3.1 Causes and Pathogenesis of Liver Cancer

3.1 Causes and Pathogenesis of Liver Cancer

The development and progression of tumors is a complex, multi-factorial, multi-stage process, potentially related to the following factors:

3.1.1 Viral Hepatitis

In China, chronic viral hepatitis, especially hepatitis B, is the most significant risk factor for primary liver cancer. Among liver cancer patients, the HBsAg positivity rate ranges from 60% to 80%, while the positive rate for hepatitis B virus (HBV) markers in liver cancer tissues exceeds 80%[20–24]. The incidence of PLC is significantly lower among individuals vaccinated against hepatitis B, indicating a close association between HBV and the high prevalence of liver cancer. It has now been confirmed that the HBx gene is the most closely related oncogene to liver cancer development; its encoded product, HBxAg, can specifically bind to the P53 protein, causing P53 mutation and loss of its normal function in inducing apoptosis. In Western countries, the main causes of liver cancer are hepatitis C and alcohol consumption.

3.1.2 Aflatoxin

The metabolite of aflatoxin, aflatoxin B (AFB1), exhibits strong carcinogenic properties. After human ingestion, AFB1 is converted in the liver into aflatoxin M (AFM), which is less toxic and less carcinogenic, and then excreted in urine. Since the liver is the body's primary detoxification organ, prolonged and continuous intake of AFB1 can lead to constant stimulation and damage to the liver, resulting in a high incidence of liver cancer[25]. Currently, it is widely believed that aflatoxin is closely associated with mutations in the tumor suppressor gene P53. Studies have also shown that regions with higher consumption of moldy foods often detect P53 gene mutations in liver cancer patients, with the main mutation sites being positions 249 and 254[26].

3.1.3 Drinking Water and Liver Cancer

According to reports from Qidong, Jiangsu Province—a high-incidence area for liver cancer in China—residents who drink pond water have a significantly higher incidence of liver cancer (60–101 per 100,000) compared to those who drink well water (0–19 per 100,000). The reason may be that microcystin produced by blue-green algae growing in ponds promotes point mutations in exon 8 of the P53 gene, causing it to lose its positive regulatory role in apoptosis and thus leading to the development of liver cancer[19,27].

Research on Pei Zhengxue’s series of formulas and medicines

3.1.4 Alcohol Consumption and Smoking

Studies have found that smoking and drinking are high-risk factors for liver cancer and exhibit synergistic carcinogenic effects when combined with HBV infection[28].

Epidemiological surveys indicate a clear positive correlation between the amount of alcohol consumed and smoking and the incidence of liver cancer[29,30]. Alcohol can induce liver cancer through two pathways: first, long-term accumulation of ethanol in the body can activate various carcinogenic factors, ultimately leading to liver cancer via cirrhosis; second, ethanol itself possesses carcinogenic and mutagenic properties, exacerbating liver damage, impairing the immune system, and synergizing with other liver cancer risk factors such as HBV and HCV to promote carcinogenesis. Chemical substances in tobacco, such as nicotine and nitrosamines, can directly damage the liver, causing hepatic lobule necrosis, pseudolobule hyperplasia, and ultimately liver cancer.

3.1.5 Genetic Factors

The incidence of liver cancer varies across different ethnic groups. Epidemiological studies show that liver cancer exhibits obvious familial clustering and genetic susceptibility. Li Suiping et al.[1] conducted a study on the heritability of liver cancer in Taixing City and found a heritability rate of 35.74%, with males showing a significantly lower rate than females, indicating that genetic factors are one of the risk factors for liver cancer. Meanwhile, Meng Wei et al.'s research suggests that the occurrence of liver cancer is influenced by a combination of genetic and environmental factors.

3.1.6 Other Factors

Certain chemicals, such as nitrosamines, azo mustard compounds, and organochlorine pesticides, are all suspected carcinogens for liver cancer. Infection with Clonorchis sinensis in small bile ducts can stimulate proliferation of bile duct epithelium, serving as one of the causes of primary cholangiocarcinoma[19].

3.2 VEGF, P53, and Liver Cancer

Vascular endothelial growth factor (VEGF) is a key regulator of liver cancer growth and metastasis, playing an important role in tumor growth, invasion, metastasis, and promotion of angiogenesis in liver cancer[3]. As early as 1989, Ferrara et al.[4] isolated, purified, and sequenced VEGF from bovine pituitary cells, identifying it as a factor promoting endothelial cell division. Subsequent studies revealed that VEGF can be produced by both normal and tumor cells, making it the most important among over 30 pro-angiogenic factors, with high VEGF expression detected in many human tumors[35,36].

