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Discov Oncol
Discov Oncol
Discover Oncology
2730-6011
Springer US New York

1347
10.1007/s12672-024-01347-9
Research
Ailanthone suppresses cell proliferation of renal cell carcinoma partially via inhibition of EZH2
Zhu Jianbing 12
Dai Guangcheng 3
Chen Ting 4
Zhou Yibin 3
Zang Yachen 3
Xu Lijun 3
Jin Lu docjin@126.com

3
Zhu Jin oceanzhu79@qq.com

3
1 https://ror.org/01rxvg760 grid.41156.37 0000 0001 2314 964X Department of Radiology, Suzhou Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu China
2 Key Laboratory of Functional Genomic and Molecular Diagnosis of Gansu Province, Lanzhou, Gansu China
3 https://ror.org/02xjrkt08 grid.452666.5 0000 0004 1762 8363 Department of Urology, The Second Affiliated Hospital of Soochow University, No.1055, Sanxiang Road, Suzhou, Jiangsu China
4 grid.452253.7 0000 0004 1804 524X Department of Pathology, Children’s Hospital of Soochow University, Suzhou, Jiangsu China
19 9 2024
19 9 2024
12 2024
15 46425 6 2024
13 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Ailanthone (Ail) extracted from medicinal plants has played an anticancer role in multiple cancers, while there is no research about Ail in renal cell carcinoma (RCC).

Methods

In the present study, we performed CCK-8 and flow cytometry to assess the effect of Ail on cell viability, apoptosis and cycle. We also performed tandem mass tags (TMT)-labeled quantitative proteomic technology and bioinformatic analysis to identify the functional pathway and proteins of Ail in RCC.

Results

The results showed Ail could inhibit cell viability and induce cell apoptosis. Proteomic profiling identified 1732 differentially expressed proteins in cells treated with Ail, compared to the negative control group. Gene ontology function annotation and Gene Set Enrichment Analysis (GSEA) were performed to identified the involved biological processes, molecular function and pathway. Results of GSEA proved the enrichment of Deps in EZH2 targets. The comparison between Deps and EZH2 co-expressed genes revealed 44 overlapped genes and we identified 4 hub genes (CDC20, CEP55, TOP2A, and UBE2C) associated with RCC progression. The molecular docking study revealed a moderate to tight binding potential of Ail and EZH2, and western blotting showed EZH2 was suppressed after cells treated with Ail.

Conclusion

Altogether, we identified the anticancer role of Ail in RCC, including inhibition of cell proliferation and induction of apoptosis. The results also screened the key proteins mediate the function of Ail, which have laid a theoretical foundation for elucidating the applications of Ail in clinical research.

the Suzhou Science & Technology Projects for People’s LivelihoodSKY2021031 Zhu Jianbing the Open Project of Key Laboratory of Functional Genomics and Molecular Diagnosis of Gansu Province2021BYGT-004 Zhu Jianbing the Urological Cancer Research Foundation of China031 Jin Lu the Natural Science Foundation of the Jiangsu Higher Education Institutions23KJB320015 Jin Lu the Suzhou Medical Research Key Talents ProgramGSWS2021016 Zhu Jin issue-copyright-statement© Springer Science+Business Media, LLC 2024
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pmcIntroduction

Clear cell renal cell carcinoma (ccRCC), which arises from the renal cortex or the renal tubular epithelial cells, is the most common type of RCC and represents the sixth most frequently diagnosed cancer in men and 9th in women [1, 2]. In recent years, the incidence of RCC increased stablely, which may be attributed to the incidental diagnosis. About 60% of the cases are diagnosed incidentally and 17% are metastatic RCC at the first time of diagnosis [3]. The 5-year survival rate of localized RCC is about 92.6%, and 11.7 for advanced RCC [4]. Surgical resection is the golden treatment for local RCC, while local advanced and metastatic RCC should accept comprehensive treatment, including immunotherapy, molecular targeted therapy, etc. [2, 5]. Based on developments in molecular biology and medical oncology, it is essential to explore a kind of new therapy with more effective treatment and fewer side effect.

Ailanthone (Ail), a kind of quassinoid extracted from traditional Chinese medicine plant Ailanthus altissima, has been employed to treat gastrointestinal diseases, bleeding, and inflammation, etc. [6, 7]. Previous studies demonstrated that Ail can be used to inhibit cancer growth in vitro and in vivo, including gastric cancer, osteosarcoma and bladder cancer [8–10]. The probable antitumor mechanism has been shown to be related to PI3K/Akt, JAK/STAT3, Ras/Raf and so on [10, 11]. However, no study about the role of Ail in RCC has been performed. In the present study, we assessed the antitumor role and explored the potential target of Ail in RCC.

Materials and methods

Ail extracted

Ail (purity > 98%) was supplied by Nanjing DASF Biotechnology and used in the present study. Ail was extracted from Ailanthus altissima as following: Ailanthus altissima was crushed and mixed with 10 times the amount of 80% ethanol. After sono-extraction, the extract was kept warm and decolorized with an appropriate amount of diatomite until condensed. Then appropriate amount of methanol was added, warming dissolved. The extraction solution was cooled to 0 °C and kept at the temperature of 0 °C overnight until white crystals were precipitated. The white crystals obtained were filtered, washed with a small amount of anhydrous ethanol, and dried to obtain the extraction. Finally, the dried extraction was purified with high-speed countercurrent chromatography and high performance liquid chromatography (HPLC) was used to detect the purity of extraction.

