m6A modification mediates SLC3A2/SLC7A5 translation in 3-methylcholanthrene-induced uroepithelial transformation.

3-Methylcholanthracene (3-MC) is one of the most carcinogenic polycyclic aromatic hydrocarbons (PAHs). Long-term exposure to PAHs has been thought of as an important factor in urothelial tumorigenesis. N6-methyladenosine (m6A) exists widely in eukaryotic organisms and regulates the expression level of specific genes by regulating mRNA stability, translation efficiency, and nuclear export efficiency. Currently, the potential molecular mechanisms that regulate m6A modification for 3-MC carcinogenesis remain unclear. Here, we profiled mRNA, m6A, translation and protein level using "-omics" methodologies, including transcriptomes, m6A profile, translatomes, and proteomics in 3-MC-transformed urothelial cells and control cells. The key molecules SLC3A2/SLC7A5 were screened and identified in 3-MC-induced uroepithelial transformation. Moreover, SLC7A5/SLC3A2 promoted uroepithelial cells malignant phenotype in vitro and in vivo. Mechanically, METTL3 and ALKBH5 mediated m6A modification of SLC3A2/SLC7A5 mRNA in 3-MC-induced uroepithelial transformation by upregulating the translation of SLC3A2/SLC7A5. Furthermore, programmable m6A modification of SLC3A2/SLC7A5 mRNA affected the expression of its proteins. Taken together, our results revealed that the m6A modification-mediated SLC3A2/SLC7A5 translation promoted 3-MC-induced uroepithelial transformation, suggesting that targeting m6A modification of SLC3A2/SLC7A5 may be a potential therapeutic strategy for bladder cancer related to PAHs.

[Screening of risky substances in sea cucumbers based on gas chromatography-orbitrap high-resolution mass spectrometry].

A new method for sample pretreatment using improved QuEChERs was established, and 289 organic pollutants with health risks could be identified and quantified through gas chromatography-orbitrap high-resolution mass spectrometry (GC-Orbitrap HRMS). A high-resolution database of 289 environmental pollutants belonging to ten categories, including organochlorine pesticides (OCPs), polycyclic aromatic hydrocarbons (PAHs), phthalates (PAEs), polychlorinated biphenyls (PCBs), and other agricultural chemicals (ACs), was established for the non-targeted screening and quantitative analysis. A simple method for biological sample preparation using improved QuEChERs was proposed by combining a conventional QuEChERs method and a column purification method. After purification using a Florisil column, the lipid content was reduced by 99.9%, which significantly reduced the interference of the matrix effect observed during the analysis. Furthermore, simultaneous high-accuracy qualitative screening and quantitative analysis of the target compounds were performed through high-resolution mass spectrometry (60000 resolution) conducted in the full scan mode. The limits of quantification were 0.56-57.8 pg/g, presenting a large linear range (~106), and the recovery range was 40%-120%. Due to the high-resolution and sensitivity of Q Exactive GC-Orbitrap HRMS, the limits of quantification of the target compounds were significantly lower than those achieved through methods based on conventional chromatography and mass spectrometry. Moreover, ultratrace organic contaminants that cannot be detected by conventional methods can be accurately quantified by the proposed method. Sea cucumber samples collected at the breeding site were analyzed using the proposed high-coverage multi-objective analytical method, and more than 100 types of organic pollutants were detected; the mean contents of PAHs, ACs, PAEs, and OCPs were 157.8, 153.2, 64.4, and 46.4 ng/g dw, respectively, which were higher than those of other pollutants. Some new contaminants, such as 9-chlorofluorene, 5-chloroacenaphthene, and 3-methylcholanthrene, were detected at very low contents for the first time in the sea cucumber samples. The proposed method is simple and efficient, allows the detection of pollutants at very low contents, and provides accurate and reliable results. Thus, this high-coverage multi-objective analytical method can be widely used for broad-spectrum screening and accurate quantification of contaminants in various aquatic products, providing technical support for food safety control.

EMAP II Expression Is Increased on Peripheral Blood Cells from Non-Hodgkin Lymphoma.

