Hyperoside induces cell cycle arrest and suppresses tumorigenesis in bladder cancer through the interaction of EGFR-Ras and Fas signaling pathways

doi:10.21203/rs.3.rs-3291461/v1

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Hyperoside induces cell cycle arrest and suppresses tumorigenesis in bladder cancer through the interaction of EGFR-Ras and Fas signaling pathways

https://doi.org/10.21203/rs.3.rs-3291461/v1

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Background

Hyperoside is a natural flavonol glycoside widely found in plants and has been reported to have a variety of pharmacological effects, including anticancer abilities. However, the antitumor effect of hyperoside on bladder cancer has not been studied, and its exact mechanism and targets remain unclear.

Methods

The human bladder cancer cells T24 and 5637 were treated by hyperoside and evaluated by MTT assay and flow cytometry. The underlying mechanisms were investigated by quantitative proteomics and bioinformatics analyses. The variation of proteins was confirmed by Western blot. In vivo studies were conducted using tumor-bearing mice to evaluate the anti-tumor effects of hyperoside in bladder cancer.

Results

We demonstrated for the first time that hyperoside repressed the proliferation of bladder cancer cells in vitro and in vivo. Moreover, hyperoside could not only induce cell cycle arrest, but also cause apoptosis of a few bladder cancer cells. Specifically, hyperoside induced overexpression of EGFR, Ras and Fas proteins, which affects a variety of synergistic and antagonistic downstream signaling pathways, including MAPKs and Akt, ultimately contributing to its anticancer effects in bladder cancer cells.

Conclusions

This study reveals that hyperoside could be a promising therapeutic strategy for the prevention of bladder cancer.

bladder cancer

hyperoside

cell cycle arrest

Ras

Fas

MAPK

Bladder cancer is the fourth most common cancer in men in the United States. In fact, there were an estimated 81,180 new cases and 17,100 deaths from bladder cancer in the United States in 2022 [1]. In addition, 573,278 new cases of bladder cancer and 212,536 bladder cancer deaths were predicted to occur globally in 2020 [2]. Although chemotherapy has revolutionized the treatment of advanced tumors [3], the median survival of cisplatin combined chemotherapy for metastatic disease can only be extended to 14 months [4], and the associated side effects due to the lack of specificity to tumor cells remain a challenging issue. Therefore, there is an urgent need for new therapeutic strategies to treat advanced bladder cancer.

The incidence of bladder cancer varies across countries and regions, with the highest rates in Southern Europe (Greece, Spain, and Italy), Western Europe (Belgium and the Netherlands), and Northern America [2]. However, the regions with the lowest incidence, such as Western and Central Africa and Southeastern Asia, have rates five to ten times lower than those in the United States and Europe [2]. Due to geographic discrepancies in incidence, more and more scholars have begun to pay attention to the effects of diet, nutritional intake, and some chemopreventive drugs on tumorigenesis.

Flavonoids, a class of polyphenolic compounds, are common bioactive secondary metabolites and components in vegetables and fruits [5], and hold great promise in cancer chemoprevention and chemotherapy due to their high availability, strong anticancer efficiency and low toxicity [6]. Hyperoside (quercetin-3-O-galactoside) is a natural flavonol glycoside widely found in Hypericaceae, Rosaceae, and other plants [7, 8], and has a variety of pharmacological effects such as anticancer, anti-inflammatory, antioxidant, organ protection, antibacterial, antiviral, etc. [9, 10].

However, the antitumor effect of hyperoside on bladder cancer has not been studied, and its exact mechanism and targets remain unclear [9, 10]. In this study, we reported for the first time that hyperoside induced cell cycle arrest and a small amount of apoptosis of bladder cancer cells, and inhibited tumor growth in xenografted tumor models in nude mice.

Reagents and antibodies

Hyperoside (> 98% pure) was obtained from Tanmo Quality Control Technology Co. (Changzhou, China) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The annexin V-FITC apoptosis detection kit was purchased from BD Biosciences (San Jose, CA, USA). The bicinchoninic acid protein assay kit was purchased from Pierce Biotechnology (Rockford, IL, USA). The Coulter DNA Prep™ Reagents Kit was purchased from Beckman Coulter (Fullerton, CA, USA). Antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell Culture

Human bladder cancer cell lines T24 and 5637 were derived from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in RPMI 1640 medium (HyClone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 mg/L) in a humidified atmosphere containing 5% CO₂ at 37°C. The day before treatment, the cells were inoculated in an antibiotic-free growth medium at a density of 30–40%. Cell images were taken using a phase contrast microscope (Olympus, Japan) with 100× magnification.