Cell culture and transfection

RCC cell lines (786-O, ACHN and OS-RC-2) were obtained from CCTCC (China Center for Type Culture Collection) and cultured as the previous protocol [12]. The pcDNA6.2-myc construct containing EZH2 or null control was transiently transfected into the cultured cells. All transfections were performed with FuGene 6 transfection reagent (Roche Diagnostics).

Cell viability assay

Cell viability is detected with cell counting kit-8 (CCK-8) assay. Cells were seeded into the 96-well plate (1 * 103 cells per well). After cultured for 24 h, 48 h or 72 h, the CCK-8 assay was performed as previous described. The OD value is recorded at 490 nm.

Cell apoptosis assay and cycle analysis

Cell apoptosis rare and cell cycle analysis were investigated with flow cytometry. For cell cycle analysis, cells were washed with PBS and 70% ethanol was used to fix cells overnight at − 20 °C. Then cells were treated with DNA staining solution containing 3.4 mM Tris–Cl (PH = 7.4), propodium iodide (PI), 0.1% triton X-100 buffer and 100 mg/ml RNase A. Then cells were scanned and cell cycle was analyzed in flow cytometer (BD, USA). For cell apoptosis, cells were washed with PBS and subsequently cultured for 30 min at 37 °C after adding 5 µl Annexin V-phycoerythrin and 10 µl PI. Then the cells would be scanned and apoptosis rate was analyzed with flow cytometry.

Protein digestion and TMT labeling

After treatment with 1 μm Ail for 24 h, SDT lysis was performed to extract the proteins as described in the previous literature [13]. The extracted protein was quantified by the BCA protein assay method. The peptides were prepared through the filter-aided proteome preparation (FASP) method and quantified at OD280. 100 ug peptides of each sample was labeled according to the instruction of the TMT Labeling Kit (ThermoFisher Scientific, USA), and the labeled peptide samples were mixed in the same quantities and separated according to the High pH Reversed-Phase Peptide Fractionation Kit. After that, each sample was separated with an Easy nLC High-Performance Liquid Chromatography (HPLC) liquid phase system (ThermoFisher Scientific, USA) at a nanoliter flow rate. The separated samples were kept for further analysis.

Protein data analysis

The separated samples were analyzed by mass spectrometry with Q Exactive plus mass spectrometer (ThermoFisher Scientific, USA). The original mass spectrometry data were output as RAW files. Proteome Discoverer 2.1 (ThermoFisher Scientific) and Mascot 2.6 were used to identify the library and perform quantitative analysis. A fold change > 1.2 or < 0.83 and p value < 0.05 (t-test) between two comparable groups (the Ail treatment groups and control groups) were considered to indicate significantly differentially abundant proteins.

Bioinformatics analysis

The Complexheatmap R package (R Version 3.4) was used to simultaneously classify the samples and protein expression in two dimensions, and a hierarchical clustering heatmap was generated. GO mapping and annotation were performed using Blast2GO. Gene Set Enrichment Analysis (GSEA) (http://www.broad.mit.edu/gsea) was performed for pathway analysis. The criterion for significance was set at a normalized enrichment score (NES) 1.0 and a p-value less than 0.05.

GEPIA database

The Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/index.html) was used to analyze the correlation between gene expression and survival rate in different groups.

Western blotting

Cells were collected and protein was extracted with RIPA lysis buffer (Beyotime, Beijing, China). Western blotting was performed as described previously. A total of 30 mg protein was used for the blotting. GAPDH was used for the loading control and antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

Molecular docking study

To analyze the binding affinities and modes of interaction between Ail and the potential targets, AutodockVina 1.2.2, a silico protein–ligand docking software was employed. The molecular structures of Ail was retrieved from PubChem Compound (https://pubchem.ncbi.nlm.nih.gov/). The structures of protein were downloaded from the PDB (http://www.rcsb.org/pdb/home/home.do). The protein and molecular files were converted into PDBQT format and the binding energy from the molecular docking tests was showed as a docking score, which can be used to access the protein–ligand binding potential. The value of docking score ≤ − 5 was considered as moderate to tight binding potential, and smaller docking score value mean better molecules binding.

Statistical analysis

The statistical analysis was performed with SPSS 21.0 (SPSS, Chicago, USA), and the data was showed as mean ± standard deviation (SD). T test or paired t test was performed to compare the difference between groups and P < 0.05 was considered statistically significant. All the experiments were repeated at least 3 times.

