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

doi:10.21203/rs.3.rs-2089441/v1

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

https://doi.org/10.21203/rs.3.rs-2089441/v1

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Background Gemcitabine (GEM) plays an important role in the chemotherapy of bladder cancer（BCa）. However, the chemoresistance and adverse effects of gemcitabine limit its effectiveness. Ursolic acid (UA) is a natural compound that exists in many natural medicinal plants and fruits, and has been demonstrated to enhance the efficacy of chemotherapy in multiple cancers. The present study aimed to observe the antitumor effects of a combination of GEM and UA in human bladder cancer cell lines, and to investigate the possible underlying mechanisms.

Methods The human bladder cancer 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 by Hoechst 33258 staining, western blot and flow cytometry. Protein expression of signaling pathways was detected by western blot.

Results UA synergistically inhibited proliferation with GEM in human bladder cancer cells. Compared with GEM treatment alone in T24 and 5637 cells, the combination of GEM and UA can enhance the antitumor effect. The PI3K/AKT and JNK signaling pathways are involved in human bladder cancer cells treated with GEM and UA. Both the Akt activator SC79 and the JNK inhibitor SP600125 reduced the expression of cleaved-PARP and cleaved-caspase3.

Conclusions our present data demonstrated that UA enhanced GEM-induced apoptosis by inactivating the PI3K/AKT signaling pathway and activating the JNK signaling pathway. The combinational treatment strategy of GEM and UA may provide a potential rational basis for the clinical treatment of BCa.

Ursolic acid

Gemcitabine

Bladder cancer

apoptosis

PI3K/AKT

JNK

According to the latest statistics, BCa is the 10th most common malignancy in the globe and is also the most common malignancy of the urinary tract. The incidence of BCa has increased in recent years, and it is estimated that there were approximately 573,000 new cases and 213,000 deaths from BCa worldwide in 2020[1, 2]. Currently, Surgical treatment is still the main treatment for BCa. Because BCa has the characteristic of polycentric growth, there is a high recurrence rate after surgery. Many studies and authoritative guidelines recommend perioperative chemotherapy to improve the effect of surgical treatment and reduce recurrence [3]. In pathology, more than 90% of BCa cases are urothelial carcinoma, which is responsive to therapy with gemcitabine (GEM), cisplatin, and doxorubicin [4]. Therefore, chemotherapy plays an important role in the treatment of BCa. GEM is a difluorinated analog of deoxycytidine, that can be activated by deoxycytosine nucleoside kinase and metabolized by cytidine deaminase, the triphosphate metabolite (dFdCTP) that blocks DNA synthesis cell stagnation in the G1 /S phase, resulting in tumor cell death [5]. Thus, GEM is widely used for BCa treatment and has been listed as a first-line chemotherapy agent for muscle invasive bladder cancer. However, chemoresistance and toxic side effects of GEM limit its long-term efficacy. Therefore, it is urgent to find more efficient and less toxic chemotherapy in the treatment of BCa.

Ursolic acid (UA) is a pentacyclic triterpenoid compound, that exists in many natural medicinal plants and fruits, such as hedyotic diffusa, ligustrum lucidum and bearberry [6]. It was reported that UA has a wide range of pharmacological effects, such as hepatoprotection, anti-oxidant, anti-inflammatory, and immune regulation[7, 8]. In addition, UA has been demonstrated to possess multiple antitumor activities including inhibiting tumor cell proliferation, promoting apoptosis, and reversing tumor chemoresistance [9, 10]. Studies have revealed that UA can enhance the efficacy of chemotherapy in many cancers, such as pancreatic cancer, colorectal cancer, and breast cancer [11, 12]. Nevertheless, whether UA can improve the chemotherapy effect of GEM in bladder cancer has not been reported so far. Hence, this study aims to investigate whether UA can enhance GEM-induced cytotoxicity in human bladder cancer cells, and to explore the possible underlying mechanisms.

