Novel Polystyrene-Binding Nanobody for Enhancing Immunoassays: Insights into Affinity, Immobilization, and Application Potential.

Nanobodies, which represent the next generation of antibodies due to their unique properties, face a significant limitation in their poor physical adsorption on solid supports. In this study, we successfully discovered polystyrene binding nanobodies from a synthetic nanobody library. Notably, bivalent nanobody B2 exhibited high affinity for polystyrene (0.7 nM for ELISA saturation binding analysis and 15.6 nM for isothermal titration calorimetry), displaying a pH-dependent behavior. Remarkably, hydrophobic and electrostatic interactions contribute minimally to the binding process. Molecular modeling provided insights into the interaction between B2 and polystyrene, revealing that the Trp51 residue within the CDR2 loop formed an aromatic H-bond with polystyrene at a distance of 2.74 Å, thus explaining the observed reduction in B2 affinity caused by Trp51 mutations. To explore B2's potential in protein immobilization, we constructed a bispecific nanobody by fusing B2 to an anticarcinoembryonic antigen nanobody 11C12, which cannot be immobilized on polystyrene through passive adsorption. Remarkably, the fusion construct achieved effective immobilization on polystyrene within 5 min by passing the need for periplasmic protein purification despite its low expression level. Moreover, the fusion construct demonstrated excellent linearity in the chemiluminescent enzyme immunoassay. For the first time, this study reports a simplified and seamless platform for the oriented immobilization of nanobody. Importantly, the entire process eliminated the need for protein purification, enabling efficient and rapid immobilization of fusion proteins directly from crude cell extracts, even when the expression level was low. Our developed process dramatically reduced the processing time from 2.5 days to just 5 min.

Tumor microenvironment-mediated immune tolerance in development and treatment of gastric cancer.

Tumor microenvironment is the general term for all non-cancer components and their metabolites in tumor tissue. These components include the extracellular matrix, fibroblasts, immune cells, and endothelial cells. In the early stages of tumors, the tumor microenvironment has a tumor suppressor function. As the tumor progresses, tumor immune tolerance is induced under the action of various factors, such that the tumor suppressor microenvironment is continuously transformed into a tumor-promoting microenvironment, which promotes tumor immune escape. Eventually, tumor cells manifest the characteristics of malignant proliferation, invasion, metastasis, and drug resistance. In recent years, stress effects of the extracellular matrix, metabolic and phenotypic changes of innate immune cells (such as neutrophils, mast cells), and adaptive immune cells in the tumor microenvironment have been revealed to mediate the emerging mechanisms of immune tolerance, providing us with a large number of emerging therapeutic targets to relieve tumor immune tolerance. Gastric cancer is one of the most common digestive tract malignancies worldwide, whose mortality rate remains high. According to latest guidelines, the first-line chemotherapy of advanced gastric cancer is the traditional platinum and fluorouracil therapy, while immunotherapy for gastric cancer is extremely limited, including only Human epidermal growth factor receptor 2 (HER-2) and programmed death ligand 1 (PD-L1) targeted drugs, whose benefits are limited. Clinical experiments confirmed that cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), vascular endothelial growth factor receptor (VEGFR) and other targeted drugs alone or in combination with other drugs have limited efficacy in patients with advanced gastric cancer, far less than in lung cancer, colon cancer, and other tumors. The failure of immunotherapy is mainly related to the induction of immune tolerance in the tumor microenvironment of gastric cancer. Therefore, solving the immune tolerance of tumors is key to the success of gastric cancer immunotherapy. In this study, we summarize the latest mechanisms of various components of the tumor microenvironment in gastric cancer for inducing immune tolerance and promoting the formation of the malignant phenotype of gastric cancer, as well as the research progress of targeting the tumor microenvironment to overcome immune tolerance in the treatment of gastric cancer.

Aire and Fezf2, two regulators in medullary thymic epithelial cells, control autoimmune diseases by regulating TSAs: Partner or complementer?

