Curcumin reversed cigarette smoke extract-induced epithelial-to-mesenchymal transition of human bladder cancer cell UMUC3 through suppression of ERK1/2 pathway

Curcumin reversed cigarette smoke extract-induced epithelial-to-mesenchymal transition of human bladder cancer cell UMUC3 through suppression of ERK1/2 pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Curcumin reversed cigarette smoke extract-induced epithelial-to-mesenchymal transition of human bladder cancer cell UMUC3 through suppression of ERK1/2 pathway Xin Sun, Tao Zhang, Liang Tang, Jie Min, Dexin Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1841893/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cigarette smoke (CS) is a main risk factor for bladder cancer (BC). The epithelial-to-mesenchymal transition (EMT), termed as an embryonic program, participates in the development process of CS-associated BC. Over the last decade, the effects and mechanisms of curcumin on CS-related BC have been extensively investigated. However, it's still elusive if MAPK pathways participate in curcumin-associated protective effect against CS-related promotive role in BC. In this study, our results showed that the exposure of BC cells UMUC3 to cigarette smoke extract (CSE) induced morphology variation, reinforced migratory and invasive abilities, decreased epithelium biomarker expression and increased mesenchyme biomarker expression. Meanwhile, CSE administration led to the stimulation of extracellular regulated protein kinases 1 and 2 (ERK1/2) and activator protein 1 (AP-1) proteins, without validly activating the Jun N-terminal kinase (JNK), p38 or ERK5 pathway. Moreover, CSE-elicited EMT and ERK1/2/AP-1 stimulation were completely diminished by U0126, a pharmacological inhibitor targeting ERK1/2. Furthermore, the co-treatment with low concentrations of curcumin significantly abolished CSE-elicited EMT in UMUC3 cells, and the ERK1/2/AP-1 pathway was implicated in the suppressive effect. In summary, these data indicated the modulatory effects of ERK1/2 on CSE-elicited urocystic EMT in vitro and the chemoprevention efficacy of curcumin. Cigarette smoke extract Bladder cancer Epithelial-to-mesenchymal transition ERK1/2 Curcumin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction BC is a commonly seen malignant cancer worldwide, which accounts for about 500000 newly diagnoses and 200000 mortalities [ 1 ]. The estimated incidence and mortality of BC in the United States are 81180 and 17100, respectively [ 2 ]. CS is a pivotal risk factor for BC, with a population-attributable risk of nearly 50% [ 3 ]. A previous population-based case-control research indicated that CS enhanced the risks of superficial and aggressive BC, and if the the staging was more advanced, the effect would be more obvious [ 4 ]. In addition, CS was involved in the chemotherapy resistance, disease recurrence, metastasis, and mortality of BC [ 5 , 6 ]. To date, the positive relationship between CS and BC has been demonstrated by numerous epidemiologic and experiment-based studies [ 3 – 8 ]. Nonetheless, the molecule-level causal links for valid diagnoses and treatment strategies against CS-related BC are still elusive. EMT is a cell process where cells lose their epithelium features and obtain mesenchyme traits. Mounting evidences have suggested that EMT is related to tumor initiation, invasion, migration, and resistance to therapies [ 9 ]. Several active compounds found in CS, such as nicotine [ 10 ], polycyclic aromatic hydrocarbons [ 11 ], and nicotine-originated nitrosamine ketone [ 12 ], have been discovered to promote EMT in vivo and in vitro through various signaling pathways. Moreover, the effect of CS-related EMT on BC has also been noted [ 7 , 8 , 13 , 14 ]. Previously, we reported that CSE exposure resulted in the EMT changes of human BC cells T24 [ 8 ]. However, the latent causal links of CS-elicited EMT are still elusive. The following four MAPK cascades have been observed in eukaryotic cells: ERK1/2, C-JNK, p38 MAPK, and ERK5 [ 15 ]. The facts that these signaling pathways lead to altered gene expression and regulate multiple biological processes are well established in human beings [ 16 ]. Researches have revealed that the dysfunction of ERK1/2 pathway is associated with the pathogenesis, progression, and oncogenic behaviour of BC [ 7 , 8 , 16 , 17 ]. The ERK1/2 pathway is one of the most important pathways for EMT [ 17 ]. Mounting proofs have revealed that the stimulation of ERK1/2 is crucial for bladder epithelial differentiation [ 8 , 9 , 14 , 15 ]. In addition, numerous researches have unveiled the significant effects of ERK1/2 on CS-induced pathophysiological processes in vitro, such as cell proliferation [ 18 ], inflammation [ 19 ], oxidative stress injury[ 20 ], autophagy[ 21 ], and EMT[ 7 , 8 ]. Nevertheless, the roles of ERK1/2 in CS-related BC are still unknown. Curcumin is an active compound derived from the turmeric rhizome curcuma longa. Curcumin has anti-oxidation, antiinflammation, anti-microbe and anti-virus attributes [ 22 ]. Over the past decade, the inhibition effect of curcumin on EMT triggered by various stimuli, such as CS, has been extensively investigated both in vitro and in vivo [ 23 ]. Liang and colleagues found that curcumin reversed chronic tobacco smoke-triggered EMT in SV-HUC-1 cells and murine bladders [ 24 , 25 ]. Alike outcomes were found in mankind pulmonary carcinoma lineage cells ( H1299 and A549) [ 26 ], kidney cell cancer lineage cells (786-O and ACHN) [ 27 ], mouse lungs [ 28 ], mouse livers [ 29 ], and mouse stomach tissues [ 30 ]. It was reported that to evade chemoresistance, curcumin suppressed EMT and enhanced the antiproliferative capacity of conventional chemotherapeutics [ 23 ]. The chemo-preventive ability of curcumin in targeting EMT and modulating associated pathways, such as ERK1/2, has also been proposed in several studies [ 23 , 25 , 28 – 30 ]. We sought to assess the association between CS and BC progression, as well as the protective effect of curcumin in vitro. In the present study, we discovered that CSE induced EMT changes in human BC cells UMUC3. Moreover, CSE-elicited EMT was linked with the stimulation of the ERK/2/AP-1 pathway, which could be restored by U0126. Furthermore, our study indicated that curcumin avoided CSE-elicited ERK1/2/AP-1 stimulation and abolished CSE-elicited EMT. Collectively, these data suggested the significance of ERK1/2 in the regulation of CSE-activated EMT and CS-associated BC facilitation effect, and our results revealed the chemopreventive effects of curcumin. Materials And Methods Materials and reagents Curcumin was obtained from Sigma (America, purity: 99.0%). DMSO and methyl alcohol were provided by Merck (Reading Township, America). The first antibodies for phosphorylated ERK1/2, JNK, p38, ERK5, c-Jun, and c-Fos were provided by CST (America). Polyclone antibodies against ZO-1, E-cadherin, N-cadherin, Snail, U0126 and GAPDH were bought from Santa Cruz (America). Primers for ZO-1, E-cadherin, N-cadherin, Snail and GAPDH were produced by Invitrogen (America). Cellular cultivation and assay Human BC lineage cell UMUC-3 was purchased from American Type Culture Collection (ATCC, America). UMUC3 cells were cultured in DMEM added with 10% FBS (HyClone, America), and then incubated at 37 °C in a 5% CO 2 environment. Cells were placed in 25-cm 2 flasks, and the intermediary was changed every other day. When 80-90% confluency was reached, cells were exposed to indicated levels of CSE for 