Effect Of Anlotinib On Lipid Metabolism And Its Underlying Mechanism

Objective:Anlotinib is a novel oral small molecule multi-target tyrosine kinase inhibitor with dual effects of anti-tumor angiogenesis and tumor growth inhibition.In May2018,anlotinib was approved by the State Drug Administration for the treatment of advanced or metastatic non-small-cell lung cancer(NSCLC).The results of this phase III clinical study(ALTER 0303,NCT02388919)were published in the JAMA Oncology Journal in November 2018.By analyzing adverse events in ALTER 0303 clinical trial,we found that the OS and PFS in the anlotinib-treated patients were significanctly prolonged while the lipid metabolism events such as hypertriglyceridemia and hypercholesterolemia occured.The incidences of hypertriglyceridemia and hypercholesterolemia were 44.56% and41.84% in the anlotinib-treated patients,respectively,which were statistically different from the placebo group.This suggests that anlotinib can regulate cancer-related siganling pathways as well as lipid metabolism-related pathways.Therefore,the purpose of this study is to further study the relationship between the adverse events of hyperlipidemia and the survival time of patients with NSCLC,to clarify the role of anlotinib in the regulation of lipid metabolism,and to explore the possible mechanism.It provides experimental basis for further elucidating the mechanism of the action of anlotinib.Methods:1.The common adverse event evaluation criteria(CTCAE 4.03)were used to evaluate and grade the adverse events of hyperlipidemia.The side effects of hyperlipidemia were observed and analyzed in anlotinib group and placebo group,and χ2 test was performed.Kaplan-Meier curve was used to analyze the survival of patients in the anlotinib group to determine the relationship between the adverse events of hyperlipidemia and the efficacy of anlotinib.2.Oil red O staining and Western blot were used to detect the deposition of lipid droplets and the expression levels of peroxisome proliferator-activated receptor γ (PPARγ)protein in 3T3-L1 cells,respectively.The m RNA expression levels of PPARγ and CCAAT enhancer binding protein α(C/EBPα)in 3T3-L1 cells were detected by RT-q PCR.3.C57BL/6J mice were randomly divided into three groups: saline group(control group),anlotinib(experiment group)(3 mg/kg),rapamycin(positive control group).)(2mg/kg).Each group of mice was intragastrically administered once a day for 21 days.During the administration,the body weight,feeding changes and the vital signs of the mice were observed and recorded.At the end of the administration,serum triacylglycerol(TG),cholesterol(totalcholesterol,TC),low density lipoprotein-cholesterol(LDL-C),and high-density lipoprotein(HDL-C)were measured.Meanwhile,the mice were dissected and the changes of the organs in each group were observed.The histiocytic morphologies of liver and adipose tissues were observed by HE staining.In order to study the mechanism of hyperlipidemia in vivo,the total protein and total RNA of mouse liver were extracted.Western blot was used to detect the expression levels of hydroxymethylglutaryl Co A reductase(HMGCR),LDL receptor(LDLR),AMP-activated protein kinase(AMPK),p-AMPK,mammalian target of rapamycin(m TOR)and p-m TOR protein as well as the key transcription factors sterol regulatory element binding protein(SREBP1),.The m RNA expression levels of HMGCR,AMPK,m TOR,SREBP1,SREBP2,LDLR,acetyl-Co A carboxylase(ACC),fatty acid synthase(FASN)and Stearoyl Co A desaturase 1,SCD1)were detected by RT-q PCR.Results:1.We performed a χ2 test on the incidence of hypertriglyceridemia and hypercholesterolemia events in 294 anlotinib-treated patients and in 143placebo-treated patients.This result showed that the incidence of hyperlipidemia in the anlotinib-treated group was significantly higher than that in the placebo group,and the difference was statistically significant.Meanwhile,we calculated the progression-free survival(PFS)and overall survival(OS)of 277 patients in the anlotinib-treated group.The results showed that the PFS in patients without and with hyperlipidemia were 131 days and 195 days,respectively,and the OS were 239 days and 378 days,respectively.As shown in Kaplan-Meier curve,the PFS and OS in patients