Mechanism And Intervention Strategy Of Vandetanib-Induced Cutaneous Toxicity

Objectives:Vandetanib was developed by Astrazeneca,which is a multi-target kinase inhibitor that mainly inhibits vascular endothelial growth factor receptor 1,2,3(VEGFR 1,2,3),epidermal growth factor receptor(EGFR),rearrangement during transfection(RET),and indicated for the treatment of unresectable medullary thyroid carcinoma.High incidence of cutaneous toxicity ranging from 29.2% to 71.2% has been reported.During clinical use of vandetanib,the cutaneous toxicity of vandetanib has limited its clinical benefits.Due to the underlying mechanisms and protective strategies are not well studied,hence there is still no clinical treatment for the cutaneous adverse reactions caused by vandetanib.This study focused on cutaneous toxicity induced by vandetanib and identified the underlying toxic mechanisms to develop intervention strategies,and ultimately provided clinicians with effective combination treatment options.Methods:C57BL/6J mice,HaCaT cells and NHEK cells were used to study the cutaneous toxicity induced by vandetanib in vitro and in vivo.(1)C57BL / 6 mice were given 55 mg/kg/d vandetanib by gavage daily for 21 days.The skin changes of chin,face and armpit were observed.(2)The skin tissues of mouse chin,face and armpit were collected,stained with HE stain and observed under optical microscope to quantify the changes of skin cuticle thickness.(3)Immunohistochemistry was used to detect the expression level of proliferation marker Ki-67 in skin tissue.(4)The effect of vandetanib on HaCaT and NHEK cell proliferation was detected by sulforhodamine B(SRB)assay,and the cell survival rate(%)was calculated.Meanwhile,the changes of HaCaT cells stimulated by vandetanib was observed under light microscope.In this study,C57BL/6J mice and HaCaT cells were used to investigate the mechanism of keratinocyte death induced by vandetanib in vitro and in vivo.(1)We performed the classic apoptotic experiment TUNEL to detect the apoptotic cell population in skin tissue of C57BL/6J mice.(2)Using western blot,detect he protein levels of cleaved-PARP,cleaved-Caspase3,Bax,Bcl2,MCL1 and Cyto C.(3)The apoptosis of HaCaT cells was detected by flow cytometry at different concentrations(0,2.5,5,10 μM)of vandetanib and at different times(0,12,24,48 h)after PI/Annexin V double staining.(4)HaCaT cells were treated with irreversible Caspase apoptosis inhibitor Z-Vad-FMK and vandetanib,and the expression levels of apoptosis-related proteins were determined by Western Blotting.The apoptosis rate(%)was determined by flow cytometry with PI/Annexin V double staining.(5)The changes of mitochondrial membrane potential were detected by JC-1 staining after vandetanib stimulation in HaCaT cell model.(6)HaCaT cell model was used to detect the changes of mitochondrial reactive oxygen species(ROS)by MitoSOX probe staining and fluorescence confocal microscopy after vandetanib stimulation.(7)HaCaT cells were treated with vandetanib,γ-H2 A.X nuclear aggregation was observed by immunofluorescence,and DNA damage was detected via single cell gel electrophoresis.(8)Using HaCaT cell model,the expression levels of autophagy markers LC3 and p62 were detected by Western Blotting after vandetanib stimulation,and the changes of LC3 protein aggregates were observed combined with immunofluorescence to investigate the autophagy activation in HaCaT cells after vandetanib stimulation.(9)HaCaT cells treated with Different autophagy inhibitors CQ,Baf A1 or 3-MA and vandetanib,c-PARP and c-Caspase 3 were detected by Western Blotting,and the incidence of apoptosis was detected by flow cytometry with PI/Annexin V double staining.To further determine the relationship between autophagy and vandetanib-induced keratinocytes apoptosis.In this study,HaCaT cells and C57BL/6J mice were used to investigate the effect of the autophagy activator didemethoxycurcumin(BDMC)on vandetanib cutaneous toxicity in vivo and in vitro.(1)The expression of autophagy marker LC3 was detected by Western Blotting in HaCaT cell stumulated by BDMC.(2)After HaCaT cells were stimulated by BDMC combined with vandetanib,c-PARP and c-Caspase 3 in HaCaT cells were detected by Western Blotting and apoptosis was also detected by PI/Annexin V double staining by flow cytometry.(3)C57BL/6J mice were dosed with50 mg/kg BDMC,55 mg/kg vandetanib,or 50 mg/kg BDMC + 55 mg/kg vandetanib by intragastric administration for 21 days,respectively,to verify the intervention effect of BDMC on vandetanib cutaneous toxicity in vivo.(4)Skin tissues from the jaw,face,armpit and other parts of the mice were collected,stained by HE,and observed under light microscope.Changes in skin cuticle thickness of the mice after BDMC intervention were quantified.(5)Skin tissues were collected from the chin,face,armpit and other parts of mice,and the expression changes of proliferation marker Ki-67 in skin tissues after BDMC intervention were detected by immunohistochemistry.