The Different Role Of Keratinocytes In Hfs Induced By Capecitabine And Sorafenib

Objectives:Hand and-foot syndrome(HFS)or hand foot skin reaction(HFSR),also known as palmoplantar erythrodysesthesia,is a well-documented adverse effect of numerous anti-tumor drugs mainly occurred in the compression area.Hand-foot-syndrome(HFS)is caused by traditional chemotherapy drugs,and hand-foot-skin-reaction(HFSR)is caused by small-molecule targeted anti-cancer drugs due to the clinical phenotype which is not exactly the same.However the mechanism of these two kinds drugs caused toxicology is rarely.The most common causes is Capecitabine,causes the continual incidence of hand-foot-syndrome(HFS)up to 60%which seriously affects the quality of life of patients.Capecitabine is one of the most widely used chemotherapy drugs which is registered for many tumor types including gastric,rectal cancer,breast cancer,colorectal as first-line treatment.Sorafenib,a molecular targeted drug has a significant anti-tumor effect,also as the first-line treatment for advanced liver cancer,however,the incidence of hand foot syndrome can be as high as 40%.Currently,the mechanism of Capecitabine and Sorafenib induced HFS is rarely known.There are no effective strategies to solve this bottleneck problem.The only choice for oncologists is to drugs dose modification or interruption which will not be conducive to the treatment of patients.To further study the mechanism of hand foot syndrome and hand foot skin reaction is very important.Therefore,this study aims to explore the mechanisms searching for the difference between the two drugs to provide clinical guidance.Methods:ICR mouse model is used to simulate the occurrence of HFS and HFSR induced by Capecitabine and the multikinase inhibitors(MKI)Sorafenib,searching for pathological difference.(1)ICR mice were respectively given Capecitabine 200mg/Kg,Sorafenib 100mg/Kg with a dose in clinical treatment for 30 days.Mice were administered at the same time every five days.The mice limbs were observed took pictures every five days,then were evaluated whether occurred HFS and HFSR through swell appearance.(2)Capecitabine and Sorafenib were also investigated to compare the incidence of HFS and HFSR.(3)the pathology changes was assessed by hematoxylin and eosin(HE)staining.Using the human keratinocyte line HaCaT and animal model to further explore the mechanism of HFS and HFSR induced by Capecitabine and Sorafenib.The changes of the related gene about the process of cornification by tissue microarray technology:(1)Through tissue microarray to explore the change of the keratin and keratin associated genes.The mechanism for Capecitabine:(1)S9-Capecitabine incubation system was used to simulate the metabolic process of drugs in vivo,then to investigate the effects on the human keratinocyte line HaCaT.(2)SRB was used to investigate the effects of S9-Capecitabine on the the human keratinocyte line HaCaT.(3)Using V-FITC/Propidium iodide Annexin(PI)staining to detect the effects of S9-Capecitabine on the human keratinocyte line HaCaT.(4)Western Blotting method was used to analysis the changes of apoptosis related proteins on the human keratinocyte line HaCaT.(5)JC-1 staining was used to detect the changes of mitochondrial membrane potential of S9-Capecitabine.(6)Western Blotting method was used to analysis the changes of Bcl-2 and Bax.The mechanism for Sorafenib:(1)Using immunofluorescence technique to study the blood vessel and the presence of hypoxia in mice limbs(2)SRB assay was used to explore the effect of Sorafenib on the human keratinocytes HaCaT.(3)RT-PCR was used to study the effect of Sorafenib on the expression of keratin associated genes in human keratinocytes HaCaT.Results1)Capecitabine and the multikinase inhibitors(MKI)Sorafenib were able to induce HFS and HFSR in mice.We treated ICR mice(6-8 weeks old)with clinical dose of Capecitabine 200mg/kg,Sorafenib 100mg/Kg for 30 days.The mice limbs were observed by taking pictures every five days,then were evaluated whether occurred HFS through swell appearance.After 5 days three of six mice treated with Capecitabine had occurred swell,finally almost all the mice occurred HFS.In the same time mice treated with Sorafenib also occurred HFSR.Meanwhile,histopathologic findings in mice limbs exhibited a few mastocyte infiltrate,just like several lymphocytic in clinic,indicating that our animal model was highly matching clinic.2)Capecitabine and the multikinase inhibitors(MKI)Sorafenib induce HFS and HFSR had pathological difference in mice.Capecitabine treated mice exhibited corneous layer became obvious thin and loosen compared with control group.But multikinase inhibitors(MKI)Sorafenib could induce the thickening of the stratum corneum in mouse claw.3)The changes of the related gene about the process of cornification by tissue microarray technology.Through microarray data analysis,we found that keratin and keratin associated proteins were down-regulated in Capecitabine treated mice.While in Sorafenib treated mice Gene chip data showed that the keratin and keratin associated proteins were up-regulated.4)The mechanism of Capecitabine caused HFS is that Capecitabine triggered mitochondria dysfunction,activating caspase-dependent apoptosis,inhibiting the proliferation of keratinocytes,leading to corneous layer reducing and finally give rise to HFS.S9-Capecitabine incubation system was used to simulate the metabolic process of drugs in vivo.S9-Capecitabine could obviously inhibited HaCaT cells proliferation.V-FITC/Propidium iodide(PI)assay was used to detect the effect which showed that cells treated with S9-Capecitabine occurred markedly apoptosis,exerting a dose-dependent increase in S9-Capecitabine treated group.Furthermore,Western blot analysis revealed an increase of PARP cleavage fragment expression when treated with S9-Capecitabine,JC-1 staining combined with flow cytometry was conducted after treated with S9-Capecitabine and the results showed that S9-Capecitabine could induced the loss of mitochondrial membrane potential.Western blot analysis revealed that the mitochondrial-associated Bcl-2 was down-regulated and Bax was up-regulated in the S9-Capecitabine group.These data further implicated that S9-Capecitabine induced mitochondrial associated-apoptosis.5)The mechanism of Sorafenib induced HFSR is that Sorafenib could destroy the capillary of mice's jaw,resulting in low oxygen environment,promoting the excessive proliferation of keratinocytes and the expression of keratin,aggravating the process of cornification,giving rise to the incidence of hand foot syndrome.Using Immunofluorescence to detect CD31,a blood vessel markers and hypoxia probe pimonidazole,the results show that Sorafenib could destroy the blood capillary in mice claw,resulting in a tissue hypoxia.SRB results showed that Sorafenib could increase the proliferation of keratinocytes in hypoxia environment.While the RT-PCR results showed that Keratin 76,Keratin6b were also up-regulation,showed that the process of cornification was aggravating.Conclusion:For the widely used Capecitabine and Sorafenib,this study reveals the difference of pathological characteristics and mechanisms of Capecitabine and Sorafenib induced HFS and HFSR,aiming to provide clinical guidance and theoretical basis for further intervening measures and mechanism research.Capecitabine could induce mitochondrial associated-apoptosis of keratinocytes,resulting in the reduction of corneous layer,which partly contribute to elucidate the causative factors of Capecitabine-induced HFS.Due to drug targets,Sorafenib could destroy the blood capillary in mice claw,resulting in a tissue hypoxia.Sorafenib could increase the proliferation of keratinocytes in hypoxia environment.Gene chip data and the RT-PCR results show that the keratin and keratin associated proteins are up-regulated,indicating the process of comification is aggravating.The results are consistent with clinical reports of Sorafenib induced hyperkeratosis which provide experimental data for explaining the reason of Sorafenib induced HFSR.