| Liver cancer is one of the deadliest malignancies worldwide,and there is no effective therapeutic schedules due to insufficient understanding of mechanisms in tumorigenesis.Recently,several studies aim at exploring molecular mechanism during hepatocarcinogenesis.Except to different kinds of cancer cell lines,rats,mice and zebrafish are commonly used as animal models.But these studies are predominantly conducted in diethylnitrosamine-initiated carcinoma of rats,orthotopic cancers of nude mice or transgenic mice and zebrafish due to their long lifespan.Studies used animal models to investigate underlying mechanisms of spontaneous neoplasms in liver are invisible.Compared to rats,mice and zebrafish,N.guentheri is used usually to study the process of aging.Our previously studies found that oxidative damage in muscle and senescence-associated beta-galactosidase in skin increased as aging.Recent studies in N.guentheri,N.furzeri and N.korthausae demonstrate that the genus Nothobranchius can also be used to study spontaneous neoplasms.These results just focus on neoplasm incidence at different developmental stages,but underlying mechanisms of tumorigenesis remain unknown.In the present study,we focused on the molecular mechanisms during spontaneous hepatic neoplasms in N.guentheri.Resveratrol,a nonflavanoids,occurs naturally in different plants.In different cells and organisms,resveratrol exhibits anti-oxidant,anti-inflammation,anti-aging and anti-tumor effects.SIRT1 is a NAD-dependent histone deacetylase.As an activator of SIRT1,resveratrol exhibits these benefit effects in SIRT1-dependent manner.However,resveratrol inhibits carcinogenesis predominantly performed in transgenic animal models,orthotopic cancers of nude mice or different cancer cell lines,but its effects during process of spontaneous tumors using vertebrate models remain untested.In the study,we aimed to explore effects of resveratrol on spontaneous neoplasms and underlying mechanisms in liver of N.guentheri.Totally,362 fish(with 181 fed with resveratrol and another 181 fish for controls)were used in the present study.The mean lifespan of N.guentheri is 12-month-old,so we examined fish in three stages,6-,9-and 12-month-old,which represented young,middle-aged and old fish respectively.When the fish grew to sexual maturity at 4-month-old,they were separated randomly into control group with standard food and resveratrol feeding group with resveratrol-supplemented food(200 μg/ gram food,Sigma)made as previously until fish were sacrificed at 6-,9-and 12-month-old.Approximately,the dose was 25 μg resveratrol/fish/day.At 6-,9-and 12-month-old,relative liver weight,activity of serum hepatic marker enzymes,incidence of spontaneous neoplasms,nodular size and malignancy level of spontaneous neoplasms,proliferation and apoptosis of cell in hepatic neoplasms,and expression of SIRT1,PCNA,K-Ras,PI3 K,p-AKT,Fox O1,Fox O3 a,Ac-Fox O1,p-Fox O3 a,DLC1,Bcl-2,Bax and cleaved caspase-3/7/9 were estimated both in control and resveratrol-fed groups by realtime PCR,immunoblot,immunohistochemical analysis and co-immunoprecipitation.Our results showed:1.Resveratrol decreased relative liver weight and activity of serum hepatic marker enzymes.Liver weight showed an upward trend from young to old age in control group with macroscopic dots by naked eyes(histopathological analysis revealed these macroscopical dots were neoplasms),and decreased in fish fed with resveratrol at 9-and 12-month-old.Relative liver weight was calculated and it significantly increased with age in control group and decreased in resveratrol(RES)group at middle and old stages.Body weight had no significant difference between resveratrol-treated and control fish.Activitiy of AST and ALT at 9-and 12-month-old was much higher than that at 6-month-old in control fish,and resveratrol contributed to the reduction of aging-induced incremental trend of AST and ALT activity significantly at the two stages.2.Resveratrol decreased incidence of spontaneous neoplasms in liver.Spontaneous neoplasms were found at 9-and 12-month-old,but not at 6-month-old.As aging,the percentage of neoplasms was 86.67%(13/15)at 9-month-old and it approached to 100%(10/10)at 12-month-old.Resveratrol decreased percentage of spontaneous neoplasm both at 9-month-old(60%,9/15)and 