6 days, with or without U0126 (5 μΜ) or curcumin (1 μΜ). CSE was produced every day prior to utilization, as per a method described in the past [7, 8]. MTT assay Cell viability was identified via MTT analysis. UMUC3 cells (2.0×103 cells/well) were inoculated into 96-well dishes and cultivated for one night in DMEM involving 10% FBS. Subsequently, cells were incubated with different levels of CSE (0%-5%) in the absence or presence of curcumin (0.1, 1, 10 μM) for 6 days. After treatments, cells were incubated with solution for 4 hours. Posterior to the removal of the MTT liquor and the solubilisation of crystals in DMSO, the OD was identified via a micro-plate reading device (Thermo Scientific, China) at a wave length of 490 nm. Transwell assay Cell migration assay was completed in transwell chambers (Millipore, America) using an 8 µm filter. Briefly, UMUC3 cells were subjected to pretreatment for 6 days, afterwards they were subjected to resuspension in 100 μl intermediary with 2% serum (1 × 10 4 cells), and they were cultivated upper chambers with 800 μl intermediary involving 10% serum in lower chambers. Afterwards, methyl alcohol was utilized to realize the fixation of cells, and cells were dyed in Giemsa. After that, cells on the top surface of the film were removed, and cellular samples on the lower surface were subjected to counting and imaging in 5 stochastic fields of 100 amplification under a microscopic device. Cellular quantity was subsequently analyzed statistically. Moreover, 50 µl matrigel (BD Bioscience, America) was supplemented into upper chambers for invasion analyses. Western blotting (WB) analysis UMUC3 cells were washed twice by pre-cooled PBS, and subjected to lysis in RIPA buffering solution (Thermo Scientifific, China). The BCA protein quantitative tool (Pierce, America) was utilized to identify protein contents. Then, protein specimens were subjected to 10% SDS-PAGE and moved to PVDF films (Millipore, America). These films were subjected to blockade in 5% skinmilk and afterwards probed with the first antibody for one night under 4°C and subsequently cultivated with HRP-conjugated second antibody. The blots were afterwards studied via a reinforced chemiluminescent identification tool (Amersham Bioscience, America). GAPDH was the load control. For densitometry assay, protein bands on the blots were identified via the ImageJ program 1.4 (America). Quantitative Real-time PCR (qRT-PCR ) Overall RNA was separated via RNAiso Plus as per the supplier's protocol (TaKaRa, Japan). Subsequently, 2 mg overall RNA was converted to cDNA via reverse transcription through AMV reversed transcriptive enzyme (TaKaRa). qRT-PCR was completed via the Power SYBR Green Master Mix (TaKaRa) and an ABI 7300 real-time PCR identification apparatus (Applied Biosystem) under default status: 95℃ for 10 s, and 40 cycles of 95℃ for 10 s and 72℃ for 0.5 min. The primers utilized are: GAPDH, forward 5′-GCTGCCCAACGCACCGAATA-3′ and reverse 5′-GAGTCAACGGATTTGGTCGT-3′; ZO-1, forward 5′-GCAGCCACAACCAATTCATAG-3′ and reverse 5′-GCAGACGATGTTCATAGTTTC-3′; E-cadherin, forward 5′-TCGACACCCGATTCAAAGTGG-3′ and reverse 5′-TTCCAGAAACGGAGGCCTGAT-3′; N-cadherin forward 5′-ATCAAGTGCCATTAGCCAAG-3′ and reverse 5′-CTGAGCAG TGAATGTTGTCA-3′; Snail, forward 5′-TTCCAGCAGCCCTACGACCAG-3′ and reverse 5′-CGGACTCTTGGTGCTTGTGGA-3′. The expressing levels of mRNA for every gene were normalised by its GAPDH respectively. Fold changes in genetic expression were computed through the comparative threshold cycle (Ct) approach via the 2 -(ΔΔCt) . Statistical Analysis Data were analyzed by the SPSS 22.0 software (SPSS, USA), and shown as mean±standard deviation. Unpaired Student’s t-test was utilized to compare the diversities of samples between 2 groups. One-way ANOVA was utilized for multi-group comparison, before the LSD significant difference test. P<0.05 had significance on statistics. Results CSE elicited EMT in UMUC3 cells To investigate the association between CSE and BC metastases, human BC cells UMUC3 were cultivated with indicated levels of CSE for 6 days. Cellular activity was reduced under 5% CSE content posterior to the 6-day treatment (Fig. 1A). Hence, CSE concentrations of 0.5%, 0.75% were chosen for the subsequent procedures in our study. CS contributes to BC development and can also induce EMT of cells [7, 8, 13, 14]. In our study, CSE exposure resulted in an evident morphologic variation, from round to spindle-shaped mesenchyme cells (Fig. 1B). Transwell assay were completed, and the outcomes revealed that CSE treatment significantly enhanced UMUC3 cellular migratory and invasive abilities (Fig. 1C, D). Hence, our team studied the effects of CSE on EMT via identifying the expressing levels of EMT biomarkers. WB analysis unveiled that CSE treatment reduced the expressing levels of epithelium indicator proteins, like E-cadherin and ZO-1, and elevated the contents of mesenchyme indicator proteins, like N-cadherin and Snail (Fig. 2A, B), which coincided with the results of qRT-PCR analyses (Fig. 2C). Collectively, those results revealed that CSE induced EMT in UMUC3 cells. CSE increased ERK/AP-1 activation in UMUC3 cells To evaluate whether CSE-triggered EMT variations was related to the variations in MAPK stimulation, the expressing levels of overall and phosphorylated ERK1/2, p38, JNK, ERK5 were identified via WB analyses. It was found that CSE significantly elevated the expressing level of p-ERK1/2, whereas it exerted little influence on p-JNK, p-p38, and p-ERK5 in UMUC3 cells (Fig. 3A, B). Moreover, WB analyses unveiled increased expressing levels of down stream target AP-1 proteins, like p-c-Fos and p-c-Jun (Fig. 3C, D). Inhibition of ERK1/2 reversed CSE-triggered EMT in UMUC3 cells The aforementioned outcomes demonstrated that CSE-elicited EMT were related to the stimulation of ERK1/2/AP-1 in UMUC3 cells. To explored the role of ERK1/2/AP-1 pathway in this process, a specific ERK1/2 suppressor, U0126, was employed to realize the pretreatment of cells. Results revealed that U0126 reduced the expression levels of p-ERK1/2, p-c-Fos and p-c-Jun proteins in UMUC3 cells, as revealed by WB analyses (Fig. 4A, B). The morphologic assessment showed that U0126 decreased the mensenchyme morphology variations (Fig. 4C). Meanwhile, U0126 attenuated CSE-mediated migratory and invasive abilities of UMUC3 cells (Fig. 4D, E). Moreover, U0126 weakened the CSE-elicited reduction of E-cadherin and ZO-1 contents and attenuated the elevation of N-cadherin and Snail in UMUC3 cells (Fig. 5A, B). Alike outcomes were identified in qRT-PCR tests as well (Fig. 5C). Curcumin suppressed ERK1/2 pathway to prevent CSE-induced EMT in UMUC3 cells Previous studies reported that high concentrations of curcumin exerted anti-proliferative or cytotoxic effect on UMUC3 cells [31, 32]. The results of MTT showed that curcumin at a concentration of 10 μM exerted an evident impact on cellular activity (Fig. 6A). Consequently, 1 μΜ curcumin was selected for subsequent experiments. We found that curcumin exposure abolished the mensenchyme morphology variation in these cells (Fig. 6B). Besides, curcumin restored the migratory and invasive abilities of UMUC3 cells, as unveiled by transwell tests (Fig. 6C, D). In addition, our team discovered that curcumin reversed the CSE-activated decrease of E-cadherin and ZO-1 contents and the reinforcement of N-cadherin and Snail in UMUC3 cells via WB (Fig. 7A, B) and qRT-PCR analyses (Fig. 7C). Those results revealed that curcumin prevented CSE-elicited EMT