Identification of Bacterial Metabolites Modulating Breast Cancer Cell Proliferation and Epithelial-Mesenchymal Transition.

Breast cancer patients are characterized by the oncobiotic transformation of multiple microbiome communities, including the gut microbiome. Oncobiotic transformation of the gut microbiome impairs the production of antineoplastic bacterial metabolites. The goal of this study was to identify bacterial metabolites with antineoplastic properties. We constructed a 30-member bacterial metabolite library and screened the library compounds for effects on cell proliferation and epithelial-mesenchymal transition. The metabolites were applied to 4T1 murine breast cancer cells in concentrations corresponding to the reference serum concentrations. However, yric acid, glycolic acid, d-mannitol, 2,3-butanediol, and trans-ferulic acid exerted cytostatic effects, and 3-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, and vanillic acid exerted hyperproliferative effects. Furthermore, 3-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, 2,3-butanediol, and hydrocinnamic acid inhibited epithelial-to-mesenchymal (EMT) transition. We identified redox sets among the metabolites (d-mannitol-d-mannose, 1-butanol-butyric acid, ethylene glycol-glycolic acid-oxalic acid), wherein only one partner within the set (d-mannitol, butyric acid, glycolic acid) possessed bioactivity in our system, suggesting that changes to the local redox potential may affect the bacterial secretome. Of the nine bioactive metabolites, 2,3-butanediol was the only compound with both cytostatic and anti-EMT properties.

Effect of Surface Modifications on Surface Roughness of Ti6Al4V Alloy Manufactured by 3D Printing, Casting, and Wrought.

This work aimed to comprehensively evaluate the influence of different surface modifications on the surface roughness of Ti6Al4V alloys produced by selective laser melting (SLM), casting and wrought. The Ti6Al4V surface was treated using blasting with Al2O3 (70-100 µm) and ZrO2 (50-130 µm) particles, acid etching with 0.017 mol/dm3 hydrofluoric acids (HF) for 120 s, and a combination of blasting and acid etching (SLA). It was found that the optimization of the surface roughness of Ti6Al4V parts produced by SLM differs significantly from those produced by casting or wrought processes. Experimental results showed that Ti6Al4V alloys produced by SLM and blasting with Al2O3 followed by HF etching had a higher surface roughness (Ra = 2.043 µm, Rz = 11.742 µm), whereas cast and wrought Ti6Al4V components had surface roughness values of (Ra = 1.466, Rz = 9.428 m) and (Ra = 0.940, Rz = 7.963 m), respectively. For Ti6Al4V parts blasted with ZrO2 and then etched by HF, the wrought Ti6Al4V parts exhibited higher surface roughness (Ra = 1.631 µm, Rz = 10.953 µm) than the SLM Ti6Al4V parts (Ra = 1.336 µm, Rz = 10.353 µm) and the cast Ti6Al4V parts (Ra = 1.075 µm, Rz = 8.904 µm).

The pro- and antineoplastic effects of deoxycholic acid in pancreatic adenocarcinoma cell models.

BACKGROUND: Commensal bacteria secrete metabolites that reach distant cancer cells through the circulation and influence cancer behavior. Deoxycholic acid (DCA), a hormone-like metabolite, is a secondary bile acid specifically synthesized by intestinal microbes. DCA may have both pro- and antineoplastic effects in cancers. METHODS AND RESULTS: The pancreatic adenocarcinoma cell lines, Capan-2 and BxPC-3, were treated with 0.7 µM DCA, which corresponds to the reference concentration of DCA in human serum. DCA influenced the expression of epithelial to mesenchymal transition (EMT)-related genes, significantly decreased the expression level of the mesenchymal markers, transcription factor 7- like 2 (TCF7L2), snail family transcriptional repressor 2 (SLUG), CLAUDIN-1, and increased the expression of the epithelial genes, zona occludens 1 (ZO-1) and E-CADHERIN, as shown by real-time PCR and Western blotting. Consequently, DCA reduced the invasion capacity of pancreatic adenocarcinoma cells in Boyden chamber experiments. DCA induced the protein expression of oxidative/nitrosative stress markers. Moreover, DCA reduced aldehyde dehydrogenase 1 (ALDH1) activity in an Aldefluor assay and ALDH1 protein level, suggesting that DCA reduced stemness in pancreatic adenocarcinoma. In Seahorse experiments, DCA induced all fractions of mitochondrial respiration and glycolytic flux. The ratio of mitochondrial oxidation and glycolysis did not change after DCA treatment, suggesting that cells became hypermetabolic. CONCLUSION: DCA induced antineoplastic effects in pancreatic adenocarcinoma cells by inhibiting EMT, reducing cancer stemness, and inducing oxidative/nitrosative stress and procarcinogenic effects such as hypermetabolic bioenergetics.