Cell growth/viability assay

The cell proliferation was measured by MTT assay. Approximately 3000 to 8000 cells (depending on their culture time) were plated in each well of the 96-well plate. After incubation overnight, cells were treated with different concentrations of hyperoside (0-800 µM) for 24 h. The medium was removed at different times after treatment, and plates were incubated at 37°C for 4 hours after adding 20 µl MTT (5 mg/ml) into each well. After that, the purple formazan precipitate in each well was dissolved in 150 µl dimethyl sulfoxide while the plates were spun in a centrifuge. Measurement of absorbance at 490 nm was performed on an absorbance reader (MRX II; DYNEX Technologies, Chantilly, VA, USA). The reduction in cell viability of each group was expressed as a percentage of control cells, which were considered to be 100% viable.

Cell cycle analysis by flow cytometry

Cell cycle analysis was performed using the Coulter DNA Prep™ Reagents Kit. Cells were plated in six-well plates and incubated overnight before treatment. After treatment for 24h, the cells were harvested, washed twice with prechilled PBS, and resuspended with 100 µl PBS at a concentration of 1×10⁶ cells/ml. Each cell sample was mixed with 100 µl DNA Prep LPR, gently mixed with a vortex, and incubated at room temperature (25°C) in the dark for 20 minutes. Then, each sample was mixed with 1 ml of DNA Prep Stain, gently mixed by vortexing, and incubated again at room temperature (25°C) in the dark for 20 minutes. Finally, the flow cytometry system (Beckman Coulter FC500 Flow Cytometry System with CXP Software; Beckman Coulter, Fullerton, CA, USA) was used for cell cycle analysis within 1 hour, and the raw data was analyzed by Multicycle for Windows (Beckman Coulter).

Detection of apoptotic cells by flow cytometry

Apoptosis was quantitatively assessed by determining the percentage of cells with highly condensed or fragmented nuclei. Cells were plated in six-well plates and incubated overnight before treatment. As mentioned above, the cells were harvested 24 hours after hyperoside treatment, washed twice with prechilled PBS, and resuspended with 100 µl 1× binding buffer at a concentration of 1×10⁶ cells/ml. Double staining was performed with fluorescein isothiocyanate (FITC) conjugated annexin V and propidium iodide (PI) (Annexin V-FITC Apoptosis Detection Kit) in accordance with the manufacturer’s protocol. Cell apoptosis was analyzed by flow cytometry within 1 hour.

Western blotting analysis

Briefly, cells were harvested at 24 hours following hyperoside treatment as described above, washed and lysed with lysis buffer. The protein concentration in the lysate was determined using the bicinchoninic acid protein assay kit. An appropriate amount of protein (30–50 µg) was isolated by electrophoresis in 10–15% Tris-glycine polyacrylamide gel and transferred to nitrocellulose membrane. The membrane was blocked and then incubated overnight with the appropriate primary antibody at a dilution specified by the manufacturer. The membrane was then washed three times in 15 ml TBST and incubated with the corresponding horseradish peroxidase (HRP) conjugated secondary antibody at a 1:1000 dilution in TBST for 1 hour. The combined secondary antibody was detected using an enhanced chemiluminescence (ECL) system (Pierce Biotechnology Inc., Rockford, IL, USA) after three washes of 15ml TBST for 5 minutes each.

Mass spectrometry and bioinformatics analyses

In brief, T24 cells were treated with 400 µM hyperoside for 24 h and then homogenized in SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl pH 8.0). The supernatant was quantified with BCA Protein Assay Kit (P0012, Beyotime). After further digestion by trypsin using filter-assisted sample preparation, the peptide content was estimated by UV spectral density at 280 nm using an extinction coefficient of 1.1 of 0.1% (g/l) solution. A 100 µg peptide mixture of each sample was labeled using TMT reagent according to the manufacturer’s instructions (Thermo Fisher Scientific) and fractionated by reverse-phase chromatography using Agilent 1260 infinity II high performance liquid chromatography (HPLC). Then, each fraction was injected for nano LC-MS/MS analysis. LC-MS/MS analysis was performed on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific), which was coupled to the Easy nLC (Thermo Fisher Scientific) for 60/90 min. MS/MS raw files were processed using the MASCOT engine (Matrix Science, London, UK; version 2.6) embedded into Proteome Discoverer 2.2. A 1% false discovery rate (FDR) was set to identify proteins. The Gene Ontology (GO) annotation was completed by Blast2GO Command Line (www.geneontology.org). Pathway enrichment analysis was performed using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.kegg.jp/). Protein-protein interaction prediction was conducted using the STRING Version 11.0 (https://string-db.org/).

