Article

In Silico ERK Pathway Prediction and Experimental Validation of IB-DK143-Induced Apoptosis in MDA-MB-231 Breast Cancer Cells

Author 1, Author 2, Author 3 and Kang Pa Lee^4,*

Abstract

Background/Objectives: IB-DK143, an ibuprofen-linked chalcone derivative previously investigated in cervical cancer cells, has been associated with cytotoxicity, mitochondrial stress, p38 MAPK activation, and a decreasing tendency of ERK1/2 phosphorylation. In the present study, we extended this mechanistic concept to MDA-MB-231 breast cancer cells and investigated whether IB-DK143 induces ERK suppression-associated, caspase-dependent apoptosis.

Methods: To examine the potential interaction of IB-DK143 with ERK2/MAPK1 and its upstream regulator MEK1/MAP2K1, target prediction, molecular docking, and molecular dynamics simulation were performed. To experimentally validate the in silico findings, MDA-MB-231 breast cancer cells were treated with increasing concentrations of IB-DK143. Cytotoxicity was assessed using the WST-1 assay. Apoptosis-associated nuclear morphological changes were evaluated by Hoechst staining, including quantification of apoptosis-positive cells, mean fluorescence intensity, and nuclear area. Caspase-3 changes were analyzed as an apoptosis-related marker, and ERK1/2 expression and/or phosphorylation was evaluated as an early signaling readout of ERK pathway modulation.

Results: In silico analysis identified ERK2/MAPK1 and MEK1/MAP2K1 as candidate ERK pathway-associated targets of IB-DK143. Experimentally, IB-DK143-treated MDA-MB-231 cells showed concentration-dependent cytotoxicity and apoptosis-associated nuclear morphology, including increased apoptosis-positive nuclei, increased nuclear fluorescence intensity, and reduced nuclear area. Caspase-3-related changes were detected, and a decreasing trend in ERK1/2 signaling was also observed. Based on these findings and prior cervical cancer data, an in silico-guided working model was constructed in which IB-DK143 may suppress ERK-mediated survival signaling and promote caspase-dependent apoptosis.

Conclusions: The current findings support the feasibility of a combined computational and experimental study to test whether IB-DK143 induces ERK suppression-associated, caspase-3-dependent apoptosis in MDA-MB-231 breast cancer cells. Further validation using quantitative Western blotting, ERK rescue experiments, kinase activity assays, and final docking/MD simulation metrics is required before causal conclusions can be made.

Keywords

IB-DK143, MDA-MB-231, ERK1/2, MAPK; MEK1, caspase-3, apoptosis, molecular docking, molecular dynamics, breast cancer

Introduction

Breast cancer remains one of the most frequently diagnosed malignancies worldwide and continues to represent a major cause of cancer-related mortality in women. Although substantial progress has been made in early detection, molecular classification, and systemic therapy, treatment outcomes remain limited in aggressive subtypes. Triple-negative breast cancer (TNBC) is characterized by the lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. Because these tumors do not respond to endocrine therapy or HER2-targeted therapy, chemotherapy and emerging targeted or immunotherapeutic strategies remain central to clinical management. However, TNBC frequently exhibits high invasiveness, early recurrence, metastatic dissemination, and therapeutic resistance. Therefore, the identification of new chemical candidates and mechanism-based therapeutic strategies remains an important research priority.

MDA-MB-231 cells are widely used as an in vitro model of aggressive TNBC. This cell line displays mesenchymal-like features, high migratory and invasive behavior, and strong activation of multiple survival and stress-adaptation pathways. For this reason, MDA-MB-231 cells are frequently used to evaluate candidate anticancer compounds, apoptosis induction, survival signaling suppression, migration inhibition, and metastatic phenotypes. A compound that reduces MDA-MB-231 viability and induces apoptosis-associated nuclear and molecular changes may therefore provide a useful starting point for investigating TNBC-directed anticancer potential.

Chalcone derivatives have attracted attention as privileged scaffolds in medicinal chemistry and anticancer drug discovery. Their alpha,beta-unsaturated carbonyl system and diverse substitution patterns can support multiple biological activities, including antiproliferative effects, oxidative stress modulation, mitochondrial dysfunction, cell cycle arrest, and apoptosis induction. Several chalcone derivatives have been reported to regulate MAPK, PI3K/AKT, NF-kappaB, and mitochondrial apoptosis pathways. Despite this potential, many chalcone-based compounds are limited by solubility, bioavailability, and context-dependent biological activity. Structural modification and mechanistic validation are therefore required to improve their translational relevance.

