and f). These results suggested that bestatin may inhibit cell transformation and proliferation by targeting the LTA4H/BLT1 pathway.
      Figure thumbnail gr3
      Figure 3Bestatin inhibits anchorage-independent growth and proliferation of skin cancer cells. (a) JB6 Cl 41 cells were exposed to TPA (10 ng/ml) and treated with bestatin; the colony numbers were counted and analyzed. Bar = 250 μm. (b) A431 and (c) SCC12 cells were treated with bestatin, and cell proliferation was detected by crystal violet staining assay. (d) JB6 Cl 41 cells were harvested to detect the expression levels of phosphorylated and total proteins by western blot analysis. (e) A431 and (f) SCC12 were treated with different concentrations of bestatin and then harvested for western blot analysis. Data are presented as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Akt, protein kinase B; BLT1, leukotriene B4 receptor type 1; h, hour; LTA4H, leukotriene A4 hydrolase; p-Akt, phosphorylated protein kinase B; p-mTOR, phosphorylated mTOR; p- p70S6K, phosphorylated p70S6K; SCC, squamous cell carcinoma; SUV, solar UV; T-Akt, total protein kinase B; T-mTOR, total mTOR; T-p70S6K, total p70S6K; TPA, 12-O-tetradecanoylphorbol-13-acetate.

      Bestatin induces apoptosis of skin cancer cells

      To assess whether bestatin could induce skin cancer cell apoptosis, we performed an apoptosis assay. The results showed that bestatin significantly induced apoptosis of A431 and SCC12 cells (Figure 4a and b). Furthermore, the anti-apoptotic protein Bcl-2 was markedly inhibited by bestatin treatment. Apoptosis-related proteins, including cleaved PARP, cleaved caspase 3, and pro-apoptotic BAX, were increased in a dose-dependent manner when treated with bestatin (Figure 4c and d). These results indicated that bestatin also inhibited skin carcinogenesis by inducing apoptosis.
      Figure thumbnail gr4
      Figure 4Bestatin induces apoptosis in A431 and SCC12 skin cancer cells. (a) A431 and (b) SCC12 cells were treated with bestatin for 72 h and stained with annexin V. Apoptosis was measured by flow cytometry. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 indicate that bestatin significantly induced apoptosis of A431 and SCC12 cells. (c) A431 and (d) SCC12 cells were treated with bestatin for 72 h, and then cells were harvested to detect apoptosis-related protein expression. Data are presented as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. h, hour; SCC, squamous cell carcinoma.

      Potential preventive value of bestatin against skin carcinogenesis

      To evaluate the preventive efficiency of bestatin on skin carcinogenesis, we assessed its efficacy in attenuating chronic SSL‒induced mouse skin tumorigenesis (Figure 5a). Bestatin pretreatment resulted in decreased tumor volumes and numbers in the chronic SSL‒induced mice (Figure 5b and c). Bestatin also reduced LTB4 concentration in mouse serum (Figure 5d) and inhibited the expression of BLT1 and phosphorylation of Akt, mTOR, and p70S6K (Figure 5e). In addition, immunohistochemistry staining also showed that bestatin suppressed the expression of BLT1, phosphorylated Akt, NF-κB p65, Bcl-2, and PCNA (Figure 5f). Overall, these results indicated that bestatin may exert a strong preventive effect against SSL-induced mouse skin carcinogenesis by targeting LTA4H.
      Figure thumbnail gr5
      Figure 5Bestatin suppresses SSL-induced skin carcinogenesis in an SKH-1 hairless mouse prevention model. (a) Representative images of the tumor. (b) Tumor volumes and (c) tumor numbers were recorded and compared in different groups. (d) The LTB4 concentration in mice serum was measured using an ELISA assay. (e) Skin tissues were collected, and BLT1, p-Akt, p-mTOR, p-p70S6K, T-Akt, mTOR, and p70S6K were detected by western blot analysis. (f) The expressions of BLT1, p-Akt, NF-κB p65, Bcl-2, and PCNA were detected by IHC. The sum of IOD units was counted using the Image-Pro Plus software program. Data are presented as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 indicate a significant difference in bestatin-treated groups compared with that in the vehicle-treated group. Black Bar = 100 μm. Akt, protein kinase B; BLT1, leukotriene B4 receptor type 1; IHC, immunohistochemistry; IOD, integrated optical density; LTB4, leukotriene B4; p-Akt, phosphorylated protein kinase B; p-mTOR, phosphorylated mTOR; p-p70S6K, phosphorylated p70S6K; SSL, solar simulated light; T-Akt, total protein kinase B; T-mTOR, total mTOR; T-p70S6K, total p70S6K.