The expression of tissue-specific antigens (TSAs) in medullary thymic epithelial cells (mTECs) is believed to be responsible for the elimination of autoreactive T cells, a critical process in the maintenance of central immune tolerance. The transcription factor autoimmune regulator (Aire) and FEZ family zinc finger 2(Fezf2) play an essential role in driving the expression of TSAs in mTECs, while their deficiency in humans and mice causes a range of autoimmune manifestations, such as type 1 diabetes, Sjögren's syndrome and rheumatoid arthritis. However, because of their regulatory mechanisms, the expression profile of TSAs and their relationship with special autoimmune diseases are still in dispute. In this review, we compare the roles of Aire and Fezf2 in regulating TSAs, with an emphasis on their molecular mechanisms in autoimmune diseases, which provides the foundation for devising improved diagnostic and therapeutic approaches for patients.

Epigenome, Transcriptome, and Protection by Sulforaphane at Different Stages of UVB-Induced Skin Carcinogenesis.

Sulforaphane (SFN), a potent antioxidant and antiinflammatory agent, has been shown to protect against cancers especially at early stages. However, how SFN affects UVB-mediated epigenome/DNA methylome and transcriptome changes in skin photodamage has not been fully assessed. Herein, we investigated the transcriptomic and DNA methylomic changes during tumor initiation, promotion, and progression and its impact and reversal by SFN using next-generation sequencing (NGS) technology. The results show that SFN reduced tumor incidence and tumor number. SFN's protective effects were more dramatic in the early stages than with later stages. Bioinformatic analysis of RNA sequencing (RNA-seq) data shows differential expressed genes and identifies the top canonical pathways related to SFN treatment of UVB-induced different stages of epidermal carcinogenesis. These pathways include p53 signaling, cell cycle: G2-M DNA damage checkpoint regulation, Th1, and Th2 activation pathway, and PTEN signaling pathways. The top upstream regulators related to UVB and SFN treatment as time progressed include dextran sulfate, TP53, NFE2L2 (Nrf2), IFNB1, and IL10RA. Bioinformatic analysis of Methyl-seq data shows several differential methylation regions induced by UVB were attenuated by SFN. These include Notch1, Smad6, Gnai3, and Apc2 Integrative analysis of RNA-seq and DNA-seq/CpG methylome yields a subgroup of genes associated with ultraviolet B (UVB) and SFN treatment. The changes in gene expression were inversely correlated with promoter CpG methylation status. These genes include Pik3cd, Matk, and Adm2 In conclusion, our study provides novel insights on the impact of SFN on the transcriptomic and DNA methylomic of UVB-induced different stages of skin cancer in mice.

DNA methylome and transcriptome alterations and cancer prevention by triterpenoid ursolic acid in UVB-induced skin tumor in mice.

Nonmelanoma skin cancers (NMSCs) are the most common type of skin cancers. Major risk factors for NMSCs include exposure to ultraviolet (UV) irradiation. Ursolic acid (UA) is a natural triterpenoid enriched in blueberries and herbal medicinal products, and possess anticancer activities. This study focuses on the impact of UA on epigenomic, genomic mechanisms and prevention of UVB-mediated NMSC. CpG methylome and RNA transcriptome alterations of early, promotion and late stages of UA treated on UVB-induced NMSC in SKH-1 hairless mice were conducted using CpG methyl-seq and RNA-seq. Samples were collected at weeks 2, 15, and 25, and integrated bioinformatic analyses were performed to identify key pathways and genes modified by UA against UVB-induced NMSC. Morphologically, UA significantly reduced NMSC tumor volume and tumor number. DNA methylome showed inflammatory pathways IL-8, NF-κB, and Nrf2 pathways were highly involved. Antioxidative stress master regulator Nrf2, cyclin D1, DNA damage, and anti-inflammatory pathways were induced by UA. Nrf2, cyclin D1, TNFrsf1b, and Mybl1 at early (2 weeks) and late (25 weeks) stages were identified and validated by quantitative polymerase chain reaction. In summary, integration of CpG methylome and RNA transcriptome studies show UA alters antioxidative, anti-inflammatory, and anticancer pathways in UVB-induced NMSC carcinogenesis. Particularly, UA appears to drive Nrf2 and its upstream/downstream genes, anti-inflammatory (at early stages) and cell cycle regulatory (both early and late stages) genes, of which might contribute to the overall chemopreventive effects of UVB-induced MNSC. This study may provide potential biomarkers/targets for chemoprevention of early stage of UVB-induced NMSC in human.

