Prostate cancer (PCa) is initially driven by excessive androgen receptor (AR) signaling with androgen deprivation therapy (ADT) being a major therapeutic approach to its treatment. However, the development of drug resistance is a significant limitation on the effectiveness of both first-line and more recently developed second-line ADTs. There is a need then to study AR signaling within the context of other oncogenic signaling pathways that likely mediate this resistance. This review focuses on interactions between AR signaling, the well-known phosphatidylinositol-3-kinase/AKT pathway, and an emerging mediator of these pathways, the Hippo/YAP1 axis in metastatic castrate-resistant PCa, and their involvement in the regulation of epithelial-mesenchymal transition (EMT), a feature of disease progression and ADT resistance. Analysis of these pathways in circulating tumor cells (CTCs) may provide an opportunity to evaluate their utility as biomarkers and address their importance in the development of resistance to current ADT with potential to guide future therapies. Prostate cancer (PCa) is highly prevalent in the Western world; it ranks sixth among cancers in regard to mortality among men . There wvia AR signaling, this suggests, in general, the common ongoing involvement of AR signaling in disease progression to mCRPC [On the molecular level, PCa is almost always initially driven by excessive signaling through the androgen receptor (AR) pathway (reviewed in ). Conseqto mCRPC –7. Abirato mCRPC , 9 and eto mCRPC , 11 haveto mCRPC  suggeststo mCRPC , 12–14, to mCRPC , 16. OurMetastasis in PCa is integrally linked to mCRPC. At the cellular level, metastasis involves a sequence of steps, and current evidence suggests that EMT and the reverse process mesenchymal-to-epithelial transition (MET) (reviewed in ) are impvia blood/lymphatic circulation, (v) leave the bloodstream or lymphatic vessels at distant organs, (vi) form a micrometastatic core, and finally (vii) adjust and reprogram the surrounding stroma to form detectable macrometastases [Metastatic spread of cancer is thought to involve different stages  in whichtastases . At a motastases , 25.α stimulates transcription factors (TFs), such as Snail and Twist, to trigger EMT [EMT is inducible by environmental factors such as radiation or hypoxia , and thegger EMT , 29. EMTgger EMT . Mechaniβ, and HGF in turn induces signaling via NF-κB, MAPK, PI3K/AKT, or Wnt/β-catenin pathways to regulate EMT-TFs and ultimately induce EMT phenotypic changes. More recently, the Hippo pathway has been implicated in regulating EMT via its downstream transcriptional modulator Yes-associated protein (YAP) and the transcriptional coactivator TAZ [Regulation by signaling cascades and signaling molecules including EGF, Hedgehog, Wnt, FGF, Notch, TGF-ator TAZ , 30–38. in vitro, and consistent with these findings, E-cadherin staining in tumor tissue correlates with longer overall survival [Several studies have assessed EMT markers for their clinical importance at various stages of human PCa. survival . Howeversurvival , suggestsurvival . Differesurvival .via the AR ligand-binding domain (LBD)  , a strucIn the nucleus, AR acts as a transcription factor by binding to androgen-response elements (AREs) in the promoter region of androgen-regulated genes , 96. AR The expression of alternative AR splice variants has been proposed as a mechanism underlying resistance to ADT , 100. Moviz cell migration and invasion, and mesenchymal marker proteins associated with EMT, while increasing the epithelial marker E-cadherin. These effects were recapitulated by treatment with the antiandrogen bicalutamide [There is no clear consensus with respect to the role of androgen signaling in the regulation of EMT. An early study using cell lines showed that androgen stimulation promoted EMT in both LNCaP and PC-3 cells but that there was an inverse relationship between AR receptor levels and androgen-mediated EMT marker expression and EMT-associated cytoskeletal changes. Low levels of AR induced by shRNA promoted PCa cell metastatic ability by inducing EMT while high levels did not . In contlutamide . Thus, iStudies with both normal mouse prostate and human prostate tumor models in mice have shown that androgen deprivation through surgical castration, while suppressing tumor growth, induces mesenchymal markers of EMT and markers of a stem cell phenotype, while suppressing epithelial markers. These changes were also seen in tissues of patients treated with ADT , supportIn further support of this view, ADT with enzalutamide in C4-2 cells, but not in PC-3 cells, induced EMT markers in a Snail-dependent fashion. Induction of EMT required both suppression of AR signaling and activation of Snail. Interestingly, Snail was downregulated by androgen in AR expressing C4-2 and VCaP cells but again, not in PC-3 cells. Importantly, the inverse correlation between AR signaling and Snail expression observed in C4-2 xenografts and castration-resistant patient-derived metastases in mice and in clinical samples supports the view that the induction of EMT is an adaptive response to ADT with enzalutamide . ADT mayOther data suggest that AR splice variants are involved in the development of drug resistance in PCa , 115–117PTEN has been associated with EMT driven through the AKT pathway or in cooperation with RAS signaling; thereby, lack of PTEN function could promote metastasis [As indicated above, due to the hormone-independent nature of mCRPC, it is unresponsive to all current forms of ADT. At this stage, AR expression may even be completely lost –124, raitastasis , 137.As indicated above, several signaling pathways may contribute to the induction of EMT and ultimately metastasis, with the AKT pathway of importance in the context of PCa. More recently, the YAP1 transcriptional coactivator regulated by the Hippo pathway has emerged as an important player in this scenario and in regulating PCa cell motility . In the via dephosphorylation, which is required for translocation into the nucleus. Although TAZ and YAP lack intrinsic DNA-binding domains, they are recruited by and enhance the activity of other transcription factors at their target promoters [Inactivation of the Hippo pathway allows for YAP and TAZ activation romoters , 146.YAP1 gene amplification. YAP1 is the most studied YAP isoform, and aberrant YAP1 activation is associated with the etiology of various malignancies including stomach [Hippo signaling can act as a tumor suppressor. Functional impairment of Hippo signaling is often due to the loss of MST1/2 or LATS1/2 function or due to  stomach , thyroid stomach , lung [1 stomach , colon [ stomach , head an stomach  ovarian  stomach , liver [ stomach , and pro stomach .Most interestingly, YAP1 and AR directly interact in PCa cells. One study demonstrates that unlike in hormone-sensitive prostate cancer cells, YAP1-AR interactions are androgen-insensitive and may cause resistance to enzalutamide in mCRPC cells. The WW/SH3 domain of YAP1 most likely facilitates the interaction with the AR amino terminal domain (NTD) .One study proposes that increased nuclear YAP1, possibly due to the loss of Hippo signaling, may lead to increased complex formation between AR and YAP1 leading to androgen-independent binding of the complex to AREs in AR-driven promoters resulting in aberrant AR target gene expression possibly promoting mCRPC .SLUG [via protein degradation due to activation of the Hippo pathway, while direct interaction of YAP with Snail and with Slug was shown to alter YAP/TEAD transcriptional activity [via PAR-1/-3 in this context, although this was not examined [β1-integrin-mediated matrix stiffness prevented both Twist and YAP nuclear translocation albeit, interestingly, by different mechanisms [Importantly, YAP has been shown to promote metastasis through several mechanisms including EMT, and there is some evidence that the PTEN-AKT axis is involved in YAP1-induced EMT , 157. ThSLUG . Focusinactivity . Anotherexamined . Anotherchanisms .In epithelial cells, cells are connected to each other by membrane structures called tight junctions, adherens junctions, and desmosomes. Any dysregulation in these junctions is implicated in metastasis and EMT , 164. ZoYAP (but not TAZ) has been shown to interact directly with ZEB1 and, remarkably, this interaction turns this transcriptional repressor into an activator. This is highlighted by the fact that ZEB1-mediated CDH1 (E-cadherin) repression is independent of YAP binding. Critically, gene upregulation by the ZEB1-YAP complex correlated with gene expression signatures of claudin-low breast cancer, a breast cancer subtype overall exhibiting an EMT phenotype. More importantly, ZEB1-YAP complex-mediated gene expression was related to poor patient survival in hormone-independent breast cancers and linked to drug resistance and metastasis . ZEB1 isCDH1 (E-cadherin) promoter and repress its transcription. These data, together with confirmation that E-cadherin inhibition upregulates YAP levels, indicate a double-negative feedback where E-cadherin and YAP mutually inhibit each other. This may be part of a switch between EMT and MET, thus potentially explaining the plasticity of the EMT process [Not surprisingly, another study showed that inhibiting a key characteristic of epithelial tissue, namely, E-cadherin-mediated cell-cell interaction, resulted in EMT and increased dissemination of Madin–Darby canine kidney cells. Interestingly, dissemination could be partially prevented by YAP knockdown. The same study found that not only is YAP required to allow nuclear entry of the MET initiating Wilms tumor protein 1 (WT1), but both WT1 and YAP form a complex at the  process .Interestingly, one possible mechanism for PTEN loss of function is mediated by YAP. The pathway involves nuclear YAP-mediated activation of the TEAD family of transcription factors, leading to synthesis of the PTEN transcriptional repressor miRNA29c. Conversely, when YAP is inactivated via phosphorylation, PTEN levels are restored and the oncogenic function of YAP is inhibited . MoreoveOn the other hand, PTEN was identified as a negative regulator of AR activity such that the AR/PTEN interaction may mediate a tumor suppressor role for PTEN via suppression of AR and apoptosis induction in PCa cells . Howevervia EMT  [The evaluation of molecular pathways underlying mCRPC is challenging because tissue biopsies are generally not available from late disease stages and animal models; further, although examination of tissue can provide some signaling pathway information, this mode of studying PCa has limitations. Liquid biopsies, and analysis of mCRPC CTCs, may be an alternative. While diagnostic CTC analysis in PCa is still in its infancy, there is ample evidence of its utility in this disease. Certainly, CTCs have been investigated by imaging and molecular technologies for expression of proteins, gene amplifications, mutations, and transcript expression on both targeted and comprehensive levels . For PCaals.gov) .in situ hybridization (FISH) and other molecular approaches. In general, these studies were able to validate the association of CTC-based AR amplification or mutation with mCRPC, while the relevance of AR cellular localisation in CTCs was shown in mCRPC and in response to taxanes [mCRPC-associated AR amplification and mutation analysis have been performed in CTCs using hybridization techniques such as fluorescent  taxanes , 183–186 taxanes , 188. In taxanes , 190. Wi taxanes , 192. In taxanes . In CRPC taxanes . In any PTEN and gain of AR copy numbers were reported in PCa CTCs [Several studies have also analysed PTEN loss in CTCs, which, as outlined above, may allow oncogenic activation of the AKT pathway and is an important PCa biomarker. Loss of PCa CTCs –197, whiPCa CTCs .Reports on hippo signaling and YAP1 analysis in CTCs, by contrast, are still scarce. One study assessed expression of TAZ using RNA in situ hybridization (RNAish) probing of NSCLC CTCs. TAZ expression was detected more frequently in EGFR wild-type cancers while its expression in CTCs was associated with lymph node status of the disease . It is lTaken together, the reviewed data suggest that AR-AKT-YAP1 network can be analysed in CTCs. Since tumor tissue is rarely available in the mCRPC setting, and blood samples can be easily taken, future endeavours in CTC analysis could open the way to better understand ADT resistance and thereby inform the development of improved diagnostic, prognostic, and therapeutic capabilities.Analysis of CTCs has provided a foundation for liquid biopsy, especially in the absence of biopsy tissue. However, there are serious challenges with CTC isolation, detection, and downstream analysis. One is that CTC numbers are relatively small within large populations of blood cells and the volume of blood that can be taken depends on the patient's general condition. CTCs are quite heterogeneous in terms of physical properties , biological characteristics, and expression of different tumor markers making enrichment or isolation of all CTCs difficult (reviewed in ). In genHere, we reviewed connections between the AR pathway and the AKT and Hippo pathways, exploring a potential role for this signaling nexus in EMT and mCRPC. Though current literature supports the importance of this tripartite relationship, further study is now needed to better evaluate its importance in PCa, as well as its clinical potential in defining biomarkers or drug targets. Analysis of PCa CTCs may facilitate deeper investigations into AR/AKT/Hippo pathway interactions, and how these drive EMT as well as ADT resistance. Such analyses may ultimately mediate the emergence of new diagnostic/prognostic assays directed towards PCa, though at this time insufficient data are available to establish feasibility of this concept. Indeed, while some aspects of these pathways have already been investigated in CTCs, optimisation of more comprehensive CTC analysis methods is now needed to permit the dissection of these pathway interactions, as a precursor to this significant goal.


