Allergic rhinitis (AR) and rhinosinusitis (RS) negatively impact psychological well-being and health-related quality of life (HRQoL). However, few population-based studies have investigated the effects of these conditions on mental health and HRQoL.To explore independent associations of AR and/or RS with mental health and HRQoL using data from the 2013–2015 Korea National Health and Nutrition Examination Survey (KNHANES).The KNHANES is a nationwide cross-sectional survey of the non-institutionalized population of Korea. A total of 15,441 adults completed the clinical examination and the health questionnaire. We divided all participants into four groups: AR-/RS-, AR-/RS+, AR+/RS-, and AR+/RS+. Logistic regression analyses were performed after adjustment for sociodemographic characteristics, general health behaviors, and other comorbidities.The AR+/RS+ group contained the highest proportion of subjects with perceived stress and depressed mood. Subjects with AR+/RS+ also had more frequent problems in terms of pain/discomfort and anxiety/depression. After adjusting for all confounders, the odds ratios (ORs) were 2.96 (p = 0.009) for depressed mood and 3.17 (p = 0.013) for suicidal ideation in the AR+/RS+ group compared with in the AR-/RS- group. The AR+/RS- group reported more perceived stress  and depression  compared with the AR-/RS- group. In terms of the ORs for HRQoL, the AR+/RS+ group reported more problems in terms of self-care  and more pain/discomfort  compared with the AR-/RS- group.In the Korean population, AR and RS exerted a synergistic negative impact on mental health and HRQoL, especially suicidal ideation. Most patients seek help from clinicians for impaired HRQoL. Therefore, clinicians should consider the underlying mental health and HRQoL of patients with AR and/or RS, as these may be impaired by their conditions. Clinicians evaluate the health problems of patients from the viewpoint of anatomical/functional impairment or disability/handicap. The latter aspect of health-related problems is difficult to qualify but is directly related to health and life. Therefore, the World Health Organization Quality of Life (WHOQOL) Group has defined quality of life (QoL) as an individual’s perception of his/her position in life in the context of the culture and value systems in which that individual lives and as it relates to that individual’s goals, expectations, standards, and concerns . This coAllergic rhinitis (AR) is an IgE-mediated inflammatory disease of the nasal mucosa. AR is one of the most common and burdensome medical conditions in many countries. AR affects 10–40% of the population worldwide, and its prevalence has recently rapidly increased. The socioeconomic burden imposed by AR is significant, as AR increases both direct and indirect costs, burdening hospitals and compromising work and academic performance –9.Rhinosinusitis (RS) is an inflammation of the nose and the paranasal sinuses. RS is categorized according to disease duration as acute rhinosinusitis (ARS) or chronic rhinosinusitis (CRS) . CRS is Many clinicians report that they frequently encounter AR and RS in the same patients but cannot tell which condition is more problematic. Although a mutual relationship may exist between AR and RS, little evidence supports this hypothesis. Additionally, the co-existence of AR and RS is associated with more severe symptoms, compromising psychological health and QoL –21. HoweThe KNHANES is a nationwide, population-based, cross-sectional health examination and survey that has been conducted by the Division of Chronic Disease Surveillance under the auspices of the Korea Centers for Disease Control and Prevention (KCDC) since 1998. The KNHANES was designed to assess the health and nutritional status of Koreans, to monitor trends in health risk factors and the prevalence of major chronic diseases, and to provide data allowing the development and evaluation of health policies and programs in Korea. The target population is non-institutionalized Korean citizens residing in Korea. The survey consists of three components; a health interview, a nutritional survey, and a health examination survey. The health interview and health examination are performed by trained medical staff and interviewers in mobile examination centers. The nutritional survey is conducted in the homes of subjects 1 week after the health interview.We used data collected during the 2013–2015 KNHANES. Of the 18,034 adults (> 19 years of age) who participated in the survey, 15,441 completed both the clinical examination and the health questionnaire. All subjects provided written informed consent for use of their data prior to survey commencement. The study was approved by the Institutional Review Board of the Korea Centers for Disease Control and Prevention.All subjects were asked about their AR and RS histories. AR was addressed with the following question: “Have you ever been diagnosed with allergic rhinitis by a doctor?” Subjects who answered “yes” were considered to have AR. RS was addressed with a similar question: “Have you ever been diagnosed with rhinosinusitis by a doctor?” Subjects who answered “yes” were considered to have RS. We divided all subjects into the following four groups: AR-/RS-, AR-/RS+, AR+/RS-, and AR+/RS+.The mental health survey included three dimensions: perceived stress, depressed mood, and suicidal ideation. Each subject reported the level of stress experienced as none, mild, moderate, or severe. Subjects with moderate-to-severe stress were considered to exhibit stress. Each subject answered the following question to assess depressed mood: “Have you ever felt sad or despair for 2 consecutive weeks during the past year such that your daily life was hindered?” Subjects who answered “yes” were considered to exhibit a depressed mood. To assess suicidal ideation, each subject was asked the following question: “Have you ever thought about committing suicide in the last year?” Subjects who answered “yes” were considered to exhibit suicidal ideation.The EuroQoL 5-dimension (EQ-5D) is a brief, self-report, standardized, non-disease-specific instrument used to measure HRQoL along the following five dimensions: mobility, self-care, ability to perform usual activities, pain/discomfort, and anxiety/depression. Each dimension has three levels: no problems, some problems, and extreme problems. All subjects were asked to indicate their health status by checking the box next to the most appropriate statement in each of the five dimensions , 23. Sub2). Obesity was defined as a BMI ≥ 25 kg/ m2.During the health interviews, data were collected using self-report questionnaires. The health interview evaluated educational level, occupation, household income, marital status, residential area, smoking status, alcohol consumption, exercise status, previous and present diseases, activity limitations, sleep duration, and subjective health. Smoking history was categorized as current smoker, ex-smoker, or nonsmoker. Alcohol use was based on the amount of alcohol consumed per day during the month before the interview. Male subjects who drank seven or more glasses of alcohol more than twice per week and female subjects who drank five or more glasses more than twice per week were categorized as heavy drinkers. Regular exercise was defined as moderate physical activity performed for at least 30 minutes at a time at least five times a week. Height, weight, and waist circumference were measured by well-trained medical professionals. The body mass index (BMI) was the body weight (kg) divided by the height squared  to reflect the complex sampling design and sampling weights of the KNHANES and to provide nationally representative prevalence estimates. Results are presented as percentages ± standard errors for categorical variables and as estimated means ± standard errors for continuous variables. To compare categorical and continuous variables, the chi-square test and a general linear model, respectively, were used. The multivariate analysis employed logistic regression to explore associations of AR and RS with mental health and EQ-5D score. We also calculated adjusted odds ratios (ORs) for mental health and the EQ-5D score. We adjusted for age and gender (model 1) and then for these variables plus educational level, marital status, household income, occupation, residential area, smoking status, alcohol consumption, exercise status, sleep duration, and obesity (model 2). Next, we adjusted for all covariates in model 2 plus comorbidities , activity limitations, subjective health, and total cholesterol and triglyceride levels (model 3).Of the 15,441 subjects, 2,286 were diagnosed with AR and 1,189 with RS, yielding weighted prevalences of 14.7% and 7.8%, respectively. The weighted prevalences of AR-/RS-, AR-/RS+, AR+/RS-, and AR+/RS+ status were 80.6%, 4.6%, 11.7%, and 3.1%, respectively.Based on age at examination, all subjects were divided into the following six age groups: 19–29, 30–39, 40–49, 50–59, 60–69, and 70 years and older. The proportions of subjects in the AR+/RS- and AR+/RS+ groups tended to decrease as a function of age . The aveMales constituted a higher proportion of the AR-/RS- group (81.7% vs. 79.6%) and a lower proportion of the AR+/RS- group (10.0% vs. 13.2%) than females .The sociodemographic and clinical characteristics by group are shown in In terms of the HRQoL, This population-based nationwide study examined the impact of AR and/or RS in terms of mental health and HRQoL. Several studies have investigated mental health or QoL in patients with AR or RS. However, as many of these were cohort studies, they may have been affected selection bias; also, they did not include non-referred subjects. Additionally, although AR and RS comorbidity is common , 25, fewIn the present study, the prevalence of AR in Korean subjects was 14.7%, and it decreased with age during 2013–2015. The prevalence was thus slightly less than the 16.2% of the 2010 KNHANES . In anotWe found that 40.2% of subjects with RS also had AR. In two Korean population-based studies, the proportions of patients with comorbid AR and RS were 33.6%  and 18.4In the present study, we found that AR and RS negatively affected mental health. The AR+/RS+ group contained the highest proportions of subjects with perceived stress  and depressed mood . The AR-only group contained slightly higher proportions of subjects with perceived stress and depressed mood than did the RS-only group. However, the extent of suicidal ideation did not differ among the groups.We also used multivariate logistic regression analysis to examine the association(s) of AR and/or RS with mental health. After adjusting for all confounders, we found that the AR+/RS- group exhibited more positive associations with perceived stress and depressed mood compared with the AR-/RS- group. Thus, AR had a more negative effect on perceived stress and depressed mood than RS. More surprisingly, the AR+/RS+ group exhibited about three-fold the proportions of those with depressed mood and suicidal ideation than did the AR-/RS- group . Thus, AR and RS seemed to exert a negative synergistic effect on mental health. Additionally, our finding that comorbid AR and RS increased the risk of suicidal ideation has major implications for Korea, because South Korea’s suicide rate is the highest among member countries of the Organization for Economic Co-operation and Development (OECD) .Previous studies found that AR and RS compromised mental health. In addition to nasal symptoms, AR patients experience fatigue, headache, mood and sleep disturbances, physical and mental problems, and cognitive dysfunction , 36. A rIn the present study, we found that AR and RS both exerted negative impacts on HRQoL. Subjects without AR, with or without RS, reported more problems in terms of mobility and the performance of usual activities than did those with AR. However, subjects in the AR-/RS- group had a higher proportion of self-care problems than did those in the AR+/RS- group, which may have been attributable to differences in age distribution. As the AR-/RS- group had the oldest average age, such subjects may have experienced more difficulties in terms of self-care than those in other groups. Also, the AR-/RS+ group (the next-oldest group) reported problems with mobility and the