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Sorafenib (SF), a multi-kinase inhibitor, is the first FDA-approved systemic chemotherapy drug for advanced hepatocellular carcinoma (HCC). However, its clinical application is limited by severe toxicity and side effects associated with high applied doses. Sophora alopecuroides L. is traditionally used as Chinese herbal medicine for treating gastrointestinal diseases, bacillary dysentery, viral hepatitis, and other diseases, and exerts an important role in anti-tumor. Hence, we investigated the synergistic actions of seventeen flavonoids from this herb combined with SF against HCC cell lines and their primary mechanism. In the experiment, most compounds were found to prominently enhance the inhibitory effects of SF on HCC cells than their alone treatment. Among them, three compounds leachianone A (1), sophoraflavanone G (3), and trifolirhizin (17) exhibited significantly synergistic anticancer activities against MHCC97H cells at low concentration with IC 50 of SF reduced by 5.8-fold, 3.6-fold, and 3.5-fold corresponding their CI values of 0.49, 0.66, and 0.46 respectively. Importantly, compounds 3 or 17 combined with SF could synergistically induce MHCC97H cells apoptosis via the endogenously mitochondrial-mediated apoptotic pathway, involving higher Bax/Bcl-2 expressions with the activation of caspase-9 and -3, and arrest the cell cycle in G1 phases. Strikingly, this synergistic effect was also closely related to the co-suppression of ERK and AKT signaling pathways. Furthermore, compound 3 significantly enhanced the suppression of SF on tumor growth in the HepG2 xenograft model, with a 79.3% inhibition ratio at high concentration, without systemic toxicity, compared to either agent alone. These results demonstrate that the combination treatment of flavonoid 3 and SF at low doses exert synergistic anticancer effects on HCC cells in vitro and in vivo., (© 2022 John Wiley & Sons Ltd.)

In this study, 39 human hepatocellular carcinoma (HCC) tissues and 7 normal adult liver tissues were screened for heterozygous polymorphisms in IGF2, H19, and the differentially methylated region of H19 (H19DMR) using PCR-RFLP and PCR sequencing. The imprinting of IGF2 and H19 was examined by RT-PCR-RFLP, while the methylation profile of H19DMR was detected by bisulfite sequencing from every informative sample. Of the informative HCC samples 47.06% (8 of 17) demonstrated a gain of imprinting of IGF2, and 21.74% (5 of 23) of the informative HCC samples demonstrated a loss of imprinting of H19. Interestingly, we found three methylation profiles for H19DMR in the informative HCC samples: hyper-, medium-, and hypomethylated profiles. Furthermore, the hypomethylated and hypermethylated profiles were immediately associated with aberrant imprinting of IGF2 and H19.

Objective: To construct a combination method of methylation sensitive restriction enzyme and semi-nested touch down PCR assay for studying the promoter region methylation status of P16 gene in human hepatocellular carcinoma., Methods: According to the sequence of CpG rich promoter region of P16 gene, three primers were designed and synthesized for semi-nested touch down PCR assay to examine the promoter region methylation status of P16 gene. 340 bp segment of this region was cloned into vector pMD18-T; the plasmid was transformed into E. coli JM109 to harvest an extended quantity, then the plasmid was treated by CpG methylase M. Sss I, the methylated plasmid was named P16Pm+. This P16Pm+ is validated by digestion of Hpa II and is employed in studying the specificity and sensitivity of this constructed method. After construction, the method was used to examine the promoter region methylation status in P16 gene of 40 DNA samples from human HCCs and three DNA samples from normal human liver tissue., Results: It was confirmed that the specificity and sensitivity of this method are solid and reliable (100 fg). It was found that 12/40 (30%) of hepatocellular carcinoma showed promoter region methylation in P16 gene whereas none (0/3) of the normal tissues was methylated in the promoter region in P16 gene., Conclusion: Promoter region methylation in P16 gene may take part in human hepatocellular carcinogenesis. The constructed method is simple, cost-effective and is of high specificity and sensitivity, thus suggesting its potential application to detecting promoter methylation in population-based studies.

