| Esophageal cancer, a highly aggressive digestive neoplasm, is the sixth-mostcommon cause of cancer death in the world. Based on histological examination,esophageal cancer includes two histological types: adenocarcinoma and squamouscell carcinoma, both with an equally poor prognosis. Although the incidence of theformer has increased in the United States and Europe over the past three decades,esophageal squamous cell carcinoma (ESCC) is still the dominant histological typeworldwide. Moreover, there is wide geographic variation in the incidence ofesophageal cancer, with the highest rates occurring in China, South-Central Asia, andEastern and Southern Africa, especially in Linzhou and Anyang city of Henanprovince, China. Currently,5-year survival rates of ESCC are also dismal (5–10%),with over50%of patients harboring distant metastases at the time of presentation.Therefore, it is particularly important to seek new powerful molecular markers todiagnose ESCC and improve the prognosis of patients with ESCC.Human Cervical Cancer Oncogene1(HCCR-1) was found to be associated withthe occurrence and development of a variety of tumors. Ko et al have screened thecandidates of differentially expressed genes in primary cancer tissues, metastaticfocus, and cancer cell lines by using DDRT-PCR. We have identified several noveloncogenes including HCCR which were overexpressed in primary cervical cancers and cervical cancer cell lines. HCCR oncogenes are classified into two types,HCCR-1and HCCR-2, according to molecular characteristics. These two proteins arealternative splicing variants. HCCR1encodes360amino acids (~42kDa) andHCCR2encodes304amino acids (~36kDa) molecules. There are two potentialNmyristylation sites, two potential phosphorylation sites for protein kinase C,N-glycosylation site, and hydrophobic trans-membrane domain comprising20aminoacids, implying the diversity of its function in tumors. Currently, more and moreevidence has demonstrated that HCCR-1as oncogene plays an essential role in theoccurrence and development of tumors and is overexpressed in many tumors,including pancreatic cancer, colon carcinoma and hepatocellular carcinoma, etc,indicating HCCR-1may be a molecular target for the diagnosis and therapy of thesetumors.Most importantly, HCCR-1affected tumor occurrence and development bymany different molecular mechanisms. Studies have revealed that TCF/beta-cateninsignaling pathway plays an important role in HCCR-1oncogene expression, andepidermal growth factor induces HCCR expression via PI3K/Akt/mTOR signalingpathway in pancreatic cancer PANC-1cells. These studies suggest that HCCR-1isimplicated in many molecular mechanisms in the occurrence and development, andthus reveal its importance.Gefitinib is an orally active EGFR tyrosine kinase inhibitor, which hasdemonstrated antitumor activity in vitro or in vivo against various kinds of humancancer cell lines, including head/neck, ovary, breast, lung, bladder, malignantmesothelioma, and colon cancer. Recently, the Food and Drug Administrationapproved gefitinib for previously treated, advanced non-small cell lung cancer andclinical trials in other tumors are ongoing. Phase I and II studies indicate that gefitinibis generally well tolerated and has strong evidence of clinical antitumor activity.Moreover, gefitinib as selective inhibitor of EGFR blocked extraneous EGF-inducedVEGF-C and VEGF-A expressions in head and neck neoplasm. These studies abovesuggests the internal correlations among HCCR-1, gefitinib and EGF, therefore, inthe current study, we elucidated the underlying role of HCCR-1and gefitinib in theoccurrence and development of ESCC and its molecular regulation mechanisms in our following three parts research, which will provide theoretical basis for themolecular target therapy of ESCC.Part I Expression of HCCR-1in esophageal squamous cell carcinomatissues and its association with the prognosis of the patientsMethods(1) HCCR-1mRNA expression was detected by in situ hybridization technologyin158cases of ESCC tissues,105cases of paracancerous dysplasia tissues and158cases of normal esophageal epithelial tissues.(2) HCCR-1protein expression was detected by immunohistochemistry methodin158cases of ESCC tissues,105cases of paracancerous dysplasia tissues and158cases of normal esophageal epithelial tissues.(3) HCCR-1mRNA level was detected by real-time quantitative PCR inrandomly selected6cases of ESCC tissues and corresponding normal esophagealepithelial tissues.(4) HCCR-1protein level was detected by Western blotting in randomly selected6cases of ESCC tissues and corresponding normal esophageal epithelial tissues.(5) Kaplan-Meier survival curve was utilized to investigate the betweenexpressions of HCCR-1mRNA and protein and the survival time of the patients withESCC.(6) Statistical analysis: Statistical analysis was performed by SPSS17.0software,all data were expressed as x s. The results of in situ hybridization andimmunohistochemistry were evaluated by chi-square, the results of real-timequantitative PCR and Western blotting were carried out by One-way ANOVA, and theassociations of HCCR-1mRNA and protein expressions with the prognosis of thepatients with ESCC were investigated by Kaplan-Meier survival curve. A P value lessthan0.05was considered as statistical significance. Results(1) HCCR-1mRNA was mainly localized in the cytoplasm of esophageal cancercells, positive ratio of HCCR-1mRNA in ESCC tissues (77.22%) was significantlyhigher than in paracancerous dysplasia tissues (36.19%) or normal esophagealepithelial tissues (3.16%), and the differences were statistical significance (P<0.05).