The Effect of Heat Treatment on a 3D-Printed PLA Polymer's Mechanical Properties.

Three-dimensional printing is a useful and common process in additive manufacturing nowadays. The advantage of additive polymer technology is its rapidity and design freedom. Polymer materials' mechanical properties depend on the process parameters and the chemical composition of the polymer used. Mechanical properties are very important in product applicability. The mechanical properties of polymers can be enhanced by heat treatment. Additive-manufactured PLA's mechanical properties and structure can be modified via heat treatment after the 3D printing process. The goal of this research was to test the effect of heat treatment on the mechanical and structural parameters of additive-manufactured PLA. This was achieved via the FDM processing of standard PLA tensile test specimens with longitudinal and vertical printing orientations. After printing, the test specimens were heat-treated at 55 °C, 65 °C and 80 °C for 5 h and after being held at 20 °C for 15 h. The printed and heat-treated specimens were tested using tensile tests and microscopy. Based on the test results, we can conclude that the optimal heat treatment process temperature was 65 °C for 5 h. Under the heat treatment, the test specimens did not show any deformation, the tensile strength increased by 35% and the porosity of the PLA structure decreased.

Methods to Assess the Role of PARPs in Regulating Mitochondrial Oxidative Function.

PARP enzymes are involved in metabolic regulation and impact on a plethora of cellular metabolic pathways, among them, mitochondrial oxidative metabolism. The detrimental effects of PARP1 overactivation upon oxidative stress on mitochondrial oxidative metabolism was discovered in 1998. Since then, there was an enormous blooming in the understanding of the interplay between PARPs and mitochondria. Mitochondrial activity can be assessed by a comprehensive set of methods that we aim to introduce here.

The role of bile acids in carcinogenesis.

Bile acids are soluble derivatives of cholesterol produced in the liver that subsequently undergo bacterial transformation yielding a diverse array of metabolites. The bulk of bile acid synthesis takes place in the liver yielding primary bile acids; however, other tissues have also the capacity to generate bile acids (e.g. ovaries). Hepatic bile acids are then transported to bile and are subsequently released into the intestines. In the large intestine, a fraction of primary bile acids is converted to secondary bile acids by gut bacteria. The majority of the intestinal bile acids undergo reuptake and return to the liver. A small fraction of secondary and primary bile acids remains in the circulation and exert receptor-mediated and pure chemical effects (e.g. acidic bile in oesophageal cancer) on cancer cells. In this review, we assess how changes to bile acid biosynthesis, bile acid flux and local bile acid concentration modulate the behavior of different cancers. Here, we present in-depth the involvement of bile acids in oesophageal, gastric, hepatocellular, pancreatic, colorectal, breast, prostate, ovarian cancer. Previous studies often used bile acids in supraphysiological concentration, sometimes in concentrations 1000 times higher than the highest reported tissue or serum concentrations likely eliciting unspecific effects, a practice that we advocate against in this review. Furthermore, we show that, although bile acids were classically considered as pro-carcinogenic agents (e.g. oesophageal cancer), the dogma that switch, as lower concentrations of bile acids that correspond to their serum or tissue reference concentration possess anticancer activity in a subset of cancers. Differences in the response of cancers to bile acids lie in the differential expression of bile acid receptors between cancers (e.g. FXR vs. TGR5). UDCA, a bile acid that is sold as a generic medication against cholestasis or biliary surge, and its conjugates were identified with almost purely anticancer features suggesting a possibility for drug repurposing. Taken together, bile acids were considered as tumor inducers or tumor promoter molecules; nevertheless, in certain cancers, like breast cancer, bile acids in their reference concentrations may act as tumor suppressors suggesting a Janus-faced nature of bile acids in carcinogenesis.