performance of usual activities. In the pain/discomfort and anxiety/depression dimensions, the AR+/RS+ group had the highest proportions of participants reporting problems. The groups with RS, with or without AR, contained higher proportions of subjects with problems in the pain/discomfort and anxiety/depression dimensions compared to AR subjects with or without RS. According to the logistic regression analysis, the AR+/RS+ group faced increased risks of poor self-care and pain/discomfort compared with the AR-/RS- group, with ORs of 3.73 and 2.19, respectively, after adjusting for all confounders. These results also emphasized the synergistic effect of AR and RS on self-care problems and pain/discomfort, as was also true in terms of depressed mood and suicidal ideation.Previous studies also found that AR or RS impaired HRQoL. A systematic review found that the symptoms of AR negatively affected HRQoL . Crump eThe mechanisms by which AR and CRS negatively affect mental health and HRQoL are not clear. Previous studies found that some nasal and extra-nasal symptoms had negative impacts on mental health and HRQoL. Olfactory loss, a severe symptom of AR or CRS, was associated with anxiety, depressed mood, and suicidal ideation , 43. TheMeanwhile, we assumed the mechanisms of deterioration effect of AR and RS on mental health and HRQoL, in terms of immunologic reaction. Several studies have investigated the associations of the cytokines involved in allergic and RS-related inflammation with mental health and HRQoL. Well-known inflammatory cytokines characteristic of AR or RS, such as IL-6 and IFN-γ , 47, havBefore performing this study, we hypothesized that subjects with AR or RS might have poorer mental health and HRQoL than subjects with only AR or RS. Our hypothesis proved largely correct; we found that AR and RS exerted a negative synergistic impact on mental health  and HRQoL (pain/discomfort and anxiety/depression). Patients with both AR and RS exhibited more severe nasal and extra-nasal symptoms induced by both allergic reactions and RS-related inflammation. Additionally, allergic patients tend to be hypersensitive to somatic pain because pro-inflammatory cytokines induced by allergic processes play crucial roles in the modulation of central and peripheral nociception . Thus, tIn this study, AR and/or RS showed a negative impact on mental health and HRQoL. Because the patients with AR and/or RS may have several psychological problems, clinicians should thoroughly treat patients by easier and more comfortable methods. And they should also assess the patient’s psychological problems using validated tools. If the patients seems to have psychological problems, clinicians should help them receive psychological intervention.A strength of our study is its design. As the KNHANES is a nationwide, complex, stratified, multistage probability-cluster survey of a representative sample of the non-institutionalized civilian population of Korea, the participants are representative the general Korean population. Second, the relatively large number of study participants (compared with other studies) afforded strong statistical power. Third, we considered a wide variety of data, including demographic characteristics, socioeconomic status, health behaviors, and other comorbidities, thus mitigating any probable bias by adjusting for confounding effects. Therefore, the association of AR and RS with mental health and HRQoL, as identified in this study, can be generalized. Last, we examined the mental health and HRQoL of subjects with both AR and RS; few previous studies have examined such subjects despite the fact that the diseases are frequently comorbid.However, our study had several limitations. First, although this was a population-based nationwide study, a cross-sectional survey cannot identify causal relationships between AR and/or RS and mental health and HRQoL. Second, as diagnoses of AR and RS were self-reported, some recall bias may have been in play. However, other large population-based surveys used similar questionnaires to diagnose AR and sinus infections –59. ThirOur population-based, nationwide cross-sectional study showed that AR and RS synergistically compromised mental health and HRQoL. Importantly, combined AR and RS exhibited a strong association with suicidal ideation, which is the most dangerous consequence of poor mental health and also a major social problem. Therefore, physicians who manage patients with AR and/or RS should consider their mental health and HRQoL, especially when patients have both AR and RS.


Allergic rhinitis (AR) is sub-optimally managed in the community and is responsible for a significant health and economic burden. Uncontrolled AR increases the risk of poorly controlled asthma and presents an increased susceptibility to thunderstorm asthma. With the availability of treatments over-the-counter, bypassing the health care professional (HCP), the role of the patient is paramount. Research on the role of the patient in AR management in the current environment is limited. This study aims to explore the patient perspective of AR management and understand why it is sub-optimally managed in the community. Patient perspectives of AR management were explored utilizing a qualitative, phenomenological approach. Adults with AR were included in the study and interviewed. Transcripts were analyzed for recurrent themes and emergent concepts. Forty-seven participants with AR were interviewed about their experiences. Patient reports of delayed diagnosis, treatment fatigue and confidence in the ability to manage their AR themselves, heavily influenced their management preferences. Patients also described barriers associated with AR management including financial expense as well as being mistaken for having an infectious disease. Patients described examples of the impact on their quality of life caused by their AR, yet they strongly believed they could manage it themselves. This belief that AR is a condition that should be entirely self-managed, contributes to its burden. It amplifies patients’ separation from HCPs and having access to guidelines aimed at optimizing their AR control. Healthcare professionals must engage fully with allergic rhinitis patients to optimize treatment and avoid issues triggered by constant self-care. Allergic rhinitis, or hay fever, can impact hugely on individuals’ health but remains poorly managed, partly because of the multiple medications available that allow people to bypass doctors and treat themselves. Biljana Cvetkovski at the University of Sydney, Australia, and co-workers, interviewed 47 adults with hay fever to analyze perceptions of the condition and its management. Many patients reported disenchantment with professional guidance for hay fever, often citing delayed diagnosis and endless tests leading to long-term ‘treatment fatigue’ and a loss of confidence in healthcare services. Patients often prefer self-treatment with limited medical help, repeatedly switching between medications and opting for ‘quick fixes’ rather than treating the chronic condition, with sometimes serious consequences. However, despite this multidimensional approach, the reality is that allergen avoidance is not effective for most patients and immunotherapy is limited to only a very select group of patients, thereby the mainstay treatment of AR relies on medication management.28 While this has been explored in the related field of asthma, in which patient medication self-management has been shown to play a significant role in overall treatment success;29 our understanding of medication self management in AR is limited. While large-scale studies in Europe and the Americas have provided us with detailed information about patient medication preferences along with the co-existence of bothersome symptoms and loss of productivity,31 they fail to explain why patients chose to self-medicate in the way they do, despite often living with sub-optimally managed AR.It is well recognized that patient self-management plays a major role in the successful management of chronic illnesses.The aim of this study is to gain an in-depth understanding of the experiences, beliefs and reasons behind the medication self-management practices of patients with AR.Fifty-seven people contacted the research team expressing interest in participating in the study. Of these, forty-seven people with AR from New South Wales, Australia, were interviewed for this study, with the remainder not being able to be contacted by the research team following initial expression of interest. The 47 participants, were aged from 18 to 86 years of age with a median age of 39 and 28 were female. Eight participants were from rural communities in NSW and 39 from the Sydney metropolitan area. Interview duration ranged from 9 to 67 min with an average duration of 23 min. Four interviews were conducted face-to-face at the Woolcock Institute and the remainder were conducted over the telephone. Participants who chose to participate over the telephone were sent participant information and consent forms via email or post based on their preference. The majority of recruited participants were on the Woolcock’s volunteers database and had responded to the letters of invitation or had heard of the study through snowballing.Data saturation was obtained following the forty-second interview after which no new information was described by the subsequent participants. However all forty-seven volunteers were interviewed to ensure saturation.Seven themes were identified that best represented the patient’s perspective of AR management. The semi-structured interview guide appears in Table Why do I always have a cold? P13A large proportion of participants reported a challenge associated with getting a correct diagnosis. In most of these cases, they had experienced symptoms from childhood and reported symptoms for many years. They were assumed to have a cold or flu and expressed relief at the point when it had been diagnosed as AR. A huge burden being lifted as a result of knowing what their symptoms were caused by, rather than relief of symptoms. They reported seeing multiple HCPs before a suitable treatment was found.I’m 52 and I didn’t get asthma until I was in my 40 s and then a few years ago I had what I felt was a cough that just kept going on and on. The doctor said no, it was hay fever. So it was allergic rhinitis. P10There was also a subset of participants who had identified as having developed AR in adulthood. They reported a delay in diagnosis, they did not expect to develop AR later in life, often they had never experienced any symptoms earlier in life and although less common, some participants reported having being treated for asthma and only later being diagnosed as having AR instead.My parents did everything under the sun. They’ve gone everywhere. P31The delay in diagnosis of AR, especially in childhood often meant participants had been exposed to a gamut of tests and treatments prior to and following their diagnosis of AR. They described a childhood filled with many investigations, medications, doctor’s appointments and alternative treatment options.I keep my ears open for breakthroughs, but there aren’t any. P1There’s just nothing I can do to relieve it and the other thing too is I’ve given up buying drugs over the counter because I know they don’t work. P10As a result of this, they reported a sense of treatment fatigue, i.e., a sense of having explored all available options with regards to treating their AR and having exhausted any desire to pursue further investigations or treatment options. They had concluded that there was nothing available to eliminate their symptoms. They had also developed skepticism about products that claimed to be “new” and would eliminate AR. Participants no longer believed that there were any options that they had not explored and that all new products were similar to old products, just in new packaging or marketed differently. They felt they could no longer generate energy to explore “new” treatments as they assumed they would be disappointed with the efficacy as they had been in the past with other products with similar claims. Consequently, these participants no longer enquired about treatments with HCPs nor did they do their own research about AR. They