The Traditional Chinese Medicine Formula Modified Wu Bao San Induces Cell Cycle Arrest and Apoptosis through Restoration of Retinoblastoma Protein Function and Inhibition of Aurora Kinase Activity

Abstract

Background: Traditional Chinese Medicine (TCM) has a long-standing history in treating various diseases, including cancer. Wu Bao San is a traditional formula known for its ability to clear heat, detoxify, relieve cough and phlegm and treat tumours. The modified Wu Bao San (MWBS) is a conventional medicine prescribed by doctors of Chinese medicine. However, the molecular mechanisms underlying the anticancer effects remain unclear owing to the complexity of the components of traditional Chinese medicine, making it challenging to pinpoint specific molecular targets or pathways underlying their clinical effect. Methods: In this study, we used an MTT assay, flow cytometry, indirect immunofluoresence imaging, and western blotting to analyse the viability, cell cycle profiles, morphology, and protein levels of cells after MWBSE treatment. Results: Our results showed that MWBSE has multiple anticancer effects. First, MWBSE induces cytokinesis failure by inhibiting Aurora kinase, leading cells to commit to cell death. Additionally, MWBSE reduces the phosphorylation of Rb, restoring its function and resulting in cell cycle arrest. The overexpression of Aurora kinases plays a pivotal role in sustaining malignant cell behaviors such as uncontrolled growth, colony formation, and tumor development. The retinoblastoma protein (Rb) is a key tumor suppressor that regulates the cell cycle, particularly the transition from the G1 to the S phase due to its significant involvement in tumor biology. Aurora kinases and Rb have emerged as promising targets for cancer therapy. Conclusion: Our findings provide a mechanistic explanation of the targets and anti-tumor pathways of MWBSE.

Keywords: Aurora kinases; Chia Wei Wu Pao San; Wu Bao San; apoptosis; cancer; cell cycle; retinoblastoma; traditional Chinese medicine.

Figure 1

MWBSE inhibits cell viability of three cancer cell lines. (A-C) The human carcinoma cell lines HeLa, HuH7, and MDA-MB-231 were treated with various concentrations of MWBSE (0, 12.5, 25, 50, 100, or 150 μg/mL). DMSO was used as a control. The cells were harvested at different time points for MTT assay. (D) The IC₅₀ values for MWBSE in the three cancer cell lines after 72 hours of treatment. The results were based on at least three independent experiments.

Figure 2

MWBSE induces cell cycle arrest in HeLa cells. (A) Flow cytometry analysis of the cell cycle distribution of HeLa cells treated with 100-300 μg/mL MWBSE for 24 hours, revealing shifts in cell cycle phases. (B) Western blot analysis showing a reduction in Cyclin B1 levels in MWBSE-treated HeLa cells, with GAPDH as the loading control. The western blot data represents averages from three independent experiments. The flow cytometry data represents averages from two independent experiments.

Figure 3

Induction of apoptosis by MWBSE in HeLa cells. (A) HeLa cells were treated with various concentrations of MWBSE for 24 hours, and apoptosis was assessed using the Annexin V: AbFluor™ 488/PI double staining assay via flow cytometry. (B) Western blot analysis showing increased cleavage of caspase-3, caspase-9, and PARP in response to MWBSE. Treatment with MWBSE resulted in increased cleavage of caspase-3, caspase-9, and PARP, indicating activation of the caspase-dependent apoptotic pathway. GAPDH served as the loading control. The western blot data were from three independent experiments. The data are presented as the means ± SEMs (*p < 0.05, **p < 0.01, ***p < 0.001 compared with controls).

Figure 4

MWBSE induces cytokinesis failure in HeLa cells. HeLa cells were treated with MWBSE (100-300 μg/mL) for 24 hours and then subjected to immunostaining. DAPI was used for DNA visualization, and anti-tubulin staining was used to highlight the microtubule structure. Yellow arrows indicate examples of multi-nuclei cells.

Figure 5

MWBSE inhibits aurora kinase phosphorylation in HeLa cells. HeLa cells were arrested in mitosis and treated with MWBSE for 24 hours. Western blot analysis shows total protein and phosphorylated (active) levels of Aurora kinases after treatment. The data were averaged from three independent experiments, with β-actin used as a loading control. The data are presented as the means ± SEMs (*p < 0.05, **p < 0.01, ***p < 0.001 compared with controls).

Figure 6

Reduction in Rb phosphorylation and increase in the Bax P18 protein by MWBSE. HeLa cells were treated with MWBSE for 24 hours. (A) Western blot showing reduced phosphorylation of Rb at the Ser807/811 and Ser795 sites. (B) Increased levels of the Bax P18 protein following MWBSE treatment. GAPDH and β-actin were used as loading controls. Data represent averages from three experiments, presented as the means ± SEMs (*p < 0.05, **p < 0.01, ***p < 0.001 compared with controls).

Figure 7

Proposed model of the cell death pathways induced by MWBSE. This schematic illustrates the proposed mechanisms through which MWBSE induces cell death, involving the inhibition of Aurora kinases, cytokinesis failure, cell cycle arrest, Rb dephosphorylation, and the activation of apoptosis pathways.