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. 2025 Oct 28;14(10):2902–2916. doi: 10.21037/tau-2025-383

Ursolic acid sensitizes bladder cancer to gemcitabine chemotherapy by concurrently targeting PI3K/AKT and JNK pathways

Xiaolong Huang 1,2, Yan Sun 1, Hang Tong 1, Junlong Zhu 1, Juncheng Pan 2, Peng Wen 2, Weiyang He 1,✉
PMCID: PMC12603850  PMID: 41230145

Abstract

Background

Gemcitabine (GEM) is a first-line chemotherapy for bladder cancer (BCa), but its efficacy is limited by drug resistance and side effects. Ursolic acid (UA), a natural compound from medicinal herbs, has shown potential to enhance chemotherapy. This study investigates whether UA synergizes with GEM against BCa and explores the underlying mechanisms.

Methods

Human BCa cell lines (T24 and 5637) were treated with GEM and/or UA in vitro. Cell viability was assessed via Cell Counting Kit-8 (CCK-8) assay; apoptosis was evaluated using Hoechst 33258 staining, flow cytometry, and western blotting. A xenograft mouse model was employed for in vivo validation. Signaling pathways [phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT), c-Jun N-terminal kinase (JNK)] were analyzed by western blot. Pharmacological modulators (SC79, SP600125) were used to verify pathway roles.

Results

UA synergistically enhanced GEM’s antitumor effects in BCa cells, significantly reducing viability and increasing apoptosis compared to GEM alone. In vivo, UA potentiated GEM’s growth inhibition in xenografts. Mechanistically, UA augmented GEM-induced apoptosis by suppressing PI3K/AKT and activating JNK signaling pathways in vitro and in vivo. Both SC79 (AKT activator) and SP600125 (JNK inhibitor) attenuated apoptosis markers (cleaved PARP, cleaved caspase-3).

Conclusions

UA sensitizes BCa to GEM chemotherapy by promoting apoptosis, mediated through PI3K/AKT inactivation and JNK activation. These findings highlight UA as a promising adjunct to GEM therapy, warranting further clinical exploration.

Keywords: Ursolic acid (UA), gemcitabine (GEM), bladder cancer (BCa), phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT), c-Jun N-terminal kinase (JNK)

Highlight box.

Key findings

• Ursolic acid (UA) sensitizes bladder cancer (BCa) to gemcitabine (GEM) chemotherapy, which is associated with apoptosis induction. We further reveal that UA augments GEM-induced apoptosis by inactivating the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway and activating the c-Jun N-terminal kinase (JNK) signaling pathway in human BCa cells.

What is known and what is new?

• UA is a natural compound that exists in various kinds of Chinese medicinal herbs, which has been demonstrated to enhance the efficacy of chemotherapy in multiple types of cancer.

• Whether UA can enhance the chemotherapeutic effects of GEM in BCa has not been reported to date. This current research aims to investigate whether UA can enhance the anticancer activity of GEM in BCa in vitro and in vivo, and to explore the possible underlying mechanisms.

What is the implication, and what should change now?

• We demonstrate that UA synergistically enhances the anticancer activity of GEM in human BCa. The combined treatment with UA and GEM may provide an experimental basis for the clinical treatment of BCa.

Introduction

Bladder cancer (BCa) ranks as the ninth most prevalent malignancy globally and is the most common urinary tract cancer, with an estimated 613,000 new cases and 220,000 deaths reported in 2022 (1). Clinically, BCa is classified into non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) subtypes based on tumor invasion depth. Its pathogenesis involves complex interactions between genetic predisposition and environmental risk factors, particularly smoking and occupational exposure to carcinogens (2). Although surgical resection remains the primary treatment, BCa exhibits a high recurrence rate due to its multifocal growth pattern. To mitigate this, perioperative chemotherapy including gemcitabine (GEM) based regimens is recommended to improve outcomes and reduce relapse (3). GEM, a deoxycytidine analog, exerts antitumor effects by incorporating its triphosphate metabolite (dFdCTP) into DNA during the G1/S phase, thereby inhibiting replication and inducing apoptosis (4). Despite its widespread use in BCa therapy, chemoresistance and dose-limiting toxicities (e.g., myelosuppression) hinder its long-term efficacy (5). Thus, identifying low-toxicity adjuvants to enhance GEM’s therapeutic index is urgently needed.

