The potential role of Listeria monocytogenes in promoting colorectal adenocarcinoma tumorigenic process.

BACKGROUND: Listeria monocytogenes is a foodborne pathogen, which can cause a severe illness, especially in people with a weakened immune system or comorbidities. The interactions between host and pathogens and between pathogens and tumor cells have been debated in recent years. However, it is still unclear how bacteria can interact with tumor cells, and if this interaction can affect tumor progression and therapy. METHODS: In this study, we evaluated the involvement of L. monocytogenes in pre-neoplastic and colorectal cancer cell proliferation and tumorigenic potential. RESULTS: Our findings showed that the interaction between heat-killed L. monocytogenes and pre-neoplastic or colorectal cancer cells led to a proliferative induction; furthermore, by using a three-dimensional cell culture model, the obtained data indicated that L. monocytogenes was able to increase the tumorigenic potential of both pre-neoplastic and colorectal cancer cells. The observed effects were then confirmed as L. monocytogenes-specific, using Listeria innocua as negative control. Lastly, data suggested the Insulin Growth Factor 1 Receptor (IGF1R) cascade as one of the possible mechanisms involved in the effects induced by L. monocytogenes in the human colorectal adenocarcinoma cell line. CONCLUSIONS: These findings, although preliminary, suggest that the presence of pathogenic bacterial cells in the tumor niches may directly induce, increase, and stimulate tumor progression.

A home-based lifestyle intervention program reduces the tumorigenic potential of triple-negative breast cancer cells.

Translational research for the evaluation of physical activity habits and lifestyle modifications based on nutrition and exercise has recently gained attention. In this study, we evaluated the effects of serum samples obtained before and after a 12-week home-based lifestyle intervention based on nutrition and exercise in breast cancer survivors in terms of modulation of the tumorigenic potential of breast cancer cells. The home-based lifestyle intervention proposed in this work consisted of educational counselling on exercise and nutritional behaviors and in 12 weeks of structured home-based exercise. Triple-negative breast cancer cell line MDA-MB-231 was cultured in semi-solid medium (3D culture) with sera collected before (PRE) and after (POST) the lifestyle intervention program. Spheroid formation was evaluated by counting cell colonies after 3 weeks of incubation. Results show a slight but significant reduction of spheroid formation induced by serum collected POST in comparison to those obtained PRE. Moreover, statistical analyses aimed to find physiologic and metabolic parameters associated with 3D cell proliferation revealed the proliferative inducer IGF-1 as the only predictor of cell tumorigenic potential. These results highlight the importance of lifestyle changes for cancer progression control in a tertiary prevention context. Translational research could offer a useful tool to identify metabolic and physiological changes induced by exercise and nutritional behaviors associated with cancer progression and recurrence risk.

Association between metabolic syndrome, insulin resistance, and IGF-1 in breast cancer survivors of DIANA-5 study.

PURPOSE: Circulating insulin-like growth factor-1 (IGF-1) is positively associated with the risk of BC recurrence, and is more frequently dysregulated in older people, especially in those with metabolic syndrome (MetS) and obesity. This study aimed to analyze the association between IGF-1 levels and indices of MetS and insulin resistance in BC survivors. METHODS: Baseline data of 563 BC survivors enrolled in the DIet and ANdrogen-5 (DIANA-5; NCT05019989) study were analyzed. RESULTS: Lower circulating IGF-1 levels in subjects with MetS than in those without MetS were found. After stratification of the patients according to the diagnosis of MetS, we highlighted that the insulin was the main predictor of elevated IGF-1 levels only in subjects without MetS. Moreover, we found an interaction between high-density lipoprotein cholesterol (HDL-C), glycemia, and IGF-1 levels, showing a positive correlation between HDL-C and IGF-1, especially in subjects with higher values of glycemia and without a diagnosis of MetS. CONCLUSIONS: While IGF-1 levels appear to be much more impaired in subjects diagnosed with MetS, in non-MetS subjects, IGF-1 levels may respond better to metabolic parameters and lifestyle changes. Further studies are needed to analyze the role of physical activity and/or dietary intervention in modulating IGF-1 concentrations in BC survivors. IMPLICATIONS FOR CANCER SURVIVORS: These results could have important clinical implications for planning customized strategies aimed at modulating IGF-1 levels in BC survivors. In fact, while the IGF-1 system seems to be much more compromised in subjects with a diagnosis of MetS, in noMetS subjects, IGF-1 levels could better respond to lifestyle changes.