Mean CT dose index decreased from 28.7 mGy without AR to 16.9 mGy with AR (p = 0.009). Mean procedure time reduced from 8.93 min without AR to 4.42 min with AR (p = 0.027). Puncture rate of a nontargeted lesion decreased from 11.9% without AR (7/59 passes) to 0% with AR (0/27 passes). First needle passes were closer to the ideal target trajectory with AR versus without AR . AR reduced variability and elevated the performance of all operators to the same level irrespective of prior clinical experience. AR guidance can provide significant improvements in procedural efficiency and radiation dose savings for targeting out-of-plane lesions.Out-of-plane lesions pose challenges for CT-guided interventions. Augmented reality (AR) headsets are capable to provide holographic 3D guidance to assist CT-guided targeting. A prospective trial was performed assessing CT-guided lesion targeting on an abdominal phantom with and without AR guidance using HoloLens 2. Eight operators performed a cumulative total of 86 needle passes. Total needle redirections, radiation dose, procedure time, and puncture rates of nontargeted lesions were compared with and without AR. Mean number of needle passes to reach the target reduced from 7.4 passes without AR to 3.4 passes with AR ( Since then, many other applications have been developed with AR devices to enhance training and image-guided procedures3.Augmented reality (AR) technologies are able to seamlessly merge virtual objects with the surrounding environment. Extensive technological progress has been made with AR headset devices since 2006 with the development of one of the first AR guidance systems, called Reality Augmentation for Medical Procedures (RAMP), for CT-guided interventions via custom headset with video see-through overlay5. Out-of-plane approaches, in particular, are often required for challenging lesions6. These may entail a greater number of needle misplacements, repeated skin punctures, and unintended traversal of critical structures along the trajectory path. As a consequence, the patient can experience increased pain and discomfort, bleeding complications, or inadvertent organ injury and perforation requiring additional interventions.However, despite technical achievements with commercial headsets now capable of projecting 3D holograms merged with transparent views of the real world, or optical see-through, clinical utility and adoption of AR technologies have made only marginal progress since RAMP. Still, the goals of any navigation system for improving CT-guided interventions remain the same. Challenging lesions and anatomy can make targeting difficult and result in prolonged procedure times, increased radiation exposure, and more complications8. 3D AR-assisted navigation systems using HoloLens  have received unanimously positive feedback among operators for its potential to enhance safety, aid in execution, and improve depth perception and spatial understanding10. Although such systems show promise, no prior study to date has demonstrated the procedural effects of AR guidance using an optical see-through headset device through a systematic trial.Many AR-assisted guidance systems have been recently developed for percutaneous needle-based interventions with the widespread availability of commercial AR devices. Smartphone or tablet-based AR navigation platforms for CT-guided needle insertion have demonstrated sub-5 mm accuracies, decreased procedure times, and fewer intermediate CT scans3. Evaluation was performed through a preclinical trial simulating CT-guided needle targeting of an out-of-plane lesion in an abdominal phantom with and without AR guidance. The number of needle redirections, radiation dose, procedure time, and puncture rates of nontargeted lesions are compared.This study describes the design of a 3D AR-assisted navigation system using the next-generation HoloLens 2  headset device. Unlike other existing AR-assisted navigation systems, no additional or extra hardware components are needed aside from the headset. Registration was performed automatically to a CT grid routinely used in clinical practice, as opposed to using separate external image-based markers or matrix barcodes required in other systemsThis study is Institutional Review Board exempt from the University of Pennsylvania as no actual patient data was obtained or analyzed. CT-guided percutaneous needle targeting was simulated on a phantom model  containing multiple targets of various sizes. A CT grid  commonly used in clinical practice was placed on the anterior surface of the phantom for planning and to serve as a fiducial target for registration.11. Segmentation meshes were exported in STL file format followed by mesh decimation using Meshmixer  to eliminate redundant vertices and reduce mesh size to improve 3D rendering performance. Reduced meshes were then exported in OBJ file format and material textures, including colors and transparencies, were applied using Blender . The target lesion was colored in green; all other nontargeted lesions were colored in red. The final 3D surface-rendered model was exported in FBX file format 17. Registration accuracies were not directly validated in this study; registration fidelity was confirmed visually by the operator based on complete alignment of the virtual gridlines with the physical gridlines. A virtual needle trajectory was added into the 3D model based on the ideal trajectory. This virtual guide allowed the user to easily trace the ideal trajectory using a real needle  residents, and 3 medical students. Both attendings had greater than 5 years of experience. 2 residents were in their final year of training. All 3 medical students had never previously seen nor performed a CT-guided intervention. Aside from 1 resident, all other participants had no prior experience wearing or interacting with HoloLens 2. In order to limit bias, participants were randomized into cohorts: CT-guided targeting 1) without AR and then repeated with AR or 2) with AR and then repeated without AR  and dose-length product (DLP) were obtained from the CT dose report. Procedure duration was measured from the acquisition time, or image metadata DICOM tag , of the CT scan following the 1med Fig. . These CVector analyses, means, paired t-tests, and F-tests were performed using Google Sheets . Post hoc power analysis suggested a total sample size of 8 for a power of 0.8 and effect size of 1 to achieve a statistical significance level of 0.05.p = 0.011). Radiation dose savings were also significant with mean CTDIvol decreasing from 28.7 mGy without AR to 16.9 mGy with AR , and DLP decreasing from 538 mGy-cm without AR to 318 mGy-cm with AR . Mean procedure duration significantly reduced from 8.93 min without AR to 4.42 min with AR . The puncture rate of a nonselected target also decreased from 11.9% (7/59 passes) without AR to 0% with AR (0/27 passes).A comparison of CT-guided needle targeting performed with and without AR is summarized in Table p = 0.018). In addition, the 1st pass distance traveled was deeper and the needle tip was closer to the target with AR versus without AR but did not reach statistical significance .The first, initial needle pass angle was more aligned with the ideal trajectory with AR guidance compared to needle insertion without using any guidance, as is common standard clinical practice . All subgroups performed similarly with the mean number of passes ranging between 3–4 passes and the 1st pass distance averaging approximately 10 cm among each subgroup. Additionally, there were no punctures of a nonselected target with AR. Medical students with no prior clinical experience performed at the same level as experienced attendings with AR.Using 3D AR guidance, participant performances were more homogeneous regardless of experience. Collectively, there were significant reductions in the variability in the number of passes and 1st pass distances with AR . This suggests that the benefits of having real-time 3D navigation likely supersede advantages associated with prior experience or recall. This contention is further supported by the fact that medical students performed at the same level as experienced attendings with 3D AR guidance.Some degree of order bias and recall did occur during non-AR-assisted simulations. The cohort that performed targeting with HoloLens 2 first and then repeated without AR had fewer total needle passes without AR (22 passes) compared to the cohort that performed targeting without AR first (37 passes). These data suggest that visually seeing the trajectory in 3D in addition to physically performing the procedure concurrently may enhance spatial understanding and innate recallGenerally, medical students were aggressive on their first needle pass without AR, advancing the needle a distance of over twice that of residents and attendings with a goal of getting close to the target as opposed to multiple smaller passes to ensure the trajectory is on course, which may come with clinical experience. As expected, medical students had the greatest number of unintentional punctures of adjacent nontargeted lesions. Without AR, residents and attendings took more conservative 1st initial passes and required a greater number of overall passes but had fewer punctures of nontargeted lesions, with attendings having none. With AR guidance, there was significantly less variability in performance. Attendings, residents, and students all performed at the same level with similar total passes and 1st-pass distances as well as having zero punctures of nontargeted lesions.20 or high frequency jet ventilation under general anesthesia21. Soft tissue deformation can be compensated by deformable modeling9, and needle bending can be extrapolated using a shape sensing needle22. Further developments will incorporate some of these features prior to evaluation with live subjects.The primary limitation of this study was the evaluation of this system using a stationary, inanimate phantom. As with any navigation system, patient motion, respiratory breathing, soft tissue deformation, and needle bending are important factors that can affect navigational performance. Breathing can be compensated by techniques such as simple respiratory gatingIn summary, holographic 3D AR guidance using an optical see-through headset device can decrease needle redirections, reduce radiation dose, shorten procedure time, and minimize punctures of nontargeted structures during CT-guided interventions. The use of 3D AR guidance may thus facilitate the treatment of challenging, hard-to-reach, or out-of-plane lesions. Additionally, these data suggest that AR guidance may immediately help to elevate the performance of inexperienced operators, providing added opportunities to treat challenging lesions that were previously declined due to limited operator experience. Although this preclinical trial shows promising and translatable benefits of 3D AR guidance, further developments and robust clinical testing will be needed for adoption into actual practice.