(6)And keratinocytes apoptosis in C57BL/6J mice skin after BDMC intervention was detect by TUNEL assay.Results:Cutaneous toxicity induced by vandetanib in vivo C57BL/6J mice and in vitro HaCaT cells and NHEK cells.C57BL/6J mice were administrated with vandetanib at55 mg/kg/d,the rashes on chin,face and armpit were induced.Under light microscope the stratum corneum thickness of mice was measured.Compared with the control group(19.9 ± 4.6 μm),the stratum corneum thickness of the vandetanib group(4.4 ± 1.2 μm)was significantly thinner(p < 0.001).Immunohistochemical analysis showed that proliferation marker Ki-67 in mouse skin tissue was obviously decreased.SRB assay showed that the survival rate of HaCaT cells and NHEK cells decreased with the increasing of vandetanib concentration and the prolongation of treatment time,in a time and concentration dependent relationship.The number of HaCaT cells decreased under light microscope.Vandetanib induces caspase-dependent apoptosis of keratinocytes and induces autophagy in vivo and in vitro.In TUNEL apoptosis assay,DNA fragments occurred around the nucleus in keratinocytes mice after vandetanib dosing.The western blot results showed that Vandetanib increased the expression level of apoptotic-related protein,c-PARP,c-Caspase 3 and Bax,and decreased the anti-apoptotic protein Bcl2 and MCL1 level,and increased release of Cytochrome C.Applying the PI/Annexin V staining with flow cytometry,apoptotic keratinocytes stimulated with vandetanib and apoptosis rates were significantly elevated at a time-and concentration-dependent manner.Western Blotting showed that the expression levels of c-PARP and c-Caspase 3in HaCaT cells were significantly reduced after combined treatment with Z-VAD-FMK.By combined administration of Z-VAD-FMK and vandetanib,Western Blotting revealed that the expression levels of c-PARP and c-Caspase 3 in HaCaT cells were significantly reduced.Flow cytometry indicated that apoptotic HaCaT cells was significantly decreased after Z-VAD-FMK combined treatment.By JC-1 staining,mitochondrial membrane potential(MMP)decreased in HaCaT cells treated with vandetanib.The level of mitochondrial reactive oxygen species(ROS)in HaCaT cells increased after vandetanib treatment by MitoSOX.DNA fragment Tail was prolonged and Tail DNA%increased significantly after treatment with vandetanib after electrophoresis.Meanwhile,immunofluorescence assay showed that vandetanib increased the expression level ofγ-H2 a.X,a marker of DNA double-strand damage.Moreover,nuclei stained with DAPI(blue)showed increased DNA fragmentation and nuclear pyknosis after vandetanib treatment at different concentrations and time.By western blot,the expression level of LC3,an autophagy marker,was up-regulated after vandetanib stimulation in HaCaT cells in a concentration and time dependent manner.By immunofluorescence assay,we observed that LC3 gradually appeared obvious punctate aggregation from the initial diffuse state,which were also a distinctive hallmarker for autophagy.By western blot,we found that a significant upregulation of apoptosis markers c-PARP,c-Caspase 3 were emerged in combined treatment group,compared with the vandetanib monotreatment group with CQ,Baf A1 and 3-MA,respectively.Via flow cytometry,the apoptosis rate(%)of HaCaT cells was further increased after the combination of vandetanib and autophagy inhibitors.Vandetanib-induced cutaneous toxicity was alleviated by the autophagy activator didemethoxy curcumin(BDMC)in vivo and in vitro.By Western blot,BDMC promoted the expression level of LC3 in HaCaT cells,indicating that BDMC could activate autophagy in HaCaT cells.Compared with vandetanib alone,the expression levels of c-PARP and c-Caspase 3 decreased after vandetanib combined with BDMC.Also,BDMC reduced vandetanib-induced apoptosis,which reflected by flow cytometry.C57BL/6J mice were orally administered with 50 mg/kg BDMC,55 mg/kg vandetanib,or 50 mg/kg BDMC + 55 mg/kg vandetanib BDMC respectively for 21 days.Compared with vandetanib mono-treatment,BDMC alleviated markedly the vandetanib-induced skin rash on chin,face and armpit.The stratum corneum thickness of the solvent control group,vandetanib group,BDMC group,vandetanib and BDMC combined group was 21.3 ± 5.8 μm,4.2 ± 1.4 μm,20.1 ± 4.2 μm and 16.3 ± 3.4 μm,respectively.After treatment with BDMC,the proliferation marker Ki-67 was obviously increased in mouse keratinocytes.TUNEL apoptosis assay showed that the DNA fragments in keratinocytes decreased after combined treatment with BDMC.Conclusions:In this study,using C57BL/6J mice as an in vivo model and HaCaT,NHEK cells as in vitro models,we investigated the potential mechanisms of vandetanib-induced cutaneous toxicity.Firstly,the mitochondrial pathway dependent apoptosis of keratinocytes induced by vandetanib was identified,accompanied by an increasing amount of mitochondrial ROS and DNA damage.Further studies showed that vandetanib could induce autophagy in keratinocytes.Furthermore,the combination of autophagy inhibitors can aggravate vandetanib induced keratinocyte apoptosis.Finally,this study confirmed that the natural medicine BDMC can activate autophagy of keratinocytes,Thus effectively alleviated vandetanib cutaneous toxicity.This study provides a promising therapeutic strategy for the treatment of cutaneous toxicity induced by vandetanib.