      Potential therapeutic value of bestatin against skin carcinogenesis

      We next investigated whether bestatin could be therapeutically used for SSL-induced mouse skin carcinogenesis (Figure 6a). The mice were treated with bestatin after tumor formation. The tumor volumes and numbers were significantly suppressed after bestatin treatment (Figure 6b and c). Bestatin treatment also markedly reduced LTB4 concentration in mouse serum (Figure 6d). The results of western blot analysis showed that bestatin inhibited the expression of BLT1 and phosphorylated Akt, phosphorylated mTOR, and phosphorylated p70S6K (Figure 6e). Bestatin also suppressed the expression of BLT1, phosphorylated Akt, NF-κB p65, Bcl-2, and PCNA in immunohistochemistry assay (Figure 6f). These results showed that SSL induced the activation of the LTA4H‒LTB4‒BLT1‒Akt‒NF-κB p65 pathway, which prompts the initiation and progression of skin carcinogenesis. Bestatin treatment showed a strong inhibitory effect on the LTA4H pathway, which may attenuate SSL-induced skin carcinogenesis.
      Figure thumbnail gr6
      Figure 6Bestatin suppresses SSL-induced skin carcinogenesis in an SKH-1 hairless mouse therapeutic model. (a) Representative images of the tumor. (b) Tumor volumes and (c) tumor numbers were recorded and compared. (d) LTB4 concentrations were detected in mice serum. (e) At week 30, skin tissues were harvested; the expressions of BLT1, p-Akt, p-mTOR, and p-p70S6K were detected by western blot analysis. (f) After fixing, the skin tissues were subjected to IHC, and the expressions of BLT1, p-Akt, NF-κB p65, Bcl-2, and PCNA were detected. The sum of IOD units was counted using the Image-Pro Plus software program. Data are presented as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 indicate a significant difference in bestatin-treated groups compared with that in the vehicle-treated group. Bar = 100 μm. Akt, protein kinase B; BLT1, leukotriene B4 receptor type 1; IOD, integrated optical density; IHC, immunohistochemistry; LTB4, leukotriene B4; p-Akt, phosphorylated protein kinase B; p-mTOR, phosphorylated mTOR; p-p70S6K, phosphorylated p70S6K; T-Akt, total protein kinase B; T-mTOR, total mTOR; T-p70S6K, total p70S6K.

