Design and Synthesis of sEH/HDAC6 Dual-Targeting Inhibitors for the Treatment of Inflammatory Pain.

Soluble epoxide hydrolase (sEH) and HDAC6 mediate the NF-κB pathway in inflammatory responses, and their inhibitors exhibit powerful anti-inflammatory and analgesic activities in treating both inflammation and pain. Therefore, a series of dual-targeting inhibitors containing urea or squaramide and hydroxamic acid moieties were designed and synthesized, and their role as a new sEH/HDAC6 dual-targeting inhibitor in inflammatory pain was evaluated in a formalin-induced mice model and a xylene-induced mouse ear swelling model. Among them, compounds 28g and 28j showed the best inhibitory and selectivity of sEH and HDAC6. Compound 28g had satisfactory pharmacokinetic characteristics in rats. Following administration at 30 mg/kg, compound 28g exhibited more effective analgesic activity than either an sEH inhibitor (GL-B437) or an HDAC6 inhibitor (Rocilinostat) alone and coadministration of both inhibitors. Thus, these novel sEH/HDAC6 dual-targeting inhibitors exhibited powerful analgesic activity in nociceptive behavior and are worthy of further development.

Ultrathin, Mechanically Robust Quasi-Solid Composite Electrolyte for Solid-State Lithium Metal Batteries.

Herein, we present the preparation and properties of an ultrathin, mechanically robust, quasi-solid composite electrolyte (SEO-QSCE) for solid-state lithium metal battery (SLB) from a well-defined polystyrene-b-poly(ethylene oxide) diblock copolymer (SEO), Li6.75La3Zr1.75Ta0.25O12 nanofiller, and fluoroethylene carbonate plasticizer. Compared with the ordered lamellar microphase separation of SEO, the SEO-QSCE displays bicontinuous phases, consisting of a Li+ ion conductive poly(ethylene oxide) domain and a mechanically robust framework of the polystyrene domain. Therefore, the 12 µm-thick SEO-QSCE membrane exhibits an exceptional ionic conductivity of 1.3 × 10-3 S cm-1 at 30 °C, along with a remarkable tensile strength of 5.1 MPa and an elastic modulus of 2.7 GPa. The high mechanical robustness and the self-generated LiF-rich SEI enable the SEO-QSCE to have an extraordinary lithium dendrite prohibition effect. The SLB of Li|SEO-QSCE|LiFePO4 reveals superior cycling performances at 30 °C for over 600 cycles, maintaining an initial discharge capacity of 145 mAh g-1 and a remarkable capacity retention of 81% (117 mAh g-1) after 400 cycles at 0.5 C. The high-voltage SLB of Li|SEO-QSCE|LiNi0.5Co0.3Mn0.2O2 displays good cycling stability for over 150 cycles at 30 °C. Moreover, the exceptional robustness of SEO-QSCE enables the high-voltage solid-state pouch cell of Li|SEO-QSCE|LiNi0.5Co0.3Mn0.2O2 with high flexibility and excellent safety features. The current investigation delivers a promising and innovative approach for preparing quasi-solid electrolytes with features of ultrathin design, mechanical robustness, and exceptional electrochemical performance for high-voltage SLBs.

Pickering emulsion stabilized by grass carp myofibrillar protein via one-step: Study on microstructure, processing stability and stabilization mechanism.

In this study, edible Pickering emulsions stabilized with GMP particles were prepared for the first time using low-value grass carp myofibrillar protein (GMP). To elucidate the emulsion formation mechanism, the grass carp myofibrillar protein particles were first characterized, and the results showed that the lyophilized GMP particles had an irregular polyhedral structure and were amphiphilic nanoparticles. The stability of GMP-based emulsions tended to stabilize with the increase of GMP concentration at an appropriate oil-to-water volume ratio, with a decrease in droplet particle size and an increase in water-holding capacity, storage stability, and rheological stability, whereas the phenomenon of droplet aggregation after storage appeared at 4.0% additive level. Therefore, appropriate concentrations of GMP at a certain oil-to-water volume ratio can stabilize Pickering emulsions, which have an important future as a potential food-grade active substance delivery carrier for biological applications.

Regulating Electrolyte Solvation Structures via Diluent-Solvent Interactions for Safe High-Voltage Lithium Metal Batteries.