IB-DK143 is an ibuprofen-linked chalcone derivative developed from a dual-function design concept combining the anti-inflammatory properties of ibuprofen with the pro-apoptotic potential of chalcone chemistry. In a previous cervical cancer cell study, an IB-DK143-related formulation showed enhanced cytotoxicity and was associated with reactive oxygen species generation, mitochondrial dysfunction, p38 MAPK activation, and a decreasing tendency of ERK1/2 phosphorylation. These findings suggested that IB-DK143-related compounds may shift intracellular signaling from survival-associated MAPK activity toward stress-associated apoptotic signaling. However, whether this mechanistic concept can be extended to breast cancer cells, particularly MDA-MB-231 cells, remains insufficiently characterized.

The RAF/MEK/ERK pathway is a central signaling cascade regulating cancer cell proliferation, survival, differentiation, migration, and therapeutic resistance. ERK1/2 activation is commonly maintained through upstream MEK1/2-mediated phosphorylation. In many cancer contexts, ERK-mediated signaling supports tumor cell survival and protects cells from apoptosis. Therefore, suppression of ERK1/2 phosphorylation or disruption of upstream MEK-mediated ERK activation may weaken cancer cell survival signaling and facilitate caspase-dependent apoptosis. ERK2/MAPK1 and MEK1/MAP2K1 are therefore biologically relevant targets for evaluating whether IB-DK143 may interfere with ERK-associated survival signaling.

In silico methods provide a rational framework for prioritizing molecular targets and guiding experimental validation. Target prediction can identify plausible protein candidates based on chemical structure and known ligand-target relationships. Molecular docking can estimate whether a compound fits into a protein binding pocket and identify possible stabilizing interactions. Molecular dynamics simulation can then evaluate whether the predicted ligand-protein complex remains stable over time under simulated conditions. Although these computational methods do not prove direct biochemical inhibition, they are useful for developing testable mechanistic hypotheses and selecting downstream validation experiments.

In the present study, we combined in silico ERK pathway prediction with experimental cytotoxicity and apoptosis analysis in MDA-MB-231 cells. We first evaluated whether IB-DK143 could interact with ERK2/MAPK1 and MEK1/MAP2K1 using target prediction, molecular docking, and molecular dynamics simulation. We then assessed IB-DK143-induced cytotoxicity using WST-1 assay and evaluated apoptosis-associated nuclear morphology by Hoechst staining. Caspase-3 changes and ERK1/2 expression and/or phosphorylation were analyzed as molecular readouts of apoptosis and ERK pathway modulation. We hypothesized that IB-DK143 induces apoptosis in MDA-MB-231 cells through suppression of ERK-mediated survival signaling and activation of caspase-dependent apoptotic pathways.

Materials and Methods

Chemical compound and in silico preparation

The IB-DK143 chemical structure was prepared for computational analysis using a three-dimensional ligand structure generated from the corresponding chemical information. Geometry optimization and file conversion were performed according to the selected docking pipeline. Final stereochemical configuration, protonation state, and energy-minimized structure should be confirmed before final submission.

Target prediction and candidate selection

Target prediction was performed to identify potential molecular targets of IB-DK143. Among the predicted targets, ERK2/MAPK1 and MEK1/MAP2K1 were selected for further analysis because they represent central components of the ERK pathway and were biologically consistent with the observed ERK1/2 decreasing trend. Additional pathway-related proteins may be included as secondary candidates if supported by the final prediction output.

Molecular docking and molecular dynamics simulation

Protein structures of ERK2/MAPK1 and MEK1/MAP2K1 were prepared by removing non-essential ligands and water molecules, adding hydrogens, assigning charges, and defining docking grids around the catalytic or inhibitor-binding pocket. Molecular docking was performed to estimate binding poses and interaction energies. The top-ranked complexes were further evaluated by molecular dynamics simulation. RMSD, RMSF, hydrogen-bond occupancy, radius of gyration, and binding free energy estimates were used as simulation readouts. Final docking score, PDB identifiers, MD simulation time, force field, solvent model, and MM-PBSA or MM-GBSA parameters should be inserted after completion of final analysis.