Movement and health beyond care, MoviS: study protocol for a randomized clinical trial on nutrition and exercise educational programs for breast cancer survivors.

BACKGROUND: Breast cancer (BC) is the most common invasive cancer in women, and exercise can significantly improve the outcomes of BC survivors. MoviS (Movement and Health Beyond Care) is a randomized controlled trial aimed to evaluate the potential health benefits of exercise and proper nutritional habits. This study aims to assess the efficacy of aerobic exercise training in improving quality of life (QoL) and health-related factors in high-risk BC. METHODS: One hundred seventy-two BC survivor women, aged 30-70 years, non-metastatic, stage 0-III, non-physically active, 6-12 months post-surgery, and post chemo- or radiotherapy, will be recruited in this study. Women will be randomly allocated to the intervention arm (lifestyle recommendations and MoviS Training) or control arm (lifestyle recommendations). The MoviS training consists of 12 weeks of aerobic exercise training (2 days/week of supervised and 1 day/week of unsupervised exercise) with a progressive increase in exercise intensity (40-70% of heart rate reserve) and duration (20-60 min). Both arms will receive counseling on healthy lifestyle habits (nutrition and exercise) based on the World Cancer Research Fund International (WCRF) 2018 guidelines. The primary outcome is the improvement of the QoL. The secondary outcomes are improvement of health-related parameters such as Mediterranean diet adherence, physical activity level, flexibility, muscular fitness, fatigue, cardiorespiratory fitness (estimated maximal oxygen uptake), echocardiographic parameters, heart rate variability (average of the standard deviations of all 5 min normal to normal intervals (ASDNN/5 min) and 24 h very low and low frequency), and metabolic, endocrine, and inflammatory serum biomarkers (glycemia, insulin resistance, progesterone, testosterone, and high-sensitivity C-reactive protein). DISCUSSION: This trial aims to evaluate if supervised exercise may improve QoL and health-related factors of BC survivors with a high risk of recurrence. Findings from this project could provide knowledge improvement in the field of exercise oncology through the participation of a multidisciplinary team that will provide a coordinated program of cancer care to improve healthcare quality, improve prognosis, increase survival times and QoL, and reduce the risk of BC recurrence. TRIAL REGISTRATION: ClinicalTrials.gov  NCT04818359 . Retrospectively registered on March 26, 2021.

Seroprevalence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection in an Italian cohort in Marche Region, Italy.

BACKGROUND AND AIM: The COVID-19 is an infectious disease caused by the novel coronavirus SARS-CoV-2, declared a public health emergency by the World Health Organization. In this study, we evaluated the seroconversion of SARS-CoV-2 antibodies to find predictors of infection in terms of symptoms, health status, and professions. METHODS: Serological samples of 341 volunteers in a cohort in Marche Region, Italy, were analyzed for the presence of IgM and/or IgG immunoglobulins specific for the SARS-CoV-2. Contextually, an anamnestic questionnaire was administered. The binary logistic regression analysis was used to find the predictors of seroconversion. RESULTS: Forty-nine subjects (14.4 %) were found positive, without significant differences between gender and age groups. The predictors identified inside the variable categories "symptoms," "risk factors" (smoking habit and established pathologies), and "professions" were the loss of taste and smell (OR, 8.563), cardiovascular diseases (OR, 2.912), and policeman profession (OR, 3.875), respectively. CONCLUSIONS: Although the limited number of subjects recruited in this study, our results could give important findings to be considered for planning preventive strategies in the view of the next COVID-19 waves.

Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment.

Listeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.

A dataset on the effect of exercise-conditioned human sera in three-dimensional breast cancer cell culture.

