JS-K—a NO prodrug—inhibits migration, invasion, and bone metastasis of renal cell carcinoma through the NF-κB signaling pathway

JS-K—a NO prodrug—inhibits migration, invasion, and bone metastasis of renal cell carcinoma through the NF-κB signaling 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 JS-K—a NO prodrug—inhibits migration, invasion, and bone metastasis of renal cell carcinoma through the NF-κB signaling pathway Yuwan Zhao, Xingzhang Qin, Lugang Zhu, Xinghua Lin, Bailiang Miu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6460853/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 Background O2-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1, 2-diolate (JS-K)—a nitric oxide prodrug—inhibits the proliferation and migration of breast cancer, non-small cell lung cancer, and liver cancer cells. However, its mechanism of action in renal cell carcinoma (RCC) remains elusive. This study seeks to investigate the effect of JS-K on RCC migration, invasion, and bone metastasisand elucidate the underlying molecular mechanisms. Methods Human RCC cell lines were treated with different concentrations of JS-K, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor Bay-11-7082, or receptor activator of nuclear factor-kappa beta ligand (RANKL), per experimental requirements. In vitro migration, invasion, adhesion, and bone metastasis were assessed by the wound healing assay, Transwell assay, and hanging drop assay. Western blotting and immunofluorescence staining were conducted to evaluate the expression of specific proteins. Changes in gene expression were analyzed by RNA sequencing and quantitative polymerase chain reaction. Results JS-K treatment inhibited the epithelial-mesenchymal transition and extracellular matrix remodeling of RCC in a concentration-dependent manner. Subsequently, the migration, invasion, bone metastasis, and adhesion abilities of RCC cells significantly decreased after JK-S treatment. At the molecular basis, JS-K upregulated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha while inhibiting the nuclear translocation of p65 and p50, suggesting its involvement in the NF-κB signaling pathway. Pharmacological inhibition of NF-κB with either Bay-11-7082 or RANKL influenced the effects of JS-K on RCC migration, invasion, and bone metastasis. Conclusion JS-K treatment inhibits RCC migration, invasion, and bone metastasis by targeting the NF-κB signaling pathway. JS-K renal cell carcinoma migration and invasion bone metastasis EMT NF-κB signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Renal cell carcinoma (RCC) is a prevalent malignancy of the urinary system, accounting for up to 85% of urological tumors. Over 400,000 newly diagnosed cases of RCC and 175,000 RCC-related deaths are reported worldwide annually [ 1 , 2 ] . Surgery remains the mainstay treatment for localized RCC. Nevertheless, approximately 30% ofpatients develop metastatic RCC (mRCC) after the surgical removal of the localized tumor [ 3 ] . Furthermore, RCC progresses slowly, with some cases remaining asymptomatic until the tumor metastasizes to the lymph nodes, lungs, liver, and bones [ 4 ] . Despite advancements in the systemic response to mRCC because of novel therapeutic approaches, metastasis remains the leading cause of death among patients with RCC [ 5 , 6 ] . The 5-year survival rate for patients with primary RCC is over 90%, compared with 70% in patients with localized spread (stage I/II) and 10% in patients with recurrent or distant metastases (stage IV) [ 7 , 8 ] . Because the early clinical manifestations of RCC are mostly non-specific, patients are often diagnosed at an advanced stage [ 9 ] . Standard treatments, such as chemotherapy, hormonal therapy, radiotherapy, and surgery, extend the overall survival [ 10 ] . Nevertheless, recurrence is frequently observed during follow-up, particularly in patients with clear cell RCC (ccRCC). Moreover, ccRCC is associated with worse prognosis than non-ccRCC [ 11 ] , along with a 20–40% risk of postoperative recurrence [ 12 ] . Despite the advent of novel targeted therapies,RCC tumors often acquire resistance by activating alternative pathways. For instance, activation of upstream receptor tyrosine kinases can trigger signaling cascades leading to continuous tumor growth [ 13 – 15 ] . The low effective rate of chemotherapy alone (4–6%) warrants an in-depth exploration of the molecular mechanisms underlying RCC progression and metastasis to identify novel therapeutic targets. Bone metastasis is a frequent and severe complication of RCC. Approximately 30% of patients develop bone metastasis, which leads to substantial bone pain, skeletal complications, and even death [ 3 ] . Tumor cells release various factors, including transforming growth factor (TGF)-β, to stimulate osteoclasts, resulting in increased secretion of receptor activator of nuclear factor-kappa B ligand (RANKL). Overexpression of RANKL promotes the formation, activation, and survival of osteoclasts, finally increasing bone resorption [ 16 ] . Increased osteoclast activity and subsequent bone destruction have been associated with bone metastasis. Additionally, bone resorption triggers the release of growth factors, such as platelet-derived growth factor, bone morphogenetic protein, and transforming growth factor beta, which increase the production of parathyroid hormone and promote tumor growth [ 16 ] . Wittrant et al. [ 17 , 18 ] demonstrated that blocking the NF-κB (RANK)/RANKL interaction prevented bone metastases in prostate cancer. Furthermore, RANKL and RANK mRNA levels are upregulated in RCC tissues than in non-neoplastic renal tissues [ 19 ] . These levels are elevated in high-grade versus low-grade ccRCC and in metastatic versus primary RCC. Elevated RANKL and RANK expression predict cancer recurrence, bone metastasis, and poor prognosis [ 19 ] . Overall, RANKL-RANK signaling promotes the migration and metastasis of cancer cells to distant organs. Thus, accelerated bone remodeling because of tumor cell-released factorsresults in a vicious cycle of bone destruction and tumor growth, culminating in bone metastasis. JS-K—a nitric oxide (NO) prodrug—was developed at the National Cancer Institute in 2008 and is a promising class of NO-based therapeutics. In reactions catalyzed by glutathione-S-transferases (GSTs), JS-K replaces glutathione (GSH) with nucleophilic aromatics to form diazonium anions and spontaneously release NO [ 20 ] . Considering high levels of GST in tumor cells, JS-K can exert anti-cancer effects [ 21 ] . JS-K is selectively toxic to RCC cell lines at concentrations that do not considerably affect the proliferation of normal renal epithelial cells [ 20 ] . NO mediates multiple physiological processes, such as smooth muscle relaxation, platelet aggregation, vasodilation, neurotransmission, cell migration, immune responses, and tumorigenesis [ 21 – 24 ] . Its precise biological mechanism of action has not been completely elucidated; nonetheless, the physiological effects of NO depend on its local concentration and exposure time [ 25 , 26 ] . The association between NO and cancer is complex [ 27 – 30 ] . Simeone et al. reported that JS-K decreases the invasiveness of breast cancer cells through the basement membrane by upregulating tissue inhibitor of metalloproteinase-2 (TIMP-2). However, TIMP-2 inhibition does not completely block JS-K, indicating the role of other mechanisms in its anti-tumor effects [ 31 ] . Tanyel et al. reported that JS-K may inhibit the proliferation and migration of endothelial cells and the formation of new blood vessels by regulating angiogenic factors [ 32 ] . Maciag et al. [ 33 ] demonstrated that JS-K releases NO in non-small cell lung cancer cell lines (H1703 and H1944) through nucleophilic aromatic substitution, increases reactive oxygen species (ROS) production, and oxidizes GSH to glutathione disulfide (GSSG). The reduction in the intracellular GSH/GSSG ratio triggers apoptosis. Liu et al. [ 34 ] reported that JS-K inhibits the growth, colony formation capacity, and migration of hepatitis B virus-positive HepG2 cells. Additionally, Qiu et al. [ 31 ] demonstrated that JS-K enhances the anti-tumor activity of doxorubicin by upregulating the p53 pathway and mitigating its cardiotoxicity. However, the effects of JS-K on the invasion and metastasis of RCC remain elusive. In summary, this study seeks to explore explore changes in the hierarchy of RCC induced by JS-K through experimental methods, such as the wound healing assay and Transwell assay. Additionally, the effects of JS-K on the genes and protein molecules of RCC were investigated at the microscopic level through experimental methods, such as Western blotting, immunofluorescence staining, RNA sequencing, and quantitative polymerase chain reaction. This study may provide valuable reference data for the treatment of metastatic RCC.. Materials and methods Cell culture Human RCC786-O and A498 cells were obtained from the cell bank of the Chinese Academy of Science (Shanghai, China). The cells were cultured in Roswell Park Memorial Institute-1640 (RPMI 1640) medium (GIBCO, Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% (v/v) fetal bovine serum (FBS; GIBCO, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO 2 . Nuclear and cytoplasmic protein extraction First, the culture dish was washed with phosphate-buffered saline (PBS), and trypsin culture fluid was added to digest the cells. The digested cells were centrifuged, and the supernatant was discarded. Approximately 1 mL of PBS was added to the centrifuged cells, and the centrifugation and supernatant discarding steps were repeated. After the second centrifugation, 100 µL of 3,6,9-trioxaundecan-1-{4-[(2-Chloroethyl)Ethylamino)]-Benzylamino},11-Azide (CEB-A) reagent (Nuclear and Cytoplasmic Protein Extraction Kit P1200, Beyotime Institute of Biotechnology) was added to the cell pellet, which, which was transferred to a pre-cooled 1.5 mL Eppendorf (EP) tube. Second, 1.5 mL EP tube was vortexed vigorously for 30 s and placed in an ice bath for 10 to 15 min, with vortexing for 15 s every 5 min. Third, the EP tube was centrifuged to resuspend (12,000 g , 4°C, 5 min). Without disturbing the pellet, the supernatant was transferred to a new EP tube (the cytoplasmic protein, to be measured quantitatively by bicinchoninic acid assay (BCA)). Fourth, the maximum supernatant was discarded from the EP tube, and the pellet was retained. Subsequently, 100 µL of CEB-A reagent and 5 µL of CEB-B reagent were added, with vortexing for 10 s to resuspend the pellet. The tube was placed in an ice bath for 1 min and centrifuged again (1,000 g , 5 min). The supernatant was completely discarded, and the pellet was retained. Fifth, 50 to 100 µL of pre-cooled NEB was added to the retained pellet to resuspend it. The tube was vortexed vigorously for 15 s, placed in an ice bath for 30 min, and vortexed for 15 s every 10 min. It was centrifuged (12,000 g , 5 min), and the supernatant was retained (the nuclear protein, to be measured quantitatively by BCA). After sample extraction, adjustments, adjustments were made per the protein concentration measured by BCA. Finally, 1/4 volume of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (5×) was added. The sample was boiled for 10 min at 100°C and stored frozen. Western blotting Total protein was extracted from the cultured cells using radioimmuno precipitation assay