in UMUC3 cells. For the sake of investigating the roles of curcumin in the CS-mediated stimulation of the MAPK path, our team studied the variations in MAPK stimulation after curcumin exposure. WB tests demonstrated that curcumin restored CSE-enhanced ERK1/2 activity and suppressed CSE-induced p-c-Fos and p-c-Jun stimulation. Moreover, we noted that curcumin had little effect on the protein expressions of p38, JNK, and ERK5 in UMUC3 cells (Fig. 7D, E). Discussion CS consists of over 5000 diverse xenobiotics chemical substances, namely, nicotine, polycyclic aromatic hydrocarbons, tar, nitrosamines, CO 2 , NO, and phenolics hydrocarbon. Amongst those CS components, almost 150 chemical substances are toxicants, and 60 chemical substances are carcinogenic agents [33]. CS is a major risk factor for BC progression, as shown repeatedly in multiple reports [8, 13, 34, 35]. Pietzak et al. indicated that compared with non-smokers at their initial presentation, heavy smokers were more likely to have a high-grade tumor and muscle-invasive BC [34]. The results of a meta-analysis suggested that long-term smokers were at elevated risks of having a more invasive BC related to poorer prognoses [35]. Similarly, some evidences have supported the cancer-promoting effect of CS on BC both in vitro and in vivo [8, 13, 36]. EMT is a pivotal cell program enabling polarised epitheliums to shift towards a mesenchyme phenotype with elevated cell migration ability. Proofs have revealed that EMT participates in facilitating cancer aggression and migration [37]. The effects of CS on EMT and tumor developmental process have been discussed previously [7, 8, 10-14, 24-30]. As aforementioned, CSE elicited EMT in UMUC3 cells, as indicated by the morphologic alteration from epitheliums to mesenchyme cells. It enhanced migratory and invasive abilities and caused variations in the expressing levels of EMT biomarker mRNAs and proteins, like E-cadherin, ZO-1, Snail and N-cadherin. Our data are consistent with previous findings, which unveiled that CSE elicited EMT in vitro. A commonly seen signal transduction by which the exocellular regulation of cell-surface acceptor activities is transmitted into a specific cell reaction is the three-tiered MAPK cascade, and the ERK1/2 path is the most broadly studied [38]. Abnormal ERK1/2 stimulation induces cancer development in various models and is usually found in mankind tumors [8, 24-30, 39-46]. ERK1/2 over expression in oncocytes, like human BC T24 cells [8], and mammary carcinoma cells (MDA-MB-231, BT-549, tamoxifen-resistant MCF-7) [39], displayed a metastasis and aggression phenotype, thus leading to enhanced migration abilities of cells. Moreover, the pharmacological inhibition of ERK1/2 suppressed cellular metastasis and aggression abilities in multiple types of oncocytes in vitro [8, 39], and it reduced the migration abilities of pancreatic carcinoma heterografts in vivo [40]. Mounting studies have found that ERK1/2 participates in regulating CS-induced urocystic, gastric, hepatic, pulmonary EMT in vivo [7, 25, 28-30]. In addition, abnormal ERK1/2 expression was associated with the poor prognosis of tumor sufferers [41], prostate cancer [42], type I endometrial cancer [43], multiple myeloma[44], hepatocellular carcinoma [45], breast cancer [39], and BC [46]. The Jun and Fos subfamilies are major AP-1 proteins [47]. Previous studies found that AP-1 overexpression existed in multiple tumor types, like colorectal carcinoma [48], mammary carcinoma [49], and so on. Moreover, AP-1 activation was discovered in CS-elicited EMT in vivo and in vitro [7, 8, 25, 28-30]. As a downstream target of the MAPK pathway, AP-1 participates in the mechanisms of cancer cell invasion, EMT, and metastases via facilitating the expressing of EMT-triggering transcriptional factors, cell factors, and miRNAs, therefore contributing to cancer progression [47]. In the present study, our results showed that the CSE-induced EMT of UMUC3 cells was related to the stimulation of ERK1/2 and AP-1 proteins (p-c-Fos, and p-c-Jun). The utilization of a pharmacological inhibitor of ERK1/2, namely U0126, terminated ERK1/2/AP-1 stimulation and abolished CSE-activated EMT. Those discoveries unveiled the latent causal links of CSE-triggered metastasis, aggression, and EMT in UMUC3 cells. Clinically, the secondary chemoprevention is most widely used in patients with precancerous lesions that may develop into invasive cancer. The definition of cancer chemoprevention, namely, utilizing synthetic, natural, or biological agents to minimize the incidence of cancer in healthy individuals [50]. A large amount of studies have reported the satisfactory results of utilizing traditional plant-derived chemicals to treat diseases. Curcumin is a natural compound, which has aroused scientists’ interest worldwide due to its biological activities, and it's one of the most prospective chemoprevention agents. Extant data strongly suggest that curcumin has protection efficacy against pathophysiologic pathways responsible for BC [22]. In recent years, mounting evidences have demonstrated that the strong chemoprevention effect of curcumin might be partially originated from the suppression of EMT [23]. Previous studies showed that curcumin suppressed long-term tobacco smoke-induced MAPK stimulation and EMT variations in various organs in vivo [25, 28-30]. Moreover, curcumin weakened CSE-elicited EMT in several types of cancer cells [26, 27]. High concerntrations of curcumin were identified to decrease the growth of BC cells in vivo and in vitro [31, 32, 51-52]. Alike outcomes were identified in our MTT assay as well. Herein, 1 μM curcumin was chosen to explore the underlying prevention efficacy against CSE-elicited EMT. A similar non-cytotoxic dosage of curcumin has also been reported in several experimental studies [31, 32]. We found that curcumin inhibited CSE-induced morphological alterations as well as the enhancement of cellular migratory and invasive abilities in UMUC3. Meanwhile, the deregulated expressions of EMT-related proteins, like E-cadherin, ZO-1, N-cadherin, and Snail, were reversed by curcumin. Moreover, CSE-induced ERK1/2/AP-1 stimulation was reduced after curcumin administration. The above data demonstrate the effects of ERK1/2/AP-1 on curcumin-based protection efficacy against CSE-triggered EMT in human BC cells UMUC3. In conclusion, our study demonstrated the effects of ERK1/2 pathway on regulating CSE-triggered EMT and revealed the protection efficacy of curcumin against CSE-induced EMT via ERK1/2 suppression. Those discoveries offer a novel method for preventing CS-associated BC. Declarations Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding This study was financed by the Provincial Natural Science Foundation of Anhui, China(No. 2018085MH290) and Natural science fund for colleges and universities in Anhui Province (No. KJ2021A0312). Author contributions Dexin Yu contributed to the study conception and design. Material preparation was performed by Xin Sun, data collection and analysis were performed by Tao Zhang, Liang Tang, and Jie Min. The first draft of the manuscript was written by Xin Sun and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. References Richters, A., Aben, K., & Kiemeney, L. (2020). The global burden of urinary bladder cancer: an update. World journal of urology, 38(8), 1895–1904. Siegel, R. L., Miller, K. D., Fuchs, H. E., & Jemal, A. (2022). Cancer statistics, 2022. CA: a cancer journal for clinicians , 72 (1), 7–33. 