Tumor immune evasion is a lineament of cancer. Endothelial monocyte activating polypeptide-II (EMAP II) has been assumed to impact tumor immune escape significantly. EMAP II was first reported in the murine methylcholanthrene A-induced fibrosarcoma supernatant and identified as a tumor-derived cytokine. This study evaluated EMAP II expression in peripheral blood cells and its association with treatment outcome, lactate dehydrogenase (LDH) levels, and clinical criteria in non-Hodgkin's lymphoma (NHL) patients. EMAP II expression on different blood cells obtained from the peripheral blood of 80 NHL patients was evaluated by two-color flow cytometry. The study reported that EMAP II expression was significantly increased in peripheral blood cells in patients with NHL compared to normal volunteers (P < 0.001). Additionally, EMAP II expression levels on blood cells decreased in complete remission (CR) while they increased in relapse. This study showed coexpression of EMAP II and CD36 on peripheral lymphocytes in NHL patients but not in healthy controls (P < 0.001). EMAP II expression on blood cells was associated with increased serum LDH levels. Furthermore, the percentages of EMAP II+/CD36+ peripheral lymphocytes were significantly higher in relapse than in CR and healthy controls. Analyses revealed that higher percentages of EMAP II+CD36+ cells were positively correlated with hepatomegaly, splenomegaly, and an advanced (intermediate and high risk) NHL stage. The results assume that EMAP II might be involved in NHL development and pathogenesis.

Aryl Hydrocarbon Receptor Directly Regulates VTCN1 Gene Expression in MCF-7 Cells.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxins and polycyclic aromatic hydrocarbons. Recent studies have suggested that AhR is involved in cancer immunity. In the present study, we examined whether AhR regulates the expression of immune checkpoint genes in breast cancer cells. We discovered that the mRNA expression of V-set domain containing T cell activation inhibitor 1 (VTCN1) that negatively regulates T cell immunity was upregulated by AhR agonists in breast cancer cell lines, MCF-7 and T47D. Furthermore, AhR knockout or knockdown experiments clearly demonstrated that upregulation of VTCN1 gene expression by 3-methylcholanthrene was AhR dependent. Luciferase reporter and chromatin immunoprecipitation assays revealed that this upregulation of VTCN1 gene expression was induced by the recruitment of AhR to the AhR responsive element in the VTCN1 gene promoter in MCF-7 cells. Taken together, AhR directly regulates VTCN1 gene expression in MCF-7 cells.

Parental exposure to 3-methylcholanthrene before gestation adversely affected the endocrine system and spermatogenesis in male F1 offspring.

The compound 3-methylcholanthrene (3-MC) is an environmental pollutant belonging to the PAHs, which reportedly have the potential to disrupt the endocrine systems of animals. In the present study, 4-week-old male and female mice were given 3-MC through their diet at a dose of 0.5 mg/kg of chow for 6 weeks before pregnancy. The first filial (F1) generation offspring of exposed or unexposed parental mice were sacrificed at the age of 5 or 10 weeks (F1-5 W or F1-10 W), and the potential effects on the F0 and F1 offspring were evaluated. The results showed that the serum and testicular testosterone (T) levels and the genes involved in T synthesis in F0 males and male F1-5 W individuals born from female mice exposed to 3-MC were significantly decreased. In addition, histological analysis suggested that exposure to 3-MC significantly disrupted testicular morphology in F0 mice and in the offspring of female mice exposed to 3-MC. Further investigation revealed that genes involved in spermatogenesis, such as Phosphoglycerate kinase 2 (Pgk2), Glial cell derived neurotrophic factor (Gdnf), Myeloblastosis oncogene (Myb), DEAD box helicase 4 (Ddx4) and KIT proto-oncogene receptor tyrosine kinase (Kit), were suppressed in these mice. However, the adverse effects of parental 3-MC exposure on the adolescent mice were mitigated when they grew to adulthood, which was verified by studies on F1-10 W mice. Our results suggest that female exposure to 3-MC has the potential to disrupt the endocrine system and spermatogenesis in male offspring; nevertheless, the adverse effects might be mitigated with age.

Increased Expression of Flightless I in Cutaneous Squamous Cell Carcinoma Affects Wnt/ß-Catenin Signaling Pathway.