Results

Ail inhibits RCC cell viability

The Molecular structure of Ail is shown in Fig. 1A. To evaluate whether Ail has the effect on RCC cell viability, we detected cell viability treated with Ail in different dose or time. As shown in Fig. 1, Ail can inhibit RCC cell (786-O, 1B; ACHN 1C; and OS-RC-2, 1D) viability significantly and the effect is dose dependent. Cells treated with higher dose Ail showed a lower viability. Similarly, Cell viability is inhibited when treated with 1 μM Ail and the effect is time dependent (Fig. 1E–G). Thus, Ail has an inhibitory effect on RCC cells and the effect is dose and time dependent.Fig. 1 Ail inhibits RCC cell viability. A The molecular structure of Ail. B–D Ail inhibits RCC cells viability in a concentration-dependent manner (786-O, B; ACHN C; and OS-RC-2, D). E–G. Ail inhibits RCC cells viability in a time-dependent manner (786-O, E; ACHN F; and OS-RC-2, G). ***P < 0.001

Ail induced cell apoptosis and S phase arrest in the earlier stage

To access the effect of Ail on cell apoptosis and cell cycle distribution, flow cytometry was performed. In Fig. 2A and B, the early apoptosis rate of cells treated with Ail is significantly higher than which in NC group (from 24 to 96 h), and increases time-dependently in 72 h. However, the apoptosis rate did not increase after 72 h, which is probably attributed to the maximum effect of drug. In the cell cycle analysis (Fig. 2C and D), percentage of cells in G1 phase is reduced after treated with Ail. Cell cycle is mainly arrested in S phase at the time of 24 h and 48 h. However, the percentage of cells at S and G2/M phase is higher than the control group at 72 h and 96 h. The results suggested Ail can mediate S phase arrest in the earlier stage of treatment.Fig. 2 Ail induces cell apoptosis, and cell cycle arrest in 48 h. A, B Ail induces 786-O cells apoptosis compared with NC group. C, D Ail induces 786-O cells cycle arrest in S phase at the time of 24 h and 48 h, while cells at S and G2/M phase were higher than the NC group at 72 h and 96 h. NC: Negative control. *P < 0.05, **P < 0.01

Proteomics analysis and GO/GSEA pathway enrichment

To analyze the change of proteins after cells treated with Ail, TMT protein labeling proteomic profiling was performed (Fig. 3A). The fold change > 1.2 or < 0.83 and P value < 0.05 (t-test) between two comparable groups (the Ail treatment groups and control groups) were considered differentially expressed proteins. 1732 different expressed proteins (Deps) were identified (Fig. 3B and C). Go pathway enrichment was performed based on the 1732 different genes, results shown in Fig. 3D. The top 5 cellular component (CC), molecular function (MF) and biological process (BP) were screened in Fig. 3D. The enriched CC terms included melanosome, endoplasmic reticulum lumen, keratin filament, T-tubule and integral component of membrane. The 5 MFs included structural constituent of epidermis, protein disulfide isomerase activity, DNA polymerase binding, cytochrome-b5 reductase activity, acting on NAD(P)H and L-aspartate transmembrane transporter activity. The enriched BP terms included cornification, xenobiotic metabolic process, positive regulation of DNA binding, inflammatory response to antigenic stimulus and negative regulation of DNA-binding transcription factor activity. GSEA indicated that the genes were enriched in metabolism of RNA (Fig. 3E), mRNA splicing (Fig. 3F), cell cycle (Fig. 3G), glycolysis (Fig. 3H), EZH2 targets (Fig. 3I and J). EZH2 is widely known as an oncogene, which can catalyze methylation of histone 3 on lysine 27 (H3K27) and induce transcriptional repression.Fig. 3 Proteomics analysis and GO/GSEA pathway enrichment. A TMT protein labeling proteomic profiling is performed to define the DEPs. Volcano plot (B) and hot map (C) of DEPs. D Go pathway enrichment of DEPs. E–J Results of GSEA, the DEPs are enriched in metabolism of RNA (E), mRNA splicing (F), cell cycle (G), glycolysis (H), EZH2 targets (I and J)

Ail suppressed EZH2 expression

Based on the GSEA results, we found the potential correlation between Ail and EZH2. Our previous research had published EZH2 co-expressed genes [14]. To confirm the probable hub genes mediated by EZH2 and Ail, we compared the genes library from Deps, EZH2 co-expressed genes and 6 hub genes associated with RCC progression [15]. 44 overlapped genes were identified both in Deps and EZH2 co-expressed genes, and 4 key genes were shown in the venn diagram (Fig. 4A), including CDC20, CEP55, TOP2A and UBE2C, which might be regulated by both Ail and EZH2. The results demonstrated Ail might function via targeting EZH2, and/or the 4 key genes. Furtherly, molecular docking was performed with EZH2 and the 4 hub genes. The results showed that Ail had moderate to tight binding potential to some proteins (Fig. 4B, only EZH2 binding targets showed, docking score of the targets: − 6.325 for EZH2, -8.911 for CDC 20, − 8.76 for TOP2A and − 9.032 for UBE2C). Further results of western blot showed Ail can suppress expression of EZH2, CDC20, CEP55, TOP2A and UBE2C in both 786-O and ACHN cells (Fig. 4C and D). The results indicated that the 5 genes were regulated by Ail, and EZH2 probably played a more important role as a key mediator. To evaluate the role of EZH2 in Ail working, we performed rescue experiment. The results demonstrated EZH2 can promote cell viability and partially rescue the inhibiting effect of Ail on RCC cells (Fig. 4E).Fig. 4 Ail suppressed EZH2 expression. A Venn Diagram of DEPs, EZH2 co-expressed genes and 6 hub genes in RCC (4 overlapped genes: CDC20, CEP55, TOP2A and UBE2C). B Molecular docking of Ail and EZH2. C, D Ail can suppress expression of EZH2, CDC20, CEP55, TOP2A and UBE2C in 786-O and CHN cells. E EZH2 can promote 786-O cells viability and partially rescue the inhibiting effect of Ail. ***P < 0.001