buffer (Beyotime, Shanghai, China) supplemented with 1 mM phenylmethanesulfonyl fluoride (Beyotime, Shanghai China). Equal amounts of protein per sample were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated overnight at 4°C with primary antibodies specific for E-cadherin (#3195S), N-cadherin (#4061S), matrix metallo proteinase (MMP)-2 (#87809S), MMP-9 (#2270S), TIMP-1 (#2270S), P65 (#4695S), p50 (#4370S), and nuclear factor of kappa light poly peptide gene enhancer in B-cells inhibitor alpha (IκBα) (#4359S). All primary antibodies were from Proteintech (Chicago, USA) and diluted 1:1000 in Primary Antibody Dilution Buffer (Beyotime, Shanghai China). The membranes were washed three times with Tris-buffered saline and Tween 20for 10 min each and incubated with immunoglobulin G-horseradish peroxidase secondary antibody (EarthOx, USA) for 1 h at room temperature. Protein bands were detected using an enhanced chemiluminescence kit (EMD Millipore, Billerica, MA, USA) and the Tanon 5200 chemiluminescent imaging system (Shanghai, China). In order to save the antibody reagent, we cut the band according to the instructions of the protein marker, and then incubated and exposed the internal reference protein and the target protein band respectively. If the reference protein bands were not neat, we discarded the protein extract of this batch, re-extracted the protein, and repeated the above process. Therefore, the protein bands presented in the original data were the bands after the first cleavage. The bands presented in Fig were the final results. Wound healing assay Cells in the logarithmic growth phase were harvested and seeded into six-well plates. After reaching 100% confluence (approximately 24 h), the monolayer was scratched with a sterile pipette tip to create a “wound.” The plates were washed three times with PBS to remove the dislodged cells, and a serum-free medium containing suitable drugs was added. Images were captured at 0 h and at different time points under 10x magnification to evaluate the rate of wound healing. Transwell assay Cell migration, invasion, and bone metastasis were analyzed using Transwell inserts. The chambers were coated with Matrigel and type I collagen for invasion and metastasis assays, respectively. Matrigel was thawed overnight at 4°C and diluted 6:1 with pre-cooled serum-free RPMI-1640, and 100 µL of the diluted gel was applied to the Transwell membrane. The inserts were incubated at 37°C for at least 4 h to solidify the gel. Type I collagen (1 mg/mL) was diluted 49:1 with serum-free RPMI-1640, and 100 µL of the diluted gel was spread on the Transwell membrane. The inserts were placed in an incubator for 40 min to allow solidification. Suitably treated cells were harvested, resuspended in serum-free medium, and seeded in the upper chambers of the inserts at the density of 2 × 10 4 cells per well. The lower chambers were filled with 800 µL complete medium (20% FBS). After incubation for 24 h, the inserts were removed, and cells remaining on the upper surface of the membranes were wiped off using a dry cotton swab. Cells on the lower surface were fixed with 800 µL of 1% paraformaldehyde for 20 min, washed twice with PBS (10 min each), andstained with 500 µL Crystal Violet solution for 20 min. The membranes were washed with PBS, air dried, and viewed under an inverted microscope. The number of cells were counted in five randomly selected fields of view, and the average was calculated. Each experiment was repeated three times. Immunofluorescence RCC cells were harvested and seeded onto coverslips in confocal culture dishes at a density of 1.2 × 10 4 cells/mL. After overnight incubation, the cells were washed three times with PBS, and cultured with respective drugs for 24 h. The culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 30 min. The cells were washed three times with PBS, permeabilized with 0.5% Triton-X 100 (diluted with PBS) for 30 min, and again washed three times with PBS. These cells were blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature andincubated overnight at 4°C with anti-p65 or anti-p50 primary antibody (diluted 1:100 with 1% BSA, 80 µL). After three washes with PBS, they were incubated in the dark for 2 h with fluorescein 488-labeled goat anti-rabbit/mouse secondary antibody (diluted with PBS, 80 µL). The coverslips were washed three times with PBS, stained with one to two drops of 4′,6-diamidino-2-phenylindole, and viewed under a fluorescence microscope. Quantitative real-time polymerase chain reaction Total RNA was extracted from tissue samples and cultured cells using TRIzol reagent (Invitrogen, Carlsbad CA, USA) and reverse transcribed to cDNA using Prime Script TM RT reagent Kit with g DNA Eraser (Takara, Japan) per the manufacturer's instructions. Quantitative real-time PCR was conducted using TB Green & Premix Ex Taq TM (Tli RNase H Plus) (Takara, Japan) on a continuous fluorescence detector 480II Real-Time PCR system. The cycling conditions were as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, and annealing at 60°C for 30 s. Relative gene expression was determined using the 2 −ΔΔCt method, with GAPDH as the internal reference [ 13 , 14 ] . The primer sequences were as follows: p65: forward: 5′-CTGCCGCCTGTCCTTTCTCATC-3′, reverse: 5′-ATGTCCTCTTTCTGCACCTTGTCAC-3′; P50: forward: 5′-TCACTTGAACACATCAAACGAC-3′, reverse:: 5′-AGTGATTACAATTTCCCCGTCT-3′; IKBa: forward: 5′-GAGACTTTCGAGGAAATACCCC-3′, reverse: 5′-GTAGCCATGGATAGAGGCTAAG-3′; and GAPDH: forward: 5′-GGTGAAGGTCGGAGTCAACGG-3′, reverse: 5′-CCTGGAAGATGGTGATGGGATT-3′. Primers for all genes and GAPDH were synthesized by Sangon Biotech (Shanghai, China). RNA sequencing RCC 786-O cells were treated with 0 and 1 µM JS-K, and cell suspensions were collected after 24 h of treatment. Three samples were acquired for each drug concentration. The cell suspensions were collected using trypsin treatment as follows: The culture medium was discarded, and the cells were washed with sterile 1×PBS solution. Sufficient trypsin solution was added to cover the monolayer of cells. For example, a 150 mm culture flask required 2 mL of trypsin solution, and a 100 mm culture dish required 1 mL of trypsin solution. The vessel was gently shaken to evenly distribute the trypsin over the cell layer until the cells became loose (usually within 12 min). Once the cells were loose, the trypsin solution was quickly discarded by tilting the dish or flask to remove the excess solution with a pipette. AA 1×PBS solution was added, and a pipette was used to dislodge the adherent cells.. Along with the PBS solution, the cells were collected into nucleasenuclease-free, screw-capped EP tubes and centrifuged at 300 to 500 g (1,100–1,500 rpm) for 5 min. The supernatant was discarded, and the cell pellet was collected. The collected cells were quick-frozen in liquid nitrogen and then to -80°C for storage. Dry ice was used for transporting the samples. These samples were sent to the Beijing Genomics Institute for RNA sequencing, and relevant data were provided by the company. Through KEGG analysis, we can gain a deep understanding of the roles of genes in complex biological systems such as metabolic pathways and signaling pathways. A large number of genes or proteins are matched with the standard metabolic pathways or signaling pathways in the KEGG database to identify pathways that are significantly enriched in specific biological functions. Enrichment statistical methods (such as hypergeometric test, Fisher's exact test, etc.) are usually used to determine whether a specific pathway is significantly enriched. The x-axis is a ratio (Rich Factor/GeneRatio/(GeneRatio/BgRatio)), or fold change of differential expression. The higher the value, the higher the enrichment degree of differential metabolites/proteins/genes in the pathway. The y-axis represents the names of the enriched pathways, and the top 20 enriched pathways are selected for plotting. The size of the dot indicates the number of genes. The larger the dot, the more genes are enriched in the pathway. The color represents the level of the p-value. The smaller the p-value, the more significant the pathway is. Statistical analysis Graph Pad Prism 7.0 and SPSS 19.0 software were used for all statistical analyses. Data are presented as the mean ± standard deviation of three experiments. One-way analysis of variance was used to compare multi-group differences and the least significant difference method was used for pairwise comparisons. Comparisons between two groups were conducted using the Student’s t-test, with P < 0.05, **P < 0.01, and ***P < 0.001 indicating statistical significance. Results JS-K inhibits RCC migration, invasion, and adhesion RCC cell lines (786-O and A498) were incubated with different concentrations of JS-K (0, 0.25, 0.5, and 1 µM) for 24 h, and the expression of epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin, and vimentin) and extracellular matrix (ECM)-remodeling proteins (MMP-9, MMP-2, and TIMP-1) were analyzed. JS-K treatment downregulated the expression of N-cadherin, vimentin, MMP-2, and MMP-9, while upregulating the expression of E-cadherin and TIMP-1 (Figs. 1 A and B). Furthermore, JS-K suppresses the EMT process by visually capturing the morphological changes in cells in each group under bright-field microscopy (Fig. 1 C). JS-K treatment significantly reduced the migration rates of 786-O and A498 cells in the wound healing assay after 6 and 12 h, respectively (Fig. 1 D). Furthermore, the inhibitory effect was concentration-dependent (Fig. 1 E). In the Transwell assay, JS-K-treated cells showed a significant decline in their migration and invasion abilities after 24 h, compared with untreated controls (Figs. 1 F and 1 G). JS-K targets the NF-kB signaling pathway in RCC To determine the molecular mechanisms underlying the effects of JS-K, 786-O cells were treated with 1 µM of JS-K for 24 h. RNA sequencing was conducted to assess transcriptional changes in relevant signaling pathways. Genes involved in the NF-κB signaling pathway showed significant expression changes after JS-K treatment, compared with untreated controls (Fig. 2 A). qPCR confirmed changes in the expression of the p65, p50, and IκBα genes were confirmed in A498 and 786-O cells treated with different concentrations of JS-K (0, 0.25, 0.5, and 1 µM). JS-K treatment significantly upregulated IκBα mRNA expression, whereas p65 and p50 mRNA expression remained unchanged (Fig. 2 B). Similar changes were observed in protein expression analysis of IκBα, p65, and p50 after treatment with different concentrations of JS-K (Figs. 2 C and 2 D). Interestingly, JS-K-treated cells showed increased cytoplasmic expression of p65 and p50 proteins and relatively lower expression in the nuclear fraction (Figs. 2 E and 2 F). Therefore, JS-K may inhibit the nuclear translocation of p65 and p50 in RCC cells. Additionally, immunofluorescence staining of the 786-Oand A498 cells was conducted to determine protein localization and confirm the above hypothesis. Fluorescence signals corresponding to p65 and p50 were stronger in the cytoplasm than in the nuclei of RCC cells after JS-K treatment (Fig. 2 G). Figure 2 H illustrates the nucleocytoplasmic ratios of p65 and p50. Taken together, JS-K modulates the NF-κB signaling pathway in RCC by preventing the nuclear translocation of p65 and p50. Effects of JS-K combined with NF-κB inhibitors and growth factors