Effects of Annealing and Solution Treatments on the Microstructure and Mechanical Properties of Ti6Al4V Manufactured by Selective Laser Melting.

Ti6Al4V (Ti64) alloys manufactured by selective laser melting (SLM) are well known for their susceptibility to failure at a low ductility of less than 10% due to the formation of an (α') martensitic structure. Annealing and solution treatments as post-heat treatments of α' are considered a good way to improve the mechanical performance of SLM-manufactured Ti64 parts. In this research, the effect of heat treatment parameters such as temperature (850 °C and 1020 °C) and cooling rate (furnace and water cooling) on the microstructure and mechanical properties of the SLM Ti64 structure was investigated. It was shown that the tensile strength/ductility of the Ti64 alloy produced by SLM was determined by the post-heat treatment. The experimental results revealed that heat treatment at 850 °C followed by furnace cooling resulted in the best possible combination of ductility (13%) and tensile strength (σy = 932, σu = 986 MPa) with a microstructure consisting mainly of 78.71% α and 21.29% ß. Heat treatment at 850 °C followed by water cooling was characterized by a reduction in hardness and the formation of predominantly α plus α'' and a small amount of ß. HT850WC exhibited yield and tensile strengths of about 870 and 930 MPa, respectively, and an elongation at fracture of 10.4%. Heat treatment at 1020 °C and subsequent cooling in the furnace was characterized by the formation of an α + ß lamellar microstructure. In contrast, heat treatment at 1020 °C and subsequent water cooling formed semi-equiaxial ß grains of about 170 µm in diameter with longer elongated α grains and basket-weave α'. Post-treatment at 1020 °C followed by furnace cooling showed high ductility with an elongation of 14.5% but low tensile strength (σy = 748, σu = 833 MPa). In contrast, post-treatment at 1020 °C followed by water cooling showed poor ductility with elongation of 8.6% but high tensile strength (σy = 878, σu = 990 MPa). The effect of aging at 550 °C for 3 h and cooling in a furnace on the microstructure and mechanical properties of the specimens cooled with water was also studied. It was found that aging influenced the microstructure of the Ti6Al4V parts, including ß, α, and α″ precipitation and fragmentation or globularization of elongated α grains. The aging process at 550 °C leads to an increase in tensile strength and a decrease in ductility.

Diagnostic challenges, management, and outcome of infants born to mothers with COVID 19 during the first wave of the pandemic.

Severe acute respiratory distress syndrome with coronavirus 2 (SARS-CoV-2) infection affected pregnant women during the pandemic. Immunological particularity of this population and the increased need for medical assistance placed this population in a high-risk category for SARS-Cov-2 infection. Owing to high contamination risk and limited studies regarding vertical transmission, the labor and delivery of positive women required particular conditions. Cesarean section probably proved to be the optimal option for delivery of infants to reduce the risk of infection during birth. The aim of the present study was to present the management and outcome of infants born to mothers confirmed with coronavirus disease 2019 (COVID 19) prior to delivery. This is a longitudinal, retrospective study, analyzing demographics, laboratory data and management of neonates born to mothers with diagnosis of SARS-Cov-2 infection. The results showed that 5 neonates were born to SARS-Cov-2-positive mothers, all by Cesarean section and had a negative reverse transcription-quantitative polymerase chain reaction (RT-qPCR) test. None of the women breastfed during the hospital stay. The negative RT-qPCR test allowed us to reduce the hospital stay of infants and care in non-isolated areas. In summary, in the present study, vertical or perinatal transmission of the infection was not present. The testing of the pregnant women, their isolation and delivery in safe conditions for the medical staff were possible, with the latter using adequate protection equipment to limit their infection and the risk for the newborns.