Fortunately, some plant-derived medicinal compounds have been found to possess significant anticancer activity and low toxic. Besides, a study reported that a combination of chemotherapy with natural compounds can improve the clinical treatment response to tumors and reduce side-effects (6). Ursolic acid (UA) is a pentacyclic triterpenoid compound that exists in a number of natural medicinal plants, such as Hedyotis diffusa, Ligustrum lucidum and Tripterygium Radix (7). It has been reported that UA has a wide range of pharmacological effects, such as hepatoprotective, antioxidant, anti-inflammatory and immunoregulatory effects (8,9). In addition, UA has been demonstrated to possess multiple antitumor activities, including the inhibition of tumor cell proliferation, the promotion of apoptosis and the reversal of tumor chemoresistance. More importantly, UA has the natural advantage of low toxicity (10,11). Studies have revealed that UA can enhance the efficacy of chemotherapy in certain types of cancer, such as pancreatic, colorectal and breast cancer (12,13). Nevertheless, whether UA can enhance the chemotherapeutic effects of GEM in BCa has not been reported to date, at least to the best of our knowledge. The chemical structures of UA and GEM are presented in Figure 1. Therefore, this current research aims to investigate whether UA enhances GEM’s anticancer activity in BCa and elucidate the underlying mechanisms, providing novel insights into a natural product-based combinatorial strategy to improve BCa therapy. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-383/rc).

Figure 1.

Figure 1

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The chemical structures of UA and GEM. GEM, gemcitabine; UA, ursolic acid.

Methods

Reagents and antibodies

UA (cat. no. U820363) and GEM (cat. no. G824361) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). UA was dissolved in dimethyl sulfoxide, and GEM was dissolved in PBS, aliquoted and stored at −20 ℃. The final concentration of dimethyl sulfoxide in the culture was <0.1% in all the experiments. Antibodies against cleaved caspase‑3 (cat. no. 9664), phosphorylated (p-)c-Jun N-terminal kinase (JNK) (cat. no. 4668), p-phosphatidylinositol-3-kinase (PI3K) (cat. no. 4228) and p-protein kinase B (AKT) (cat. no. 13038) were purchased from Cell Signaling Technology, Inc. (Boston, USA). Anti-poly(ADP-ribose) polymerase (PARP; cat. no. 556494) antibody was purchased from BD Biosciences (New York, USA). Anti-PI3K (cat. no. 20584-1-AP) and anti-AKT (cat. no. 60203-2-Ig) antibodies were purchased from Proteintech Group, Inc. (Wuhan, China). Anti-JNK (cat. no. D120893) antibody was obtained from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Anti-β-actin (cat. no. ABM‑0001) antibody was obtained from Nanjing Zoonbio Biotecnology Co., Ltd. (Nanjing, China). Horseradish peroxidase‑conjugated goat anti‑rabbit (cat. no. 7074) and goat anti‑mouse (cat. no. 7076) were obtained from Cell Signaling Technology, Inc. The AKT activator, SC79 (cat. no. SF2730), was purchased from the Beyotime Institute of Biotechnology (Beijing, China). The JNK inhibitor, SP600125 (cat. no. s1460), was obtained from Selleck Chemicals. Fetal bovine serum (FBS) (cat. no. 16010159) was purchased from Gibco (Waltham, USA); Thermo Fisher Scientific, Inc. (Waltham, USA). RPMI-1640 medium (cat. no. SH30809.01B) and trypsin (cat. no. SH30042.01) were obtained from HyClone; Cytiva (Logan, USA). The Cell Counting Kit‑8 (CCK‑8) (cat. no. C0037) and the Hoechst 33258 stain (cat. no. C1017) were purchased from the Beyotime Institute of Biotechnology.

Cells and cell culture

The human BCa cell lines, T24 and 5637, were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. These cells were cultured in RPMI-1640 supplemented with 10% FBS, and 100 mg/mL penicillin‑streptomycin at 37 ℃ in a humidified atmosphere containing 5% CO2.

