Artesunate induces tumor cell cycle arrest in the G0/G1 phase by targeting cyclin-dependent kinase 4 (CDK4)
XiaosuZou1,2
QicongChen1
GangWang1
HonglinLuo1
ShenhongQu1✉Emailshqu@gxams.org.cn
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Institute of OncologyGuangxi Academy of Medical Sciences and the People’s Hospital of Guangxi Zhuang Autonomous Region6 Taoyuan Road530021NanningChina2Guangxi Key Laboratory of Eye HealthGuangxi Academy of Medical Sciences and the People’s Hospital of Guangxi Zhuang Autonomous Region530021NanningChina
Xiaosu Zou1,2, Qicong Chen1,Gang Wang1, Honglin Luo1, Shenhong Qu1*
1 Institute of Oncology, Guangxi Academy of Medical Sciences and the People's Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, China.
2 Guangxi Key Laboratory of Eye Health, Guangxi Academy of Medical Sciences and the People's Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, China.
*Corresponding author: Prof. Shenhong Qu, Institute of Oncology, Guangxi Academy of Medical Sciences and the People's Hospital of Guangxi Zhuang Autonomous Region, 6 Taoyuan Road, Nanning 530021, China.
Email: shqu@gxams.org.cn
Abstract
Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality worldwide, highlighting the urgent need for novel therapeutic targets. Cyclin-dependent kinase 4 (CDK4) plays a critical oncogenic role in NSCLC; however, current CDK4 inhibitors face limitations, including structural homogeneity and acquired resistance. Through HuProt™ human proteome microarray screening, we identified CDK4 as a direct cellular target of artesunate, which was validated by molecular docking, with a binding energy of -7.069 kcal/mol. Artesunate induced profound G0/G1 cell cycle arrest in both p53-wild (A549) and p53-deficient (H1299) NSCLC models via inhibition of the CDK4–Rb–E2F signaling axis, as demonstrated by the dose-dependent suppression of Rb phosphorylation at Ser780/Ser795. Collectively, these results establish artesunate as a structurally distinct CDK4 inhibitor scaffold, providing a strategic template for overcoming the limitations of aminopyrimidine-based kinase inhibitors.
Keywords:
Artesunate
Lung cancer
Human proteome microarray
Cell cycle arrest
CDK4
Introduction
Lung cancer remains the leading cause of cancer-related mortality worldwide, accounting for approximately 20% of all cancer deaths, with a dismal 5-year overall survival rate of 17.8% (1). Non-small cell lung cancer (NSCLC) constitutes 80–85% of lung cancer cases. CDK4/6 drives cell cycle progression via RB phosphorylation and shows broad therapeutic potential owing to its dual role in NSCLC and SCLC (2, 3). The CDK4/6–Rb–E2F pathway is hyperactivated in > 68% of adenocarcinoma cases (4, 5). The conserved role of CDK4/6 in cell cycle control across lung cancer subtypes supports its therapeutic relevance in NSCLC. Recent clinical evidence has demonstrated that CDK4/6 inhibitors significantly increase the efficacy of chemotherapy in treating lung cancer by reversing T-cell exhaustion and promoting antitumor immunity (6). CDK4 plays a critical cancer-driving role in NSCLC, making the development of effective targeted therapeutics against this protein a major research focus in NSCLC treatment.
Artemisinin derivatives offer structurally distinct alternatives as sesquiterpene lactones containing unique endoperoxide bridges. Numerous studies have shown that these compounds have excellent antitumor effects and should thus be a focus of research (7–9). Artesunate exhibits favorable pharmacokinetic properties, including high efficacy, rapid action, and low toxicity, and has been repurposed as a potential anticancer candidate (10, 11). Some experiments have shown that artesunate can inhibit the proliferation of lung cancer cells (12, 13) and induce colon cancer cell apoptosis (14). Furthermore, studies have shown that artesunate can inhibit the proliferation and metastasis of breast cancer cells by acting on the PI3K/Akt signaling pathway (15). Although numerous studies have investigated the mechanism of artesunate, these works have focused primarily on the pathways involved, and the direct binding target still needs to be investigated.
In the present study, CDK4 was identified as a primary direct cellular target protein of artesunate using HuProt™ human proteome microarray screening involving > 20,000 human proteins, with the highest binding signal intensity (Z score = 5.2) among the investigated targets. We verified the biological activity of artesunate in two types of NSCLC cells, namely, the A549 and H1299 cell lines. Notably, the H1299 cell line is a p53-deficient model. We found that artesunate suppressed the CDK4/6–Cyclin D1–Rb–E2F1 signaling axis, thereby inducing cell cycle arrest and inhibiting cell proliferation. Overall, we demonstrate that CDK4 is a promising pharmacological therapeutic target for lung cancer and that the natural small-molecule artesunate is a CDK4-targeted inhibitor of lung cancer.
