| Part one CMSP induces the differentiation of ESCC cellsObjective: p-Hydroxylcin-namaldehyde(CMSP)is a novel member of the ethanol extracts of CMS(CMSE).This study reports that CMSP can inhibit viability,invasion and expression of malignant biomarkers in ESCC cells via inducing differentiation,and the underlying mechanism of the anti-cancer effect of the CMSP on ESCC cell lines was investigated.These research findings will provide scientific support for the novel use of CMSP as an differentiation inducer for clinical reatment of ESCC.Methods:1 Effect of CMSP on cell viability of ESCC(Kyse30?TE-13?Eca109? Kyse180)and normal oesophageal(HEEC)was determined by the MTS assay.2 Effect of CMSP at different concentration on cell cycle distribution and apoptosis was assessed by flow cytometry analysis of cells.3 The morphologic changes in the Kyse30 and TE-13 cells were observed by opitic and scanning electronic microscopy(SEM).4 Reverse transcription-quantitative polymerase chain reaction(RTqPCR)and ELISA assays were used to detect the effect of CMSP on expression of tumor related antigens(CEA,SCC)and malignant biomarkers(IL-6,MIC-1)of ESCC cells at mRNA and protein secretion level.5 Western blotting assay was used to detect the effect of CMSP on expression of C-myc and N-myc,the two malignant biomarkers of ESCC cells.6 The function of CMSP in the colony formation,migration and invasion of Kyse30 and TE-13 cells was determined by colony-formation,wound healing and transwell assays.And the Western blotting assay was aslo used to detect the effect of CMSP on expression of MMP2,E-cadherin,N-cadherin and Vimentin,the epithelial-to-mesenchymal transition(EMT)related biomarkers of ESCC cells.Results:1 Our results showed that the growth of ESCC cells(Kyse30,TE-13,Eca109 and Kyse180)was significantly inhibited by CMSP in a dose-and time-dependent manner(P<0.01).The numbers of these cells treated with CMSP were decreased significantly.By contrast,the growth and morphology of the normal oesophageal cell line HEEC were not changed(P>0.05).2 The results of flow cytometry revealed that CMSP increased the number of cells in G0/G1 phase and decreased the number of cells in S phase in Kyse30 and TE-13 cells,showing that CMSP significantly prevented cell cycle exit from G1 phase arrest(P<0.01).And,CMSP treatment did not significantly increase the number of Annexin V-FITC-positive cells,compared with that in the control group.Thus,apoptosis may not be involved in the growth arrest of Kyse30 and TE-13 cells treated with 10-40 ?g/ml CMSP(P>0.05).3 10-40 ?g/ml CMSP-treated cells showed typical dendrite-like cellular protrusions,and the percentage of such elongated cells was significantly and progressively increased with the increase in CMSP concentration(P<0.01).4 CMSP could decrease the expression of CEA,SCC,IL-6 and MIC-1 both in mRNA and protein secretion levels significantly in a dose-and timedependent manner(P<0.01).5 Western blotting analysis showed that C-myc and N-myc proteins were all decreased significantly after treatment with CMSP in a dose-and timedependent manner(P<0.01).6 The the colony formation,migration and invasion abilities of Kyse30 and TE-13 cells in vitro were reduced after treatment with CMSP(P<0.01).The levels of the expression of N-cadherin,vimentin and MMP2 proteins were reduced,whereas the level of the epithelial marker protein E-cadherin was increased in CMSP-treated Kyse30 and TE-13 cells,compared with the control(P<0.05).Conclusions:1 The growth,colony formation,migration and invasion abilities of Kyse30 and TE-13 cells in vitro can be obviously inhibited by CMSP.2 CMSP can inihibit the activity of ESCC cells through inducing differentiation,whereas not apoptosis.3 No inhibitory effect of CMSP was noticed on normal oesophageal cell line HEEC indicating that there might be less toxicity and damage of this novel drug.Part two The mechanism of the differentiation inducing effect of CMSP on ESCC cellsObjective: The aim of the present study was to characterize the mechanism in the differentiation of ESCC cells treated with CMSP,which