Measurement of cell viability

Cell viability was assessed using CCK-8 assay. The cells were seeded into 96-well plates at 5×103 cells/well and cultured at 37 ℃ with 5% CO2 for 24 h. The cells were then treated with various concentrations of GEM and/or UA for a further 24 h. CCK‑8 reagent was added to the medium at a ratio of 1:10 followed by incubation at 37 ℃ for 2 h. The absorbance at 450 nm was measured using a Tecan Infinite F200/M200 multifunction microplate reader (Tecan Group, Ltd., Männedorf, Switzerland). The viability rate of cells = [the optical density values (OD) of experimental group/OD of control group] × 100%. The index analysis of the UA and GEM combination was calculated according to the following formulas: the combinational index (CI) = (IRUA + IRGEM − IRUA × IRGEM)/IR(UA + GEM), and the inhibition rate (IR) = [(OD of control group − OD of experimental groups)/OD of control group] × 100%, CI <1 indicates a synergistic effect, CI =1 indicates an additive effect, and CI >1 indicates an antagonistic effect (14).

Hoechst 33258 staining

The T24 and 5637 cells were seeded in six‑well plates at 5×104 cells/well and incubated at 37 ℃ with 5% CO2 for 24 h. Following 24 h of adherence, the cells were treated with GEM and/or UA for a further 24 h. The cells were then washed three times with phosphate-buffered saline (PBS) and incubated with Hoechst 33258 (10 µg/mL) in the dark at room temperature for 10 min. The observation of cell morphology was performed using a fluorescence microscope with a blue filter.

Apoptosis analysis using flow cytometry

The T24 and 5637 cells were inoculated into six‑well plates at a density of 5×104 cells/well and cultured for 24 h. Following treatment with GEM and/or UA for 24 h, the cells were collected, washed with PBS and suspended in 195 µL Annexin V-FITC binding buffer containing 5 µL Annexin V-FITC and 10 µL propidium iodide (cat. no. C1062M; Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Following incubation for 10–20 min at room temperature in the dark, flow cytometry (Gallios, Miami, USA) was performed for detection.

Western blot analysis

Total protein was extracted from the cells using RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing 1 mmol/L phenylmethanesulfonyl fluoride (PMSF) (cat. no.ST506; Beyotime Institute of Biotechnology). The protein concentration was measured using a BCA kit (cat. no. P0010S; Beyotime Institute of Biotechnology). A 10–12% SDS-polyacrylamide gel (cat. no. P0012A; Beyotime Institute of Biotechnology) was used to separate the same amount of protein sample (40 µg) followed by transfer onto a nitrocellulose membrane. After blocking with 5% non-fat dried milk for 1 h at room temperature, the PVDF membranes were incubated with primary antibodies overnight at 4 ℃. The PVDF membranes were then exposed for an additional 2 h with horseradish peroxidase-conjugated secondary antibodies at room temperature, followed by chemiluminescence (Amersham; Cytiva).

Tumorigenicity assay in nude mice

All animal experiments were performed under a project license (approval No. IACUC-CQMU-2024-0634) granted by the Animal Ethics Committee of Chongqing Medical University, in compliance with national or institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration. To adhere to the 4R principles, we used BALB/C female nude mice (Charles River; Beijing, China) aged 4 to 6 weeks, as alternative non-animal models were not suitable for studying tumorigenicity. Prior to the experiment, a power analysis was performed to ensure that the sample size was adequate for detecting significant differences with 80% power at a 0.05 significance level, minimizing animal use while maintaining statistical rigor. Mice were housed in the Animal Care facility at Chongqing Medical University for 1 week to acclimate, with free access to food and water. Tumorigenicity assays were performed by subcutaneously injecting 5×107/mL T24 cells into the flanks of the mice. Once tumors were palpable, mice were randomly assigned to four groups and administered drugs via intraperitoneal injection (five mice per group): the Control group (PBS of equal volume to the UA/day), UA group (UA at 10 mg/kg/day), the GEM group (GEM at 150 mg/kg/week), and the GEM + UA group (UA at 10 mg/kg/day + GEM at 150 mg/kg/week). Tumor size was measured every 3 days using an electronic vernier caliper, and tumor volume was calculated using the formula 0.52 × (L × W2). At the study endpoint, after 21 days, the mice were anesthetized and euthanized by cervical dislocation, and tumors were harvested for analysis. All procedures followed strict ethical guidelines, including appropriate anesthesia, analgesia, and minimizing animal distress through proper care and management.