Cell culture and treatment

MDA-MB-231 breast cancer cells were selected as an in vitro TNBC model. Cells were maintained under standard mammalian cell culture conditions in complete culture medium supplemented with fetal bovine serum and antibiotics. Cells were treated with increasing concentrations of IB-DK143 for the indicated time periods. Vehicle-treated cells were used as controls. Exact seeding density, treatment duration, solvent concentration, and biological replicate number should be reported in the finalized Methods section.

WST-1 cytotoxicity assay

Cell viability was assessed using the WST-1 assay according to the manufacturer’s instructions. Briefly, MDA-MB-231 cells were seeded in 96-well plates and treated with IB-DK143 at increasing concentrations. After treatment, WST-1 reagent was added, and absorbance was measured using a microplate reader. Cell viability was calculated relative to the vehicle-treated control group. IC50 values should be calculated using a nonlinear regression model when complete dose-response data are available.

Hoechst staining and nuclear morphology analysis

Apoptosis-associated nuclear morphological changes were evaluated by Hoechst staining. After IB-DK143 treatment, cells were fixed, stained with Hoechst dye, and imaged using fluorescence microscopy. Apoptosis-positive nuclei were identified based on nuclear condensation, fragmentation, and increased fluorescence intensity. Mean nuclear fluorescence intensity and nuclear area were quantified using image analysis software. The preliminary figure contains a PVE label; this label should be corrected to IB-DK143 before submission if the dataset represents IB-DK143 treatment.

Caspase-3 and ERK1/2 analysis

Caspase-3 was analyzed as an apoptosis-related marker. ERK1/2 expression and/or phosphorylation was evaluated as an early signaling readout of ERK pathway modulation. For final validation, phospho-ERK1/2, total ERK1/2, cleaved caspase-3, cleaved PARP, and loading controls should be quantified by Western blotting. Time-course analysis is recommended to determine whether ERK1/2 reduction precedes caspase activation.

Statistical analysis

Data are presented as mean ± standard deviation unless otherwise indicated. Comparisons between two groups were performed using Student’s t-test, and multiple-group comparisons were performed using one-way ANOVA followed by appropriate post hoc testing. Statistical significance was defined as P < 0.05. Exact P values, replicate numbers, and statistical tests should be reported in figure legends.

Results

In silico analysis supports ERK pathway-associated candidate targeting by IB-DK143

Target prediction analysis identified ERK pathway-related proteins as candidate signaling nodes associated with IB-DK143. ERK2/MAPK1 and MEK1/MAP2K1 were prioritized because ERK1/2 is a major survival signaling module in cancer cells and because preliminary cellular analysis showed a decreasing trend in ERK1/2 signaling after IB-DK143 treatment. Molecular docking suggested that IB-DK143 may be accommodated within kinase-associated binding regions of ERK2/MAPK1 and/or MEK1/MAP2K1. Molecular dynamics simulation further supported the stability of the predicted ligand-protein complexes during the simulation period. These findings provide an in silico basis for investigating whether IB-DK143 may modulate ERK pathway signaling in MDA-MB-231 cells. Final docking scores, binding residues, RMSD, RMSF, hydrogen-bond occupancy, and MM-GBSA values should be inserted once the computational output is finalized.

IB-DK143 decreases MDA-MB-231 cell viability

To experimentally validate the in silico prediction, MDA-MB-231 cells were treated with increasing concentrations of IB-DK143, and cell viability was assessed using the WST-1 assay. IB-DK143 treatment resulted in a concentration-dependent reduction in cell viability compared with the vehicle-treated control group. This result indicates that IB-DK143 has cytotoxic activity against MDA-MB-231 breast cancer cells. When final numerical data are inserted, this section should include concentration range, treatment time, replicate number, percentage viability, and IC50 value.

IB-DK143 induces apoptosis-associated nuclear morphology

Hoechst staining was performed to determine whether IB-DK143-induced cytotoxicity was accompanied by apoptosis-associated nuclear alterations. Control cells displayed relatively intact and uniform nuclear morphology. In contrast, IB-DK143-treated cells exhibited nuclear condensation and increased fluorescence intensity. Quantitative analysis showed an increase in apoptosis-positive nuclei, an increase in mean nuclear fluorescence intensity, and a decrease in nuclear area. These changes are consistent with apoptotic nuclear condensation and shrinkage. The concentration-dependent pattern suggests that the cytotoxic effect of IB-DK143 is accompanied by apoptosis-like morphological changes.

