Adipocytes Promote Hepatocellular Carcinoma Progression And The Inhibitory Effects Of Pentamethylquercetin

Obesity is a major risk of incidences of cancer and cancer-related mortality.Programmed death protein 1（PD-1）and its ligand Programmed cell death ligand 1（PD-L1）are deeply concerned checkpoints,overexpreesion of PD-1/PD-L1 may lead tumor to escape from immune system.Relationship between obesity and checkpoints is little known.The present study established monosodium glutamate-induced obesity mice model to drive tumor progression and PD-L1 expression in H22-xenografted tumor.Further to study the mechanism of adipocytes-induced PD-L1 expression in vitro.Effect of pentamethylquercetin（PMQ）on tumor progression is also obserced.PART 1 Obesity promotes tumor prograssionObjective:To study the mechanism of obesity promotes tumor growth in vivo and vitro.Method:To build a appropriate obese model,male neonatal CD1 mice were subcutaneously injected with MSG（3 mg/kg/d）and control group with the vehicle（saline）from day 1 to day 7.At week of 15,checkpoints of liver,spleen and epididymal fat of some mice were tested,rest animals were injected with 1×10⁵H22cells at right armpit.Tumors were measured when it is palpable.At 17th day after inoculation,all mice were sacrificed,and tumor tissues and epididymis fat were dissected and weighed.For following research,tissues were fixed in formalin or frozen in-80°C.PD-L1 m RNA and protein level were tested by PCR and westernblot,respectively.In vitro,adipocytes conditional media was collected to treat Hep G2,SMMC-7721 cells and B16F1 cells,anti-TNF-αand anti-IL-6 neutralizing antibody,and inhibitor of STAT3 or NF-κB were used to check the mechanism.Exosomes from adipocytes were extracted and treat Hep G2 cells.Resulted:Compared with control mice,MSG-IO mice exhibited increased bodyweight,waistline,Lee index,adipose tissue,thymus weight and liver weight;Tumor tissue grow faster in NSG-IO mice;PD-L1 protein level were increased in MSG-IO mice tumor tissues and tumor infiltrating lymphocyte were reduced;Adipocytes conditional media upregulated PD-L1 expression of Hep G2,SMMC-7721cells and B16F1 cells;Anti-TNF-αand anti-IL-6 neutralizing antibody could reverse the effect of Adipocytes conditional media;inhibitor of STAT3 or NF-κB reduced PD-L1 level in adi-CM treated HCC cells;Exosomes from adipocytes induced PD-L1protein level of Hep G2 cells.Conclusion:MSG-IO mice exhibited obvious obese state and tumor tissues express higher PD-L1 level and grow faster in MSG-IO mice.TNF-αand IL-6 secreted from adipocytes upregulate PD-L1 of Hep G2,SMMC-7721 and B16F1 cells.Adipocytes-derived exosomes induce Hep G2 cells PD-L1 protein level.PART 2 the mechanism of PMQ in inhibiting tumor growth of MSG miceObjective:to study the effect of PMQ on tumor growth and its potential mechanism.Method:Male neonatal KM mice were subcutaneously treated with MSG（3mg/kg/d）and control group with saline from day 1 to day 7 after birth.At the age of 5weeks,MSG-IO mice were randomly divided into MSG-IO group,PMQ 5,10,20mg/kg groups.All mice were daily administrated by gastric gavage from 5^th to 24^thweek.At the age of 22 week,all mice were injected with 1×10⁶H22 cells.At 17th day after inoculation,all mice were sacrificed,and tumor tissues were dissected and weighed.For following research,tissues were fixed in formalin or frozen in-80°C.In vitro,Hep G2 cells were treated with PMQ or adipocytes conditional media for 48hours.PD-L1 protein level was tested by westernblot.Secreted IFN-γwas detected by ELISA kit.Result:PMQ inhibited H22 tumor proliferation in MSG-IO mice,and reduced PD-L1 level and promoted CD8⁺T cell infiltratioin;adipocytes derived IFN-γinduced PD-L1 protein level of Hep G2 cells;soluble IFN-γlevel from adipocytes was decreased after PMQ treated.Conclusion:PMQ down regulated PD-L1 level and inhibited H22 tumor proliferation.PMQ decreased lipogenesis during differentiation of adipocytes,and reduce secreted IFN-γ,thus reversed PD-L1 level in adipocytes conditional media treated Hep G2 cells.