Results
The artesunate–biotin probe retained the ability to inhibit cell proliferation
The chemical structure of the artesunate–biotin probe is shown in Fig. 1A. The inhibition of cancer cell proliferation was determined by a CCK8 assay. The results of the experiment showed that artemisinin and the artemisinin–biotin probe exerted comparable inhibitory effects on the proliferation of cancer cells. After 72 hours of treatment, the IC50 values of artemisinin for A549 and H1299 lung cancer cells were 0.2620 µM, 0.2626 µM, and 0.4127 µM, respectively. Similarly, the IC50 values of the artemisinin–biotin probe for A549 and H1299 cells were 0.2924 µM and 0.3820 µM, respectively. The results are shown in Fig. 1B.
Fig. 1
Chemical structures and equipotent antiproliferative activity of artesunate and the artesunate–biotin probe. (A) Chemical structures of artesunate and the artesunate–biotin probe. (B) Comparison of the antiproliferative effects of artesunate and the artesunate–biotin probe against NSCLC cells after 72 h of treatment (CCK8 assay, n = 3; mean ± SD).
Identification of artesunate targets
Protein microarray technology, a versatile platform for identifying target proteins of bioactive small molecules in an unbiased and high-throughput manner (16), was used to screen potential artesunate-binding proteins. We applied a HuProt™ human proteome microarray containing more than 20,000 human proteins to systematically identify artesunate-binding proteins. The artesunate–biotin probe was incubated with the human proteome microarray, and proteins with artesunate-binding capacity were identified via the addition of Cy5-Streptavidin (Cy5-SA). The experimental flow chart is shown in Fig. 2A. To avoid false-positive results, biotin was used as a negative control to exclude nonspecific ligand‒protein interactions.
Fig. 2
CDK4 is directly targeted by artesunate. (A) Schematic of artesunate target screening using a HuProt™ proteome microarray. (B) Venn diagram analysis. (C) CDK4 had the highest IMean_Ratio value. (D) Molecular docking analysis.
We calculated the Z score for each spot (17), and we selected the protein candidates whose Z scores were ≥ 2.8. The protein candidates detected for the biotin and artesunate–biotin groups were compared, and the results were visualized using a Venn diagram; a total of 122 proteins were identified as specifically binding to the artesunate–biotin probe (Fig. 2B). The IMean_Ratio values (IMean_Artesunate-Bio vs. IMean_Biotin) for all 122 proteins were determined. Our results revealed that CDK4 had the highest value (IMean_Ratio = 2.3707), indicating that CDK4 was selectively targeted by artesunate (Fig. 2C).
To further investigate the binding mode of artesunate with CDK4, we performed a docking simulation using AutoDock Vina. We found that the stereo conformation of artesunate fit well with the binding sites around TYR-180, THR-177, and TRP-179, and the binding energy was − 7.069 kcal/mol− 1 (Fig. 2D).
CDK4 is a direct target protein of artesunate
We next explored whether artesunate interacts with CDK4 to promote the proliferation of tumor cells through the CDK4–Cyclin D1–Rb–E2F1 pathway. Cell cycle assays revealed that artesunate induced G0/G1 phase arrest in all A549 and H1299 cells (Fig. 3A). We also found that artesunate decreased the protein expression levels of cyclin D1, p-RbThr821, Rb, p-E2F1S364 and E2F1 (Fig. 3B). These results clearly indicated that the ability of artesunate to promote tumor cell proliferation is dependent on the CDK4–Cyclin D1–Rb–E2F1 pathway.
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Fig. 3
Artesunate induces G0/G1 phase arrest and suppresses the CDK4–Cyclin D1–Rb–E2F1 pathway in NSCLC cells. (A) Flow cytometry analysis showing G0/G1 phase accumulation in A549 and H1299 cells after 48 h of treatment with different concentrations of artesunate. (B) Western blot analysis of key CDK4/6–Cyclin D1–Rb–E2F1 pathway proteins in treated A549 and H1299 cells. The data are presented as the mean ± SD. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t test.