VEGF is currently recognized as the most potent and specific cytokine promoting vascular endothelial growth, considered a specific source of endothelial cell division and angiogenesis within the body. Its mechanism of stimulating blood vessel formation includes: ① binding to the corresponding VEGF receptor and immediate phosphorylation, activating phosphatase C and phosphatidylinositol-3-kinase; these two enzymes serve as signal couplers for KDR and flk-1, further enhancing their binding to the receptor and exerting positive regulation on endothelial cells; ② effectively promoting Ca²⁺ influx, which directly stimulates mitosis of vascular endothelial cells; ③ significantly enhancing the activity of plasminogen activator (PA), raising PA and its inhibitor-1 (PAI-1) mRNA levels, regulating urokinase-type plasminogen activator (UPA) expression, degrading extracellular matrix, facilitating angiogenesis; ④ increasing vascular permeability by activating intracellular vesicles (VVO) in endothelial cells, allowing plasma proteins and other large molecules to leak out of vessels, with fibrin forming a temporary matrix outside the vessels. This matrix not only provides a fibrous network for blood vessel formation but also encourages some interstitial cells to further develop mature vascular matrices, thereby inducing angiogenesis[37].

The regulation of VEGF expression is quite complex, mainly related to hypoxia, P53 mutation, increased estrogen levels, NO stimulation, mTOR activation, initiation of liver cancer cell cycle, endocrine hormone disorders, renin-angiotensin-converting enzyme activation, and MAPK activation. Marschall et al.[8] believe that as liver cancer progresses and the portal vein can no longer supply the tumor with necessary nutrients and oxygen, hypoxia-inducible factor-1 (HIF-1) binds to the hypoxic enhancer sequence in liver cancer cells, initiating and strengthening VEGF gene transcription, upregulating VEGF mRNA expression and enhancing its stability, thereby synthesizing large amounts of VEGF to promote new blood vessel formation. P53 mutation is also an important factor promoting angiogenesis[9], and wild-type P53 inhibits VEGF promoter activity in a dose-dependent manner[40]. However, after P53 mutation, it loses its tumor-suppressing function and can even upregulate VEGF expression to induce angiogenesis[41].

The P53 gene is currently the most extensively studied and highly correlated gene with human tumors, closely linked to cell proliferation, differentiation, apoptosis, invasion, metastasis, and metabolism[42]. Under normal circumstances, P53 activity is very low. When the body encounters strong stimuli causing DNA damage, P53 is activated through three pathways: phosphorylation, acetylation, and ubiquitination. The body completes phosphorylation by disrupting the interaction between P53 and its negative regulator MDM2, stabilizing P53; however, the mechanisms of acetylation and ubiquitination remain incompletely understood. Once activated, P53's activity and protein levels rise rapidly, producing different responses depending on stress signals and tissue/cell types. Any disruption in these regulatory steps can cause genomic damage, leading to liver cancer.

Activated P53 is an active transcription factor that regulates gene expression by binding to the regulatory regions of downstream target genes. These target genes include those involved in cell cycle control, apoptosis, differentiation, DNA repair, vascular regeneration, oxidative stress, chemotaxis, and immune surveillance. When cells suffer toxic damage, P53 accumulates to halt cell proliferation, initiates and participates in DNA repair, and if repair fails, activates apoptosis through both intracellular and extracellular pathways, preventing the damage from being passed on to daughter cells and maintaining genomic stability[43,44]. Conversely, if P53's structure or function is abnormal and unable to effectively regulate downstream genes' responses to genotoxic damage, it may lead to cellular transformation into cancer.

P53 is divided into two types: wild-type P53 (wt-P53) and mutant P53 (mt-P53). wt-P53 is considered the most important tumor-suppressing gene during hepatocellular carcinogenesis[45], playing a crucial role in DNA repair, transcription, cell growth, proliferation, apoptosis, and many other metabolic processes. Hence, it is hailed as the guardian of the genome or the molecular police. However, once mutated, P53 loses the aforementioned functions. mt-P53 allows cells to bypass the G1/S checkpoint of the cell cycle, resulting in continuous and rapid proliferation and resistance to apoptosis, which plays a key role in the development and progression of liver cancer. Possible mechanisms include: P53²⁴⁹ mutation located in the P53 DNA-binding domain, directly affecting P53's function as a transcription factor; wt-P53 inhibits IGF-II transcription, whereas mt-P53 greatly enhances IGF-II transcription activity, leading to continuous hepatocyte proliferation; due to P53 mutation, cell cycle-related genes such as CKI (P21, Waf1, P27) and apoptosis-related genes like BAX and BCL-X are significantly downregulated, halting hepatocyte proliferation and preventing apoptosis; moreover, mutated P53 genes, on the one hand, acquire oncogenic functions leading to cellular transformation into cancer, while on the other hand, upregulate growth factor and receptor gene expression to promote cell proliferation, including PCNA, MDR, EGFR, VEGF, IGF-IR, bFGF, C-myc, and C-fos; P53 mutation can also affect the function of other signaling pathways[46–49].

Numerous experiments have proven that wt-P53 can inhibit VEGF expression[50,51], and the expression levels of the two are significantly positively correlated. The mechanism may involve mt-P53 downregulating thrombospondin (TSP) expression or enhancing protease C activity, thereby increasing tumor VEGF expression[40]. Therefore, the synergistic action of VEGF and mt-P53 not only leads to the development of liver cancer but also promotes its invasion and metastasis.

3.3 Treatment of Liver Cancer