Statistical analysis

SPSS 22.0 statistical software (IBM Corp., Armonk, USA) and Graph-Pad Prism v. 8.0.1 (GraphPad Software, San Diego, USA) ware used to perform statistical analyses. Before conducting statistical analysis, normality and homogeneity of variance tests were conducted first. Unpaired two-tailed Student’s t-tests were used to analyze two unpaired samples. One-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test were used to analyze multiple unpaired samples. The data are presented as the mean ± standard deviation (SD). The data for each group were obtained from at least three independent experiments. In all analyses, P<0.05 was considered to indicate a statistically significant difference.

Results

UA and GEM synergistically reduce cell viability of human BCa cells in vitro

The T24 and 5637 cells were treated with a series of concentrations of UA (0, 10, 20, 30, 40 and 50 µM) or GEM (0, 0.01, 0.1, 1.0, 10 and 100 µg/mL) for 24 h, and cell viability was measured using CCK-8 assay. The results revealed that UA or GEM reduced cell viability of the BCa cells lines T24 and 5637 in a concentration-dependent manner (Figure 2A,2B). The 50% inhibitory concentration (IC50) of GEM for the T24 and 5637 cells was 3.98 and 2.53 µg/mL, respectively. The IC50 of UA was calculated to be 34.11 µM for T24 cells and 30.89 µM for 5637 cells. In the present study, to achieve obvious and stable effects, the concentrations of 4.0 and 2.5 µg/mL GEM were selected for the T24 and 5637 cells, respectively. The concentrations of UA 10, 20, 30 µM which were below the IC50 were selected in combination with GEM to treat the BCa cells. The results revealed that UA enhanced the cytotoxic effects of GEM on T24 and 5637 cells (Figure 3A,3B). The CI of T24 and 5637 cells was calculated in Table 1, both <1, suggesting that the combination of UA and GEM exerted a synergistic antitumor effect. In addition, the results showed that the CI of T24 and 5637 cells were lower when UA was 10 µM, which indicated 10 µM UA could exert a stronger synergistic effect. Thus, 10 µM UA was chosen in combination with GEM in subsequent experiments.

Figure 2.

Figure 2

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UA or GEM reduced cell viability of human BCa cells. (A) Cell viability was detected by CCK-8 assay. T24 and 5637 cells were treated with different concentrations of UA for 24 h. (B) T24 and 5637 cells were treated with different concentrations GEM for 24 h. *, P<0.05; **, P<0.01; ***, P<0.001 vs. the control group. BCa, bladder cancer; CCK-8, Cell Counting Kit-8; GEM, gemcitabine; UA, ursolic acid.

Figure 3.

Figure 3

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UA synergistically enhances the cytotoxic effects of GEM in human BCa cells. (A) T24 cells were treated with GEM (4.0 µg/mL) and/or UA (10, 20, 30 µM) for 24 h and subjected to CCK-8 assay. (B) 5637 cells were treated with GEM (2.5 µg/mL) and/or UA (10, 20, 30 µM) for 24 h and subjected to CCK-8 assay. *, P<0.05; **, P<0.01; ***, P<0.001 vs. the control group. #, P<0.05; ##, P<0.01 vs. GEM group. BCa, bladder cancer; CCK-8, Cell Counting Kit-8; GEM, gemcitabine; UA, ursolic acid.

Table 1. The CI of BCa cells in combination with GEM and UA at different concentrations.

GEM and UA in different concentrations CI
T24: GEM (4.0 μg/mL) + UA (10 μM) 0.8711
T24: GEM (4.0 μg/mL) + UA (20 μM) 0.8923
T24: GEM (4.0 μg/mL) + UA (30 μM) 0.9227
5637: GEM (2.5 μg/mL) + UA (10 μM) 0.9122
5637: GEM (2.5 μg/mL) + UA (20 μM) 0.9374
5637: GEM (2.5 μg/mL) + UA (30 μM) 0.9686
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BCa, bladder cancer; CI, combinational index; GEM, gemcitabine; UA, ursolic acid.