Chlamydophila pneumoniae, Haemophilus influenzae, and Streptococcus pneumoniae, among others) and viruses . Although the precise mechanisms that link glycemia to the exacerbated infections remain elusive, hyperglycemia is known to induce a wide array of changes in the immune system activity, including alterations in: (i) the microenvironment of immune cells ; (ii) the supply of energy to infectious bacteria; (iii) the inflammatory response; and (iv) oxidative stress as a result of bacterial proliferative metabolism. Consistent with this evidence, some bacterial infections are typical (and/or have a worse prognosis) in patients with hypercaloric diets and a stressful lifestyle (conditions that promote hyperglycemic episodes). On this basis, the present review is particularly focused on: (i) the role of diabetes in the development of some bacterial and viral infections by analyzing preclinical and clinical findings; (ii) discussing the possible mechanisms by which hyperglycemia may increase the susceptibility for developing infections; and (iii) further understanding the impact of hyperglycemia on the immune system.Uncontrolled diabetes results in several metabolic alterations including hyperglycemia. Indeed, several preclinical and clinical studies have suggested that this condition may induce susceptibility and the development of more aggressive infectious diseases, especially those caused by some bacteria (including  Diabetes mellitus is a chronic and complex illness characterized by several metabolic alterations including dyslipidemia and hyperglycemia, among others . AccordiPeople with metabolic impairments  show greater ranges of glucose levels . Indeed,Listeria monocytogenes) was dramatically decreased when they were gavage-fed. In contrast, if these animals received glucose (i.p.), all animals died  may induce inhibition of anorexia via Salmonella leucine rich repeat protein (SlrP) which inhibits interleukin-1β (IL-1β). This effect seems to maintain the conditions for increasing the opportunity for Salmonella to infect other hosts . To focus on these aspects, the present review has considered information on: (i) the effect of glycemia on infection outcome and immune cells physiology; (ii) the biochemical alterations in cell physiology during diabetes and/or hyperglycemia; and (iii) the impact of pharmaceutical care interventions for glycemia control on some of the most frequent emerging infectious diseases.To consider the relevant literature in this theoretical review, we searched for studies published in various databases such as Science direct, Pubmed central and Google Scholar. These databases included any combination of the main key terms “bacterial infections,” “COVID-19,” “diabetes,” “influenza A virus,” “hepatitis B and C viruses,” “human immunodeficiency virus,” and “hyperglycemia” among themselves and with important topics such as: “rheological properties of blood,” “biochemical alterations of diabetic immune cells,” “immune response on hyperglycemic environment,” “hypoglycemic drugs,” “bacterial infection outcome,” and “comorbidities with COVID-19.” Around 600 articles published from 1966 up to 2020 were perused, and only 260 of those articles with experimental and/or theoretical information which related hyperglycemia and/or diabetes to bacterial infections and/or COVID-19 and some other viruses were included in this review.In general, alterations in the immune system during hyperglycemia seem to be associated with mechanisms that include lower secretion of inflammatory cytokines, depression in neutrophils and T cells function, as well as decreases in humoral immunity , 22. MorMycobacterium tuberculosis, which facilitates infection and its symptomatic progression  event  should be quite different from the changes induced by a chronic hyperglycemia. Nevertheless, in any hyperglycemic condition , patients may be susceptible to some of the same clinical complications, including poor wound healing and an increased rate of infection . In factBesides diabetes, another condition that commonly predisposes to hyperglycemia is stress. The stress-induced hyperglycemia (SIH) generally refers to a metabolic condition with a transient hyperglycemia associated with clinical illness . The SIHThe SIH is especially dangerous in chronic critical illnesses , as the A stress response is associated with an increased pro-inflammatory response characterized by the release of several cytokines, including tumor necrosis factor-alpha (TNF-α), IL-1 and IL-6, which are related to insulin resistance . Indeed:Additionally, there may be an integration of a vicious cycle when taking a hypercaloric fatty diet, which can also induce an increase in catecholamines' release , in combThe contribution of SIH in the diabetic patient is a complex issue that seems to worsen the glycemic status of patients with type 2 diabetes; whereas the autonomic damage induced by neuropathy in type 1 diabetes leads to contradictory and non-conclusive data . PatientDiabetes is a condition that may potentiate infectious diseases and predispose patients to acquiring more severe diseases. To support this notion, a recent matched cohort study analyzed the incidence infection rate from 306,011 patients , and reported that patients with diabetes  are more susceptible to developing severe infectious diseases . In addiAccording to epidemiological studies, the most common infectious diseases of hospitalization in children, adolescents and adults with diabetes are lower tract respiratory infections , diabetic foot infection, skin and soft tissue abscesses, and urinary tract infections , 108. ThChlamydophila pneumoniae, Legionella pneumophila, methicillin-resistant Staphylococcus aureus, Haemophilus influenzae, and Streptococcus pneumoniae  in a recent metanalysis reported by Langford et al. . NotorioDM, hypertension, cardiovascular diseases, and obesity are the top four comorbidities worldwide associated to critically ill patients with COVID-19 and mortality , 119–121Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Enterobacteriaceae, Pseudomonas aeruginosa, Moraxella catarrhalis, and Acinetobacter baumannii were the 28% of bacterial strains isolated by experimental laboratory procedures (cultures or PCRs assays)  . ImportaAdmittedly, pathogenesis of COVID-19 viremia remains unclear. However, some lines of evidence suggest that high levels of systemic glucose increase glucose concentration in the epithelial secretion of the respiratory tract, disrupting the orchestration of the innate and humoral immunological response. This includes, in particular, hyperglycemia-induced changes in coagulation, worsening of endothelial function, and reproduction of inflammatory cytokines .Data about hospitalization for infectious diseases in diabetic and non-diabetic subjects have been associated with various hyperglycemic conditions on admission, increasing poor outcomes and mortality rates. Moreover, hyperglycemia on admission was clearly associated with undiagnosed DM, strongly suggesting that an optimal glycemic control that reduces glycemic fluctuations during hospitalization should be a beneficial clinical practice for viremia control .The American Diabetes Association (A.D.A.) recommends a blood glucose level of 140 to 180 mg /dL (7.8 to 10.0 mmol/L) for most critically ill patients and patients who are not in good health. Glycemic control during clinical procedures could be accompanied by insulin therapy if the hyperglycemia persists starting at a threshold ≥ 180 mg/dL . Thus, pin vitro.The influenza A virus (IAV) induces a self-limited infection in most patients, which is characterized by several symptoms such as myalgia, fever, and dry cough . Neverthin vitro and in vivo model of pulmonary epithelial-endothelial cells exposed to a high glucose concentration (12 mM) demonstrated an increased barrier damage after co-cultured cells were infected with IAV; this, in turn, augmented pulmonary edema associated with a pro-inflammatory response  and Hepatitis B virus (HBV) are known causes of hepatic decompensation, liver cirrhosis, and hepatocellular carcinoma (HCC), being two major public health problems worldwide –147. TheHuman immunodeficiency virus (HIV) increases the risk for developing T2DM , 156. LipH   . DKA reslow 7.3) . This, ilow 7.3) , 179. Keacidosis , 180.As the most severe complication of DM, patients with DKA have more difficulty to handle infections , 181. AdpH in the immune response. For example, with a pH below 6.5: (i) the mobility of polymorphonuclear leukocytes was impaired  an increase in lymphocytes mobilization was reported  (see below).Other important alterations induced by hyperglycemia in the circulatory system are related to a miss-functionality of the enzymatic machinery of blood cells, including Na+/K+-ATPase is a transmembrane protein responsible for maintaining intracellular Na+/K+ balance by generating the gradients of Na+ and K+ (+ gradient) in distal convoluted tubule, motility in sperm cells, action potentials in synaptic neurons, etc.   . Besidesronment) , such assylation , supportGlucose-6-phosphate dehydrogenase (G6PD) is an enzyme expressed ubiquitously in all mammalian tissues. It plays an important role in the pentose pathway catalyzing the first reaction in this metabolic route, which is necessary to convert glucose into pentose sugars . This paMany studies have reported the importance of G6PD in antioxidant defense against toxicity of reactive oxygen species (ROS) , 213. InAdmittedly, the specific molecular mechanisms that explain the effects of chronic hyperglycemia on G6PD activity in immune cells remain uncertain. For example, Xu et al.  showed eIn summary, the above physicochemical alterations resulting from hyperglycemia impair the immune response, predisposing diabetic subjects to acquire infections as well as exacerbates them.An uncontrolled blood glucose level is associated with an increase in microvascular and macrovascular complications in diabetic patients . LikewisStaphylococcus aureus (S. aureus) induced by hyperglycemia  and decreased glucose flux in an epithelial cell culture model  metformin pre-treatment of hyperglycemic animals reduced both airway glucose and bacterial load ; (ii) thConsistent with the above findings, diabetic patients with community-acquired pneumonia (CAP) developed worse results and longer hospital stays in comparison to patients with CAP without diabetes ; accordiMoreover, Mendy et al.  analyzedLikewise, Pan et al.  investigFurthermore, Shih et al.  reportedP. aeruginosa CFU in leptin receptor-deficient (db/db) mice, as compared to those seen in wild type (WT) mice  mice .In summary, the available evidence thus far has established the increased susceptibility to certain types of infections related to hyperglycemia in T2DM. Clearly, further studies on the mechanisms regulating OADs and bacterial action on specific tissues/organs are