Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 64  |  Issue : 2  |  Page : 294--301

Xanthoceraside induces cell apoptosis through downregulation of the PI3K/Akt/Bcl-2/Bax signaling pathway in cell lines of human bladder cancer


Xue Chai1, Jian-Wu Zhang2, Sheng-Hui Li1, Qing-Shui Cheng1, Ming-Ming Qin3, Chun-Yan Yang4, Jia-Lin Gao5, Hou-Bao Huang1,  
1 Department of Urology, The First Affiliated Hospital of Wannan Medical College, Wuhu, Anhui, China
2 Department of Pharmacology, North Sichuan Medical College, Nanchong, Sichuan, China
3 Clinical Laboratory, The First Affiliated Hospital of Wannan Medical College, Wuhu, Anhui, China
4 Institute of Materia Medica, North Sichuan Medical College, Nanchong, Sichuan, China
5 Department of Endocrinology, The First Affiliated Hospital of Wannan Medical College, Wuhu, Anhui, China

Correspondence Address:
Hou-Bao Huang
Department of Urology, The First Affiliated Hospital of Wannan Medical College, Wuhu, Anhui - 241001
China

Abstract

Background: Xanthoceraside is a component obtained in the husks of Xanthoceras sorbifolia Bunge. Series of researches proved that xanthoceraside had functions of anti-inflammation and anti-tumor effects. However, the mechanisms of xanthoceraside against bladder cancer are unclear. Accordingly, we proposed to investigate xanthoceraside's impacts and potential mechanisms in cells of bladder cancer. Methods: By using the CCK-8 assay, we measured the viability of cells. With the use of 4,6-diamidino-2-phenylindole (DAPI) staining, we examined nuclear fragmentation and chromatin condensation in the nuclei of apoptotic cells. By using flow cytometry, we measured cell apoptosis. By using Western blotting, we tested the expressions of Caspase-9, Caspase-8, Caspase-3, Bcl-xL, P53, and PI3K/Akt/Bcl-2/Bax. Results: The proliferation of cell lines of human bladder cancer T24 and 5637 was suppressed by xanthoceraside significantly in a time- and concentration-dependent way. When cell lines 5637 and T24 were incubated as the xanthoceraside dose increased, the rates of cell apoptosis were upregulated, which was dependent on dose. According to further analysis, xanthoceraside induced apoptosis by upregulating Bax and downregulating the expression of Bcl-xL and Bcl-2. However, xanthoceraside did not change the expression of Caspase-9, Caspase-8, and Caspase-3. Interestingly, xanthoceraside also downregulated the expression of p-PI3K and p-Akt, and upregulated P53. Conclusions: Xanthoceraside induces cell apoptosis through downregulation of the PI3K/Akt/Bcl-2/Bax signaling pathway in cell lines of human bladder cancer.



How to cite this article:
Chai X, Zhang JW, Li SH, Cheng QS, Qin MM, Yang CY, Gao JL, Huang HB. Xanthoceraside induces cell apoptosis through downregulation of the PI3K/Akt/Bcl-2/Bax signaling pathway in cell lines of human bladder cancer.Indian J Pathol Microbiol 2021;64:294-301


How to cite this URL:
Chai X, Zhang JW, Li SH, Cheng QS, Qin MM, Yang CY, Gao JL, Huang HB. Xanthoceraside induces cell apoptosis through downregulation of the PI3K/Akt/Bcl-2/Bax signaling pathway in cell lines of human bladder cancer. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Jun 17 ];64:294-301
Available from: https://www.ijpmonline.org/text.asp?2021/64/2/294/313297


Full Text



 Background



Bladder cancer represents a common malignancy in urinary system, and its morbidity tends to yearly grow. Each year, more than 400,000 new bladder cancer cases are reported around the world.[1] The statistics of National Cancer Institute of China estimated that new cases of bladder cancer were 80,500, and death of bladder cancer were 32,900 in 2015 in China.[2] Bladder cancer is originally derived from the transitional cell epithelium (urothelium) of the lining of the bladder and is directly exposed to urine, also known as transitional cell carcinoma. Nearly, 75% of newly diagnosed bladder cancers are non-muscle invasive (NMIBC).[3] NMIBC particularly receives the treatment with endoscopic transurethral resection (TUR), probably followed by an intravesical therapy, which is dependent on the carcinoma presence, tumor grade and the extent of the cancer in situ. In view of the high risk of recurrence and disease progression, careful monitoring of tumor resection by cystoscope is now standard clinical practice. Intravesical therapies with chemotherapeutic agents (e.g., mitomycin C) or Bacillus Calmette–Guerin (BCG), delivered via a urethral catheter, are used to prevent or delay recurrence and progression after TUR. A meta-analysis on the published results of seven randomized clinical trials showed that immediate instillation of chemotherapy following TUR can decrease the risk of recurrence in patients with single tumor or multiple tumors by 11%.[4] Current drugs for installation of chemotherapy comprise BCG, mitomycin C, and gemcitabine and so on. Although these drugs can be curative effect, however, they are associated with certain side effects. For instance, treatment-related adverse events of BCG can involve serious local and systemic response, such as hyperthermia, discomfort, and transient influenza symptoms, particularly the strong local stimulus similar to urinary tract infection following bladder instillation.[5]

