4.6 Pei's Ruan Gan Xiao Pi Wan Inhibits Mutant P53 Expression

4.6 Pei's Ruan Gan Xiao Pi Wan Inhibits Mutant P53 Expression

P53 protein was first discovered in 1979 in SV40-transformed cells and is currently the gene most closely associated with human tumors, as well as one of the most widely and deeply studied tumor suppressor genes. It is closely related to cell proliferation, differentiation, apoptosis, invasion, metastasis, and metabolism. Human P53 protein consists of 393 amino acids, approximately 16–20 kb in length, containing 11 exons and 10 introns, with mRNA measuring 2.8 kb and a molecular weight of about 53 kD, hence the name P53. P53 is located on chromosome 17p13.1 and structurally divided into the NH2-terminal transactivation domain, the central DNA-binding domain, the COOH-terminal tetramer polymerization domain, and the regulatory domain [103]. It participates in many cellular functions, such as cell cycle regulation, DNA repair, cell differentiation, genomic plasticity, and programmed cell death [104].

P53 is classified into two types: wild-type P53 (wt-P53) and mutant P53 (mt-P53). wt-P53 is considered the most important tumor suppressor gene during the process of hepatocellular carcinogenesis [4], maintaining genomic stability and inhibiting or preventing cellular transformation, thus earning the title of "guardian of the genome" or "molecular police." wt-P53 resides in the nucleus and is a nuclear-binding protein; under normal circumstances, P53 activity is very low. However, when DNA damage or hypoxia occurs, P53 is activated through three pathways: phosphorylation, acetylation, and ubiquitination. The body stabilizes P53 by disrupting the interaction between P53 and its negative regulator MDM2, thereby completing the phosphorylation process; the mechanisms of acetylation and ubiquitination regulating P53 remain incompletely understood. Once activated, P53 activity and protein levels rise rapidly, causing cyclin-dependent kinases (CDK) dependent on P53 to inhibit the upregulation of P21 and DNA repair genes (GADD45), resulting in cell growth arrest during the G1 phase and subsequent DNA repair. If repair is successful, the cell enters the S phase; if repair fails, the bax gene triggers apoptosis, ensuring genomic genetic stability. In contrast, mt-P53 cannot enter G1 arrest and undergo DNA repair via P53-mediated pathways after DNA damage, ultimately leading to uncontrolled cell proliferation and the development of malignant tumors [105].

Research has found that the P53 gene contains three main functional regions: ① the NH2-terminal transcription activation region, which can activate transcription and mediate protein-protein interactions, and can also bind to P53's negative regulatory factors; ② the central DNA core binding region, which has specific DNA-binding capabilities and is a hotspot for mutations in tumor cells; ③ the COOH-terminal tetramer polymerization and regulatory domain, a non-specific DNA-binding region that includes the nuclear localization signal (NLS) and nuclear export signal (NES). P53 has five highly conserved regions, among which amino acids numbered 132–143, 174–179, 236–248, and 272–281 are mutation hotspots, accounting for approximately 86.0% of all P53 gene mutations [106]. Mutant P53 not only loses its tumor-suppressive activity but may also accelerate tumor development by upregulating VEGF expression [107]. The half-life of wt-P53 in cells is about 20 minutes, whereas the half-life of mutated P53 protein can extend to 1.4–7 hours [108], making the protein more stable and capable of accumulating, thus allowing it to be detected by immunohistochemistry [109].

Research on Pei Zhengxue's series of prescriptions

Therefore, detecting P53 protein by immunohistochemistry is a method that can indicate whether P53 has undergone mutation, even more sensitive than gene sequence analysis. The results of this experiment show that the expression of mt-P53 in the treatment group decreased to varying degrees compared with the control group (p<0.05), indicating that Ruan Gan Xiao Pi Wan downregulates mt-P53 expression, possibly by inhibiting mt-P53's regulation of cell growth at the DNA replication level, thereby suppressing or blocking cell transcription and achieving an anti-cancer effect.

In summary, the following conclusions are drawn: Pei's Ruan Gan Xiao Pi Wan has a significant inhibitory effect on the growth of H22 hepatocellular carcinoma in mice; it increases the weight of immune organs—thymus and spleen—in tumor-bearing mice, thereby enhancing the body's non-specific immune function; and it significantly inhibits the expression of VEGF and wt-P53 in tumor tissues of tumor-bearing mice. This suggests that one of the mechanisms underlying Pei's Ruan Gan Xiao Pi Wan's anti-tumor effects may be achieved by downregulating the expression of VEGF and wt-P53 in tumor tissues.

Conclusion