Local high concentration electrolytes (LHCEs) have been proved to be one of the most promising systems to stabilize both high voltage cathodes and Li metal anode for next-generation batteries. However, the solvation structures and interactions among different species in LHCEs are still convoluted, which bottlenecks the further breakthrough on electrolyte development. Here, it is demonstrated that the hydrogen bonding interaction between diluent and solvent is crucial for the construction of LHCEs and corresponding interphase chemistries. The 2,2,2-trifluoroethyl trifluoromethane sulfonate (TFSF) is selected as diluent with the solvent dimethoxy-ethane (DME) to prepare a non-flammable LHCE for high voltage LMBs. This is first find that the hydrogen bonding interaction between TFSF and DME solvent tailors the electrolyte solvation structures by weakening the coordination of DME molecules to Li+ cations and allows more participation of anions in the first solvation shell, leading to the formation of aggregates (AGGs) clusters which are conducive to generating inorganic solid/cathodic electrolyte interphases (SEI/CEIs). The proposed TFSF based LHCE enables the Li||NCM811 (LiNi0.8 Mn0.1 O2 ) batteries to realize >80% capacity retention with a high average Coulombic efficiency of 99.8% for 230 cycles under aggressive conditions (NCM811 cathode: 3.4 mAh cm-2 , cut-off voltage: 4.4 V, and 20 µm Li foil).

Integrative high-throughput enhancer surveying and functional verification divulges a YY2-condensed regulatory axis conferring risk for osteoporosis.

The precise roles of chromatin organization at osteoporosis risk loci remain largely elusive. Here, we combined chromatin interaction conformation (Hi-C) profiling and self-transcribing active regulatory region sequencing (STARR-seq) to qualify enhancer activities of prioritized osteoporosis-associated single-nucleotide polymorphisms (SNPs). We identified 319 SNPs with biased allelic enhancer activity effect (baaSNPs) that linked to hundreds of candidate target genes through chromatin interactions across 146 loci. Functional characterizations revealed active epigenetic enrichment for baaSNPs and prevailing osteoporosis-relevant regulatory roles for their chromatin interaction genes. Further motif enrichment and network mapping prioritized several putative, key transcription factors (TFs) controlling osteoporosis binding to baaSNPs. Specifically, we selected one top-ranked TF and deciphered that an intronic baaSNP (rs11202530) could allele-preferentially bind to YY2 to augment PAPSS2 expression through chromatin interactions and promote osteoblast differentiation. Our results underline the roles of TF-mediated enhancer-promoter contacts for osteoporosis, which may help to better understand the intricate molecular regulatory mechanisms underlying osteoporosis risk loci.

Design and Synthesis of Dual-Targeting Inhibitors of sEH and HDAC6 for the Treatment of Neuropathic Pain and Lipopolysaccharide-Induced Mortality.

Epoxyeicosatrienoic acids with anti-inflammatory effects are inactivated by soluble epoxide hydrolase (sEH). Both sEH and histone deacetylase 6 (HDAC6) inhibitors are being developed as neuropathic pain relieving agents. Based on the structural similarity, we designed a new group of compounds with inhibition of both HDAC6 and sEH and obtained compound M9. M9 exhibits selective inhibition of HDAC6 over class I HDACs in cells. M9 shows good microsomal stability, moderate plasma protein binding rate, and oral bioavailability. M9 exhibited a strong analgesic effect in vivo, and its analgesic tolerance was better than gabapentin. M9 improved the survival time of mice treated with lipopolysaccharide (LPS) and reversed the levels of inflammatory factors induced by LPS in mouse plasma. M9 represents the first sEH/HDAC6 dual inhibitors with in vivo antineuropathic pain and anti-inflammation.

Journey of Mineral Precursors in Bone Mineralization: Evolution and Inspiration for Biomimetic Design.

Bone mineralization is a ubiquitous process among vertebrates that involves a dynamic physical/chemical interplay between the organic and inorganic components of bone tissues. It is now well documented that carbonated apatite, an inorganic component of bone, is proceeded through transient amorphous mineral precursors that transforms into the crystalline mineral phase. Here, the evolution on mineral precursors from their sources to the terminus in the bone mineralization process is reviewed. How organisms tightly control each step of mineralization to drive the formation, stabilization, and phase transformation of amorphous mineral precursors in the right place, at the right time, and rate are highlighted. The paradigm shifts in biomineralization and biomaterial design strategies are intertwined, which promotes breakthroughs in biomineralization-inspired material. The design principles and implementation methods of mineral precursor-based biomaterials in bone graft materials such as implant coatings, bone cements, hydrogels, and nanoparticles are detailed in the present manuscript. The biologically controlled mineralization mechanisms will hold promise for overcoming the barriers to the application of biomineralization-inspired biomaterials.