UA enhances the GEM-induced apoptosis of human BCa cells in vitro

Our study demonstrated UA and GEM synergistically reduced cell viability of human BCa cells. In order to explore UA synergistically enhances the anticancer effect of GEM was associated with apoptosis induction, we further investigated whether UA could augment the GEM-induced apoptosis of human BCa cells by Hoechst 33258 staining, western blot and flow cytometry. Fluorescence microscopy of Hoechst 33258 staining revealed that nuclear pyknosis or fragmentation in the GEM + UA group was significantly increased, compared with that in the GEM group (Figure 4A). The results of western blot indicated that the activation of caspase-3 and the cleavage of PARP were increased following treatment with GEM + UA compared with GEM alone (Figure 4B), which was consistent with the results of the apoptotic rate evaluated by flow cytometry (Figure 4C). These results suggested that UA enhanced the GEM-induced apoptosis of T24 and 5637 cells.

Figure 4.

Figure 4

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UA increased GEM-induced apoptosis in human BCa cells. (A) Human BCa cells were treated with GEM (4.0 µg/mL for T24 cells, 2.5 µg/mL for 5637 cells) and/or UA (10 µM) for 24 h. Human BCa cells were stained with Hoechst 33258 and observed under a fluorescence microscope (×100). (B) The indicated proteins were detected by western blot. β-actin was detected as a loading control. (C) The apoptotic rates were measured by Annexin‑V/propidium iodide staining and analyzed by flow cytometry. Annexin V−/PI− represents normal cells. Annexin V−/PI+ represents necrotic cells. Annexin V+/PI− represents the early apoptotic stage, whereas Annexin V+/PI+ represents apoptotic cells in the terminal stage. The percentages of early and terminal stage apoptotic cells were calculated and presented in the histogram. *, P<0.05; **, P<0.01 vs. the control group. ##, P<0.01 vs. GEM group. BCa, bladder cancer; GEM, gemcitabine; PARP, poly(ADP-ribose) polymerase; UA, ursolic acid.

UA enhances the GEM-induced apoptosis of BCa cells by inactivating the PI3K/AKT pathway and activating the JNK pathway in vitro

GEM is widely used in chemotherapy for BCa. Previous studies have reported that PI3K/AKT and JNK signaling pathways play crucial roles in GEM-induced apoptosis of BCa cells (15,16). Because UA possesses the potent effect in anti-tumor therapy, and the relevant researches on UA have gradually increased in recent years. It has been reported that UA could induce apoptosis of tumor cells through multiple signaling pathways (17). In the present study, in order to investigate whether the PI3K/AKT and JNK signaling pathways are involved in the treatment of GEM and UA on human BCa cells, western blot analysis was used to evaluate the expression levels of proteins related to these signaling pathways. It was found that the phosphorylation levels of PI3K and AKT were significantly reduced in the GEM + UA group compared with the GEM group. In addition, it was found the expression of p-JNK was markedly increased following combined treatment with GEM and UA compared to treatment with GEM alone (Figure 5). To determine the roles of the inactivation of the PI3K/AKT pathway and the activation of the JNK pathway in UA-induced apoptosis, the selective AKT activator (SC79) and the JNK inhibitor (SP600125) were used to activate AKT and to inhibit JNK, respectively. It was found that SC79 reduced the levels of cleaved caspase-3 and cleaved PARP (Figure 6A). Besides, CCK8 results showed that cell viability was increased after AKT activation (Figure 6B). Correspondingly, we found that SP600125 decreased the expression of cleaved caspase-3 and cleaved PARP (Figure 7A), and the cell viability was increased after JNK inhibition (Figure 7B).

Figure 5.

Figure 5

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UA inactivated the PI3K/AKT pathway and activated the JNK pathway in human BCa cells. T24 and 5637 cells were treated with GEM, UA and GEM + UA for 24 h. The indicated proteins were detected by western blot. β-actin was detected as a loading control. *, P<0.05; **, P<0.01 vs. the control group. ##, P<0.01; ###, P<0.001 vs. GEM group. AKT, protein kinase B; BCa, bladder cancer; GEM, gemcitabine; JNK, c-Jun N-terminal kinase; PI3K, phosphatidylinositol-3-kinase; UA, ursolic acid.