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High Expression of Phosphorylated Extracellular Signal-Regulated Kinase (ERK1/2) is Associated with Poor Prognosis in Newly Diagnosed Patients with Multiple Myeloma. Medical science monitor , 23 , 2636–2643. Gao, X., Shan, W., Liu, X., Zhang, J., Zheng, J., & Yao, H. (2018). JNK1/2 and ERK1/2 provides vital clues about tumor recurrence and survival in hepatocellular carcinoma patients. Future oncology (London, England) , 14 (24), 2471–2481. Karlou, M., Saetta, A. A., Korkolopoulou, P., Levidou, G., Papanastasiou, P., & Boltetsou, E., et al. (2009). Activation of extracellular regulated kinases (ERK1/2) predicts poor prognosis in urothelial bladder carcinoma and is not associated with B-Raf gene mutations. Pathology , 41 (4), 327–334. Trop-Steinberg, S., & Azar, Y. (2017). AP-1 Expression and its Clinical Relevance in Immune Disorders and Cancer. The American journal of the medical sciences , 353 (5), 474–483. Leupold, J. H., Asangani, I., Maurer, G. D., Lengyel, E., Post, S., & Allgayer, H. (2007). Src induces urokinase receptor gene expression and invasion/intravasation via activator protein-1/p-c-Jun in colorectal cancer. Molecular cancer research : MCR , 5 (5), 485–496. Tolza, C., Bejjani, F., Evanno, E., Mahfoud, S., Moquet-Torcy, G., & Gostan, T., et al. (2019). AP-1 Signaling by Fra-1 Directly Regulates HMGA1 Oncogene Transcription in Triple-Negative Breast Cancers. Molecular cancer research , 17 (10), 1999–2014. Bachmeier, B. E., Killian, P. H., & Melchart, D. (2018). The Role of Curcumin in Prevention and Management of Metastatic Disease. International journal of molecular sciences , 19 (6), 1716. Tian, B., Wang, Z., Zhao, Y., Wang, D., Li, Y., Ma, L., et al. (2008). Effects of curcumin on bladder cancer cells and development of urothelial tumors in a rat bladder carcinogenesis model. Cancer letters , 264 (2), 299–308. Pan, Z. J., Deng, N., Zou, Z. H., & Chen, G. X. (2017). The effect of curcumin on bladder tumor in rat model. European review for medical and pharmacological sciences , 21 (4), 884–889. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1841893","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":120069190,"identity":"1b1cf040-dd1d-4bb8-b300-fe8691c8874c","order_by":0,"name":"Xin Sun","email":"","orcid":"","institution":"the Second Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Sun","suffix":""},{"id":120069191,"identity":"2fd1b385-6664-4a34-b43c-e2f5b6aa8f8c","order_by":1,"name":"Tao Zhang","email":"","orcid":"","institution":"the Second Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Zhang","suffix":""},{"id":120069192,"identity":"70a190ef-1e97-42c3-b9e1-d976c4dde1d3","order_by":2,"name":"Liang Tang","email":"","orcid":"","institution":"the Second Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Tang","suffix":""},{"id":120069193,"identity":"66cf5b55-ab52-41ff-8564-4ecbdc4a4ae0","order_by":3,"name":"Jie Min","email":"","orcid":"","institution":"the Second Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Min","suffix":""},{"id":120069194,"identity":"bc4370e7-8150-47f3-b4af-c403d0aa8f4c","order_by":4,"name":"Dexin Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYHACAzDJByI+GNjIEa+FDYgZZxSkGZOmhZnnw+FEwupvJG+TYGy7J8fG3n5N2saAOYGB/fDRDfi1pBUbMLYVG7PxnCmTzjFgy2PgSUu7gV9LjuEDxraExDaJnDSgFp5iBgkeM0JaDA7AtVgYSCQ2EKEFZkv6MWkGAwPCWiTPPCs2YDiXAPILs2WPAYhBwC98x4EhxlCWIMfP3v7wxo8//4GMw8fwalE4AIyOP2Amj4kEiGLDpxwE5BvgTPbHHwipHgWjYBSMgpEJAL38RLcDdnBPAAAAAElFTkSuQmCC","orcid":"","institution":"the Second Hospital of Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Dexin","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2022-07-09 14:14:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1841893/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1841893/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23995540,"identity":"d70e53f6-f466-4da1-9efa-f7bdffdbb4e8","added_by":"auto","created_at":"2022-07-18 16:49:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1836149,"visible":true,"origin":"","legend":"\u003cp\u003eCSE elicited EMT morphologic changes and elevated migratory and invasive capabilities of UMUC3 cells. \u003cstrong\u003eA\u003c/strong\u003e Cells viabilities were examined after the 6-day exposure to diverse contents of CSE. \u003cstrong\u003eB\u003c/strong\u003e CSE induced morphologic variations from epitheliums to spindle-shaped mesenchyme cells. \u003cstrong\u003eC, D\u003c/strong\u003e CSE reinforced migratory and invasive capabilities of UMUC3 cells, as revealed by transwell migration and invasion tests. \u003cem\u003e**p \u003c/em\u003e\u0026lt; 0.01, \u003cem\u003e***p \u003c/em\u003e\u0026lt; 0.001, in contrast to the controls.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1841893/v1/ac12be72824d0ba4c8655d77.png"},{"id":23995975,"identity":"3a31fff0-3d08-4397-a3e9-7bebe8e09b77","added_by":"auto","created_at":"2022-07-18 16:54:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":632426,"visible":true,"origin":"","legend":"\u003cp\u003eCSE modulated the expressions of EMT biomarkers in UMUC3 cells. \u003cstrong\u003eA, B \u003c/strong\u003eCSE reduced the expressing levels of epithelium biomarkers, and elevated the expressing levels of mesenchyme cell biomarkers. \u003cstrong\u003eC\u003c/strong\u003e mRNA contents of ZO-1, E-cadherin, N-cadherin and Snail via qRT-PCR analyses, posterior to normalisation to GAPDH. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, in contrast to the controls.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1841893/v1/08015f4ba7be810f93a5a905.png"},{"id":23995974,"identity":"ba5205f6-1fd2-4952-a5c5-58e15d57e494","added_by":"auto","created_at":"2022-07-18 16:53:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1419165,"visible":true,"origin":"","legend":"\u003cp\u003eCSE-elicited EMT was associated with ERK1/2/AP-1 stimulation in UMUC3 cells. \u003cstrong\u003eA, B\u003c/strong\u003e CSE stimulated ERK1/2 in UMUC3 cells after the 6-day CSE exposure. Meanwhile, CSE had little effect on the activation of p38, JNK, or ERK5 in UMUC3. \u003cstrong\u003eC, D\u003c/strong\u003e CSE increased the expressing levels of AP-1 proteins (p-c-Fos and p-c-Jun). *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, in contrast to the controls.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1841893/v1/10b9d469cc5a90bf7ca72c0e.png"},{"id":23995537,"identity":"58aa0937-5156-4f2e-9705-360444be57b9","added_by":"auto","created_at":"2022-07-18 16:49:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2278716,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of ERK1/2 blocked CSE-triggered morphological alterations and migratory and invasive capabilities of UMUC3 cells. \u003cstrong\u003eA, B\u003c/strong\u003e Cells were exposed to diverse contents of CSE with/with no 5 μΜ U0126 for 6 days, followed by Western blot assay, which revealed that U0126 repressed CSE-induced ERK1/2 and AP-1 protein stimulation. \u003cstrong\u003eC\u003c/strong\u003e U0126 weakened the morphologic variation of UMUC3 triggered by CSE. \u003cstrong\u003eD, E \u003c/strong\u003eCSE-reinforced migratory and invasive capabilities of UMUCE cells were restored by U0126. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, in contrast to the controls; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, in contrast to the 0.75% CSE-exposed group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1841893/v1/224d41bc17a1665accab9c94.png"},{"id":23995535,"identity":"420705c0-a2e8-4e3e-a7f1-8a6eadb1670a","added_by":"auto","created_at":"2022-07-18 16:48:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":666517,"visible":true,"origin":"","legend":"\u003cp\u003eERK1/2 repression reversed CSE-elicited variations of EMT biomarkers in UMUC3 cells. \u003cstrong\u003eA, B \u003c/strong\u003eWestern blotting analyses showed that U0126 ameliorated the CSE-elicited downregulation of protein contents of ZO-1 and E-cadherin and that it ameliorated the upregulated expressing levels of N-cadherin and Snail proteins. \u003cstrong\u003eC \u003c/strong\u003eU0126 attenuated CSE-induced alterations of ZO-1, E-cadherin, N-cadherin and Snail mRNAs. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, in contrast to the controls; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, in contrast to the 0.75% CSE-exposed group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-1841893/v1/96cc8f51c2b3e5cf644a12ea.png"},{"id":23995976,"identity":"07aa56c5-253d-47f8-a922-ced3806ab251","added_by":"auto","created_at":"2022-07-18 16:54:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1871317,"visible":true,"origin":"","legend":"\u003cp\u003eCurcumin repressed CSE-induced morphologic variations and migratory and invasive capabilities of UMUC3 cells. \u003cstrong\u003eA\u003c/strong\u003e MTT analysis revealed that cellular activity was below 50% when UMUC3 cells were treated with 10 μM curcumin for 6 days. \u003cstrong\u003eB \u003c/strong\u003eMorphologic variations of UMUC3 cells caused by CSE were weakened by curcumin.\u003cstrong\u003e C, D \u003c/strong\u003eTranswell analysis unveiled that curcumin reduced the CSE-elicited migratory and invasive capabilities of UMUC3 cells. **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, in contrast to the controls; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, in contrast to the 0.75% CSE-exposed group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-1841893/v1/99ac7e59e0b25efa6fd2667b.png"},{"id":23995539,"identity":"fb95b66d-a670-47ac-a84e-03b92fe505b1","added_by":"auto","created_at":"2022-07-18 16:49:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":831286,"visible":true,"origin":"","legend":"\u003cp\u003eCurcumin rescued CSE-triggered changes of EMT markers’ expression and ERK1/2/AP-1 stimulation. \u003cstrong\u003eA, B\u003c/strong\u003e After UMUC3 cells were exposed to diverse contents of CSE with/with no 1 μΜ curcumin for 6 days, WB analysis revealed that curcumin repressed the CSE-elicited downregulation of protein contents of ZO-1 and E-cadherin, and it inhibited the upregulated expressing levels of N-cadherin and Snail proteins. \u003cstrong\u003eC \u003c/strong\u003eThe expression levels of ZO-1, E-cadherin, N-cadherin, and Snail were identified by qRT-PCR, after the normalization to GAPDH.\u003cstrong\u003e D, E \u003c/strong\u003eCurcumin abolished CSE-elicited ERK1/2/AP-1 stimulation, while it had little influence on p38, JNK, or ERK5, as indicated by WB analyses. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, in contrast to the controls; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, in contrast to the 0.75% CSE-exposed group.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-1841893/v1/312df6b363bd82347a8fa256.png"},{"id":24114340,"identity":"a7c1866b-338b-42af-932d-9f0666dbe80b","added_by":"auto","created_at":"2022-07-20 22:59:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8666108,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1841893/v1/ce3f619c-20c1-4059-8c85-053859939b66.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Curcumin reversed cigarette smoke extract-induced epithelial-to-mesenchymal transition of human bladder cancer cell UMUC3 through suppression of ERK1/2 pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBC is a commonly seen malignant cancer worldwide, which accounts for about 500000 newly diagnoses and 200000 mortalities [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The estimated incidence and mortality of BC in the United States are 81180 and 17100, respectively [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. CS is a pivotal risk factor for BC, with a population-attributable risk of nearly 50% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A previous population-based case-control research indicated that CS enhanced the risks of superficial and aggressive BC, and if the the staging was more advanced, the effect would be more obvious [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, CS was involved in the chemotherapy resistance, disease recurrence, metastasis, and mortality of BC [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. To date, the positive relationship between CS and BC has been demonstrated by numerous epidemiologic and experiment-based studies [\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Nonetheless, the molecule-level causal links for valid diagnoses and treatment strategies against CS-related BC are still elusive.\u003c/p\u003e \u003cp\u003eEMT is a cell process where cells lose their epithelium features and obtain mesenchyme traits. Mounting evidences have suggested that EMT is related to tumor initiation, invasion, migration, and resistance to therapies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Several active compounds found in CS, such as nicotine [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], polycyclic aromatic hydrocarbons [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and nicotine-originated nitrosamine ketone [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], have been discovered to promote EMT in vivo and in vitro through various signaling pathways. Moreover, the effect of CS-related EMT on BC has also been noted [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Previously, we reported that CSE exposure resulted in the EMT changes of human BC cells T24 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the latent causal links of CS-elicited EMT are still elusive.\u003c/p\u003e \u003cp\u003eThe following four MAPK cascades have been observed in eukaryotic cells: ERK1/2, C-JNK, p38 MAPK, and ERK5 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The facts that these signaling pathways lead to altered gene expression and regulate multiple biological processes are well established in human beings [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Researches have revealed that the dysfunction of ERK1/2 pathway is associated with the pathogenesis, progression, and oncogenic behaviour of BC [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The ERK1/2 pathway is one of the most important pathways for EMT [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Mounting proofs have revealed that the stimulation of ERK1/2 is crucial for bladder epithelial differentiation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, numerous researches have unveiled the significant effects of ERK1/2 on CS-induced pathophysiological processes in vitro, such as cell proliferation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], inflammation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], oxidative stress injury[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], autophagy[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and EMT[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Nevertheless, the roles of ERK1/2 in CS-related BC are still unknown.