Xanthoceraside [Figure 1], a triterpene extracted from the husk of Xanthoceras sorbifolia Bunge,[6] can improve the learning and memory impairment,[7] as well as possesses anti-inflammatory roles and anti-oxidative activities.[8] Previous studies indicated that xanthoceraside exhibited the cell growth inhibition activity in human neuroblastoma SH-SY5Y cells and induces apoptosis in melanoma cells through a mitochondria-mediated apoptotic pathway.[9] However, the effect and the mechanisms of xanthoceraside against bladder cancer is unclear.{Figure 1}

In the present study, the antitumor effects and the molecular mechanisms of xanthoceraside in cell lines of human bladder cancer T24 and 5637 were first investigated.

 Materials and Methods



Xanthoceraside and extraction procedure

Xanthoceraside was provided by the Institute of Materia Medica, North Sichuan Medical College (Nanchong, China), identified and voucher specimen (CBY-2016-0006) was deposited in the herbarium of the same institution. The preparation procedure is described briefly: Dry powder was prepared from the husks of Xanthoceras sorbifolia, and refluxed with 70% aqueous ethanol to obtain XST (the ethanol extract of Xanthoceras sorbifolia husks). The extract was suspended in distilled water and successively partitioned with chloroform and water-saturated n-butanol to yield XSC (the chloroform fraction of Xanthoceras sorbifolia husks), XSB (the n-Butanol fraction of Xanthoceras sorbifolia husks) and XSW (the water fraction of Xanthoceras sorbifolia husks). The XSB part was loaded into a reverse-phase silica gel column with a MeOH: water elution gradient of 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80% to gain eight fractions, in which 70% methanol elution was chromatographed on a Sephadex LH-20 column eluted with CHCl3: MeOH (1: 1) to obtain xanthoceraside.

Materials

We dissolved xanthoceraside in RPMI-1640 medium (HyClone, USA) and dimethyl sulfoxide (DMSO) (Sigma, USA) at a concentration of 100 mM and stored at -20°C. DMSO's final concentration in all assays was less than 0.1%.[8] CCK-8 Cell Proliferation Detection Kit and Annexin V-FITC Apoptosis Detection Kit were provided by KeyGEN Bio TECH (Nanjing, China); 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma (USA). The rabbit polyclonal antibodies against Caspase-3, Bcl-xL, Bax, P53, PI3K, Akt, p-Akt, and the mouse polyclonal antibodies against Caspase-8, Caspase-9, Bcl-2, β-actin were provided by Cell Signaling Technology (Danvers, MA, USA). The rabbit polyclonal antibodies against p-PI3K were provided by Abcam (Cambridge, UK).

Cell lines and culture

The cell lines of human bladder cancer T24 and 5637 were purchased from the Chinese Academy of Sciences (Shanghai, China). The cells were routinely cultured in the RPMI-1640 medium with 100 U/mL of penicillin, 100 U/mL of streptomycin, and 10% fetal bovine serum (GIBCO, USA) in a humidified cell incubator with 5% CO2 at 37°C.

Cell viability assay

By using the CCK-8 assay, we measured the cells' viability. We planted the cells in 96-well flat-bottom plates at a density of 2 × 103 cells per well. After 24 h in a CO2 incubator at 37°C, we treated the cells with different concentrations of xanthoceraside and cultured them for 24 h (or we treated the cells with the same concentrations of xanthoceraside and cultured them for different times). Next, CCK-8 testing liquid was added to each well. Four hours later, by using an enzyme-linked immunosorbent assay reader (BIO-RAD680, USA), we measured the absorbance at 450 nm. The percentage of cell viability was determined as: cell viability (%) = [A450 (xanthoceraside)/A450 (control)] ×100%. IC50 was calculated in accordance with the xanthoceraside dose and cell survival rate.

Nuclear staining with DAPI

By using DAPI (Sigma, USA) staining, Chromatin condensation and nuclear fragmentation in the nuclei of apoptotic cells were examined. The cells were harvested, washed with phosphate buffer saline (PBS) twice, and fixed with 4% paraformaldehyde (Sigma, USA) in PBS for 10 min at 25°C. The fixed cells were washed with PBS and stained with 1 mg/ml DAPI solution for 10 min. Subsequently, we washed the cells twice with PBS and observed them under a fluorescence microscope (Nikon Eclipse E200, Japan).

