| Hepatocellular carcinoma(HCC)is one of the most lethal and prevalent malignancies,which often occurs as a subsequent lesion from chronic liver diseases caused by a variety of risk factors including infection with viruses like hepatitis B and hepatitis C,aflatoxin,obesity,alcohol consumption,tobacco usage and so on.Currently,there are only very limited therapeutic measures with poor clinical outcomes for the patients with HCC,which resulted in huge burden on the medical industry and incredible pain and financial burden on the patients and their family,It has been proved that dietary restriction(DR)without malnutrition could prevent spontaneous and chemically induced tumors,but there are no exhaustive researches on its positive role in cancer and the mechanism of this protective effect remains unclear.Therefore,it is very meaningful to explore the underlying mechanism of the inhibitory effects of DR on liver cancer in a suitable animal model to provide useful guidance for clinical treatments and prevention of liver disease for all human beings.To detect the preventive effects of DR and uncover its mechanism,we utilized the diethylnitrosamine(DEN)-induced HCC mice model.Two-week-old male C57BL/6 mice were randomly distributed into the control,DR,DEN,DEN+DR groups,mice inDEN and DEN+DR groups were injected intraperitoneally with 25 mg/kg body weight of DEN.Mice in control and DR groups were injected with corresponding volume of saline.DR was carried out from age of 3 weeks for mice in DR and DEN+DR groups according to the control and DEN group respectively in a multistep-advanced process(90%of AL-fed for one week followed by 80%for one week and finally stabilized at 70%till the ending point).Mice in control and DEN groups were on an ad libitum diet,and all animals in four groups had free access to water.Mice were sacrificed for sample collection 30 weeks after DEN administration.Compared with the DEN group,we found the mice in DEN+DR group were with less tumors at liver surface and the volumes of these tumors were much smaller with relative lower-grade pathological characteristics,in addition,the livers of DEN+DR mice demonstrated reduced DNA damage,proliferation and inflammation accompanied with increased apoptosis and autophagy.Meanwhile,we detected that DR inhibited the activation of the HCC stimulative signaling pathway mTOR and NF-?B,but promoted the Keapl-Nrf2 signaling pathway that has been proven to be important in the anticancer effects of DR.Furthermore,we confirmed the role of DR in apoptosis and proliferation in Hepal-6 cell in vitro.By RNA Sequencing and differential expression analysis,we revealed DR reversed the effects of DEN on transcriptome of mice liver.According to the clues from the sequencing,we discovered the mutation of Braf gene and the activation of the ERK signaling in DEN mice liver,which was not observed in DEN+DR mice.Further,we found ERK signaling pathway affected the cell viability of Hepal-6 cells and played an important role in the inhibition role of DR in Hepal-6 cell in vitro by the agonist of ERK(TPA)and inhibitor of ERK(SCH772984).To clarify the role of DR-induced autophagy in the preventive effects of DR on cancer,we used the induced-autophagy defective mice by knockout of Ulk1.The age-matched Ulk1KO mice were subjected to DEN administration and DR intervention under the same experimental settings.We found DR prevented DEN induced HCC in Ulk1KOmice,but this inhibitory effects of DR was not as much as that in the wide type mice.More importantly,we observed that the deletion of Ulk1 prevented the DEN induced HCC,and there was interaction effects between the anticancer effects of DR and deletion of Ulkl.Taken together,we confirmed the preventive role of DR in DEN induced HCC,suggetsed its anti-tumor effects was mainly associated with the regulatory role in proliferation and apoptosis rather than autophagy and recommended the Braf/MEK/ERK signaling pathway was importan for the inhibitory role of DR in cancer. |
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