BFTC909 cells were purchased from Shanghai Chuanqiu Biotechnology Co., Ltd. and incubated in DMEM/F12 (HyClone; Cytiva) supplemented with 10% fetal bovine serum (FBS; HyClone; Cytiva) at 37°C with 5% CO₂. UM-UC-14 cells were purchased from Shanghai Fusheng Industrial Co., Ltd. and cultured in Eagle's minimum essential medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 2 mM L-glutamine, 10% FBS, antibiotic-antimycotic and 1% non-essential amino acids at 37°C with 5% CO₂.

The cells were incubated with high-efficiency RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) on ice for 5 min. After centrifugation at 11,000 × g for 20 min at 4°C, the supernatant was collected, and the protein concentration was determined using a BCA kit (Beijing Solarbio Science & Technology Co., Ltd.). Then, 20 μg/lane of sample was separated by 12.5% SDS-PAGE and transferred to a PVDF membrane, followed by incubation with 8% skim milk powder (Thermo Fisher Scientific, Inc.) at room temperature for 2 h. After washing with PBST (0.1% Tween) three times, the PVDF membrane was incubated with the following primary antibodies: Anti-PKA (cat. no. ab75991; 1:1,000), anti-COX2 (cat. no. ab179800; 1:1,000), anti-MMP9 (cat. no. ab76003; 1:1,000), anti-VEGF (cat. no. ab32152; 1:1,000) and anti-GAPDH (cat. no. ab8245; 1:3,000) (all primary antibodies were purchased from Abcam) at 4°C overnight. After three washes with PBST, the PVDF membranes were incubated with HRP-labeled secondary antibodies [1:5,000; cat. nos. SE131 (anti-mouse) and SE134 (anti-rabbit); Beijing Solarbio Science & Technology Co., Ltd.] at room temperature for 2 h. Subsequently, the protein expression signals were detected with ECL Western Blotting Substrate (Beijing Solarbio Science & Technology Co., Ltd.). GAPDH was used as an internal reference and ImageJ software (version 1.53a; National Institutes of Health; https://imagej.nih.gov/ij/) was used for analysis.

BFTC909 and UM-UC-14 cells were inoculated into 96-well cell culture plates at 1,000 cells per well and incubated at 37°C overnight. Subsequently, the cells were divided into the control, BZ (Sigma-Aldrich; Merck KGaA; 1, 5, 10 and 50 nM), and emodin (Sigma-Aldrich; Merck KGaA; 12.5, 25, 50, 100 and 200 μM) groups and incubated at 37°C for 48 h. Then, 10 μl CCK-8 solution (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well and the cells were incubated at 37°C for 4 h. Finally, the absorbance was measured at 450 nm, and three replicate wells were set up for each group.

BFTC909 and UM-UC-14 cells were lysed by sonication in an ice bath and centrifuged at 5,000 × g for 20 min at 4°C. After the supernatant was collected, the intracellular cAMP and LDH concentrations were analyzed using the cAMP Assay Kit (cat. no. ab65355; Abcam) and LDH Assay Kit (cat. no. ab102526; Abcam) according to the manufacturer's instructions.

BFTC909 and UM-UC-14 cells were inoculated at 5×10⁵ cells/well in 6-well plates and incubated overnight. The next day, the cells were gently scraped with a 200 μl pipette tip, then washed well with PBS (this was noted as 0 h). Next, the BFTC909 and UM-UC-14 cells were treated with 10 nM BZ or 50 μM emodin at 37°C for 24 h in medium containing 2% serum. Representative images at 0 and 24 h were collected using a light microscope (Olympus Corporation), and the wound healing area was recorded (S1 represented the initial wound area, and S2 represented the wound area after 24 h of treatment). The cell migration rate (%) was calculated as [(S1-S2)/S1] ×100%, and each set of experiments was repeated three times. ImageJ software (version 1.53a; National Institutes of Health; https://imagej.nih.gov/ij/) was used for analysis.

In brief, both BFTC909 and UM-UC-14 cells were seeded at a concentration of 1×10⁴ in the upper chamber with 200 μl serum-free DMEM. Then, 600 μl DMEM containing 10% FBS was added to the lower chambers at 37°C for 24 h. Subsequently, the cells that migrated to the lower chamber were fixed with 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 20 min and stained with 0.1% crystal violet for 20 min at room temperature. Representative images were observed under a light microscope (Olympus Corporation). ImageJ software (version 1.53a; National Institutes of Health; https://imagej.nih.gov/ij/) was used for analysis.

