| Objectives:Sorafenib is a multikinase inhibitor targeted at VEGFR-2,VEGFR-3,RAF-1 and FLT-3 etc,which has been applied widely as the first-line drug for advanced renal cell carcinoma and hepatocellular carcinoma.However,hand-foot skin reaction(HFSR)is a common side effect in patients receiving sorafenib,the incidence is up to 60%.Although HFSR does not directly affect patients' survival,it can impact quality of life and lead to sorafenib dose modification or interruption,potentially limiting the antitumor activity.Currently,there is short of effective therapy towards HFSR which is a tough clinical problem and now the underlying mechanism of sorafenib-induced hand-foot skin reaction is unclear.Therefore,studying molecular mechanism of sorafenib-induced HFSR is necessary for figuring out the feasible intervention strategy.In this paper,we hope to explore the mechanism of sorafenib-iduced HFSR,and further provide new strategies and basis for the treatment of HFSR in clinic.Methods:Using human umbilical vein endothelial cells(HUVEC)and human keratinocytes(HaCaT)to confirm the cross talk between the two cell lines cause hyperkeratosis of HaCaT cells.(1)HaCaT cells were treated with sorafenib,real-time quantitative PCR was used to detect the mRNA levels of keratin 1,keratin 10 and Loricrin,exploring the effect of sorafenib on differentiation of HaCaT cells.(2)A:Collected HUVEC cells' conditioned medium.HUVEC cells were treated with 15?M sorafenib,then collected its' medium as conditioned medium.B:Explored the effect of HUVEC cells' conditioned medium on hyperkeratosis.Treated HaCaT cells with conditioned medium,real-time quantitative PCR was used to detect the mRNA levels of keratin 1,keratin 10 and Loricrin,exploring the effect of HUVEC cells' conditioned medium on differentiation of HaCaT cells.Using human umbilical vein endothelial cells(HUVEC)and human keratinocytes(HaCaT)to confirm the role of HUVEC-secreted HB-EGF on the hyperkeratosis of HaCaT cells by sorafenib.(1)Treated HUVEC cells with sorafenib,collected the medium to detect the expression of heparin-binding epidermal growth factor(HB-EGF)by western blot and ELISA.(2)ICR mice were given sorafenib for 30 days,then collected blood sample and test the quantity of HB-EGF by ELISA.(3)Detected the content of HB-EGF in the blood sample of sorafenib-induced HFSR patients by ELISA.(4)Treated HaCaT cells with HUVEC cells' conditioned medium with or without HB-EGF antibody,real-time quantitative PCR was used to detect the mRNA levels of keratin 1,keratin 10 and Loricrin,investigating the connection between HB-EGF and sorafenib-induced hyperkeratosis.(5)A:Collected HB-EGF silenced-HUVEC cells' conditioned medium.Silenced HB-EGF by short hairpin RNA in HUVEC cells,then gave HUVEC cells 15 ?M sorafenib,collected its medium.B:Explored the effect of HB-EGF silenced-HUVEC cells' conditioned medium on hyperkeratosis.Treat HaCaT cells with conditioned medium,real-time quantitative PCR was used to detect the mRNA levels of keratin 1,keratin 10 and Loricrin,further confirm the connection between HB-EGF and sorafenib-induced hyperkeratosis.(6)Treated HaCaT cells with 2.5 ng/mL HB-EGF recombinant protein,detected the mRNA levels of keratin 1,keratin 10 and Loricrin by real-time quantitative PCR to further verify the connection between HB-EGF and hyperkeratosis.Using human umbilical vein endothelial cells(HUVEC)and human keratinocytes(HaCaT)to explore the mechanism of sorafenib-induced HFSR.(1)Treated HaCaT cells with 2.5 ng/mL HB-EGF recombinant protein,western blot was used to detect the expression of p-JNK2.(2)Treated HaCaT cells with HUVEC cells'conditioned medium,western blot was used to detect the expression of p-JNK2.(3)Silenced JNK2 of HaCaT cells by small interfering RNA,then treated HaCaT cells with HUVEC cells' conditioned medium,24 hours later detected the mRNA levels of keratin 1,keratin 10 and Loricrin by real-time quantitative PCR to explore the correlation between p-JNK2 and sorafenib-induced hyperkeratosis.(4)Silenced JNK2 of HaCaT cell by small interfering RNA,then treated HaCaT cells with 2.5 ng/mL HB-EGF recombinant protein,detected the mRNA levels of keratin 1,keratin 10 and Loricrin by real-time quantitative PCR to explore the correlation between p-JNK2 and HB-EGF-induced hyperkeratosis.Using human umbilical vein endothelial cells(HUVEC)and human keratinocytes(HaCaT)to confirm the effect of p-JNK2/SIRT1 axis on hyperkeratosis.