\u003c/p\u003e \u003cp\u003eCurcumin is an active compound derived from the turmeric rhizome curcuma longa. Curcumin has anti-oxidation, antiinflammation, anti-microbe and anti-virus attributes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Over the past decade, the inhibition effect of curcumin on EMT triggered by various stimuli, such as CS, has been extensively investigated both in vitro and in vivo [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Liang and colleagues found that curcumin reversed chronic tobacco smoke-triggered EMT in SV-HUC-1 cells and murine bladders [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Alike outcomes were found in mankind pulmonary carcinoma lineage cells ( H1299 and A549) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], kidney cell cancer lineage cells (786-O and ACHN) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], mouse lungs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], mouse livers [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and mouse stomach tissues [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It was reported that to evade chemoresistance, curcumin suppressed EMT and enhanced the antiproliferative capacity of conventional chemotherapeutics [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The chemo-preventive ability of curcumin in targeting EMT and modulating associated pathways, such as ERK1/2, has also been proposed in several studies [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe sought to assess the association between CS and BC progression, as well as the protective effect of curcumin in vitro. In the present study, we discovered that CSE induced EMT changes in human BC cells UMUC3. Moreover, CSE-elicited EMT was linked with the stimulation of the ERK/2/AP-1 pathway, which could be restored by U0126. Furthermore, our study indicated that curcumin avoided CSE-elicited ERK1/2/AP-1 stimulation and abolished CSE-elicited EMT. Collectively, these data suggested the significance of ERK1/2 in the regulation of CSE-activated EMT and CS-associated BC facilitation effect, and our results revealed the chemopreventive effects of curcumin.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCurcumin was obtained from Sigma (America, purity: 99.0%). DMSO and methyl alcohol were provided by Merck (Reading Township, America). The first antibodies for phosphorylated ERK1/2, JNK, p38, ERK5, c-Jun, and c-Fos were provided by CST (America). Polyclone antibodies against ZO-1, E-cadherin, N-cadherin, Snail, U0126 and GAPDH were bought from Santa Cruz (America). Primers for ZO-1, E-cadherin, N-cadherin, Snail and GAPDH were produced by Invitrogen (America).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellular cultivation and assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman BC lineage cell UMUC-3 was purchased from American Type Culture Collection (ATCC, America). UMUC3 cells were cultured in DMEM added with 10% FBS (HyClone, America), and then incubated at 37 \u0026deg;C in a 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eenvironment. Cells were placed in 25-cm\u003csup\u003e2\u003c/sup\u003e flasks, and the intermediary was changed every other day. When 80-90% confluency was reached, cells were exposed to indicated levels of CSE for 6 days, with or without U0126 (5 \u0026mu;\u0026Mu;) or curcumin (1 \u0026mu;\u0026Mu;). CSE was produced every day prior to utilization, as per a method described in the past [7, 8].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMTT assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was identified via MTT analysis. UMUC3 cells (2.0\u0026times;103 cells/well) were inoculated into 96-well dishes and cultivated for one night in DMEM involving 10% FBS. Subsequently, cells were incubated with different levels of CSE (0%-5%) in the absence or presence of curcumin (0.1, 1, 10 \u0026mu;M) for 6 days. After treatments, cells were incubated with solution for 4 hours. Posterior to the removal of the MTT liquor and the solubilisation of crystals in DMSO, the OD was identified via a micro-plate reading device (Thermo Scientific, China) at a wave length of 490 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell migration assay was completed in transwell chambers (Millipore, America) using an 8 \u0026micro;m filter. Briefly, UMUC3 cells were subjected to pretreatment for 6 days, afterwards they were subjected to resuspension in 100 \u0026mu;l intermediary with 2% serum (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells), and they were cultivated upper chambers with 800 \u0026mu;l intermediary involving 10% serum in lower chambers. Afterwards, methyl alcohol was utilized to realize the fixation of cells, and cells were dyed in Giemsa. After that, cells on the top surface of the film were removed, and cellular samples on the lower surface were subjected to counting and imaging in 5 stochastic fields of 100 amplification under a microscopic device. Cellular quantity was subsequently analyzed statistically. Moreover, 50 \u0026micro;l matrigel (BD Bioscience, America) was supplemented into upper chambers for invasion analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting (WB) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUMUC3 cells were washed twice by pre-cooled PBS, and subjected to lysis in RIPA buffering solution (Thermo Scientifific, China). The BCA protein quantitative tool (Pierce, America) was utilized to identify protein contents. Then, protein specimens were subjected to 10% SDS-PAGE and moved to PVDF films (Millipore, America). These films were subjected to blockade in 5% skinmilk and afterwards probed with the first antibody for one night under 4\u0026deg;C and subsequently cultivated with HRP-conjugated second antibody. The blots were afterwards studied via a reinforced chemiluminescent identification tool (Amersham Bioscience, America). GAPDH was the load control. For densitometry assay, protein bands on the blots were identified via the ImageJ program 1.4 (America).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative Real-time PCR (qRT-PCR\u003c/strong\u003e \u003cstrong\u003e)\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall RNA was separated via RNAiso Plus as per the supplier\u0026apos;s protocol (TaKaRa, Japan). Subsequently, 2 mg overall RNA was converted to cDNA via reverse transcription through AMV reversed transcriptive enzyme (TaKaRa). qRT-PCR was completed via the Power SYBR Green Master Mix (TaKaRa) and an ABI 7300 real-time PCR identification apparatus (Applied Biosystem) under default status: 95℃ for 10 s, and 40 cycles of 95℃ for 10 s and 72℃ for 0.5 min. The primers utilized are: GAPDH, forward 5\u0026prime;-GCTGCCCAACGCACCGAATA-3\u0026prime; and reverse 5\u0026prime;-GAGTCAACGGATTTGGTCGT-3\u0026prime;; ZO-1, forward 5\u0026prime;-GCAGCCACAACCAATTCATAG-3\u0026prime; and reverse 5\u0026prime;-GCAGACGATGTTCATAGTTTC-3\u0026prime;; E-cadherin, forward 5\u0026prime;-TCGACACCCGATTCAAAGTGG-3\u0026prime; and reverse 5\u0026prime;-TTCCAGAAACGGAGGCCTGAT-3\u0026prime;; N-cadherin forward 5\u0026prime;-ATCAAGTGCCATTAGCCAAG-3\u0026prime; and reverse 5\u0026prime;-CTGAGCAG TGAATGTTGTCA-3\u0026prime;; Snail, forward 5\u0026prime;-TTCCAGCAGCCCTACGACCAG-3\u0026prime; and reverse 5\u0026prime;-CGGACTCTTGGTGCTTGTGGA-3\u0026prime;. The expressing levels of mRNA for every gene were normalised by its GAPDH respectively. Fold changes in genetic expression were computed through the comparative threshold cycle (Ct) approach via the 2\u003csup\u003e-(\u0026Delta;\u0026Delta;Ct)\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed by the SPSS 22.0 software (SPSS, USA), and shown as mean\u0026plusmn;standard deviation. Unpaired Student\u0026rsquo;s t-test was utilized to compare the diversities of samples between 2 groups. One-way ANOVA was utilized for multi-group comparison, before the LSD significant difference test. P\u0026lt;0.05 had significance on statistics.