on RCC invasion and migration To determine whether the NF-κB pathway is involved in the anti-tumor effects of JS-K, RCC cells were co-treated with JS-K (1 µM) and either the NF-κB inhibitor Bay-11-7082 (4 µM) or the NF-κB pathway activator RANKL (20 µg/mL). Pharmacological blockade of NF-κB augmented the inhibitory effects of JS-K on RCC migration rates. Contrarily, NF-kB pathway activation rescued the cells from the effects of JS-K and increased their migration rates (Figs. 3 A and 3 B). The Transwell assay yielded consistent results. Co-treatment with JK-S and Bay-11-7082 significantly decreased the migration and invasion rates, compared with either drug alone, whereas co-treatment with JK-S and RANKL increased RCC migration and invasion (Figs. 3 E and 3 F). Furthermore, co-treatment with and either Bay-11-7082 or RANKL increased the cytoplasmic levels of p65 and p50 while reducing their nuclear levels, compared with single-drug treatment (Figs. 3 C and 3 D). The metastatic ability of RCC cells was assessed by the Transwell assay using type I collagen-coated membranes. JS-K reduced RCC invasiveness (Figs. 4 A and B).) Furthermore, NK-κB inhibition or RANKL co-treatment intensified the inhibitory effects of JK-S on the migration rates (Fig. 4 C), and the differences were statistically significant (Fig. 4 D). In summary, JS-K treatment inhibits RCC inhibits RCC migration and invasion through the NF-κB signaling pathway, while preventing RCC bone metastasis. Discussion RCC is one of the most prevalent malignancies of the urinary system, and approximately 30% of advanced cases progress to lung, bone, lymph node, liver, adrenal gland, and brain metastases [ 35 ] . General surgery remains a key therapeutic approach for early-stage RCC. However, the prognosis for advanced RCC is poor because of the high probability of metastases. Additionally, mRCC is highly recalcitrant to chemotherapy, and immunotherapies, such as interferon-alpha and interleukin-2, yield partial or complete remissions for only 10–20% of patients [ 36 ] . This is particularly true for bone metastasis, for which no effective therapy has been identified. Thus, treatment goals for bone metastases are restricted to mitigating skeletal complications, minimizing pain, and sustaining the quality of life. This necessitates developing novel therapeutic targets for RCC. JS-K effectively releases NO in tumor cells through the action of GSTs, thereby inhibiting tumor growth [ 16 , 17 ] . There are also studies indicating that JS-K can inhibit the growth of renal cancer cells through the induction of DNA damage [ 45 ] . Despite its benefits, only a few studies have examined the effects of JS-K on RCC. In this study, JS-K exerted limited impact on RCC proliferation at concentrations ranging from 0.25 to 1 µM. Nevertheless, it significantly inhibited RCC migration and invasion in a concentration-dependent manner. During EMT, cancer cells lose intercellular adhesion and polarity, acquiring a mesenchymal phenotype that enhances their invasive capacity [ 37 ] . JS-K significantly downregulated N-cadherin and vimentin (mesenchymal markers)) and upregulated E-cadherin (epithelial marker), suggesting that it may impede EMT in RCC. Furthermore, ECM breakdown by MMPs facilitates cancer cell invasion during metastasis [ 38 ] . JS-K treatment significantly decreased MMP-2 and MMP-9 protein expression while increasing TIMP-1 protein expression. Thus, JS-K can inhibit EMT and ECM remodeling processes in RCC, thus impairing their invasive and migratory capacities. The NF-κB signaling pathway is central to inflammation, immune responses, and cancer invasion. JS-K-treated RCC cells expressed higher IκBα mRNA and protein. However, JS-K did not significantly alter the expression of p65 and p50 and inhibited their nuclear translocation. The involvement of NF-κB pathway in the anti-tumor action of JS-K was confirmed through the pharmacological inhibition of NF-κB or with RANKL. Bone remains a frequent site of metastasis in RCC, with approximately 20–35% of patients developing bone metastases [ 39 , 40 ] . Metastatic RCC cells frequently affect bones in the pelvis, spine, and ribs [ 41 ] . Furthermore, RCC-related bone metastases are associated with a higher incidence of skeletal-related events, such as radiation therapy for bone pain, bone surgery, pathological fractures, spinal cord, nerve root compression, and hypercalcemia, compared with bone metastases in other tumor types [ 42 ] . JS-K blocks the invasion of breast cancer cells through the matrix rather than type I collagen, suggesting a likelihood of targeting the basement membrane over the bone matrix [ 43 ] . Nonetheless, previous studies had prolonged experimental period and inadequate processing of breast cancer cells, which may have affected their capacity to traverse type I collagen. Through the osteoprotegerin (OPG)-RANKL-RANK regulatory axis, JS-K treatment upregulated the RANKL gene in RCC cell lines and activated the downstream NK-κB pathway. RCC is characterized by high levels of RANK but low levels of RANKL [ 44 ] . The OPG-RANKL-RANK pathway is strongly associated with bone metastasis. Osteoblasts express higher levels of RANKL, compared with RCC [ 44 ] . Therefore, RCC mutate and autosecrete the RANKL factor, inducing bone metastasis. Osteoblasts expressing higher levels of RANKL promote RCC colonization and subsequent proliferation. Therefore, RCC canmetastasize to the bone. Furthermore, type I collagen is an important component of the periosteum. For RCC to metastasize to the bone, it needs to evade the periosteal barrier composed of type I collagen. In this study, JS-K likely inhibited the breach of the periosteal barrier by modulating the NK-κB pathway, thereby affecting RCC invasion. To summarize, JS-K inhibited RCC migration and invasion by interfering with the EMT and ECM remodeling of tumor cells through the NF-κB pathway. Because JS-K may inhibit bone metastasis, it warrants further investigation as a therapeutic agent for mRCC. Declarations Author Contribution declaration Yuwan Zhao: Design; Data curation; Formal analysis; Investigation; Writing-Original draft. Xingzhang Qin: Data curation; Investigation; Methodology; Writing Original draft. Lugang Zhu: Data curation; Formal analysis; Methodology. Xinghua Lin: Software; Supervision. Bailiang Miu: Project administration; Resources; Visualization. Sheng Gao: Conceptualization; Resources. Jianjun Liu: Conceptualization; Funding acquisition; Project administration; Huanshu Tian: Writing-review and editing; Project administration; Conceptualization. Acknowledgements We would like to thank MogoEdit (https://www.mogoedit.com) for its English editing during the preparation of this manuscript. Funding This work was supported by the National Natural Science Funds (No. 81272833) of China. Data Availability declaration All data generated during this study are included in this published article [and its supplementary information files], and the original data are available on request from the corresponding author. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Clinical trial number Not applicable. References Bray F, Ferlay J, Soerjomataram I, Jemal A, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394–424. Barata PC, Rini BI. Treatment of renal cell carcinoma: Current status and future directions. CA Cancer J Clin. 2017;67(6):507–24. Rabinovitch RA, Zelefsky MJ, Gaynor JJ, et al. Patterns of failure following surgical resection of renal cell carcinoma: implications for adjuvant local and systemic therapy. J Clin oncology: official J Am Soc Clin Oncol. 1994;12:206–12. Franzen AM, Glitzky S, Moganti A, et al. Metastases to Both Parotid Glands Six and Twelve Years after Resection of Renal Cell Carcinoma. Case Rep Med. 2018;2018:7301727. Lalani AA, McGregor BA, Albiges L, et al. Systemic Treatment of Metastatic Clear Cell Renal Cell Carcinoma in 2018: Current Paradigms, Use of Immunotherapy, and Future Directions. Eur Urol. 2019;75(1):100–10. Hakimi AA, Voss MH, Kuo F. Transcriptomic Profiling of the Tumor Microenvironment Reveals Distinct Subgroups of Clear Cell Renal Cell Cancer: Data from a Randomized Phase III Trial. Cancer Discov. 2019;9(4):510–25. Cao C, Bi X, Liang J, et al. Long-term survival and prognostic factors for locally advanced renal cell carcinoma with renal vein tumor thrombus. BMC Cancer. 2019;19:144. Ha US, Lee KW, Jung JH, et al. Renalcapsular invasion is a prognostic biomarker in localized clear cell renal cell carcinoma. Sci Rep. 2018;8:202. Masannat J, Purayil HT, Zhang Y, et al. βArrestin2 Mediates Renal Cell Carcinoma Tumor Growth. Sci Rep. 2018;20(1):4879. Ma YL, Chen F, Shi J. Rhein inhibits malignant phenotypes of human renal cell carcinoma by impacting on MAPK/NF-κB signaling pathways. Onco Targets Ther. 2018;14:11:1385–94. Jung K, Lein M, Laube C, et al. Blood specimen collection methods influence the concentration and the diagnostic validity of matrix metalloproteinase 9 in blood. Clin Chim Acta. 2001;314(1–2):241–4. Chen P, Quan J, Jin L, et al. miR-216a-5p acts as an oncogene in renal cell carcinoma. Exp Ther Med. 2018;15(4):4039–46. Joosten SC, Hamming L, Soetekouw PM, et al. Resistance to sunitinib in renal cell carcinoma: From molecular mechanisms to predictive markers and future perspectives. Biochim Biophys Acta. 2015;1855(1):1–16. Santoni M, Pantano F, Amantini C, et al. Emerging strategies to overcome the resistance to current mTOR inhibitors in renal cell carcinoma. Biochim Biophys Acta. 2014;1845(2):221–31. Zhou L, Liu XD, Sun M, et al. Targeting MET and AXL overcomes resistance to sunitinib therapy in renal cell carcinoma. Oncogene. 2016;35(21):2687–97. Dougall WC. Molecular pathways: osteoclast-dependent and osteoclast-independent roles of the RANKL/RANK/OPG pathway in tumorigenesis and metastasis. Clin Cancer Res. 2012;18(2):326–35. Wittrant Y, Théoleyre S, Chipoy C, et al. RANKL/RANK/OPG: new therapeutic targets in bone tumours and associated osteolysis. Biochim Biophys Acta. 2004;1704(2):49–57. Zhang J, Dai J, Yao Z, Keller ET, et al. Soluble receptor activator of nuclear factor jB Fc diminishes prostate cancer progression in bone. Cancer Res. 2003;63:7883–90. Mikami S, Katsube K, Oya M, et al. Increased RANKL expression is related to tumour migration and metastasis of renal cell carcinomas. J Pathol. 2009;218(4):530–9. Chakrapani H, Kalathur RC, Maciag AE, et al. Synthesis, mechanistic studies, and anti-proliferative activity of glutathione/glutathione S-transferase-activated nitric oxide prodrugs. Bioorg Med Chem. 2008;16(22):9764–71. Maciag AE, Saavedra JE, Chakrapani H. The nitric oxide prodrug JS-K and its structural analogues as cancer therapeutic agents. Anticancer Agents Med Chem. 2009;9(7):798–803. Furchgott RF. Endothelium-Derived Relaxing Factor: Discovery, Early Studies, and Identification as Nitric Oxide (Nobel Lecture). Angew Chem Int Ed Engl. 1999;38(13–14):1870–80. Ignarro LJ. Nitric Oxide: A Unique Endogenous Signaling Molecule in Vascular Biology (Nobel Lecture). Angew Chem Int Ed Engl. 1999;38(13–14):1882–92. Murad F. Discovery of Some of the Biological Effects of Nitric Oxide and Its Role in Cell Signaling (Nobel Lecture). Angew Chem Int Ed Engl. 1999;38(13–14):1856–68. Qiu M, Ke L, Zhang S, et al. JS-K, a GST-activated nitric oxide donor prodrug, enhances chemo-sensitivity in renal carcinoma cells and prevents cardiac myocytes toxicity induced by Doxorubicin. Cancer Chemother Pharmacol. 