Clinical significance of EZH2 and 4 hub genes

To evaluate the clinical significance of EZH2 and the 4 genes, we analyzed the correlation of EZH2 and the 4 genes expression in TCGA database and GSE2748 datasets, and performed Kaplan–Meier curve to assess the predicting roles in overall survival (OS) and disease free survival (DFS). The results showed expression of EZH2 in RCC was positively correlated with CDC20, CEP55, TOP2A and UBE2C in both TCGA database (Fig. 5A–D) and GSE2748 datasets (Fig. 5E–H). The results of survival analysis showed higher expression of the 5 genes (EZH2, CDC20, CEP55, TOP2A and UBE2C) predicted worse OS (Fig. 5I–M) and DFS rates (Fig. 5N–R).Fig. 5 Clinical significance of EZH2 and 4 hub genes. Expression of EZH2 is positively correlated with CDC20, CEP55, TOP2A and UBE2C in TCGA database (A–D) and GSE2748 datasets (E–H). Higher expression of the 5 genes (EZH2, CDC20, CEP55, TOP2A and UBE2C) predict worse OS (I–M) and DFS rates (N–R) in RCC

Discussion

Systemic therapy should be considered to treat local advanced and matastatic RCC. Several targeting drugs and immune checkpoint inhibitors (ICIs) were approved for the treatment of RCC, such as mechanistic target of rapamycin (mTOR) inhibitors, tyrosine kinase inhibitors (TKIs), and bevacizumab (a VEGF antibody) [2]. In China, traditional Chinese medicine (TCM) prescriptions are always be used to treat some diseases, including cancers. Ailanthus altissima, a kind of TCM, has been utilized as a traditional herbal medicine for the treatment of asthma, epilepsy, ascariasis, bleeding, and spermatorrhea diseases in China for more than 2000 years [16, 17]. Analysis of components in ailanthus altissima demonstrated more than 200 compounds, such as quassinoids, alkaloids, triterpenoids, phenylpropanoids and volatile oils [18]. In modern pharmacological studies, these isolated compounds have effects of anti-inflammatory, anti-tumor and insecticidal [16, 19–22]. However, the bioactivities have been explored only in a few components extracted from ailanthus altissima.

Ail is a quassinoid product isolated from Ailanthus altissima. Recent researches indicated Ail had pharmacological properties of anticancer and antiviral effects. The anticancer roles displayed as inhibition of cell proliferation, induction of cell apoptosis and sensitization of anticancer treatment. In hepatocellular carcinoma cell lines, Ail inhibited cell proliferation and caused G0/G1 cell cycle arrest with activation of DNA damage signaling pathway (ATM/ATR) and inhibition of cyclins D, E, and CDK2-, -4 and -6 [23]. In non-small cell lung cancer (NSCLC) cells, it was demonstrated that Ail could promote GAS5 expression by inhibiting UPF1-mediated nonsense-mediated mRNA decay, which then caused the repression cell autophagy mediated by ULK1 and subsequent inhibitory effects on NSCLC cells [21]. In another research, Ni et al. found Ali inhibited NSCLC cell proliferation and tumor growth in xenografted and orthotopic lung tumor models, through the repression of DNA replication via downregulating RPA1 [24]. In the combination therapy of a xenografted melanoma model [6], Ail promoted c-Jun degradation and inhibited c-Jun induced PD-L1 expression. What’s more, Treg differentiation was attenuated in tumor microenvironment (TME) after treatment with Ail. Altogether, Ail presented anticancer effect in multiple cancers with different working ways.

In the present research, we found Ail could inhibit RCC cell viability, induce cell apoptosis, and induce cycle arrest in the earlier stage of treatment. The proteomics analysis in cells treated with Ail showed an enrichment of EZH2 pathway, and 44 different expressed genes overlapped with EZH2 co-expressed genes. 4 of the 44 overlapped genes, were identified as key genes when compared with 6 hub genes [15] associated with RCC progression. EZH2 is the enzyme catalytic subunit, which can inhibit gene expression by trimethylating Lys at the 27th position of histone H3 (H3K27) [25, 26]. Previous studies have demonstrated EZH2 is overexpressed in RCC and can promote RCC development and metastasis partially via inhibition of large tumor suppressor kinase 1 (LATS1) [27, 28]. Abnormal increase of EZH2 in RCC can lead to suppression of some tumor-suppressor genes (such as E-cadherin, p27Kip1) and increases of some oncogenes (such as H3K27me3), and then promote cell proliferation, migration and inhibit cell apoptosis [29]. Suppression of EZH2 with siRNA or pharmaceutically can inhibit RCC growth and development both in vitro and vivo [30, 31]. In hepatocellular carcinoma, it is found that EZH2 mediating miR-139-5p silencing can inhibit cell via targeting TOP2A [32]. Our results showed the inhibition of EZH2 after RCC cell treated with Ail, and the molecular docking results had demonstrated a moderate to tight binding potential between Ail and EZH2, which indicated Ail might mediate the stability of EZH2. However, the mechanism how EZH2 regulating CDC20, CEP55 and UBE2C remains unclear, further research should focus on the relationship between EZH2 and the hub genes.