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCSE elicited EMT in UMUC3 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the association between CSE and BC metastases, human BC cells UMUC3 were cultivated with indicated levels of CSE for 6 days. Cellular activity was reduced under 5% CSE content posterior to the 6-day treatment (Fig. 1A). Hence, CSE concentrations of 0.5%, 0.75% were chosen for the subsequent procedures in our study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCS contributes to BC development and can also induce EMT of cells [7, 8, 13, 14]. In our study, CSE exposure resulted in an evident morphologic variation, from round to spindle-shaped mesenchyme cells (Fig. 1B). Transwell assay were completed, and the outcomes revealed that CSE treatment significantly enhanced UMUC3 cellular\u0026nbsp;migratory and invasive\u0026nbsp;abilities (Fig. 1C, D). Hence, our team studied the effects of CSE on EMT via identifying the expressing levels of EMT biomarkers. WB analysis unveiled that CSE treatment reduced the expressing levels of epithelium indicator proteins, like E-cadherin and ZO-1, and elevated the contents of mesenchyme indicator proteins, like N-cadherin and Snail (Fig. 2A, B), which coincided with the results of qRT-PCR analyses (Fig. 2C). Collectively, those results revealed that CSE induced EMT in UMUC3 cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCSE increased ERK/AP-1 activation in UMUC3 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate whether CSE-triggered EMT variations was related to the variations in MAPK stimulation, the expressing levels of overall and phosphorylated ERK1/2, p38, JNK, ERK5 were identified via WB analyses. It was found that CSE significantly elevated the expressing level of p-ERK1/2, whereas it exerted little influence on p-JNK, p-p38, and p-ERK5 in UMUC3 cells (Fig. 3A, B). Moreover, WB analyses unveiled increased expressing levels of down stream target AP-1 proteins, like p-c-Fos and p-c-Jun (Fig. 3C, D). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of ERK1/2 reversed CSE-triggered EMT in UMUC3 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe aforementioned outcomes demonstrated that CSE-elicited EMT were related to the stimulation of ERK1/2/AP-1 in UMUC3 cells. To explored the role of ERK1/2/AP-1 pathway in this process, a specific ERK1/2 suppressor, U0126, was employed to realize the pretreatment of cells. Results revealed that U0126 reduced the expression levels of p-ERK1/2, p-c-Fos and p-c-Jun proteins in UMUC3 cells, as revealed by WB analyses (Fig. 4A, B). The morphologic assessment showed that U0126 decreased the mensenchyme morphology variations (Fig. 4C). Meanwhile, U0126 attenuated CSE-mediated\u0026nbsp;migratory and invasive\u0026nbsp;abilities of UMUC3 cells (Fig. 4D, E). Moreover, U0126 weakened the CSE-elicited reduction of E-cadherin and ZO-1 contents and attenuated the elevation of N-cadherin and Snail in UMUC3 cells (Fig. 5A, B). Alike outcomes were identified in qRT-PCR tests as well (Fig. 5C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCurcumin suppressed ERK1/2 pathway to prevent CSE-induced EMT in UMUC3 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies reported that high concentrations of curcumin exerted anti-proliferative or cytotoxic effect on UMUC3 cells [31, 32]. The results of MTT showed that curcumin at a concentration of 10 \u0026mu;M exerted an evident impact on cellular activity (Fig. 6A). Consequently, 1 \u0026mu;\u0026Mu; curcumin was selected for subsequent experiments. We found that curcumin exposure abolished the mensenchyme morphology variation in these cells (Fig. 6B). Besides, curcumin restored the\u0026nbsp;migratory and invasive\u0026nbsp;abilities of UMUC3 cells, as unveiled by transwell tests (Fig. 6C, D). In addition, our team discovered that curcumin reversed the CSE-activated decrease of E-cadherin and ZO-1 contents and the reinforcement of N-cadherin and Snail in UMUC3 cells via WB (Fig. 7A, B) and qRT-PCR analyses (Fig. 7C). Those results revealed that curcumin prevented CSE-elicited EMT in UMUC3 cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the sake of investigating the roles of curcumin in the CS-mediated stimulation of the MAPK path, our team studied the variations in MAPK stimulation after curcumin exposure. WB tests demonstrated that curcumin restored CSE-enhanced ERK1/2 activity and suppressed CSE-induced p-c-Fos and p-c-Jun stimulation. Moreover, we noted that curcumin had little effect on the protein expressions of p38, JNK, and ERK5 in UMUC3 cells (Fig. 7D, E).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCS consists of over 5000 diverse xenobiotics chemical substances, namely, nicotine, polycyclic aromatic hydrocarbons, tar, nitrosamines, CO\u003csub\u003e2\u003c/sub\u003e, NO, and phenolics hydrocarbon. Amongst those CS components, almost 150 chemical substances are toxicants, and 60 chemical substances are carcinogenic agents [33]. CS is a major risk factor for BC progression, as shown repeatedly in multiple reports [8, 13, 34, 35]. Pietzak et al. indicated that compared with non-smokers at their initial presentation, heavy smokers were more likely to have a high-grade tumor and muscle-invasive BC [34]. The results of a meta-analysis suggested that long-term smokers were at elevated risks of having a more invasive BC related to poorer prognoses [35]. Similarly, some evidences have supported the cancer-promoting effect of CS on BC both in vitro and in vivo [8, 13, 36].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEMT is a pivotal cell program enabling polarised epitheliums to shift towards a mesenchyme phenotype with elevated cell migration ability. Proofs have revealed that EMT participates in facilitating cancer aggression and migration [37]. The effects of CS on EMT and tumor developmental process have been discussed previously [7, 8, 10-14, 24-30]. As aforementioned, CSE elicited EMT in UMUC3 cells, as indicated by the morphologic alteration from epitheliums to mesenchyme cells. It enhanced\u0026nbsp;migratory and invasive\u0026nbsp;abilities and caused variations in the expressing levels of EMT biomarker mRNAs and proteins, like E-cadherin, ZO-1, Snail and N-cadherin. Our data are consistent with previous findings, which unveiled that CSE elicited EMT in vitro.