Figure 6.

Figure 6

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Activation of PI3K/AKT reduced apoptosis in human BCa cells. T24 and 5637 cells were pretreated with SC79 (10 µM) for 30 min, and then treated with GEM + UA for 24 h. (A) The indicated proteins were detected by western blot. β-actin was detected as a loading control. (B) Cell viability was detected by CCK-8 assay. *, P<0.05; **, P<0.01; ***, P<0.001 vs. the control group. ns, P>0.05; #, P<0.05; ##, P<0.01 vs. GEM + UA group. AKT, protein kinase B; BCa, bladder cancer; GEM, gemcitabine; PARP, poly(ADP-ribose) polymerase; PI3K, phosphatidylinositol-3-kinase; UA, ursolic acid.

Figure 7.

Figure 7

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Inhibition of JNK signaling reduced apoptosis in human BCa cells. T24 and 5637 cells were pretreated with SP600125 (10 µM) for 30 min, and then treated with GEM + UA for another 24 h. (A) The indicated proteins were detected by western blot. β-actin was detected as a loading control. (B) Cell viability was detected by CCK-8 assay. ns, P>0.05; **, P<0.01; ***, P<0.001 vs. the control group. #, P<0.05; ##, P<0.01 vs. GEM + UA group. BCa, bladder cancer; GEM, gemcitabine; JNK, c-Jun N-terminal kinase; PARP, poly(ADP-ribose) polymerase; UA, ursolic acid.

UA enhances the growth inhibition of GEM on BCa in vivo

Our studies in vitro have demonstrated that UA enhanced the anticancer activity of GEM in BCa cell lines. We aimed to detect whether UA also enhanced the growth inhibition of GEM in BCa though a xenograft mouse model in vivo. After 21 days, the nude mice were euthanized, and the tumors were analyzed. As showed in Figure 8A, both the UA and GEM groups demonstrated a reduction in xenograft tumor volume. Notably, the tumor volume in the GEM + UA group was significantly decreased compared to GEM group. Additionally, tumor weight analysis revealed that the tumor weight in GEM + UA group was significantly smaller than the group treated with GEM (Figure 8B). Furthermore, we discovered that GEM exerted a noticeable therapeutic effect beginning at Day 9, with a marked enhancement by Day 21, and GEM + UA treatment significantly reduced the tumor growth rate compared to GEM treatment alone (Figure 8C). To investigate the mechanism underlying the tumor growth inhibition, which we hypothesized to be associated with apoptosis induced by the PI3K/AKT and JNK signaling pathways, we examined the levels of key pathway and apoptotic proteins via western blot analysis. As showed in Figure 9, we found UA could reduce the level of P-AKT and increase the expression of p-JNK. In addition, we also found that compared to the GEM group, the GEM + UA group was associated with a decrease in p-AKT, an increase in p-JNK, and a corresponding upregulation of cleaved caspase-3. These results suggested that UA enhanced the chemotherapeutic efficacy of GEM on BCa in vivo by inactivating the PI3K/AKT pathway and concurrently activating the JNK pathway.

Figure 8.

Figure 8

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UA enhances the growth inhibition of GEM in nude mice xenograft model. (A) Tumor volume in different groups was measured. (B) Tumor weight was measured in different groups. (C) Tumor growth curve was measured on different days. *, P<0.05; **, P<0.01; ***, P<0.001 vs. the control group. ##, P<0.01; ###, P<0.001 vs. GEM group. GEM, gemcitabine; UA, ursolic acid.

Figure 9.

Figure 9

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UA augments GEM’s efficacy against BCa via modulation of the PI3K/AKT and JNK pathways in xenograft model. The expression of the indicated proteins in tumor tissues from the xenograft model was assayed by western blot. β-actin was detected as a loading control. *, P<0.05; **, P<0.01 vs. the control group. ##, P<0.01 vs. GEM group. AKT, protein kinase B; BCa, bladder cancer; GEM, gemcitabine; JNK, c-Jun N-terminal kinase; UA, ursolic acid.