2017;80(2):275–86. Zekri J, Ahmed N, Coleman RE, Hancock BW. The skeletal metastatic complications of renal cell carcinoma. Int J Oncol. 2001;19:379–82. Chakrapani H, Kalathur RC, Maciag AE, et al. Synthesis, mechanistic studies, and anti-proliferative activity of glutathione/glutathione S-transferase-activated nitric oxide prodrugs. Bioorg Med Chem. 2008;16(22):9764–71. Wink DA, Mitchell JB. Nitric oxide and cancer: an introduction. Free Radic Biol Med. 2003;34(8):951–4. Wink DA, Vodovotz Y, Laval J, et al. Carcinogenesis. 1998;19:711–21. Wink DA, Mitchell JB. Free Rad. Biol Med. 1998;25:434–56. Simeone AM, McMurtry V, Nieves-Alicea R, et al. TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells. Breast Cancer Res. 2008;10(3):R44. Kiziltepe T, Anderson KC, Kutok JL, et al. JS-K has potent anti-angiogenic activity in vitro and inhibits tumour angiogenesis in a multiple myeloma model in vivo. J Pharm Pharmacol. 2010;62(1):145–51. Maciag AE, Chakrapani H, Saavedra JE, et al. The nitric oxide prodrug JS-K is effective against non-small-cell lung cancer cells in vitro and in vivo: involvement of reactive oxygen species. Pharmacol Exp Ther. 2011;336(2):313–20. Liu Z, Li G, Gou Y, et al. JS-K, a nitric oxide prodrug, induces DNA damage and apoptosis in HBV-positive hepatocellular carcinoma HepG2.2.15 cell. Biomed Pharmacother. 2017;92:989–97. Rini BI. Stabilization of disease in patients with metastatic renal cell carcinoma using sorafenib. Nat Clin Pract Oncol. 2006;3(11):602–3. Motzer RJ, Bacik J, Mariani T, et al. Treatment outcome and survival associated with metastatic renal cell carcinoma of non-clear-cell histology. J Clin Oncol. 2002;20(9):2376–81. Yeung KT, Yang J. Epithelial-mesenchymal transition in tumor metastasis. Mol Oncol. 2017;11(1):28–39. Roy R, Yang J, Moses MA. Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer. J Clin Oncol. 2009;27:5287–97. Kozlowski J. Management of distant solitary recurrence in the patient with renal cancer. Urol Clin North Am. 2004;21:601–24. Lipton A, Colombo-Berra A, Bukowski RM, et al. Skeletal complications in patients with bone metastases from renal cell carcinoma and therapeutic benefits of zoledronic acid. Clin Cancer Res. 2004;10(18 Pt 2):S6397–403. Zekri J, Ahmed N, Coleman RE, et al. The skeletal metastatic complications of renal cell carcinoma. Int J Oncol. 2001;19:379–82. Smith MR. Zoledronic acid to prevent skeletal complications in cancer: corroborating the evidence. Cancer Treat Rev. 2005;31(Suppl 3):19–25. Simeone AM, McMurtry V, Nieves-Alicea R, et al. TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells. Breast Cancer Res. 2008;10(3):R44. Mikami S, Katsube K, Oya M, et al. Increased RANKL expression is related to tumour migration and metastasis of renal cell carcinomas. J Pathol. 2009;218(4):530–9. Yuwan Z, Qiuming L, Jierong M, et al. Metformin in combination with JS-K inhibits growth of renal cell carcinoma cells via reactive oxygen species activation and inducing DNA breaks[. J] J Cancer. 2020;11:0. Additional Declarations No competing interests reported. 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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-6460853","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472848309,"identity":"a11d69e2-5af5-41d2-9900-40c8f5689fc0","order_by":0,"name":"Yuwan Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYDACCTBpw8PG3tj44AMJWtLk+HgONxvOIEHLYWM5ifQ2aQ5idMjPbn74mLeNObFN8mGDNAODnZxuAwEtjHOOGRvztrEltkknNhgXMCQbmx0goIVZIsFMmncbD1hL8gyGA4nbCGlhk0j/BtQiAXTYwYbDPMRo4ZHIAdliYMwmwdjYTJQWCYmcYsO5/xLk2HgSmxlnGBDhF/kZ6RsfvDnzn0e+/fjzHx8q7OQIagEBJh4404AI5SDA+INIhaNgFIyCUTBCAQAjtz4xL7ZkZQAAAABJRU5ErkJggg==","orcid":"","institution":"Laboratory of Urology, Affiliated Hospital of Guangdong Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yuwan","middleName":"","lastName":"Zhao","suffix":""},{"id":472848310,"identity":"740cd7bb-9464-4900-9e2e-31c058ba9d33","order_by":1,"name":"Xingzhang Qin","email":"","orcid":"","institution":"Laboratory of Urology, Affiliated Hospital of Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xingzhang","middleName":"","lastName":"Qin","suffix":""},{"id":472848311,"identity":"9878ab7a-d642-4c60-9ef9-e6db92e73e10","order_by":2,"name":"Lugang Zhu","email":"","orcid":"","institution":"Laboratory of Urology, Affiliated Hospital of Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lugang","middleName":"","lastName":"Zhu","suffix":""},{"id":472848312,"identity":"aa33cff1-5154-45f9-8b7e-a1f1f2000709","order_by":3,"name":"Xinghua Lin","email":"","orcid":"","institution":"Laboratory of Urology, Affiliated Hospital of Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinghua","middleName":"","lastName":"Lin","suffix":""},{"id":472848313,"identity":"f17a1d38-fde1-4b79-b5fc-5d5cc8b7c89b","order_by":4,"name":"Bailiang Miu","email":"","orcid":"","institution":"Laboratory of Urology, Affiliated Hospital of Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bailiang","middleName":"","lastName":"Miu","suffix":""},{"id":472848314,"identity":"93f9c818-7cd8-4ede-8218-00c416d86830","order_by":5,"name":"Sheng Gao","email":"","orcid":"","institution":"Laboratory of Urology, Affiliated Hospital of Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sheng","middleName":"","lastName":"Gao","suffix":""},{"id":472848315,"identity":"a86813c7-c50b-4a55-b2ce-6bd9f0a3d2d4","order_by":6,"name":"Jianjun Liu","email":"","orcid":"","institution":"Laboratory of Urology, Affiliated Hospital of Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianjun","middleName":"","lastName":"Liu","suffix":""},{"id":472848316,"identity":"fee04a7f-4211-4be1-9533-24cc8ba4ef67","order_by":7,"name":"Huanshu Tian","email":"","orcid":"","institution":"Laboratory of Urology, Affiliated Hospital of Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huanshu","middleName":"","lastName":"Tian","suffix":""}],"badges":[],"createdAt":"2025-04-16 07:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6460853/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6460853/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85081049,"identity":"cb691e45-9a3c-423e-b77f-13b05608c5b4","added_by":"auto","created_at":"2025-06-20 17:45:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":20686230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of JS-K on the migration, invasion, and adhesion in RCC 786-O and A498 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot shows the effects of JS-K on the expression of EMT and ECM remodeling-related proteins; (B) Quantitative statistics of A; (C) the effects JS-K on affects the EMT process adhesion of 786-O and A498 cells; (D, E) Wound healing assay shows the effect of JS-K on RCC migration. (F and G) Transwell assay shows the effects of JS-K on RCC invasion and migration. All data are presented as the mean ± SD based on one-way ANOVA (n = 3, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001 versus negative control).\u003c/p\u003e\n\u003cp\u003eJS-K, O2-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1, 2-diolate; RCC, renal cell carcinoma; EMT, epithelial-mesenchymal transition; ECM, extracellular matrix; SD, standard deviation; and ANOVA, analysis of variance.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6460853/v1/e70773822fe62d3659a39282.png"},{"id":85081038,"identity":"e33b833d-5a24-4c8a-8da1-625e07b14223","added_by":"auto","created_at":"2025-06-20 17:45:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6902966,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJS-K affects RCC through the NF-kB signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) RNA-seq detects the difference in mRNA expression of signaling pathway-related genes after JS-K treatment of RCC; (B) qPCR confirms the mRNA levels of key genes in the NK-κB signaling pathway; (C and D) Western blot shows the expression of NF-κB signaling pathway-related proteins after JS-K treatment. (E and F ) Western blot shows the expression of NF-κB signaling pathway-related proteins in the nucleus and cytoplasm after JS-K treatment; (G and H) Immunofluorescence staining detects the distribution and changes in p65 and p50 levels in the cytoplasm and nucleus after 24 h.. All data are presented as the mean ± SD based on one-way ANOVA (n = 3, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus negative control).\u003c/p\u003e\n\u003cp\u003eJS-K, O2-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1, 2-diolate; RCC, renal cell carcinoma; RNA-seq, RNA sequencing; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; qPCR, quantitative polymerase chain reaction; SD, standard deviation; and ANOVA, analysis of variance.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6460853/v1/b5455fbde0dbb2b2104883da.png"},{"id":85081634,"identity":"f877a15d-40a4-4f53-a6c4-01b1fe6c5708","added_by":"auto","created_at":"2025-06-20 17:53:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60209109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of JS-K combined with NF-κB inhibitors and growth factors on RCC invasion and migration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A and B) Wound healing assay shows changes in RCC migration ability after treatment with JS-K (1 μM) alone or in combination with NF-κB inhibitors Bay-11-7082 (4 μM) and RANKL (20 μg/mL); (C and D) Western blot detects changes in NF-κB pathway-related proteins in RCC cell lines treated with JS-K (1 μM) alone or in combination with NF-κB inhibitors Bay-11-7082 (4 μM) and RANKL (20 μg/mL); and (E and F) Transwell assay shows changes in RCC invasive ability after treatment with JS-K (1 μM) alone or in combination with NF-κB inhibitors Bay-11-7082 (4 μM) and RANKL (20 μg/mL). All data are presented as the mean ± SD based on one-way ANOVA (n = 3, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus negative control).\u003c/p\u003e\n\u003cp\u003eJS-K, O2-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1, 2-diolate; RCC, renal cell carcinoma; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; RANKL, receptor activator of nuclear factor-kappa B ligand; SD, standard deviation; and ANOVA, analysis of variance.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6460853/v1/6787218f6384dca7438125f5.png"},{"id":85081638,"identity":"60ce8ac5-e8a2-46eb-869a-16a5a4848214","added_by":"auto","created_at":"2025-06-20 17:54:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10014774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJS-K inhibits RCC bone metastasis through the NF-kB pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Transwell assay detects the effect of JS-K on 786-O and A498 cells in invading ; \u003ca href=\"https://www.thermofisher.cn/order/catalog/product/cn/en/A1048301\"\u003ecollagen I\u003c/a\u003e; (B) Statistical analysis of A; (C) Transwell assay detects the effect of JS-K on RCCRCC invasion ability after treatment with NF-κB inhibitor Bay-11-7082 (4 μM) and NF-κB activator (20 μg/mL); and (D) Statistical analysis of CC. All data are presented as the mean ± SD based on one-way ANOVA (n = 3, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 versus negative control).\u003c/p\u003e\n\u003cp\u003eJS-K, O2-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1, 2-diolate; RCC, renal cell carcinoma; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; RANKL, receptor activator of nuclear factor-kappa B ligand; SD, standard deviation; ANOVA, analysis of variance.