The Interplay Between HIF-1α and EZH2 in Lung Cancer and Dual-Targeted Drug Therapy.

Interactions between oncogenic proteins contribute to the phenotype and drug resistance. Here, EZH2 (enhancer of zest homolog 2) is identified as a crucial factor that mediates HIF-1 (hypoxia-inducible factor) inhibitor resistance. Mechanistically, targeting HIF-1 enhanced the activity of EZH2 through transcription activation of SUZ12 (suppressor of zest 12 protein homolog). Conversely, inhibiting EZH2 increased HIF-1α transcription, but not the transcription of other HIF family members. Additionally, the negative feedback regulation between EZH2 and HIF-1α is confirmed in lung cancer patient tissues and a database of cell lines. Moreover, molecular prediction showed that a newly screened dual-target compound, DYB-03, forms multiple hydrogen bonds with HIF-1α and EZH2 to effectively inhibit the activity of both targets. Subsequent studies revealed that DYB-03 could better inhibit migration, invasion, and angiogenesis of lung cancer cells and HUVECs in vitro and in vivo compared to single agent. DYB-03 showed promising antitumor activity in a xenograft tumor model by promoting apoptosis and inhibiting angiogenesis, which could be almost abolished by the deletion of HIF-1α and EZH2. Notably, DYB-03 could reverse 2-ME2 and GSK126-resistance in lung cancer. These findings clarified the molecular mechanism of cross-regulation of HIF-1α and EZH2, and the potential of DYB-03 for clinical combination target therapy.

Synthesis and biological evaluations of 8-biaryl-2,2-dimethylbenzopyranamide derivatives against Alzheimer's disease and ischemic stroke.

Alzheimer's disease, the commonest cause of dementia, is a growing global health concern with huge implications for individuals and society. Stroke has still been a significant challenge in clinics for a long time, which is the second leading cause of death in the world, especially ischemic stroke. Both Alzheimer's disease and stroke are closely related to oxidative stress and HIF-1 signaling pathways in nerve cells. Herein, we describe our structure-based design, synthesis, and biological evaluation of a new class of 8-biaryl-2,2-dimethylbenzopyranamide derivatives as natural product derivatives. Our efforts have resulted in the discovery of highly potent neuroprotective agents, as exemplified by compound D13 as a HIF-1α inhibitor, which significant improvement in the behavior of Alzheimer's disease mice and shows great potential improvement of brain infarct volume in pMCAO model rats, improves the increase of blood-brain barrier permeability after cerebral ischemia in rats, neuroprotective effect, reduce the level of apoptotic cells in rats after cerebral ischemia, better than Edaravone.

The Potent Anti-Tumor Effects of Rhodiola Drinking Are Associated with the Inhibition of the mTOR Pathway and Modification of Tumor Metabolism in the UPII-Mutant Ha-Ras Model.

Background: SHR-5 has been used as an "adaptogen" for enhancing physical and mental performance and for fighting stress in the healthy population. The purpose of this study is to determine the chemopreventive efficacy of SHR-5 for superficial bladder cancer and to investigate the underlying mechanisms of action. Methods: UPII-mutant Ha-ras bladder-cancer-transgenic mice, that developed low-grade and noninvasive papillary transitional urothelial cell carcinoma, were fed with 1.25 and 6.25 mg/mL SHR-5 in drinking water for 6 months. The survival of the mice, obstructive uropathy, tumor burden and morphology, and proliferation were evaluated by pathological, molecular, metabolic, and statistical analyses. Results: Approximately 95% or more of the male UPII-mutant Ha-ras mice that drank SHR-5 daily survived over 6 months of age, while only 33.3% of those mice that drank normal water survived over 6 months of age (p < 0.0001); SHR-5 drinking exposure also reduced tumor-bearing bladder weight and urinary tract obstruction and inhibited mTOR signaling in neoplastic tissues. Global metabolic analysis revealed that SHR-5 resulted in increased phenolic metabolites and decreased CoA, a critical metabolic cofactor for lipid metabolism. Conclusions: Our findings highlight the potential of SHR-5 as an anti-aging agent for bladder cancer prevention through reshaping tumor metabolism via the inhibition of the mTOR signaling. Global metabolomics profiling provides a unique and efficient tool for studying the mechanisms of complex herb extracts' action.