BFTC909 and UM-UC-14 cells were inoculated at a density of 5×10⁵ cells/well in 6-well plates and incubated overnight. Next, 10 μl siRNA targeting COX2 (sense, 5′-AAAUUUGAACAAUAAUUUGGU-3′; antisense, 5′-CAAAUUAUUGUUCAAAUUUAG-3′) or negative control (NC; sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT-3′) (Shanghai GenePharma Co., Ltd.) was added to serum-free medium to achieve a final concentration of 20 nM. Then, 10 μl HiPerFect Transfection Reagent (Qiagen GmbH) was added to the siRNA solution. The mixture was incubated at room temperature for 15 min to form siRNA-HiPerFect complexes. The complexes were carefully added to the respective wells and incubated at 37°C in a 5% CO₂ incubator for 24 h. After the incubation period, the cells were collected for subsequent experiments.

The cells were divided into four groups for treatment: i) Control (Con) group, cells were treated with 10 nM BZ alone for 48 h; ii) emodin group, cells were pretreated with 50 μM emodin for 1 h, followed by the addition of 10 nM BZ for 48 h; iii) Sp-8-CPT-cAMPS group, cells were pretreated with 10 μM Sp-8-CPT-cAMPS (cat. no. HY-120994B; MedChemExpress) for 1 h, followed by the addition of 10 nM BZ for 48 h; and iv) emodin + Sp-8-CPT-cAMPS group, cells were pretreated with a combination of 10 μM Sp-8-CPT-cAMPS and 50 μM emodin for 1 h, followed by the addition of 10 nM BZ for 48 h. Then, the cells were collected for further assay.

Intracellular MDA, SOD, ROS and total antioxidant capacity were analyzed using a Lipid Peroxidation MDA Assay Kit (cat. no. S0131M; Beyotime Institute of Biotechnology), Reactive Oxygen Species Assay Kit (cat. no. S0033S; Beyotime Institute of Biotechnology), Total Superoxide Dismutase Assay Kit (cat. no. S0101S; Beyotime Institute of Biotechnology) and Total Antioxidant Capacity Assay Kit (cat. no. S0121; Beyotime Institute of Biotechnology), respectively. All operations were carried out in strict accordance with the instruction manuals.

Detection of intracellular calcium ion levels was accomplished using an Intracellular Ca²⁺ Calcium Ion Assay Kit (cat. no. HR8227; Beijing Biolab Technology Co., Ltd.). Briefly, BFTC909 and UM-UC-14 cells were collected, and F04 staining solution was incubated with the cells at 37°C for 30 min, after which the intracellular Ca²⁺ content was detected (excitation wavelength of 488-495 nm and emission wavelength of 516 nm).

The cells were initially divided into four groups: The control, emodin (50 μM), BZ (10 nM) and BZ + emodin groups. BFTC909 and UM-UC-14 cells were then treated with the indicated drugs for 24 h. Subsequently, the cells were co-incubated with a final concentration of 1 μM JC-1 dye at 37°C for 30 min. After staining, the cells were washed with PBS and observed under a fluorescence microscope (Olympus Corporation) to assess changes in the mitochondrial membrane potential. At low membrane potentials, JC-1 emits green fluorescence as a monomer, whereas at higher potentials, JC-1 forms 'J-aggregates', emitting red fluorescence.