Epidemiological evidence shows that physical activity lowers the risk of developing breast cancer and decreases the risk of disease recurrence [1,2]. The main hypothesis on the positive effects of exercise-oncology has focused on lowering the basal systemic levels of cancer risk factors with exercise training. Recently, the effects of cancer progression control by components released after acute exercise bouts have gained attention [3,4]. However, the evaluation of the antiproliferative potential of a single exercise bout needs technical improvement. Here, we present data of a pilot study showing how to evaluate the anti-cancer potential of single exercise bouts with an in vitro three-dimensional cell growth assay, using a triple-negative breast cancer cell line cultured with exercise-conditioned serum.

Chemopreventive Potential of Apple Pulp Callus Against Colorectal Cancer Cell Proliferation and Tumorigenesis.

This study focused on the evaluation of the chemopreventive potential of tissue in vitro culture of the "Mela Rosa Marchigiana" apple (MRM callus) that allows the amplification of secondary metabolites. The MRM pulp and MRM callus chemopreventive potential was evaluated in terms of antiproliferative activity, inhibition of tumorigenesis in soft agar cultures, cell cycle and western blotting analyses in CaCo2 and LoVo colon cancer cell lines and in JB6 promotion-sensitive (JB6 P+) cells. MRM callus induced a strong concentration-dependent inhibition of colon cancer cell proliferation and suppressed 12-o-tetra-decanoyl-phorbol-13-acetate-induced tumorigenesis of JB6 P+ cells in soft agar cultures. MRM callus inhibited the phosphorylation of JNK, p38, and eIF2alpha. Our data indicate that the MRM callus exerts a good antiproliferative and antitumorigenic potential through the MAP kinase inhibition and could provide natural compounds with chemopreventive properties.

Metformin prevents cell tumorigenesis through autophagy-related cell death.

Autophagy is a cellular mechanism by which cells degrade intracellular components in lysosomes, maintaining cellular homeostasis. It has been hypothesized that autophagy could have a role in cancer prevention through the elimination of damaged proteins and organelles; this could explain epidemiological evidence showing the chemopreventive properties of the autophagy-inducer metformin. In this study, we analyzed the autophagy-related effect of metformin in both cancer initiation and progression in non-tumorigenic cells. We also analyzed the induction of tumorigenesis in autophagy-deficient cells, and its correlation with the ER stress. Our results showed that metformin induced massive cell death in preneoplastic JB6 Cl 41-5a cells treated with tumor promoter (phorbol) and in NIH/3T3 treated with H2O2. Inhibiting autophagy with wortmannin or ATG7 silencing, the effect of metformin decreased, indicating an autophagy-related cytotoxic activity under stress conditions. We also found an induction of tumorigenesis in ATG7-silenced NIH/3T3 cell clone (3T3-619C3 cells), but not in wild-type and in scrambled transfected cells, and an upregulation of unfolded protein response (UPR) markers in 3T3-619C3 cells treated with H2O2. These findings suggest that autophagic cell death could be considered as a new mechanism by which eliminate damaged cells, representing an attractive strategy to eliminate potential tumorigenic cells.

New Insights into the Role of Exercise in Inhibiting mTOR Signaling in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) does not express estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 and is characterized by its aggressive nature, lack of targets for targeted therapies, and early peak of recurrence. Due to these specific characteristics, chemotherapy does not usually yield substantial improvements and new target therapies and alternative strategies are needed. The beneficial responses of TNBC survivors to regular exercise, including a reduction in the rate of tumor growth, are becoming increasingly apparent. Physiological adaptations to exercise occur in skeletal muscle but have an impact on the entire body through systemic control of energy homeostasis and metabolism, which in turn influence the TNBC tumor microenvironment. Gaining insights into the causal mechanisms of the therapeutic cancer control properties of regular exercise is important to improve the prescription and implementation of exercise and training in TNBC survivors. Here, we provide new evidence of the effects of exercise on TNBC prevention, control, and outcomes, based on the inhibition of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB also known as Akt)/mammalian target of rapamycin (mTOR) (PI3K-Akt-mTOR) signaling. These findings have wide-ranging clinical implications for cancer treatment, including recurrence and case management.