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6460853/v1/6b2ff926ff750e49bc706212.png"},{"id":85081057,"identity":"bd39d799-e61b-4b09-81cb-742956e42170","added_by":"auto","created_at":"2025-06-20 17:46:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10677887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell signaling pathway diagram\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJS-K inhibits RCC migration, invasion, and bone metastasis through the NF-kB pathway.\u003c/p\u003e\n\u003cp\u003eRCC, renal cell carcinoma; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6460853/v1/339e9fe025503cbb84ddb432.png"},{"id":90509330,"identity":"8ab81ba5-c8f3-44e3-ab54-3e28a5d07dd2","added_by":"auto","created_at":"2025-09-03 13:17:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":95032652,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6460853/v1/3c1b5a0d-112d-4c25-8cca-a34cb201848e.pdf"},{"id":85081056,"identity":"e0479b58-eaed-415d-a674-b7dac66fd200","added_by":"auto","created_at":"2025-06-20 17:46:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":273940,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6460853/v1/a78a333e4e1fee82d15dcfa0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"JS-K—a NO prodrug—inhibits migration, invasion, and bone metastasis of renal cell carcinoma through the NF-κB signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRenal cell carcinoma (RCC) is a prevalent malignancy of the urinary system, accounting for up to 85% of urological tumors. Over 400,000 newly diagnosed cases of RCC and 175,000 RCC-related deaths are reported worldwide annually\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Surgery remains the mainstay treatment for localized RCC. Nevertheless, approximately 30% ofpatients develop metastatic RCC (mRCC) after the surgical removal of the localized tumor\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Furthermore, RCC progresses slowly, with some cases remaining asymptomatic until the tumor metastasizes to the lymph nodes, lungs, liver, and bones\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Despite advancements in the systemic response to mRCC because of novel therapeutic approaches, metastasis remains the leading cause of death among patients with RCC\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. The 5-year survival rate for patients with primary RCC is over 90%, compared with 70% in patients with localized spread (stage I/II) and 10% in patients with recurrent or distant metastases (stage IV)\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Because the early clinical manifestations of RCC are mostly non-specific, patients are often diagnosed at an advanced stage\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Standard treatments, such as chemotherapy, hormonal therapy, radiotherapy, and surgery, extend the overall survival\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, recurrence is frequently observed during follow-up, particularly in patients with clear cell RCC (ccRCC). Moreover, ccRCC is associated with worse prognosis than non-ccRCC\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, along with a 20\u0026ndash;40% risk of postoperative recurrence\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Despite the advent of novel targeted therapies,RCC tumors often acquire resistance by activating alternative pathways. For instance, activation of upstream receptor tyrosine kinases can trigger signaling cascades leading to continuous tumor growth\u003csup\u003e[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The low effective rate of chemotherapy alone (4\u0026ndash;6%) warrants an in-depth exploration of the molecular mechanisms underlying RCC progression and metastasis to identify novel therapeutic targets.\u003c/p\u003e \u003cp\u003eBone metastasis is a frequent and severe complication of RCC. Approximately 30% of patients develop bone metastasis, which leads to substantial bone pain, skeletal complications, and even death\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Tumor cells release various factors, including transforming growth factor (TGF)-β, to stimulate osteoclasts, resulting in increased secretion of receptor activator of nuclear factor-kappa B ligand (RANKL). Overexpression of RANKL promotes the formation, activation, and survival of osteoclasts, finally increasing bone resorption\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Increased osteoclast activity and subsequent bone destruction have been associated with bone metastasis. Additionally, bone resorption triggers the release of growth factors, such as platelet-derived growth factor, bone morphogenetic protein, and transforming growth factor beta, which increase the production of parathyroid hormone and promote tumor growth\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Wittrant et al.\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e demonstrated that blocking the NF-κB (RANK)/RANKL interaction prevented bone metastases in prostate cancer. Furthermore, RANKL and RANK mRNA levels are upregulated in RCC tissues than in non-neoplastic renal tissues\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. These levels are elevated in high-grade versus low-grade ccRCC and in metastatic versus primary RCC. Elevated RANKL and RANK expression predict cancer recurrence, bone metastasis, and poor prognosis\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Overall, RANKL-RANK signaling promotes the migration and metastasis of cancer cells to distant organs. Thus, accelerated bone remodeling because of tumor cell-released factorsresults in a vicious cycle of bone destruction and tumor growth, culminating in bone metastasis.\u003c/p\u003e \u003cp\u003eJS-K\u0026mdash;a nitric oxide (NO) prodrug\u0026mdash;was developed at the National Cancer Institute in 2008 and is a promising class of NO-based therapeutics. In reactions catalyzed by glutathione-S-transferases (GSTs), JS-K replaces glutathione (GSH) with nucleophilic aromatics to form diazonium anions and spontaneously release NO\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Considering high levels of GST in tumor cells, JS-K can exert anti-cancer effects\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. JS-K is selectively toxic to RCC cell lines at concentrations that do not considerably affect the proliferation of normal renal epithelial cells\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. NO mediates multiple physiological processes, such as smooth muscle relaxation, platelet aggregation, vasodilation, neurotransmission, cell migration, immune responses, and tumorigenesis\u003csup\u003e[\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Its precise biological mechanism of action has not been completely elucidated; nonetheless, the physiological effects of NO depend on its local concentration and exposure time\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. The association between NO and cancer is complex\u003csup\u003e[\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Simeone et al. reported that JS-K decreases the invasiveness of breast cancer cells through the basement membrane by upregulating tissue inhibitor of metalloproteinase-2 (TIMP-2). However, TIMP-2 inhibition does not completely block JS-K, indicating the role of other mechanisms in its anti-tumor effects\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Tanyel et al. reported that JS-K may inhibit the proliferation and migration of endothelial cells and the formation of new blood vessels by regulating angiogenic factors\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Maciag et al.\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e demonstrated that JS-K releases NO in non-small cell lung cancer cell lines (H1703 and H1944) through nucleophilic aromatic substitution, increases reactive oxygen species (ROS) production, and oxidizes GSH to glutathione disulfide (GSSG). The reduction in the intracellular GSH/GSSG ratio triggers apoptosis. Liu et al.\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e reported that JS-K inhibits the growth, colony formation capacity, and migration of hepatitis B virus-positive HepG2 cells. Additionally, Qiu et al.\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e demonstrated that JS-K enhances the anti-tumor activity of doxorubicin by upregulating the p53 pathway and mitigating its cardiotoxicity. However, the effects of JS-K on the invasion and metastasis of RCC remain elusive.\u003c/p\u003e \u003cp\u003eIn summary, this study seeks to explore explore changes in the hierarchy of RCC induced by JS-K through experimental methods, such as the wound healing assay and Transwell assay. Additionally, the effects of JS-K on the genes and protein molecules of RCC were investigated at the microscopic level through experimental methods, such as Western blotting, immunofluorescence staining, RNA sequencing, and quantitative polymerase chain reaction. This study may provide valuable reference data for the treatment of metastatic RCC..\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHuman RCC786-O and A498 cells were obtained from the cell bank of the Chinese Academy of Science (Shanghai, China). The cells were cultured in Roswell Park Memorial Institute-1640 (RPMI 1640) medium (GIBCO, Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% (v/v) fetal bovine serum (FBS; GIBCO, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNuclear and cytoplasmic protein extraction\u003c/h3\u003e\n\u003cp\u003eFirst, the culture dish was washed with phosphate-buffered saline (PBS), and trypsin culture fluid was added to digest the cells. The digested cells were centrifuged, and the supernatant was discarded. Approximately 1 mL of PBS was added to the centrifuged cells, and the centrifugation and supernatant discarding steps were repeated. After the second centrifugation, 100 \u0026micro;L of 3,6,9-trioxaundecan-1-{4-[(2-Chloroethyl)Ethylamino)]-Benzylamino},11-Azide (CEB-A) reagent (Nuclear and Cytoplasmic Protein Extraction Kit P1200, Beyotime Institute of Biotechnology) was added to the cell pellet, which, which was transferred to a pre-cooled 1.5 mL Eppendorf (EP) tube. Second, 1.5 mL EP tube was vortexed vigorously for 30 s and placed in an ice bath for 10 to 15 min, with vortexing for 15 s every 5 min. Third, the EP tube was centrifuged to resuspend (12,000 \u003cem\u003eg\u003c/em\u003e, 4\u0026deg;C, 5 min). Without disturbing the pellet, the supernatant was transferred to a new EP tube (the cytoplasmic protein, to be measured quantitatively by bicinchoninic acid assay (BCA)). Fourth, the maximum supernatant was discarded from the EP tube, and the pellet was retained. Subsequently, 100 \u0026micro;L of CEB-A reagent and 5 \u0026micro;L of CEB-B reagent were added, with vortexing for 10 s to resuspend the pellet. The tube was placed in an ice bath for 1 min and centrifuged again (1,000 \u003cem\u003eg\u003c/em\u003e, 5 min). The supernatant was completely discarded, and the pellet was retained. Fifth, 50 to 100 \u0026micro;L of pre-cooled NEB was added to the retained pellet to resuspend it. The tube was vortexed vigorously for 15 s, placed in an ice bath for 30 min, and vortexed for 15 s every 10 min. It was centrifuged (12,000 \u003cem\u003eg\u003c/em\u003e, 5 min), and the supernatant was retained (the nuclear protein, to be measured quantitatively by BCA). After sample extraction, adjustments, adjustments were made per the protein concentration measured by BCA. Finally, 1/4 volume of sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (5\u0026times;) was added. The sample was boiled for 10 min at 100\u0026deg;C and stored frozen.