(1)Silenced JNK2 of HaCaT cell by small interfering RNA,then treated HaCaT cells with HB-EGF recombinant protein,detected the protein level of SIRT1 by western blot,exploring the correlation between the phosphorylation of JNK2 and the expression of SIRT1 in the presence of HB-EGF.(2)Treated HaCaT cells with HUVEC cells' conditioned medium,detected the expression of SIRT1 by western blot to explore the relation between HB-EGF and SIRT1.(3)Treated HaCaT cells with 2.5 ng/mL HB-EGF recombinant protein,detected the expression of SIRT1 by western blot to further verify the relation between HB-EGF and SIRT1.(4)ICR mice were given 100 mg/kg sorafenib per day,30 days later,detected the expression of SIRT1 in claw by immunohistochemical to verify the correlation between SIRT1 and sorafenib-induced hyperkeratosis.(5)Silenced SIRT1 of HaCaT cells by small interfering RNAs,then treated HaCaT cells with HUVEC cells' conditioned medium,detected the mRNA levels of keratin 1,keratin 10 and Loricrin by real-time quantitative PCR to explore the effect of HB-EGF/SIRT1 axis on sorafenib-induced hyperkeratosis.(6)Silenced SIRT1 of HaCaT cells by small interfering RNAs,then treated HaCaT cells with 2.5 ng/mL HB-EGF recombinant protein,detected the mRNA levels of keratin 1,keratin 10 and Loricrin by real-time quantitative PCR to explore the effect of HB-EGF/SIRT1 axis on HB-EGF-induced hyperkeratosis.Using human umbilical vein endothelial cells(HUVEC),human keratinocytes(HaCaT)and ICR mice to detect the protective effects of SIRT1 inhibitor nicotinamide(NAM)on sorafenib-induced HFSR.(1)Gave HaCaT cells conditioned medium with or without NAM,detected the mRNA levels of keratin 1,keratin 10 and Loricrin by real-time quantitative PCR,exploring the protective effects of NAM on HFSR.(2)ICR mice were given 100 mg/kg sorafenib,100 mg/kg NAM,100 mg/kg sorafenib and 100 mg/kg NAM by gastric perfusion per day,after 30 days sacrificed mice,H&E staining was used to detect the thickness of corneum of claw.Results:Sorafenib-treated HUVEC cells' conditioned medium causes hyperkeratosis of HaCaT cells.HaCaT cells were treated with sorafenib under gradient concentration,real-time quantitative PCR results showed that the mRNA levels of differentation bio-marker keratin 1,keratin 10 and Loricrin had little change(p>0.05).Treated HaCaT cells with conditioned medium,real-time quantitative PCR results showed that the mRNA levels of keratin 1,keratin 10 and Loricrin increased to 5.5 ± 0.8,6.0 ± 1.9 and 10.9 ? 4.2 folds(p values are 0.001,0.02 and 0.03 respectively).Sorafenib induces the release of HB-EGF in HUVEC cells,which cause HaCaT cells hyperkeratosis.Treated HUVEC cells with sorafenib,collected its medium to detect the expression of HB-EGF by western blot and ELISA,western blot result showed the release of HB-EGF up-regulated after treated with sorafenib;ELISA result revealed the protein level of HB-EGF increased from 1.2 ± 0.1 ng/mL to 2.4 ± 0.2 ng/mL(p<0.05).ICR mice were given sorafenib by gastric perfusion for 30 days,then collected blood sample and found the quantity of HB-EGF of normal mice was 1.2 ± 0.1 ng/mL,while the quantity of HB-EGF of HFSR mice was 1.6 ± 0.2 ng/mL(p = 0.002).Besides the contents of HB-EGF in blood sample of HFSR patients increased as well,the contents of healthy people were 0.5 ± 0.1 ng/mL,while it up to 1.3 ? 0.5 ng/mL of HFSR patients.Treated HUVEC cells with sorafenib for 24 hours,collected its medium as conditioned medium,then gave HaCaT cells conditioned medium with/without HB-EGF antibody,real-time quantitative PCR showed the mRNA levels of keratinl,keratinlO and Loricrin decreased treated with conditioned medium and HB-EGF recombinant protein,from 2.4 ± 0.3,3.9 ± 1.0 and 5.4 ± 0.8 folds to 0.4 ± 0.2,1.4 ± 0.3 and 1.9 ± 0.8 folds(p values are 0.008,0.04 and 0.002 respectively).Treated HB-EGF silenced-HUVEC cells with sorafenib,collected its medium to treat HaCaT cells,real-time quantitative PCR results showed that the mRNA levels of keratin 1,keratin 10 and Loricrin down-regulated after silencing HB-EGF,from 2.6 ± 0.3,8.5 ± 3.6 and 7.5± 1.2 folds to 1.4 ± 0.1,1.1 ± 0.3 and 1.3 ± 0.3 folds(p values are 0.001,0.02,0.005 respectively).Treated HaCaT cells with