UVB drives different stages of epigenome alterations during progression of skin cancer.

Exposure to ultraviolet B (UVB) irradiation results in multitude of cellular responses including generation of reactive oxygen species and DNA damage and is responsible for non-melanoma skin cancers (NMSCs). Although genetic mutation is well documented, the epi-mutation, the alteration in epigenetics, remains elusive. In this study, we utilized CpG Methyl-seq to identify a genome-wide DNA CpG methylation, to profile the DNA methylation in UVB-irradiated SKH-1 mouse skin epidermis and non-melanoma skin papillomas at various stages. Methyl-seq and RNA-seq were performed to examine the methylation and corresponding transcriptome alterations. The methylation profiles in mouse epidermis were altered by UVB-irradiation as time progresses. Ingenuity Pathways Analysis (IPA) identified many cancer related pathways including PTEN, p53, Nrf2 and inflammatory signaling in UVB-irradiation induced carcinogenesis. Additionally, some novel genes involved in skin carcinogenesis that were not previously reported were differentially methylated, including Enf2, Mgst2, Vegfa, and Cdk4. Taken together, the current study provides novel profiles and insights of methylation and transcriptomic changes at different stages of carcinogenesis in UVB-irradiation induced NMSC and offers potential targets for prevention and treatment of NMSC at different stages of human skin cancer.

PDE2 is a novel target for attenuating tumor formation in a mouse model of UVB-induced skin carcinogenesis.

Our previous studies demonstrated that the topical application of caffeine is a potent inhibitor of UVB-induced carcinogenesis and selectively increases apoptosis in tumors but not in non-tumor areas of the epidermis in mice that are at a high risk for developing skin cancer. While this effect is mainly through a p53 independent pathway, the mechanism by which caffeine inhibits skin tumor formation has not been fully elucidated. Since caffeine is a non-specific phosphodiesterase inhibitor, we investigated the effects of several PDE inhibitors on the formation of sunburn cells in mouse skin after an acute exposure to ultraviolet light B (UVB). The topical application of a PDE2 inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA hydrochloride), stimulated epidermal apoptosis compared to control (P<0.01) and to a greater extent than caffeine whereas a PDE4 inhibitor attenuated the epidermal apoptosis compared to control (P<0.01). Since PDE2 hydrolyzes cyclic nucleotides, mainly cGMP, the effects of EHNA hydrochloride on epidermal apoptosis following UVB exposure may be mediated, in part, by increased cGMP signaling. Data demonstrated that the topical application of dibutyryl cGMP stimulated epidermal apoptosis (P<0.01) following an acute exposure to UVB. Treating UVB-pretreated mice topically with 3.1 µmole or 0.8 µmole of EHNA hydrochloride attenuated tumor formation to a greater extent than treating with 6.2 µmole caffeine when these compounds were applied once a day, five days a week for 18 weeks. These observations suggest a novel role for PDE2 in UVB-induced tumorigenesis and that PDE2 inhibitors that mediate cGMP signaling may be useful for the prevention and treatment of skin cancer.

Nrf2 null enhances UVB-induced skin inflammation and extracellular matrix damages.