Objective: To investigate the allelic expression of IGF2/H19 in human hepatocellular carcinoma and to analyze the relationship between the imprint status of IGF2 and that of H19., Methods: Heterozygotes of IGF2 and of H19 were identified by restriction fragment length polymorphism (RFLP). Allelic expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and RFLP., Results: Forty seven point one percent (47.1%, 8 of 17) of HCC samples were demonstrated to have the gain of imprinting (GOI) of in IGF2 gene, and 45.5% (5 of 11) of HCC samples were found to have the loss of imprinting (LOI) for H19 gene. No relationship was observed between the imprinting status of IGF2 and that of H19., Conclusions: IGF2 GOI and H19 LOI are common in HCC, and the imprinting of IGF2 could be independent from that of H19 in adult liver.

Objective: To investigate the inhibition of tumor cell lines' growth and the demethylation of p16 gene by a component of natural drug, CDP., Methods: Human liver cancer cell line Huh-7 and breast cancer cell line T47D were treated with the CDP and DNA methyltransferase inhibitor, 5-azacytidine (5-aza-C). The inhibition of growth was assayed by MTT; the methylation status of p16 gene promoter was analyzed by MSP., Results: 1. The CpG islands in promoter of p16 gene were identified as completely methylated in Huh-7 and T47D. 2. CDP and 5-aza-C inhibited the proliferation of Huh-7 and T47D cell lines in a dose, time-dependent mode. 3. Methylation-specific bands of CpG islands in p16 gene' promoter were still existed in Huh-7 and T47D, while unmethylation-specific bands appeared in Huh-7 after the cells being treated with 25, 50, 75 and 100 micromol/ L of CDP for 6 days as well as in T47D after the cells being treated with 50, 75 and 100 micromol/L of CDP for 6 days. 4. After the cells being treated with 50 micromol/L of CDP, the methylation-specific bands of CpG island in p16 gene' promoter were still existed in Huh-7 and T47D; unmethylation-specific bands appeared at 12 h and lasted to 144 h in Huh-7 while at 72 h and lasted to 144 h in T47D., Conclusion: CDP has inhibition effect on the cancer cell lines and has the ability to reverse the hypermethylated p16 gene promoter. These results suggest that CDP has great potential in the development of effective anticancer drugs.

Objective: To detect the aberrant promoter CpG islands hypermethylation status of GSTP1 gene in hepatocellular carcinomas (HCC) and to assess its significant role in hepatocarcinogenesis., Methods: Surgically resected cancerous and non-cancerous liver tissues of 26 hepatocellular carcinoma patients were obtained from West China Hospital, and 11 peripheral blood samples from healthy donors as negative control were collected. Breast cancer cell line MCF-7 with CpG islands hypermethylation of GSTP1 as positive control was obtained from the Cell Bank of Chinese Academy of Sciences in Shanghai. All genomic DNA were extracted using common phenolchloroform approach, and the 5' CpG islands methylation status of GSTP1 gene was studied by methylation-specific polymerase chain reaction (MSP)., Results: GSTP1 gene promoter CpG island hyperthylation was detected in 88.5% (23/26) of cancerous tissues and in 69% (18/26) of corresponding non-cancerous tissues from the 26 HCC patients. None of the 11 control samples were methylation positive., Conclusion: The data indicates that the detection of GSTP1 gene methyl-lation may be a valuable biomarker by MSP for HCC early diagnosis and disease monitoring.