stated that the only thing that sparked their interest to reconsider their AR management were media reports about a “breakthrough” in AR treatment.As soon as I take them all the symptoms dissipate. They won’t certainly go away. But they certainly dissipate. P30So Zyrtec, Beconase, the sprays. Like the nasal sprays and the tablets, whatever brand really. Claratyne, I’ve tried all of them. P57Participants reported confidence in their ability to manage their AR independently. After years of trying different medications and consulting HCPs, patients felt that they had learnt enough and tried enough treatments to determine for themselves what was best. This was especially the case when considering symptom relief, identifying risk factors and preferences with regards to mode of treatment delivery. With regards to symptom relief, the participants expressed a high tolerance of their symptoms and were satisfied with a small reduction in symptom severity.I usually get itchy eyes when the pollen count is really high and also when it’s dusty and I sneeze a lot as well. P18That is more of a preventative measure - I stock up on it 2 weeks in advance - which minimises the effect of the symptoms. Heading up to spring and so the body, I believe, builds up a sort of immunisation or builds up a resistance to the hay fever. It allows me to deal with it on a - in a much better fashion. P31The chronic nature of AR allowed them to recognize patterns of symptoms, identify triggers and develop avoidance strategies. Patients felt that HCP appointments were a significant impingement on their own time and that their own efforts in managing their AR were a more efficient use of their time. Using their own skills to manage their AR let them accommodate their treatment preferences best, both with respect to method of delivery and expectation regarding symptom relief. Some participants prepared for a predicted worsening of symptoms due to an upcoming exposure to triggers by taking antihistamines preventatively, based on their own experimentation and belief for what is best for them.There were three predominant sentiments that were reported by participants when describing their preferences for managing their AR, all of which related to medication use and selection. Firstly, a preference not to medicate at all with the exception of a nasal saline wash or mist. Secondly, those who reluctantly medicate, when they felt the symptoms were otherwise intolerable. Thirdly, those who want to take any form of treatment in order to be symptom free.So I don’t really like sticking chemicals up my nose. P25I want someone to tell me that just having a carrot a day or something gross like a zucchini will fix everything. I’ll be like ‘ok, I’ll do it if I really have to’. P17I don’t particularly like taking medication. P19The first group that had a preference not to medicate, represented people who did not believe that medication worked and those that had beliefs against medication. A subset of participants felt that they had tried all available AR treatments and nothing cured or completely eliminated their symptoms, therefore taking symptom-relieving medicines was a waste of time and an unnecessary risk of experiencing adverse effects. Other participants who wished not to medicate, said that they ideologically preferred not to use medicines for their AR.I wait for the symptoms to occur because I’m very anti-drug and the symptoms are so severe to the point where I actually can’t work, because it is like a head cold. P30I have to take something otherwise I am a mess. Only when it gets really really bad. A couple of times a week. P56I’d use them just while there is a lot of flowers and dust in the air, the change of season in springtime and that sort of thing. If it started coming on then I’d use it for a week or something and see how I went without it. P50The second group medicated when they perceived it necessary, sharing some of the sentiments of participants who preferred not to medicate at all. They too felt that no treatment completely eliminated their symptoms, hence did not sense a purpose in using regular medicines and experiencing the expense and commitment associated with it. They preferred not to have to use any medicines, but in some circumstances perceived their symptoms to be intolerable and unacceptably impeding on their productivity or quality of life. In such circumstances, this cohort of participants had a preference for using non-sedating oral antihistamines for their ease of use, availability, onset of action and perceived effectiveness. The majority of participants had at some point used an intranasal corticosteroid [INCS], however very few were still using it. The main reason for discontinuation was the perception that it did not work, even though they had used it for weeks or months at a time. Some participants reported they had undergone immunotherapy at some stage in their lives. For some, this meant a significant reduction in their symptoms and symptoms thereafter were manageable with the occasional use of oral antihistamines. For others, it cemented the ideology that nothing would cure their AR and these participants were not motivated to try any further treatments.[What prompted you to see a naturopath in the first place?]Just desperation I think. P21I’d have to rely on them daily. To be able to cope, yeah…to work I’d have to rely on them - even the eye sprays - to get me through the day because it gets really bad, really irritating. They [symptoms] just reduce for a few hours. P39The final group, which medicated to be symptom free, used more medicines more frequently than the other two groups. AR presented a significant burden to them and their quality of life that they were willing to take regular medicines preventatively to try and control their symptoms. This cohort of participants reported an exploration of alternate medicines in order to relieve the symptoms of AR. They explored alternate therapies in circumstances where traditional medicines had not met their needs. These participants were most likely to have undergone immunotherapy.I alternated between them, but I found the Claratyne was the most effective, so I ended up just buying Claratyne from then on. P56No it[non-sedating antihistamine] seems to be working well, cost effective, I don’t get any side effects from it, but yeah. P44Because [antihistamines] are smaller and I can swallow them without water. P13I didn’t find them [INCS] that effective. I still use them if I feel like I have to.But I just don’t find them that effective. So I don’t really rely on them to dowhat they need to do. P57An individuals’ approach/attitude to taking AR medicine was not influenced by the severity of symptoms or the impact on quality of life. Regardless of the participant’s severity or medication preferences, there was an overwhelming preference for use of oral medicines due to ease of administration and use, portability, palatability, perceived effectiveness and onset of action (compared with INCS). Participants reported that when they were using a medicine, which they perceived as adequately relieving their symptoms, they would not explore alternative options. There was however a belief among the majority of participants that they would develop a tolerance to oral antihistamine medicines if they repeatedly used the same product. They believed that this could be avoided if they occasionally were to switch brands.Some, participants had consulted an ear, nose, and throat surgeon. Where additional nasal issues were identified and ear-marked as potentially benefitting from surgical intervention , there were mixed responses in the uptake. There was almost an equal distribution between people who were willing to explore surgery as an option versus those who preferred to avoid it. Some thought a one-off intervention was worth considering where others felt that the risk and significance of surgery substantially outweighed any potential benefit. Some participants reported a significant improvement in the ability to breathe after they had surgery, only then realizing that they had accepted a poor standard of breathing in the past.[Preference for medication], yeah by price basically. P34A lot of it depends on how expensive they are as well. P41That’s always been a goal of mine to be able to have the pre-test to find out exactly what the triggers are. But I pretty much know what they are-know not specifically, but I know what they are. Pollens and dust pretty much. It doesn’t really bother me, so I haven’t done it and it’s expensive. P50It worked quite good[INCS] but at the time I didn’t have the money to buy it because it was so expensive. P25I had to use it-to do one spray of the steroid one and another spray of the other one otherwise it wouldn’t work. Until eventually the steroid did take over but it didn’t happen straight away. It was really annoying because I’d spent all this money on the spray but I had to use the other one. P25Participants reported that in some circumstances their choices regarding AR management were based on cost. Cost arose as an issue when describing treatment selections, visits to a doctor and procedures to formally diagnose AR. The perception of financial burden of AR treatments was evaluated based on the person’s perception of its monetary value versus perceived effectiveness. In some instances further investigation, such as a skin prick test, was deemed expensive and were not perceived as necessary. Participants felt they had identified their triggers through observation of pattern and exposure history. Similarly, the financial expense of medication was questioned based on how effective it was perceived to be and the difference it made to the person’s quality of life.The whole idea is, in front of people I want to just come across as unaffected. P47There was a strong sentiment among the participants that they did not want to be labeled as having an illness. They referred to it as nuisance or an irritation, rather than a chronic respiratory disease. For some, this meant that they deliberately did not discuss their AR with anyone and internalized their management entirely. They felt that discussing it with others would not achieve anything except bring them more unwarranted attention.There’s a big stigma and focus on people sneezing…so I often say to people I don’t have a cold, it’s hay fever. P14It’s embarrassing you know, having these symptoms. P14People think I’ve got a cold. PHWe’ve got a bridge club and they always say, ‘if you’ve got a cold, stay home, we don’t want you here’. I’ve had to tell people, ‘well it isn’t a cold that I have. I’ve just got it all the time’. P7Participants reported that the symptoms of AR brought unwarranted attention, particularly sneezing and nasal congestion. They felt that there is a stigma associated with sneezing and nasal congestion due to others misinterpreting the symptoms as belonging to a contagious cold or flu. Participants reported having to constantly explain to people that they have AR and are not infectious and do not need to be isolated or kept away from school or the workplace. The symptoms of AR also caused them to feel embarrassed among their colleagues and peers.You get not depressed but kind of like depressed I suppose the word is. It’s very debilitating. You can’t believe it but it’s true. P33It is an annoyance, but it’s not a lethal, life threatening disease or anything. It’s not life crippling. It’s just inconvenient, really. P56No, no that bad [not symptoms everyday]. I’d neck myself if it was every day. P2A theme that repeatedly displayed variation among participants was the perception of the impact AR on their life. Responses ranged from AR being regarded as an “inconvenience” to something that if persistent, can make them feel depressed. Participants who felt AR was a nuisance, regardless of its severity, did so because they compared it to having a life-threatening condition. Ultimately, they felt that AR did not warrant more attention than they had given it because it would not end their life.I have a young family now so sleep is a little more precious to me . P8Many identified the impact of AR on daily activities extended beyond themselves and their immediate family.I’m tired and it’s hard to concentrate and feel unwell. P14To go to work, I’d