      Discussion

      Inflammation functions to eliminate invading pathogens and tissue damage, thereby facilitating the regeneration of damaged host tissue (
      • Kotas M.E.
      • Medzhitov R.
      Homeostasis, inflammation, and disease susceptibility.
      ). However, inflammation is involved in the process of carcinogenesis (
      • Bottazzi B.
      • Riboli E.
      • Mantovani A.
      Aging, inflammation and cancer.
      ). Recent data have suggested that many cancers originate from sites of infection, chronic irritation, and inflammation (
      • Hensler S.
      • Mueller M.M.
      Inflammation and skin cancer: old pals telling new stories.
      ;
      • van Elsland D.
      • Neefjes J.
      Bacterial infections and cancer.
      ). The skin is the largest physical and immunological barrier to the external environment. Evidence has shown that chronic inflammation might be one of the enabling characteristics in SUV-induced skin cancer (
      • Pasparakis M.
      • Haase I.
      • Nestle F.O.
      Mechanisms regulating skin immunity and inflammation.
      ;
      • Proksch E.
      • Brandner J.M.
      • Jensen J.M.
      The skin: an indispensable barrier.
      ). Therefore, identifying the major inflammatory pathways in SSL-induced skin cancer is a critically important task for skin cancer prevention and therapy. LTA4H is highly expressed in several human colorectal cancer cell lines, and [6]-gingerol effectively suppresses colon carcinogenesis by targeting LTA4H (
      • Jeong C.H.
      • Bode A.M.
      • Pugliese A.
      • Cho Y.Y.
      • Kim H.G.
      • Shim J.H.
      • et al.
      [6]-Gingerol suppresses colon cancer growth by targeting leukotriene A4 hydrolase.
      ;
      • Zhao S.
      • Yao K.
      • Li D.
      • Liu K.
      • Jin G.
      • Yan M.
      • et al.
      Inhibition of LTA4H by bestatin in human and mouse colorectal cancer.
      ). Targeting LTA4H could suppress pancreatic cancer cell proliferation and anchorage-independent growth (
      • Oi N.
      • Jeong C.H.
      • Nadas J.
      • Cho Y.Y.
      • Pugliese A.
      • Bode A.M.
      • et al.
      Resveratrol, a red wine polyphenol, suppresses pancreatic cancer by inhibiting leukotriene A4hydrolase.
      ). However, the mechanistic roles of LTA4H and its catalytic products in inflammation-induced skin carcinogenesis are still unclear. Our human clinical skin tissues showed that BLT1 expression was increased in SSL-induced acute inflammatory responses. The mouse model also showed increased LTB4 and BLT1 but not LTA4H after acute SSL exposure. We also confirmed that BLT1 expression increased after LTB4 induction in a time-dependent manner, which is consistent with the literature (Supplementary Figure S4) (
      • Qiu H.
      • Johansson A.S.
      • Sjöström M.
      • Wan M.
      • Schröder O.
      • Palmblad J.
      • et al.
      Differential induction of BLT receptor expression on human endothelial cells by lipopolysaccharide, cytokines, and leukotriene B4.
      ). Furthermore, BLT1 was highly expressed in chronic inflammation and skin cancer (Figure 1). We therefore suggest that the LAT4H pathway may be a promising target pathway for skin cancer.
      Bestatin therapies for anti-inflammatory and anticancer functions have achieved clinical success in the process of a challenge to various cancers, including lung cancer, gastric cancer, melanoma, and breast cancer (
      • Aozuka Y.
      • Koizumi K.
      • Saitoh Y.
      • Ueda Y.
      • Sakurai H.
      • Saiki I.
      Anti-tumor angiogenesis effect of aminopeptidase inhibitor bestatin against B16-BL6 melanoma cells orthotopically implanted into syngeneic mice.
      ;
      • Ezawa K.
      • Minato K.
      • Dobashi K.
      Induction of apoptosis by ubenimex (bestatin) in human non-small-cell lung cancer cell lines.
      ;
      • Fujioka S.
      • Kohno N.
      • Hiwada K.
      Ubenimex activates the E-cadherin-mediated adhesion of a breast cancer cell line YMB-S.
      ;
      • Yabusaki H.
      • Aizawa K.
      • Tanaka N.
      • Suzuki T.
      • Tanaka O.
      Inhibition of tumor-cell invasion and gelatinase production in metastatic human gastric-carcinoma cells by retinoic acid and bestatin.
      ). However, the prevention or therapeutic effects of bestatin against SUV-induced skin carcinogenesis have not been reported. Our in vivo data showed that bestatin inhibited BLT1 and NF-κB p65 expression in mouse skin tissues induced by acute SSL. Topical application of bestatin significantly inhibited the tumor volumes and numbers in both prevention and therapy skin carcinogenesis models (Figures 5 and 6). Most importantly, topical administration of bestatin reversed acute SSL‒induced BLT1 expression in the human skin tissue. Our results offer insight into the effect of bestatin as a promising drug for SSL-induced acute inflammation and skin carcinogenesis. Because the bestatin cream is developed in our laboratory and is easily absorbed into the skin, we will later conduct skin cancer clinical trials. This may provide a rationale for the clinical use of bestatin in SSL-induced skin carcinogenesis.
      This study indicated that LTA4H was involved in SSL-induced skin carcinogenesis and that bestatin might inhibit cell proliferation and induce apoptosis by targeting LTA4H. Bestatin could also attenuate SSL-induced acute inflammatory response and skin carcinogenesis. Collectively, these results support the idea that bestatin could serve as a promising compound for prevention and treatment of skin carcinogenesis.