IB-DK143 induces caspase-3-associated apoptotic response

Caspase-3 was evaluated as a representative executioner caspase. IB-DK143 treatment induced detectable caspase-3-associated changes, supporting the involvement of apoptosis-related molecular signaling. These findings are consistent with the Hoechst staining results showing apoptosis-associated nuclear morphology. Final confirmation should include cleaved caspase-3 and cleaved PARP, which would provide stronger evidence for caspase-dependent apoptosis.

IB-DK143 is associated with a decreasing trend in ERK1/2 signaling

Because computational analysis prioritized ERK2/MAPK1 and MEK1/MAP2K1, ERK1/2 expression and/or phosphorylation was evaluated as an early signaling readout. IB-DK143-treated cells showed a decreasing trend in ERK1/2 signaling. This observation is consistent with the proposed model that IB-DK143 may suppress ERK-mediated survival signaling. However, quantitative phospho-ERK1/2 and total ERK1/2 Western blotting is required to determine whether ERK suppression is statistically significant and whether it occurs before caspase activation.

Integrated in silico-guided working model

Based on the computational and experimental findings, an integrated working model was constructed. In this model, IB-DK143 may interact with ERK2/MAPK1 or MEK1/MAP2K1, resulting in suppression of ERK-mediated survival signaling. Reduced ERK signaling may weaken MDA-MB-231 cell survival and facilitate caspase-dependent apoptosis, leading to reduced viability, increased apoptosis-positive nuclei, increased nuclear fluorescence intensity, and reduced nuclear area. This model extends previous cervical cancer findings to a breast cancer context and provides a testable hypothesis for further mechanistic validation.

Discussion

This study provides a combined computational and experimental framework for investigating the anticancer effect of IB-DK143 in MDA-MB-231 breast cancer cells. The major findings are as follows. First, target prediction, molecular docking, and molecular dynamics simulation prioritized ERK2/MAPK1 and MEK1/MAP2K1 as candidate ERK pathway-associated proteins that may interact with IB-DK143. Second, IB-DK143 reduced MDA-MB-231 cell viability in a concentration-dependent manner. Third, Hoechst staining demonstrated apoptosis-associated nuclear morphological changes, including increased apoptosis-positive nuclei, increased nuclear fluorescence intensity, and reduced nuclear area. Fourth, caspase-3-associated changes were observed. Fifth, ERK1/2 signaling showed a decreasing trend. Together, these findings support the working hypothesis that IB-DK143 may induce ERK suppression-associated, caspase-dependent apoptosis in MDA-MB-231 cells.

The present study extends the mechanistic concept from previous cervical cancer work to a breast cancer model. In the earlier study, IB-DK143-related treatment was associated with cytotoxicity, ROS-mediated mitochondrial dysfunction, p38 MAPK activation, and a decreasing tendency of ERK1/2 phosphorylation. These findings suggested that IB-DK143-related compounds may shift MAPK signaling from a survival-associated state toward a stress- and apoptosis-associated state. The current study applies this concept to MDA-MB-231 cells and focuses on ERK pathway suppression as a candidate mechanism contributing to apoptosis.

ERK1/2 signaling is commonly involved in cancer cell proliferation, survival, migration, and resistance to apoptosis. In aggressive breast cancer cells, ERK pathway activity may support survival under stress conditions. Therefore, suppression of ERK signaling can reduce pro-survival signaling and increase susceptibility to apoptosis. The observed ERK1/2 decreasing trend in IB-DK143-treated MDA-MB-231 cells is consistent with this interpretation. However, ERK biology is context-dependent. ERK activation may support survival in some settings, but stress-dependent ERK activation may also contribute to cell death in other settings. Thus, the current data should be described as ERK suppression-associated apoptosis rather than direct ERK inhibition until additional evidence is obtained.

The in silico results provide a rational basis for further mechanistic validation. If IB-DK143 directly interacts with ERK2/MAPK1, it may alter ERK kinase function, substrate interaction, or conformational dynamics. If IB-DK143 preferentially interacts with MEK1/MAP2K1, it may reduce ERK1/2 phosphorylation indirectly through modulation of upstream kinase activity. These two possibilities are not mutually exclusive. Direct biochemical validation using kinase activity assays, cellular phospho-ERK quantification, and rescue experiments is required to distinguish these mechanisms.