Krukenberg tumor in pregnancy: a rare case and review of the literature.

Krukenberg's tumor diagnosed in pregnancy is an uncommon situation that raises both diagnosis and medical management issues. We performed a review of the existing literature regarding this pathology, diagnostic means and therapeutic approaches, motivated by a case in our own practice. A 35-year-old primigravida was diagnosed with an adnexal mass during the first trimester prenatal ultrasound. Ultrasound revealed a 10 cm right adnexal mass with multiple septae, richly vascularized, whose presence and characteristics were confirmed by magnetic resonance imaging. Due to the progressively increasing tumor size, laparoscopy was performed with right adnexectomy and peritoneal biopsies. Histopathology diagnosed a metastatic ovarian tumor from a mucinous colorectal adenocarcinoma. After delivery the patient was further investigated and diagnosed with sigmoid cancer. Even though ovarian cancer in pregnancy is rare, adnexal ultrasound is mandatory when scanning during the first trimester to rule out the presence of associated fallopian or ovarian masses.

The involvement of oncobiosis and bacterial metabolite signaling in metastasis formation in breast cancer.

Breast cancer, the most frequent cancer in women, is characterized by pathological changes to the microbiome of breast tissue, the tumor, the gut, and the urinary tract. Changes to the microbiome are determined by the stage, grade, origin (NST/lobular), and receptor status of the tumor. This year is the 50th anniversary of when Hill and colleagues first showed that changes to the gut microbiome can support breast cancer growth, namely that the oncobiome can reactivate excreted estrogens. The currently available human and murine data suggest that oncobiosis is not a cause of breast cancer, but can support its growth. Furthermore, preexisting dysbiosis and the predisposition to cancer are transplantable. The breast's and breast cancer's inherent microbiome and the gut microbiome promote breast cancer growth by reactivating estrogens, rearranging cancer cell metabolism, bringing about a more inflammatory microenvironment, and reducing the number of tumor-infiltrating lymphocytes. Furthermore, the gut microbiome can produce cytostatic metabolites, the production of which decreases or blunts breast cancer. The role of oncobiosis in the urinary tract is largely uncharted. Oncobiosis in breast cancer supports invasion, metastasis, and recurrence by supporting cellular movement, epithelial-to-mesenchymal transition, cancer stem cell function, and diapedesis. Finally, the oncobiome can modify the pharmacokinetics of chemotherapeutic drugs. The microbiome provides novel leverage on breast cancer that should be exploited for better management of the disease.

Silencing of Poly(ADP-Ribose) Polymerase-2 Induces Mitochondrial Reactive Species Production and Mitochondrial Fragmentation.

PARP2 is a DNA repair protein. The deletion of PARP2 induces mitochondrial biogenesis and mitochondrial activity by increasing NAD+ levels and inducing SIRT1 activity. We show that the silencing of PARP2 causes mitochondrial fragmentation in myoblasts. We assessed multiple pathways that can lead to mitochondrial fragmentation and ruled out the involvement of mitophagy, the fusion-fission machinery, SIRT1, and mitochondrial unfolded protein response. Nevertheless, mitochondrial fragmentation was reversed by treatment with strong reductants, such as reduced glutathione (GSH), N-acetyl-cysteine (NAC), and a mitochondria-specific antioxidant MitoTEMPO. The effect of MitoTEMPO on mitochondrial morphology indicates the production of reactive oxygen species of mitochondrial origin. Elimination of reactive oxygen species reversed mitochondrial fragmentation in PARP2-silenced cells.

Functional outcome after lower limb periprosthetic fractures.