      Materials and Methods

      Human skin tissues

      SSL-exposed human skin tissues were provided by the University of Arizona Cancer Center (Tucson, AZ). This study was approved by The University of Arizona Institutional Review Board (FWA0 0004218/IRB000 00291). All participants provided written informed consent. The participants were determined to receive the minimal erythema dose of SSL using the Multiport UV Solar Simulator Model 600 (Solar Light, Philadelphia, PA) and a reflectance spectrophotometer (Minolta Chroma Meter Model CR-20; Minolta, Osaka, Japan). The 6-mm skin punch biopsy was taken at 5 minutes, 1 hour, 5 hours, and 24 hours after SSL irradiation. The details of the experiment are described in a previous study (
      • Roh E.
      • Lee M.H.
      • Zykova T.A.
      • Zhu F.
      • Nadas J.
      • Kim H.G.
      • et al.
      Targeting PRPK and TOPK for skin cancer prevention and therapy.
      ). A human skin cancer tissue array (SK2081) was purchased from US Biomax (Rockville, MD).

      Acute SSL‒induced inflammation in human skin‒transplanted mouse model

      Transplanted healthy human skin tissues were donated by China-US (Henan) Hormel Cancer Institute (Zhengzhou, China). This study was approved by China-US (Henan) Hormel Cancer Institute Ethics Committee. After anesthesia, a circular piece of skin (1 cm diameter) was removed on the back of the nude mice. Human skin was cut into 1 cm diameter/0.5 mm thick pieces to fit the skin gaps. Surgical adhesive and elastic bandage were used to fix the mouse skin graft (
      • Boudry I.
      • Blanck O.
      • Cruz C.
      • Blanck M.
      • Vallet V.
      • Bazire A.
      • et al.
      Percutaneous penetration and absorption of parathion using human and pig skin models in vitro and human skin grafted onto nude mouse skin model in vivo.
      ). The human skin graft mice were used for the acute SSL‒induced assay. The mice were pretreated with bestatin and then induced with acute SSL (149 kJ/m2 UVA/7.2 kJ/m2 UVB). The mice were euthanized at different time points, and the skin tissues were excised.

      Chemicals and reagents

      Bestatin was purchased from BOC Sciences (Shirley, NY). LTB4 was purchased from Cayman Chemical (Ann Arbor, MI). Antibodies to detect LTA4H (160250) and BLT1 (120114) were obtained from Cayman Chemical. Antibodies to detect β-actin (sc-47778), GAPDH (sc-25778), and Bcl-2 (sc-7382) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to detect NF-κB p65 (#8242), phosphorylated Akt (#9271), Akt (#9272), phosphorylated mTOR (#5536), mTOR (#2983), phosphorylated p70S6K (#9234), p70S6K (#2708), BAX (#2772), PARP (#9532), caspase 3 (#9665), cleaved PARP (#5625), and cleaved caspase 3 (#9664) were purchased from Cell Signaling Technology (Danvers, MA).

      Cell culture

      The HaCaT cells, JB6 Cl 41 mouse epidermal cells, and human epidermoid carcinoma A431 cells were purchased from ATCC (Manassas, VA). The human SCC12 cells were obtained from Thermo Fisher Scientific (Waltham, MA). The identities of all cells were confirmed using Short-Tandem Repeat profiling, and the cells were confirmed free of mycoplasma before freezing. Each vial was thawed and maintained in culture for no more than 10 passages. All cell culture conditions were performed following ATCC's instructions.

      Crystal violet staining assay

      Cells (2.4 × 104/well) were seeded into 24-well plates and incubated overnight. Cells were then treated with various concentrations of bestatin and incubated for 0 hours, 24 hours, 48 hours, or 72 hours. Absorbance was read at 540 nm using the Thermo Multiskan plate-reader (Thermo Fisher Scientific).

      Anchorage-independent cell growth assay

      Cells (8 × 103/well) were seeded into 6-well plates with 0.3% Basal Medium Eagle agar containing 10% fetal bovine serum with or without 12-O-tetradecanoylphorbol-13-acetate (10 ng/ml) and different concentrations of bestatin (0, 10, 20, 50, 100 μM) and then cultured for 1‒2 weeks. Colonies were quantified using the Image-Pro Plus (version 6) computer software program (Media Cybernetics, Rockville, MA).