might provide new potential strategies for ESCC treatment.Methods:1 The Relative isotopic labeling and absolute quantification(iTRAQ)analysis,as well as the GO(Gene Ontology)and pathway analysis were performed to detect the change of the proteins(>3 fold)and then the the key signalling pathway regulated by CMSP in ESCC cells was sifted out.2 ELISA assay was used to detect the effect of CMSP on cAMP secretion of ESCC cells.3 Western blotting and Pull down assays were used to detect the effect of CMSP on expression of RhoA,GTP-RhoA,P-38,p-P38,ERK1/2,p-ERK1/2,SAPK/JNK and p-SAPK/JNK in CMSP treated Kyse30 and TE-13 cells.4 To elucidate the underlying mechanism of signalling pathway in CMSP-induced cells differentiation,the effect of specific cAMP inhibitor(SQ22536)affecting on CMSP-induced differentiation of Kyse30 and TE-13 were investigated.5 Furthermore,the effect of specific cAMP enhancer(Forskolin),the inhibitor of ERK signalling pathway(PD98059)or the inhibitor of JNK signalling pathway(SP600125)affecting on differentiation of Kyse30 and TE-13 were investigated.Results:1 The result of iTRAQ analysis indicated that the 79 proteins were downreglated,whereas the 103 proteins were upregulated,in CMSP treated Kyse30 cells.After GO and pathway analysis,we found that there were 176 proteins can be characterized,which were related to various cellular activities and biochemical processes,and the pathways affected by CMSP were regulation of actin cytokeleton,cAMP,Insulin,splicesome,MAPK,Rho and so on.2 Increased expression of cAMP,p-P38 and decreased expression of ERK1/2,SAPK/JNK and GTP-RhoA,were detected after treatment with CMSP(P<0.05).3 The cAMP inhibitor SQ22536 blocked cells proliferative inhibition and G0/G1 phase arrest in Kyse30 and TE-13 cells treated with CMSP(P<0.01).SQ22536 aslo impeded the morphologic changes induced by CMSP(P<0.01).Moreover,block of cAMP by SQ22536 markedly increased the expression of CEA,SCC,IL-6 and MIC-1 in Kyse30 and TE-13 cells treated with CMSP in protein secretion level(P<0.01).The expression of C-myc and N-myc was aslo upregulated significantly by SQ22536 in Kyse30 and TE-13 cells treated with CMSP(P<0.01).Furthermore,ELISA revealed that the cAMP content was significantly decreased by SQ22536 in the culture supernatant of Kyse30 and TE-13 cells treated with CMSP(P<0.01).And the expression of GTP-Rho A,p-ERK1/2 and p-SAPK/JNK were upregulated significantly by SQ22536(P<0.01),while the expression of p-P38 was not changed in Kyse30 and TE-13 cells treated with CMSP(P>0.05).Taken together,SQ22536 can partly reverse the differentiation induced by CMSP.4 Forskolin,PD98059 and SP600125 can led to differentiation of Kyse30 and TE-13 cells,whose appearance was similar to the ESCC treated with CMSP.Taken together,the above data indicated that there is an axis of cAMP-RhoA-ERK/JNK regulating the differentiation of Kyse30 and TE-13 cells,which could be affected by CMSP.However,the mechanism underlying the activation of the P38 pathway remains unknown.Conclusions: In the present study,we found that decreased GTP-Rho A,p-ERK1/2,and p-SAPK/JNK and increased cAMP in CMSP-treated ESCC cells,indicated that cAMP-induced RhoA activation blockade and down regulation of ERK/JNK signalling pathway might be the key factor affected by CMSP regulating the differentiation of ESCC cells.Part three Effect of CMSP on the growth and differentiation of ESCC cells in vivoObjective: To investigate the effect of CMSP on viability of ESCC in vivo,the Kyse 30 nude mice model was used to evaluate the effect of CMSP on Kyse30 cell viability in vivo.Methods:1 Balb-c/null mice were used in the in vivo tumor growth assay.Kyse30 cells were harvested with trypsin solution and resuspended in PBS.Cells(1×106 cells/mouse)in 0.1 ml were injected subcutaneously into balb-c/null mice,after the formation of tumor,the Kyse 30 cells(2×105 cells/mouse)were injected into caudal vein.The stock solution of CMSP,CDDP and ATRA was resolved in PBS,and the final concentration of ethanol was less than 0.5%.The mice were divided randomly to five groups(6 