Animal experiments

Male BALB/c-nude mice weighing 18–20 g each (4 weeks old, provided by the Shanghai Experimental Animal Center, Chinese Academy of Sciences, Shanghai, China) were used for the experiments. The mice ate and drank freely and were fed in a 12:12 h light:dark cycle with room temperature maintained at 21℃. T24 bladder cancer cells (1×10⁵ in 10 µL PBS) were injected s.c. into the right flank of mice. The growth rate of tumors was monitored and measured by a blinded observer. The formula for calculating tumor volume is: volume (mm³) = width² (mm²) × length (mm) × 0.52, where length and width were tumor diameters measured with calipers in mutually perpendicular directions. When the tumor size reached about 30 mm³, the mice were divided into three groups and treated with an i.p. injection of propylene glycol (vehicle, 0.1 mL), 20 mg/kg hyperoside (in 0.1 mL propylene glycol), or 40 mg/kg hyperoside (in 0.1 mL propylene glycol) once daily for 3 weeks. Tumor size was measured every two days for 21 days.

Statistical analysis

All in vitro experiments were performed in triplicate. GraphPad Prism software (San Diego, CA, USA) was used to calculate statistically significant differences with Student's t test. All values are expressed as means ± SDs, and P < 0.05 was considered statistically significant.

Hyperoside inhibits cell growth in bladder cancer cells

To investigate the effects of hyperoside on human bladder cancer cells, T24 and 5637 cells were treated with 400 or 800 µM hyperoside for 12 to 48 hours and found to gradually lose their viability and stop proliferating (Fig. 1a and 1b). The cytotoxic effects of hyperoside on T24 and 5637 cells at varying concentrations and times (12–72 h) were determined by MTT assay. As shown in Fig. 1, the effects of hyperoside on cell viability, which were dose and time dependent, occurred within 12 h and at concentrations as low as 25 µM. Compared with the control cells treated with 0.1% DMSO, the viability in T24 cells treated with hyperoside at concentrations of 25–800 µM decreased by 4.2–36.0% after 12 h, whereas it deceased by 10.4–65.8%, 14.8–72.7% and 17.7–92.2% after 24, 48 and 72 h, respectively (Fig. 1c). Similarly, the reduction in viability in 5637 cells ranged from 2.7–40.2%, 1.8–67.5% and 3.3–88.2% after hyperoside treatment for 12, 24 and 48 h, respectively (Fig. 1d). The inhibitory concentration 50% (IC50) of hyperoside treatment on T24 cells were about 629, 330, 252 and 159 µM for 12, 24, 48 and 72 h, respectively. Moreover, the IC50 values of hyperoside in 5637 cells were approximately 667, 431 and 250 µM at 12, 24 and 48 h, respectively.

Hyperoside induces cell cycle arrest in bladder cancer cells

Flow cytometry was used to study the relationship between hyperoside-mediated inhibition of cell proliferation and cell cycle arrest. As shown in Fig. 2a, G1 and G2 phase arrest was observed in 400–800 µM hyperoside-treated T24 cells. The number of G1 phase cells treated with 400 and 800 µM hyperoside was approximately 55% at 48 h following treatment and increased by more than 10% compared to the control group. In addition, the number of G2 phase cells treated with 400–800 µM hyperoside also increased by 3% − 12%. The increase in the G1 and G2 phase cells was found to be associated with a concomitant significant decrease in the S phase population (Fig. 2a and 2c). A similar phenomenon of G1 and G2 phase cell cycle arrest was also observed in 5637 bladder cancer cells (Fig. 2b and 2d).