required. Such studies could yield potential alternatives to prevent/suppress hyperglycemia and bacterial infections.Moreover, the risk for developing infections is increased in hyperglycemic environments, where there is a lower production of interleukins, a reduced chemotaxis and phagocytic activity, and a gastrointestinal dysmotility . The useTo conclude this section, it is to be noted that experimental anorexia seems to play an important protective role in supporting the recuperation of bacterial infections . MoreoveA higher risk of CAP was found with other OADs, except with dipeptidyl-peptidase 4 (DPP-4) inhibitors . Indeed,In view that hyperglycemia may be a determinant factor in the outcome for bacterial infections, any effort for controlling the increases glucose levels is valuable. Another interesting approach may be the supplementation with calcium and vitamin D because it decreases insulin resistance and hyperglycemia ; neverthHypoglycemia occurs when there exists a lack of adequate food intake, excessive exercise, a stressful experience, excessive alcohol consumption, concurrent infections, severe digestive and urologic diseases, and/or after taking antidiabetic medications . This suConsidering that fasting plasma glucose is normally maintained between 70 and 99 mg/dL , a biomaGlycemic control and reduction of hyperglycemia or hypoglycemia events are the main challenges in the clinical experience to achieve decreases of blood glucose variability . Indeed,Glucose variability is currently considered more deleterious than chronic hyperglycemia in the development of diabetes-related complications . HoweverAnother consideration is that under normal and pathophysiological metabolic functions, individuals course with glucose swings during the day , correlaDuring physiological gastric emptying, carbohydrates and proteins are evacuated faster than lipids for their caloric content. The evacuation of these macronutrients begins at 20 to 40 min after food intake and when they reach the intestine, incretin hormones are secreted to blood. Glucagon-like peptide-1 (GLP-1) is an incretin hormone that stimulates insulin secretion, reduces glucagon secretion, and delays gastric emptying in a glucose-dependent manner , 258.Significantly, glucose-lowering therapies through the use of diverse drug classes have been reported as an important source of heart failure risk, particularly with differential effects on insulin . ConsequHyperglycemia clearly induces physiological and immunological disorders in body tissues/organs that may predispose and exacerbate some infectious diseases. Therefore, the control of glucose levels could be an alternative tool to contribute to the fight against infections not only in diabetic patients, but also in other conditions that induce hyperglycemia, such as SIH. In addition, several studies have shown the potential benefits of controlling it , opening a new option to improve the outcome of some infections . It is worth noting that the authors of this review agree that glycemic control is necessary as part of good intervention strategies to treat current and emerging infectious diseases. Admittedly, the clinical evidence for reducing glycemic exposure requires more supportive data, specifically for hypoglycemia as a tool to fight infections in humans. Notwithstanding, this review summarizes enough preclinical evidence to increase our chances of beating infections by focusing on the key role of glycemic control.JC-R, CE-G, and BM-C developed the central idea of this article and wrote the manuscript. BM-C proposed the central idea, made the graphs, and obtained funding. PV-L, EC-S, AL-B, and AQ-S provided original ideas, developed some sections, and reviewed the manuscript. CMV and JM-R discussed the central ideas, reviewed, edited, and corrected the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor and reviewer AV declared a shared affiliation with one of the authors EC-S at time of review.


These results provide preclinical proof of principle for Zeta55 as a promising therapeutic in prostate cancer treatment.Androgen receptor (AR) and histone deacetylase 6 (HDAC6) are important targets for cancer therapy. Given that both AR antagonists and HDAC6 inhibitors modulate AR signaling, a novel AR/HDAC6 dual inhibitor is investigated for its anticancer effects in castration-resistant prostate cancer (CRPC). Zeta55 inhibits nuclear translocation of AR and suppresses androgen-induced PSA and TMPRSS2 expression. Meanwhile, Zeta55 selectively inhibits HDAC6 activity, leading to AR degradation. Zeta55 reduces the growth of AR-overexpressing VCaP prostate cancer cells both  Castration-resistant prostate cancer (CRPC) is the second leading cause of cancer death among men in the United States  –6. HowevHistone deacetylases (HDACs) are involved in epigenetic regulation of gene expression and protein activity by removing acetyl groups from histone or nonhistone proteins . It has HDAC6 is primarily localized in the cytoplasm and deacetylates a variety of cytoplasmic proteins, which are involved in protein degradation, protein trafficking, cell migration, and metastasis . Overexpin vitro and in vivo. These data support future clinical development of Zeta55 for the treatment of prostate cancer.In this study, we describe the discovery and preclinical development of Zeta55, a dual inhibitor of AR and HADC6 by integrating HDACi zinc-binding group into the optimized skeleton of clinical AR antagonist. Mechanistic studies reveal that Zeta55 not only inhibits AR and HDAC6 activities, but also induces AR degradation. In addition, our work shows that Zeta55 potently inhibits the proliferation of prostate cancer cells We designed a dual-target inhibitor, Zeta55, which integrates AR and HDAC6 inhibitory functional groups into one single small molecule , which was reacted with cyclobutanone and KCN via Strecker reaction to generate the cyanoamine (compound 3). The cyclization of cyanoamine with 4-cyano-3-trifluomethylbenzamine in the presence of thiophosgene to give the desired thiohydantoin (compound 4) [Zeta55 was synthesized according to pound 4) , which wpound 4) .50 value of 0.63 μM, which is slightly less potent than MDV3100 (IC50: 0.42 μM), suggesting that the substitution of methyl group with hydroxyl group could slightly decrease the binding affinity to AR. An AR competitive binding experiment using fluorescence polarization also reached the same conclusion . Both compounds seem ineffective against HDAC4. These results suggest that Zeta55 is a HDAC6-selective HDAC inhibitor.To evaluate the AR inhibition activities of Zeta55, an AR luciferase activity assay was performed . Zeta55 nclusion . To evalnclusion . As a paWe further assessed both agonist and antagonist activity of AR signaling in AR-dependent VCaP prostate cancer cell line. Without DHT, 10 μM Zeta55 did not induce the expression of reporter gene, indicating that Zeta55 does not have AR agonist activity . In the We then assessed the effects of Zeta55 on the expression of Prostate-specific antigen (PSA) and transmembrane protease serine 2 (TMPRSS2), two endogenous AR-regulated gene. We evaluated both mRNA and protein levels of PSA and TMPRSS2 in VCaP cells. Similar to MDV3100, Zeta55 significantly suppressed DHT-induced endogenous mRNA expression and protein expression of PSA and TMPRSS2 –3F.To evaluate the HDAC6 inhibition ability of Zeta55 in VCaP cells, the acetylation status of α-tubulin protein (a HDAC6 substrate) was assessed using western blot. Zeta55 enhanced α-tubulin acetylation in a dose-dependent manner, while MDV3100 did not . HDAC6 iAn immunofluorescence experiment of AR and DAPI  was performed to study the effects of Zeta55 on the localization of AR in VCaP cells. As shown in the 50 value of 2.47 μM, and the inhibition ability was stronger than MDV3100 (IC50: 11.04 μM) and SAHA (IC50: 4.10 μM). Only SAHA potently inhibited the cell proliferation of AR-negative DU145 cells (IC50: 4.02 μM) and HEK293 cells (IC50: 4.43 μM). IC50 value of Zeta55 on LNCaP cells is higher than that on VCaP cells, which may due to the lower expression of AR in LNCaP cells , DU145 cells (AR negative prostate cancer cell line) and HEK293 cells  were performed using SAHA and MDV3100 as controls . Zeta55 aP cells . These din vivo pharmacodynamic activity of Zeta55, castrated male immunodeficient mice harboring VCaP xenograft tumors were orally treated with vehicle, MDV3100 , Zeta55  or Zeta55 . Zeta55 (30 mg/kg/d) showed slightly better antitumor activity than MDV3100 (30 mg/kg/d). A higher Zeta55 dose (60 mg/kg/d) led to increased efficacy (5 of 6 tumors with tumor regression)  , 4B. Durression) .in vitro.Ki-67 and TUNEL staining of tumor xenografts showed that Zeta55 inhibited tumor cell proliferation and induced apoptosis . Immunohin vivo, rats were orally (20 mg/kg) or intravenously (5 mg/kg) administrated with Zeta55, and the plasma concentration of Zeta55 was measured using LC-MS/MS. The key pharmacokinetic parameters of Zeta55 are summarized in t1/2) of Zeta55 in rats were about 0.8 hour for intravenous administration, and 2.9 hours for oral administration. The oral bioavailability of Zeta55 in rats was 17.6%.To evaluate the pharmacokinetic properties of Zeta55 in vitro and in vivo. Compared to single-target drugs, this multiple-target drug Zeta55 exhibits stronger anti-proliferation activity than MDV3100 and SAHA in VCaP cell experiments and better antitumor activity than MDV3100 in CRPC xenograft model. Compared to the combination of existing drugs in prostate cancer, this multiple-target drug Zeta55 may provide higher therapeutic effects and fewer side effects, as well as more predictable pharmacokinetic profiles and better patient compliance [The combination therapy that two or more drugs are used in combination to treat disease attract increasing attention in new drug discovery and development . In prosmpliance , 24.Other multiple-target drugs have also been developed for treating prostate cancers. For example, CFG920, a dual inhibitor of CYP17 and CYP11B2, is in clinical phase II trials , 26. Galin vitro and in vivo functional assays. The AR binding activity of Zeta55 was slightly weaker than MDV3100, indicating that substituting a methyl group with a hydroxyl group affects the binding affinity to AR. Meanwhile, Zeta55 has less potent HDAC6 inhibition than SAHA, with an IC50 of 0.98 μM. However, combining these two suboptimal activities appears to have a synergistic effect on inhibiting the cell proliferation in prostate cancer. In the cell proliferation assays of VCaP cells, with 60% AR binding affinity of MDV3100, Zeta55 exhibits 4.5-fold stronger anti-proliferation activity than MDV3100.Coincident with the design strategy, Zeta55 inhibits both AR and HDAC6 activities in both The potent anti-proliferation activity of Zeta55 could be attributed to its unique combination of multiple mechanisms . First, Previous