      Apoptosis assay

      Skin cancer cells (1 × 105) were plated in 60-mm dishes and then treated for 72 hours with bestatin. The cells were fixed with ice-cold 70% ethanol at –20 °C overnight. After staining with Annexin V, apoptosis was analyzed by two-color flow cytometry.

      Constructed LTA4H-knockdown cells

      The 293T cells were transfected with 2 μg PLKO.1-shLTA4H, 2 μg pMD2.0G, and 2 μg psPAX using iMFectin Poly DNA transfection reagent following the manufacturer’s instructions. The medium was replaced with a fresh complete medium containing serum and antibiotics after 24 hours. The viral supernatant fractions were collected at 24 hours and 48 hours. Target cells were infected with virus-containing media supplemented with polybrene. The positive LTA4H-knockdown cells were screened with puromycin.

      SSL irradiation system

      The SSL irradiation system (UVA-340 lamps) provided UVA (94.5%) and UVB (5.5%) irradiation. This system was purchased from Q-Lab (Cleveland, OH). With this system, cells were exposed to the simulated sunlight, including UVA and UVB, in the critical short wavelength region from 365 nm down to the solar cutoff of 295 nm, with a peak emission at 340 nm. This system is described in our previous study (
      • Roh E.
      • Lee M.H.
      • Zykova T.A.
      • Zhu F.
      • Nadas J.
      • Kim H.G.
      • et al.
      Targeting PRPK and TOPK for skin cancer prevention and therapy.
      ).

      Acute SSL‒induced inflammation in Lta4h-KO mice

      Lta4h-KO mice were purchased from Beijing Vitalstar Biotechnology (Beijing, China). After homozygous mice were screened, Lta4h-KO mice and WT mice were randomly grouped for the acute SSL‒induced study. The hair on the back of the mice was shaved, and the mice were treated topically with cream only or cream containing bestatin as indicated. After 1 hour, the mice were induced with acute SSL (149 kJ/m2 UVA/7.2 kJ/m2 UVB). Skin tissues were collected 24 hours and 48 hours after acute SSL treatment. This study was approved by China-US (Henan) Hormel Cancer Institute Ethics Committee.

      SSL-induced acute inflammation in SKH-1 hairless mice and BALB/C mice

      SKH-1 hairless mice and BALB/C mice were respectively divided into nine groups: group 1, no treatment; group 2, cream-only treatment; group 3, treated with 1 mg bestatin only; group 4, treated 24 hours after SSL with cream; group 5, treated 24 hours after SSL with 0.1% bestatin; group 6, treated 24 hours after SSL with 1% bestatin; group 7, treated 48 hours after SSL with cream only; group 8, treated 48 hours after SSL with 0.1% bestatin; and group 9, treated 48 hours after SSL with 1% bestatin. The mice were treated topically with cream only or cream containing bestatin as indicated. After 1 hour of incubation, the dorsal skin of the mice was treated with SSL (149 kJ/m2 UVA and 7.2 kJ/m2 UVB). Twenty-four hours and 48 hours after SSL irradiation, mouse tissues were collected and analyzed. This study was approved by China-US (Henan) Hormel Cancer Institute Ethics Committee.

      Chronic SSL‒induced mouse skin tumorigenesis studies

      For the prevention study, SKH-1 mice were divided into six groups: group 1, no treatment; group 2, cream-only treatment; group 3, bestatin-only treatment; group 4, treated with cream and SSL; group 5, treated with 0.1% bestatin and SSL; and group 6, treated with 1% bestatin and SSL. The mice were treated topically with vehicle or bestatin on the dorsal skin and then were exposed to SSL irradiation after 1-hour incubation. The drug treatment and SSL irradiation were applied three times per week for a total of 15 weeks. After 15 weeks, SSL was stopped, and the mice were only treated with the drug until 25 weeks. For the therapeutic study, SKH-1 mice were divided into five groups: group 1, no treatment; group 2, cream-only treatment; group 3, bestatin-only treatment; group 4, treated with cream and SSL; and group 5, treated with 1% bestatin and SSL. The mice were exposed to SSL irradiation three times per week for 15 weeks, and then the mice were treated with bestatin from week 25 to week 30. The mice weight, tumor incidence, and tumor multiplicity were recorded weekly when a tumor was observed. Tumor volume was calculated as tumor volume (mm3) = length × width × height × 0.52. At the end of the studies, the mice were killed, and the blood and dorsal area of skin were collected. This study was approved by China-US (Henan) Hormel Cancer Institute Ethics Committee.