We then examined the expression of several cell cycle-related proteins. Accordingly, interference with the cell cycle was associated with decreased expression of cyclin D1 and reduced phosphorylation of CDK2 and CDK1 in hyperoside-treated T24 cells. We also found a small decrease in cyclin B1 expression in hyperoside-treated cells, but no alteration in cyclin E and CDK4 expression (Fig. 3a-3c). As a result, the phosphorylation level of Rb protein, a key point in the cell cycle to propel cell proliferation [11], is significantly reduced (Fig. 3d and 3e).

Hyperoside induces apoptosis in a small number of bladder cancer cells

We next analyzed the relationship between hyperoside-mediated loss of cell viability and apoptosis by flow cytometry, and found that hyperoside induced mild apoptosis in bladder cancer cells T24 and 5637 after 400 µM hyperoside treatment for 48 h. As shown in Fig. 4a-4d, the proportion of late apoptotic cells (UR quadrant) increased to more than 10%, and the early apoptotic cells (LR quadrant) also increased slightly compared with the control group.

Caspase-3 and poly (ADP-ribose) polymerase (PARP) play a central role in apoptosis. Accordingly, significantly reduced levels of pro-caspase-3 were observed in T24 cells treated with 200–400 µM hyperoside for 48 h (Fig. 4e and 4f). Moreover, 89 kDa cleaved PARP fragments were detected in the hyperoside-treated samples. Thus, the evident changes in apoptosis-related proteins affected by hyperoside treatment confirmed the ongoing apoptosis and the antitumor effects on human bladder cancer cells.

Quantitative proteomics and bioinformatics analyses suggest the involvement of EGFR-RAS and Fas signaling induced by hyperoside

To explore the potential mechanism responsible for hyperoside-induced cell cycle arrest and apoptosis, quantitative proteomics was used to detect the proteomic alterations in T24 cells treated with 400 µM hyperoside for 24 h. Total cell proteins were collected and digested with trypsin for LC-MS/MS proteomics analysis based on tandem mass tag (TMT) (Fig. 5a). As shown in Fig. 5b, 246,507 spectra were obtained from TMT-based proteomics analysis. After data filtering, the low-scoring spectra was eliminated and 88,026 spectra were matched to 52,354 peptides in the database, of which 48,619 were unique peptides. Finally, 6,593 proteins were identified. For comparison of hyperoside-treated cells and control groups, a protein was regarded as a differentially expressed protein (DEP) if the fold change is > 1.5 or < 0.67 and the P value of < 0.05. Based on these two criteria, 1,627 DEPs were identified, of which 874 were significantly upregulated and 753 were downregulated in hyperoside-treated T24 bladder cancer cells compared with controls (Fig. 5c). These DEPs were mainly clustered into 59 GO functional categories, involving 26 biological processes, 19 cellular components, and 14 molecular functions (Fig. 5d).

To further understand the function of these proteins, we used WoLF PSORT to predict the subcellular localization of these DEPs. The results indicated that the largest subcellular fraction was cytosol, accounting for 30.6% of the DEPs, and other significant subcellular location sites were nucleus (20.8%) and plasma membrane (17.6%) (Fig. 5e). GO enrichment analysis is shown in Fig. 5f, and the most significantly enriched cellular components were integral components of the membrane. Then, KEGG analysis was conducted to study the enriched pathways related to the dysregulated proteins, and the most enriched pathways were found to be the metabolic pathways (Fig. 5g).

To better understand the potential protein-protein interactions (PPIs) of the DEPs and the related unaltered proteins, we subsequently performed a PPI proteomics network analysis using STRING software. The interactions of robust and crosstalk signaling are mapped in Fig. 6. Network analysis of the differentially expressed proteins suggested that the EGFR-Ras and Fas signaling pathways might play a key role in mediating the effects of hyperoside on bladder cancer cells.

The alteration of Fas and EGFR-Ras related signaling pathways induced by hyperoside

In order to confirm the involvement of Fas and EGFR-Ras signaling pathways in the treatment of hyperoside in human bladder cancer cells, Western blotting was performed to detect the expression levels of Fas, EGFR and Ras, which are upstream proteins in the MAPK and Akt signaling pathways. The results showed that hyperoside significantly increased the expression of Fas, EGFR and Ras at the protein level (Fig. 7a and 7b).