study reported that MDV3100 has an oral bioavailability of 97% and a plasma half-life of 8.56 hours in rats . Our phain vitro and in vivo studies of CRPC. Zeta55, with its unique and novel multitarget activities, is suitable for future clinical development in CRPC treatment.In summary, our findings show that Zeta55 is more efficacious than MDV3100 in nonclinical in vitro assays. For in vivo studies in the mouse model, Zeta55 and MDV3100 were dissolved in a formulation of 1% carboxymethyl cellulose, 0.1% Tween-80 and 5% DMSO.Zeta55 and MDV3100 were synthesized in laboratory. SAHA was purchased from ApexBio Technology, and dihydrotestosterone (DHT) was purchased from Sigma-Aldrich. All compounds were dissolved in dimethyl sulfoxide (DMSO) for VCaP, LNCaP, DU145 and HEK293 cells were obtained from the Cell Bank of the Chinese Academy of Sciences  with STR Authentication. For subculturing, VCaP cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and digested by phenol-red-free TrypLE Express (Gibco). For AR antagonist or agonist assays, VCaP cells were cultured in steroid-free assay medium with or without 10 nM DHT. The steroid-free assay medium consisted of phenol-red-free DMEM (Gibco) and 5% charcoal-stripped fetal bovine serum (HyClone). LNCaP, DU145 and HEK293 cells were cultured in DMEM with 10% FBS.Computational docking was performed to predict the binding of Zeta55 to AR and HDAC6. The chemical structure of Zeta55 was drawn using ChemDraw and the 3D coordinate was generated in PRODRG . AutoDocVCaP cells were seeded in 96-well plates and cultured in steroid-free assay medium for 24 hours. After adherence, VCaP cells were transfected with MMTV-LUC using Lipofectamine 3000 (Invitrogen) for 24 hours and then treated with Zeta55 or MDV3100 together with 10 nM DHT for AR antagonist assays or without DHT for AR agonist assays. After 48 hours, the cell extracts were tested for luciferase activity using a Steady-Lumi™ Firefly Luciferase Assay Kit (Beyotime Biotechnology).Commercial fluorescent assay kits were used to measure the activities of HDAC (BPS Bioscience). Briefly, the tested compounds were diluted in DMSO into a 96-well plate, and then assay buffer, HDAC, and substrate were added. The plate was incubated at 37° C for 30 minutes for enzymatic reactions, and the developer was added. After 10 minutes of incubation, the generated fluorescence was measured at an excitation of 380 nm and an emission of 460 nm.To measure AR-mediated cell proliferation, 10,000 VCaP cells (AR positive prostate cancer cells) were plated in each well of a 96-well plate and incubated in steroid-free assay medium for 12 hours. After adherence, VCaP cells were treated with increasing concentrations of Zeta55, MDV3100 or SAHA together with 10 nM DHT for AR antagonist assays or without DHT for AR agonist assays. After 7 days, cell viability was measured using a CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega). 8,000 LNCaP cells, 5,000 DU145 cells (AR negative prostate cancer cells) or HEK293 cells (AR negative noncancerous cells) per well were cultured in DMEM with 10% FBS for 12 hours. After treatment with increasing concentrations of Zeta55, MDV3100 or SAHA for 3 days, the cell viability was measured using a CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega).The RNA of VCaP cells was extracted using a Simply P Total RNA Extraction kit (Bioer technology) and cDNA was synthesized using a SMARTer PCR cDNA Synthesis Kit (Clontech). PSA and β-actin primers were synthesized by Sangon Biotech according to previous literature . Real-tiThe whole-cell extracts were prepared in lysis buffer with a protease inhibitor cocktail (Bimake), while the xenograft tissue extracts were prepared in 0.5% Nonidet P 40 Substitute solution (Biosharp) with cocktail protease inhibitor (Bimake). Each sample containing 30 μg of total protein was loaded onto 10% SDS-PAGE and electrotransferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skimmed milk in PBST at room temperature for 2 hours and incubated with primary antibodies at 4° C overnight. After the membranes were washed with PBST three times, they were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody at room temperature for 1 hour. Chemiluminescent detection was performed with WesternBright ECL (Advansta). The immunoblots were visualized using the FluorChem FC3 system (ProteinSimple). Antibodies: AR , PSA , acetylated α-tubulin , and α-tubulin .6 cells/well) were seeded on coverslips in 6-well plates and treated by Zeta55 (10 μM), MDV3100 (10μM) or DMSO for 24 hours. Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with immunostaining permeate for 10 minutes and blocked with 5% BSA containing 1% Tween-20 for 1 hour. After overnight Immunostain with AR Rabbit monoclonal antibody  at 4° C, a Cy3 conjugated Goat Anti-Rabbit IgG  reacted for 1 hour. DAPI  stained the nuclear to bule for 10 minutes in the dark. Washing three times with PBS between each step. Immunofluorescence images were acquired by fluorescence microscopy using a NIKON ECLIPSE CI microscope .VCaP cells , all NOD-SCID mice were castrated under anesthesia. When the xenografts resumed growth after 21 days of recovery, the castrated mice were randomly divided into four groups (n=6) and were orally treated with vehicle, MDV3100  or Zeta55  for 49 days. VCaP xenografts were measured twice per week in two dimensions (L and W) by a Vernier caliper, and the tumor volume was calculated as W2×L/2 (mm3).The animal protocols used in this study were evaluated and approved by the Xiangya Hospital Medical ethics committee of Central South University. NOD-SCID mice were purchased from Hunan SJA Laboratory Animal Co. Ltd. with a quality license and raised in the SPF Laboratory Animal Room of the Department of Laboratory Zoology at Central South University. Briefly, VCaP cells were suspended in DMEM and Matrigel (BD) (1:1), and 5×10Formalin-fixed, paraffin-embedded xenograft tissue sections were used for immunohistochemistry. TUNEL staining was performed according to the instructions in the TUNEL kit (KeyGEN). Tissue sections were deparaffinized and then boiled in 0.01 mol/L sodium citrate buffer (pH 6.0) in a kilowatt microwave oven for 10 minutes to retrieve cell antigens. Ki67  or AR  was used as the primary antibody. All tissue sections were immunohistochemically stained using the avidin-biotin-peroxidase method and were counterstained with hematoxylin. The staining intensities were evaluated at 100× magnification.0-t, AUC0-∞, t1/2, Tmax, Vz/F, and Cl/F were analyzed by Phoenix winnonlin 8.0.SD male rats weighing about 250 g were used for the pharmacokinetics study of Zeta55. After a fast of 12 hours, four rats were oral administrated with 20 mg/kg Zeta55 and one rat was tail vein injected with 5 mg/kg Zeta55. Plasma samples of 0.2 ml were taken at 0, 0.083, 0.25, 0.5, 1, 2, 3, 5, 8, 10, 24, 48, and 72 hours after administration and then measured by LC-MS/MS. AUCSupplementary Figures


The putative signal transduction systems of A2A-AR in cells in the living heart, as well as the known interactions of A2A-AR with membrane-bound receptors, will be addressed. The possible role that the receptors play in some relevant cardiac pathologies, such as persistent or transient ischemia, hypoxia, sepsis, hypertension, cardiac hypertrophy, and arrhythmias, will be reviewed. Moreover, the cardiac utility of A2A-AR as therapeutic targets for agonistic and antagonistic drugs will be discussed. Gaps in our knowledge about the cardiac function of A2A-AR and future research needs will be identified and formulated.This review presents an overview of cardiac A The present work attempts to close this gap in the literature.There have been many reviews on adenosine receptors (AR), specifically A2A-AR . HoweverIn their pioneering work on the pharmacology of adenosine in the heart, 1, A2A, A2B, and A3. In general, A1-AR and A3-AR inhibit adenylyl cyclase, while A2A-AR and A2B-AR stimulate adenylyl cyclase activities in the heart  in peripheral tissues   levels, stimulated cAMP-dependent protein kinase (PKA) and phosphorylated cAMP response element-binding protein (CREB)  . Due to n (CREB) , and actn (CREB) , and p38n (CREB) .2A-AR can also exert inhibitory effects in signal transduction; for example, it can inhibit thrombin-induced ERK1/2 phosphorylation  mice with a cardiac overexpression of A2A-AR, but not in WT mice (2+ can be released from the sarcoplasmic reticulum (SR) because activation of the A2A-AR via PKA increases the phosphorylation state of the cardiac ryanodine receptor (RYR2), which opens the RYR2  receptors and cannabinoid receptors  of isoproterenol, which is a β-adrenoceptor agonist. This well-known effect was attenuated in perfused rat hearts through the additional stimulation of the A2A-AR   .2A-AR might be relevant for both primary arrhythmias due to inborn errors and secondary arrhythmias from ischemia, hypertrophy, drug treatment, or aging. However, more work on this topic needs to be undertaken.There are several types of cardiomyocytes in the heart: atrial cardiomyocytes form the main bulk of atrial muscle; ventricular cardiomyocytes; cardiomyocytes that form the path of the system that propagates depolarization from the sinus node, specialized cells in the atrium (Bachmann bundles), the sinus node (SA) node, Tawara branches, and Purkinje bundles . Alterat2A-AR in the heart, these receptors are known to be present and functional in blood cells that are continuously transported to and from the heart by the circulatory system  pathway  in neuroblastoma cells , which can lead to cellular depolarization, initiate afterdepolarizations, and cause cardiac arrhythmias  and lead to arrhythmias  in the mammalian heart; the effect of A2A-AR may also be species- or method-dependent. Studies have reported a lack of effect in the rat  mouse hearts clearly established that the A2A-AR agonist CGS21680 was selective; <1 µM CGS21680 increased contractility in WT mouse hearts, but not in hearts from A2A-AR KO mice , as the A2A-AR will increase cAMP and cAMP increase can lead to arrhythmias. Another caveat is in order: to the best of our knowledge, it has not been reported that A2A-AR agonist can increase force of contraction in the human ventricle, though this is usually taken for granted  and might be used in the future as positive inotropic agents. A2A-AR antagonist might become useful as novel options in some subtypes of heart failure.The AJN initiated the project. BH, NZ, and UG added own topics and aspects. UG, PB, and JN finalized the figures and body text. All authors read and approved submission of this version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Castration-resistant prostate cancer (CRPC) remains prostate cancer research and treatment bottleneck. Abnormal androgen receptor (AR) activation still has a pivotal role in CRPC. Multiple mechanisms involve the process, of