Ursolic acid synergistically enhances gemcitabine-induced apoptosis in bladder cancer via the PI3K/AKT and JNK signaling pathways

doi:10.21203/rs.3.rs-2089441/v2

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Ursolic acid synergistically enhances gemcitabine-induced apoptosis in bladder cancer via the PI3K/AKT and JNK signaling pathways

https://doi.org/10.21203/rs.3.rs-2089441/v2

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Ursolic acid (UA) is a natural compound that exists in a number of Chinese medicinal herbs, which has been demonstrated to enhance the efficacy of chemotherapy in multiple types of cancer. The present study aimed to observe whether UA enhances the antitumor effects of gemcitabine (GEM) in human bladder cancer (BCa) cell lines, and to investigate the possible underlying mechanisms. The human BCa cell lines, T24 and 5637, were treated with GEM and/or UA in vitro. Cell viability was measured by the Cell Counting Kit-8 assay. Apoptosis was detected using Hoechst 33258 staining, western blot analysis and flow cytometry. The expression levels of signaling pathway-related proteins were detected using western blot analysis. UA and GEM synergistically inhibited the proliferation of human BCa cells. Compared with GEM treatment alone, the combination of GEM and UA led to enhanced the antitumor effects, which were associated with the induction of apoptosis. The PI3K/AKT and JNK signaling pathways were involved in human BCa cells treated with GEM and UA. Both the AKT activator, SC79, and the JNK inhibitor, SP600125, reduced the expression of cleaved PARP and cleaved caspase-3. On the whole, the results of the present study demonstrate that UA enhances GEM-induced apoptosis by inactivating the PI3K/AKT signaling pathway and activating the JNK signaling pathway in human BCa cells.

Oncology

Cancer Biology

Chemical Biology

Ursolic acid

Gemcitabine

Bladder cancer

apoptosis

PI3K/AKT

JNK

According to the latest statistics, bladder cancer (BCa) is the 10th most common malignancy worldwide and is also the most common malignancy of the urinary tract; it was estimated that there were ~573,000 new cases and 213,000 deaths from BCa worldwide in 2020 (1,2). Currently, Surgical treatment is the main treatment strategy for BCa. As BCa has the characteristic of polycentric growth, there is a high recurrence rate following surgery. Previous studies and authoritative guidelines recommend perioperative chemotherapy to improve the efficacy of surgical treatment and to reduce the recurrence rate (3). As regards the disease pathology, >90% of BCa cases are urothelial carcinoma, which is responsive to therapy with gemcitabine (GEM), cisplatin and doxorubicin (4). Therefore, chemotherapy plays a key role in the treatment of BCa. GEM is a difluorinated analog of deoxycytidine, that can be activated by deoxycytidine nucleoside kinase and metabolized by cytidine deaminase, the triphosphate metabolite (dFdCTP) that blocks DNA synthesis in the G1/S phase, resulting in tumor cell death (5). Thus, GEM is widely used in the treatment of BCa and has been listed as a first-line chemotherapeutic agent for muscle-invasive BCa. However, chemoresistance and the side-effects of GEM limit its long-term efficacy. Thus, the identification of a more efficient and less toxic treatment agent for BCa is of utmost urgency.

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 (6). It has been reported that UA has a wide range of pharmacological effects, such as hepatoprotective, antioxidant, anti-inflammatory and immunoregulatory effects (7,8). 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 (9,10). Studies have revealed that UA can enhance the efficacy of chemotherapy in certain types of cancer, such as pancreatic, colorectal and breast cancer (11,12). 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. Hence, the present study aimed to investigate whether UA can enhance GEM-induced apoptosis in human BCa cells, and to explore the possible underlying mechanisms. The chemical structures of UA and GEM are presented in Fig. 1.