Abnormalities in the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathways contribute to the carcinogenesis process, including cell proliferation, differentiation, invasion, angiogenesis, apoptosis, and metastasis [12, 13]. Thus, the primary subgroups of MAPKs engaged in carcinogenesis, including extracellular signal-regulated kinase (ERK), c-Jun NH2- terminal kinase (JNK) and p38 MAPK, were detected and found to be more phosphorylated in hyperoside-treated T24 cells (Fig. 7C-D), which was consistent with the activation of Ras and Fas signaling. Moreover, the expression and phosphorylation levels of Akt were investigated after hyperoside treatment for 24 h. As shown in Fig. 7c and 7d, the phosphorylation of Akt at Ser473 was evidently upregulated, which was consistent with Ras overexpression.

Therefore, in contrast to previous studies [9, 10] that hyperoside exerts its anticancer effects through a certain pathway, these phosphorylated MAPK and Akt proteins possess complicated and conflicting downstream signaling pathways, which lead to the ultimate scenario of cell cycle arrest and a small amount of cell apoptosis in T24 bladder cancer cells (Fig. 9).

Hyperoside suppresses the growth of bladder tumor xenografts in vivo

To evaluate the therapeutic potential of hyperoside in cancer treatment, 1×10⁵ T24 cells were subcutaneously injected into the flanks of nude mice. When the tumor size reached about 30 mm³, the mice were randomized into three groups and i.p. injected with hyperoside (20 mg/kg and 40 mg/kg) or vehicle control once daily for 3 weeks. As shown in Fig. 8, the tumors treated with hyperoside grew more slowly than those in the control group. In the first week, there was no difference in tumor volume between the hyperoside-treated groups and the control group (P > 0.05). In contrast, the tumor volumes of hyperoside-treated groups were significantly smaller than those of the control group from days 11 to 21 (P < 0.05). The results indicated that hyperoside treatment (20 or 40 mg/kg) had antitumor effects on bladder cancer xenografts in vivo.

Hyperoside, also known as quercetin 3-O-beta-D-galactopyranoside, has recently received a lot of attention for its anticancer, anti-inflammatory, antioxidant and organ protective activities. In particular, hyperoside can inhibit cell growth by inducing cell apoptosis and cell cycle arrest in several cancer cells [14–19]. However, the exact mechanism of its antitumor effects remains unclear. Some studies attributed this effect to the PI3K/Akt signaling pathway, but others suggested that MAPKs, Fas or NF-κB were also involved in its antitumor activities [9, 10].

Unscheduled proliferation is a hallmark of cancer. Akt and MAPK signaling pathways regulate cell proliferation, survival and differentiation at different transition points, which have a profound impact on the development of various cancers. It is well established that hyperactivation of Akt kinases is a common event in many human cancers, including bladder cancer [20], leading to tumor cell survival and enhanced resistance to apoptosis through multiple mechanisms [21]. Overexpression of upstream activators, including epidermal growth factor receptor (EGFR; also known as ERBB1), ERBB2 and/or ERBB3, are associated with grade, stage and outcome in subsets of bladder cancer, and EGFR induces PI3K/Akt activation via RAS activation [22, 23]. In our study, hyperoside activated the EGFR-Ras signaling and induced the phosphorylation of Akt at Ser473 in T24 bladder cancer cells, thereby promoting cell proliferation and preventing cell apoptosis.

Unlike Akt, MAPKs have cell type- and context-dependent actions in different cancers, with ERK inhibiting apoptosis and increasing cell proliferation, while JNK and/or p38 are c-Jun/AP-1 and/or p53-mediated apoptotic regulators [24]. However, the role of MAPK signaling and its relationship to key mutations in bladder tumors remain unclear. Current data suggest that the majority of bladder cancers may be highly dependent on ERK [25], while both FGFR3 mutations and PIK3CA mutations commonly cooccur in NMIBC, suggesting synergistic activation of MAPK and PI3K pathways [26, 27]. Our data showed that all three MAPKs were phosphorylated by hyperoside induced upstream signals in T24 bladder cancer cells. Taken together, hyperphosphorylated Akt and ERK by the upregulation of EGFR-Ras expression might promote cell proliferation and resist apoptosis, but phosphorylation of JNK and p38 by the activation of Ras or Fas signaling could promote cell cycle arrest and apoptosis conversely.