In summary, we clarified the anti-cancer role of Ail in RCC cells via cell viability, apoptosis and cycle analysis. The proteomics analysis and gene enrichment showed EZH2 pathway might mediate the anti-cancer role of Ail. It is also confirmed that Ail could inhibit expression of EZH2 and 4 hub genes associated with RCC progression, including CDC20, CEP55, TOP2A and UBE2C. The findings indicate Ail can repress RCC development partially via EZH2 and may be used as a novel anticancer drug.

Abbreviations

Ail Ailanthone

RCC Renal cell carcinoma

ccRCC Clear cell renal cell carcinoma

TMT Tandem mass tags

GSEA Gene Set Enrichment Analysis

CCTCC China Center for Type Culture Collection

CCK-8 Cell counting kit-8

FASP Filter-aided proteome preparation

HPLC High-Performance Liquid Chromatography

GEPIA Gene Expression Profiling Interactive Analysis

NES Normalized enrichment score

dep Different expressed proteins

CC Cellular component

MF Molecular function

BP Biological process

OS Overall survival

DFS Disease free survival

ICIs Immune checkpoint inhibitors

mTOR Mechanistic target of rapamycin

TKIs Tyrosine kinase inhibitors

TCM Traditional Chinese medicine

NSCLC Non-small cell lung cancer

Author contributions

JBZ: writing–original draft, investigation, methodology, funding acquisition. GD: writing–review and editing, investigation, software, data curation, formal analysis. TC: investigation, visualization. YBZ: writing–review and editing, investigation, methodology. YCZ: writing–review and editing, software, formal analysis. LX: writing–review and editing, methodology, visualization. LJ: writing–review and editing, validation, funding acquisition, resources. JZ: writing–review and editing, conceptualization, validation, visualization, project administration, validation, supervision, funding acquisition, resources.

Funding

This work was partially supported by the Suzhou Science & Technology Projects for People’s Livelihood (SKY2021031), the Open Project of Key Laboratory of Functional Genomics and Molecular Diagnosis of Gansu Province (2021BYGT-004), the Suzhou Medical Research Key Talents Program (GSWS2021016), the Urological Cancer Research Foundation of China (031) and the Natural Science Foundation of Jiangsu Higher Education Institutions (23KJB320015).

Data availability

The data is provided within the manuscript or supplementary information files, further inquiries can be directed to the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jianbing Zhu and Guangcheng Dai contributed equally to the manuscript.
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References