Discussion
Through HuProt™ human proteome microarray screening, we identified CDK4 as a direct target of artesunate, with molecular docking simulations confirming a binding energy of -7.069 kcal/mol. Consistent G0/G1 phase arrest was observed in both A549 and H1299 NSCLC cells, accompanied by dose-dependent suppression of the CDK4/6–Cyclin D1–Rb–E2F1 signaling axis (Fig. 4).
Fig. 4
Schematic diagram showing the mechanism by which artesunate regulates the CDK4–Cyclin D1–Rb–E2F1 pathway.
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Current CDK4-targeted therapeutics face significant limitations, with all three clinically approved inhibitors (palbociclib, ribociclib, and abemaciclib) sharing an identical 2-aminopyrimidine core scaffold, resulting in structural homogeneity and potential cross-resistance (18–20). In contrast, artesunate features a sesquiterpene lactone scaffold with an endoperoxide bridge, and this non-aminopyrimidine scaffold not only circumvents resistance-conferring mutations through alternative binding modes but also enables multitarget engagement, which cannot be realized with current therapeutics. This structural diversification addresses a critical bottleneck in kinase drug development and positions artesunate as a pioneering template for the development of next-generation CDK4 inhibitors.Defective p53 is a common factor that makes it difficult to control cancer (21). As cell cycle inhibition operates via p53-independent mechanisms, artesunate is an effective treatment against p53-mutant tumors that are resistant to conventional CDK4/6 inhibitors such as palbociclib (22). In our study, artesunate induced G0/G1 phase arrest in both A549 and H1299 (p53-deficient model) cells and inhibited the downstream pathways of CDK4. These findings collectively demonstrate that artesunate exerts its antitumor effects via a p53-independent mechanism, as evidenced by conserved G0/G1 cell cycle arrest and CDK4/Rb pathway suppression in both p53-wild and p53-deficient NSCLC models. This p53-independent mechanism positions artesunate as a promising therapeutic for the > 60% of patients with NSCLC with TP53 mutations, as current targeted therapies frequently fail for these individuals (23, 24).
We identified CDK4 as a primary target of artesunate through proteome microarray and cellular approaches; however, there are three limitations that should be addressed. First, although molecular docking simulations predicted that artesunate competitively binds to CDK4 (-7.069 kcal/mol) and downstream p-RbThr821, Rb, and p-E2F1S364, these results strongly suggest kinase inhibition, and CDK4 enzymatic suppression was not directly validated. Second, the absence of in vivo data restricts the generalizability of these results to clinical settings. Third, the HuProt™ microarray and computational docking data require biochemical confirmation of direct binding.
To address these limitations, in future studies, we will conduct in vitro kinase assays using the CDK4/cyclin D1 complex and perform in vivo targeted tracking experiments using a patient-derived xenograft model with high CDK4 expression and an artesunate–biotin probe. Furthermore, surface plasmon resonance will be employed to assess the target-binding abilities of artesunate and CDK4. Although these experiments were not conducted in this study, these limitations do not diminish the immediate value of confirming artesunate as a non-amino pyrimidine-type CDK4 scaffold, which provides a practical solution to address the problem of drug resistance caused by p53 deficiency.
Materials and methods
Chemicals and reagents
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Artesunate-biotin was purchased from QI YUE Biology (Xian, China).Dulbecco’s modified Eagle medium (DMEM), Fetal bovine serum (FBS), antibiotics, and trypsin were from Gibco (Shanghai, China). Cell Counting Kit-8 (CCK8) was from DOJINDO (Shanghai, China). Cell Cycle and Apoptosis Analysis Kit and Propidium staining were from Beyotime (Shanghai, China). CDK4 polyclonal antibody (ab108357), Rabbit polyclonal to E2F1 (phospho S364) antibody, E2F1 rabbit polyclonal antibody (ab137415) ,and HRP Rabbit polyclonal to beta Tubulin antibody (ab21058) were from Abcam (Shanghai, China). Cyclin D1 Recombinant Rabbit Monoclonal Antibody (MA5-14512), Rb Recombinant Rabbit Monoclonal Antibody (MA5-32103), Phospho-Rb (Thr821) Rabbit Polyclonal Antibody (44-582G), Goat anti-Rabbit IgG (H + L) Secondary Antibody (31460), and Goat anti-Mouse IgG (H + L) Secondary Antibody (31430) were from Invitrogen (Shanghai, China). Polyvinylidene difluoride (PVDF) membranes were from Merck Millipore. SuperSignal West Femto Maximum Sensitivity Substrate were from Thermo Scientific (Shanghai, China).