12-month-old(80%,8/10).Nodular size and malignancy level of neoplasms showed a significant decrease in resveratrol-fed fish compared with control group.Immunoreactivity indicated that only locations of neoplasm nodules showed mutant p53 positive signal,and hepatic neoplasms are malignant tumors.Resveratrol decreased mutant p53 staining at 9-and 12-month-old.3.Resveratrol increased SIRT1 expression in liver.In control group,SIRT1 expression was present at a much lower level at 12-month-old than that at 6-and 9-month-old.Using the annual fish at three different ages we showed for the first time that SIRT1 expression decreased over time.Age-dependent increase of neoplasm accompanied to SIRT1 down-regulation.After long-term resveratrol supplementation,m RNA and protein of SIRT1 elevated significantly at 6-,9-and 12-month-old.Immunohistochemical staining showed SIRT1 level decreased in partial neolpasms of control fish,but increased in neolpasms of resveratrol-fed fish.It confirmed that SIRT1 served as a tumor suppressor and resveratrol improved its expression apparently in the fish.4.Resveratrol inhibited proliferation through inactivating K-Ras/PI3K/AKT pathway probably mediated by SIRT1.Less PCNA protein was found in RES group than in control group by immunoblot,and decreased PCNA staining was observed both at neolpasms and pericarcinous tissues in resveratrol-fed fish by immunohistochemistry,confirming that formation of hepatic neoplasms was accompanied with activation of liver cell proliferation.Resveratrol decreased protein levels of K-Ras,PI3 K and p-AKT at 9-and 12-month-old.Co-IP assay showed two endogenous proteins(SIRT1 and K-Ras)interacted with each other in liver of the annual fish.Acetylation of K-Ras was decreased by long-term resveratrol feeding,suggesting that resveratrol regulated K-Ras mediated pathway through deacetylation activity of SIRT1.In spontaneous neoplasms of the annual fish,resveratrol down-regulated acetylation of K-Ras by SIRT1 deacetylase and further suppresses K-Ras/PI3K/AKT pathway.Resveratrol inhibited proliferation by SIRT1-mediated post-translational modulations.5.Resveratrol significantly induced apoptosis in spontaneous liver neoplasms by Fox Os-and DLC1-mediated pathways.Apoptotic cells were observed rarely in the liver at 6-month-old.Liver sections displayed fewer apoptotic cells in control group both at 9-month-old(0.216%±0.016%)and 12-month-old(1.497%±0.118%),and a dramatic increase of apoptotic cells in RES group(1.630%±0.165% at 9-month-old and 3.965%±0.341% at 12-month-old),especially apoptotic foci distributed widely in neoplasms at 12-momth-old.Resveratrol caused a steady reduction of Fox O1 and Fox O3 a with increase of SIRT1 at 9-and 12-month-old,and resveratrol negatively regulated protein levels of Ac-Fox O1 and p-Fox O3 a.DLC1 protein was markedly lower in the liver of control group than in RES group both at 9-and 12-month-old.Endogenous SIRT1 and DLC1 interacted with each other and resveratrol enhanced the interaction.Resveratrol decreased DLC1 phosphorylation in spontaneous neoplasms.Levels of Bax,cleaved caspase-3/7/9 became dramatically higher in resveratrol feeding fish than that in control fish,whereas Bcl-2 displayed the opposite results with above proapoptotic proteins at 12-month-old.Apoptosis induced by resveratrol might be resulted from deacetylation and dephosphorylation of Fox Os,up-regulation of DLC1 and enhancement of interaction between SIRT1 and DLC1 in the fish.In conclusion,spontaneous hepatic neoplasms were observed at 9-month-old and approached to 100% incidence at 12-month-old in N.guentheri.Age-dependent increase of spontaneous liver neoplasms might result from aging-induced down-regulation of SIRT1.Resveratrol inhibited age-dependent liver spontaneous neoplasms and up-regulated SIRT1.Resveratrol decreased the formation and growth of liver neoplasms by reduction of K-Ras,PI3 K and AKT mediated proliferation,and promotion of Fox Os-and DLC1-mediated apoptosis with up-regulation of SIRT1.These results highlighted the essential role of SIRT1 in development of spontaneous neoplasms and resveratrol may be a promising therapy for liver neoplasms of vertebrates and provided evidence for the annual fishes to act as an excellent vertebrate model in tumorigenesis related studies. |