1. Siegel RL Miller KD Wagle NS Jemal A Cancer statistics, 2023 CA Cancer J Clin 2023 73 17 48 10.3322/caac.21763 36633525
Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin. 2023;73:17–48. 10.3322/caac.21763.36633525
2. Ljungberg B Albiges L Abu-Ghanem Y Bedke J Capitanio U Dabestani S European association of urology guidelines on renal cell carcinoma: the 2022 update Eur Urol 2022 82 399 410 10.1016/j.eururo.2022.03.006 35346519
Ljungberg B, Albiges L, Abu-Ghanem Y, Bedke J, Capitanio U, Dabestani S, et al. European association of urology guidelines on renal cell carcinoma: the 2022 update. Eur Urol. 2022;82:399–410. 10.1016/j.eururo.2022.03.006.35346519
3. Novacescu D Feciche BO Cumpanas AA Bardan R Rusmir AV Bitar YA Contemporary clinical definitions, differential diagnosis, and novel predictive tools for renal cell carcinoma Biomedicines 2022 10.3390/biomedicines10112926 36428491
Novacescu D, Feciche BO, Cumpanas AA, Bardan R, Rusmir AV, Bitar YA, et al. Contemporary clinical definitions, differential diagnosis, and novel predictive tools for renal cell carcinoma. Biomedicines. 2022. 10.3390/biomedicines10112926.36428491
4. Motzer RJ Jonasch E Agarwal N Alva A Baine M Beckermann K Kidney cancer, version 3.2022, NCCN clinical practice guidelines in oncology J Natl Compr Canc Netw 2022 20 71 90 10.6004/jnccn.2022.0001 34991070
Motzer RJ, Jonasch E, Agarwal N, Alva A, Baine M, Beckermann K, et al. Kidney cancer, version 3.2022, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2022;20:71–90. 10.6004/jnccn.2022.0001.34991070
5. Soares A Monteiro FSM Maluf FC Bastos DA Jardim DL Sasse AD Advanced renal cell carcinoma (RCC) management: an expert panel recommendation from the Latin American Cooperative Oncology Group (LACOG) and the Latin American Renal Cancer Group (LARCG) J Cancer Res Clin Oncol 2020 146 1829 1845 10.1007/s00432-020-03236-4 32410064
Soares A, Monteiro FSM, Maluf FC, Bastos DA, Jardim DL, Sasse AD, et al. Advanced renal cell carcinoma (RCC) management: an expert panel recommendation from the Latin American Cooperative Oncology Group (LACOG) and the Latin American Renal Cancer Group (LARCG). J Cancer Res Clin Oncol. 2020;146:1829–45. 10.1007/s00432-020-03236-4.32410064
6. Yu P Wei H Li K Zhu S Li J Chen C The traditional Chinese medicine monomer Ailanthone improves the therapeutic efficacy of anti-PD-L1 in melanoma cells by targeting c-Jun J Exp Clin Cancer Res 2022 41 346 10.1186/s13046-022-02559-z 36522774
Yu P, Wei H, Li K, Zhu S, Li J, Chen C, et al. The traditional Chinese medicine monomer Ailanthone improves the therapeutic efficacy of anti-PD-L1 in melanoma cells by targeting c-Jun. J Exp Clin Cancer Res. 2022;41:346. 10.1186/s13046-022-02559-z.36522774
7. Wang S Cui Q Chen X Zhu X Lin K Zheng Q Ailanthone inhibits cell proliferation in tongue squamous cell carcinoma via PI3K/AKT pathway Evid Based Complement Alternat Med 2022 2022 3859489 10.1155/2022/3859489 36387351
Wang S, Cui Q, Chen X, Zhu X, Lin K, Zheng Q, et al. Ailanthone inhibits cell proliferation in tongue squamous cell carcinoma via PI3K/AKT pathway. Evid Based Complement Alternat Med. 2022;2022:3859489. 10.1155/2022/3859489.36387351
8. Zhang Y Gong R Liu Y Sun X Liang J Zhou Y Ailanthone inhibits proliferation, migration and invasion of osteosarcoma cells by downregulating the serine biosynthetic pathway Front Oncol 2022 12 842406 10.3389/fonc.2022.842406 35186770
Zhang Y, Gong R, Liu Y, Sun X, Liang J, Zhou Y, et al. Ailanthone inhibits proliferation, migration and invasion of osteosarcoma cells by downregulating the serine biosynthetic pathway. Front Oncol. 2022;12:842406. 10.3389/fonc.2022.842406.35186770
9. Wang C Yi T Li X Cui J Li B Qin Y Ailanthone synergizes with PARP1 inhibitor in tumour growth inhibition through crosstalk of DNA repair pathways in gastric cancer J Cell Mol Med 2024 28 e18033 10.1111/jcmm.18033 38009603
Wang C, Yi T, Li X, Cui J, Li B, Qin Y, et al. Ailanthone synergizes with PARP1 inhibitor in tumour growth inhibition through crosstalk of DNA repair pathways in gastric cancer. J Cell Mol Med. 2024;28: e18033. 10.1111/jcmm.18033.38009603
10. Bailly C Anticancer properties and mechanism of action of the quassinoid ailanthone Phytother Res 2020 34 2203 2213 10.1002/ptr.6681 32239572
Bailly C. Anticancer properties and mechanism of action of the quassinoid ailanthone. Phytother Res. 2020;34:2203–13. 10.1002/ptr.6681.32239572
11. Ding H Yu X Hang C Gao K Lao X Jia Y Ailanthone: a novel potential drug for treating human cancer Oncol Lett 2020 20 1489 1503 10.3892/ol.2020.11710 32724391
Ding H, Yu X, Hang C, Gao K, Lao X, Jia Y, et al. Ailanthone: a novel potential drug for treating human cancer. Oncol Lett. 2020;20:1489–503. 10.3892/ol.2020.11710.32724391