INTRODUCTION: Periprosthetic fractures (PPFx) occur more and more often following joint replacement. The aim of this study was to investigate the functional outcome of patients treated with lower limb periprosthetic fractures in our institution. METHODS: Between 2007 and 2016 75 patients were managed with 13 intraoperative and 62 postoperative fractures. Fifty-nine fractures occurred around THR and 16 around TKR. Fractures were classified according to Unified Classification System (UCS) and the treatment was according to this algorithm. Functional outcome was assessed with the Harris Hip Scores and the Knee Society Score (KSS). RESULTS: Follow up time was 52.9 months (range 12-100 m.). The mean age of patients was 75.1 years (range 54-87 years), there were 6 males and 69 females. Harris Hip Scores were available for 42 patients and the average score was 82 (range 68-96). The KSS was available in nine patients and the average score was 124 (range 102-141). Eventually, radiographic union was observed in all cases. CONCLUSION: In this series of patients, fixation of lower limb periprosthetic fractures was found to be associated with good results despite the different fracture patterns studied. Larger studies with subgroup analysis of different fracture patterns are desirable to throw more light in this complex group of patients.

Indoxylsulfate, a Metabolite of the Microbiome, Has Cytostatic Effects in Breast Cancer via Activation of AHR and PXR Receptors and Induction of Oxidative Stress.

Changes to bacterial metabolite-elicited signaling, in oncobiosis associated with breast cancer, plays a role in facilitating the progression of the disease. We show that indoxyl-sulfate (IS), a tryptophan metabolite, has cytostatic properties in models of breast cancer. IS supplementation, in concentrations corresponding to the human serum reference range, suppressed tumor infiltration to the surrounding tissues and metastasis formation in a murine model of breast cancer. In cellular models, IS suppressed NRF2 and induced iNOS, leading to induction of oxidative and nitrosative stress, and, consequently, reduction of cell proliferation; enhanced oxidative and nitrosative stress are crucial in the subsequent cytostasis. IS also suppressed epithelial-to-mesenchymal transition vital for suppressing cellular movement and diapedesis. Furthermore, IS rendered cells hypometabolic, leading to a reduction in aldehyde-dehydrogenase positive cells. Pharmacological inhibition of the pregnane-X receptor using CH223191 and the aryl-hydrocarbon receptor using ketoconazole diminished the IS-elicited effects, suggesting that these receptors were the major receptors of IS in these models. Finally, we showed that increased expression of the human enzymes that form IS (Cyp2E1, Sult1A1, and Sult1A2) is associated with better survival in breast cancer, an effect that is lost in triple negative cases. Taken together, IS, similar to indolepropionic acid (another tryptophan metabolite), has cytostatic properties and higher expression of the metabolic machinery responsible for the formation of IS supports survival in breast cancer.

Glycogen phosphorylase inhibitor, 2,3-bis[(2E)-3-(4-hydroxyphenyl)prop-2-enamido] butanedioic acid (BF142), improves baseline insulin secretion of MIN6 insulinoma cells.

Type 2 diabetes mellitus (T2DM), one of the most common metabolic diseases, is characterized by insulin resistance and inadequate insulin secretion of ß cells. Glycogen phosphorylase (GP) is the key enzyme in glycogen breakdown, and contributes to hepatic glucose production during fasting or during insulin resistance. Pharmacological GP inhibitors are potential glucose lowering agents, which may be used in T2DM therapy. A natural product isolated from the cultured broth of the fungal strain No. 138354, called 2,3-bis(4-hydroxycinnamoyloxy)glutaric acid (FR258900), was discovered a decade ago. In vivo studies showed that FR258900 significantly reduced blood glucose levels in diabetic mice. We previously showed that GP inhibitors can potently enhance the function of ß cells. The purpose of this study was to assess whether an analogue of FR258900 can influence ß cell function. BF142 (Meso-Dimethyl 2,3-bis[(E)-3-(4-acetoxyphenyl)prop-2-enamido]butanedioate) treatment activated the glucose-stimulated insulin secretion pathway, as indicated by enhanced glycolysis, increased mitochondrial oxidation, significantly increased ATP production, and elevated calcium influx in MIN6 cells. Furthermore, BF142 induced mTORC1-specific phosphorylation of S6K, increased levels of PDX1 and insulin protein, and increased insulin secretion. Our data suggest that BF142 can influence ß cell function and can support the insulin producing ability of ß cells.