Leishmania infantum Induces Mild Unfolded Protein Response in Infected Macrophages.

The Leishmaniases are a group of parasitic diseases caused by protozoa of the Leishmania genus affecting both humans and other vertebrates. Leishmania is an intracellular pathogen able to confer resistance to apoptosis in the early phase of macrophages infection by activation of host PI3K/Akt pathway and inhibition of caspase-3 activation. Intracellular pathogens hijack organelles such as ER to facilitate survival and replication, thus eliciting ER stress and activating/modulating the unfolded protein response (UPR) in the host cell. The UPR is aimed to mitigate ER stress, thereby promoting cell survival. However, prolonged ER stress will activate the apoptotic pathway. The aim of this study was to investigate the ER stress response in Leishmania-infected macrophages to gain insights about the mechanisms underlying the apoptosis resistance in parasitized cells. Macrophages differentiated from human monocytic cell lines (U937 and THP-1) and murine primary macrophages were infected with Leishmania infantum MHOM/TN/80/IPT1 (WHO international reference strain). Several ER stress/autophagy expression markers, as well as cell survival/apoptosis markers (phospho-Akt and cleaved caspase-3) were evaluated by qPCR and/or by western blotting. As ER stress positive control, cells were treated with tunicamycin or dithiothreitol (DTT). The gene expression analyses showed a mild but significant induction of the ER stress/autophagy markers. The western blot analyses revealed that the Leishmania infection induced Akt phosphorylation and significantly inhibited the induction of caspase-3 cleavage, eIF2α phosphorylation and DDIT3/CHOP expression in tunicamycin and DTT treated cells. The mild but significant increase in ER stress expression markers and the delay/attenuation of the effects of ER stress inducers in infected cells support the hypothesis that L. infantum could promote survival of host cells by inducing a mild ER stress response. The host ER stress response could be not only a common pathogenic mechanism among Leishmania species but also a target for development of new drugs.

Human IGF1 pro-forms induce breast cancer cell proliferation via the IGF1 receptor.

BACKGROUND: IGF1 is a key regulator of tissue growth and development and has been implicated in the initiation and progression of various cancers, including breast cancer. Through IGF1 mRNA splicing different precursor pro-peptides, i.e., the IGF1Ea, IGF1Eb and IGF1Ec pro-forms, are formed whose biological roles in the pathogenesis of breast cancer have not been established yet. The objective of this study was to assess the biological activity of the IGF1 pro-forms in human breast cancer-derived cells. METHODS: The different IGF1 pro-forms were generated through transient transfection of HEK293 cells with the respective vector constructs. The resulting conditioned media were applied in vitro to MCF7, T47D and ZR751 breast cancer-derived cell cultures. The recombinant human IGF1 pro-forms were also tested for their binding affinity to an anti-IGF1 specific antibody by immunoprecipitation. To determine whether the IGF1 pro-forms induce cell proliferation, mature IGF1 was neutralised in HEK293-derived conditioned media. RESULTS: We found that the IGF1 pro-forms were the only forms that were produced intra-cellularly, whereas both mature IGF1 and the IGF1 pro-forms were detected extra-cellularly. We also found that E peptides can impair the IGF1 pro-form binding affinity for the anti-IGF1 antibody and, thus, hamper an accurate measurement of the IGF1 pro-forms. Additionally, we found that the IGF1 antibody can completely inhibit IGF1-induced breast cancer cell proliferation and IGF1 receptor (IGF1R) phosphorylation, wheras the same antibody was found to only partially inhibit the biological activity of the pro-forms. Moreover, we found that the IGF1 pro-form activities can completely be inhibited by neutralising the IGF1R. Finally, we compared the bioactivity of the IGF1 pro-forms to that of mature IGF1, and found that the IGF1 pro-forms were less capable of phosphorylating the IGF1R in the breast cancer-derived cells tested. CONCLUSIONS: Our data indicate that IGF1 pro-forms can induce breast cancer cell proliferation via the IGF1R, independent from the mature IGF1 form. These results underline the importance of an accurate assessment of the presence of IGF1 pro-forms within the breast cancer microenvironment.