Cutaneous squamous cell carcinoma (cSCC) accounts for 25% of cutaneous malignancies diagnosed in Caucasian populations. Surgical removal in combination with radiation and chemotherapy are effective treatments for cSCC. Nevertheless, the aggressive metastatic forms of cSCC still have a relatively poor patient outcome. Studies have linked actin cytoskeletal dynamics and the Wnt/ß-catenin signaling pathway as important modulators of cSCC pathogenesis. Previous studies have also shown that the actin-remodeling protein Flightless (Flii) is a negative regulator of cSCC. The aim of this study was to investigate if the functional effects of Flii on cSCC involve the Wnt/ß-catenin signaling pathway. Flii knockdown was performed using siRNA in a human late stage aggressive metastatic cSCC cell line (MET-1) alongside analysis of Flii genetic murine models of 3-methylcholanthrene induced cSCC. Flii was increased in a MET-1 cSCC cell line and reducing Flii expression led to fewer PCNA positive cells and a concomitant reduction in cellular proliferation and symmetrical division. Knockdown of Flii led to decreased ß-catenin and a decrease in the expression of the downstream effector of ß-catenin signaling protein SOX9. 3-Methylcholanthrene (MCA)-induced cSCC in Flii overexpressing mice showed increased markers of cancer metastasis including talin and keratin-14 and a significant increase in SOX9 alongside a reduction in Flii associated protein (Flap-1). Taken together, this study demonstrates a role for Flii in regulating proteins involved in cSCC proliferation and tumor progression and suggests a potential role for Flii in aggressive metastatic cSCC.

Decoupling tumor cell metastasis from growth by cellular pilot protein TNFAIP8.

Cancer metastasis accounts for nearly 90% of all cancer deaths. Metastatic cancer progression requires both cancer cell migration to the site of the metastasis and subsequent proliferation after colonization. However, it has long been recognized that cancer cell migration and proliferation can be uncoupled; but the mechanism underlying this paradox is not well understood. Here we report that TNFAIP8 (tumor necrosis factor-α-induced protein 8), a "professional" transfer protein of phosphoinositide second messengers, promotes cancer cell migration or metastasis but inhibits its proliferation or cancer growth. TNFAIP8-deficient mice developed larger tumors, but TNFAIP8-deficient tumor cells completely lost their ability to migrate toward chemoattractants and were defective in colonizing lung tissues as compared to wild-type counterparts. Mechanistically, TNFAIP8 served as a cellular "pilot" of tumor cell migration by locally amplifying PI3K-AKT and Rac signals on the cell membrane facing chemoattractant; at the same time, TNFAIP8 also acted as a global inhibitor of tumor cell growth and proliferation by regulating Hippo signaling pathway. These findings help explain the migration-proliferation paradox of cancer cells that characterizes many cancers.

Activation of the aryl hydrocarbon receptor by 3-methylcholanthrene, but not by indirubin, suppresses mammosphere formation via downregulation of CDC20 expression in breast cancer cells.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates various toxicological and biological functions. We reported previously that 3-methylcholanthrene (3MC), an exogenous AhR agonist, inhibited tumorsphere (mammosphere) formation from breast cancer cell lines, while the endogenous AhR agonist, indirubin, very weakly inhibited this process. However, the difference in inhibition mechanism of mammosphere formation by 3MC or indirubin is still unknown. In this study, we established AhR-re-expressing (KOTR-AhR) cells from AhR knockout MCF-7 cells using the tetracycline (Tet)-inducible gene expression systems. To identify any difference in inhibition of mammosphere formation by 3MC or indirubin, RNA-sequencing (RNA-seq) experiments were performed using KOTR-AhR cells. RNA-seq experiments revealed that cell division cycle 20 (CDC20), which regulates the cell cycle and mitosis, was decreased by 3MC, but not by indirubin, in the presence of AhR expression. Furthermore, the mRNA and protein levels of CDC20 were decreased by 3MC in MCF-7 cells via the AhR. In addition, mammosphere formation was suppressed by small interfering RNA-mediated CDC20 knockdown compared to the negative control in MCF-7 cells. These results suggest that AhR activation by 3MC suppresses mammosphere formation via downregulation of CDC20 expression in breast cancer cells. This study provides useful information for the development of AhR-targeted anti-cancer drugs.

Metformin alters therapeutic effects in the BALB/c tumor therapy model.