12. Xu M Wang Y Zhou LN Xu LJ Jin ZC Yang DR The therapeutic value of SC66 in human renal cell carcinoma cells Cell Death Dis 2020 11 353 10.1038/s41419-020-2566-1 32393791
Xu M, Wang Y, Zhou LN, Xu LJ, Jin ZC, Yang DR, et al. The therapeutic value of SC66 in human renal cell carcinoma cells. Cell Death Dis. 2020;11:353. 10.1038/s41419-020-2566-1.32393791
13. Xu D Zhu X Ren J Huang S Xiao Z Jiang H Quantitative proteomic analysis of cervical cancer based on TMT-labeled quantitative proteomics J Proteomics 2022 252 104453 10.1016/j.jprot.2021.104453 34915198
Xu D, Zhu X, Ren J, Huang S, Xiao Z, Jiang H, et al. Quantitative proteomic analysis of cervical cancer based on TMT-labeled quantitative proteomics. J Proteomics. 2022;252: 104453. 10.1016/j.jprot.2021.104453.34915198
14. Zhu J Jin L Zhang A Gao P Dai G Xu M Coexpression analysis of the EZH2 gene using the cancer genome atlas and oncomine databases identifies coexpressed genes involved in biological networks in breast cancer, glioblastoma, and prostate cancer Med Sci Monit 2020 26 e922346 10.12659/msm.922346 32595202
Zhu J, Jin L, Zhang A, Gao P, Dai G, Xu M, et al. Coexpression analysis of the EZH2 gene using the cancer genome atlas and oncomine databases identifies coexpressed genes involved in biological networks in breast cancer, glioblastoma, and prostate cancer. Med Sci Monit. 2020;26: e922346. 10.12659/msm.922346.32595202
15. Yuan L Chen L Qian K Qian G Wu CL Wang X Co-expression network analysis identified six hub genes in association with progression and prognosis in human clear cell renal cell carcinoma (ccRCC) Genom Data 2017 14 132 140 10.1016/j.gdata.2017.10.006 29159069
Yuan L, Chen L, Qian K, Qian G, Wu CL, Wang X, et al. Co-expression network analysis identified six hub genes in association with progression and prognosis in human clear cell renal cell carcinoma (ccRCC). Genom Data. 2017;14:132–40. 10.1016/j.gdata.2017.10.006.29159069
16. Li X Li Y Ma S Zhao Q Wu J Duan L Traditional uses, phytochemistry, and pharmacology of Ailanthus altissima (Mill.) Swingle bark: a comprehensive review J Ethnopharmacol 2021 275 114121 10.1016/j.jep.2021.114121 33862103
Li X, Li Y, Ma S, Zhao Q, Wu J, Duan L, et al. Traditional uses, phytochemistry, and pharmacology of Ailanthus altissima (Mill.) Swingle bark: a comprehensive review. J Ethnopharmacol. 2021;275: 114121. 10.1016/j.jep.2021.114121.33862103
17. Kim SR Park Y Li M Kim YK Lee S Son SY Anti-inflammatory effect of Ailanthus altissima (Mill.) Swingle leaves in lipopolysaccharide-stimulated astrocytes J Ethnopharmacol 2022 286 114258 10.1016/j.jep.2021.114258 34271112
Kim SR, Park Y, Li M, Kim YK, Lee S, Son SY, et al. Anti-inflammatory effect of Ailanthus altissima (Mill.) Swingle leaves in lipopolysaccharide-stimulated astrocytes. J Ethnopharmacol. 2022;286: 114258. 10.1016/j.jep.2021.114258.34271112
18. Yan ZY Lv TM Wang YX Shi SC Chen JJ Bin L Terpenylated coumarins from the root bark of Ailanthus altissima (Mill.) Swingle Phytochemistry 2020 175 112361 10.1016/j.phytochem.2020.112361 32289598
Yan ZY, Lv TM, Wang YX, Shi SC, Chen JJ, Bin L, et al. Terpenylated coumarins from the root bark of Ailanthus altissima (Mill.) Swingle. Phytochemistry. 2020;175: 112361. 10.1016/j.phytochem.2020.112361.32289598
19. Cho SK Jeong M Jang DS Choi JH Anti-inflammatory effects of canthin-6-one alkaloids from Ailanthus altissima Planta Med 2018 84 527 535 10.1055/s-0043-123349 29179245
Cho SK, Jeong M, Jang DS, Choi JH. Anti-inflammatory effects of canthin-6-one alkaloids from Ailanthus altissima. Planta Med. 2018;84:527–35. 10.1055/s-0043-123349.29179245
20. Ma SB Liu L Li X Xie YH Shi XP Wang SW Virtual screening-molecular docking-activity evaluation of Ailanthus altissima (Mill.) swingle bark in the treatment of ulcerative colitis BMC Complement Med Ther 2023 23 197 10.1186/s12906-023-03991-0 37322476
Ma SB, Liu L, Li X, Xie YH, Shi XP, Wang SW. Virtual screening-molecular docking-activity evaluation of Ailanthus altissima (Mill.) swingle bark in the treatment of ulcerative colitis. BMC Complement Med Ther. 2023;23:197. 10.1186/s12906-023-03991-0.37322476
21. Fang C Wu W Ni Z Liu Y Luo J Zhou Y Ailanthone inhibits non-small cell lung cancer growth and metastasis through targeting UPF1/GAS5/ULK1 signaling pathway Phytomedicine 2023 128 155333 10.1016/j.phymed.2023.155333 38518633
Fang C, Wu W, Ni Z, Liu Y, Luo J, Zhou Y, et al. Ailanthone inhibits non-small cell lung cancer growth and metastasis through targeting UPF1/GAS5/ULK1 signaling pathway. Phytomedicine. 2023;128: 155333. 10.1016/j.phymed.2023.155333.38518633
22. Cucci MA Grattarola M Dianzani C Damia G Ricci F Roetto A Ailanthone increases oxidative stress in CDDP-resistant ovarian and bladder cancer cells by inhibiting of Nrf2 and YAP expression through a post-translational mechanism Free Radic Biol Med 2020 150 125 135 10.1016/j.freeradbiomed.2020.02.021 32101771