Previous studies [9, 10] only focused on and verified the involvement of a single pathway in several cancer cell lines and have not systematically analyzed the role of hyperoside using quantitative proteomics and bioinformatics analysis. In fact, the addition of drug to cells can cause changes in a variety of cellular pathways, eventually resulting in a certain phenotype in a specific cellular and environmental context. In our study, hyperoside induced cell cycle arrest and only a small amount of apoptosis in bladder cancer cells, mainly as a result of the phosphorylation of JNK and p38. The inconspicuous cell apoptosis may be related to the antiapoptotic effects of the phosphorylation of Akt and ERK. Since the hyperactivation of Akt and ERK is already present in most bladder cancers [20, 25], the phosphorylation of Akt and ERK by hyperoside might not significantly enhance their activity further, which could explain why they do not dominatet in the story of hyperoside.

Although adjuvant therapy can improve treatment outcomes, especially with the great advances in the development of combined therapeutics such as immune checkpoint inhibitors PD-1/PD-L1 inhibitor combined chemotherapy or antibody-drug conjugates (ADCs) or CTLA-4 inhibitors [4, 28], advanced and metastatic bladder cancer can develop resistance to these drugs when treated for a period. Therefore, identification of novel targets and development of effective drugs are urgently needed for bladder cancer treatment. Natural products, especially flavonoids, are an abundant source for screening antitumor drugs due to their remarkable efficacy and low toxicity [6]. Our results indicated that hyperoside not only inhibited the proliferation of bladder cancer cells in vitro, but also suppressed the tumorigenesis of bladder cancer in vivo. Molecularly, well-known cell cycle arrest markers, including cyclins and phosphorylation of CDKs and Rb proteins, were significantly reduced by hyperoside treatment, suggesting that hyperoside might exert its antitumor effects by inducing cell cycle arrest. Moreover, caspase-3 activation plays a central role in apoptosis by cleaving intracellular proteins vital for cell survival and growth, such as PARP [30, 31], leading to the completion of apoptosis and reinforcing the anticancer abilities of hyperoside in bladder cancer cells.

Taken together, we revealed for the first time that hyperoside has significant suppressive effects on bladder tumorigenesis in vitro and in vivo by activating the EGFR-Ras and Fas signaling pathways. In contrast to previous studies on a single pathway, proteomics and bioinformatics analysis revealed that hyperoside affects multiple signaling pathways, whose synergistic and antagonistic effects ultimately lead to cell cycle arrest in bladder cancer cells under specific contexts and conditions. Regardless, hyperoside offers a new approach to comprehensively prevent bladder cancer. Despite the promise, further studies are needed to screen more natural compounds for cancer treatment and delineate their exact mechanism.

μM, μmol/l; IC50: inhibitory concentration 50%; CDK: cyclin-dependent kinase; PARP: poly (ADP-ribose) polymerase; TMT: tandem mass tag; HPLC: high performance liquid chromatography; DEP: differentially expressed protein; PPIs: protein-protein interactions; EGFR: epidermal growth factor receptor; MAPK: mitogen-activated protein kinase; PI3K/Akt: phosphatidylinositol-3-kinase/protein kinase B; ERK: extracellular signal-regulated kinase; JNK: c-Jun NH2-terminal kinase.

Ethics approval and consent to participate

All experiments were approved by the Animal Experimental Ethical Inspection of the First Affiliated Hospital, College of Medicine, Zhejiang University (Reference number: 2020-340).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Funding

This study was supported by grants from the National Natural Science Foundation of China (Grant No. 81101718) and the Natural Science Foundation of Zhejiang Province (Grant No. LY13H160009).

Authors’ contributions

KY was responsible for conceptualization, formal analysis, and writing the original draft. KY and ZQ performed experiments. MS was responsible for validation. LX was responsible for reviewing, editing and supervision; All authors have read and approved the final manuscript.

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Hyperoside induces cell cycle arrest and suppresses tumorigenesis in bladder cancer through the interaction of EGFR-Ras and Fas signaling pathways

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Reagents and antibodies

Cell Culture

Cell growth/viability assay

Cell cycle analysis by flow cytometry

Detection of apoptotic cells by flow cytometry

Western blotting analysis

Mass spectrometry and bioinformatics analyses

Animal experiments

Statistical analysis

Results

Hyperoside inhibits cell growth in bladder cancer cells

Hyperoside induces cell cycle arrest in bladder cancer cells

Hyperoside induces apoptosis in a small number of bladder cancer cells

The alteration of Fas and EGFR-Ras related signaling pathways induced by hyperoside

Hyperoside suppresses the growth of bladder tumor xenografts in vivo

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

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