BACKGROUND: Despite considerable medical proceedings, cancer is still a leading cause of death. Major problems for tumor therapy are chemoresistance as well as toxic side effects. In recent years, the additional treatment with the antidiabetic drug metformin during chemotherapy showed promising results in some cases. The aim of this study was to develop an in vitro tumor therapy model in order to further investigate the potential of a combined chemotherapy with metformin. METHODS: Cytotoxic effects of a combined treatment on BALB/c fibroblasts were proven by the resazurin assay. Based on the BALB/c cell transformation assay, the BALB/c tumor therapy model was established successfully with four different and widely used chemotherapeutics from different categories. Namely, Doxorubicin as a type-II isomerase inhibitor, Docetaxel as a spindle toxin, Mitomycin C as an alkylating agent and 5-Fluorouracil as an antimetabolite. Moreover, glucose consumption in the medium supernatant was measured and protein expressions were determined by Western Blotting. RESULTS: Initial tests for the combined treatment with metformin indicated unexpected results as metformin could partly mitigate the cytotoxic effects of the chemotherapeutic agents. These results were further confirmed as metformin induced resistance to some of the drugs when applied simultaneously in the tumor therapy model. Mechanistically, an increased glucose consumption was observed in non-transformed cells as well as in the mixed population of malignant transformed cell foci and non-transformed monolayer cells, suggesting that metformin could also increase glucose consumption in transformed cells. CONCLUSION: In conclusion, this study suggests a cautious use of metformin during chemotherapy. Moreover, the BALB/c tumor therapy model offers a potent tool for further mechanistic studies of drug-drug interactions during cancer therapy.

Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells.

This study evaluates the impact of physiologically relevant oxygen tensions on the response of HepG2 cells to known inducers and hepatotoxic drugs. We compared transcriptional regulation and CYP1A activity after a 48 h exposure at atmospheric culture conditions (20% O2) with representative periportal (8% O2) and perivenous (3% O2) oxygen tensions. We evaluated cellular responses in 2D and 3D cultures at each oxygen tension in parallel, using monolayers and a paper-based culture platform that supports cells suspended in a collagen-rich environment. Our findings highlight that the toxicity, potency, and mechanism of action of drugs are dependent on both culture format and oxygen tension. HepG2 cells in 3D environments at physiologic oxygen tensions better matched primary human hepatocyte data than HepG2 cells cultured under standard conditions. Despite altered transcriptional regulation with decreasing oxygen tensions, we did not observe the zonation patterns of drug-metabolizing enzymes found in vivo. Our approach demonstrates that oxygen is an important regulator of liver function but it is not the sole regulator. It also highlights the utility of the 3D paper-based culture platform for continued mechanistic studies of microenvironmental influences on cellular responses.

PD-1-specific "Blocking" antibodies that deplete PD-1+ T cells present an inconvenient variable in preclinical immunotherapy experiments.

Therapeutic antibodies blocking PD-1-/PD-L1 interaction have achieved remarkable clinical success in cancer. In addition to blocking a target molecule, some isotypes of antibodies can activate complement, NK cells or phagocytes, resulting in death of the cell expressing the antibody's target. Human anti-PD-1 therapeutics use antibody isotypes designed to minimize such antibody-dependent lysis. In contrast, anti-PD-1 reagents used in mice are derived from multiple species, with different isotypes, and are not engineered to reduce target cell death: few studies analyze or discuss how antibody species and isotype may impact data interpretation. We demonstrate here that anti-PD-1 therapy to promote activation and proliferation of murine PD-1-expressing CD8 T cells sometimes led instead to a loss of antigen specific cells. This phenomenon was seen in two tumor models and a model of virus infection, and varied with the clone of anti-PD-1 antibody. Additionally, we compared competition among anti-PD-1 clones to find a combination that allows detection of PD-1-expressing cells despite the presence of blocking anti-PD1 antibodies in vivo. These data bring attention to the possibility of unintended target cell depletion with some commonly used anti-mouse PD-1 clones, and should provide a valuable resource for the design and interpretation of anti-PD-1 studies in mice.

Two-stage 3-methylcholanthrene and butylated hydroxytoluene-induced lung carcinogenesis in mice.