Nrf2 plays a critical role in defending against oxidative stress and inflammation. We previously reported that Nrf2 confers protection against ultraviolet-B (UVB)-induced inflammation, sunburn reaction, and is involved in sulforaphane-mediated photo-protective effects in the skin. In this study, we aimed to demonstrate the protective role of Nrf2 against inflammation-mediated extracellular matrix (ECM) damage induced by UVB irradiation. Ear biopsy weights were significantly increased in both Nrf2 wild-type (Nrf2 WT) and knockout (Nrf2 KO) mice one week after UVB irradiation. However, these weights increased more significantly in KO mice compared to WT mice, suggesting a greater inflammatory response in KO mice. In addition, we analyzed the protein expression of numerous markers, including macrophage inflammatory protein-2 (MIP-2), pro-matrix metalloproteinase-9 (MMP-9), and p53. p53, a regulator of DNA repair, was overexpressed in Nrf2 KO mice, indicating that the absence of Nrf2 led to more sustained DNA damage. There was also more substantial ECM degradation and increased inflammation in UVB-irradiated Nrf2 KO mice compared to UVB-irradiated WT mice. Furthermore, the protective effects of Nrf2 in response to UVB irradiation were mediated by increased HO-1 protein expression. Collectively, our results show that Nrf2 plays a key role in protecting against UVB irradiation and that the photo-protective effect of Nrf2 is closely related to the inhibition of ECM degradation and inflammation.

Genome-wide analysis of DNA methylation in UVB- and DMBA/TPA-induced mouse skin cancer models.

AIMS: Ultraviolet irradiation and carcinogens have been reported to induce epigenetic alterations, which potentially contribute to the development of skin cancer. We aimed to study the genome-wide DNA methylation profiles of skin cancers induced by ultraviolet B (UVB) irradiation and 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-1,3-acetate (TPA). MAIN METHODS: Methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing was utilized to ascertain the DNA methylation profiles in the following common mouse skin cancer models: SKH-1 mice treated with UVB irradiation and CD-1 mice treated with DMBA/TPA. Ingenuity® Pathway Analysis (IPA) software was utilized to analyze the data and to identify gene interactions among the different pathways. KEY FINDINGS: 6003 genes in the UVB group and 5424 genes in the DMBA/TPA group exhibited a greater than 2-fold change in CpG methylation as mapped by the IPA software. The top canonical pathways identified by IPA after the two treatments were ranked were pathways related to cancer development, cAMP-mediated signaling, G protein-coupled receptor signaling and PTEN signaling associated with UVB treatment, whereas protein kinase A signaling and xenobiotic metabolism signaling were associated with DMBA/TPA treatment. In addition, the mapped IL-6-related inflammatory pathways displayed alterations in the methylation profiles of inflammation-related genes linked to UVB treatment. SIGNIFICANCE: Genes with altered methylation were ranked in the UVB and DMBA/TPA models, and the molecular interaction networks of those genes were identified by the IPA software. The genome-wide DNA methylation profiles of skin cancers induced by UV irradiation or by DMBA/TPA will be useful for future studies on epigenetic gene regulation in skin carcinogenesis.

Requirement and epigenetics reprogramming of Nrf2 in suppression of tumor promoter TPA-induced mouse skin cell transformation by sulforaphane.

Nrf2 is a transcription factor that plays critical roles in regulating the expression of cellular defensive antioxidants and detoxification enzymes. However, the role of Nrf2 and Nrf2's epigenetics reprogramming in skin tumor transformation is unknown. In this study, we investigated the inhibitory role and epigenetics of Nrf2 on tumor transformation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin epidermal JB6 (JB6 P+) cells and the anticancer effect of sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables. After five days of treatment, SFN significantly inhibited TPA-induced JB6 cellular transformation and SFN enhanced the nuclear translocation of Nrf2 and increased the mRNA and protein levels of the Nrf2 target genes HO-1, NQO1 and UGT1A1. Knockdown of Nrf2 attenuated the induction of Nrf2, HO-1 and NQO1 by SFN, enhanced TPA-induced colony formation and dampened the inhibitory effect of SFN on TPA-induced JB6 transformation. Epigenetics investigation using bisulfite genomic sequencing showed that SFN decreased the methylation ratio of the first 15 CpGs of the Nrf2 gene promoter, which was corroborated by increased Nrf2 mRNA expression. Furthermore, SFN strongly reduced the protein expression of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b). SFN also inhibited the total histone deacetylase (HDAC) activity and decreased the protein expression of HDAC1, HDAC2, HDAC3 and HDAC4. Collectively, these results suggest that the anti-cancer effect of SFN against TPA-induced neoplastic transformation of mouse skin could involve the epigenetic reprogramming of anti-cancer genes such as Nrf2, leading to the epigenetic reactivation of Nrf2 and the subsequent induction of downstream target genes involved in cellular protection.