which overabundant AR-V7 mRNA splicing production is currently focused and increasingly studied. However, factually, there is no definite conclusion about regulation of AR-V7 mRNA splicing. Recently developed knowledge has demonstrated that JMJD6 and U2AF65 as a hopeful approach in mRNA splicing regulation. The authors propose a novel possible mechanism elucidating AR mRNA splicing for CRPC progression using dual-function enzyme JMJD6 and its induced JMJD6/U2AF65/AR-V7 axis. In this hypothesis JMJD6 introduces to AR promoter to demethylate H3R or H4R and promotes AR mRNA transcription via its demethylase activity and interaction with U2AF65. It is expected that JMJD6 could further effectively perform U2AF65 hydroxylation to achieve AR-V7 mRNA splicing via its hydroxylase activity. Prostate cancer has become one of the most frequently diagnosed cancers of men in the western countries and the second-leading cause of male cancer death. Androgen-deprivation therapy (ADT), as the mainstay and milestone of PC treatment, remains standard selection for both advanced and metastatic prostate cancer patients. Unfortunately, although most patients are effective to ADT in early-stage, almost all of PC finally develop from androgen-dependent prostate cancer to castration-resistant prostate cancer (CRPC) after a median-survival time.A panel of studies have confirmed that the androgen receptor (AR) still has a crucial role even in CRPC. Numerous mechanisms have been proposed to involve in the role of AR in CRPC progression, containing generation of truncated AR mutations and spliceosomes such as AR-V4, AR-V7, ARv567s, AR-V9 , 2, locaU2AF65, as the important component of U2 mRNA spliceosome, used to assist U2 performing mRNA splicing by recognizing and binding with splicing identification sequence GU-AG. In aspects of molecular structure, U2AF65 is composed of an N-terminal arginine/serine-rich domain, two RNA-recognition motifs and C-terminal noncanonical RNA-recognition motifs , 5. ThesJMJD6 is upregulated and over-activated in a series of cancers including breast, lung, neuroglioma and melanoma . PreviouAltered splicing machinery based on RNA-binding protein splicing factor proline- and glutamine-rich results in a dysregulated AR splicing process and the expression of AR transcript variants including AR-V7. AR-V7 mRNA production requires the removal of alternative intron sequences via special RNA site recognition. The spliceosome excises the AR mRNA 3′ SS and ligates exon 3 with special exon 3B. Among these splicers, the recruitment of U2AF65 to the 3′ splice site of AR-V7 enhanced by ADT is of vital importance for increased expression of AR-V7 , 15. ColJMJD6, as dual-function regulators of both arginine demethylase and lysine hydroxylase, can regulate the alternative splicing through U2AF65 hydroxylation, suggesting the possibility of JMJD6 as splicing factors. Accordingly, the knock-down of JMJD6 in HEK-293T cells resulted in a significant change of the outcome for 14% of all alternative splicing events (n = 3710), and 74% of those were co-regulated with U2AF65 . CombineWe hypothesized that JMJD6 can play the role of dual-functional enzyme in initiating AR mRNA transcription activation by demethylating histone in the manner of its demethylase activity and further promoting alternative AR-V7 mRNA splicing by hydroxylating and activating U2AF65 in the manner of its hydrolase activity. Upregulated JMJD6 usually progressively induce AR mRNA transcription, U2AF65 activation and ultimately special AR-V7 mRNA splicing. Therefore, the JMJD6/U2AF65/AR-V7 axis was established and it plays important regulatory roles in CRPC progression. We evaluated the current evidence supporting this hypothesis in the following part.According to our bioinformatic analysis from TCGA database, JMJD6 has upregulated expression in prostate cancer patient samples, and shows co-expression and parallel upregulation with AR-V7 Figs.  and 2. TRecent studies specially focus on AR splicing regulation including U2AF65 as one of representatives. Elevated U2AF65 could significantly increase AR-V7 protein expression but no effect on steroidogenesis gene AKR1C3, indicating that AR gene splicing is highly active and plays important role in CRPC. Under ADT conditions, recruitment of RNA splicing factors to the 3′ splicing site of AR-V7 was increased. U2AF65 and ASF/SF2 binding with two kinds of RNA splicing enhancers called intronic splicing enhancer (ISE) and exonic splicing enhancer (ESE) near AR-V7 splicing domain, 3′ss of exon 3B, were identified to play critical roles in AR pre-mRNA splicing to generate AR-V7. The ISE was predicted to bind U2AF65 . AccordiJMJD6 may be a dual enzyme guiding initial AR mRNA transcription via demethylase activity and further AR mRNA splicing to AR-V7 mRNA via hydroxylating U2AF65 Fig. . The rolAccording to our hypothesis, activated U2AF65 by JMJD6 plays key roles in regulating AR-V7 splicing. Testing the hypothesis will be challenging. The first topic needed to be addressed is that JMJD6 binds with and catalyzes U2AF65, which further conducts AR-V7 splicing. The complex of JMJD6, U2AF65 and AR mRNA should be verified via IP and RNA-IP assays. Furthermore, suppression and activation of JMJD6 regulates AR-V7 expression by regulation of U2AF65 should be confirmed.Among JMJD family members, JMJD1A was reported to promote the alternative splicing of AR-V7 by JMJD1A–HNRNPF interaction mechanistically –20. JustJMJD6 includes four annotated transcripts or isoforms like JMJD6-1, JMJD6-2, JMJD6-3(JMJD6-Ex5) and JMJD6-4 due to diverse expression of exons. JMJD6-2 located in nucleoplasm and JMJD6-Ex5 in nucleolus and nuclear speckles are the most abundant variants in many human tissues and cell lines , 22. JMJCombating AR-V7 is challenging due to lack of targeting drugs and therapy resistance. Some drugs like niclosamide and ONC201 could inhibit AR-V7 signaling , 28. How


Enzalutamide (ENZ) is an important drug used to treat castration-resistant prostate cancer (CRPC), which inhibits androgen receptor (AR) signaling. Previous study showed that 3,3′-diindolylmethane (DIM) is an AR antagonist that also inhibits Wnt signaling and epithelial-mesenchymal transition (EMT). To investigate whether combined treatment with ENZ and DIM can overcome ENZ resistance by regulating Wnt signaling to inhibit AR signaling and EMT in ENZ-resistant prostate cancer cells, 22Rv1 cells were cultured in normal medium and treated with ENZ, DIM, and DIM with ENZ. Exposure of ENZ-resistant cells to both DIM and ENZ significantly inhibited cell proliferation without cytotoxicity and invasion in comparison with the control. DIM significantly increased the E-cadherin expression and inhibited the expressions of Vimentin and Fibronectin, subsequently inhibiting EMT. Co-treatment with ENZ and DIM significantly increased the expressions of GSK3β and APC and decreased the β-catenin protein expression, causing inhibition of Wnt signaling and AR expression, it also significantly decreased the AR-v7 expression and down-regulated AR signaling. Via suppression of Wnt and AR signaling, co-treatment increased the E-cadherin and decreased the Vimentin and Fibronectin RNA and protein expressions, then inhibited EMT. Co-treatment with DIM and ENZ regulated Wnt signaling to reduce not only the AR expression, but also the AR-v7 expression, indicating suppression of EMT that inhibits cancer cell proliferation, invasion and migration to ameliorate ENZ resistance. In Taiwan, the prevalence and mortality of PCa are increasing, and around 50% of patients are diagnosed with PCa at the late stage2. At the late stage, androgen deprivation therapy (ADT) is considered the standard therapy3; however, after the beginning of ADT, the duration of response to castration is short and, in almost all patients, is followed by the emergence of a castration-resistant phenotype4. Recurrence of PCa is regarded as castration-resistant prostate cancer (CRPC). It has been confirmed that CRPC is still an androgen-dependent disease that relies on androgen receptor (AR) signaling5. AR signaling can be activated through various mechanisms, such as AR overexpression, binding of alternate AR ligands, upregulation of AR coactivators and downregulation of AR corepressors, steroidogenesis, inflammation pathways, growth-factor signaling, and Wnt/β-catenin signaling6. Therefore, second-generation anti-androgen drugs, such as abiraterone and enzalutamide (ENZ), were developed as new therapies for CRPC7.Prostate cancer (PCa) is not only the second most common cancer in men; it is also the fifth leading cause of cancer mortality among men worldwide8. ENZ has been proven to increase the overall survival duration and improve quality of life of PCa patients. Owing to its safety characteristics, it has become a critical drug for use at different stages of PCa9. Although ENZ has many benefits, 20–40% of patients have primary resistance, and nearly all treated CRPC patients develop ENZ resistance eventually10. Recent evidence has shown that several mechanisms are involved in ENZ resistance, including AR mutation, the existence of AR splice variants (AR-Vs), AR and glucocorticoid receptor (GR) overexpression, autophagy, intracrine androgen biosynthesis, and epithelial-mesenchymal transition (EMT)13.ENZ, also named MDV3100, is an AR antagonist that competitively antagonizes androgen binding to AR and inhibits nuclear translocation of AR, recruitment of AR co-factors, and AR binding to DNA14. Our previous studies have shown that dysregulation of Wnt signaling is associated with hepatoma15, ovarian cancer17, and cervical cancer18, while other studies have demonstrated its involvement in prostate cancer19 and colorectal cancer20 in terms of affecting cell proliferation, invasion or migration. The two distinct pathways of Wnt signals are a canonical or Wnt/β‐catenin pathway and a noncanonical pathway or pathways that are β-catenin-independent21. The stabilization and nuclear translocation of β-catenin play crucial roles while Wnt signaling is engaged. β-catenin no longer binds to some proteins, such as adenomatous polyposis coli (APC) protein and glycogen synthase kinase‑3 (GSK3), but does bind to T-cell factor (TCF) family transcription factors, whereupon it activates target genes determining carcinogenesis, including MYC, MMP7, VEGF and AR19.Wnt proteins are cysteine-rich, secreted lipoglycoproteins that play critical roles in normal embryonic development, and are involved in a variety of biological processes, but aberrant activation of the Wnt signaling pathway has been noted in carcinogenesis22. Of several mechanisms involved in ENZ-resistant CRPC, aberrant AR signaling is a crucial one. In general, AR signaling is activated while AR is bound to androgen, and then translocates to the nucleus to bind with androgen-responsive elements (ARE) in the promoter regions of target genes6. AR-Vs