Lung cancer is one of the deadliest types of cancer and as such requires disease models that are useful for identification of novel pathways for biomarkers as well as to test therapeutic agents. Adenocarcinoma (ADC), the most prevalent type of lung cancer, is a subtype of non-small cell lung carcinoma (NSCLC) and a disease driven mainly by smoking. However, it is also the most common subtype of lung cancer found in non-smokers with environmental exposures. Chemically driven models of lung cancer, also called primary models of lung cancer, are important because they do not overexpress or delete oncogenes or tumor suppressor genes, respectively, to increase oncogenesis. Instead these models test tumor development without forcing a specific pathway (i.e., Kras). The primary focus of this chapter is to discuss a well-established 2-stage mouse model of lung adenocarcinomas. The initiator (3-methylcholanthrene, MCA) does not elicit many, if any, tumors if not followed by exposure to the tumor promoter (butylated hydroxytoluene, BHT). In sensitive strains, such as A/J, FVB, and BALB, significantly greater numbers of tumors develop following the MCA/BHT protocol compared to MCA alone. BHT does not elicit tumors on its own; it is a non-genotoxic carcinogen and promoter. In these sensitive strains, promotion is also associated with inflammation characterized by infiltrating macrophages, lymphocytes, and neutrophils, and other inflammatory cell types in addition to increases in total protein content reflective of lung hyperpermeability. This 2-stage model is a useful tool to identify unique promotion specific events to then test in future intervention studies.

Using methylcholanthrene-induced fibrosarcomas to study tumor immunology.

Mouse models of cancer are essential in furthering our understanding both of the mechanisms that drive tumor development and the immune response that develops in parallel, and also in providing a platform for testing novel anti-cancer therapies. The majority of solid tumor models available rely on the injection of existing cancer cell lines into naïve hosts which, while providing quick and reproducible model systems, typically lack the development of a tumor microenvironment that recapitulates those seen in human cancers. Administration of the carcinogen 3-methylcholanthrene (MCA), allows tumors to develop in situ, forming a tumor microenvironment with an established stroma and vasculature. This article provides a detailed set of protocols for the administration of MCA into mice and the subsequent monitoring of tumors. Protocols are also provided for some of the routinely used downstream applications that can be used for MCA tumors.

DNA methylation and hydroxymethylation associated with gene expression regulatory network during 3-methylcholanthrene induced lung cell malignant transformation.

3-methylcholanthrene (3-MCA) is a typical representative PAH. It has strong toxicity and is a typical chemical carcinogen. However, the epigenetic mechanisms underlying 3-MCA-induced tumourigenesis are largely unknown. In this study, a model of the 3-MCA-induced malignant transformation of human bronchial epithelial (HBE) cells was established successfully. The profiles of gene expression and DNA methylation and hydroxymethylation were obtained and analysed with an Illumina HiSeq 4000. A total of 707 genes were found to be significantly up-regulated, and 686 genes were found to be significantly down-regulated. Compared to control cells, 8545 mRNA-associated differentially methylated regions and 15,121 mRNA-associated differentially hydroxymethylated regions in promoters were found to be significantly altered in transformed cells. By using mRNA expression and DNA methylation and hydroxymethylation interaction analysis, 99 differentially expressed genes were identified. Among them, CA9 and EGLN3 were verified to be significantly down-regulated, and CARD6 and LCP1 were shown to be significantly up-regulated, and these genes mainly participated in cell growth, migration and invasion, indicating that these genes were key genes involved in the 3-MCA-induced malignant transformation of HBE cells. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that a large number of differentially expressed genes (DEGs) were involved mainly in RNA polymerase II transcription factor activity, chemical carcinogenesis, base-excision repair (BER), cytokine-cytokine receptor interactions, glycerolipid metabolism, steroid hormone biosynthesis, cAMP signalling pathways and other signalling pathways. Our study suggested that characteristic gene alterations associated with DNA methylation and hydroxymethylation could play important roles in environmental 3-MCA-induced lung carcinogenesis.

TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy.

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer's disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2-/- mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

SQSTM1/p62 and PPARGC1A/PGC-1alpha at the interface of autophagy and vascular senescence.