Cucci MA, Grattarola M, Dianzani C, Damia G, Ricci F, Roetto A, et al. Ailanthone increases oxidative stress in CDDP-resistant ovarian and bladder cancer cells by inhibiting of Nrf2 and YAP expression through a post-translational mechanism. Free Radic Biol Med. 2020;150:125–35. 10.1016/j.freeradbiomed.2020.02.021.32101771
23. Zhuo Z Hu J Yang X Chen M Lei X Deng L Ailanthone inhibits Huh7 cancer cell growth via cell cycle arrest and apoptosis in vitro and in vivo Sci Rep 2015 5 16185 10.1038/srep16185 26525771
Zhuo Z, Hu J, Yang X, Chen M, Lei X, Deng L, et al. Ailanthone inhibits Huh7 cancer cell growth via cell cycle arrest and apoptosis in vitro and in vivo. Sci Rep. 2015;5:16185. 10.1038/srep16185.26525771
24. Ni Z Yao C Zhu X Gong C Xu Z Wang L Ailanthone inhibits non-small cell lung cancer cell growth through repressing DNA replication via downregulating RPA1 Br J Cancer 2017 117 1621 1630 10.1038/bjc.2017.319 29024939
Ni Z, Yao C, Zhu X, Gong C, Xu Z, Wang L, et al. Ailanthone inhibits non-small cell lung cancer cell growth through repressing DNA replication via downregulating RPA1. Br J Cancer. 2017;117:1621–30. 10.1038/bjc.2017.319.29024939
25. Zeng J Zhang J Sun Y Wang J Ren C Banerjee S Targeting EZH2 for cancer therapy: from current progress to novel strategies Eur J Med Chem 2022 238 114419 10.1016/j.ejmech.2022.114419 35569264
Zeng J, Zhang J, Sun Y, Wang J, Ren C, Banerjee S, et al. Targeting EZH2 for cancer therapy: from current progress to novel strategies. Eur J Med Chem. 2022;238: 114419. 10.1016/j.ejmech.2022.114419.35569264
26. Xia J Li J Tian L Ren X Liu C Liang C Targeting enhancer of zeste homolog 2 for the treatment of hematological malignancies and solid tumors: candidate structure-activity relationships insights and evolution prospects J Med Chem 2022 65 7016 7043 10.1021/acs.jmedchem.2c00047 35531606
Xia J, Li J, Tian L, Ren X, Liu C, Liang C. Targeting enhancer of zeste homolog 2 for the treatment of hematological malignancies and solid tumors: candidate structure-activity relationships insights and evolution prospects. J Med Chem. 2022;65:7016–43. 10.1021/acs.jmedchem.2c00047.35531606
27. Hong SH Hwang HJ Son DH Kim ES Park SY Yoon YE Inhibition of EZH2 exerts antitumorigenic effects in renal cell carcinoma via LATS1 FEBS Open Bio 2023 13 4 724 735 10.1002/2211-5463.13579 36808829
Hong SH, Hwang HJ, Son DH, Kim ES, Park SY, Yoon YE. Inhibition of EZH2 exerts antitumorigenic effects in renal cell carcinoma via LATS1. FEBS Open Bio. 2023;13(4):724–35. 10.1002/2211-5463.13579.36808829
28. Xu S Ma B Feng X Yao C Jian Y Chen Y EZH2-regulated immune risk score prognostic model predicts outcome of clear cell renal cell carcinoma Transl Androl Urol 2023 12 71 82 10.21037/tau-22-817 36760869
Xu S, Ma B, Feng X, Yao C, Jian Y, Chen Y, et al. EZH2-regulated immune risk score prognostic model predicts outcome of clear cell renal cell carcinoma. Transl Androl Urol. 2023;12:71–82. 10.21037/tau-22-817.36760869
29. Li T Yu C Zhuang S Histone methyltransferase EZH2: a potential therapeutic target for kidney diseases Front Physiol 2021 12 640700 10.3389/fphys.2021.640700 33679454
Li T, Yu C, Zhuang S. Histone methyltransferase EZH2: a potential therapeutic target for kidney diseases. Front Physiol. 2021;12: 640700. 10.3389/fphys.2021.640700.33679454
30. Sun C Zhao C Li S Wang J Zhou Q Sun J EZH2 Expression is increased in BAP1-mutant renal clear cell carcinoma and is related to poor prognosis J Cancer 2018 9 3787 3796 10.7150/jca.26275 30405850
Sun C, Zhao C, Li S, Wang J, Zhou Q, Sun J, et al. EZH2 Expression is increased in BAP1-mutant renal clear cell carcinoma and is related to poor prognosis. J Cancer. 2018;9:3787–96. 10.7150/jca.26275.30405850
31. Liu L Xu Z Zhong L Wang H Jiang S Long Q Enhancer of zeste homolog 2 (EZH2) promotes tumour cell migration and invasion via epigenetic repression of E-cadherin in renal cell carcinoma BJU Int 2016 117 351 362 10.1111/bju.12702 24612432
Liu L, Xu Z, Zhong L, Wang H, Jiang S, Long Q, et al. Enhancer of zeste homolog 2 (EZH2) promotes tumour cell migration and invasion via epigenetic repression of E-cadherin in renal cell carcinoma. BJU Int. 2016;117:351–62. 10.1111/bju.12702.24612432
32. Wang K Jiang X Jiang Y Liu J Du Y Zhang Z Li Y Zhao X Li J Zhang R EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A J Exp Clin Cancer Res 2023 42 1 320 10.1186/s13046-023-02855-2 38008711
Wang K, Jiang X, Jiang Y, Liu J, Du Y, Zhang Z, Li Y, Zhao X, Li J, Zhang R. EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A. J Exp Clin Cancer Res. 2023;42(1):320. 10.1186/s13046-023-02855-2.38008711