Inhibition of Breast Cancer Cell Proliferation and In Vitro Tumorigenesis by a New Red Apple Cultivar.

PURPOSE: The aim of this study was to evaluate the antiproliferative activity in breast cancer cells and the inhibition of tumorigenesis in pre-neoplastic cells of a new apple cultivar with reddish pulp, called the Pelingo apple. METHODS: The antiproliferative activity was evaluated in MCF-7 and MDA-MB-231 human breast cancer cells. The inhibition of tumorigenesis was performed in JB6 promotion-sensitive (P+) cells. RESULTS: Results showed that Pelingo apple juice is characterized by a very high polyphenol content and strongly inhibited breast cancer cell proliferation. Its antiproliferative activity was found to be higher than the other five apple juices tested. Pelingo juice induced cell accumulation in the G2/M phase of the cell cycle and autophagy through overexpression of p21, inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) activity and an increase in lipidated microtubule-associated protein-1 light chain-3 beta (LC3B). Remarkably, Pelingo juice inhibited the 12-o-tetra-decanoyl-phorbol-13-acetate (TPA)-induced tumorigenesis of JB6 P+ cells, suppressing colony formation in semi-solid medium and TPA-induced ERK1/2 phosphorylation. CONCLUSIONS: Our data indicate that the Pelingo apple is rich in food components that can markedly inhibit in vitro tumorigenesis and growth of human breast cancer cells and could provide natural bioactive non-nutrient compounds, with potential chemopreventive activity.

Inhibition of Testosterone Aromatization by the Indole-3-carbinol Derivative CTet in CYP19A1-overexpressing MCF-7 Breast Cancer Cells.

Natural products such as aromatase inhibitors have been the object of growing attention in recent years because of their potential to inhibit aromatase with fewer side effects and the possible translation of their current use as chemotherapeutic agents to future clinical applications in breast cancer chemoprevention. We have previously investigated CTet, a novel anticancer agent obtained from the broccoli-derived compound indole-3- carbinol (I3C), that shows great anticancer potential in both in vitro and in vivo studies. Here we evaluated the potential of CTet as a chemopreventive agent in aromatase expressing MCF-7/AROM-1 breast cancer cells. The testosterone (TE) aromatization in estradiol (E2) was indirectly evaluated in terms of inhibition of TE-induced cell proliferation, ERα phosphorylation/activation and Bcl-2 and IGF-1R ERE-regulated protein accumulation. Our results showed that CTet inhibited TE-driven ERα phosphorylation of both cytosolic and nuclear ERα pools, suggesting an inhibitory effect of TE aromatization in E2. CTet did not inhibit E2-driven nuclear ERα phosphorylation, but partially inhibited E2-driven cytosolic ERα phosphorylation. Moreover, CTet inhibited Bcl-2 and IGF-1R accumulation induced by TE but not that which was induced by E2. A cell-free enzymatic assay showed that CTet did not inhibit aromatase activity directly; however, since CTet treatment induced endoplasmic reticulum stress, the TE aromatization could be affected because the aromatase enzyme is located within the endoplasmic reticulum. Finally, CTet and letrozole synergistically inhibited TE-induced cell proliferation. These results showed the potential of the I3C derivative CTet as a chemopreventive agent that interferes with aromatase activity.

Inactivation of pathogenic dermatophytes by ultraviolet irradiation in swimming pool thermal water.

The effect of ultraviolet (UV) radiation from low-pressure mercury lamp against some pathogenic dermatophytes species such as Epidermophyton floccosum, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton tonsurans and Trichophyton violaceum suspended in thermal water was evaluated in laboratory-scale condition at various times. The main results showed that within 120 s of exposure, all species of dermatophytes are completely inactivated, which was evidenced by the absence of fungal regrowth, while after 60 s only T. tonsurans was recovered, with a reduction of 3.28 log. Shorter exposure times were not enough to completely inactivate all dermatophytes species. The samples treated with UV radiation for 120 s did not give evidence of fungal regrowth indicating that this disinfectant action is persistent over time. In conclusion, UV radiation can be proposed to reduce the risk of infection by dermatophytes eventually present in swimming pools that use thermal water.