Inverse relationship between p53 and phospho-Chk1 (Ser317) protein expression in UVB-induced skin tumors in SKH-1 mice.

Immunohistochemical evaluation of serial stored paraffin sections from 42 keratoacanthomas and 11 squamous cell carcinomas demonstrated that skin tumors from UVB-exposed mice showed an inverse relationship (>95%) between p53 protein expression and phospho-Chk1 (Ser317), but not phospho-Chk1 (Ser345) protein expression. Tumors expressing high levels and large areas of p53 protein had no detectable phospho-Chk1 (Ser317), whereas tumors expressing high levels and large areas of phospho-Chk1 (Ser317) protein had no detectable p53. Squamous cell carcinomas that demonstrated heterogeneous p53 and phospho-Chk1 (Ser317) protein expression within the same tumor showed that areas expressing p53 were negative for phospho-Chk1 (Ser317) immunostaining while areas expressing phospho-Chk1 (Ser317) were negative for p53. Similar patterns were observed for keratoacanthomas. These findings were also observed in epidermal areas distant from tumors that demonstrated no detectable phospho-Chk1 (Ser317), but appreciable p53 protein in the basal layer. Tumors from congenic hairless p53 knockout mice had elevated levels of phospho-Chk1 (Ser317) compared to tumors from p53 wild-type SKH-1 controls. After a single exposure to UVB, normal epidermal cells from a p53 knockout mouse expressed a relatively high level of phospho-Chk1 (Ser317) whereas epidermal cells from a p53 wild-type littermate induced p53 protein and expressed a relatively low level of phospho-Chk1 (Ser317). These data illustrate the dynamic regulation of checkpoint function, suggesting that phosphorylation of Chk1 on Serine 317 is regulated by p53 status and that p53 may act as a molecular on/off switch for phosphorylation at this site.

Parametrial fat tissue from high fat diet-treated SKH-1 mice stimulates transformation of mouse epidermal JB6 cells.

Our previous studies indicated that decreasing visceral adipose tissue by surgical removal of the parametrial fat pads inhibited UVB-induced carcinogenesis in SKH-1 mice fed a high fat diet (HFD), but not a low fat diet (LFD) indicating that the parametrial fat tissue from mice fed a HFD played a role in skin carcinogenesis. OBJECTIVE: In the present study, we sought to investigate how a HFD may influence the intrinsic properties of the parametrial fat tissue to influence UVB-induced skin tumor formation. METHODS AND RESULTS: Immunohistochemical staining, adipokine array, and flow cytometry showed that parametrial fat tissue from mice fed a HFD had a higher density of macrophage-fused dead adipocytes (crown-like structures), more adipokines, and stimulated the production of more reactive oxygen species compared with parametrial fat tissue from mice fed a LFD. These differences between parametrial fat tissue from mice fed a HFD and LFD were associated with their effect on the in vitro transformation of mouse epidermal JB6 cells. Our results indicated that fat tissue filtrate (an aqueous filtrate made from the parametrial fat pad) from mice fed a HFD enhanced the conversion of JB6 cells from an epithelial-like morphology to cells with a fibroblast-like morphology to a greater extent than fat tissue filtrate from mice fed a LFD. Studies indicated that the fibroblast-like cells had decreased levels of E-cadherin, increased levels of Twist as assayed by western blot. Fat tissue filtrate made from the parametrial fat tissue of mice fed a HFD had 160% more transforming activity than that from mice fed a LFD and formed malignant mesenchymal tumors in vivo. CONCLUSION: These studies provide the first in vitro demonstration of a parametrial fat tissue-induced transformation of an epidermal cell.

Oral caffeine during voluntary exercise markedly inhibits skin carcinogenesis and decreases inflammatory cytokines in UVB-treated mice.