with hyperlipidemia were significantly longer than those with normal blood lipid levels.2.The results of oil red O staining showed that anlotinib had no obvious inhibitory effect on the deposition of lipid droplets in 3T3-L1 cells.Western blot results showed that anlotinib could not inhibit the expression of PPARγ protein in 3T3-L1 cells.The results of RT-q PCR showed that anlotinib could not inhibit the expression of PPARγ and C/EBPα m RNA in cells.3.The results of C57BL/6J mice showed that there was no significant difference in body weight,feeding and vital signs between the mice in the anlotinib-treated group compared with the control group.However,in rapamycin-treated group,the weight and intake of mice decreased significantly,but there was no significant change in vital signs compared with the control group.Compared with the control group,the serum levels of TC and LDL-C in the anlotinib and rapamycin groups were significantly Significant increased and there was an increasing trend with the serum levels of TG,however there was no significant change in the content of HDL-C in the serum of the mice compared with the control group.Compared with the control group,the hepatic weight coefficient of the mice in the anlotinib and rapamycin groups was significantly increased.The results of HE staining showed that both anlotinib and rapamycin could promote the increase of fat vacuoles in the liver of mice.There was no significant change in the morphology of adipose tissue cells in the mice of the anlotinib group,while the adipose tissue cells in the rapamyc-treated group were significantly smaller.Western blot results showed that compared with the control group,the expression level of SREBP1 protein was significantly increased in the anlotinib group and rapamycin group,while the expression levels of LDLR,p AMPKα and pm TOR protein were significantly decreased,and the expression levels of HMGCR,AMPKα and m TOR were no significant change.The results of RT-q PCR showed that compared with the control group,the expression of HMGCR m RNA in the anlotinib group were significantly increased,and the expression of ACC,SCD1 and FASN m RNA was increased,but the difference were not statistically significant,while the expression of LDLR and AMPKα m RNA were decreased significantly.The expression of m TOR,SREBP1 and SREBP2 m RNA were no changed significantly.Compared with the control group,the expression of HMGCR,ACC and FASN m RNA in rapamycin group increased significantly,and the expression of SREBP1,SREBP2 and SCD1 m RNA increased,but the difference were not statistically significant,while the expression of AMPKα and LDLR m RNA were significantly decreased,and there was no significant change in the expression of m TOR m RNA.Conclusion:1.The incidence of hyperlipidemia adverse events in the anlotinib-treated group was significantly higher than the placebo group.It is suggested that anlotinib can regulate cancer-related siganling pathways as well as lipid metabolism-related pathways.Compared with patients without adverse events of hyperlipidemia,PFS and OS were significantly prolonged in patients with hyperlipidemia in the anlotinib-treated group.It is suggested that hyperlipidemia may become one of the predictive biomarker for anlotinib.2.There were no significant effect on the expression of key lipid transcription factors and lipid transcription factors in 3T3-L1 cells with anlotinib.It indicated that anlotinib had no significant effect on the differentiation of 3T3-L1 cells.Anlotinib may not affect lipid metabolism by promoting adipocyte differentiation.3.Higher serum TC and LDL-C levels were observed in the anlotinib-treated group.Anlotinib may promote liver TC synthesis by inhibiting AMPK phosphorylation and up-regulating HMGCR expression.At the same time,anlotinib can also reduce liver LDLR expression and reduce LDL-C uptake.Anlotinib may cause hypercholesterolemia through the above dual effects.On the other hand,anlotinib may promote the synthesis of FA in the liver by inhibiting the phosphorylation of AMPK and enhancing the expression of SREBP1.In addition,anlotinib may also affect lipid metabolism by inhibiting liver m TOR phosphorylation by regulating downstream lipid metabolism-related pathways.