H4K20me3 upregulated by reactive oxygen species is associated with tumor progression and poor prognosis in patients with hepatocellular carcinoma.

Epigenetic alteration by oxidative stress is vitally involved in carcinogenesis and cancer progression. Previously, we demonstrated that oxidative stress was increased in hepatocellular carcinoma (HCC) patients and associated with tumor aggressiveness. Herein, we immunohistochemically investigated whether histone methylation, specifically H4K20me3, was upregulated in human hepatic tissues obtained from HCC patients (n = 100). Also, we experimentally explored if the H4K20me3 was upregulated by reactive oxygen species (ROS) and contributed to tumor progression in HCC cell lines. We found that H4K20me3 level was increased in HCC tissues compared with the adjacent noncancerous liver tissues. H3K9me3 and H3K4me3 levels were also increased in HCC tissues. Cox regression analysis revealed that the elevated H4K20me3 level was associated with tumor recurrence and short survival in HCC patients. Experimentally, H2O2 provoked oxidative stress and induced H4K20me3 formation in HepG2 and Huh7 cells. Transcript expression of histone methyltransferase Suv420h2 (for H4K20me3), Suv39h1 (for H3K9me3), and Smyd3 (for H3K4me3) were upregulated in H2O2-treated HCC cells. H2O2 also induced epithelial-mesenchymal transition (EMT) in HCC cells, indicated by decreased E-cadherin but increased α-SMA and MMP-9 mRNA expression. Migration, invasion, and colony formation in HCC cells were markedly increased following the H2O2 exposure. Inhibition of H4K20me3 formation by A196 (a selective inhibitor of Suv420h2) attenuated EMT and reduced tumor migration in H2O2-treated HCC cells. In conclusion, we demonstrated for the first time that H4K20me3 level was increased in human HCC tissues, and it was independently associated with poor prognosis in HCC patients. ROS upregulated H4K20me3 formation, induced mRNA expression of EMT markers, and promoted tumor progression in human HCC cells. Inhibition of H4K20me3 formation reduced EMT and tumor aggressive phenotypes in ROS-treated HCC cells. Possibly, ROS-induced EMT and tumor progression in HCC cells was epigenetically mediated through an increased formation of repressive chromatin H4K20me3.

HydroZitLa inhibits calcium oxalate stone formation in nephrolithic rats and promotes longevity in nematode Caenorhabditis elegans.

Low fluid intake, low urinary citrate excretion, and high oxidative stress are main causative factors of calcium oxalate (CaOx) nephrolithiasis. HydroZitLa contains citrate and natural antioxidants and is developed to correct these three factors simultaneously. Antioxidants theoretically can prolong the lifespan of organisms. In this study, we preclinically investigated the antilithogenic, lifespan-extending and anti-aging effects of HydroZitLa in HK-2 cells, male Wistar rats, and Caenorhabditis elegans. HydroZitLa significantly inhibited CaOx crystal aggregation in vitro and reduced oxidative stress in HK-2 cells challenged with lithogenic factors. For experimental nephrolithiasis, rats were divided into four groups: ethylene glycol (EG), EG + HydroZitLa, EG + Uralyt-U, and untreated control. CaOx deposits in kidneys of EG + HydroZitLa and EG + Uralyt-U rats were significantly lower than those of EG rats. Intrarenal expression of 4-hydroxynonenal in EG + HydroZitLa rats was significantly lower than that of EG rats. The urinary oxalate levels of EG + HydroZitLa and EG + Uralyt-U rats were significantly lower than those of EG rats. The urinary citrate levels of EG + HydroZitLa and EG + Uralyt-U rats were restored to the level in normal control rats. In C. elegans, HydroZitLa supplementation significantly extended the median lifespan of nematodes up to 34% without altering feeding ability. Lipofuscin accumulation in HydroZitLa-supplemented nematodes was significantly lower than that of non-supplemented control. Additionally, HydroZitLa inhibited telomere shortening, p16 upregulation, and premature senescence in HK-2 cells exposed to lithogenic stressors. Conclusions, HydroZitLa inhibited oxidative stress and CaOx formation both in vitro and in vivo. HydroZitLa extended the lifespan and delayed the onset of aging in C. elegans and human kidney cells. This preclinical evidence suggests that HydroZitLa is beneficial for inhibiting CaOx stone formation, promoting longevity, and slowing down aging.

Oxidative stress indicated by elevated expression of Nrf2 and 8-OHdG promotes hepatocellular carcinoma progression.

Reactive oxygen species (ROS) is excessively generated in tumors creating an oxidative stress in tumor microenvironment. We investigated hepatic expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and 8-hydroxydeoxyguanosine (8-OHdG) in hepatocellular carcinoma (HCC) patients, and asked if ROS epigenetically upregulated Nrf2 and enhanced aggressiveness in HCC cells. Expression of Nrf2 (n = 100) and 8-OHdG (n = 53) was remarkably increased in HCC tissues compared with the noncancerous hepatic tissues. Elevated expression of 8-OHdG was associated with poor survival in HCC patients. H2O2, as ROS representative, provoked oxidative stress in HepG2 cells, indicated by increased protein carbonyl content and decreased total antioxidant capacity. Nrf2 expression and 8-OHdG formation were markedly increased in the H2O2-treated cells compared with the untreated control. Co-treatment with antioxidants, tocopheryl acetate (TA) and S-adenosylmethionine (SAM) effectively attenuated expression of Nrf2 and 8-OHdG in H2O2-treated cells. HepG2 cells treated with H2O2 had significantly higher migration and invasion capabilities than the untreated control cells, and this aggressiveness was significantly inhibited by TA and SAM. Bisulfite sequencing revealed that CpG dinucleotides in Nrf2 promoter were unmethylated in the H2O2-treated cells similar to the untreated control. In conclusion, robust histological evidence of increased antioxidative response and oxidative DNA damage in human HCC tissues was demonstrated. Elevated oxidative DNA lesion 8-OHdG was associated with shorter survival. Experimentally, ROS enhanced Nrf2 expression, 8-OHdG formation and tumor progression in HCC cells. These effects were inhibited by antioxidants. Therefore, oxidative stress-reducing regimens might be beneficial to diminish the ROS-induced HCC progression.