have to rely on them [antihistamines],even the eye sprays, to get me through the day because it gets really bad, really irritating. P39I can’t work, now I’m allergic to rats… I couldn’t continue with research in that area. A career working with animals would have been out for sure. P14People with AR were more likely to notice the impact of AR on their life when it interfered with their workplace productivity. Treatment options were explored and determined based on how AR affected their ability to perform at work. Participants became tolerant to a certain impact on their quality of life brought upon by AR. This tolerance would be challenged and further treatment would be sought when the impact on quality of life was heightened, sometimes due to factors unrelated to AR, .32 and by self-managing their AR in isolation, patients are moving further away from the reach of these guidelines and into unguided territory, compounding the burden it has on the community.This research provides important fresh insights to the reasons why people with AR sub-optimally manage their condition and goes to explain what has led to patients choosing to carry the burden of AR management themselves. This study of patient’s perspective has identified that patient’s choice to self-manage their AR is linked to sub-optimal experiences with HCPs and confidence in their own abilities to treat their AR with OTC medicines. AR guidelines are developed for use by HCPs33 and includes elements of trigger avoidance and monitoring in addition to medication management, the patient’s perspective of management and their role in it is focused on pharmaceuticals and the options they have. There are several aspects that impact on this, as highlighted in the following discussion.In conducting this research, one striking point was noted; when patients consider the management of their AR, there is a dominant focus on medication management. While self-management is multifaceted3 a finding similar to that experienced by patients with chronic rhinosinusitus.34The delayed diagnosis/misdiagnosis of AR plays a formative role in the patient’s sub-optimal management. Disenchantment with HCPs’ ability to identify their problem steers them on the path of self-management, in a sense, towards a “self-help” approach. This goes to set up a vicious cycle, and increased burden of AR, as delayed diagnosis results in a delay in seeking medical advice until symptoms are intolerable,35 treatment fatigue in AR seems to be less about adherence and more about disillusionment with multiple failed attempts to control their AR with the treatments available. Treatment fatigue in AR also expands to the patient’s rigmarole of health care consultations and investigative procedures, without AR symptom resolution. To overcome treatment fatigue, patients must be offered support and counseling regarding their AR as has been shown for other chronic respiratory disorders such as asthma.36Treatment fatigue (defined as a decreased desire to explore further treatment based on exhaustion from pursuing previous unsuccessful pathways) was synonymous with and often a consequence of delayed diagnosis. Treatment fatigue caused patients to be reluctant to explore further therapies or consult HCPs. While treatment fatigue is often associated with poor adherence,37 facilitates them in accessing their preferred oral, quick relief, occasional-use medicine and avoid the disliked intranasal sprays.38 The preference for quick relief occasional-use medicine is evidence of their perception of AR as an acute condition, rather than a chronic disease.Patients believe that AR is a condition that they should be self-managing. This is influenced by the availability of medicines over the counter (OTC). As patients try different medicines, they develop confidence in their ability to do so, continuing the cycle of self-management and treatment trials. Being able to choose their medicines OTC41 and should be taken into account when developing their management plan.This research highlights that it is not just the availability of OTC medicines that drives the patient to self-manage their AR. These patients reported that they know what works best for them and suggested that their HCPs have not entirely understood their goals for their AR. Understanding of patients’ treatment aims and goals has been shown to improve their AR management42 and the development of tools and support mechanism for patients through therapeutic partnerships with HCPs.43 Of critical importance, is that tools available to patients should be dynamic, accommodate for changing AR severities and provide guidance in instances of a “flare up” as seen in written asthma action plans.33 The MACVIA-Allergy Diary App44 is able to provide the patient with some guidance on self-managing their AR but further research needs to be conducted to investigate how HCPs can best collaborate with patients to optimize its use.In considering these findings it is important to consider possible solutions. Possible solutions should include simple pathways for recognition and diagnosis of AR in primary care,38This study was potentially limited by the sample, which is likely to under-represent patients with mild AR. While a majority of participants reported being diagnosed with AR, the inability to confirm the patient’s diagnosis of AR is a potential limitation. It should be noted however that the demographics of the study population were consistent with that reported previously for the Asia-Pacific region.In conclusion, our findings strongly demonstrate that the patient’s experience with AR is unique, varied and very much a condition in which patients self-manage with medications that are easily and abundantly available to them. AR management is influenced by patients’ experiences prior to receiving a diagnosis following which their knowledge and beliefs about AR and its treatments are formed. These results highlight the critical need to increase the dialog between HCPs and their patients regarding AR, improve the understanding of the significance of AR and optimize its management and for HCPs to acknowledge patients’ expectations and beliefs with regards to treatment. HCPs must engage patients, embrace AR self-management and work collaboratively with patients to optimize their AR management.This study took the form of a qualitative exploration of patients’ perspectives of AR management and was conducted as a part of a larger study exploring AR management in the community.A phenomenological approach was utilized to explore the phenomenon of AR. Participants were asked to describe their experiences with AR and given “free range” to voice what AR meant to them.Inclusion criteria: People aged 18 years or older, who identified themselves as having AR and were able to speak English, were eligible for inclusion in the study.Recruitment: Advertisements for the study were placed on the website and Facebook page of the Woolcock Institute of Medical Research, Sydney, Australia. Letters of invitation were sent to individuals with AR on a volunteers’ database at the Woolcock Institute . People with AR, who expressed interest in participating, were required to contact the research team to give consent and make an appointment to be interviewed. Snowballing was welcomed and participants were permitted to alert others with AR about the study. Participants did not receive any reward or remuneration for participating in the study.Participants were given the option of being interviewed in person at the Woolcock Institute or over the telephone. Interviews were conducted by one researcher (BC). They were audio recorded on a digital audio recording device, transcribed verbatim and stored electronically. Participants were informed that their responses were completely de-identified and would not be traced back to them as individuals in any circumstances. Written participant consent was obtained prior to the commencement of the study.Sample size: Phenomenological research requires interviews to be conducted with 5–25 people.45 Participant recruitment continued until data saturation was achieved with consideration of the phenomenological research sampling frame guidelines.46Semi-structured interview guide: The semi-structured interview guide, based on empirical data and a phenomenological framework48 covered a range of topics/facets of AR management  and served to guide the qualitative framework of the research.45The interview commenced with open ended questioning in which participants voiced their initial thoughts or their most pertinent perceived issues. Secondary and clarifying questions were asked to encourage further thoughts and experiences to be articulated.The specific questions included in the interview guide are displayed in Table Qualitative data analysis and representation: In phenomenological data analysis, “significant statements” that represent how the participant experienced the phenomenon are identified as “codes”. These codes are then categorized into themes that are used to write a description of the experiences of the participants with the phenomenon being researched.4545A mixed deductive—inductive approach was utilized in the qualitative analysis of the interview transcripts. The mixed analysis approach sought to identify themes arising from the identification of codes in AR literature (deductive) as well as identification of codes from the analytical reading of the data (inductive). The deductive analysis identified the participants’ responses to themes that were known to be important with regards to AR management in the literature. Such codes included treatment preferences, symptoms, investigations and HCP consultations. These themes were often prompted with the interview guide if not immediately described by the participant. Inductive analysis of the transcripts included the identification of codes that were presented organically through the interview process but were not necessarily explored by the interview guide.The researchers  met weekly to review a summary of the interviews conducted during the week based on notes taken by BC, while conducting the interviews. Data saturation was identified following reports of no new perspectives being provided by participants with regards to their AR management.The initial process of analysis involved a detailed description of what BC and SBA independently observed in the transcripts and categorizing those observations into codes. Some of these codes represented deductive analysis e.g. descriptions of medicines used while others were inductive and were observed without pre-determined identification of the code, e.g. being mistaken for having a cold. Following the independent categorization conducted by BC and SBA, meetings were held with VK and KY to discuss the codes and the transcripts, look for common elements and categorize them into agreed themes. The breadth and scale of interviews meant that there were many individual and unique perspectives of AR management described by the participants, where the volume is prohibitive of all being reported in this paper. BC and SBA found that participants gave very detailed accounts of the pharmaceutical regimens they use for managing their AR. Codes describing participants pharmaceutical management of AR were categorized into themes by BC and SBA, describing their preferences and the reasons behind them, rather than detailed descriptions of the regimen. In addition to the descriptions of pharmaceutical regimens BC and SBA identified common themes overarching many of the transcripts. These themes were chosen to be reported in this manuscript based on what BC, SBA, VK, and KY perceived to be most important to the participants during their interviews. Further results not reported here will be reported at later stage in subsequent manuscripts.49ATLAS.ti was used to facilitate the qualitative analysis by providing an organizational structure to link the codes, themes and their supporting quotes among the interview transcripts.Once analysis was complete, the participants were not given the opportunity to provide feedback on the findings but were able to request a summary of the results.50This study was approved by the University of Sydney Human Ethics Committee. Methods were performed in accordance with COREQ regulations and guidelines.