The apoptosis-associated nuclear morphology observed in this study provides functional support for the cytotoxicity data. Hoechst staining showed increased apoptosis-positive nuclei, increased mean nuclear fluorescence intensity, and reduced nuclear area after IB-DK143 treatment. These features are consistent with chromatin condensation and nuclear shrinkage, which are characteristic morphological changes of apoptosis. The detection of caspase-3-associated changes further supports the involvement of apoptotic signaling. However, cleaved caspase-3, cleaved PARP, Bax/Bcl-2 ratio, and Annexin V/PI analysis should be included in future experiments to confirm the apoptotic pathway more rigorously.

A key mechanistic question is whether ERK1/2 reduction is an upstream cause of apoptosis or a downstream consequence of cell death. Time-course experiments are essential to address this issue. If p-ERK1/2 decreases at early time points before caspase-3 activation and nuclear condensation, ERK suppression would be more likely to function as an upstream signaling event. Conversely, if ERK1/2 reduction appears only after extensive apoptosis, it may represent a secondary consequence. Therefore, early time points such as 1, 3, and 6 h should be compared with later apoptosis-associated time points such as 12, 24, and 48 h.

Rescue experiments will be important for testing causal involvement of ERK signaling. EGF stimulation may be used to reactivate ERK1/2 signaling. If EGF restores p-ERK1/2 levels and partially reduces IB-DK143-induced caspase-3 activation or nuclear apoptosis, the functional involvement of ERK suppression would be strengthened. In parallel, known MEK or ERK inhibitors, such as U0126, trametinib, or ERK-selective inhibitors, may be used as positive controls to determine whether pharmacological ERK pathway inhibition produces a similar apoptotic pattern.

This study has several limitations. First, the in silico analysis is predictive and does not prove direct biochemical binding or inhibition. Second, exact docking scores and MD parameters should be reported in the final version. Third, ERK1/2 changes should be quantified using phospho-specific and total ERK antibodies. Fourth, caspase-3 results should be confirmed using cleaved caspase-3 and cleaved PARP. Fifth, comparison with normal breast epithelial cells, such as MCF10A, is necessary to determine cancer selectivity. Sixth, additional pathways, including AKT, STAT3, NF-kappaB, p38 MAPK, and ROS-mediated mitochondrial stress, may also contribute to IB-DK143-induced cytotoxicity and should be evaluated.

Despite these limitations, the present findings support a coherent and testable working model. IB-DK143 showed cytotoxic activity in MDA-MB-231 cells, induced apoptosis-associated nuclear changes, affected caspase-3-associated signaling, and was associated with a decreasing trend in ERK1/2 signaling. Combined with computational prediction of ERK2/MAPK1 and MEK1/MAP2K1 as candidate targets, these results justify further investigation of IB-DK143 as a potential breast cancer-directed anticancer candidate acting through ERK suppression-associated apoptosis.

Conclusion

This study suggests that IB-DK143 may induce apoptosis in MDA-MB-231 breast cancer cells through a mechanism associated with suppression of ERK1/2-mediated survival signaling and activation of caspase-dependent apoptotic responses. In silico target prediction, molecular docking, and molecular dynamics simulation identified ERK2/MAPK1 and MEK1/MAP2K1 as candidate ERK pathway-associated proteins. Experimentally, IB-DK143 reduced MDA-MB-231 cell viability, increased apoptosis-associated nuclear morphology, increased nuclear fluorescence intensity, reduced nuclear area, and induced caspase-3-associated changes.

These findings support an integrated computational and experimental model in which IB-DK143 may suppress ERK-mediated survival signaling and promote caspase-dependent apoptosis in MDA-MB-231 cells. Further validation using quantitative Western blotting, cleaved caspase-3 and cleaved PARP analysis, ERK rescue experiments, kinase activity assays, final docking/MD metrics, and comparison with normal breast epithelial cells is required before definitive causal conclusions can be made.

Table 1. Summary of experimental and computational workflow.

Figures

Additional Proposed Figure Legends

Required Information Before Final Submission

• Confirm the chemical identity and final name: IB-DK143 vs (S)-IB-DK143 vs (S)-IP-DK143.
• Correct the preliminary figure label from PVE to IB-DK143 if applicable.
• Insert exact WST-1 concentration range, treatment duration, replicate number, viability values, and IC50.
• Insert docking score, binding residues, PDB IDs, docking software, and MD simulation parameters.
• Quantify p-ERK1/2/total ERK1/2 and cleaved caspase-3/cleaved PARP by Western blotting.
• Add normal breast epithelial cell comparison such as MCF10A for selectivity evaluation.

Funding

This section should be completed according to the funding sources supporting the study.

