Molecular mechanism of modified Yigong San formula against colorectal cancer via EZH2/METTL3/SOX4 pathway-mediated apoptosis

Abstract

BACKGROUND

Colorectal cancer (CRC) is a malignant tumor characterized by high global incidence and mortality rates. Contemporary therapeutic modalities remain limited by suboptimal efficacy and adverse effects, thereby necessitating the pursuit of more efficacious treatment strategies. Within traditional Chinese medicine, spleen deficiency is regarded as a central pathogenic mechanism in CRC, persisting throughout the entire disease course.

AIM

To elucidate the mechanism by which modified Yigong San confers therapeutic efficacy against CRC, potentially exerting its effects through apoptosis regulation mediated by the enhancer of zeste homolog 2 (EZH2)/methyltransferase-like 3 (METTL3)/SRY-box transcription factor 4 (SOX4) axis.

METHODS

In the clinical study, CRC tissues and corresponding adjacent normal samples that fulfilled inclusion criteria were procured. Quantitative reverse transcription polymerase chain reaction was employed to determine the transcriptional expression of EZH2 and METTL3 mRNA. For in vitro experimentation, SW-480 cells were allocated into five experimental conditions: Control, control + serum, control + negative control, control + overexpressing-EZH2, and control + overexpressing-EZH2 + serum. The mRNA expression levels of EZH2, METTL3, SOX4, B-cell lymphoma 2, and Bax across groups were quantified via quantitative reverse transcription polymerase chain reaction, while protein levels were assessed using western blot analysis. The presence of EZH2 binding sites within the METTL3 promoter region was verified through chromatin immunoprecipitation polymerase chain reaction. The optimal concentration of drug-containing serum (5%, 10%, 15%) was determined using the Cell Counting Kit-8 assay. Cell migratory ability was evaluated via scratch assays, and apoptotic activity was quantified by flow cytometry.

RESULTS

The clinical findings demonstrated significantly elevated transcriptional levels of METTL3 and EZH2 mRNA in tumor tissues compared to their adjacent normal counterparts (P < 0.05). In vitro, cells treated with modified Yigong San exhibited a substantial downregulation of EZH2, METTL3, SOX4, B-cell lymphoma 2, and Bax mRNA and protein levels (P < 0.05), relative to the control group. Apoptotic rates were markedly increased, while migratory capacity was significantly attenuated. Furthermore, in EZH2-overexpressing cells treated with modified Yigong San, similar reductions in both mRNA and protein levels of the aforementioned targets were observed (P < 0.05), concomitant with enhanced apoptosis and reduced migration. Chromatin immunoprecipitation polymerase chain reaction analysis confirmed EZH2 occupancy at specific loci within the METTL3 promoter.

CONCLUSION

Modified Yigong San exhibits both preventive and therapeutic potential against CRC, likely mediated through the regulation of apoptosis via the EZH2/METTL3/SOX4 signaling pathway.

Keywords: Apoptosis; Colorectal cancer; Enhancer of zeste homolog 2/methyltransferase-like 3/SRY-box transcription factor 4 pathway; Yigong San; Cell proliferation; Traditional Chinese medicine

Core Tip: In this study, SW-480 cells were used as experimental subjects. Quantitative reverse transcription polymerase chain reaction, western blot, cell scratch assay, flow cytometry and other experimental methods were used to explore the expression of genes such as methyltransferase-like 3 (METTL3), enhancer of zeste homolog 2 (EZH2), SRY-box transcription factor 4 (SOX4), B-cell lymphoma 2, Bax and other genes as well as proteins, and to analyze that Isokongsan plus formula mediated the cellular apoptosis through EZH2/METTL3/SOX4 apoptosis against colorectal cancer through EZH2/METTL3/SOX4.