Defective macroautophagy/autophagy and mitochondrial dysfunction are known to stimulate senescence. The mitochondrial regulator PPARGC1A (peroxisome proliferator activated receptor gamma, coactivator 1 alpha) regulates mitochondrial biogenesis, reducing senescence of vascular smooth muscle cells (VSMCs); however, it is unknown whether autophagy mediates PPARGC1A-protective effects on senescence. Using ppargc1a-/- VSMCs, we identified the autophagy receptor SQSTM1/p62 (sequestosome 1) as a major regulator of autophagy and senescence of VSMCs. Abnormal autophagosomes were observed in VSMCs in aortas of ppargc1a-/- mice. ppargc1a-/- VSMCs in culture presented reductions in LC3-II levels; in autophagosome number; and in the expression of SQSTM1 (protein and mRNA), LAMP2 (lysosomal-associated membrane protein 2), CTSD (cathepsin D), and TFRC (transferrin receptor). Reduced SQSTM1 protein expression was also observed in aortas of ppargc1a-/- mice and was upregulated by PPARGC1A overexpression, suggesting that SQSTM1 is a direct target of PPARGC1A. Inhibition of autophagy by 3-MA (3 methyladenine), spautin-1 or Atg5 (autophagy related 5) siRNA stimulated senescence. Rapamycin rescued the effect of Atg5 siRNA in Ppargc1a+/+ , but not in ppargc1a-/- VSMCs, suggesting that other targets of MTOR (mechanistic target of rapamycin kinase), in addition to autophagy, also contribute to senescence. Sqstm1 siRNA increased senescence basally and in response to AGT II (angiotensin II) and zinc overload, two known inducers of senescence. Furthermore, Sqstm1 gene deficiency mimicked the phenotype of Ppargc1a depletion by presenting reduced autophagy and increased senescence in vitro and in vivo. Thus, PPARGC1A upregulates autophagy reducing senescence by a SQSTM1-dependent mechanism. We propose SQSTM1 as a novel target in therapeutic interventions reducing senescence. ABBREVIATIONS: 3-MA: 3 methyladenine; ACTA2/SM-actin: actin, alpha 2, smooth muscle, aorta; ACTB/ß-actin: actin beta; AGT II: angiotensin II; ATG5: autophagy related 5; BECN1: beclin 1; CAT: catalase; CDKN1A: cyclin-dependent kinase inhibitor 1A (P21); Chl: chloroquine; CTSD: cathepsin D; CYCS: cytochrome C, somatic; DHE: dihydroethidium; DPBS: Dulbecco's phosphate-buffered saline; EL: elastic lamina; EM: extracellular matrix; FDG: fluorescein-di-ß-D-galactopyranoside; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; γH2AFX: phosphorylated H2A histone family, member X, H2DCFDA: 2',7'-dichlorodihydrofluorescein diacetate; LAMP2: lysosomal-associated membrane protein 2; MASMs: mouse vascular smooth muscle cells; MEF: mouse embryonic fibroblast; NBR1: NBR1, autophagy cargo receptor; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; MTOR: mechanistic target of rapamycin kinase; NFE2L2: nuclear factor, erythroid derived 2, like 2; NOX1: NADPH oxidase 1; OPTN: optineurin; PFA: paraformaldehyde; PFU: plaque-forming units; PPARGC1A/PGC-1α: peroxisome proliferator activated receptor, gamma, coactivator 1 alpha; Ptdln3K: phosphatidylinositol 3-kinase; RASMs: rat vascular smooth muscle cells; ROS: reactive oxygen species; SA-GLB1/ß-gal: senescence-associated galactosidase, beta 1; SASP: senescence-associated secretory phenotype; SIRT1: sirtuin 1; Spautin 1: specific and potent autophagy inhibitor 1; SQSTM1/p62: sequestosome 1; SOD: superoxide dismutase; TEM: transmission electron microscopy; TFEB: transcription factor EB; TFRC: transferrin receptor; TRP53/p53: transformation related protein 53; TUBG1: tubulin gamma 1; VSMCs: vascular smooth muscle cells; WT: wild type.

3-Methylcholanthrene impacts on the female germ cells of rats without causing systemic toxicity.