Reagents and antibodies

UA and GEM 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°C. The final concentration of dimethyl sulfoxide in the culture was less than 0.1% in all experiments. Antibodies cleaved caspase‑3 (#9664), phospho-c-Jun N-terminal kinase (JNK) (#4668), phospho-PI3K (#4228), phospho-AKT (#13038) were purchased from Cell Signaling Technoogy (Danvers, Massachusetts,USA).Anti-poly-ADP-ribosepolymerase(PARP)( #556494 ) was purchased from BD Biosciences (New York, USA). Anti-PI3K(#20584-1-AP) and Anti-AKT(60203-2-Ig) were purchased from Proteintech Group(Wuhan, China ).Anti-JNK(#D120893) was obtained from Sangon Biotech(Shanghai,China). Anti-β-actin ༈#ABM‑0001༉was obtained from Zoonbio Biotecnology(Nanjing, China). All the secondary antibodies were obtained from Abgent (San Diego, California, USA). The AKT activator SC79(SF2730) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). The JNK inhibitor SP600125 (s1460) was obtained from Selleckchem (Houston,Texas, USA). Fetal bovine serum (FBS) was purchased from Gibco (Thermo FisherScientific, Waltham, Massachusetts, USA). RPMI-1640 medium and trypsin were obtained from HyClone (Logan, Utah, USA). Cell Counting Kit‑8 (CCK‑8) and Hoechst33258 staining were purchased from Beyotime Institute of Biotechnology༈Shanghai, China༉.

Cell culture

The human bladder cancer cell lines T24 and 5637 were purchased from the Chinese Academy of Sciences(Beijing, China). These cells were cultured in RPMI-1640 supplemented with 10% FBS, and 100mg/ml penicillin‑streptomycin at 37°C in a humidified atmosphere containing 5% CO₂.

Measurement of cell viability

Cell viability was assessed using the Cell Counting Kit-8(CCK-8) assay. Cells were seeded into 96-well plates at 1 × 10⁴cells/cm² and cultured at 37˚C with 5% CO₂ for 24 h. Then treated with different concentrations of GEM and/or UA for another 24 h. CCK‑8 reagent was added to the medium at a ratio of 1:10 and incubated at 37˚C for 2h. 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]× 100%. 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 × IR_GEM)/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[13].

Hoechst 33258 staining

T24 and 5637 cells were seeded into 6‑well plates at a density of 5x10⁴ cells/well and cultured at 37˚C with 5% CO2. After 24 h of adherence, the cells were treated with GEM and/or UA for another 24h. Then, the cells were washed three times with PBS and incubated with Hoechst 33258 (10 µg/ml) at room temperature in the dark for 10 min. The cell morphology was observed under a fluorescence microscope using a blue filter.

Apoptosis analysis by flow cytometry

T24 and 5637 cells were inoculated into 6‑well plates at a density of 5x10⁴ cells/well and cultured for 24 h. After 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 of annexin V-FITC

and 10 µl of propidium iodide (PI; Beyotime, Shanghai,China) according to the manufacturer's instructions. After incubation for 10∼20 min at room temperature in the dark, flow cytometry (Gallios, USA) was performed for detection.

Western blot analysis

The total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai,China) containing 1mmol/L phenylmethane-sulfonyl fluoride (PMSF)( Beyotime), and then centrifuged at 12000 rpm/min for 15 min at 4°C to remove debris. The protein concentration was measured using a BCA kit (Beyotime). A 10–12% SDS-polyacrylamide gel (Beyotime) was used to separate the same amount of protein sample (40 µg) and transferred to a nitrocellulose membrane. After blocking with 5% nonfat dried milk for 1 h at room temperature,the nitrocellulose membrane was incubated with primary antibodies overnight at 4˚C. The nitrocellulose membranes were then exposed for an additional 2 h with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology) at room temperature, followed by chemiluminescence (Amersham Biosciences).

Statistical analysis

All data were presented as the mean ± SD and were obtained from at least 3 independent experiments. SPSS 22.0 statistical software was used to carry out all statistical analyses. Comparisons between multiple groups were performed by one-way analysis of variance (ANOVA). In all analyses, P < 0.05 was termed as statistically significant.