Ultraviolet B (UVB)-pretreated SKH-1 mice were treated with water, caffeine (0.1 mg/ml), voluntary running wheel exercise (RW) or caffeine together with RW for 14 wk. Treatment of the mice with caffeine, RW, or caffeine plus RW decreased skin tumors per mouse by 27%, 35%, and 62%, respectively, and the tumor volume per mouse was decreased by 61%, 70%, and 85%, respectively. In mechanistic studies, mice were treated with water, caffeine, RW, or caffeine plus RW for 2 wk prior to a single irradiation with UVB. Caffeine plus RW increased RW activity by 22% when compared with RW alone. Caffeine ingestion was not significantly different between groups. Treatment of mice with caffeine plus RW for 2 wk decreased the weight of the parametrial fat pads and stimulated the formation of UVB-induced apoptosis to a greater extent than treatment with caffeine or RW alone. An antibody array revealed that caffeine plus RW administered to mice fed a high-fat diet and irradiated with UVB decreased the epidermal levels of lipopolysaccharide-induced CXC chemokine, soluble TNF alpha receptor-1, and macrophage inflammatory protein-1γ. Overall, caffeine during RW exerts a stronger effect than either treatment alone for decreasing tissue fat, increasing UVB-induced apoptosis, lowering the levels of cytokines associated with inflammation and for inhibiting UVB-induced carcinogenesis.

Mechanisms of Caffeine-Induced Inhibition of UVB Carcinogenesis.

Sunlight-induced non-melanoma skin cancer is the most prevalent cancer in the United States with more than two million cases per year. Several studies have shown an inhibitory effect of caffeine administration on UVB-induced skin cancer in mice, and these studies are paralleled by epidemiology studies that indicate an inhibitory effect of coffee drinking on non-melanoma skin cancer in humans. Strikingly, decaffeinated coffee consumption had no such inhibitory effect. Mechanism studies indicate that caffeine has a sunscreen effect that inhibits UVB-induced formation of thymine dimers and sunburn lesions in the epidermis of mice. In addition, caffeine administration has a biological effect that enhances UVB-induced apoptosis thereby enhancing the elimination of damaged precancerous cells, and caffeine administration also enhances apoptosis in tumors. Caffeine administration enhances UVB-induced apoptosis by p53-dependent and p53-independent mechanisms. Exploration of the p53-independent effect indicated that caffeine administration enhanced UVB-induced apoptosis by inhibiting the UVB-induced increase in ATR-mediated formation of phospho-Chk1 (Ser345) and abolishing the UVB-induced decrease in cyclin B1 which resulted in caffeine-induced premature and lethal mitosis in mouse skin. In studies with cultured primary human keratinocytes, inhibition of ATR with siRNA against ATR inhibited Chk1 phosphorylation and enhanced UVB-induced apoptosis. Transgenic mice with decreased epidermal ATR function that were irradiated chronically with UVB had 69% fewer tumors at the end of the study compared with irradiated littermate controls with normal ATR function. These results, which indicate that genetic inhibition of ATR (like pharmacologic inhibition of ATR via caffeine) inhibits UVB-induced carcinogenesis support the concept that ATR-mediated phosphorylation of Chk1 is an important target for caffeine's inhibitory effect on UVB-induced carcinogenesis.

Inhibition of UVB-induced nonmelanoma skin cancer: a path from tea to caffeine to exercise to decreased tissue fat.

Oral administration of green tea, black tea, or caffeine (but not the decaffeinated teas) inhibited ultraviolet B radiation (UVB)-induced skin carcinogenesis in SKH-1 mice. Studies with caffeine indicated that its inhibitory effect on the ATR/Chk1 pathway is an important mechanism for caffeine's inhibition of UVB-induced carcinogenesis. The regular teas or caffeine increased locomotor activity and decreased tissue fat. In these studies, decreased dermal fat thickness was associated with a decrease in the number of tumors per mouse. Administration of caffeine, voluntary exercise, and removal of the parametrial fat pads all stimulated UVB-induced apoptosis, inhibited UVB-induced carcinogenesis, and stimulated apoptosis in UVB-induced tumors. These results suggest that caffeine administration, voluntary exercise, and removal of the parametrial fat pads inhibit UVB-induced carcinogenesis by stimulating UVB-induced apoptosis and by enhancing apoptosis in DNA-damaged precancer cells and in cancer cells. We hypothesize that tissue fat secretes antiapoptotic adipokines that have a tumor promoting effect.