Annexin V-FITC labeling

After being treated with xanthoceraside, cell lines T24 and 5637 were collected and washed twice with ice-cold PBS. Next, we added Annexin V-FITC to each sample and incubated them in dark for 15 min at ambient temperature. We analyzed the fluorescent signal in cells by flow cytometry (CytomicsTM FC 500, USA) within 1 h.

Western blot assay

Cell lines T24 and 5637 treated with xanthoceraside in dose of 0, 6, 8, and 10 μM, respectively for 24 h were collected and lysed in 1× Laemmli sample buffer (Sigma, USA) on ice. After the lysate boiled, we fractionated protein samples (5 μl) in 10% SDS-polyacrylamide gel and subsequently transferred them to nitrocellulose (NC) membrane (GE Healthcare, Piscataway, NJ, USA). The cell membrane was blocked with 10% skim milk, washed with TBST, and then incubated with primary antibody (dilution at 1:1000) at 4°C for 12 h. The NC film was washed three times, and then incubated with the second level antibody of horseradish peroxidase conjugate in mice or rabbits. After removing the secondary antibody, the membrane was scanned by fluorescent chemistry FC3 (Protein Simple, San Jose, CA, USA).

Statistical analysis

We performed each experiment in triplicate. Mean ± standard deviation noted the continuous variables. We compared the difference between groups and within groups by single factor analysis of variance or t-test. All data were analyzed with software SPSS for windows (Ver. 20.0, Inc., Chicago, IL, USA). A two-sided P value <0.05 was considered with statistical significance.

 Results



Xanthoceraside suppressed bladder cancer cell lines T24 and 5637 proliferation

With concentrations ranging from 0 to 10 μM, xanthoceraside suppressed the growth of cell lines T24 and 5637 in a concentration-dependent way and the IC50 was 8.39 μM for T24 cell and 10.77 μM for 5637 cells at 24 h. The 10 μM xanthoceraside significantly suppressed the growth of T24 and 5637 at 24 h. Accordingly, we used a single dose of 10 μM for different time points in the subsequent experiments. With times ranging from 0 to 48 h, xanthoceraside suppressed the of T24's and 5637's growth in a time-dependent way [Figure 2].{Figure 2}

DAPI Staining of cell lines T24 and 5637 with xanthoceraside

According to DAPI staining, nuclei of chromatin aggregates and apoptotic bodies are formed in cells cultured with xanthine alcohol [Figure 3]. Living cells with complete DNA (control group), with light activation under fluorescence microscopy, were negative for DAPI. Besides, DAPI positive cells and their strength were all increased in a dose-dependent manner of xanthine dinucleotide. This reveals that most cells apoptosis primarily through the treatment of xanthan gum.{Figure 3}

Xanthoceraside-induced apoptosis in cell lines T24 and 5637

The rates of cell apoptosis would be increased in a dose-dependent way when cell lines T24 and 5637 were incubated as the xanthoceraside dose increased. The apoptotic cells upon treatment with 0, 6, 8, and 10 μM of xanthoceraside in T24 cells were (3.72 ± 0.53) %, (18.97 ± 1.24) %, (25.30 ± 2.02) % and (38.97 ± 0.76) % in percentage, respectively, after 24 h incubation [Figure 4]. Treatment with 0, 6, 8, and 10 μM of xanthoceraside in 5637 cells caused (2.82 ± 0.44) %, (4.51 ± 0.60) %, (13.60 ± 1.11) %, and (23.13 ± 1.99) % apoptotic cells [Figure 4].{Figure 4}

Xanthoceraside regulated the protein expressions

Treatment with xanthoceraside for 24 h led to the change of protein expression [Figure 5]. Xanthoceraside treated cell lines T24 and 5637 induced the protein Bcl-2, Bcl-xL and Bax, and significant differences were shown between control and xanthoceraside-treated group, indicating the apoptosis induction effects of xanthoceraside in bladder cancer cell lines T24 and 5637. No obvious difference was shown in Caspase-9, Caspase-8, and Caspase-3 in both cell lines T24 and 5637. Yet xanthoceraside upregulates the expression of P53. Xanthoceraside had no significant effect on the expression of PI3K and Akt, but decreased the expression of phosphorylated PI3K and phosphorylated Akt in T24 and 5637 cell lines, indicating xanthoceraside induces apoptosis in cell lines of human bladder cancer T24 and 5637 through downregulation of the PI3K/Akt/Bcl-2/Bax signaling pathway [Figure 6].{Figure 5}{Figure 6}

 Discussion



Natural products have always been an important source of effective drugs for the prevention and treatment of diseases. The ingredients used in traditional medicine not only come from natural compounds, but also more than 30% of modern medical preparations are chemical derivatives of herbal substances. Currently, more drugs are developed on natural product scaffolds basis by modifying or altering the chemical configuration.[10] Chan et al. investigated the cytotoxicity of Xanthoceras sorbifolia plant extract in different cancer cell lines determined by the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and found that some cell lines were sensitive, whereas others were not.[11] Xanthoceraside is a type of triterpene saponin monomer compound extracted from the husks of Xanthoceras sorbifolia Bunge, a plant native to China.[9]