Reagents and antibodies. UA and GEM were purchased from Shanghai Macklin Biochemical Co., Ltd. UA was dissolved in dimethyl sulfoxide, and GEM was dissolved in PBS, aliquoted and stored at -20˚C. 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-PI3K (cat. no. 4228) and p-AKT (cat. no. 13038) were purchased from Cell Signaling Technology, Inc. Anti-poly(ADP-ribose) polymerase (PARP; cat. no. 556494) antibody was purchased from BD Biosciences. Anti-PI3K (cat. no. 20584-1-AP) and anti-AKT (cat. no. 60203-2-Ig) antibodies were purchased from Proteintech Group, Inc. Anti-JNK (cat. no. D120893) antibody was obtained from Sangon Biotech (Shanghai) Co., Ltd. Anti-β-actin (cat. no. ABM‑0001) antibody was obtained from Nanjing Zoonbio Biotecnology Co., Ltd. All the secondary antibodies were obtained from Abgent, Inc. The AKT activator, SC79 (cat. no. SF2730), was purchased from the Beyotime Institute of Biotechnology. The JNK inhibitor, SP600125 (cat. no. s1460), was obtained from Selleck Chemicals. Fetal bovine serum (FBS) was purchased from Gibco; Thermo Fisher Scientific, Inc. RPMI-1640 medium and trypsin were obtained from HyClone; Cytiva. The Cell Counting Kit‑8 (CCK‑8) and the Hoechst 33258 stain 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. These cells were cultured in RPMI-1640 supplemented with 10% FBS, and 100 mg/ml penicillin‑streptomycin at 37˚C 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 5x10³cells/well and cultured at 37˚C 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˚C for 2 h. The absorbance at 450 nm was measured using a Tecan Infinite F200/M200 multifunction microplate reader (Tecan Group, Ltd.). The viability rate of cells = [the optical density values (OD) of experimental group/OD of control group] x100%. The index analysis of the UA and GEM combination was calculated according to the following formulas: The combinational index (CI) = (IR_UA+ IR_GEM - IR_UA x IR_GEM)/IR_{(UA +GEM)}, and the inhibition rate (IR) = [( OD of control group - OD of experimental groups)/OD of control group] x100%, CI <1 indicates a synergistic effect, CI = 1 indicates an additive effect, and CI > 1 indicates an antagonistic effect (13).

Hoechst 33258 staining. The T24 and 5637 cells were seeded in six‑well plates at 5x10⁴cells/well and incubated at 37˚C 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 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 5x10⁴cells/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 (PI; 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) was performed for detection.

Western blot analysis. Total protein was extracted from the cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) containing 1 mmol/l phenylmethanesulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology). The protein concentration was measured using a BCA kit (Beyotime Institute of Biotechnology). A 10-12% SDS-polyacrylamide gel (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˚C. The PVDF membranes were then exposed for an additional 2 h with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, Inc.) at room temperature, followed by chemiluminescence (Amersham; Cytiva).

Statistical analysis. The data are presented as the mean ± SD. SPSS 22.0 statistical software (IBM Corp.) was used to perform all statistical analyses. The data for each group were obtained from three independent experiments. One-way analysis of variance was used to compare multiple groups. In all analyses, P<0.05 was considered to indicate a statistically significant difference.

UA and GEM synergistically inhibit the proliferation of human BCa cells. 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 proliferation was measured using CCK-8 assay. The results revealed that UA or GEM inhibited the proliferation of the lines T24 and 5637 BCa cells in a concentration-dependent manner (Fig. 2A and B). The 50% inhibitory concentration (IC₅₀) of GEM for the T24 and 5637 cells was 3.9753±0.1313 and 2.5293±0.3432 μg/ml, respectively. 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 in subsequent experiments. In the present study, 10 μM UA exhibited a low cytotoxicity in both the T24 and 5637 cells. Thus, 10 μM UA was used in combination with GEM to treat the T24 and 5637 cells. The results revealed that UA enhanced the inhibitory effects of GEM on the proliferation of the T24 and 5637 cells (Fig. 3A); this finding was consistent with the results observed under an inverted microscope (Fig. 3B). The CI of T24 and 5637 cells was 0.871 and 0.912, respectively, both <1, suggesting that the combination of UA and GEM exerted a synergistic antitumor effect.

UA enhances the GEM-induced apoptosis of human BCa cells. It has been reported that UA enhances the sensitivity of tumor cells to chemotherapeutic drugs in several tumor types (14,15). The present study further investigated whether UA can enhance the GEM-induced apoptosis of human BCa cells. 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 (Fig. 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 (Fig. 4B), which was consistent with the results of the apoptotic rate evaluated by flow cytometry (Fig. 4C). These results suggested that UA enhances the GEM-induced apoptosis of T24 and 5637 cells.