have been found to translocate into the nucleus without androgen, and can form homodimers or heterodimers with androgen-bound AR23. AR-Vs may be associated with the initiation and progression of PCa. Evidence has shown that AR-V expressions are involved in EMT, which plays important roles in cancer progression and drug resistance25. Among approximately 20 different variants, increased AR-v7 expression has been identified in CRPC patients, and is related to ENZ resistance27. Moreover, interaction of AR with Wnt signaling has been suggested, and both could activate the process of EMT29. Therefore, a new adjuvant therapy might be helpful in improving the treatment of ENZ-resistant CRPC.Relatively high gene expression levels of both β-catenin and AR have been observed in CRPC32, and dietary and plant-based drugs have been suggested as possible alternative strategies in the treatment of cancer. The phytochemical indole-3-carbinol (I3C) and its metabolite 3,3′-diindolylmethane (DIM) are present in high levels in cruciferous vegetables, and have been shown to exert anticancer effects, but DIM has a greater bioactivity and is safer than I3C34. Previous studies have shown that DIM induces cell-cycle arrest in AR positive, p53 wild-type LNCaP and AR negative, p53 mutant DU145 human prostate cancer cell lines35. It also has inhibitory effects on AR signaling, Wnt signaling and the process of EMT36. Moreover, in clinical experiments, a formulated DIM  was used as an adjuvant therapy in the early stages of PCa, and a good response was observed34. However, no research has been performed to examine the effects of DIM through Wnt/β-catenin signaling in the late stages of ENZ-resistant PCa. Therefore, in the current study, we focused on AR, AR-v7 and Wnt signaling to investigate whether combined treatment with ENZ and DIM could overcome ENZ resistance by regulating Wnt signaling to inhibit AR signaling and EMT in ENZ-resistant PCa cells. We found that co-treatment with DIM and ENZ inhibited prostate cancer cell proliferation, invasion and migration through regulating Wnt signaling to reduce the expressions of AR and AR-v7.According to epidemic evidence, high intakes of cruciferous vegetables reduce the risk of PCa, especially in the early stagesp < 0.05 vs. control), and the effect of co-treatment with DIM and 10 μM ENZ was similar to that of treatment with 40 μM ENZ. Co-exposure to DIM and ENZ resulted in significant inhibition of cell proliferation .Exposure to a low dose of ENZ for 48 and 72 h had no suppressive effect on 22Rv1 cells (data not shown). It was confirmed that 22Rv1 cells exhibited ENZ resistance. Treatment of cells with DIM alone for 48 and 72 h caused significant dose- and time-dependent inhibition of cell proliferation, with 25% suppression following 72 h of exposure to 30 μM DIM. Therefore, in order to investigate the adjuvant effects of DIM on ENZ resistance, 22Rv1 cells were treated with DIM and ENZ simultaneously, and the results showed that treatment of 22Rv1 cells with 30 μM DIM alone for 48 and 72 h significantly decreased the cell viability , while there was no suppression when cells were treated with 10 μM ENZ. In cells treated with a high concentration of ENZ, colony formation was significantly inhibited , and the inhibition was of the same magnitude as that caused by treatment with 30 μM DIM. Combined DIM and ENZ treatment significantly increased the inhibition of colony formation . The results of the present study indicated that ENZ and DIM may have a synergistic effect in suppressing cell proliferation and colony formation in PCa cells.In 22Rv1 cells, treatment with DIM alone significantly inhibited the clonogenic ability , but DIM had a better inhibition effect . In addition, exposure to DIM and ENZ in combination significantly increased the efficacy of ENZ in suppressing cell invasion . The results of the present study indicated that DIM may potentiate the efficacy of ENZ with regards to invasion of 22Rv1 cells.In order to examine whether combined treatment with DIM and ENZ resulted in stronger inhibition of invasion in PCa cells, 22Rv1 cells were treated with DIM and ENZ alone and together for 24 h. The results illustrated that both DIM and ENZ significantly inhibited cell invasion . Treatment with ENZ (10 and 40 μM) and DIM combined resulted in a stronger inhibition effect (p < 0.05 vs. control and 10 μM ENZ). Furthermore, as shown in Fig. p < 0.05 vs. control and ENZ alone). The results showed that combined treatment with DIM and ENZ resulted in better suppression of migration in PCa cells after 24 h. Therefore, a wound-healing assay was employed to detect cell migration at 48 and 72 h. Exposure to DIM and ENZ alone did not suppress migration of 22Rv1 cells . The results of the present study indicated that co-treatment with DIM and ENZ resulted in better suppression of migration of PCa cells.To investigate whether combined treatment with DIM and ENZ inhibited PCa cell migration, a wound-healing assay and a Transwell migration assay were employed. As shown in Fig. Z 10 and 0 μM and Z 10 and 0 μM and p < 0.05 vs. control). Compared with treatment of 22Rv1 cells with 10 μM ENZ alone, co-treatment with ENZ and DIM significantly upregulated the level of GSK3β and decreased the expression of β-catenin .Wnt signaling is an important mechanism in CRPC. In order to elucidate whether co-treatment with DIM and ENZ inhibits Wnt signaling, western blot analysis was employed to detect the expressions of β-catenin, APC, and GSK3β, which play important roles in Wnt signaling. After 48 h of treatment, exposure to DIM and ENZ in combination significantly increased the expressions of APC and GSK3β, and downregulated the expression of β-catenin , but there was no significant difference in the expression of β-catenin . The results demonstrated that combined treatment with DIM and ENZ may inhibit Wnt signaling.After 72 h, combined treatment with DIM and ENZ significantly increased the expressions of APC and GSK3β ; however, combined treatment with DIM and 40 μM ENZ significantly decreased the level of AR . After 72 h of treatment, exposure to DIM and 40 μM ENZ significantly downregulated the AR expression .Aberrant AR signaling is considered a critical mechanism in CRPC. Moreover, a mechanism of cross-talk between Wnt and AR signaling has been identified. Therefore, to elucidate whether AR signaling is inhibited when Wnt signaling is inhibited, western blot analysis was employed to detect the expression of AR. After 48 h of treatment, 40 μM ENZ significantly increased the expression of AR . Compared with treatment of 22Rv1 cells with 10 μM ENZ alone, co-treatment with ENZ and DIM significantly downregulated the level of AR-v7 . After 72 h of treatment, 40 μM ENZ and combined treatment with DIM and ENZ significantly decreased the AR-v7 expression . The results indicated that co-treatment with DIM and ENZ may inhibit AR signaling by downregulating the expressions of AR and AR-v7.Besides AR overexpression, a high AR-v7 expression has been found in ENZ-resistant PCa, and AR-v7 may also activate AR signaling. Therefore, to examine the effect of DIM on AR-v7 expression, western blot analysis was used to detect the protein expression of AR-v7 in 22Rv1 cells. After 48 h of treatment, exposure to ENZ alone or in combination with DIM significantly decreased the AR-v7 expression . On the other hand, cells treated with DIM alone and co-treated with DIM and ENZ exhibited significantly decreased expressions of FN1 and VIM, but cells treated with 10 μM ENZ alone exhibited a significantly increased expression of FN1 . Exposure to 40 μM ENZ significantly downregulated the level of FN1 but increased the expression of VIM . Compared with treatment of 22Rv1 cells with 10 μM ENZ alone, co-treatment with ENZ and DIM significantly upregulated the level of CDH1 and downregulated the levels of FN1 and VIM .The process of EMT plays a pivotal role in CRPC and is related to drug resistance. Moreover, Wnt signaling and AR signaling activate EMT in CRPC. To investigate the role of EMT in CRPC, qPCR was performed to examine the mRNA expressions of an epithelial marker  and mesenchymal markers . After treatment for 48 h, exposure to DIM or ENZ alone, and combined treatment with DIM and ENZ, significantly upregulated the expression of CDH1, but cells treated with ENZ alone exhibited a significantly decreased level of CDH1 . Exposure to DIM or ENZ alone, and co-treatment with DIM and ENZ, significantly inhibited the FN1 expression, but only ENZ treatment and combined treatment decreased the VIM expression . Compared with treatment of 22Rv1 cells with 10 μM ENZ alone, co-treatment with ENZ and DIM significantly upregulated the level of CDH1 and downregulated the levels of FN1 and VIM .After treatment for 72 h, exposure to DIM alone and combined treatment with DIM and ENZ significantly upregulated the expression of p < 0.05 vs. control). In contrast, cells treated with DIM alone and co-treated with DIM and 40 μM ENZ had significantly decreased expressions of FIN and VIM . Compared with treatment of 22Rv1 cells with 10 μM ENZ alone, co-treatment with DIM and ENZ significantly downregulated the expression of FIN .Furthermore, western blot analysis was employed to detect the protein expressions of E-cadherin (E-cad), Fibronectin (FIN) and Vimentin (VIM). After treatment for 48 h Fig. A, exposup < 0.05 vs. control). Exposure to DIM and 40 μM ENZ alone, and co-treatment with DIM and ENZ, significantly inhibited the FIN expression, but only combined treatment decreased the VIM expression . Compared with treatment of 22Rv1 cells with 10 μM ENZ alone, co-treatment with ENZ and DIM significantly decreased the expressions of FIN and VIM . The results revealed that co-treatment with DIM and ENZ possibly inhibits EMT and improves drug resistance.After treatment for 72 h Fig. A, exposup < 0.05 vs. control). The results showed that ENZ and DIM treatment may not enhance cell cytotoxicity.To ascertain whether combined treatment with DIM and ENZ had a cytotoxic effect on PCa cells, 22Rv1 cells were treated with DIM and ENZ alone and together for 48 and 72 h. The results showed that treatment with DIM for 48 and 72 h significantly inhibited PARP and c-Caspase 3, respectively, and treatment of 22Rv1 cells with DIM and ENZ together for 48 and 72 h significantly decreased the levels of PARP, c-PARP, Caspase 3 and c-Caspase 3  did not significantly inhibit cell proliferation, proving that 22Rv1 is ENZ-resistant7. ENZ is one of the newer and potentially more effective AR-targeting agents49; however, the response to ENZ is not permanent, and almost all patients eventually develop resistance to ENZ, demonstrating that targeting AR signaling alone is not sufficient for CRPC therapy10. Lee et al.50 revealed that Wnt signaling might be active in PCa cells after ADT, partly due to enforcement of interaction between β-catenin