      Immunohistochemistry staining

      Skin tissues were embedded in paraffin and cut into 4-μm sections. The slides were incubated with different primary antibodies (LTA4H, 1:75; BLT1, 1:50; NF-κB p65, 1:50; phosphorylated Akt, 1:200; Bcl-2, 1:100; and PCNA, 1:2,000) overnight at 4 °C. The secondary antibodies (anti-rabbit 1:150 and anti-mouse 1:150) were from Vector Laboratories (Burlingame, CA). Slides were stained using the Vectastain Elite ABC Kit from Vector Laboratories. The slides were counterstained with hematoxylin and analyzed using the Image-Pro Plus (version 6) computer software program.

      Western blot analysis

      JB6 CI41 cells were starved for 24 hours before being treated with bestatin and were then exposed to SSL (60 kJ/m2 UVA and 2.9 kJ/m2 UVB) and incubated for an additional 15 minutes. A431 and SCC12 were treated with various concentrations of bestatin for 24 hours or 48 hours. Proteins (30‒50 μg) were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA). The membranes were incubated with primary antibodies overnight at 4 °C, and protein bands were detected with a chemiluminescent reagent (GE Healthcare Biosciences AB, Uppsala, Sweden).

      LTB4 ELISA assay

      After anesthesia, whole blood was collected from the mice. After separating the serum and plasma, the serums were subjected to ELISA assay following the manufacturer’s instructions. The LTB4 ELISA kit was purchased from Cayman Chemical.

      Statistical analysis

      All quantitative data are expressed as mean values ± SD of at least three independent experiments. Significant differences were determined by a Student's t-test or one-way ANOVA. A probability value of P < 0.05 was used as the criterion for statistical significance.

      Data availability statement

      No datasets were generated or analyzed during this study.

      ORCIDs

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported by the National Natural Science Foundation of China, Beijing, China (numbers 81472324 and 81572812) and the Henan Province Science and Technology Project (number 212102310880). The authors thank Tianshun Zhang and Qiushi Wang, University of Minnesota, Austin, MN, for their help.

      Author Contributions

      Conceptualization: SZ, ZigD; Data Curation: SZ, KY, KL; Investigation: YJ, JL; Writing - Original Draft Preparation: SZ, KY, KL; Writing - Review and Editing: SZ, LH, ZimD

      Supplementary Materials

      Figure thumbnail fx2
      Supplementary Figure S1The inhibitory function of bestatin in acute SSL‒induced nude mice skin tissues. (a) BLT1 and (b) NF-κB p65 expressions were detected in bestatin-pretreated nude mice skin tissues after 48 h of SSL irradiation. (c) The mice serum LTB4 was measured by ELISA assay in an acute SSL‒induced mouse model. Data are presented as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Bar = 100 μm. BLT1, leukotriene B4 receptor type 1; h, hour; LTB4, leukotriene B4; SSL, solar simulated light.
      Figure thumbnail fx3
      Supplementary Figure S2LTA4H expression in human skin cancer cell lines. Protein levels of LTA4H were analyzed by western blotting in human skin cancer cell lines. LTA4H, leukotriene A4 hydrolase; SCC, squamous cell carcinoma.
      Figure thumbnail fx4
      Supplementary Figure S3Bestatin has no effect on the cell proliferation of shLTA4H cells. A431 cells transfected with shMOCK or shLTA4H plasmid and then treated with bestatin (100 μM). Cell proliferation was detected. The data are shown as mean values ± SE from triplicate experiments. ∗∗P < 0.01. h, hour; NS, not significant; SE, standard error; shLTA4H, short hairpin LTA4H; shMOCK, short hairpin MOCK.
      Figure thumbnail fx5
      Supplementary Figure S4LTB4 stimulation induced BLT1 protein expression. HUVECs were treated with LTB4 at different time points (0 h, 1 h, 3 h, and 6 h). Cells were collected, proteins were extracted, then the cells were subjected to western blotting, and BLT1 expression levels were analyzed. BLT1, leukotriene B4 receptor type 1; h, hour; HUVEC, human umbilical vein endothelial cell; LTB4, leukotriene B4.

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