HB-EGF recombinant protein,it was found that the mRNA levels of keratin 1,keratin 10 and Loricrin increased to 2.6 ± 0.3,3.4 ± 0.4 and 10.5 ± 5.8 folds(p values are 0.003,0.001 and 0.04 respectively)by real-time quantitative PCR.HB-EGF induced hyperkeratosis by activating phosphorylation of JNK2.Treated HaCaT cells with 2.5 ng/mL HB-EGF recombinant protein,western blot results revealed that the level of p-JNK2 increased.Treated HaCaT cells with HUVEC cells'conditioned medium,western blot results showed that the level of p-JNK2 increased.These results implied up-regulated p-JNK2 may be involved in HB-EGF-increased protein level of SIRT1.Silenced JNK2 of HaCaT cells by small interfering RNAs,then treated HaCaT cells with conditioned medium,real-time quantitative PCR results showed that the mRNA levels of keratin 1,keratin 10 and Loricrin reduced from 3.2±0.5,3.0 ± 0.2 and 8.8 ± 3.5 fold to 1.1 ± 1.2,1.5 ± 0.5 and 3.5 ± 2.2 fold(sequence#1,p values are 0.08,0.03 and 0.14 respectively)or 0.7 ± 0.2,1.2 ± 0.4 and 1.7 ± 1.0 fold(sequence#2,p values are 0.004,0.0006 and 0.04 respectively).Silenced JNK2 of HaCaT cells by small interfering RNAs,then treated HaCaT cells with HB-EGF recombinant protein,real-time quantitative PCR results showed that the mRNA levels of keratin 1,keratin 10 and Loricrin decreased after silencng JNK2,from 2.8 ± 0.5,5.1± 1.4 and 6.3 ± 2.1 fold to 1.6 ± 0.4,1.2 ± 0.5 and 1.6 ? 1.4 fold(sequence#1,p values are 0.04,0.1 and 0.05 respectively)or 1.1 ± 0.4,1.0 ? 0.7 and 0.8 ± 0.5 fold(sequence#2,p values are 0.02,0.02 and 0.02 respectively).p-JNK2/SIRT1 axis regulates sorafenib-induced HFSR.Silenced JNK2 of HaCaT cells by small interfering RNAs,then treated HaCaT cells with HB-EGF recombinant protein,western blot results showed that the expression of SIRT1 had no effectful change.Treated HaCaT cells with HUVEC cells' conditioned medium or HB-EGF recombinant protein,western blot results showed the expression of SIRT1 increased.ICR mice were treated with 100 mg/kg sorafenib per day for 30 days,immunohistochemical result showed that the expression of SIRT1 in claw decreased.Silenced SIRT1 of HaCaT cells by small interfering RNAs,then treated HaCaT cells with conditioned medium,real-time quantitative PCR results showed that the mRNA levels of keratin 1,keratin 10 and Loricrin reduced after silenced SIRT1,from 6.2 ± 1.6,6.1 ± 1.4 and 9.1 ± 3.4 fold to 1.6 ± 0.4,1.2 ± 0.5 and 1.6 ± 1.4 fold(sequence#1,p values are 0.02,0.01 and 0.04 respectively)or 1.9 ± 0.2,1.5 ± 1.1 and 1.7 ± 0.3 fold(sequence#2,p values are 0.02,0.02 and 0.04 respectively).Silenced SIRT1 of HaCaT cells by small interfering RNAs,then treated HaCaT cells with HB-EGF recombinant protein,real-time quantitative PCR results showed that the mRNA levels of keratin 1,keratin 10 and Loricrin decreased from 3.8 ± 0.2,3.6 ± 0.8 and 8.7 ± 2.2 fold to 0.9 ±0.5,0.8 ± 0.5 and 0.4 ± 0.3 fold(sequence#1,p values are 0.002,0.01 and 0.01 respectively)or 0.8 ± 0.5,1.3 ± 0.6 and 0.9 ± 0.3 fold(sequence#2,p values are 0.002,0.03 and 0.007 respectively).SIRT1 inhibitor nicotinamide(NAM)protects against sorafenib-induced HFSR.Real-time quantitative PCR results showed the mRNA levels of keratin 1,keratin 10 and Loricrin of HaCaT cells treated with HUVEC cells conditioned medium and NAM were decreased compared with HaCaT cells treated with HUVEC cells conditioned medium,from 3.6 ± 0.8,4.6 ± 1.3 and 8.5 ± 2.7 fold to 1.1 ± 0.2,1.2 ± 0.7 and 1.0 ± 0.6 fold(p values are 0.02,0.03 and 0.02 respectively).ICR mice were given 100 mg/kg sorafenib,100 mg/kg NAM,100 mg/kg sorafenib and 100 mg/kg NAM by gastric perfusion per day,after 30 days,killed mice,H&E staining showed that the thickness of corneum of claw increased after treated with sorafenib,while the thickness of corneum decreased after using NAM.Conclusions:In conclusion,the paper uncovered that sorafenib up-regulated the release of HB-EGF in HUVEC cells,which caused hyperkeratosis of HaCaT cells.We revealed that HB-EGF/SIRT1 axis regulate the crosstalk between HUVEC cells and HaCaT cells in sorafenib-induced HFSR.Mechanistically HB-EGF stablized SIRT1 through up-regulating p-JNK2 which resulted in keratinization.In addition,we found that SIRT1 inhibitor nicotanamide had protective effects on sorafenib-induced HFSR. |
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