In this study, different concentration of xanthoceraside effectively suppressed proliferation of cell lines T24 and 5637 in a dose-dependent manner. Furthermore, the survival rate of both cell lines T24 and 5637 treated with the same concentration of xanthoceraside was gradually reduced with prolonged treatment. These results suggest that xanthoceraside inhibits the growth of bladder cancer cells in a concentration and time-dependent way. Previous studies have shown that the antiproliferative effect of xanthoceraside on human melanoma A375.S2 cells, human breast cancer MCF-7 cells.[9] Our findings were consistent with these reports. It indicated that xanthoceraside had anti-tumor effect. Apoptosis or programmed cell death is considered as a natural way to remove the aged cells from the body. The potential to induce apoptosis has become an important target in the development of anti-tumor agents.[12] In our study, after treated with xanthoceraside, cell lines T24 and 5637 were stained with DAPI. This observation shows that xanthoceraside could induce apoptosis of bladder cancer cells. The results of flow cytometry also showed that xanthoceraside could induce the apoptosis of cells in bladder cancer in a concentration-dependent manner.[INLINE:1]

Apoptosis occurs primarily via the mitochondrial (intrinsic) pathway and the death receptor (extrinsic) pathway.[13] Pro-apoptotic Bax and anti-apoptotic Bcl-2 and Bcl-xL are important functional protein in mitochondrial apoptosis systems. Caspases refer to a type of aspartate-specific cysteine-dependent proteases. The activation of Caspase-9 leads to the activation of Caspases-3 and Caspases-8 and that leads to apoptosis.[14] To further confirm the mechanisms of xanthoceraside against bladder cancer in cell lines T24 and 5637, the expressions of protein were tested by Western blotting. In this study, the expression of protein Caspases-9, Caspases-8, and Caspases-3 was not changed significantly by the xanthoceraside treatment, so it was considered that there were no cooperative effects of Caspases-3, Caspases-8, and Caspases-9 on xanthoceraside cytotoxicity. Compared with cell lines T24 and 5637 without treatment of xanthoceraside, Bcl-2 and Bcl-xL protein expressions were remarkably down-regulated, whereas Bax protein expression was obviously upregulated after cell lines T24 and 5637 were treated by xanthoceraside for 24 h. The mentioned results indicate that the positive effects of xanthoceraside on apoptosis induction in bladder cancer cells may have a correlation with mitochondrial pathway. Jiao et al. described that xanthoceraside can induce apoptosis of melanoma cells via a mitochondria-mediated apoptotic pathway.[9] Our results are consistent with this report.

The development and occurrence of a variety of tumors included the PI3K/Akt signal transduction pathway.[15] Also, this pathway is important apoptotic pathway mediated by mitochondria in cell apoptosis.[16],[17],[18],[19] PI3K/Akt signaling pathway is known included in the apoptosis of epithelial cells, and Akt is the mediator for PI3K in its signaling transduction.[20] The tumor suppressor P53 is a transcription factor. Akt can phosphorylate p53 binding protein MDM2, thus affecting the activity of p53. Xanthoceraside remarkably upregulated P53 protein expression and suppressed the phosphorylation of PI3K and Akt proteins, indicating that xanthoceraside is likely to regulate proliferation and apoptosis of human bladder cancer cell through the PI3K/Akt signaling pathway.

These results provided experimental evidence that xanthoceraside had functions of anti-tumor effects in bladder cancer. Also, the mentioned findings suggest that xanthoceraside induces cell apoptosis by downregulating the PI3K/Akt/Bcl-2/Bax signaling pathway in cell lines of human bladder cancer [Figure 6]. Therefore, xanthoceraside is an antitumor drug.

 Conclusion



Xanthoceraside induces cell apoptosis through downregulation of the PI3K/Akt/Bcl-2/Bax signaling pathway in cell lines of human bladder cancer.

 Declarations



Ethics approval and consent to participate

Not applicable because we did not work with animals or humans.

Consent to publish

Not applicable.

Availability of data and material

The authors have presented all their main data in the form of figures. The datasets supporting the conclusions of this article are included within the article.

Acknowledgement

The authors would like to express their gratitude to The First Affiliated Hospital of Wannan Medical College and North Sichuan Medical College for providing administrative and technical support.

Financial support and sponsorship

This study was funded by Wuhu Municipal Science and Technology Program for Public Welfare (No.: 2016hm13) and Municipal-school cooperative scientific research project(No.: 19SXHZ0242).

Conflicts of interest

There are no conflicts of interest.

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