mice/group)and were injected paratumor since the 11 th day: GI: Control group treated with PBS once every two days;GII: CMSP(10 mg/kg)group treated once every two days;GIII: CMSP(20 mg/kg)group treated once every two days;GIV: Cisplatin(CDDP)(2 mg/kg)group treated once every two days;and GV: ARTA(10-2 mmol/kg)group treated once every two days and all mice were sacrificed by cervical dislocation on day 32.2 The volume of tumors was oberserved every two days,and the weight of tumor tissues was detected after the tumors were removed.3 Pull down and Immunohistochemical staining assays were performed to detect the expression of GTP-RhoA,p-ERK1/2,p-SAPK/JNK,MMP2,E-cadherin,N-cadherin,Vimentin,N-myc and C-myc in tumor tissues.4 The concentration of CEA,SCC,IL-6 and MIC-1 in serum of mice was detected using ELISA.5 The lungs were removed,washed with PBS,and stained with haematoxylin and eosin(HE),to observe the formation of tumor node in lung tissue.6 The liver,spleen and kidney were removed,washed with PBS,and stained with HE,to evaluate the toxicity and damage of CMSP.Results:1 The volume and weight of tumors in mice treated with CDDP,ATRA or CMSP at different doses were significantly lower than those in control mice(P<0.05).Histology showed that no apoptotic and necrotic cancer cells were found in control,CMSP-treated and ARTA-treated tumors;by contrast,conspicuous apoptotic and necrotic cancer cells were seen in CDDP-treated tumors.2 ELISA also revealed that the concentration of CEA,SCC,IL-6 and MIC-1 in the serum of CMSP-treated mice were lower than those in the serum of control mice(P<0.05).And immunohistochemistry showed that CMSPtreated tumors expressed lower levels of C-myc and N-myc(P<0.01).3 Histology showed that no metastatic cancer nodules was found in lungs of CMSP-treated mice;in contrast,several metastatic nodules were seen in control mice,indicating that CMSP was capable of impairing the metastasis ability of ESCC cells.4 Pull down assay and immunohistochemistry showed that CMSP-treated tumors expressed lower levels of GTP-RhoA,p-ERK1/2,p-SAPK/JNK,MMP2,N-cadherin,Vimentin and higher levels of E-cadherin(P<0.05).5 Moreover,no pathological changes were noticed in the liver and spleen tissues in both the control and CMSP-treated groups.Conclusions:All of the above indicated that CMSP can inhibit the viability of ESCC cells via inducing differentiation in vivo.Part four The study of the reversal effect on progression of oesophageal precancerous lesions and immunoregultion function of CMSPObjective: This study will further investigate the reversal effect of CMSP on oesophageal precancerous lesions and the specific mechanism in mice model.These research findings will provide scientific support for the novel use of CMSP as an extract from traditional Chinese medicine for ESCC precaution and treatment.Methods:1 Drinking water containing 100 ?g/ml 4NQO was used to induce esophageal precancerous lesions in C57BL/6 mice.The stock solution of CMSP,CDDP and ATRA was resolved in PBS,and the final concentration of ethanol was less than 0.5%.The mice were divided randomly to five groups(45 mice/group): GI: Control group drinked with water from the 1st week;GII: Model group drinked with water containing 100 ?g/ml 4NQO from the 1st week to the 15 th week,and drinked with water from the 16 th to 30 th week;GIII: CMSP(20 mg/kg)precaution group drinked with water containing 100 ?g/ml 4NQO from the 1st week to the 15 th week,and treated with CMSP(20 mg/kg)once every two days from 1st week;GIV: CMSP(20 mg/kg)treatment group drinked with water containing 100 ?g/ml 4NQO from the 1st week to the 15 th week,and treated with CMSP(20 mg/kg)once every two days from the 16 st week;GV: ARTA group drinked with water containing 100 ?g/ml 4NQO from the 1st week to the 15 th week,and treated with ATRA(10-2 mmol/kg)once every two days from the 16 st week.All mice were sacrificed by cervical dislocation on the 18 th,24th,and 30 th week.2 During the procession,general conditions and body weights of mice were observed.The oesophageal tissues were removed,washed