UA synergistically inhibited proliferation with GEM in human bladder cancer cells

T24 and 5637 cells were treated with a series of concentrations of GEM (0, 0.01, 0.1, 1.0, 10, and 100 μg/ml) or UA (0,10,20,30,40, and 50 μM) for 24 h, and cell proliferation was measured using the CCK-8 assay. As shown in the results, UA and GEM inhibited the proliferation of bladder cancer cell lines T24 and 5637 in a concentration-dependent manner (Fig.1A and B). The 50% inhibiting concentration (IC50) of GEM for T24 and 5637 was 3.9753±0.1313 μg/ml and 2.5293 ± 0.3432 μg/ml, respectively. To achieve obvious and stable effects, we chose GEM concentrations of 4.0 μg/ml and 2.5 μg/ml for T24 and 5637cells in subsequent experiments.

In our study,10 μM UA was not significantly cytotoxic to either T24 or 5637 cells. Thus ,10 μM UA was used in combination with GEM to treat T24 and 5637 cells. The results showed that UA enhanced GEM-mediated proliferation inhibition in T24 and 5637 cells (Fig.2A), which was consistent with the results observed under an inverted microscope (Fig.2B).The combinational index ( CI ) of T24 and 5637 was 0.8013 and 0.8594, respectively, both less than 1, suggesting that the combination of UA and GEM had a synergistic antitumor effect.

UA enhanced GEM-induced apoptosis in human bladder cancer cells

UA has been demonstrated to promote apoptosis in human bladder cancer cells, which is also the same for GEM [14,15]. In our study, we further investigated whether a dose of UA without significant cytotoxicity can enhance GEM-induced apoptosis in T24 and 5637 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.3 A). Western blotting results indicated that the activation of Caspase 3 and cleavage of PARP were increased after treatment with GEM+UA compared with GEM alone (Fig.3 B), which was consistent with the results of the apoptotic rate evaluated by flow cytometry (Fig.3 C). These results suggested that UA can enhance GEM-induced apoptosis in T24 and 5637 cells.

UA enhanced GEM-induced apoptosis by inactivating the PI3K/AKT pathway and activating the JNK pathway

Both the PI3K/AKT signaling pathway and the c-Jun N-terminal kinase（JNK）signaling pathway are classical signaling pathways, which play important roles in tumor apoptosis, metastasis and drug resistance[16,17].In order to investigate whether the PI3K/AKT and JNK signaling pathways were involved in human bladder cancer cells treated with GEM and UA, western blotting was used to evaluate the protein expression of the associated signaling pathways. In the present study, we detected phosphorylation levels of PI3K and AKT were significantly reduced in GEM+UA group than GEM group，and we also found the expression of phosphorylation-JNK were apparently increased after treated with combination treatment of GEM and UA than GEM alone(Fig.4). To determine the roles of inactivating the PI3K/AKT and activating the JNK signaling pathways induced by UA in apoptosis. The selective AKT activator SC79 was used to activate the AKT, and we found SC79 can reduce the levels of cleaved-caspase3 and cleaved- PARP(Fig.5A). Correspondingly, the JNK inhibitor SP600125 was used to inhibit JNK, we detected SP600125 can decrease the expression of cleaved-caspase3 and cleaved- PARP(Fig.5B).

GEM plays an important role in the treatment of BCa, and it is widely used in the treatment of non-muscle invasive BCa and muscle invasive BCa. However, chemotherapeutic drugs have some common disadvantages, such as poor sensitivity, chemoresistance, and adverse effects. It not only affects the treatment effect but also increases the patient's suffering. Fortunately, some plant-derived medicinal compounds are found to have high-efficiency and low-toxic antitumor activities, and a combination of chemotherapy with natural products can improve the clinical treatment response to tumors [18].UA is a pentacyclic triterpenoid compound existing in multiple Chinese herbal plants and fruits. Studies have shown that UA exhibits potent antitumor activities by inducing tumor cells apoptosis, suppressing tumor cells proliferation, and inhibiting tumor Angiogenesis [19]. UA combined with chemotherapy drugs has also achieved satisfactory results in the treatment of some tumors [20, 21].

As the application of UA combined with GEM in BCa has not been reported, our study aimed to investigate whether UA enhances GEM- induced chemotherapeutic efficacy in BCa. In the present study, a concentration of UA (10µM) was selected, which showed no significant cytotoxicity and did not increase additional adverse drug reactions. The results showed that the combinational index (CI) of UA and GEM was less than 1, suggesting that a combination of UA and GEM had a synergistic antitumor effect. Furthermore, by examining apoptosis in T24 cells and 5637 cells, we found that UA could enhance the cytotoxicity of GEM in human bladder cancer cells.