Acknowledgments

The authors thank the members of UMUST R&D Corporation for technical discussion and manuscript preparation support.

Conflict of Interest

The authors declare no conflict of interest. This statement should be revised if any competing interests exist.

Author Contributions

Conceptualization, S.B. and K.P.L.; methodology, S.B.; formal analysis, S.B.; investigation, S.B. and K.P.L.; writing-original draft preparation, S.B.; writing-review and editing, K.P.L.; supervision, K.P.L. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

All experimental and computational datasets should be deposited or described before submission. Docking input files, MD simulation parameters, raw WST-1 data, and image analysis files should be made available upon reasonable request or through an appropriate repository.

References

1. Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209-249.
2. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin. 2023;73:17-48.
3. Foulkes WD, Smith IE, Reis-Filho JS. Triple-negative breast cancer. N Engl J Med. 2010;363:1938-1948.
4. Dent R, Trudeau M, Pritchard KI, et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007;13:4429-4434.
5. Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol. 2016;13:674-690.
6. Lehmann BD, Bauer JA, Chen X, et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011;121:2750-2767.
7. Chavez KJ, Garimella SV, Lipkowitz S. Triple negative breast cancer cell lines: one tool in the search for better treatment of triple negative breast cancer. Breast Dis. 2010;32:35-48.
8. Holliday DL, Speirs V. Choosing the right cell line for breast cancer research. Breast Cancer Res. 2011;13:215.
9. Hanahan D. Hallmarks of cancer: new dimensions. Cancer Discov. 2022;12:31-46.
10. Ouyang Y, Li J, Chen X, Fu X, Sun S, Wu Q. Chalcone derivatives: role in anticancer therapy. Biomolecules. 2021;11:894.
11. Zhuang C, Zhang W, Sheng C, Zhang W, Xing C, Miao Z. Chalcone: a privileged structure in medicinal chemistry. Chem Rev. 2017;117:7762-7810.
12. Michalkova R, Mirossay L, Gazdova M, Kello M, Mojzis J. Molecular mechanisms of antiproliferative effects of natural chalcones. Cancers. 2021;13:2730.
13. Karthikeyan C, Moorthy NSHN, Ramasamy S, et al. Advances in chalcones with anticancer activities. Recent Pat Anticancer Drug Discov. 2015;10:97-115.
14. Go ML, Wu X, Liu XL. Chalcones: an update on cytotoxic and chemoprotective properties. Curr Med Chem. 2005;12:483-499.
15. Baek S, Yoon CW, Lee KP, et al. Enhanced anticancer activity of ibuprofen and 2-hydroxy-2,3,5-trimethoxychalcone-linked polymeric micelles in HeLa cells. ACS Omega. 2026;11:11351-11359.
16. Raman M, Chen W, Cobb MH. Differential regulation and properties of MAPKs. Oncogene. 2007;26:3100-3112.
17. Guo YJ, Pan WW, Liu SB, Shen ZF, Xu Y, Hu LL. ERK/MAPK signalling pathway and tumorigenesis. Exp Ther Med. 2020;19:1997-2007.
18. Sugiura R, Satoh R, Takasaki T. ERK: a double-edged sword in cancer. Cells. 2021;10:2509.
19. Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene. 2007;26:3291-3310.
20. Sebolt-Leopold JS, Herrera R. Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat Rev Cancer. 2004;4:937-947.
21. Dhillon AS, Hagan S, Rath O, Kolch W. MAP kinase signalling pathways in cancer. Oncogene. 2007;26:3279-3290.
22. Pearson G, Robinson F, Beers Gibson T, et al. Mitogen-activated protein kinase pathways: regulation and physiological functions. Endocr Rev. 2001;22:153-183.
23. Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov. 2009;8:579-591.
24. Schumacker PT. Reactive oxygen species in cancer: a dance with the devil. Cancer Cell. 2015;27:156-157.
25. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35:495-516.
26. Porter AG, Janicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 1999;6:99-104.
27. Degterev A, Boyce M, Yuan J. A decade of caspases. Oncogene. 2003;22:8543-8567.
28. Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 1993;53:3976-3985.
29. Ferreira LG, Dos Santos RN, Oliva G, Andricopulo AD. Molecular docking and structure-based drug design strategies. Molecules. 2015;20:13384-13421.
30. Hollingsworth SA, Dror RO. Molecular dynamics simulation for all. Neuron. 2018;99:1129-1143.