SPF male BALB/c-nu nude mice (6-8 weeks old; n=25) weighing 15-20 g were obtained from Jinzhou Medical University Experimental Animal Center (Jinzhou, China). All mice were kept in housing with free access to food and water, in an environment with a temperature of 20-24°C and a humidity level of 45-55% throughout a 12 h light/dark cycle. In the experiment, a BFTC909 cell suspension was prepared at a density of 5×10⁷ cells/ml, 0.1 ml was then subcutaneously inoculated into the dorsal region of the nude mice to construct UTUC transplantation tumors. The tumor formation time was 14 days. Subsequently, the nude mice were divided into two groups: The control (n=5) and BZ (n=20) groups (Fig. 1). In the BZ group, nude mice were administered BZ at a dose of 22 mg/kg body weight in 1 ml of water per dose via gavage for 5 consecutive days, based on previous studies (20,21). The control group was administered an equal volume of water. Subsequently, nude mice in the control and BZ groups (n=5 for each group) were sacrificed, and the tumor volume was evaluated as follows: tumor volume=length × width²/2. After evaluation, the remaining 15 nude mice in the BZ group were randomly divided into three groups: the Control (Con) vehicle group, the Low-dose emodin (L-emodin) group, and the High-dose emodin (H-emodin) group. The Control vehicle group received 1 ml of 2% DMSO in saline by gavage once daily. The L-emodin group was administered emodin at a dose of 40 mg/kg intraperitoneally in 1 ml of PBS once daily, while the H-emodin group received emodin at a dose of 80 mg/kg intraperitoneally in 1 ml of PBS once daily. The entire intervention process lasted for 4 weeks.

The humane endpoints used to determine when animals should be euthanized were severe behavioral abnormalities, such as persistent self-mutilation or inability to move normally. The entire intervention process lasted for 4 weeks, and the tumor formation time was 14 days. Thus, the total duration of the experiment was 6 weeks. No animals were euthanized before the end of the study. There were also no instances of animal death due to the experimental procedures. Animal health and behavior were monitored once daily. For anesthesia, the mice were anesthetized using isoflurane, with an induction concentration of 2-3% and a maintenance concentration of 1.5-2%. Deep anesthesia was confirmed by the absence of a response to a paw pinch and the absence of a corneal reflex, following ethical guidelines for animal experimentation. Once deep anesthesia was achieved, ~0.6 ml of cardiac blood was collected via cardiac puncture. Death was verified by confirming the absence of a response to a paw pinch and the absence of a corneal reflex. After blood collection, the subcutaneous xenograft tumors were harvested for further experiments, including the determination of cAMP and PGE2 levels, western blotting and immunohistochemistry (IHC).

The blood samples were centrifuged at 1,500 × g for 10 min at 4°C to separate the serum. An automated biochemical analyzer (Hitachi High-Technologies, Japan) was used to measure the ALT, AST, creatinine and urea levels in the serum, using the respective detection kits: ALT Detection Kit, AST Detection Kit, Creatinine Detection Kit and Urea Detection Kit (all from Zybio Inc.).

Tumor tissues were fixed using 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.) for 24 h at room temperature. After fixation, the tissues were dehydrated with gradient alcohol, cleared with xylene and finally embedded in paraffin. Specifically, the embedded tissues were cut into 5 μm-thick sections, sequentially placed in xylene (three times, 10 min each), gradient alcohol (100, 95, 85 and 70%, 5 min each), and finally washed with distilled water. The tissue sections were autoclave-treated in sodium citrate buffer (pH 6.0; Beijing Solarbio Science & Technology Co., Ltd.) for 15 min. After deparaffinization and rehydration, sections were incubated with 3% hydrogen peroxide (cat. no. H1009; MilliporeSigma) in methanol for 10 min at room temperature to quench endogenous peroxidase activity. Afterwards, the sections were blocked with a solution containing 5% BSA (Thermo Fisher Scientific, Inc.) for 30 min at room temperature. A Ki-67 (1:50; cat. no. 9449, CST Biological Reagents Co., Ltd.) primary antibody was added dropwise to the tissue sections and incubated overnight at 4°C in a wet box. The sections were subsequently washed three times with PBS for 5 min each. Biotinylated secondary antibodies (1:50; cat. no. SHB131; Beijing Solarbio Science & Technology Co., Ltd.) were added to the sections and incubated for 60 min at room temperature. The sections were washed three times with PBS for 5 min each. DAB color development solution (OriGene Technologies, Inc.) was added dropwise to the sections and incubated for 3 min at room temperature, then the reaction was terminated with distilled water. The nuclei were lightly stained with hematoxylin (Beijing Solarbio Science & Technology Co., Ltd.) for 30 sec at room temperature and washed with tap water. Subsequently, the sections were dehydrated with gradient alcohol, cleared with xylene, and blocked with neutral gum (Beijing Solarbio Science & Technology Co., Ltd.). The sections were finally visualized with a light microscope (BZ-X; Keyence Corporation). ImageJ software (version 1.53a; National Institutes of Health; https://imagej.nih.gov/ij/) was used for analysis. The Ki-67 positive cell rate=(Ki-67 positive cells/total cells) ×100%.