Indolepropionic Acid, a Metabolite of the Microbiome, Has Cytostatic Properties in Breast Cancer by Activating AHR and PXR Receptors and Inducing Oxidative Stress.

Oncobiotic transformation of the gut microbiome may contribute to the risk of breast cancer. Recent studies have provided evidence that the microbiome secretes cytostatic metabolites that inhibit the proliferation, movement, and metastasis formation of cancer cells. In this study, we show that indolepropionic acid (IPA), a bacterial tryptophan metabolite, has cytostatic properties. IPA selectively targeted breast cancer cells, but it had no effects on non-transformed, primary fibroblasts. In cell-based and animal experiments, we showed that IPA supplementation reduced the proportions of cancer stem cells and the proliferation, movement, and metastasis formation of cancer cells. These were achieved through inhibiting epithelial-to-mesenchymal transition, inducing oxidative and nitrosative stress, and boosting antitumor immune response. Increased oxidative/nitrosative stress was due to the IPA-mediated downregulation of nuclear factor erythroid 2-related factor 2 (NRF2), upregulation of inducible nitric oxide synthase (iNOS), and enhanced mitochondrial reactive species production. Increased oxidative/nitrosative stress led to cytostasis and reductions in cancer cell stem-ness. IPA exerted its effects through aryl hydrocarbon receptor (AHR) and pregnane X receptor (PXR) receptors. A higher expression of PXR and AHR supported better survival in human breast cancer patients, highlighting the importance of IPA-elicited pathways in cytostasis in breast cancer. Furthermore, AHR activation and PXR expression related inversely to cancer cell proliferation level and to the stage and grade of the tumor. The fecal microbiome's capacity for IPA biosynthesis was suppressed in women newly diagnosed with breast cancer, especially with stage 0. Bacterial indole biosynthesis showed correlation with lymphocyte infiltration to tumors in humans. Taken together, we found that IPA is a cytostatic bacterial metabolite, the production of which is suppressed in human breast cancer. Bacterial metabolites, among them, IPA, have a pivotal role in regulating the progression but not the initiation of the disease.

4-chloro-orto-cresol activates ryanodine receptor more selectively and potently than 4-chloro-meta-cresol.

In this study we performed the comprehensive pharmacological analysis of two stereoisomers of 4-chloro-meta-cresol (4CMC), a popular ryanodine receptor (RyR) agonist used in muscle research. Experiments investigating the Ca2+-releasing action of the isomers demonstrated that the most potent isomer was 4-chloro-orto-cresol (4COC) (EC50 = 55 ± 14 µM), although 3-chloro-para-cresol (3CPC) was more effective, as it was able to induce higher magnitude of Ca2+ flux from isolated terminal cisterna vesicles. Nevertheless, 3CPC stimulated the hydrolytic activity of the sarcoplasmic reticulum ATP-ase (SERCA) with an EC50 of 91 ± 17 µM, while 4COC affected SERCA only in the millimolar range (IC50 = 1370 ± 88 µM). IC50 of 4CMC for SERCA pump was 167 ± 8 µM, indicating that 4CMC is not a specific RyR agonist either, as it activated RyR in a similar concentration (EC50 = 121 ± 20 µM). Our data suggest that the use of 4COC might be more beneficial than 4CMC in experiments, when Ca2+ release should be triggered through RyRs without influencing SERCA activity.

The Microbiome as a Component of the Tumor Microenvironment.