Cell culture and Cell proliferation inhibition analysis
A549 and H1299 cells were obtained from ATCC (CRM-CCL-185, CRM-CRL-5803). Cells were cultured in high glucose DMEM supplement with 10% FBS, 100U/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator with 5% CO2 at 37℃.
CCK8 assay.
Cells were seeded in 96-well plates at a density of 3×103 cells/well. Plates were incubated at 37°C under 5% CO₂ for 12 hours to allow cell attachment. Cells were treated with artesunate or artesunate-biotin at designated concentrations.Treated cells were cultured for 72 hours under standard conditions (37°C, 5% CO₂). 10 µL of CCK-8 reagent was added to each well. After incubating for 1 hour, absorbance was measured at 450 nm using a microplate reader.
Identification of artesunate-binding proteins with human proteome microarray
HuProt™ human proteome microarray was from CDI company (Massachusetts, America). The proteome microarray was blocked with 5% BSA in PBS-T for 1.5 h at room temperature, followed by incubation with artesunate-biotin probe for 1 h at room temperature. After washing with PBS-T, the microarray was incubated with 0.1% Cy5-streptavidin for 20 min at room temperature. Signal detection was scanned with a GenePix 4000B microarray scanner (Axon Instruments, Silicon Valley, USA).
Molecular docking
Molecular docking was employed to validate the binding activity between the active ingredient and the key target. AutoDock Vina (Vina, version 1.1.2). The chemical structure of the small molecule Artesunate was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) for docking. The target protein structure of human CDK4 (PDB: 7SJ3) was retrieved from the Protein Data Bank (PDB). Molecular docking was performed using AutoDock Vina (version 1.5.7) (http://mgltools.scripps.edu/downloads). Interaction analysis and visualization from 3D and 2D perspectives were conducted using PyMOL (version 4.3.0) and Discovery Studio Visualizer (version 2021).
Cell cycle analysis
Cells were treated with artesunate at final concentrations of 1 µM, 2 µM and 4 µM for 48 hours, followed by trypsinization and washing with ice-cold PBS. The cell pellet was resuspended in pre-chilled 70% ethanol and fixed at 4°C for overnight. After fixation, the cells were washed with PBS and treated with RNase A (50 µg/mL) at 37°C for 30 min to remove RNA interference. The cells were then stained with PI (50 µg/mL) and incubated in the dark at room temperature for 30 minutes. The stained cell suspension was analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, USA) with an excitation wavelength of 488 nm and an emission wavelength of 617 nm. Data were analyzed using FlowJo software.
Western blot analysis
Cells were treated with artesunate at final concentrations of 1 µM, 2 µM and 4 µM for 48 hours, cells were lysed with RIPA supplemented with protease inhibitor. The lysates were then centrifuged at 12,000 rpm/min for 15 min at 4℃. Supernatants were collected for protein concentration detection with BCA protein assay kit. The proteins were separated using 8–15% SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk in 1 × Tris buffered saline with 0.1% Tween 20 for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4℃. Immunoreactive bands were detected by incubating with HRP-conjugated secondary antibodies at room temperature for 1 h. Signal detection was carried out by UVP GelStudio PLUS Touch (Analytik Jena Logo, Thuringia, Germany). The optical density of each band was analyzed by Image J software.
Statistical analysis
Data were represented as mean ± SD and analyzed by GraphPad Prism version 7.0 (GraphPad Software, La Jolla, CA, USA) using twotailed Student’s t-test for two group comparisons or one-way ANOVA with Bonferroni’s test for multiple comparisons. P < 0.05 was considered statistically significant.
Acknowledgements
This project was financially supported by the Xiaosu Zou for Research (Grant no 2023GXNSFBA026079, 82404696), Guangxi Youth Science Fund Project and National Natural Science Foundation of China
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Funding Statement
This research was supported by Guangxi Youth Science Fund Project (2023GXNSFBA026079), National Natural Science Foundation of China (NSFC, 82404696), Guangxi Key Research and Development Program (Guike AB25069036).
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Data Availability
The data generated in the present study may be requested from the corresponding author.
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Author Contribution
SQ conceived the study. XZ and QC performed the experiments, analyzed data and wrote and revised the manuscript. GW and HL analyzed and interpreted data. All authors have read and approved the final manuscript. SQ and XZ take responsibility for the integrity of data analysis. SQ and XZ confirm the authenticity of all the raw data.
Competing interests
The authors declare that they have no competing interests, and all authors should confirm its accuracy.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Electronic Supplementary Material
Below is the link to the electronic supplementary material
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