Discovery of Dual Lysine Methyltransferase G9a and EZH2 Inhibitors with In Vivo Efficacy against Malignant Rhabdoid Tumor.

In recent years, it has been proposed that G9a/EZH2 dual inhibition is a promising cancer treatment strategy. Herein, we present the discovery of G9a/EZH2 dual inhibitors that merge the pharmacophores of G9a and EZH2 inhibitors. Among them, the most promising compound 15h displayed potent inhibitory activities against G9a (IC50 = 2.90 ± 0.05 nM) and EZH2 (IC50 = 4.35 ± 0.02 nM), superior antiproliferative profiles against RD (CC50 = 19.63 ± 0.18 µM) and SW982 (CC50 = 19.91 ± 0.50 µM) cell lines. In vivo, 15h achieved significant antitumor efficacy in a xenograft mouse model of human rhabdoid tumor with a tumor growth inhibitory rate of 86.6% without causing observable toxic effects. The on-target activity assays illustrated that compound 15h can inhibit tumor growth by specifically inhibiting EZH2 and G9a. Therefore, 15h is a potential anticancer drug candidate for the treatment of malignant rhabdoid tumor.

Kavalactone Kawain Impedes Urothelial Tumorigenesis in UPII-Mutant Ha-Ras Mice via Inhibition of mTOR Signaling and Alteration of Cancer Metabolism.

UPII-mutant Ha-ras transgenic mice develop urothelial hyperplasia and low-grade papillary carcinoma, which mimics human non-muscle invasive bladder cancer (NMIBC). We investigated the effects and mechanisms of kawain, a main kavalactone in the kava plant, on oncogenic Ha-ras-driven urothelial carcinoma in these mice. The mice were fed at six weeks of age with vehicle control or kawain (6 g/kg) formulated food for approximately five months. Seventy-eight percent of the mice or more fed with kawain food survived more than six months of age, whereas only 32% control food-fed male mice survived, (p = 0.0082). The mean wet bladder weights (a surrogate for tumor burden) of UPII-mutant Ha-ras transgenic mice with kawain diet was decreased by approximately 56% compared to those fed with the control diet (p = 0.035). The kawain diet also significantly reduced the occurrence of hydronephrosis and hematuria in UPII-mutant Ha-ras transgenic mice. Histological examination and immunohistochemistry analysis revealed that vehicle control-treated mice displayed more urothelial carcinoma and Ki67-positive cells in the bladder compared to kawain treated mice. Global metabolic profiling of bladder tumor samples from mice fed with kawain food showed significantly more enrichment of serotonin and less abundance of xylulose, prostaglandin A2, D2 and E2 compared to those from control diet-fed mice, suggesting decreased shunting of glucose to the pentose phosphate pathway (PPP) and reduced inflammation. In addition, kawain selectively inhibited the growth of human bladder cancer cell lines with a significant suppression of 4E-BP1 expression and rpS6 phosphorylation. These observations indicate a potential impact of kawain consumption on bladder cancer prevention by rewiring the metabolic programs of the tumor cells.

Mitochondria transfer reverses the inhibitory effects of low stiffness on osteogenic differentiation of human mesenchymal stem cells.

Microenvironment biophysical factors such as matrix stiffness can noticeably affect the differentiation of mesenchymal stem cells (MSCs). In this mechanobiology transduction process, mitochondria are shown to be an active participant. The present study aims to systematically elucidate the phenotypic and functional changes of mitochondria during the stiffness-mediated osteogenic differentiation. Additionally, the effect of mitochondria transfer on the osteogenesis of impaired MSCs caused by stiffness was investigated. Human periodontal ligament stem cells (PDLSCs) were used as model cells in the current study. Low stiffness restrained the cell spreading and significantly inhibited the proliferation and osteogenic differentiation of PDLSCs. Mitochondria of PDLSCs cultured on low stiffness exhibited shorter length, rounded shape, fusion/fission imbalance, ROS and mitophagy level increase, and ATP production reduction. The inhibited mitochondria function and osteogenic differentiation capacity were recovered to near-normal levels after transferring the mitochondria of PDLSCs cultured on the high stiffness. This study indicated that low matrix stiffness altered the mitochondrial morphology and induced systematical mitochondrial dysfunction during the osteogenic differentiation of MSCs. Mitochondria transfer was proved to be a feasible technique for maintaining MSCs function in vitro by reversing the osteogenesis ability.