Microbes, which live in the human body, affect a large set of pathophysiological processes. Changes in the composition and proportion of the microbiome are associated with metabolic diseases (Fulbright et al., PLoS Pathog 13:e1006480, 2017; Maruvada et al., Cell Host Microbe 22:589-599, 2017), psychiatric disorders (Macfabe, Glob Adv Health Med 2:52-66, 2013; Kundu et al., Cell 171:1481-1493, 2017), and neoplastic diseases (Plottel and Blaser, Cell Host Microbe 10:324-335, 2011; Schwabe and Jobin, Nat Rev Cancer 13:800-812, 2013; Zitvogel et al., Cell 165:276-287, 2016). However, the number of directly tumorigenic bacteria is extremely low. Microbial dysbiosis is connected to cancers of the urinary tract (Yu, Arch Med Sci 11:385-394, 2015), cervix (Chase, Gynecol Oncol 138:190-200, 2015), skin (Yu et al., J Drugs Dermatol 14:461-465, 2015), airways (Gui et al., Genet Mol Res 14:5642-5651, 2015), colon (Garrett, Science 348:80-86, 2015), lymphomas (Yamamoto and Schiestl, Int J Environ Res Public Health 11:9038-9049, 2014; Yamamoto and Schiestl, Cancer J 20:190-194, 2014), prostate (Yu, Arch Med Sci 11:385-394, 2015), and breast (Flores et al., J Transl Med 10:253, 2012; Fuhrman et al., J Clin Endocrinol Metab 99:4632-4640, 2014; Xuan et al., PLoS One 9:e83744, 2014; Goedert et al., J Natl Cancer Inst 107:djv147, 2015; Chan et al., Sci Rep 6:28061, 2016; Hieken et al., Sci Rep 6:30751, 2016; Urbaniak et al., Appl Environ Microbiol 82:5039-5048, 2016; Goedert et al., Br J Cancer 118:471-479, 2018). Microbial dysbiosis can influence organs in direct contact with the microbiome and organs that are located at distant sites of the body. The altered microbiota can lead to a disruption of the mucosal barrier (Plottel and Blaser, Cell Host Microbe 10:324-335, 2011), promote or inhibit tumorigenesis through the modification of immune responses (Kawai and Akira, Int Immunol 21:317-337, 2009; Dapito et al., Cancer Cell 21:504-516, 2012) and microbiome-derived metabolites, such as estrogens (Flores et al., J Transl Med 10:253, 2012; Fuhrman et al., J Clin Endocrinol Metab 99:4632-4640, 2014), secondary bile acids (Rowland, Role of the gut flora in toxicity and cancer, Academic Press, London, p x, 517 p., 1988; Yoshimoto et al., Nature 499:97-101, 2013; Xie et al., Int J Cancer 139:1764-1775, 2016; Shellman et al., Clin Otolaryngol 42:969-973, 2017; Luu et al., Cell Oncol (Dordr) 41:13-24, 2018; Miko et al., Biochim Biophys Acta Bioenerg 1859:958-974, 2018), short-chain fatty acids (Bindels et al., Br J Cancer 107:1337-1344, 2012), lipopolysaccharides (Dapito et al., Cancer Cell 21:504-516, 2012), and genotoxins (Fulbright et al., PLoS Pathog 13:e1006480, 2017). Thus, altered gut microbiota may change the efficacy of chemotherapy and radiation therapy (McCarron et al., Br J Biomed Sci 69:14-17, 2012; Viaud et al., Science 342:971-976, 2013; Montassier et al., Aliment Pharmacol Ther 42:515-528, 2015; Buchta Rosean et al., Adv Cancer Res 143:255-294, 2019). Taken together, microbial dysbiosis has intricate connections with neoplastic diseases; hereby, we aim to highlight the major contact routes.

Silencing of PARP2 Blocks Autophagic Degradation.

Poly(ADP-Ribose) polymerases (PARPs) are enzymes that metabolize NAD+. PARP1 and PARP10 were previously implicated in the regulation of autophagy. Here we showed that cytosolic electron-dense particles appear in the cytoplasm of C2C12 myoblasts in which PARP2 is silenced by shRNA. The cytosolic electron-dense bodies resemble autophagic vesicles and, in line with that, we observed an increased number of LC3-positive and Lysotracker-stained vesicles. Silencing of PARP2 did not influence the maximal number of LC3-positive vesicles seen upon chloroquine treatment or serum starvation, suggesting that the absence of PARP2 inhibits autophagic breakdown. Silencing of PARP2 inhibited the activity of AMP-activated kinase (AMPK) and the mammalian target of rapamycin complex 2 (mTORC2). Treatment of PARP2-silenced C2C12 cells with AICAR, an AMPK activator, nicotinamide-riboside (an NAD+ precursor), or EX-527 (a SIRT1 inhibitor) decreased the number of LC3-positive vesicles cells to similar levels as in control (scPARP2) cells, suggesting that these pathways inhibit autophagic flux upon PARP2 silencing. We observed a similar increase in the number of LC3 vesicles in primary PARP2 knockout murine embryonic fibroblasts. We provided evidence that the enzymatic activity of PARP2 is important in regulating autophagy. Finally, we showed that the silencing of PARP2 induces myoblast differentiation. Taken together, PARP2 is a positive regulator of autophagic breakdown in mammalian transformed cells and its absence blocks the progression of autophagy.