Lysine-specific demethylase 1 (LSD1) has been recognized as a potential therapeutic target for acute myeloid leukemia (AML). Herein, we identified a novel LSD1 inhibitor, JL1037, via Computer Aided Drug Design technology. JL1037 is a potent, selective and reversible LSD1 inhibitor with IC50s of 0.1 μM and >1.5 μM for LSD1 and monoamine oxidases A/B (MAO-A/B), respectively. Treatment of THP-1 and Kasumi-1 cell lines with JL1037 resulted in dose dependent accumulation of H3K4me1 and H3K4me2, the major substrates of LSD1, as well as inhibition of cell proliferation, blockade of cell cycle and induction of apoptosis. Further investigations demonstrated that JL1037 could upregulate cell cycle-related proteins P21, P57, pro-apoptotic protein Bax and downregulate anti-apoptosis proteins Bcl-2 and Bcl-XL. JL1037 appeared to activate autophage response in AML cell lines as well as primary cells from AML patients by increasing LC3-II expression and the formation of autophagosomes and autolysosomes in cytoplasm. Co-treatment with autophagy inhibitor chloroquine (CQ) enhanced JL1037-induced cell apoptosis. Moreover, daily intravenous administration of JL1037 tended to reduce tumor burden and prolong the survival of t leukemia mice. In conclusion, JL1037 exhibited potent anti-leukemia effect and could be a potential therapeutic agent for AML treatment. Epigenetic abnormalities are in close relationship with the initiation and progression of leukemia , 2. SincMore and more investigators have reported that LSD1 is highly expressed and closely correlated with poor prognosis in various kinds of malignancies –16. Harrex vivo AML cell systems as well as in a mouse model harboring AML1-ETO translocation to elucidate the role of JL1037 as an effective anti-leukemia agent.In this study, using structure-based virtual screening and subsequent compound optimization, we identified and synthesized a novel reversible LSD1 inhibitor coded as JL1037, which specifically inhibited LSD1 in cell-free enzymatic assay with great potency. We further characterized JL1037 in To investigate whether LSD1 could be a valid therapeutic target for AML, the expression levels of LSD1 in a variety of AML cell lines were compared with that of normal bone marrow mononuclear cells (BMMNCs) by real-time quantitative PCR (qRT-PCR) and Western blot. The results clearly demonstrated that LSD1 expression at both mRNA and protein level was significantly higher in majority of AML cell lines, especially in Kasumi-1, THP-1 and K562 cells, compared with that of normal BMMNCs, which was hardly detectable Figure  and 1B.Increasing numbers of investigators have demonstrated LSD1 as a potentially promising drug target for AML. Herein, we successfully synthesized a novel LSD1 inhibitor, JL1037, which was originated from computational screening and designed using mainly the DOCK module of the MOE software . Molecular docking sampled conformations of small molecules in a protein binding site and found which of those shapes fit well both spatially and chemically to the protein binding site. In this study, docking was implemented with the crystal structure of the LSD1 enzyme in complex with CoREST and a substrate-like peptide (PDB ID: 2VID) as shown in Figure in vitro inhibitory activity of JL1037 on LSD1 with LSD1 Fluorimetric Drug Discovery kit . JL1037 exhibited good inhibitory potency against LSD1 with IC50 value of 110 nM  μM and (19.42 ± 3.02) μM, respectively Figure . We furtThe mechanism underlying the cell cycle arrest and apoptosis induced by JL1037 was explored. Cell cycle-related proteins P21, P27 and P57 were examined by Western blot after 24-hour treatment of the cell lines with JL1037. As shown in Figure Autophagy, a kind of non-apoptotic programmed cell death, can be induced by many cytotoxic compounds. In present study, to explore whether autophagy was involved in the process of cell death induced by JL1037, we utilized transmission electron microscopy (TEM) to analyze the ultrastructure of AML cells treated by JL1037. Both autophagosomes and autolysosomes were clearly observed in THP-1 and Kasumi-1 cells after being treated with 30 μM of JL1037 for 48 hours and neither was seen in untreated cells Figure . Under TThe conversion of LC3-I into LC3-II is an essential step in autophagy and the number of LC3-II puncta represents the number of autophagosomes. Western blot assay showed that JL1037 significantly enhanced the expression of LC3-II even at the concentration of 10 μM Figure . Immunof+ hematopoietic stem/progenitor cells were also examined. CD34+ cells were isolated from fresh umbilical cord blood samples with immunomagnetic beads and treated with different concentrations of JL1037 for 48 h, the percentage of apoptotic cells was determined with flow cytometry. The results showed that JL1037 induced apoptosis of CD34+ cells evidently at the high concentration of 50 μM, suggesting JL1037 at high concentrations might have toxic effect on hematopoietic cells , except for two mice in high-dose JL1037 group (5 mg/kg) died as the result of drug toxicity  and a small number of highly selective and potent compounds. In the present study, we disclosed a novel, potent LSD1 inhibitor JL1037 and evaluated the therapeutic effect of JL1037 on AML with both e Figure . JL1037 e Figure . In addie Figure , Table 1e Figure , which sy Figure . This wa+ hematopoietic stem/progenitor cell was not spared by the inhibition of JL1037 due to its high expression of LSD1 as shown in Figure LSD1 inhibitors are effective in a panel of leukemia cells, especially those harboring MLL-AF9 and RUNX1-RUNX1T1 translocations , 26. THP2O3 and ATRA , Caspases-3, P21, P27, P57, Bcl-xL, Bcl-2, Bax and LC3B . β-actin  and Histone H3  were used as internal controls. Then, membranes were incubated in HRP-conjugated secondary antibody and visualized using the HRP Substrate  by Image Quant LAS-4010 system .Treated cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton X-100 for 10 min and blocked with PBS containing 2% goat serum for 30 min at room temperature. Cells were then incubated with an anti-LC3B primary antibody  overnight at 4°C, washed three times with PBS and stained with an Alexa 488-labeled donkey anti-mouse IgG secondary antibody  2 h at room temperature in darkness. Nuclei were stained with DAPI for visualization. Fluorescence images were taken on a spinning disk confocal microscope using a 100X oil-immersion objective. Autophagy was evaluated by the formation of punctate fluorescent structures.After the treatment with or without JL1037 for indicated hours, cells were fixed with 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide and dehydrated by a series of ethanol washes before being embedded in Epon812. Ultra-thin sections were obtained and observed by a JEM-2100F transmission electron microscope.in vivo study, 6~8 week female C57BL/6 mice were exposed to sub-lethal of radiation (450 cGy) and transplanted with 5 × 105 GFP+ spleen cells from the third generation leukemia mouse. The mice were monitored for 10 days and then randomly divided into three groups: PBS group, low-dose JL1037 (2.5 mg/kg.d) group and high-dose JL1037 (5 mg/kg.d) group. Each group included 8 mice. The intraperitoneal administrations of drugs were initiated on day 10 after transplantation. During a 10-day administration period, the percentage of GFP+ cells in peripheral blood was measured by flow cytometry every three days to assess the development of leukemia. Body weight was measured every two days to evaluate drug toxicity. The overall survival of each group was calculated from the date of transplantation to the date of death. All animal experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of Peking Union Medical College.The AML1-ETO & C-KIT leukemia mouse model was previously established by our laboratory . For in 50 values of MTS assay was calculated using SPSS software (version 16.0). The comparisons were performed by Student's t-test analysis using GraphPad Prism (version 5.0). The lifespan of mice was analyzed by Kaplan-Meier methods and a log-rank test. P-values < 0.05 were considered statistically significant.All experiments in this study were conducted at least three times. ICThis work was supported by National Natural Science Foundation of China  and CAMS Initiative Fund for Medical Sciences (2016-I2M-1-001).


Androgen receptor (AR) is frequently over-expressed and plays a critical role in the growth and progression of human prostate cancer. The therapy attempting to target AR signalling was established in decades ago but the treatment of prostate cancer is far from being satisfactory. The assignable cause is that our understanding of the mechanism of AR regulation and re-activation remains incomplete. Increasing evidence suggests that deubiquitinases are involved in the regulation of cancer development and progression but the specific underlying mechanism often is not elucidated. In the current study, we have identified ubiquitin-specific protease 14 (USP14) as a novel regulator of AR, inhibiting the degradation of AR via deubiquitinating this oncoprotein in the androgen-responsive prostate cancer cells. We found that (i) USP14 could bind to AR, and additionally, both genetic and pharmacological inhibition of USP14 accelerated the ubiquitination and degradation of AR; (ii) downregulation or inhibition of USP14 suppressed cell proliferation and colony formation of LNcap cells and, conversely, overexpression of USP14 promoted the proliferation; and (iii) reduction or inhibition of USP14 induced G0/G1 phase arrest in LNcap prostate cancer cells. Hence, we conclude that USP14 promotes prostate cancer progression likely through stabilization of AR, suggesting that USP14 could be a promising therapeutic target for prostate cancer. Subsequently, the ligand-activated AR binds to specific DNA sequences on the target genes and initiates expression of a series of genes that promote prostate cancer progression. For instance, kallikrein-related peptidase 3  is the best characterized AR target, which is used in the clinic to monitor prostate cancer development and progression.5 In addition, AR gene amplification and mutation are associated with prostate cancer development and the progression from androgen-dependent prostate cancer to castrate-resistant prostate cancer, which renders the cancer incurable.8Androgen receptor (AR) signalling pathway dominates the survival, proliferation and growth of prostate cancer. AR is a ligand-dependent transcription factor, belonging to the nuclear receptor superfamily.2 Although numerous researches have focused on the regulation of AR synthesis in prostate cancer, the regulation of AR post-translational modification and degradation has been historically underappreciated. Nevertheless, it has been demonstrated that Akt and E3 ligase MDM2 form a complex with AR and promote phosphorylation-dependent AR ubiquitination, resulting in AR degradation by the proteasome.9 This implies that AR-centred signalling could potentially be regulated by altering AR protein stability, which prompted us to examine the potential role of deubiquitinases (DUBs) in the regulation of AR protein degradation.Given the pivotal role of AR signalling in prostate cancer development, AR-based therapy was born several decades ago. However, the current anti-prostate cancer strategies in the clinic cannot fully cure the disease. Recent studies have attached importance to the dysregulated AR expression in prostate cancer and its underlying mechanisms because these represent