Discussion

Chemotherapy after surgical operation of BCa plays an important role in reducing recurrence. GEM is widely used in the treatment of NMIBC and MIBC, and has been listed as a first-line chemotherapeutic agent for MIBC. However, chemotherapeutic drugs have some common disadvantages, such as poor sensitivity, chemoresistance and adverse effects. The adverse effects of GEM usually include bone marrow suppression, gastrointestinal reaction, liver and kidney function impairment. This not only undermines the treatment efficacy, but also increases the patient’s suffering. It has been found that some monomer compounds extracted from some Chinese herbs have significant anticancer activity and few side effects, which have become a research hotspot of anti-tumor therapy in recent years. UA is a pentacyclic triterpenoid compound existing in multiple Chinese herbal plants. It has been demonstrated that UA exhibits potent antitumor activities by inducing tumor cell apoptosis, suppressing tumor cell proliferation and inhibiting tumor angiogenesis (18). UA combined with chemotherapeutic drugs has been shown to achieve satisfactory effects in the treatment of certain types of tumors (19,20). What is more gratifying is that both the Animal studies and preclinical studies have shown that UA has an excellent safety profile, it even could reduce some side effects of chemotherapy drugs (21,22).

As the application of UA combined with GEM in BCa has not yet been reported. The aim of the present study is to investigate whether UA enhances the chemotherapeutic efficacy of GEM in BCa. In our study, the results revealed that the CI of UA and GEM was <1, suggesting the combination of UA and GEM exerted a synergistic antitumor effect. The concentration of UA (10 µM) not only exhibited better safety, but also had lower CI value in combination with GEM. Therefore, the concentration of UA (10 µM) was selected for a better synergistic anti-tumor effect. Furthermore, by examining the apoptosis of T24 cells and 5637 cells, it was found that UA enhanced the GEM-induced apoptosis of human BCa cells in vitro. The nude mice xenograft model also demonstrated that UA enhanced the chemotherapy effect of GEM for BCa in vivo.

Cell apoptosis is a highly regulated physiological mechanism of cell death. It is a key response to antitumor therapy. Nevertheless, the molecular mechanisms underlying the promoting effects of UA on the GEM-induced apoptosis of human BCa cells remain unclear. The PI3K/AKT signaling pathway is recognized as a crucial signaling pathway involved in apoptosis, invasion, cell survival and protein synthesis (23). The activation of the PI3K/AKT signaling pathway can promote cell growth and survival. Conversely, the inhibition of the expression of PI3K and AKT can increase cell death (24). PI3K is a broadly expressed lipid kinase, that can activate and phosphorylate AKT. The activation of AKT can regulate a number of downstream target molecules, such as caspase family proteins, Bcl-2 family proteins, NF-κB and glycogen synthase kinase 3, which play critical roles in cell apoptosis and survival (25). Therefore, the PI3K/AKT signaling pathway is an attractive target for antitumor therapy. It has been reported that GEM leads to the production of excess reactive oxygen species by activating the PI3K/AKT signaling pathway, which inhibits the chemotherapeutic effect and reduces the antitumor responses of pancreatic cancer cells to GEM (26,27). Therefore, the activation of PI3K/AKT signaling pathway is also considered as a regulatory factor for tumor cells to escape the killing effect of chemotherapy drugs. In the present study, we found GEM activated the PI3K/AKT signaling pathway in human BCa cells. Thus, it was hypothesized that the activation of the PI3K/AKT signaling pathway was involved in the chemoresistance of human BCa cells to GEM. It has reported that inactivation the PI3K/AKT signaling pathway could enhance the sensitivity of tumor cells to chemotherapy drugs (28). In our previous study we have demonstrated that UA could down-regulate PI3K/AKT signaling pathway in BCa (29). As was expected, the present study also demonstrated that UA significantly suppressed the activation of the PI3K/AKT signaling pathway. SC79 is a selective AKT activator. 10 µM of SC79 was informed by existing literature, which did not indicate any cytotoxic effects associated with this concentration (30,31). In this study, we found that the activation of AKT by SC79, reversed the antitumor effect. Thus, the results revealed UA can enhance GEM-induced apoptosis by inactivating the PI3K/AKT signaling pathway in human BCa cells.