Nuclear Factor Erythroid 2-Related Factor 2 in Regulating Cancer Metabolism.

Significance: Nuclear factor erythroid 2 (NFE2)-related factor 2 (NFE2L2, or NRF2) is a transcription factor predominantly affecting the expression of antioxidant genes. NRF2 plays a significant role in the control of redox balance, which is crucial in cancer cells. NRF2 activation regulates numerous cancer hallmarks, including metabolism, cancer stem cell characteristics, tumor aggressiveness, invasion, and metastasis formation. We review the molecular characteristics of the NRF2 pathway and discuss its interactions with the cancer hallmarks previously listed. Recent Advances: The noncanonical activation of NRF2 was recently discovered, and members of this pathway are involved in carcinogenesis. Further, cancer-related changes (e.g., metabolic flexibility) that support cancer progression were found to be redox- and NRF2 dependent. Critical Issues: NRF2 undergoes Janus-faced behavior in cancers. The pro- or antineoplastic effects of NRF2 are context dependent and essentially based on the specific molecular characteristics of the cancer in question. Therefore, systematic investigation of NRF2 signaling is necessary to clarify its role in cancer etiology. The biggest challenge in the NRF2 field is to determine which cancers can be targeted for better clinical outcomes. Further, large-scale genomic and transcriptomic studies are missing to correlate the clinical outcome with the activity of the NRF2 system. Future Directions: To exploit NRF2 in a clinical setting in the future, the druggable members of the NRF2 pathway should be identified. In addition, it will be important to study how the modulation of the NRF2 system interferes with cytostatic drugs and their combinations.

Lithocholic Acid, a Metabolite of the Microbiome, Increases Oxidative Stress in Breast Cancer.

In breast cancer patients, the diversity of the microbiome decreases, coinciding with decreased production of cytostatic bacterial metabolites like lithocholic acid (LCA). We hypothesized that LCA can modulate oxidative stress to exert cytostatic effects in breast cancer cells. Treatment of breast cancer cells with LCA decreased nuclear factor-2 (NRF2) expression and increased Kelch-like ECH associating protein 1 (KEAP1) expression via activation of Takeda G-protein coupled receptor (TGR5) and constitutive androstane receptor (CAR). Altered NRF2 and KEAP1 expression subsequently led to decreased expression of glutathione peroxidase 3 (GPX3), an antioxidant enzyme, and increased expression of inducible nitric oxide synthase (iNOS). The imbalance between the pro- and antioxidant enzymes increased cytostatic effects via increased levels of lipid and protein oxidation. These effects were reversed by the pharmacological induction of NRF2 with RA839, tBHQ, or by thiol antioxidants. The expression of key components of the LCA-elicited cytostatic pathway (iNOS and 4HNE) gradually decreased as the breast cancer stage advanced. The level of lipid peroxidation in tumors negatively correlated with the mitotic index. The overexpression of iNOS, nNOS, CAR, KEAP1, NOX4, and TGR5 or the downregulation of NRF2 correlated with better survival in breast cancer patients, except for triple negative cases. Taken together, LCA, a metabolite of the gut microbiome, elicits oxidative stress that slows down the proliferation of breast cancer cells. The LCA-oxidative stress protective pathway is lost as breast cancer progresses, and the loss correlates with poor prognosis.