the most therapeutically relevant targets in this disease.11 DNA stabilization,14 chromatin modification15 and various cellular signalling pathways.16 The human genome encodes approximately 100 putative DUBs, which are subdivided into six families on the basis of their sequences and structural differences. Recently, several DUBs are reported to be associated with the co-regulation, stabilization or transcription of AR. For example, USP26 physically interacts with AR and influences AR ubiquitination and transcriptional activation;17 USP12 stabilizes AR, enhances its cellular function, and thereby triggers the gene expression of PSA.18 In addition, USP10 also has been reported to bind AR, resulting in increased transcriptional activity. Overexpression of wild-type USP10 stimulated AR activity as revealed by reporter constructs harbouring selective androgen response elements, non-selective steroid response elements or the mouse mammary tumour virus promoter. USP10 reduction impaired the mouse mammary tumour virus response to androgen.20 Moreover, USP7 seems to be required for binding of the AR to chromatin and mediates its activity. USP7 was detected in the AR-containing protein complex assembled on the androgen response elements of FKBP5, PSA and PDE9A upon dihydrotestosterone stimulation, which are required for the proliferation of prostate cancer cells.21In the ubiquitin proteasome system, ubiquitination and deubiquitination are two reversible events that counter one another and control the stability of most cellular proteins. Specifically, protein ubiquitination transforms the function and location of the target protein or promotes its degradation, while this process can be reversed by deubiquitination. In eukaryotic organisms, DUBs remove ubiquitin (Ub) and ubiquitin-like (Ubl) chains from target proteins prior to their degradation and thereby participate in the regulation of multiple cellular processes, including cell cycle control,25 As a member of the ubiquitin-specific processing protease family, USP14 has been reported to be highly expressed in several kinds of carcinoma, including multiple myeloma,23 ovarian carcinoma26 and colorectal cancer.27 In this study, we have identified that USP14 promotes the cell cycle in prostate carcinoma cells by deubiquitination and stabilization of AR.To date, the regulation of AR by DUBs associated with the 19S regulatory particle of the proteasome complex remains poorly understood. There are three identified DUBs associated with the proteasome: USP14, UCHL5 and Rpn11 in mammalian cells. Rpn11 is a stoichiometric subunit of the lid subcomplex of the 19S regulatory particle whereas USP14 and UCHL5 reversibly associate with the 19S, indicative of attractive and versatile roles for these molecules.μM), a selective and potent inhibitor of USP14, on the cell growth of LNcap cells. We found that IU1 significantly decreased the cell growth in a time- and dose-dependent manner  and found that inhibition of USP14 activity dramatically induced G0/G1 cell cycle arrest at different time points   . In addi4, 48 h) , indicat4, 48 h) . To furt4, 48 h) , while t4, 48 h) , and led4, 48 h) . Taken tWe then sought to address why USP14, which is expressed in both androgen-responsive and -irresponsive prostate cancer cells, plays a starkly different role between the two types of prostate cancer cells. Generally, AR is over-expressed and the AR signalling pathway is extraordinarily activated in androgen-responsive prostate cancer cells; by contrast, the expression of AR is extremely low in androgen-irresponsive prostate cancer cells and the growth and proliferation of these cells is independent of AR. Therefore, we hypothesized that USP14 inhibition-induced cell cycle arrest is dependent on the expression and responsiveness of AR. To test this hypothesis, we examined the AR protein level in LNcap cells exposed to various concentrations of IU1 for different durations and found that IU1 decreased the protein levels of AR and PSA (a target gene of PSA) in a time- and dose-dependent manner, and markedly increased the expression and phosphorylation of MDM2, which is one of the known E3 ligases of AR that promotes AR degradation . Meanwhi2-PCR). We found that both IU1 and USP14 siRNA dramatically decreased the PSA mRNA but not the AR mRNA, indicating that USP14 inhibition or gene silence does not affect the transcription of AR , and found that USP14 inhibition or silence increased the binding of MDM2 to AR , suggeston of AR . Hence, on of AR . To furton of AR , suggest5 The androgen deprivation therapy with gonadotropin-releasing hormone agonists or bilateral orchiectomy is the mainstay of clinical management for prostate cancer.28 However, the inevitable progression to castration-resistant prostate cancer, which occurs within 2−3 years after the initiation of androgen deprivation therapy, represents a major medical challenge.29 Additionally, patients treated with androgen deprivation therapy are at increased risk of cardiovascular events, including myocardial infarction and stroke.31 Therefore, more therapeutic strategies as well as alternative targets are necessary for prostate cancer treatment.Prostate cancer remains the most frequently diagnosed non-cutaneous malignancy and a leading cause of male cancer death.1 to S phase transition in human prostate cancer cells and also identified USP14 as a potential target for prostate cancer therapy.A few DUBs were reported to be over-expressed in tumour tissues and some of them are emerging as a class of novel targets or biomarkers for anticancer strategy. The current study has discovered that USP14, one of the 19S proteasome-associated DUBs, is involved in the stabilization of AR proteins and promotes G0/G32 AR increases cyclin D mRNA and cyclin E activity, decreases p21 expression, and promotes p27 degradation. Typically, in the early G1 phase, cyclin Ds and CDK4 or 6 form complexes and thereby initiate phosphorylation/inactivation of the retinoblastoma tumour suppressor (Rb), a negative regulator for cell cycle transitions and the onset of DNA replication. Rb phosphorylation releases E2F and leads to G1-S transition. Protein p27 and p21 belong to the endogenous CDK inhibitors, which potently inhibit CDKs kinase activity in the G0/G1 phase and trigger cell cycle arrest.34 The p15 protein can bind to CDK 4 and 6 and inhibit the kinase activity of the formed complex, which similarly blocks the G1-S transtion.35Our study shows that inhibiting USP14 expression or its function leads to cell proliferation inhibition and cell cycle arrest at the G0/G1 phase in androgen-responsive prostate cancer cells but not in androgen-irresponsive prostate cancer cells. We confirmed that AR was highly expressed in the androgen-responsive prostate cancer cells (LNcap cells) but was hardly detectable in the androgen-irresponsive prostate cancer cells (DU145 and PC3 cells) tested here , implyinThe AR-dependent cell cycle arrest by inhibiting USP14 prompted us to investigate the interaction between USP14 and AR. Indeed, we found that inhibition or reduction of USP14 dramatically decreased the protein levels of AR and PSA (a target gene of AR). Our gene expression analyses of androgen-responsive prostate cancer cells exposed to IU1 or USP14 siRNA show a downregulation of PSA but not AR mRNA expression, suggesting that USP14 might not enhance the transcriptional activity of AR. In the rescue experiments, IU1-induced AR downregulation can be reversed by proteasome inhibitor bortezomib (Velcade), suggesting that reduction of AR protein levels by the inhibition of USP14 depends on proteasome activity. Thus we present a hypothesis that USP14 increases AR by inhibiting AR degradation, not by promoting AR transcription. Furthermore, we found that the binding of AR to MDM2, as well as the levels of native and phosphorylated MDM2, a known E3 ligase of AR, which assists AR ubiquitination and degradation in physiological condition, were significantly increased upon inhibiting USP14 activity or expression. The increased MDM2 can also promote AR ubiquitination and degradation. More importantly, we observed that USP14 could bind to AR and decreased AR ubiquitination, suggesting that USP14 may be another DUB of AR .In summary, via demonstrating the role of USP14 in AR stabilization in androgen-responsive prostate cancer, here we provide a potentially new strategy for inhibiting AR-mediated prostate cancer carcinogenesis or progression through inhibition of USP14.IU1, siRNA and shRNA were obtained from Santa Cruz Biotechnology . MG132 and bortezomib (Velcade) were purchased from Selleckchem . MTS assay (CellTiter 96 Aqueous One Solution reagent) was purchased from Promega Corporation . Propidium iodide (PI) and Annexin V-FITC Apoptosis Detection Kit were purchased from Keygen Company . Dynabeads antibody coupling kit was from Life technologies. Antibodies used in this study were purchased from following sources: anti-ubiquitin (P4D1) (Santa Cruz Biotechnology); anti-PARP, anti-CDK2, anti-phospho-Rb, anti-Rb, anti-PSA, anti-Bax, anti-GFP, anti-GAPDH ; anti-CDK4, anti-CDK6, anti-phospho-MDM2, anti-P53, anti-USP14, anti-Flag, anti-cyclin D1, anti-p15, anti-p27 ; anti-MDM2 and anti-AR .2.Human prostate cell lines LNcap, PC3 and DU145 were purchased from American Type Culture Collection . LNcap grown in RPMI 1640 supplemented with 10% FBS. PC3 and DU145 grown in Hyclone DMEM/F-12 supplemented with 10% FBS. Cultured cells were maintained at 37 °C and 5% CO36 In brief, exponentially growing LNcap, PC3 or DU145 cells were seeded at 2500 cells/well in a 96-well plate. After incubation for 24 h, cells were treated with IU1, usp14 siRNA or shRNA, followed by continuous incubation for 24, 48 or 72 h. 20 μl MTS reagent was directly added to each well and the incubation was continued for additional 3 h. The absorbance of optical density was measured with a microplate reader  at wavelength 490 nm. Cell viability was calculated by the following formula: cell viability (%)=(average absorbance of treated group−average absorbance of blank)/(average absorbance of untreated group−average absorbance of blank) × 100%.MTS assay (CellTiter 96 Aqueous One Solution reagent) was used to test cell viability as we previously reported.37 Briefly, cultured LNcap cells were harvested and washed with 4 °C PBS twice and resuspended with the binding buffer, followed by Annexin V-FITC incubation for 15 min and PI staining for another 15 min in dark. The stained cells were analysed with flow cytometry within 30 min.For cell cycle assay, LNcap cells were harvested and washed with 4 °C PBS twice and the precipitated cells were resuspended with 2 ml 70% ethanol at 4 °C overnight. And then the cells washed with 4 °C PBS twice again, followed by incubation with PI (50 μg/ml), RNase A (100 μg/ml) and 0.2% Triton X-100 complexes for 30 min at 4 °C in dark. The stained cells were analysed with flow cytometry. Apoptosis assay was performed as previously described.38 LNcap cells exposed to either IU1 or USP14 shRNA were suspended in 60-mm dishes carrying 30% agarose supplemented with 10% FBS RPMI-1640 medium then cultured in an atmosphere of 5% CO2 for 10 days, then stained with 0.3% crystal violet solution. The colonies >60 μM in diameter were counted under the light microscope. The experiments were done in three independent repeats.This assay was performed as we previously described.39 To knock down USP14 expression in prostate cancer cells, siRNA or shRNA targeting human USP14 were synthesized and purchased