and TCF. In androgen-independent PCa cell lines, the combination of a GSK3β inhibitor or APC knockdown with ENZ increases growth inhibition through repressing both AR signaling and Wnt signaling50. In addition, overexpressions of AR and CTNNB1 (β-catenin) were found in ENZ-resistant PCa cells, and the combination of β-catenin inhibitor ICG001 with ENZ improved ENZ resistance22. A previous study indicated that exposure of LNCaP and C4-2B PCa cells to 50 μM BR-DIM decreased the phosphorylation of GSK3β, and then inhibited nucleus translocation of β-catenin; in addition, it caused FOXO3a to bind to the p27kip1 promoter rather than the AR promoter51. Leem et al.52 showed that treatment of colon cancer cells with DIM downregulated the expressions of β-catenin, Myc and FOS, which are related to cancer prevention and prognosis. In gastric cancer cells, treatment with different concentrations of DIM produces contrary results. A high level of DIM (30 μM) resulted in inhibition of cell proliferation, but a low level (1 and 10 μM) activated Wnt4 signaling, which enhanced the progression of gastric cancer53. In the present study, we decided to use a high level of DIM to avoid any adverse influence, and our results showed that DIM did not significantly inhibit Wnt signaling, but combined treatment with DIM and ENZ significantly inhibited Wnt signaling through upregulating the expressions of APC and GSK3β and downregulating the β-catenin expression.In the clinical treatment of PCa, repressing AR signaling is a critical strategy28. However, in PCa, the increased level of β-catenin leads to the formation of β-catenin–AR complexes, and the formation of β-catenin–TCF complexes is preferred under the condition of absence of androgen, which express target genes, including AR, c-myc, MMP7, and VEGF28. DIM has been proven to be a strong androgen antagonist that is a competitive inhibitor of DHT binding to AR54. DIM binds to AR directly, and then inhibits nucleus translocation of AR and transcription activation54. However, Palomera-Sanchez et al.55 treated LNCaP cells with DIM for 48 h, and the results showed that DIM inhibited the protein expression of AR, but increased the mRNA expression of AR. Although the mRNA expression of AR increased, chromatin modifications at its promoter region had no significant effects55. In our study, DIM downregulated the protein expression of AR, but upregulated its mRNA expression (data not shown), consistent with previous studies. DIM treatment might cause AR turnover by inducing AR-protein instability. Several studies have revealed that AR overexpression and increased expressions of AR-Vs alleviate the efficacy of ENZ and induce progression of PCa57. Of the variants, AR-v7 is the most common in patients with ENZ resistance, and activates ligand-independent AR signaling60. Ong et al.61 showed that exposure of PCa cells to BR-DIM for 96 h significantly downregulated the AR-v7 mRNA expression. However, DIM did not significantly decrease the level of AR-v7 protein expression in this study. Although the effects of DIM on AR-v7 expression were not significant, combined DIM and ENZ treatment significantly inhibited the AR-v7 expression.In addition, there is a connection between AR signaling and Wnt signaling. In normal prostate epithelial cells, androgen modulates the activation of AR signaling62. There are several mechanisms, including AR signaling and Wnt signaling, that activate the process of EMT62. Numerous molecular processes mediate the process of EMT, which is associated with loss of epithelial factors  and gain of mesenchymal factors 24. In normal prostate tissue, canonical AR signaling suppresses EMT24; however, a previous study showed that androgen deprivation increased the expressions of AR and AR-Vs, and upregulated EMT markers , which caused therapy resistance63. Li et al.63 reported that BR-DIM increased the E-cadherin expression and decreased the vimentin expression by inhibiting miR-92a expression, and then suppressed the process of EMT. In addition, miR-34a, miR-27b, miR-320m, miR-200 and let-7 were re-expressed by BR-DIM, which then repressed the expressions of AR, AR-Vs and EMT marker64. In the present study, DIM increased the E-cadherin expression and decreased the expressions of Fibronectin and Vimentin, then inhibited the process of EMT, which was consistent with the results of previous studies. Compared with treatment with ENZ alone, combined treatment with DIM and EMT resulted in stronger inhibition of EMT, which confirmed that DIM may mediate ENZ resistance by inhibiting EMT.Recent studies have indicated that EMT is involved in PCa progression, migration and therapy resistance, but the effects of ADT on EMT are still unclear65. Therefore, in order to enhance bioavailability, a formulation of DIM (BR-DIM) was developed to contain small particles of DIM within a water-soluble matrix, containing α-tocopherol polyethylene glycol succinate and phosphatidyl choline, by solubility-enhancing microencapsulation technology66. Anderton et al. administered DIM and BR-DIM to mice and then analyzed the concentration of DIM in the plasma and liver, kidney and lung tissues. The results showed that BR-DIM exhibited an approximate 50% higher bioavailability than DIM66. Moreover, in clinical studies, adjuvant intervention in the early stage of PCa has been performed using BR-DIM, and was proven to be an effective method by which to improve the therapeutic outcome34.Although our results demonstrated that DIM could inhibit both Wnt signaling and AR signaling, the inhibition effects were not as obvious as those reported in previous studies, which might be because most of those studies used BR-DIM, rather than DIM. DIM is stable under acidic conditions, but the administration of DIM to female Sprague–Dawley rats by the i.p. route has better effects than the oral routeIn our study, DIM was effective in ameliorating carcinogenesis through modulating AR signaling, Wnt signaling and EMT in ENZ-resistant cells. The findings may have several implications for the use of DIM as a dietary supplement or a therapeutic agent. The results only relate to patients who have already exhibited ENZ resistance, and in future study, cells could be treated with ENZ for a long duration in order to build a model of long-term ENZ resistance to solve the clinical problem. Furthermore, transcriptional activation of ARE and TCF directly influences AR and Wnt signaling, and can therefore be analyzed to confirm the results in future study.Combined treatment with DIM and ENZ significantly inhibited the proliferation, invasion and migration of 22Rv1 cells. DIM could regulate Wnt signaling to inhibit the recurrence of AR signaling by reducing the expressions of AR and AR-v7. In addition, inhibition of these two signaling pathways is related to suppression of the EMT process. Our results demonstrated that treatment with DIM, or a combination of DIM and ENZ, could potentially represent other ADTs.2 at 37 °C. DIM  and ENZ  were dissolved in dimethyl sulfoxide (DMSO) and diluted in medium before use.22Rv1 (ATCC CRL-2505) is an androgen-independent PCa cell line that expresses AR and AR-v7, and is often used as a CRPC model. Cells were cultured in RPMI 1640  supplemented with 10% fetal bovine serum (FBS) , sodium pyruvate and 0.2% penicillin/streptomycin  in a humidified incubator containing 5% CO4 cells per well in 96-well plates and grown for 24 h, following which different concentrations of DIM and ENZ were added and incubated for 48 and 72 h. Viable concentrations were tested using a CellTiter 96 Aqueous One Solution Cell Proliferation Assay . Cell viability was analyzed from absorbance readings at 490 nm.Cells were seeded at 5 × 103 cells were mixed with 0.7% melting agar , and the mixtures were then placed on a solidified layer of 0.5% agar made from 1% melting agar, 2X RPMI and 20% FBS in a 6-well plate. The plate was incubated for 24 h, and 2 ml RPMI with different concentrations of DIM and ENZ were added. Fresh medium was added every 7 days during the different treatments. After incubation for 28 days, colonies were stained with crystal violet for 1 h, then captured and counted using Cell3iMager Neo .The anchorage independent growth (AIG) assay of colony formation is performed in soft agar, and detects malignant transformation of cells. 7 × 106 cells were seeded onto the insert. After the cells had grown in the medium until approximate confluence, the culture insert was removed and washed with PBS. Under varying conditions, different treatments were added to the well, and microscopy was employed to capture images at different time points. Images were analyzed using Image J .A wound-healing assay was employed to evaluate cell migration using a culture insert . First, the culture insert was placed on a 24-well plate, and 105 and 105 for the invasion and migration assays, respectively, were resuspended in 300 μL serum-free medium with different treatments and then added to the upper chamber. Then, 800 μL medium containing 10% FBS were added to the lower chamber. After 24 h of incubation, cells were fixed with 1% paraformaldehyde at 4 °C for 24 h and then stained with Giemsa  for 2 min. Images were captured using a microscope, and invaded cells were counted using Image J 68.Cell migration and invasion assays were performed using Transwell chambers with an 8.0-µm-pore PET membrane . Transwell chambers were coated with 15 μL Matrigel  for the invasion assay, while uncoated chambers were used for the migration assay. Cells at densities of 2 × 10−ΔΔCt method. The gene expression levels were normalized to GAPDH, and the results are presented as the relative fold change compared with the control. The sequences of the primers used are presented in Supplementary Table 167.Cells were treated with different concentrations of DIM and ENZ for 48 and 72 h, and then total RNA was extracted using a RNeasy Plus Mini Kit . cDNA was generated from 2 μg of total RNA using SuperScript III Reverse Transcriptase . qPCR analysis was performed using 2X SYBR Green I master on a LightCycler 480 system . The data were analyzed using the 269.After treatment with different concentrations of DIM and ENZ for 48 and 72 h, cells were washed with PBS three times and then lysed in RIPA buffer , protease inhibitor and phosphatase inhibitor. The lysates were cleared by centrifugation at 4 °C for 30 min at 14,000 rpm, and the concentrations of proteins were measured using a BCA protein assay kit . Equal amounts of the proteins were loaded per well, then separated using 8% SDS-PAGE gels and transferred onto polyvinyl difluoride (PVDF) membranes . The PVDF membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated with primary antibodies  with Duncan’s multiple range test to determine significant differences between groups. These analyses were performed using SAS 9.4 software . Supplementary Figures.Supplementary Tables.