Structure-Directed Discovery of Potent Soluble Epoxide Hydrolase Inhibitors for the Treatment of Inflammatory Diseases.

Soluble epoxide hydrolase (sEH) has been identified as an attractive target for anti-inflammatory drug design in recent years. Picomolar level compound G1 against sEH was obtained by introducing the hydrophilic group homopiperazine and hydrophobic fragment propionyl onto the structure of lead compound A. G1 showed good microsomal stability, a moderate plasma protein binding rate, and good oral bioavailability and was well tolerated in rats. G1 has significant analgesic effects on CFA-induced AIA mice, ameliorated the pancreatic injury in acute pancreatitis induced by l-arginine, reversed pancreatic injury, edema, and neutrophil infiltration, and increased the survival time of C57BL/6 mice in a lipopolysaccharide (LPS)-induced sepsis model. Moreover the expression levels of sEH, COX-2, NOS-2, vascular cell adhesion molecule (VCAM), IL-6, MCP-5, and tumor necrosis factor α (TNF-α) were measured by Western blot or enzyme-linked immunosorbent assay (ELISA), with varying degrees of decrease. These results suggested that G1 is a drug candidate worthy of further evaluation for the treatment of inflammation-induced diseases such as arthritis, acute pancreatitis, and sepsis.

Daphnetin Alleviates Senile and Disuse Osteoporosis by Distinct Modulations of Bone Formation and Resorption.

Senile and disuse osteoporosis have distinct bone turnover status and lack effective treatments. In this study, senescence-accelerated mouse prone 8 (SAMP8) and hindlimb unloading mouse models were used to explore the protective effects of daphnetin on these two types of osteoporosis, and primary osteoblasts and bone marrow monocyte-derived osteoclasts, as well as pre-osteoblast MC3T3-E1, and osteoclast precursor RAW264.7 cells were used to investigate the underlying mechanisms. The results showed that daphnetin administration effectively improved bone remodeling in both senile and disuse osteoporosis, but with different mechanisms. In senile osteoporosis with low bone turnover, daphnetin inhibited NOX2-mediated ROS production in osteoblasts, resulting in accelerated osteogenic differentiation and bone formation, while in disuse osteoporosis with high bone turnover, daphnetin restored SIRT3 expression, maintained mitochondrial homeostasis, and additionally upregulated SOD2 to eliminate ROS in osteoclasts, resulting in attenuation of osteoclast differentiation and bone resorption. These findings illuminated that daphnetin has promising potential for the prevention and treatment of senile and disuse osteoporosis. The different mechanisms may provide clues and basis for targeted prevention and treatment of osteoporosis according to distinct bone turnover status.

The Osteoinductivity of Calcium Phosphate-Based Biomaterials: A Tight Interaction With Bone Healing.

Calcium phosphate (CaP)-based bioceramics are the most widely used synthetic biomaterials for reconstructing damaged bone. Accompanied by bone healing process, implanted materials are gradually degraded while bone ultimately returns to its original geometry and function. In this progress report, we reviewed the complex and tight relationship between the bone healing response and CaP-based biomaterials, with the emphasis on the in vivo degradation mechanisms of such material and their osteoinductive properties mediated by immune responses, osteoclastogenesis and osteoblasts. A deep understanding of the interaction between biological healing process and biomaterials will optimize the design of CaP-based biomaterials, and further translate into effective strategies for biomaterials customization.

Structure-guided discovery of potent and oral soluble epoxide hydrolase inhibitors for the treatment of neuropathic pain.

Soluble epoxide hydrolase (sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases. A new series of memantyl urea derivatives as potent sEH inhibitors was obtained using our previous reported compound 4 as lead compound. A preferential modification of piperidinyl to 3-carbamoyl piperidinyl was identified for this series via structure-based rational drug design. Compound A20 exhibited moderate percentage plasma protein binding (88.6%) and better metabolic stability in vitro. After oral administration, the bioavailability of A20 was 28.6%. Acute toxicity test showed that A20 was well tolerated and there was no adverse event encountered at dose of 6.0 g/kg. Inhibitor A20 also displayed robust analgesic effect in vivo and dose-dependently attenuated neuropathic pain in rat model induced by spared nerve injury, which was better than gabapentin and sEH inhibitor (±)-EC-5026. In one word, the oral administration of A20 significantly alleviated pain and improved the health status of the rats, demonstrating that A20 was a promising candidate to be further evaluated for the treatment of neuropathic pain.