Objective: To study the imprinting status and expression level of insulin-like growth factor 2 (IGF2) gene in hepatocellular carcinoma and to provide a clue for elucidating the mechanism of carcinogenesis of hepatocellular carcinoma., Methods: The heterozygote status of IGF2 gene was detected by restriction fragment length polymorphism. The imprinting status and expression level of IGF2 were evaluated by semi-quantitative reverse transcription-polymerase chain reaction., Results: It was found that 10/18 (55.6%) of hepatocellular carcinoma showed the gain of imprinting (GOI), with 6/10 (60%) adjacent cirrhosis of liver tissues also displaying GOI of IGF2. Overexpression of IGF2 in cancer tissues was detected in 9/18 (50%) samples, but no significant difference was observed among each imprinting status., Conclusion: GOI of IGF2 may take part in human hepatocellular carcinogenesis.

Aim: To study the significance of p16 and p15 transcription suppression with hypermethylation of their genes' 5'CpG islands during human hepatocellular carcinogenesis., Methods: The mRNA expression levels of p16 and p15 genes were evaluated in cancerous, para-cancerous and non-cancerous tissues of 20 HCC, 3 normal liver tissues from 3 accidentally died healthy adults using semi-quantitatively Northern blot. The methylation status was also assessed with methylation specific PCR., Results: p16 mRNA expression level was decreased in the cancerous tissues in 60% (12/20) of HCC patients, of which 2 cases had no p16 mRNA detected, 5 cases (25%) displayed variation in the order of cancerous

Objective: Both tumor suppressor p16INK4A and p15INK4B are members of INK family of CDK inhibitor. Although the role of p16 has been well documented, the role of p15 and its signaling pathway remain less well studied. This study was aimed to assess the effect of p16 and p15 on hepatocarcinoma cell lines with different status of Rb gene., Methods: After identification of the genetic status of p16, p15 as well as Rb of human hepatocellular carcinoma (HCC) cell lines BEL7402, SMMC7721 with the use of multiple PCR, the eukaryotic expression p16 and p15 recombinants pXJ-p16 and pXJ-p15 were constructed, respectively. The existence of exogenous p16, p15 genes, and the expression of p16 and p15 were assayed by means of PCR and RNA dot blotting. Finally, the proliferation and apoptosis were studied by using MTT, colony formation assay and flow cytometry., Results: Neither deletion of p16 nor p15 was detected in the two cell lines. However, Rb exons 14-16 instead of exons 22-23 deletion existed in SMMC7721. The increased mRNA expression level of p16 was found in BEL7402-p16 and SMMC7721-p16, while increased mRNA expression level of p15 was found in BEL7402-p15. The endogenous p16 and p15 genes were transcripted at low level. The cell growth and colony formation were decreased in BEL7402-p15, compared with either mock cell BEL7402 or vector control cell BEL7402-pXJ. Also shown in this study were an altered G1 phase population from 37.7% to 43.6%, an S phase population from 22% to 13% (P<0.05), and a Sub G1 peak (apoptosis peak) in BEL7402-p15. Conversely, BEL7402-p16 with endogenous p16 gene showed neither difference in cell cycle population nor difference in colony formation rate, compared with control cell groups. Additionally, SMMC7721-p16 cell growth was not inhibited by exogenous p16 gene., Conclusion: p15 significantly arrested cell proliferation and induced apoptosis in BEL7402 in vitro, and the function was not influenced by endogenous p15 gene. The inhibition of cell growth by p16 on HCC cells could be dependent on intact RB pathway.

Southern blot studies on Hepatitis B Virus (HBV) DNA integration in 13 human hepatocellular carcinomas (HCCs) patients revealed the presence of several distinct HBV integration sites in different human liver disease patients. In one HCC patient the DNA fragment containing the HBV integration also hybridized to an erb B probe. The erb B/HBV co-migrating DNA fragment was cloned and sequenced, and showed that HBV DNA is integrated next to a cellular DNA fragment which is homologous to the tyrosine protein kinase domain of the human epidermal growth factor receptor gene and other cell surface receptor genes. The virus-integrated cellular DNA sequence is expressed in this HCC patient, suggesting a possible role for this gene in hepatocarcinogenesis.