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eTotal protein was extracted from the cultured cells using radioimmuno precipitation assay buffer (Beyotime, Shanghai, China) supplemented with 1 mM phenylmethanesulfonyl fluoride (Beyotime, Shanghai China). Equal amounts of protein per sample were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated overnight at 4\u0026deg;C with primary antibodies specific for E-cadherin (#3195S), N-cadherin (#4061S), matrix metallo proteinase (MMP)-2 (#87809S), MMP-9 (#2270S), TIMP-1 (#2270S), P65 (#4695S), p50 (#4370S), and nuclear factor of kappa light poly peptide gene enhancer in B-cells inhibitor alpha (IκBα) (#4359S). All primary antibodies were from Proteintech (Chicago, USA) and diluted 1:1000 in Primary Antibody Dilution Buffer (Beyotime, Shanghai China). The membranes were washed three times with Tris-buffered saline and Tween 20for 10 min each and incubated with immunoglobulin G-horseradish peroxidase secondary antibody (EarthOx, USA) for 1 h at room temperature. Protein bands were detected using an enhanced chemiluminescence kit (EMD Millipore, Billerica, MA, USA) and the Tanon 5200 chemiluminescent imaging system (Shanghai, China). In order to save the antibody reagent, we cut the band according to the instructions of the protein marker, and then incubated and exposed the internal reference protein and the target protein band respectively. If the reference protein bands were not neat, we discarded the protein extract of this batch, re-extracted the protein, and repeated the above process. Therefore, the protein bands presented in the original data were the bands after the first cleavage. The bands presented in Fig were the final results.\u003c/p\u003e\n\u003ch3\u003eWound healing assay\u003c/h3\u003e\n\u003cp\u003eCells in the logarithmic growth phase were harvested and seeded into six-well plates. After reaching 100% confluence (approximately 24 h), the monolayer was scratched with a sterile pipette tip to create a \u0026ldquo;wound.\u0026rdquo; The plates were washed three times with PBS to remove the dislodged cells, and a serum-free medium containing suitable drugs was added. Images were captured at 0 h and at different time points under 10x magnification to evaluate the rate of wound healing.\u003c/p\u003e\n\u003ch3\u003eTranswell assay\u003c/h3\u003e\n\u003cp\u003eCell migration, invasion, and bone metastasis were analyzed using Transwell inserts. The chambers were coated with Matrigel and type I collagen for invasion and metastasis assays, respectively. Matrigel was thawed overnight at 4\u0026deg;C and diluted 6:1 with pre-cooled serum-free RPMI-1640, and 100 \u0026micro;L of the diluted gel was applied to the Transwell membrane. The inserts were incubated at 37\u0026deg;C for at least 4 h to solidify the gel. Type I collagen (1 mg/mL) was diluted 49:1 with serum-free RPMI-1640, and 100 \u0026micro;L of the diluted gel was spread on the Transwell membrane. The inserts were placed in an incubator for 40 min to allow solidification.\u003c/p\u003e \u003cp\u003eSuitably treated cells were harvested, resuspended in serum-free medium, and seeded in the upper chambers of the inserts at the density of 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well. The lower chambers were filled with 800 \u0026micro;L complete medium (20% FBS). After incubation for 24 h, the inserts were removed, and cells remaining on the upper surface of the membranes were wiped off using a dry cotton swab. Cells on the lower surface were fixed with 800 \u0026micro;L of 1% paraformaldehyde for 20 min, washed twice with PBS (10 min each), andstained with 500 \u0026micro;L Crystal Violet solution for 20 min. The membranes were washed with PBS, air dried, and viewed under an inverted microscope. The number of cells were counted in five randomly selected fields of view, and the average was calculated. Each experiment was repeated three times.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eRCC cells were harvested and seeded onto coverslips in confocal culture dishes at a density of 1.2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/mL. After overnight incubation, the cells were washed three times with PBS, and cultured with respective drugs for 24 h. The culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 30 min. The cells were washed three times with PBS, permeabilized with 0.5% Triton-X 100 (diluted with PBS) for 30 min, and again washed three times with PBS. These cells were blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature andincubated overnight at 4\u0026deg;C with anti-p65 or anti-p50 primary antibody (diluted 1:100 with 1% BSA, 80 \u0026micro;L). After three washes with PBS, they were incubated in the dark for 2 h with fluorescein 488-labeled goat anti-rabbit/mouse secondary antibody (diluted with PBS, 80 \u0026micro;L). The coverslips were washed three times with PBS, stained with one to two drops of 4\u0026prime;,6-diamidino-2-phenylindole, and viewed under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantitative real-time polymerase chain reaction\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from tissue samples and cultured cells using TRIzol reagent (Invitrogen, Carlsbad CA, USA) and reverse transcribed to cDNA using Prime Script TM RT reagent Kit with g DNA Eraser (Takara, Japan) per the manufacturer's instructions. Quantitative real-time PCR was conducted using TB Green \u0026amp; Premix Ex Taq TM (Tli RNase H Plus) (Takara, Japan) on a continuous fluorescence detector 480II Real-Time PCR system. The cycling conditions were as follows: initial denaturation at 95\u0026deg;C for 30 s, followed by 40 cycles of denaturation at 95\u0026deg;C for 5 s, and annealing at 60\u0026deg;C for 30 s. Relative gene expression was determined using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method, with GAPDH as the internal reference\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The primer sequences were as follows:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ep65: forward: 5\u0026prime;-CTGCCGCCTGTCCTTTCTCATC-3\u0026prime;, reverse: 5\u0026prime;-ATGTCCTCTTTCTGCACCTTGTCAC-3\u0026prime;;\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eP50: forward: 5\u0026prime;-TCACTTGAACACATCAAACGAC-3\u0026prime;, reverse:: 5\u0026prime;-AGTGATTACAATTTCCCCGTCT-3\u0026prime;;\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eIKBa: forward: 5\u0026prime;-GAGACTTTCGAGGAAATACCCC-3\u0026prime;, reverse: 5\u0026prime;-GTAGCCATGGATAGAGGCTAAG-3\u0026prime;; and\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eGAPDH: forward: 5\u0026prime;-GGTGAAGGTCGGAGTCAACGG-3\u0026prime;, reverse: 5\u0026prime;-CCTGGAAGATGGTGATGGGATT-3\u0026prime;.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003ePrimers for all genes and GAPDH were synthesized by Sangon Biotech (Shanghai, China).\u003c/p\u003e\n\u003ch3\u003eRNA sequencing\u003c/h3\u003e\n\u003cp\u003eRCC 786-O cells were treated with 0 and 1 \u0026micro;M JS-K, and cell suspensions were collected after 24 h of treatment. Three samples were acquired for each drug concentration. The cell suspensions were collected using trypsin treatment as follows:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe culture medium was discarded, and the cells were washed with sterile 1\u0026times;PBS solution. Sufficient trypsin solution was added to cover the monolayer of cells. For example, a 150 mm culture flask required 2 mL of trypsin solution, and a 100 mm culture dish required 1 mL of trypsin solution. The vessel was gently shaken to evenly distribute the trypsin over the cell layer until the cells became loose (usually within 12 min).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eOnce the cells were loose, the trypsin solution was quickly discarded by tilting the dish or flask to remove the excess solution with a pipette. AA 1\u0026times;PBS solution was added, and a pipette was used to dislodge the adherent cells..\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAlong with the PBS solution, the cells were collected into nucleasenuclease-free, screw-capped EP tubes and centrifuged at 300 to 500 \u003cem\u003eg\u003c/em\u003e (1,100\u0026ndash;1,500 rpm) for 5 min. The supernatant was discarded, and the cell pellet was collected.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe collected cells were quick-frozen in liquid nitrogen and then to -80\u0026deg;C for storage. Dry ice was used for transporting the samples. These samples were sent to the Beijing Genomics Institute for RNA sequencing, and relevant data were provided by the company. Through KEGG analysis, we can gain a deep understanding of the roles of genes in complex biological systems such as metabolic pathways and signaling pathways. A large number of genes or proteins are matched with the standard metabolic pathways or signaling pathways in the KEGG database to identify pathways that are significantly enriched in specific biological functions. Enrichment statistical methods (such as hypergeometric test, Fisher's exact test, etc.) are usually used to determine whether a specific pathway is significantly enriched. The x-axis is a ratio (Rich Factor/GeneRatio/(GeneRatio/BgRatio)), or fold change of differential expression. The higher the value, the higher the enrichment degree of differential metabolites/proteins/genes in the pathway. The y-axis represents the names of the enriched pathways, and the top 20 enriched pathways are selected for plotting. The size of the dot indicates the number of genes. The larger the dot, the more genes are enriched in the pathway. The color represents the level of the p-value. The smaller the p-value, the more significant the pathway is.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraph Pad Prism 7.0 and SPSS 19.0 software were used for all statistical analyses. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three experiments. One-way analysis of variance was used to compare multi-group differences and the least significant difference method was used for pairwise comparisons. Comparisons between two groups were conducted using the Student\u0026rsquo;s t-test, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 indicating statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eJS-K inhibits RCC migration, invasion, and adhesion\u003c/h2\u003e \u003cp\u003eRCC cell lines (786-O and A498) were incubated with different concentrations of JS-K (0, 0.25, 0.5, and 1 \u0026micro;M) for 24 h, and the expression of epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin, and vimentin) and extracellular matrix (ECM)-remodeling proteins (MMP-9, MMP-2, and TIMP-1) were analyzed. JS-K treatment downregulated the expression of N-cadherin, vimentin, MMP-2, and MMP-9, while upregulating the expression of E-cadherin and TIMP-1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). Furthermore, JS-K suppresses the EMT process by visually capturing the morphological changes in cells in each group under bright-field microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). JS-K treatment significantly reduced the migration rates of 786-O and A498 cells in the wound healing assay after 6 and 12 h, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Furthermore, the inhibitory effect was concentration-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In the Transwell assay, JS-K-treated cells showed a significant decline in their migration and invasion abilities after 24 h, compared with untreated controls (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eJS-K targets the NF-kB signaling pathway in RCC\u003c/h2\u003e \u003cp\u003eTo determine the molecular mechanisms underlying the effects of JS-K, 786-O cells were treated with 1 \u0026micro;M of JS-K for 24 h. RNA sequencing was conducted to assess transcriptional changes in relevant signaling pathways. Genes involved in the NF-κB signaling pathway showed significant expression changes after JS-K treatment, compared with untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). qPCR confirmed changes in the expression of the p65, p50, and IκBα genes were confirmed in A498 and 786-O cells treated with different concentrations of JS-K (0, 0.25, 0.5, and 1 \u0026micro;M). JS-K treatment significantly upregulated IκBα mRNA expression, whereas p65 and p50 mRNA expression remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Similar changes were observed in protein expression analysis of IκBα, p65, and p50 after treatment with different concentrations of JS-K (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Interestingly, JS-K-treated cells showed increased cytoplasmic expression of p65 and p50 proteins and relatively lower expression in the nuclear fraction (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Therefore, JS-K may inhibit the nuclear translocation of p65 and p50 in RCC cells. Additionally, immunofluorescence staining of the 786-Oand A498 cells was conducted to determine protein localization and confirm the above hypothesis. Fluorescence signals corresponding to p65 and p50 were stronger in the cytoplasm than in the nuclei of RCC cells after JS-K treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH illustrates the nucleocytoplasmic ratios of p65 and p50. Taken together, JS-K modulates the NF-κB signaling pathway in RCC by preventing the nuclear translocation of p65 and p50.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects of JS-K combined with NF-κB inhibitors and growth factors on RCC invasion and migration\u003c/h2\u003e \u003cp\u003eTo determine whether the NF-κB pathway is involved in the anti-tumor effects of JS-K, RCC cells were co-treated with JS-K (1 \u0026micro;M) and either the NF-κB inhibitor Bay-11-7082 (4 \u0026micro;M) or the NF-κB pathway activator RANKL (20 \u0026micro;g/mL). Pharmacological blockade of NF-κB augmented the inhibitory effects of JS-K on RCC migration rates. Contrarily, NF-kB pathway activation rescued the cells from the effects of JS-K and increased their migration rates (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The Transwell assay yielded consistent results. Co-treatment with JK-S and Bay-11-7082 significantly decreased the migration and invasion rates, compared with either drug alone, whereas co-treatment with JK-S and RANKL increased RCC migration and invasion (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Furthermore, co-treatment with and either Bay-11-7082 or RANKL increased the cytoplasmic levels of p65 and p50 while reducing their nuclear levels, compared with single-drug treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The metastatic ability of RCC cells was assessed by the Transwell assay using type I collagen-coated membranes. JS-K reduced RCC invasiveness (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B).) Furthermore, NK-κB inhibition or RANKL co-treatment intensified the inhibitory effects of JK-S on the migration rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), and the differences were statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In summary, JS-K treatment inhibits RCC inhibits RCC migration and invasion through the NF-κB signaling pathway, while preventing RCC bone metastasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRCC is one of the most prevalent malignancies of the urinary system, and approximately 30% of advanced cases progress to lung, bone, lymph node, liver, adrenal gland, and brain metastases\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. General surgery remains a key therapeutic approach for early-stage RCC. However, the prognosis for advanced RCC is poor because of the high probability of metastases. Additionally, mRCC is highly recalcitrant to chemotherapy, and immunotherapies, such as interferon-alpha and interleukin-2, yield partial or complete remissions for only 10\u0026ndash;20% of patients\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. This is particularly true for bone metastasis, for which no effective therapy has been identified. Thus, treatment goals for bone metastases are restricted to mitigating skeletal complications, minimizing pain, and sustaining the quality of life. This necessitates developing novel therapeutic targets for RCC.\u003c/p\u003e \u003cp\u003eJS-K effectively releases NO in tumor cells through the action of GSTs, thereby inhibiting tumor growth\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. There are also studies indicating that JS-K can inhibit the growth of renal cancer cells through the induction of DNA damage\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Despite its benefits, only a few studies have examined the effects of JS-K on RCC. In this study, JS-K exerted limited impact on RCC proliferation at concentrations ranging from 0.25 to 1 \u0026micro;M. Nevertheless, it significantly inhibited RCC migration and invasion in a concentration-dependent manner.\u003c/p\u003e \u003cp\u003eDuring EMT, cancer cells lose intercellular adhesion and polarity, acquiring a mesenchymal phenotype that enhances their invasive capacity\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. JS-K significantly downregulated N-cadherin and vimentin (mesenchymal markers)) and upregulated E-cadherin (epithelial marker), suggesting that it may impede EMT in RCC. Furthermore, ECM breakdown by MMPs facilitates cancer cell invasion during metastasis\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. JS-K treatment significantly decreased MMP-2 and MMP-9 protein expression while increasing TIMP-1 protein expression. Thus, JS-K can inhibit EMT and ECM remodeling processes in RCC, thus impairing their invasive and migratory capacities.\u003c/p\u003e \u003cp\u003eThe NF-κB signaling pathway is central to inflammation, immune responses, and cancer invasion. JS-K-treated RCC cells expressed higher IκBα mRNA and protein. However, JS-K did not significantly alter the expression of p65 and p50 and inhibited their nuclear translocation. The involvement of NF-κB pathway in the anti-tumor action of JS-K was confirmed through the pharmacological inhibition of NF-κB or with RANKL. Bone remains a frequent site of metastasis in RCC, with approximately 20\u0026ndash;35% of patients developing bone metastases\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Metastatic RCC cells frequently affect bones in the pelvis, spine, and ribs\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Furthermore, RCC-related bone metastases are associated with a higher incidence of skeletal-related events, such as radiation therapy for bone pain, bone surgery, pathological fractures, spinal cord, nerve root compression, and hypercalcemia, compared with bone metastases in other tumor types\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. JS-K blocks the invasion of breast cancer cells through the matrix rather than type I collagen, suggesting a likelihood of targeting the basement membrane over the bone matrix\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Nonetheless, previous studies had prolonged experimental period and inadequate processing of breast cancer cells, which may have affected their capacity to traverse type I collagen. Through the osteoprotegerin (OPG)-RANKL-RANK regulatory axis, JS-K treatment upregulated the RANKL gene in RCC cell lines and activated the downstream NK-κB pathway. RCC is characterized by high levels of RANK but low levels of RANKL \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. The OPG-RANKL-RANK pathway is strongly associated with bone metastasis. Osteoblasts express higher levels of RANKL, compared with RCC\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Therefore, RCC mutate and autosecrete the RANKL factor, inducing bone metastasis. Osteoblasts expressing higher levels of RANKL promote RCC colonization and subsequent proliferation. Therefore, RCC canmetastasize to the bone. Furthermore, type I collagen is an important component of the periosteum. For RCC to metastasize to the bone, it needs to evade the periosteal barrier composed of type I collagen. In this study, JS-K likely inhibited the breach of the periosteal barrier by modulating the NK-κB pathway, thereby affecting RCC invasion. To summarize, JS-K inhibited RCC migration and invasion by interfering with the EMT and ECM remodeling of tumor cells through the NF-κB pathway. Because JS-K may inhibit bone metastasis, it warrants further investigation as a therapeutic agent for mRCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuwan Zhao: Design; Data curation; Formal analysis; Investigation; Writing-Original draft. Xingzhang Qin: Data curation; Investigation; Methodology; Writing Original draft. Lugang Zhu: Data curation; Formal analysis; Methodology. Xinghua Lin: Software; Supervision. Bailiang Miu: Project administration; Resources; Visualization. Sheng Gao: Conceptualization; Resources. Jianjun Liu: Conceptualization; Funding acquisition; Project administration; Huanshu Tian: Writing-review and editing; Project administration; Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank MogoEdit (https://www.mogoedit.com) for its English editing during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Funds (No. 81272833) of China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated during this study are included in this published article [and its supplementary information files], and the original data are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Ferlay J, Soerjomataram I, Jemal A, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394\u0026ndash;424.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarata PC, Rini BI. Treatment of renal cell carcinoma: Current status and future directions. CA Cancer J Clin. 2017;67(6):507\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRabinovitch RA, Zelefsky MJ, Gaynor JJ, et al. Patterns of failure following surgical resection of renal cell carcinoma: implications for adjuvant local and systemic therapy. J Clin oncology: official J Am Soc Clin Oncol. 1994;12:206\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranzen AM, Glitzky S, Moganti A, et al. Metastases to Both Parotid Glands Six and Twelve Years after Resection of Renal Cell Carcinoma. Case Rep Med. 2018;2018:7301727.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLalani AA, McGregor BA, Albiges L, et al. Systemic Treatment of Metastatic Clear Cell Renal Cell Carcinoma in 2018: Current Paradigms, Use of Immunotherapy, and Future Directions. Eur Urol. 2019;75(1):100\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHakimi AA, Voss MH, Kuo F. Transcriptomic Profiling of the Tumor Microenvironment Reveals Distinct Subgroups of Clear Cell Renal Cell Cancer: Data from a Randomized Phase III Trial. Cancer Discov. 