The cells were fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were subsequently washed three times with PBS for 5 min each, then permeabilized with 0.3% Triton X-100 (in PBS) for 10 min at room temperature. The cells were blocked with 5% BSA (Thermo Fisher Scientific, Inc.) for 1 h at room temperature, followed by overnight incubation with a COX2 primary antibody (1:50; cat. no. ab179800; Abcam) in a wet box at 4°C. The cells were subsequently washed three times with PBS for 5 min each. The cells were incubated with goat anti-rabbit IgG/Cy3 (1:200; cat. no. K1034G-Cy3; Beijing Solarbio Science & Technology Co., Ltd.) in a wet box protected from the light for 1 h at room temperature. The cells were incubated with Hoechst (Beijing Solarbio Science & Technology Co., Ltd.) for 10 min at room temperature, then washed three times with PBS for 5 min each time. The coverslips were sealed with a sealer containing an anti-fluorescence attenuation reagent (Beijing Solarbio Science & Technology Co., Ltd.). The cells were observed using a fluorescence microscope (Keyence Corporation).

The data are presented as the mean ± standard deviation and were analyzed using Prism 9.0 (Dotmatics). Comparisons between two groups were made using unpaired Student's t-test, and comparisons between more than two groups were made using one-way ANOVA followed by Tukey's HSD test. P<0.05 was considered to indicate statistically significant difference.

The impact of BZ on the viability of BFTC and UM-UC-14 cells was initially investigated. As shown in Fig. 2A and B, BZ significantly enhanced the viability of both cell lines at concentrations of 1, 5, 10 and 50 nM. Furthermore, the wound healing and Transwell assays demonstrated that BZ facilitated the migration of BFTC and UM-UC-14 cells (Fig. 2C-F). Subsequent in vivo experiments further validated these findings, indicating that BZ promoted subcutaneous tumor growth in nude mice (Fig. 2G). IHC staining revealed that BZ increased the percentage of Ki-67-positive cells compared with the control group (Fig. 2H).

It was next evaluated whether emodin could inhibit BZ-induced malignant cell survival in vitro. Compared with BZ alone (10 nM), emodin significantly reduced the viability of both BFTC and UM-UC-14 cells (at concentrations of 12.5, 25, 50, 100, and 200 μM) in a concentration-dependent manner (Fig. 3A and B). The LDH leakage rate, a critical indicator of cell damage, was significantly lower in the BZ-treated group than in the control group; however, emodin treatment increased the LDH leakage rate (Fig. 3C and D). Changes in mitochondrial membrane potential were assessed using the JC-1 staining method. JC-1 is a commonly used fluorescent probe for evaluating mitochondrial membrane potential; it forms aggregates and emits red fluorescence at high membrane potential, while at low membrane potential, it remains in the monomeric form and emits green fluorescence. In this experiment, strong red fluorescence was observed in the untreated control group, indicating a high mitochondrial membrane potential. Compared with the control, cells treated with emodin showed a significant decrease in red fluorescence and an increase in green fluorescence in both BFTC and UM-UC-14 cells, indicating a decrease in mitochondrial membrane potential. Even in the presence of BZ, emodin effectively lowered the mitochondrial membrane potential, as evidenced by the reduced red and increased green fluorescence (Fig. 3E and F).

The effect of BZ on the COX2/PKA signaling pathway was further investigated. It was observed that BZ significantly upregulated the protein expression of PKA and COX2 in BFTC and UM-UC-14 cells transfected with si-NC (Fig. 4A and B). To investigate the role of COX2 in BZ-induced PKA signaling activation, siRNAs specifically targeting COX2 were screened. si-COX2 effectively downregulated COX2 expression and subsequently reduced PKA expression. Furthermore, COX2 knockdown reversed the BZ-induced increase in PKA expression. These findings indicated that BZ-mediated activation of PKA signaling was achieved through the induction of COX2 (Fig. 4A and 4B). Additionally, BZ treatment led to increased levels of cAMP and PGE2 in cell lysates, but si-COX2 reversed these effects with or without BZ treatment (Fig. 4C-F). In vivo, BZ also elevated the protein expression of PKA and COX2 in tumor tissues from nude mice (Fig. 4G) and increased the levels of cAMP and PGE2 within these tumors (Fig. 4H and I).