with PBS,and the pathological changes of esophagi were evaluated by HE staining.3 Pull down,immunohistochemical staining and western blotting assays were performed to detect the expression of GTP-Rho A,p-ERK1/2,p-SAPK/ JNK,N-myc,and C-myc in oesophageal tissues.4 The concentration of CEA,SCC,IL-6 and MIC-1 in serum of mice was detected using ELISA.5 The concentration of IFN-??TNF-? and sB7-H4 in serum of mice was detected using ELISA;The percentage of various lymphocyte subtypes in the peripheral blood was detected by FCM;Immunohistochemical staining was performed to detect the expression of B7-H4?STAT3 and p-STAT3 in oesophageal tissues.6 The liver,spleen and kidney were removed,washed with PBS,and stained with haematoxylin and eosin(HE),to evaluate the toxicity and damage of this drug.Results:1 From 18 th to 30 th week,there were visible pathological changes in esophageal tissues of model mice.Moreover,with the inducing time going,the degree of the lesions increased gradually.However,the incidence and degree of the lesions of CMSP precaution,treatment and ATRA treatment groups was lower than mice in model group(P<0.05).The treatment effect of CMSP was similar to ATRA,indicating that novel use of CMSP as an differentiation inducer for ESCC precaution and treatment.2 ELISA assays also revealed that the concentration of CEA,SCC,IL-6 and MIC-1 in the serum of CMSP-treated mice were lower than those in the serum of control mice(P<0.05).Immunohistochemistry and Western blotting showed that oesophageal tissues of mice in CMSP precaution and treatment expressed lower levels of C-myc and N-myc(P<0.05).3 Immunohistochemistry,Western blotting and pull down assays showed that oesophageal tissues of mice in CMSP precaution and treatment expressed lower levels of GTP-RhoA,p-ERK1/2 and p-SAPK/JNK than model group(P<0.05).4 During the whole process of ESCC tumorigenesis,the number and activity of peripheral lymphocytes as well as the spleen index decreased gradually(P<0.05).As compared with the model group,the number and activity of peripheral lymphocytes as well as the spleen index of mice in CMSP precaution or treatment group were significantly higher(P<0.05).5 FCM showed that,during the whole process of ESCC tumorigenesis,the CD4~+T cells,CD8+T cells and NK cells ratios notably decreased,whereas CD4 + CD25 + Treg cells ratio in peripheral lymphocytes of mice in model group obviously increased gradually(P<0.05).As compared with the model group,the CD4~+T cells,CD8+T cells and NK cells ratios were significantly higher,while the CD4 + CD25 + Treg cells ratio was obviously lower in peripheral lymphocytes of mice in CMSP precaution or treatment group(P<0.05).6 During the whole process of ESCC tumorigenesis,the concentration of sB7-H4,IL-6 and MIC-1in the serum of mice in model group increased,while the concentration of IFN-? and TNF-? decreased gradually(P<0.01).However,the concentration of sB7-H4,IL-6 and MIC-1 was lower,while the concentration of IFN-? and TNF-? was higher in the serum of mice in CMSP precaution or treatment group(P<0.01).Immunohistochemistry also showed that oesophageal tissues of mice in CMSP precaution and treatment expressed lower levels of B7-H4,STAT3 and p-STAT3 than model group(P<0.01).As we have shown previously,CMSP can improve the immune function during the process of ESCC tumorigenesis;and the B7-H4/STAT3 signalling pathway might be the key target for the immune regulation function of CMSP.7 No obvious pathological changes were noticed in the liver,spleen and kidney tissues in both the model and CMSP precaution and treatment groups.Conclusions:1 CMSP can reverse the progression of esophageal precancerous lesions via inducing differentiation.2 CMSP can improve the immune function,during the process of ESCC tumorigenesis,via suppressing B7-H4/STAT3 signalling pathway.3 No pathological changes were noticed in the liver,spleen and kidney tissues in both the CMSP precaution and treatment groups.indicating that there might be less toxicity and damage of this novel drug. |
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