The JNK signaling pathway is another classic apoptotic signaling pathway. JNK is one of the MAPK family members that can dominate the apoptosis, proliferation and metastasis of tumor cells (32). Teraishi et al. (33) found that GEM activated the JNK pathway to induce the apoptosis of human lung cancer cells. A recent study also demonstrated that UA induced the activation of JNK to promote the apoptosis of multiple cancer cells (17). Accordingly, we also detected UA could activate the JNK signaling pathway in the present study. Furthermore, we found that UA could upregulate the expression of p-JNK induced by GEM. SP600125 is a JNK inhibitor, 10 µM of SP600125 was chosen with careful consideration of previous research, which also suggested its safety (34,35). When we used SP600125 to inhibit the JNK signaling pathway, the levels of cleaved PARP and cleaved caspase-3 were markedly reduced. The results thus suggested that UA contributed to GEM-induced apoptosis by activating the JNK signaling pathway in human BCa cells.

In the present study, although it was demonstrated that UA enhanced GEM-induced apoptosis through the PI3K/AKT and JNK signaling pathways, certain clarifications are still required. For instance, the downstream mechanisms involved need to be further investigated in future studies. Additionally, in our research, both T24 and 5637 exhibited CI values below 1, indicating that the amalgamation of UA and GEM yielded a synergistic antitumor impact. It is worth noting that this combination concentration might not be optimal. The more in-depth investigation of the pharmacokinetics of UA and GEM may optimize the treatment durations and concentrations (21), and it may provide further insight into enhancing the therapeutic effects of UA and GEM in BCa. Though UA possesses potent anticancer effect and the natural advantage of low toxicity. However, the poor permeability and low oral bioavailability of UA hinder its further clinical use. In recent years, nano formulations such as polymer micelles and liposomes have been regarded as potential solutions to ameliorate UA delivery to tumors (36). Some phase I clinical trials of UA nano-formulations have shown that UA nano-formulations can improve the bioavailability of UA (37). With the development of nanotechnology, it may promote the clinical use of UA in treatment of BCa.

Conclusions

In conclusion, UA is a natural compound derived from Chinese medicinal herbs, which has potent anticancer effects and deserves further exploration. The present study demonstrates that UA sensitizes BCa to GEM chemotherapy, which is associated with apoptosis induction. Furthermore, we found that UA enhances GEM-induced apoptosis by inactivating the PI3K/AKT signaling pathway and activating the JNK signaling pathway in human BCa both in vitro and in vivo. The combined treatment with UA and GEM may provide an experimental basis for the clinical treatment of BCa.

Supplementary

The article’s supplementary files as

tau-14-10-2902-rc.pdf (1.2MB, pdf)
DOI: 10.21037/tau-2025-383
tau-14-10-2902-coif.pdf (679.5KB, pdf)
DOI: 10.21037/tau-2025-383

Acknowledgments

The authors thank the Central Laboratory, The First Affiliated Hospital of Chongqing Medical University (Chongqing, China) for their technical support. This manuscript was submitted as a pre-print in the link “https://www.researchsquare.com/article/rs-2089441/v2”.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All animal experiments were performed under a project license (approval No. IACUC-CQMU-2024-0634) granted by the Animal Ethics Committee of Chongqing Medical University, in compliance with national or institutional guidelines for the care and use of animals.

Footnotes

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-383/rc

Funding: This work was supported by the Natural Science Foundation of China (No. 81874092).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-383/coif). The authors have no conflicts of interest to declare.

Data Sharing Statement

Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-383/dss

tau-14-10-2902-dss.pdf (62.8KB, pdf)
DOI: 10.21037/tau-2025-383
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Associated Data

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    Supplementary Materials

    The article’s supplementary files as

    tau-14-10-2902-rc.pdf (1.2MB, pdf)
    DOI: 10.21037/tau-2025-383
    tau-14-10-2902-coif.pdf (679.5KB, pdf)
    DOI: 10.21037/tau-2025-383

    Data Availability Statement

    Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-383/dss

    tau-14-10-2902-dss.pdf (62.8KB, pdf)
    DOI: 10.21037/tau-2025-383
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