It was also investigated whether emodin inhibits the cAMP/PKA/COX2 signaling pathway in UTUC cells in the presence of BZ. The findings indicated that emodin combined with BZ markedly decreased cAMP levels in BFTC909 and UM-UC-14 cells compared with BZ treatment alone (Fig. 5A and B). Additionally, western blotting analysis revealed that emodin significantly suppressed the expression of PKA and the downstream effector COX2 in these cells at concentrations of 25 and 50 μM, compared with BZ treatment alone (Fig. 5C and D). Additionally, IF experiments confirmed that BZ increased COX2 expression in BFTC909 and UM-UC-14 cells, while emodin treatment reduced the BZ-induced increase in COX2 expression (Fig. 5E).

To further investigate the capacity of emodin to suppress the malignant phenotype of UTUC cells by inhibiting PKA signaling, the BFTC909 and UM-UC-14 cells were preincubated with BZ before further treatment. Subsequent exposure to Sp-8-CPT-cAMPS, a PKA activator, resulted in a significant increase in PKA and COX2 protein levels, as demonstrated by western blotting analysis (Fig. 6A and B). Notably, the emodin-induced downregulation of PKA and COX2 expression was partially reversed by Sp-8-CPT-cAMPS. Additionally, while emodin significantly reduced the PGE2 levels, treatment with Sp-8-CPT-cAMPS led to an increase in PGE2 production and it significantly reversed the reduction caused by emodin (Fig. 6C and D). In terms of cell viability, compared with the control, Sp-8-CPT-cAMPS notably enhanced the survival of BFTC909 and UM-UC-14 cells, and it notably mitigated the emodin-induced decrease in cell viability (Fig. 6E and F). These findings suggest that emodin effectively inhibits the viability of UTUC cells predominantly through the inhibition of PKA signaling.

Previous studies have shown that PGE2 enhances tumor cell infiltration and metastasis by upregulating MMP9 and VEGF (22,23). In the present study, the effects of emodin on the expression of these critical proteins was investigated. The BFTC909 and UM-UC-14 cells were preincubated with 10 nM BZ before further treatment. It was observed that emodin significantly reduced the expression of MMP9 and VEGF in both BZ-treated BFTC909 and UM-UC-14 cells compared with the control cells. Conversely, treatment with Sp-8-CPT-cAMPS alone led to an increase in the expression of these proteins (Fig. 7A and B). Notably, Sp-8-CPT-cAMPS also counteracted the suppressive effect of emodin on MMP9 and VEGF expression (Fig. 7A and B). Furthermore, emodin decreased the migration of BFTC909 and UM-UC-14 cells relative to that of control cells, while Sp-8-CPT-cAMPS promoted cell migration (Fig. 7C and D). Most notably, Sp-8-CPT-cAMPS reversed the emodin-induced decrease in migration in these BZ-treated cell lines (Fig. 7C and D).

In vivo experiments involving nude mice pretreated with BZ confirmed that emodin could effectively decrease the weight and volume of tumors (Fig. 8A-C). Additionally, emodin reduced the levels of cAMP and PGE2 in tumor tissues (Fig. 8D and E). The in vivo safety of emodin was also evaluated. The experimental results showed that emodin did not have any significant toxic effects on the liver or kidneys of the treated mice. Liver function tests, including the measurement of ALT and AST enzymes, revealed no significant differences between the emodin-treated group and the control group (Fig. 8F and G). Similarly, kidney function markers, such as creatinine and UREA, remained within the normal ranges, indicating no renal toxicity (Fig. 8H and I). By contrast, emodin decreased the LDH leakage rate in tumor tissues compared with the control tissues (Fig. 8J). Ki-67 staining also showed that L-emodin and H-emodin decreased the percentage of Ki-67-positive cells (Fig. 8K). Furthermore, there was a notable decrease in the expression of MMP9 and VEGF in the tumor tissues of these nude mice (Fig. 8L). These findings collectively indicate that emodin exerts a notable inhibitory effect on tumor development in vivo.