Surgical removal of the parametrial fat pads stimulates apoptosis and inhibits UVB-induced carcinogenesis in mice fed a high-fat diet.

Removal of the parametrial fat pads (partial lipectomy) from female SKH-1 mice fed a high-fat diet inhibited UVB-induced carcinogenesis, but this was not observed in mice fed a low-fat chow diet. Partial lipectomy in high-fat-fed mice decreased the number of keratoacanthomas and squamous cell carcinomas per mouse by 76 and 79%, respectively, compared with sham-operated control mice irradiated with UVB for 33 wk. Immunohistochemical analysis indicated that partial lipectomy increased caspase 3 (active form) positive cells by 48% in precancerous epidermis away from tumors, by 68% in keratoacanthomas, and by 224% in squamous cell carcinomas compared with sham-operated control mice. In addition, partial lipectomy decreased cell proliferation away from tumors and in tumors. RT-PCR analysis for adipokines revealed that mRNAs for TIMP1, MCP1, and SerpinE1 (proinflammatory/antiapoptotic cytokines) in the parametrial fat pads of sham-operated control mice were 54- to 83-fold higher than levels in compensatory fat that returned after surgery in partially lipectomized mice at the end of the tumor study. Feeding mice high-fat diets for 2 wk increased levels of TIMP1 and other adipokines in serum and epidermis, and these increases were inhibited by removal of the parametrial fat pads. Our results are a unique demonstration that surgical removal of a specific tissue fat results in inhibition of carcinogenesis in obese mice. This inhibition was associated with an increase in apoptosis and a decrease in proliferation in tumors and in precancerous areas away from tumors.

Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice.

The aim of this study was to investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on newly developed high-fat/Western-style diet-induced obesity and symptoms of metabolic syndrome. Male C57BL/6J mice were fed a high fat/Western-style (HFW; 60% energy as fat and lower levels of calcium, vitamin D(3), folic acid, choline bitartrate, and fiber) or HFW with EGCG (HFWE; HFW with 0.32% EGCG) diet for 17 wks. As a comparison, two other groups of mice fed a low-fat diet (LF; 10% energy as fat) and high-fat diet (HF; 60% energy as fat) were also included. The HFW group developed more body weight gain and severe symptoms of metabolic syndrome than the HF group. The EGCG treatment significantly reduced body weight gain associated with increased fecal lipids and decreased blood glucose and alanine aminotransferase (ALT) levels compared to those of the HFW group. Fatty liver incidence, liver damage, and liver triglyceride levels were also decreased by the EGCG treatment. Moreover, the EGCG treatment attenuated insulin resistance and levels of plasma cholesterol, monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), interlukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). Our results demonstrate that the HFW diet produces more severe symptoms of metabolic syndrome than the HF diet and that the EGCG treatment can alleviate these symptoms and body fat accumulation. The beneficial effects of EGCG are associated with decreased lipid absorption and reduced levels of inflammatory cytokines.

Protection from UV-induced skin carcinogenesis by genetic inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase.

Multiple human epidemiologic studies link caffeinated (but not decaffeinated) beverage intake with significant decreases in several types of cancer, including highly prevalent UV-associated skin carcinomas. The mechanism by which caffeine protects against skin cancer is unknown. Ataxia telangiectasia and Rad3-related (ATR) is a replication checkpoint kinase activated by DNA stresses and is one of several targets of caffeine. Suppression of ATR, or its downstream target checkpoint kinase 1 (Chk1), selectively sensitizes DNA-damaged and malignant cells to apoptosis. Agents that target this pathway are currently in clinical trials. Conversely, inhibition of other DNA damage response pathways, such as ataxia telangiectasia mutated (ATM) and BRCA1, promotes cancer. To determine the effect of replication checkpoint inhibition on carcinogenesis, we generated transgenic mice with diminished ATR function in skin and crossed them into a UV-sensitive background, Xpc(-/-). Unlike caffeine, this genetic approach was selective and had no effect on ATM activation. These transgenic mice were viable and showed no histological abnormalities in skin. Primary keratinocytes from these mice had diminished UV-induced Chk1 phosphorylation and twofold augmentation of apoptosis after UV exposure (P = 0.006). With chronic UV treatment, transgenic mice remained tumor-free for significantly longer (P = 0.003) and had 69% fewer tumors at the end of observation of the full cohort (P = 0.019), compared with littermate controls with the same genetic background. This study suggests that inhibition of replication checkpoint function can suppress skin carcinogenesis and supports ATR inhibition as the relevant mechanism for the protective effect of caffeinated beverage intake in human epidemiologic studies.