\u003c/p\u003e\n\u003cp\u003eA commonly seen signal transduction by which the exocellular regulation of cell-surface acceptor activities is transmitted into a specific cell reaction is the three-tiered MAPK cascade, and the ERK1/2 path is the most broadly studied [38]. Abnormal ERK1/2 stimulation induces cancer development in various models and is usually found in mankind tumors [8, 24-30, 39-46]. ERK1/2 over expression in oncocytes, like human BC T24 cells [8], and mammary carcinoma cells (MDA-MB-231, BT-549, tamoxifen-resistant MCF-7) [39], displayed a metastasis and aggression phenotype, thus leading to enhanced migration abilities of cells. Moreover, the pharmacological inhibition of ERK1/2 suppressed cellular metastasis and aggression abilities in multiple types of oncocytes in vitro [8, 39], and it reduced the migration abilities of pancreatic carcinoma heterografts in vivo [40]. Mounting studies have found that ERK1/2 participates in regulating CS-induced urocystic, gastric, hepatic, pulmonary EMT in vivo [7, 25, 28-30]. In addition, abnormal ERK1/2 expression was associated with the poor prognosis of tumor sufferers [41], prostate cancer [42], type I endometrial cancer [43], multiple myeloma[44], hepatocellular carcinoma [45], breast cancer [39], and BC [46]. The Jun and Fos subfamilies are major AP-1 proteins [47]. Previous studies found that AP-1 overexpression existed in multiple tumor types, like colorectal carcinoma [48], mammary carcinoma [49], and so on. Moreover, AP-1 activation was discovered in CS-elicited EMT in vivo and in vitro [7, 8, 25, 28-30]. As a downstream target of the MAPK pathway, AP-1 participates in the mechanisms of cancer cell invasion, EMT, and metastases via facilitating the expressing of EMT-triggering transcriptional factors, cell factors, and miRNAs, therefore contributing to cancer progression [47]. In the present study, our results showed that the CSE-induced EMT of UMUC3 cells was related to the stimulation of ERK1/2 and AP-1 proteins (p-c-Fos, and p-c-Jun). The utilization of a pharmacological inhibitor of ERK1/2, namely U0126, terminated ERK1/2/AP-1 stimulation and abolished CSE-activated EMT. Those discoveries unveiled the latent causal links of CSE-triggered metastasis, aggression, and EMT in UMUC3 cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClinically, the secondary chemoprevention is most widely used in patients with precancerous lesions that may develop into invasive cancer. The definition of cancer chemoprevention, namely, utilizing synthetic, natural, or biological agents to minimize the incidence of cancer in healthy individuals [50]. A large amount of studies have reported the satisfactory results of utilizing traditional plant-derived chemicals to treat diseases. Curcumin is a natural compound, which has aroused scientists\u0026rsquo; interest worldwide due to its biological activities, and it\u0026apos;s one of the most prospective chemoprevention agents. Extant data strongly suggest that curcumin has protection efficacy against pathophysiologic pathways responsible for BC [22]. In recent years, mounting evidences have demonstrated that the strong chemoprevention effect of curcumin might be partially originated from the suppression of EMT [23]. Previous studies showed that curcumin suppressed long-term tobacco smoke-induced MAPK stimulation and EMT variations in various organs in vivo [25, 28-30]. Moreover, curcumin weakened CSE-elicited EMT in several types of cancer cells [26, 27]. High concerntrations of curcumin were identified to decrease the growth of BC cells in vivo and in vitro [31, 32, 51-52]. Alike outcomes were identified in our MTT assay as well. Herein, 1\u0026nbsp;\u0026mu;M\u0026nbsp;curcumin was chosen to explore the underlying prevention efficacy against CSE-elicited EMT. A similar non-cytotoxic dosage of curcumin has also been reported in several experimental studies [31, 32]. We found that curcumin inhibited CSE-induced\u0026nbsp;morphological alterations as well as the\u0026nbsp;enhancement of cellular\u0026nbsp;migratory and invasive\u0026nbsp;abilities in UMUC3. Meanwhile, the deregulated expressions of EMT-related proteins, like E-cadherin, ZO-1, N-cadherin, and Snail, were reversed by curcumin. Moreover, CSE-induced ERK1/2/AP-1 stimulation was reduced after curcumin administration. The above data demonstrate the effects of ERK1/2/AP-1 on curcumin-based protection efficacy against CSE-triggered EMT in human BC cells UMUC3.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study demonstrated the effects of ERK1/2 pathway on regulating CSE-triggered EMT and revealed the protection efficacy of curcumin against CSE-induced EMT via ERK1/2 suppression. Those discoveries offer a novel method for preventing CS-associated BC.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This study was financed by the Provincial Natural Science Foundation of Anhui, China(No. 2018085MH290) and Natural science fund for colleges and universities in Anhui Province (No. KJ2021A0312).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eDexin Yu contributed to the study conception and design. Material preparation was performed by Xin Sun, data collection and analysis were performed by Tao Zhang, Liang Tang, and Jie Min. The first draft of the manuscript was written by Xin Sun and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRichters, A., Aben, K., \u0026amp; Kiemeney, L. (2020). The global burden of urinary bladder cancer: an update. \u003cem\u003eWorld journal of urology, 38(8), \u003c/em\u003e1895\u0026ndash;1904.\u003c/li\u003e\n\u003cli\u003eSiegel, R. L., Miller, K. D., Fuchs, H. E., \u0026amp; Jemal, A. (2022). 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The effect of curcumin on bladder tumor in rat model. \u003cem\u003eEuropean review for medical and pharmacological sciences\u003c/em\u003e, \u003cem\u003e21\u003c/em\u003e(4), 884\u0026ndash;889.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cigarette smoke extract, Bladder cancer, Epithelial-to-mesenchymal transition, ERK1/2, Curcumin","lastPublishedDoi":"10.21203/rs.3.rs-1841893/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1841893/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCigarette smoke (CS) is a main risk factor for bladder cancer (BC). The epithelial-to-mesenchymal transition (EMT), termed as an embryonic program, participates in the development process of CS-associated BC. Over the last decade, the effects and mechanisms of curcumin on CS-related BC have been extensively investigated. However, it's still elusive if MAPK pathways participate in curcumin-associated protective effect against CS-related promotive role in BC. In this study, our results showed that the exposure of BC cells UMUC3 to cigarette smoke extract (CSE) induced morphology variation, reinforced migratory and invasive abilities, decreased epithelium biomarker expression and increased mesenchyme biomarker expression. Meanwhile, CSE administration led to the stimulation of extracellular regulated protein kinases 1 and 2 (ERK1/2) and activator protein 1 (AP-1) proteins, without validly activating the Jun N-terminal kinase (JNK), p38 or ERK5 pathway. Moreover, CSE-elicited EMT and ERK1/2/AP-1 stimulation were completely diminished by U0126, a pharmacological inhibitor targeting ERK1/2. Furthermore, the co-treatment with low concentrations of curcumin significantly abolished CSE-elicited EMT in UMUC3 cells, and the ERK1/2/AP-1 pathway was implicated in the suppressive effect. In summary, these data indicated the modulatory effects of ERK1/2 on CSE-elicited urocystic EMT in vitro and the chemoprevention efficacy of curcumin.\u003c/p\u003e","manuscriptTitle":"Curcumin reversed cigarette smoke extract-induced epithelial-to-mesenchymal transition of human bladder cancer cell UMUC3 through suppression of ERK1/2 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-18 16:48:58","doi":"10.21203/rs.3.rs-1841893/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dd2077cb-ba46-47da-a900-ee0b85d0eabd","owner":[],"postedDate":"July 18th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-07-20T22:59:10+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-18 16:48:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1841893","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1841893","identity":"rs-1841893","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}