PINK1 mediated mitophagy attenuates early apoptosis of gingival epithelial cells induced by high glucose.

BACKGROUND: Oxidative stress mediated by hyperglycemia damages cell-reparative processes such as mitophagy. Down-regulation of mitophagy is considered to be a susceptible factor for diabetes mellitus (DM) and its complications. However, the role of mitophagy in DM-associated periodontitis has not been fully elucidated. Apoptosis of human gingival epithelial cells (hGECs) is one of the representative events of DM-associated periodontitis. Thus, this study aimed to investigate PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy activated in the process of high glucose (HG)-induced hGECs apoptosis. METHODS: For dose-response studies, hGECs were incubated in different concentrations of glucose (5.5, 15, 25, and 50 mmol/L) for 48 h. Then, hGECs were challenged with 25 mmol/L glucose for 12 h and 48 h, respectively. Apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL), caspase 9 and mitochondrial membrane potential (MMP). Subsequently, autophagy was evaluated by estimating P62, LC3 II mRNA levels, LC3 fluorescent puncta and LC3-II/I ratio. Meanwhile, the involvement of PINK1-mediated mitophagy was assessed by qRT-PCR, western blotting and immunofluorescence. Finally, hGECs were transfected with shPINK1 and analyzed by MMP, caspase 9 and annexin V-FITC apoptosis. RESULTS: The number of TUNEL-positive cells and caspase 9 protein were significantly increased in cells challenged with HG (25 mmol/L) for 48 h (HG 48 h). MMP was impaired both at HG 12 h and HG 48 h, but the degree of depolarization was more serious at HG 48 h. The autophagy improved as the amount of LC3 II increased and p62 decreased in HG 12 h. During this process, HG 12 h treatment induced PINK1-mediated mitophagy. PINK1 silencing with HG 12 h resulted in MMP depolarization and cell apoptosis. CONCLUSIONS: These results suggested that loss of the PINK1 gene may cause mitochondrial dysfunction and increase sensitivity to HG-induced apoptosis of hGECs at the early stage. PINK1 mediated mitophagy attenuates early apoptosis of gingival epithelial cells induced by high glucose.

Chemoprevention of Urothelial Cell Carcinoma Tumorigenesis by Dietary Flavokawain A in UPII-Mutant Ha-ras Transgenic Mice.

Non-muscle-invasive bladder cancer (NMIBC) has one of the highest recurrence rates among all solid cancers and the highest lifetime treatment cost per patient. Therefore, the development of chemoprevention strategies for reducing the occurrence and recurrence of NMIBC as well as its burdens on the healthcare system is valuable. Our aim was to determine whether flavokawain A (FKA), a kava chalcone isolated from the kava plant, can target the in vivo activated Ha-ras pathway for prevention and treatment of NMIBC. UPII-mutant Ha-ras transgenic mice that develop papillary urothelial cell carcinoma were fed orally with vehicle control or FKA-formulated food for 6 months starting at 6 weeks of age. Seventy-nine percent (15/19) of male mice fed with 6 g FKA per kilogram (kg) of food survived beyond the 6 months of treatment, while 31.6% (6/19) of control food-fed male mice survived the 6-month treatment period (p = 0.02). The mean bladder weights in FKA vs. control food-fed mice were 0.216 ± 0.033 vs. 0.342 ± 0.039 g in male mice (p = 0.0413) and 0.043 ± 0.004 vs. 0.073 ± 0.004 g in female mice (p < 0.0001); FKA reduced bladder weight by 37% and 41%, respectively. The tumor burdens, determined by the wet bladder weight, in these mice were inversely related to plasma FKA concentrations. In addition to decreased bladder weight, FKA treatment significantly reduced the incidences of hydronephrosis and hematuria. FKA-treated mice exhibited more well-differentiated tumors in the bladder and ureter. Immunohistochemical analysis of FKA-treated tumors compared to those in the control group revealed fewer Ki-67- and survivin-positive cells and an increased number of p27- and TUNEL-positive cells, indicating that FKA inhibits proliferation and induces apoptosis. Overall, the results suggest that FKA can target the in vivo activated Ha-ras pathway for the prevention and treatment of NMIBC.