UA enhances the GEM-induced apoptosis of BCa cells by inactivating the PI3K/AKT pathway and activating the JNK pathway. The PI3K/AKT signaling pathway and the JNK signaling pathway are classical signaling pathways, which play crucial roles in tumor apoptosis, metastasis and drug resistance (16,17). In the present study, in order to investigate whether the PI3K/AKT and JNK signaling pathways are involved in the effects 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 (Fig. 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. 10μM of SC79 was informed by existing literature, which did not indicate any cytotoxic effects associated with this concentration (18,19). Similarly, 10μM of SP600125 was chosen with careful consideration of previous research, which also suggested its safety (20,21). It was found that SC79 reduced the levels of cleaved caspase-3 and cleaved PARP (Fig. 6). Correspondingly, we found that SP600125 decreased the expression of cleaved caspase-3 and cleaved PARP (Fig. 7).

GEM plays a critical role in the treatment of BCa, and it is widely used in the treatment of non-muscle invasive and muscle-invasive BCa. However, chemotherapeutic drugs have some common disadvantages, such as poor sensitivity, chemoresistance and adverse effects. This not only undermines the treatment efficacy, but also increases the patient's suffering. Fortunately, some plant-derived medicinal compounds have been found to have a high efficiency and low toxic antitumor activities, and a combination of chemotherapy with natural compounds can improve the clinical treatment response to tumors (22). 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 (23). UA combined with chemotherapeutic drugs has been shown to achieve satisfactory effects in the treatment of certain types of tumors (24,25).

As the application of UA combined with GEM in BCa has not yet been reported, at least to the best of our knowledge, the present study aimed to investigate whether UA enhances the chemotherapeutic efficacy of GEM in BCa. Within a clinical setting, doctors typically adopt a cautious approach for safety considerations. This often involves utilizing a low-concentration, extended-duration regimen. Specifically, the suggested concentration for GEM stands at 1000mg/m², but in vitro experiments conducted at this concentration might yield suboptimal results due to varying durations of drug activity. Consequently, the intervention concentration utilized in vitro is generally set at the drug's half maximal inhibitory concentration (IC50) (26). This choice forms the foundation for establishing the intervention concentration for GEM. In the present study, a concentration of UA (10 μM) was selected, which exhibited a low cytotoxicity. The results revealed that the CI of UA and GEM was <1, suggesting that the combination of UA and GEM exerted a synergistic antitumor 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.

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 (27). 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 (28). 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 (29). 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 (30,31). In the present study, it was 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. Previous studies have reported that the inactivation of the PI3K/AKT signaling pathway plays a critical role in UA-induced apoptosis in cancers, such as pancreatic and prostate cancer (32,33). As was expected, the present study demonstrated that UA significantly suppressed the activation of the PI3K/AKT signaling pathway. Furthermore, it was found that the activation of AKT by SC79, a selective AKT activator, reversed the antitumor effect. Thus, it was hypothesized that 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 (34). Teraishi et al (35) 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 (36). Accordingly, in the present study, it was demonstrated that UA upregulated the expression of p-JNK induced by GEM. When SP600125, a JNK inhibitor, we used 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's worth noting that while this combination concentration might not be optimal, the more in-depth investigation of the treatment durations and concentrations of UA and GEM used in combination may provide further insight into enhancing the therapeutic effects in human BCa cells. Studies have also reported that UA is capable of reversing chemotherapeutic drug resistance in certain types of cancer (37,38). Therefore, UA is a promising candidate for the treatment of GEM-resistant BCa, and further investigations are warranted to verify this hypothesis.

In conclusion, UA is a natural compound derived from Chinese medicinal herbs. The present study demonstrated that UA enhanced GEM-induced apoptosis by inactivating the PI3K/AKT signaling pathway and activating the JNK signaling pathway in human BCa cells. Combined treatment with UA and GEM may provide an experimental basis for the clinical treatment of BCa.

Acknowledgements

The Authors thank the Central Laboratory, The First Afliated Hospital of Chongqing Medical University (Chongqing,China) for their technical support.

Funding

This work was funded by the Natural Science Foundation of China (NO. 81874092).

Authors’ contributions

Weiyang He and Xiaolong Huang designed the research. Xiaolong Huang and Junlong Zhu conducted the experiments. Hang Tong and Peng Wen analyzed data, Xiaolong Huang and Yan Sun wrote the paper.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Patient consent for publication

Not applicable.

Conflicts of interest

The authors declare that they have no conflicts of interest.

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The authors declare no competing interests.

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Ursolic acid synergistically enhances gemcitabine-induced apoptosis in bladder cancer via the PI3K/AKT and JNK signaling pathways

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