Cell apoptosis is a highly regulated physiological mechanism of cell death. It is an important response to antitumor therapy. Nevertheless, the molecular mechanism of UA enhances GEM- induced apoptosis in human bladder cancer cells remains unclear. The PI3K/AKT signaling pathway is recognized as a crucial signaling pathway involved in apoptosis, invasion, cell survival and protein synthesis [22]. Activation of the PI3K/AKT signaling pathway can promote cell growth and survival, and inhibiting the expression of PI3K and Akt can increase cell death [23]. PI3K is a broadly expressed lipid kinase, that can activate and phosphorylate AKT. Activation of AKT can regulate many downstream target molecules, such as Caspase family proteins, Bcl-2 family proteins, NF-κB and glycogen synthase kinase 3 (GSK3), which play an important role in cell apoptosis and survival [24]. Therefore, the PI3K/AKT signaling pathway displays an attractive target for antitumor therapy. It has been reported that GEM can product excess reactive oxygen species (ROS) by activating the PI3K/AKT signaling pathway, which inhibits the chemotherapy effect and reduces the antitumor responses of pancreatic cancer cells to GEM [25, 26]. In the present study, we also demonstrated GEM activated the PI3K/AKT signaling pathway in human bladder cancer cells. Thus, we hypothesized that activation of the PI3K/AKT signaling pathway was involved in the chemoresistance of human bladder cancer cells to GEM. Many researches have reported that inactivation of the PI3K / AKT signaling pathway played an important role in UA-induced apoptosis in cancers, such as pancreatic cancer and prostate cancer [27, 28]. As expected, our research showed that UA can significantly suppress the activation of the PI3K/AKT signaling pathway. Furthermore, we proved that activating the Akt activity by SC79, a selective Akt activator, can reverse the antitumor effect. Thus, we speculate that UA can enhance GEM-induced apoptosis by inactivating the PI3K/Akt signaling pathway in human bladder cancer cells.

The JNK signaling pathway is another classic apoptosis signaling pathway. JNK is one of mitogen-activated protein kinase(MAPK)family members that can dominate the apoptosis, proliferation and metastasis of tumor cells[29]. Teraishi et al found that GEM could activate the JNK pathway to induce apoptosis in human lung cancer cells [30]. A recent review demonstrated that UA could induce the activation of JNK to promote the apoptosis of multiple cancer cells [31]. Accordingly, in our research, we showed that UA could up-regulate the expression of phosphorylated JNK induced by GEM. When we used SP600125, a JNK inhibitor, to inhibit the JNK signaling pathway, the levels of cleaved-PARP and cleaved-caspase3 were obviously reduced. Hence, we speculate that UA may contribute to GEM- induced apoptosis by activating the JNK signaling pathway in human bladder cancer cells.

In our study, although we have demonstrated that UA enhances GEM- induced apoptosis through PI3K/AKT and JNK signaling pathways. We still have some insufficiencies. For instance, the downstream mechanisms are warranted to detect by further study. In addition, deeply investigating the treatment time and concentration of UA and GEM combined application may further improve the therapeutic effects in human bladder cancer cells. In addition, it was reported that UA was capable of reversing chemotherapeutic drug resistance in some cancers [32, 33]. Therefore, UA is a promising candidate for the potential treatment of GEM-resistant BCa, and further investigations are needed to verify this hypothesis.

In conclusion, our study demonstrated for the first time that UA could enhance the antitumor effect of GEM by inactivating the PI3K/Akt signaling pathway and activating the JNK signaling pathway in human bladder cancer cells. The combinational treatment strategy of UA and GEM may provide a potential rational basis for the clinical treatment of BCa.

Author contributions

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

Funding

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

Conflicts of interest

The authors declare that they have no conflicts of interest.

Ethical approval

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

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

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Abstract

Figures

Introduction

Materials And Methods

Reagents and antibodies

Cell culture

Measurement of cell viability

Hoechst 33258 staining

Apoptosis analysis by flow cytometry

Western blot analysis

Statistical analysis

Results

Discussion

Declarations

References

Status:

Version 1