from Santa Cruz Biotechnology Inc.. siRNA or shRNA with non-specific sequences were used as control scrambled RNA. Different siRNAs and shRNAs were transfected separately into cells using Lipofecatmine 2000  reagent and the medium was replaced 6 h after the transfection.This assay was performed as we previously described.Lentivirus (pLent-EF1a-FH-CMV-GP) overexpressing Flag-USP14 (NM-005151) or containing control vector was purchased from VigeneBio . Exponentially growing LNcap cells were seeded in six-well plates. After culture overnight and reaching 50% confluence, medium containing lentiviruses and polybrene  was added at a multiplicity of infection of 10 and mixed with the cells. After incubation overnight, the supernatant at each well was replaced with RPMI 1640 containing 10% FBS and cultured for 48 h. For selection of stably transfected cells, we proceeded with puromycin selection as follows: replacing the medium with fresh medium containing puromycin (Santa Cruz) at the concentration of 1 μg/ml. Every 2 days, the medium was replaced with freshly prepared selective medium to culture the survived cells.Total RNA was isolated from the pooled cells using an RNeasy Mini Kit  according to the manufacturer's instructions. The total RNA was digested by DNase I  to remove genomic DNA contamination. The purified total RNA was measured at 260 and 280 nm using a Bio-Rad SmartSpec 3000 spectrophotometer . The 260:280 nm ratios and a 1% agarose-formaldehyde gel stained with ethidium bromide were used to verify the quality of the RNA in each sample. The first-strand cDNA was synthesized from 1 μg total RNA using a PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology). The expression levels of target genes were detected by real-time quantitative PCR, with the housekeeping gene GAPDH being used as an internal control. SYBR Green PCR Master Mix reagent kits  were used according to the manufacturer's instructions for quantification of gene expression with a 7500 real-time PCR system (Applied Biosystems Inc.). PCR primers are as following, AR forward: 5′-GGTGAGCAGAGTGCCCTATC-3′, AR reverse: 5′-GAAGACCTTGCAGCTTCCAC-3′ PSA forward: 5′-AGGCCTTCCCTGTACACCAA-3′, PSA reverse: 5′-GTCTTGGCCTGGTCATTTCC-3′ GAPDH forward: 5′-TCCCATCACCATCTTCCA-3′, GAPDH reverse: 5′-CATCACGCCACAGTTTCC-3′. The cycling conditions were as follows: 95 °C for 10 s followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. After PCR, a melting curve analysis was performed to demonstrate the PCR product specificity. Every sample was analysed in triplicate. The relative expression level of a target gene was presented as the sample versus the control.40 In brief, an equal amount of total protein extracted from cultured cells were separated by 12% SDS–PAGE and transferred to polyvinylidene difluoride membranes. The blots were blocked with 5% milk for 1 h. Primary Abs and horseradish peroxidase-conjugated secondary Abs were each incubated for 1 h. The bounded secondary antibodies were reacted to the ECL detection reagents and exposed to X-ray films .For IP and western blot, dynabeads m-270 Epoxy (Invitrogen) coupled with antibodies were prepared and then cell lysates were added, and the antibodies−lysate mixtures were rotated at 4 °C for 1 h. Immunocomplexes separated from the dynabeads were washed with lysis buffer and then suspended with SDS blue loading buffer. To detect ubiquitinated proteins, lysis was performed under 80 °C for 10 min. Western blot analysis was performed as we described previously.t-test or one-way ANOVA is used where appropriate for determining statistic probabilities. GraphPad Prism5.0 software (GraphPad Software) was used for statistical analysis. P value less than 0.05 was considered statistically significant.Mean±S.D. are presented where applicable. Unpaired Student's


LncRNA-IL7R) in cervical cancer is limited. We, here, investigated the clinical role of inflammation-related LncRNA-IL7R (Lnc-IL7R) in healthy cervical tissue (n=15), CIN 1/2/3 (n=35), cervical cancer (n=70), and clarified its function via knockdown in vitro and in vivo. The results showed that the expression of Lnc-IL7R was increased from normal tissues to neoplastic lesions and cervical cancer. Up-regulated Lnc-IL7R positively correlated to tumor size, International Federation of Gynaecology and Obstetrics (FIGO) stage, and lymph node metastasis (LNM). Patients with high expression of Lnc-IL7R had poor prognosis with short overall survival (OS) time, and Cox regression analysis revealed that Lnc-IL7R could be independent prognostic factor for cervical cancer. Moreover, knockdown of Lnc-IL7R by two different siRNAs in cervical cancer cell lines Hela and SiHa induced impaired cell vitality and caspase-3-dependent apoptosis in vitro. Furthermore, inhibition of Lnc-IL7R in vivo significantly restricted the tumor growth with decreased expressions of proliferation index Ki-67 and Lnc-IL7R. These data indicated that Lnc-IL7R predicts a poor clinical outcome of cervical cancer patients, and knockdown of Lnc-IL7R is amenable to the treatment of cervical cancer.The long-term chronic inflammation of cervical intraepithelial neoplasia (CIN) induces the initiation and progression of cervical cancer. Long non-coding RNAs (LncRNAs) are being identified to be involved into inflammation and carcinogenesis and could function as cancer biomarkers in clinical. However, the significance of inflammation-related LncRNA (e.g.  Cervical cancer is the major cause of death from gynecological cancers and is the third most common malignancy in women worldwide with a global incidence of 500000 and mortality of 250000. More than 85% of these cases and deaths occurred in developing countries, including China ,2. PersimiR-138 expression in cervical cancer cells is significantly lower than that in normal tissues, which causes telomerase activation and carcinogenesis [miR-23b, miR-143, and miR-196b were down-regulated in cell lines and tumor tissue compared with normal tissue whereas miR-21 was up-regulated [MALAT1 and HOTAIR were reported to predict the poor clinical outcome of patients with cervical cancer [The majority of transcribed RNAs are non-coding in mammalian cells, which do not contain protein-coding sequences. These transcripts are eventually, on one hand, processed into small RNAs including miRNAs, Piwi-interacting RNAs (piRNAs), tRNA-derived stress-induced fragment RNAs, and small nucleolar RNAs (snoRNAs), and, on the other hand, processed into long non-coding RNAs (LncRNAs) ,7. The mogenesis . Let-7b,egulated . Up-reguegulated . The tumegulated . SimilarLncRNA-IL7R (Lnc-IL7R) was capable of diminishing the LPS-induced inflammatory response, inhibiting the expressions of LPS-induced E-selectin, VCAM-1, IL-6, and, IL-8. Ding et al. [Lnc-IL7R was also induced in response to TLR3 stimulation and negatively regulated the TNF-α and IL-8. Lnc-IL7R has been found to participate in multiple sclerosis, chemotherapy, and acute respiratory distress syndrome (ARDS) [Lnc-IL7R levels were correlated with the severity of ARDS and could predict 28-day mortality in the patient’s cohort [Lnc-IL7R in cervical cancer. The expression of Lnc-IL7R in normal, CIN, and cervical cancer samples, and its correlation to clinical characteristics were analyzed. The functions of Lnc-IL7R in vitro and in vivo were also assessed in cervical cancer cell lines Hela and SiHa.Cui et al.  found ang et al.  reportede (ARDS) . The Lncs cohort . In the n=15), CIN 1/2/3 (n=35), cervical cancer (n=70) were collected to determine the expressions of Lnc-IL7R and TNF-α, and median age was 35, 37, and 51 years, respectively. The sample collection in the present study was approved by Yan’an People’s Hospital and all patients completed informed consent forms and the healthy individuals’ recruitment were also obtained from Yan’an People’s Hospital. All these retrospective specimens were handled and anonymized according to ethical and legal standards. The tissue samples were isolated from surgical removal and then stored at −80°C until use.Healthy cervical tissue (Lnc-IL7R were defined by the median of the expression. The three grades of Lnc-IL7R expression were defined as Grade 1 (25th percentile), Grade 2 (25–50th percentile), and Grade 3 (>50th percentile).The patients with primary cervical cancer were diagnosed by Hematoxylin and Eosin staining by experienced pathologists from Department of Pathology at Yan’an People’s Hospital. None of the patients underwent preoperative chemotherapy and/or radiotherapy. Patients with other kinds of cancer or some autoimmune disease  were absolutely excluded. Besides, pregnant and lactating individuals were also excluded from the present study. The 70 patients were followed up until 1 October 2017. The high or low expressions of CT−ΔΔ method was used to estimate relative expression changes in genes including Lnc-IL7R and TNF-α. The expression levels were normalized to GAPDH for gene expression.Total RNA was extracted using TRIzol reagent (Invitrogen) according to standard RNA isolation protocol. The conditions of reverse-transcription for cDNA with Reverse Transcription System were 42°C for 5 min, 99°C for 20 min, 4°C for 5 min. The SYBR Green PCR Master Mix (Applied Biosystems), according to the manufacturer’s instructions, was performed for quantitative real-time RT-PCR (qRT-PCR) and 22 conditions. siRNA-Lnc-IL7R or negative control was purchased from RiboBio . Anti-GAPDH, Ki-67, caspase-3, and Bcl-2 antibodies were obtained from Cell Signaling Technology  and Abcam (U.S.A.).The cervical cancer cell lines Hela and SiHa were purchased from the cell bank of the Chinese Academy of Sciences  and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS , ampicillin and streptomycin at 37°C, 5% COThe Hela and SiHa cell lines were cultured to ~80% confluence in 12/96-well plates and then, using Lipofectamine 2000 , the cells were transfected with indicated agents according to the manufacturer’s instructions. After transfection for the indicated time, the cells were harvested for further experiments.The Hela and SiHa cell lines were transfected with siRNAs and then were harvested to wash with PBS, and then cell counting kit-8  mixed with DMEM was used for cell viability assay, and the absorbance was measured at 450 nm by a microplate reader.The conditional Hela and SiHa cell lines were harvested and incubated with Hoechst 33342  for 10 min at room temperature. Following washing with 0.5% Triton X-100 in PBS, the changes in nuclear morphology were observed under a fluorescence microscope . Each experiment was performed in triplicate and repeated three times.According to the manufacturer’s protocol, cells for Western blot were collected and total protein was isolated from the cell samples. Detailed procedures for immunoblotting are described elsewhere . GAPDH wTo determine the expression of Ki-67 in tissues, 2-μm thick, formalin-fixed, and paraffin-embedded specimen sections were used. After the slides were incubated in xylene for 5 min, 100% ethanol was used for 10 min, 95% ethanol for 10 min. Antigen unmasking was performed and then the slides were blocked with 3% hydrogen peroxide for 30 min at room temperature. Then the primary antibody for Ki-67 was incubated the FFPE specimen sections at 4°C overnight, then the biotinylated horse secondary antibody and streptavidin-horseradish peroxidase (Zymed Laboratories Inc.) were used for the detection of Ki-67. After that, the EnVision Detection System kit  was used for the DAB chromogen followed by nuclear staining using