We have previously shown that daily exposure to the environmental pollutant 3-methylcholanthrene (3MC) alters the ovarian function by affecting follicle growth and ovulation. To extend our findings, the aims of this work were to study the effects of daily and non-daily exposure to 3MC on oocyte morphology and integrity and the meiosis process. To this end, immature female rats were daily (0.1-1.0 mg/kg) and non-daily (0.1 mg/kg, three times a week) exposed to 3MC and/or α-naphthoflavone (αNF) (80 mg/kg) for 19 and 20 days, respectively. The latter was used to study its ability to prevent the 3MC action. Follicular growth was examined by histology, apoptosis by in situ cell death detection, oocyte integrity by morphological parameters and fluorescent dyes, and the meiotic spindle by immunostaining. Compared with controls (C), and in a dose-dependent manner, all 3MC-treated rats showed i) increased presence of apoptotic cells in antral follicles and decreased percentage of healthy oocytes, ii) increased oocyte area, perimeter and perivitelline space and decreased thickness of the zona pellucida, and ii) increased percentage of oocytes with abnormal meiotic spindle. In addition, the non-daily dose of 3MC caused DNA damage in oocytes, but not in blood or bone marrow cells. All 3MC-induced changes were prevented with the co-treatment with αNF. These results suggest that low doses of 3MC severely disrupt the ovarian function and that germ cells seem to be more sensitive to this environmental pollutant than other cells such as peripheral blood and bone marrow cells.

Hydroxysteroid sulfotransferase 2B1 affects gastric epithelial function and carcinogenesis induced by a carcinogenic agent.

BACKGROUND: A healthy gastric mucosal epithelium exhibits tumor-suppressive properties. Gastric epithelial cell dysfunction contributes to gastric cancer development. Oxysterols provided from food or cholesterol oxidation in the gastric epithelium may be further sulfated by hydroxysteroid sulfotransferase 2B1 (SULT2B1), which is highly abundant in the gastric epithelium. However, the effects of SULT2B1 on gastric epithelial function and gastric carcinogenesis are unclear. METHODS: A mouse gastric tumor model was established using carcinogenic agent 3-methylcholanthrene (3-MCA). A SULT2B1 deletion (SULT2B1-/-) human gastric epithelial line GES-1 was constructed by CRISPR/CAS9 genome editing system. RESULTS: The gastric tumor incidence was higher in the SULT2B1-/- mice than in the wild-type (WT) mice. In gastric epithelial cells, adenovirus-mediated SULT2B1b overexpression reduced the levels of oxysterols, such as 24(R/S),25-epoxycholesterol (24(R/S),25-EC) and 27-hydroxycholesterol (27HC). This condition also increased PI3K/AKT signaling to promote gastric epithelial cell proliferation, epithelization, and epithelial development. However, SULT2B1 deletion or SULT2B1 knockdown suppressed PI3K/AKT signaling, epithelial cell epithelization, and wound healing and induced gastric epithelial cell malignant transition upon 3-MCA induction. CONCLUSIONS: The abundant SULT2B1 expression in normal gastric epithelium might maintain epithelial function via the PI3K/AKT signaling pathway and suppress gastric carcinogenesis induced by a carcinogenic agent.

AHR and GPER mediate the stimulatory effects induced by 3-methylcholanthrene in breast cancer cells and cancer-associated fibroblasts (CAFs).

BACKGROUND: The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity. METHODS: Molecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA. RESULTS: We first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC. CONCLUSIONS: In the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.

Liposome mediated-CYP1A1 gene silencing nanomedicine prepared using lipid film-coated proliposomes as a potential treatment strategy of lung cancer.

The occurrence of lung cancer is linked with tobacco smoking, mainly through the generation of polycyclic aromatic hydrocarbons (PAHs). Elevated activity of cytochrome P4501A1 (CYP1A1) plays an important role in the metabolic processing of PAHs and its carcinogenicity. The present work aimed to investigate the role of CYP1A1 gene in PAH-mediated growth and tumor development in vitro and using an in vivo animal model. RNAi strategy was utilized to inhibit the overexpression of CYP1A1 gene using cationic liposomes generated using a lipid film-coated proliposome microparticles. Treatment of PAH-induced human alveolar adenocarcinoma cell line with cationic liposomes carrying CYP1A1 siRNA resulted in down regulation of CYP1A1 mRNA, protein as well as its enzymatic activity, triggering apoptosis and inhibiting multicellular tumor spheroids formation in vitro. Furthermore, silencing of CYP1A1 gene in BALB/c nude xenografts inhibited tumor growth via down regulation of CYP1A1 expression. Altogether, our findings showed that liposome-based gene delivery technology is a viable and stable approach for targeting cancer causing genes such as CY1PA1. This technology facilitated by the use of sugar particles coated with lipid films has demonstrated ability to generate anticancer effects that might be used in the future for therapeutic intervention and treatment of lung cancer.