The Indole-3-carbinol cyclic tetrameric derivative CTet synergizes with cisplatin and doxorubicin in triple-negative breast cancer cell lines.

BACKGROUND/AIM: The indole-3-carbinol cyclic tetrameric derivative (CTet) inhibits breast cancer cell proliferation by endoplasmic reticulum stress and autophagy-related cell death induction, AKT/PKB (protein kinase B) activity inhibition and p53-independent overexpression of cyclin-dependent kinase inhibitor-1A (p21/CDKN1A). In the present study we evaluated the synergistic activity of CTet in combination with cisplatin and doxorubicin in triple-negative breast cancer cell lines. MATERIALS AND METHODS: Synergisms were evaluated in terms of cell viability, induction of autophagy and overexpression of microtubule-associated protein-1 light chain-3 beta (MAP1LC3B) autophagy-related gene in MDA-MB-231 and BT-20 triple-negative breast cancer cells. RESULTS: We demonstrated that CTet in combination with both cisplatin and doxorubicin synergistically inhibits cell viability and induces autophay. The MAP1LC3B gene was synergistically overexpressed in MDA-MB-231 cells treated with CTet-cisplatin combination. Moreover, the cytotoxic activity of CTet was improved in cells pre-treated with cisplatin and doxorubicin. CONCLUSION: This preliminary in vitro study confirms the potential of CTet as a chemopreventive agent or chemotherapeutic in combination with standard approaches for triple-negative breast cancer.

Antitumoral activity of indole-3-carbinol cyclic tri- and tetrameric derivatives mixture in human breast cancer cells: in vitro and in vivo studies.

Indole-3-carbinol (I3C) and its oligomeric derivatives have been widely studied for their chemopreventive and chemotherapeutic properties. We have previously shown that the I3C cyclic tetrameric derivative CTet inhibits breast cancer cell proliferation in vitro and in xenotrasplanted tumor. Here we report the antitumoral activity of a mixture of tri- and tetrameric cyclic I3C derivatives (CTr/CTet) both in vitro (MCF-7 and MDA-MB-231 breast cancer cell lines) and in vivo in a tumor xenograft model. CTr/CTet mixture avoids the low solubility drawbacks of CTet, thus favouring its solubilization, and reducing purification process, time and costs. CTr/CTet mixture has been shown to inhibit breast cancer cell proliferation (IC50 = 1.3 and 1.6 µg/ml in MCF-7 and MDAMB- 231, respectively) inducing the G0/1 cell cycle phase accumulation. The main molecular events related to CTr/CTet activity are the overexpression of p21, p27 and GADD45A, nuclear translocation of FOXO3a, inhibition of Akt activity and downregulation of estrogen receptor. In vivo, the growth of xenotransplanted tumor has been inhibited and the pro-tumoral low molecular weight cyclin E downregulation has been detected. Our data indicate that CTr/CTet is a potential anticancer combination agent for both hormone-responsive and triple-negative breast tumors.

Induction of endoplasmic reticulum stress response by the indole-3-carbinol cyclic tetrameric derivative CTet in human breast cancer cell lines.