Effects of high-fat diets rich in either omega-3 or omega-6 fatty acids on UVB-induced skin carcinogenesis in SKH-1 mice.

Our previous studies reported that caffeine or voluntary exercise decreased skin tumor multiplicity, in part, by decreasing fat levels in the dermis. These data suggest that tissue fat may play an important role in regulating ultraviolet light (UV) B-induced skin tumor development. In the present study, we explored the effects of high-fat diets rich in either omega-3 or omega-6 fatty acids on UVB-induced skin carcinogenesis. SKH-1 mice were irradiated with 30 mJ/cm(2) of UVB once a day, two times per week for 39 weeks. During UVB treatment, one group of mice was given a high-fat fish oil (HFFO) diet rich in omega-3 fatty acids and the other group of mice was given a high-fat mixed-lipids (HFMLs) diet rich in omega-6 fatty acids. The results showed that, compared with HFML diet, HFFO treatment (i) increased latency for the development of UVB-induced skin tumors; (ii) decreased the formation of papilloma, keratoacanthoma and carcinoma by 64, 52 and 46%, respectively and (iii) decreased the size of papilloma, keratoacanthoma and carcinoma by 98, 80 and 83%, respectively. Mechanistic studies with antibody array revealed that compared with HFML diet, administration of HFFO to the mice significantly decreased the UVB-induced increases in the levels of TIMP-1, LIX and sTNF R1 as well as other several proinflammatory cytokines and stimulated the UVB-induced apoptosis in the epidermis. Our results indicate that omega-3 fatty acids in HFFO diet have beneficial effects against UVB-induced skin carcinogenesis, and these effects may be associated with an inhibition on UVB-induced inflammatory response.

Caffeine decreases phospho-Chk1 (Ser317) and increases mitotic cells with cyclin B1 and caspase 3 in tumors from UVB-treated mice.

Oral administration of caffeine to mice inhibits UVB-induced carcinogenesis, and these results are paralleled by epidemiology studies indicating that caffeinated coffee and tea intake (but not decaffeinated beverage intake) is associated with decreased incidence of nonmelanoma skin cancer. Topical applications of caffeine to the skin of SKH-1 mice that had previously been treated with UVB inhibited subsequent skin tumor development and stimulated apoptosis in tumors but not in nontumor areas of the epidermis. This study sought to determine the basis of these differential effects on tumor versus nontumor sites that can be induced by caffeine, long after all UVB treatment has ceased. The activation status of the ATR/Chk1 pathway in UVB-induced tumors and uninvolved skin was determined by quantitating phospho-Chk1 (Ser317) and induction of lethal mitosis in vivo in the presence and absence of topical caffeine treatment. In the absence of caffeine, we found that UVB-induced tumors often had islands of phospho-Chk1 (Ser317) staining cells that were not present in nontumor areas of the epidermis. Treatment of mice with topical caffeine significantly diminished phospho-Chk1 (Ser317) staining and increased the number of mitotic cells that expressed cyclin B1 and caspase 3 in tumors, consistent with caffeine-induced lethal mitosis selectively in tumors. We hypothesize that compared with adjacent uninvolved skin, UVB-induced skin tumors have elevated activation of, and dependence on, the ATR/Chk1 pathway long after UVB exposure has ceased and that caffeine can induce apoptosis selectively in tumors by inhibiting this pathway and promoting lethal mitosis.