2019;9(4):510\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao C, Bi X, Liang J, et al. Long-term survival and prognostic factors for locally advanced renal cell carcinoma with renal vein tumor thrombus. BMC Cancer. 2019;19:144.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHa US, Lee KW, Jung JH, et al. Renalcapsular invasion is a prognostic biomarker in localized clear cell renal cell carcinoma. Sci Rep. 2018;8:202.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasannat J, Purayil HT, Zhang Y, et al. βArrestin2 Mediates Renal Cell Carcinoma Tumor Growth. Sci Rep. 2018;20(1):4879.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa YL, Chen F, Shi J. Rhein inhibits malignant phenotypes of human renal cell carcinoma by impacting on MAPK/NF-κB signaling pathways. Onco Targets Ther. 2018;14:11:1385\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJung K, Lein M, Laube C, et al. Blood specimen collection methods influence the concentration and the diagnostic validity of matrix metalloproteinase 9 in blood. Clin Chim Acta. 2001;314(1\u0026ndash;2):241\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen P, Quan J, Jin L, et al. miR-216a-5p acts as an oncogene in renal cell carcinoma. Exp Ther Med. 2018;15(4):4039\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoosten SC, Hamming L, Soetekouw PM, et al. Resistance to sunitinib in renal cell carcinoma: From molecular mechanisms to predictive markers and future perspectives. Biochim Biophys Acta. 2015;1855(1):1\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantoni M, Pantano F, Amantini C, et al. Emerging strategies to overcome the resistance to current mTOR inhibitors in renal cell carcinoma. Biochim Biophys Acta. 2014;1845(2):221\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou L, Liu XD, Sun M, et al. Targeting MET and AXL overcomes resistance to sunitinib therapy in renal cell carcinoma. Oncogene. 2016;35(21):2687\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDougall WC. Molecular pathways: osteoclast-dependent and osteoclast-independent roles of the RANKL/RANK/OPG pathway in tumorigenesis and metastasis. Clin Cancer Res. 2012;18(2):326\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWittrant Y, Th\u0026eacute;oleyre S, Chipoy C, et al. RANKL/RANK/OPG: new therapeutic targets in bone tumours and associated osteolysis. Biochim Biophys Acta. 2004;1704(2):49\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Dai J, Yao Z, Keller ET, et al. Soluble receptor activator of nuclear factor jB Fc diminishes prostate cancer progression in bone. Cancer Res. 2003;63:7883\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMikami S, Katsube K, Oya M, et al. Increased RANKL expression is related to tumour migration and metastasis of renal cell carcinomas. J Pathol. 2009;218(4):530\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChakrapani H, Kalathur RC, Maciag AE, et al. Synthesis, mechanistic studies, and anti-proliferative activity of glutathione/glutathione S-transferase-activated nitric oxide prodrugs. Bioorg Med Chem. 2008;16(22):9764\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaciag AE, Saavedra JE, Chakrapani H. The nitric oxide prodrug JS-K and its structural analogues as cancer therapeutic agents. Anticancer Agents Med Chem. 2009;9(7):798\u0026ndash;803.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFurchgott RF. Endothelium-Derived Relaxing Factor: Discovery, Early Studies, and Identification as Nitric Oxide (Nobel Lecture). Angew Chem Int Ed Engl. 1999;38(13\u0026ndash;14):1870\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIgnarro LJ. Nitric Oxide: A Unique Endogenous Signaling Molecule in Vascular Biology (Nobel Lecture). Angew Chem Int Ed Engl. 1999;38(13\u0026ndash;14):1882\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurad F. Discovery of Some of the Biological Effects of Nitric Oxide and Its Role in Cell Signaling (Nobel Lecture). Angew Chem Int Ed Engl. 1999;38(13\u0026ndash;14):1856\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu M, Ke L, Zhang S, et al. JS-K, a GST-activated nitric oxide donor prodrug, enhances chemo-sensitivity in renal carcinoma cells and prevents cardiac myocytes toxicity induced by Doxorubicin. Cancer Chemother Pharmacol. 2017;80(2):275\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZekri J, Ahmed N, Coleman RE, Hancock BW. The skeletal metastatic complications of renal cell carcinoma. Int J Oncol. 2001;19:379\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChakrapani H, Kalathur RC, Maciag AE, et al. Synthesis, mechanistic studies, and anti-proliferative activity of glutathione/glutathione S-transferase-activated nitric oxide prodrugs. Bioorg Med Chem. 2008;16(22):9764\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWink DA, Mitchell JB. Nitric oxide and cancer: an introduction. Free Radic Biol Med. 2003;34(8):951\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWink DA, Vodovotz Y, Laval J, et al. Carcinogenesis. 1998;19:711\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWink DA, Mitchell JB. Free Rad. Biol Med. 1998;25:434\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimeone AM, McMurtry V, Nieves-Alicea R, et al. TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells. Breast Cancer Res. 2008;10(3):R44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiziltepe T, Anderson KC, Kutok JL, et al. JS-K has potent anti-angiogenic activity in vitro and inhibits tumour angiogenesis in a multiple myeloma model in vivo. J Pharm Pharmacol. 2010;62(1):145\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaciag AE, Chakrapani H, Saavedra JE, et al. The nitric oxide prodrug JS-K is effective against non-small-cell lung cancer cells in vitro and in vivo: involvement of reactive oxygen species. Pharmacol Exp Ther. 2011;336(2):313\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Z, Li G, Gou Y, et al. JS-K, a nitric oxide prodrug, induces DNA damage and apoptosis in HBV-positive hepatocellular carcinoma HepG2.2.15 cell. Biomed Pharmacother. 2017;92:989\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRini BI. Stabilization of disease in patients with metastatic renal cell carcinoma using sorafenib. Nat Clin Pract Oncol. 2006;3(11):602\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMotzer RJ, Bacik J, Mariani T, et al. Treatment outcome and survival associated with metastatic renal cell carcinoma of non-clear-cell histology. J Clin Oncol. 2002;20(9):2376\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeung KT, Yang J. Epithelial-mesenchymal transition in tumor metastasis. Mol Oncol. 2017;11(1):28\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoy R, Yang J, Moses MA. Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer. J Clin Oncol. 2009;27:5287\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozlowski J. Management of distant solitary recurrence in the patient with renal cancer. Urol Clin North Am. 2004;21:601\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLipton A, Colombo-Berra A, Bukowski RM, et al. Skeletal complications in patients with bone metastases from renal cell carcinoma and therapeutic benefits of zoledronic acid. Clin Cancer Res. 2004;10(18 Pt 2):S6397\u0026ndash;403.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZekri J, Ahmed N, Coleman RE, et al. The skeletal metastatic complications of renal cell carcinoma. Int J Oncol. 2001;19:379\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith MR. Zoledronic acid to prevent skeletal complications in cancer: corroborating the evidence. Cancer Treat Rev. 2005;31(Suppl 3):19\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimeone AM, McMurtry V, Nieves-Alicea R, et al. TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells. Breast Cancer Res. 2008;10(3):R44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMikami S, Katsube K, Oya M, et al. Increased RANKL expression is related to tumour migration and metastasis of renal cell carcinomas. J Pathol. 2009;218(4):530\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuwan Z, Qiuming L, Jierong M, et al. Metformin in combination with JS-K inhibits growth of renal cell carcinoma cells via reactive oxygen species activation and inducing DNA breaks[. J] J Cancer. 2020;11:0.\u003c/span\u003e\u003c/li\u003e\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":"JS-K, renal cell carcinoma, migration and invasion, bone metastasis, EMT, NF-κB signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-6460853/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6460853/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eO2-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1, 2-diolate (JS-K)\u0026mdash;a nitric oxide prodrug\u0026mdash;inhibits the proliferation and migration of breast cancer, non-small cell lung cancer, and liver cancer cells. However, its mechanism of action in renal cell carcinoma (RCC) remains elusive. This study seeks to investigate the effect of JS-K on RCC migration, invasion, and bone metastasisand elucidate the underlying molecular mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHuman RCC cell lines were treated with different concentrations of JS-K, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor Bay-11-7082, or receptor activator of nuclear factor-kappa beta ligand (RANKL), per experimental requirements. In vitro migration, invasion, adhesion, and bone metastasis were assessed by the wound healing assay, Transwell assay, and hanging drop assay. Western blotting and immunofluorescence staining were conducted to evaluate the expression of specific proteins. Changes in gene expression were analyzed by RNA sequencing and quantitative polymerase chain reaction.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eJS-K treatment inhibited the epithelial-mesenchymal transition and extracellular matrix remodeling of RCC in a concentration-dependent manner. Subsequently, the migration, invasion, bone metastasis, and adhesion abilities of RCC cells significantly decreased after JK-S treatment. At the molecular basis, JS-K upregulated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha while inhibiting the nuclear translocation of p65 and p50, suggesting its involvement in the NF-κB signaling pathway. Pharmacological inhibition of NF-κB with either Bay-11-7082 or RANKL influenced the effects of JS-K on RCC migration, invasion, and bone metastasis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eJS-K treatment inhibits RCC migration, invasion, and bone metastasis by targeting the NF-κB signaling pathway.\u003c/p\u003e","manuscriptTitle":"JS-K—a NO prodrug—inhibits migration, invasion, and bone metastasis of renal cell carcinoma through the NF-κB signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-20 17:45:53","doi":"10.21203/rs.3.rs-6460853/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":"f9e4dc56-df9a-4339-9a6f-7324f439028f","owner":[],"postedDate":"June 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-03T13:08:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-20 17:45:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6460853","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6460853","identity":"rs-6460853","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}