BACKGROUND: Indole-3-carbinol and its metabolic products are considered promising chemopreventive and anticancer agents. Previously we have shown that the indole-3-carbinol cyclic tetrameric derivative CTet induces autophagy and inhibits cell proliferation via inhibition of Akt activity and overexpression of p21/CDKN1A and GADD45A, in both estrogen receptor-positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines. In the present study, we further characterize the autophagic response and investigate the mechanism through which CTet regulates these events. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of gene expression microarray data and subsequent confirmation by quantitative real-time PCR, showed that CTet is able to induce up-regulation of key signaling molecules involved in endoplasmic reticulum (ER) stress response (e.g. DDIT3/CHOP, CHAC1, ATF3, HSPA5/BiP/GRP78, CEBPB, ASNS) and autophagy (e.g. MAP1LC3B), in both MCF-7 and MDA-MB-231 cell lines. Moreover, the monitoring of Xbp-1 splicing confirmed the activation of IRE1/Xbp-1 ER stress response branch after CTet treatment. The role of autophagic processes (known to be induced by ER stress) was investigated further through ATG5 gene silencing and pharmacological inhibition of AVOs formation. CTet was shown to induce an autophagy-related cell death. Moreover, CTet-treated cells stained with Hoechst/PI revealed the presence of necrotic processes without evidence of apoptosis. CONCLUSIONS/SIGNIFICANCE: The ER stress response was identified as the main upstream molecular mechanism through which CTet acts in both hormone-responsive and triple-negative breast cancer cells. Because of its important role in cancer development, ER stress is a potential target in cancer therapy. The abiltiy of CTet to induce ER stress response and subsequently activate a death program in tumor cells confirms this molecule as a promising anticancer agent.

The indole-3-carbinol cyclic tetrameric derivative CTet inhibits cell proliferation via overexpression of p21/CDKN1A in both estrogen receptor-positive and triple-negative breast cancer cell lines.

INTRODUCTION: Indole-3-carbinol (I3C), an autolysis product of glucosinolates present in cruciferous vegetables, and its dimeric derivative (3,3'-DIM) have been indicated as promising agents in preventing the development and progression of breast cancer. We have recently shown that I3C cyclic tetrameric derivative CTet formulated in γ-cyclodextrin (γ-CD) efficiently inhibited cellular proliferation in breast cancer cell lines. This study aims to analyze the mechanisms involved in the in vitro inhibition of cell proliferation and to evaluate the in vivo antitumor activity of CTet in a xenograft study. METHODS: Estrogen receptor-positive MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines were exposed to CTet to evaluate cell cycle perturbation (propidium iodide staining and cytofluorimetric acquisition), induction of autophagic morphological features (co-localization of LC3b autophagosome marker and LAMP2a lysosome marker by immunofluorescence) and changes in protein expression (immunoblot and microarray-based gene expression analyses). To test the in vivo efficacy of CTet, female athymic nude mice inoculated with MCF-7 cells were i.p. treated with 5 mg/kg/day of CTet for five days/week for two weeks and the tumor mass was externally monitored. RESULTS: CTet induced accumulation in G2/M phase without evidence of apoptotic response induction in both cell lines tested. In triple-negative MDA-MB-231 the autophagic lysosomal activity was significantly up-regulated after exposure to 4 µM of CTet for 8 hours, while the highest CTet concentration was necessary to observe autophagic features in MCF-7 cells. The inhibition of Akt activity and p53-independent p21/CDKN1A and GADD45A overexpression were identified as the main molecular events responsible for CTet activity in MCF-7 and p53-mutant MDA-MB-231 cells. In vivo, CTet administration was able to significantly inhibit the growth of MCF-7 xenotransplanted into nude mice, without adverse effect on body weight or on haematological parameters. CONCLUSIONS: Our data support CTet formulated with γ-CD as a promising and injectable anticancer agent for both hormone-responsive and triple-negative breast tumors.

Characterization of the volatile organic compounds of Italian 'Fossa' cheese by solid-phase microextraction gas chromatography/mass spectrometry.

Fossa cheese is an Italian hard cheese, ripened for up to 3 months in underground pits dug into tuffaceous rock. During this period, the cheese develops a unique flavour and intense and somewhat piquant aroma. Solid-phase microextraction gas chromatography/mass spectrometry (SPME-GC/MS) was utilized to characterize the volatile organic compounds (VOCs) of Fossa cheese. A total of 75 VOCs were separated and identified; in particular, the major class of compounds found in the cheeses ripened in the pits were the esters of fatty acids. Discriminant analysis of volatile profiles allowed us to distinguish between cheeses in different stages of seasoning (60-day-old cheese and cheese ripened an additional 90 days in and out of the pits).