ABSTRACT
Background: Hepatitis B virus (HBV) infection is one of the major causes of chronic liver disease, which progresses from chronic hepatitis B (CHB) to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Early detection and laboratory-based screening of hepatocellular carcinoma are still major challenges. This study was undertaken to determine whether the cancer hallmark gene signatures that are released into circulation as circulating tumour DNA (ctDNA) can be used as a liquid biopsy marker for screening, early detection, and prognosis of HCC. Methods: A total of 130 subjects, including HBV-HCC (n = 80), HBV-cirrhotic and non-cirrhotic (n = 35), and healthy (n = 15) controls, were evaluated for TP53 and beta-catenin (CTNNB1) gene hotspot mutations in ctDNA by Sanger-based cycle sequencing and droplet digital PCR (ddPCR) assays. Mutation detection frequency, percentage mutant fractions, and their association with tumour stage, mortality, and smoking habits were determined. Results: Sanger-based cycle sequencing was carried out for 32 HCC patients. Predict SNP Tools analysis indicated several pathogenic driver mutations in the ctDNA sequence, which include p.D228N, p.C229R, p.H233R, p.Y234D, p.S240T, p.G245S, and p.R249M for TP53 gene exon 7 and p.S33T for CTNNB1 gene exon 3. The TP53 c.746G>T (p.R249M) mutation was detected predominately (25% cases) by sequencing, but there was no dominant mutation at position c.747G>T (p.R249S) that was reported for HBV-HCC patients. A dual-probe ddPCR assay was developed to determine mutant and wild-type copy numbers of TP53 (p.R249M and p.R249S) and CTNNB1 (p.S45P) and their percentage mutant fraction in all 130 subjects. The TP53 R249M and CTNNB1 S45P mutations were detected in 31.25% and 26.25% of HCC patients, respectively, with a high mutant-to-wild-type fraction percentage (1.81% and 1.73%), which is significant as compared to cirrhotic and non-cirrhotic patients. Poor survival was observed in HCC patients with combined TP53 and CTNNB1 gene driver mutations. The TP53 R249M mutation was also significantly (p < 0.0001) associated with smoking habits (OR, 11.77; 95% CI, 3.219-36.20), but not the same for the TP53 R249S mutation. Conclusion: Screening of ctDNA TP53 and CTNNB1 gene mutations by ddPCR may be helpful for early detection and identifying the risk of HCC progression.
ABSTRACT
Ultraviolet (UV) radiation-induced skin damage contributes strongly to the formation of melanoma, a highly lethal form of skin cancer. Quercetin (Qu), the most widely consumed dietary bioflavonoid and well known inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, has been reported to be chemopreventive in several forms of non-melanoma skin cancers. Here, we report that the treatment of ultraviolet (UV)-B-irradiated B16F10 melanoma cells with quercetin resulted in a dose dependent reduction in cell viability and increased apoptosis. The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM). Promotion of UVB-induced cell death by quercetin was further revealed by cleavage of chromosomal DNA, caspase activation, poly (ADP) ribose polymerase (PARP) cleavage, and an increase in sub-G1 cells. Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation. Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF). Overall, these results suggest the possibility of using quercetin in combination with UVB as a possible treatment option for melanoma in future.