Hematoxylin.Lnc-IL7R in vivo, Hela cells were transfected with lentivirus vector of siRNA-Lnc-IL7R or negative control, 2 × 106 Hela cells were subcutaneously injected in rear flank of nude mice (five per group). The tumor sizes were measured 3 days apart and the tumor volumes were calculated: V (cm3) = width2 (cm2) * length (cm)/2. On day 2, the mice were killed.To investigate the role of Lnc-IL7R in each group did not follow a normal distribution. The Mann–Whitney U-test was used to compare the two groups  and the differences between more than two groups (e.g. CIN1/2/3) were analyzed by the Kruskal–Wallis test. Kaplan–Meier survival curves and the log-rank statistic were used to analyze the prognostic significance of Lnc-IL7R. Correlations between the expression of Lnc-IL7R and TNF-α were analyzed by Spearman’s Rho analysis. Correlations of expression of Lnc-IL7R and clinicopathological characters were analyzed by Pearson chi-square. Cox proportional hazards regression was used for univariate and multivariate analysis of OS according to Lnc-IL7R expression. P<0.05 was considered statistically significant. All experiments were performed at least three times.The results were analyzed by the Statistical Package for Social Sciences version 16.0  and the Prism statistical software package . Kolmogorov–Smirnov and Shapiro–Wilk tests showed that the expression of Lnc-IL7R in cervical cancer, normal cervix (n=15), (CIN1/2/3) (n=35), and cervical cancer samples (n=70) were collected and the results of Q-PCR indicated that the expression of Lnc-IL7R was increased during the development of cervical cancer. The cancer tissues harbored the highest Lnc-IL7R level  of normal cervix showed the low expression of Lnc-IL7R and only 3 in 15 (20%) showed the high expression of Lnc-IL7R. But in the CIN samples, the high expression of Lnc-IL7R accounted for (57%) 20 in 35, which was further increased to (59%) 41 in 70 in cervical cancer  have more high-expressed Lnc-IL7R. These data implicated that, in patients with cervical cancer, Lnc-IL7R could predict a poor clinical outcome including tumor size, FIGO, and LNM.Since the Lnc-IL7R was also investigated in the present study. The 70 patients were followed up until 1 October 2017. The OS time was analyzed according to the expression of Lnc-IL7R, which indicated that patients with highly expressed Lnc-IL7R had shorter OS than those with lowly expressed Lnc-IL7R (chi square = 5.605 by log-rank test) (The prognostic role of nk test) D.Lnc-IL7R were the independent prognostic factors for the OS of patients with cervical cancer   D.Lnc-IL7R in vitro, we next estimated the efficiency of Lnc-IL7R knockdown for tumor progression by lentivirus vector of siRNA-Lnc-IL7R in vivo. The xenograft model of human HeLa was established. The results showed that the inhibition of Lnc-IL7R could effectively inhibit tumor growth  demonstrates that down-regulation of seven miRNAs  associated with advanced-stage SCCC patients (FIGO IB2-IV) compared with early-stage SCCC patients (FIGO IB1), six miRNAs with metastasis, and two with poor prognosis  [LncRNA-MALAT1 was significantly increased in cervical cancer than in normal tissues and correlated with tumor size, FIGO stage, vascular invasion, and LNM and is an independent predictor for OS of cervical cancer. LncRNA CCAT2 was also found to be up-regulated in cervical squamous cell cancer tissues, patients with high expression of lncRNA CCAT2 had poor OS and PFS rates and was an independent poor prognostic factor for cervical cancer patients [Lnc-IL7R in cervical cancer, the expression of Lnc-IL7R was elevated in CIN tissues and cervical cancers, which predicted the poor clinical outcome of patients and could be an independent factor for cervical cancer. This finding suggested that the inflammation-related Lnc-IL7R functioned as an oncogene in cervical cancer.The non-coding RNAs (ncRNAs) participate in the post-transcription of gene expressions or interact with proteins to regulate the target mRNAs and proteins that were involved into carcinogenesis . AmountiiR-125b) . Yang etiR-125b)  reportedpatients . In the in vivo was limited. Using the siHOXA11-AS-transfected HeLa cells revealed that HOXA11-AS strongly induced tumor growth in xenograft experiments with the decreased cancer stemness and triggered the EMT program [in vivo and decrease the expression of Ki67.The potential mechanisms of LncRNA in cervical cancer had been reported. Kim et al.  found th program . We founLnc-IL7R is increased during the development of cervical cancer, and could be an independent factor for the patients with cervical cancer. Knockdown of Lnc-IL7R inhibited the tumor growth in vivo, which could be a potential treatment for cervical cancer.We reported that an oncogene


In view of the fact that JS-K might regulate ubiquitin E3 ligase and that ubiquitin E3 ligase plays an important role in the mechanism of CRPC formation, the goal was to investigate the probable mechanism by which JS-K regulates prostate cancer cells.Proliferation inhibition by JS-K on prostate cancer cells was examined usingCCK-8 assays. Caspase 3/7 activity assays and flow cytometry were performed to examine whether JS-K induced apoptosis in prostate cancer cells. Western blotting and co-immunoprecipitation analyses investigated JS-K’s effects on the associated apoptosis mechanism. Real time-PCR and Western blotting were performed to assess JS-K’s effect on transcription of specific AR target genes. Western blotting was also performed to detect Siah2 and AR protein concentrations and co-immunoprecipitation to detect interactions of Siah2 and AR, NCoR1 and AR, and p300 and AR.JS-K inhibited proliferation and induced apoptosis in prostate cancer cells. JS-K increased p53 and Mdm2 concentrations and regulated the caspase cascade reaction-associated protein concentrations. JS-K inhibited transcription of AR target genes and down-regulated PSA protein concentrations. JS-K inhibited Siah2 interactions and also inhibited the ubiquitination of AR. With further investigation, JS-K was found to stabilize AR and NCoR1 interactions and diminish AR and p300 interactions.The present results suggested that JS-K might have been able to inhibit proliferation and induce apoptosis via regulation of the ubiquitin-proteasome degradation pathway, which represented a promising platform for the development of new compounds for PCa treatments.The online version of this article (doi:10.1186/s12885-017-3351-0) contains supplementary material, which is available to authorized users. Nitric oxide (NO), which was found in 1987 to be a physiological constituent, and in the following years, found to be synthesized in vivo, work as a signal molecule, toxicant, and antioxidant with a broad spectrum of actions among physiological and pathological processes , 3. As iChemical agents with stabilize NO release have been developed as NO’s limitations, such aqueous solubility and instability in the presence of various oxidants, have become better understood. One such effective NO pro-drug is JS-K-1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate), a new nitric oxide donor that belongs to the diazeniumdiolate family of compounds. It has been designed to release NO within a cell in a sustained and controlled manner during its reaction with glutathione-S-transferase (GST), which is often overexpressed in cancer cells . RecentlProstate cancer (PCa) is the most commonly diagnosed neoplasm in elderly men and the second greatest cause of cancer-related deaths in the United States . AndrogeThe ubiquitin-proteasome pathway works in multiple steps. First, ubiquitin is activated from its precursor by addition to the ubiquitin-activating enzyme (E1); second, the activated ubiquitin is transferred to the ubiquitin-conjugating enzyme (E2); third, E2 interacts with ubiquitin-protein ligase (E3) and transfers ubiquitin to the target protein and ubiquitin; and finally, selective tagging and degradation of specific intracellular proteins are allowed according to the type of ubiquitin modification on protein substrates –17. AlthOncogene has shown thatMdm2 is an ubiquitin ligase E3 that auto-ubiquitylates itself and also ubiquitylates p53, resulting in degradation of both proteins. Furthermore, JS-K inhibits Mdm2-mediated p53 ubiquitylation, leading to p53 accumulation in Tert-immortalized, human retinal pigment, epithelial (RPE) cells , and Cell lysates were cleared by centrifuging at 14,000×g for 10 min at 4 °C. After the supernatant was incubated overnight at 4 °C with suitable dilutions of the primary antibody, Protein A/G Agarose  was added, and incubated for additional 4 h at 4 °C. Washed precipitated proteins were analyzed by Western blot.Cells were washed with PBS prior to cell lysis in 1 ml of IP lysis buffer . Significant difference was inferred for 50, Fig. First, the inhibitory effects of proliferation by JS-K onC4-2 and LNCaP cells were investigated. JS-K inhibited growth of C4-2 and LNCaP cells in a time-dependent manner  were clearly decreased Fig.  and b, wIn humans, Siah2 regulates ubiquitination-dependent degradation of multiple substrates. Siah2-mediated proteasomal degradation of NCoR1-bound AR  on PSA promoter allows subsequent recruitment of p300-bound AR , leading to an increase in PSA gene transcription . In addiTo understand if JS-K affected NCoR1 and p300 concentrations, Western blotting analyses were performed and it was found that NCoR1 concentrations increased while p300 concentrations diminished Fig.  and dimiRecently, it has become known that current treatments of advanced PCa, based on androgen ablation therapies such as surgical and chemical castration, are very effective treatments initially, but almost all cases progress to CRPC eventually. Accumulating evidence has revealed that in nearly all cases resumption of AR transcription activity contributes to CRPC progression . A recenInterestingly, NO inhibition of AR-function in PCa cells was first described in vitro using the NO-donor DETA/NO. This study showed that NO inhibited AR-mediated genomic function by preventing its DNA-binding activity while not decreasing AR protein concentrations or decreasing nuclear AR translocation . JS-K, aIn this study, JS-K was shown to induce apoptosis in the PCa cell lines LNCaP and C4-2. As p53 operates as a key regulator in the apoptotic process, JS-K was reasonably expected to induce apoptosis by modulating p53. As is known, Mdm2, an ubiquitin ligase E3, is involved in p53 ubiquitin-proteasome degradation. In addition, evidence has shown that JS-K inhibits p53 degradation mediated by Mdm2 in RPE cells . HoweverA study has revealed that JS-K inhibits PCa cell proliferation through inhibition of the AR signaling pathway; this study is the only report reporting JS-K’s impact on PCa cells . CronaueThe present results suggested that JS-K was in a position to inhibit proliferation and induce apoptosis through probable regulation of the ubiquitin-proteasome degradation pathway. Taken together, it would be of high interest to further investigate whether JS-K regulates interactions between AR-bound co-regulators and AREs of specific AR target genes. These findings warrant further investigation to facilitate potential development of AR-based prognostic and therapeutic approaches.Additional file 1:50 of LNCaP and C4-2 cell lines that treated by JS-K. (XLSX 10 kb)IC50 results. The ICAdditional file 2:western blot results. The quantitation of western blot of relative protein of LNCaP and C4-2 cell lines that treated by JS-K. (XLSX 38 kb)