(2) HCCR-1protein was mainly localized in the cytoplasm of esophageal cancercells, positive ratio of HCCR-1protein in ESCC tissues (79.75%) was significantlyhigher than in paracancerous dysplasia tissues (40.95%) or normal esophagealepithelial tissues (3.80%), and the differences were statistical significance (P<0.05).(3) Expressions of HCCR-1mRNA and protein were not related to the sex andage of the patients with ESCC, but closely correlated with hislogical grade, TNMstaging and lymph node metastasis (P<0.05). Notably, positive ratios of HCCR-1mRNA and protein were both100%in80cases of metastatic ESCC patients.(4) Correlation analysis revealed that110of the122HCCR-1mRNA-positiveESCC tissues displayed the HCCR-1protein-positive expression, whereas20of36HCCR-1mRNA-negative ESCC tissues exhibited the HCCR-1protein-negativeexpression, and the expressions of HCCR-1mRNA and protein had significantcorrelation (γ=0.447, P<0.001).(5) The results of real-time quantitative PCR and Western blotting demonstratedthat HCCR-1mRNA and protein levels in randomly selected6cases of ESCC tissueswere significantly higher than that in corresponding normal esophageal epithelialtissues, and the differences were statistical significance (P<0.05).(6) During the follow-up period,72of the104HCCR-1mRNA–positive ESCCpatients (69.23%) had died, whereas17of the54HCCR-1mRNA–negative orHCCR-1mRNA–weakpositive ESCC patients (31.48%) had died, as determined bythe log-rank test (Mantel-Cox), the survival rate of patients with low-level HCCR-1mRNA staining was higher than those patients with high-level HCCR-1mRNAstaining.(7) During the follow-up period,77of the113HCCR-1protein–positive ESCCpatients (68.14%) and whereas15of the45HCCR-1protein–negative or HCCR-1protein–weak positive ESCC patients (33.33%) had died, as determined by the log-rank test (Mantel-Cox), the survival rate of patients with low-level HCCR-1protein staining was higher than those patients with high-level HCCR-1proteinstaining.(8) Multivariate analysis using Cox proportional hazards model showed thatclinical stage and lymph node metastases were independent prognostic factors ofESCC.Part II Targeting inhibition of HCCR-1expression enhances thesensitivity of gefitinib on esophageal squamous cell carcinomaMethods(1) HCCR-1mRNA and protein expressions were detected by real-timequantitative PCR and Western blotting technology in3of ESCC cell lines (Eca109,EC1and EC9706) and Non-cancerous esophageal epithelial cell Het-1A.(2) HCCR-1siRNA was used to transfect the ESCC EC1cells, and expressionsof HCCR-1mRNA and protein were investigated after transfection with HCCR-1siRNA by real-time quantitative PCR and Western blotting methods.(3) Cell proliferation was detected by CCK-8kit at24h,48h,72h and96hafter transfection with HCCR-1siRNA, meanwhile, different concentration gefitinib(0.1M,1M,10M,20M and40M) were added to EC1cells, and cellproliferation was measured at48h after treatment.(4) The changes of cell cycle and apoptosis were investigated by Flow cytometryin different treatment groups including untreated group, control siRNA group,HCCR-1siRNA group, gefitinib group and combinations of HCCR-1siRNA andgefitinib groups.(5) The changes of cell invasion were detected by Transwell chamber in differenttreatment groups.(6) Expressions of cell cycle related proteins (cyclin D1, cdk2and p21), cellapoptosis related proteins (bcl-2and bax), cell invasion associated proteins (MMP-2and MMP-9) as well as key proteins (p-Akt and total protein) of Akt signaling pathway were investigated by Western blotting in different treatment groups.(7) Statistical analysis: statistical analysis was performed by SPSS17.0software,all data were expressed as x s. The comparison of mutiple sample tests werecarried out by One-way ANOVA and a P value less than0.05was considered asstatistical significance.Results(1) Relative levels of HCCR-1mRNA and protein in3of ESCC cell lines(Eca109, EC1and EC9706) were significantly higher than that in non-cancerousesophageal epithelial cell Het-1A, whereas relative levels of HCCR-1mRNA andprotein in EC1cells were markedly higher than those in Eca109and EC9706cells,and the differences were statistical significance (P<0.05).(2) Compared with untreated group and control siRNA group, expressions ofHCCR-1mRNA and protein were significantly downregulated at24h,48h and72hafter transfection with HCCR-1siRNA, and the differences were statisticalsignificance (P<0.05), in which expressions of HCCR-1mRNA and protein reachedthe lowest level at48h after transfection with HCCR-1siRNA.(3) The results of CCK-8proliferation experiment demonstrated that comparedwith untreated group and control siRNA group, the proliferation of ESCC EC1cellswas obviously inhibited at different time point after transfection with HCCR-1siRNA,and the differences were statistical significance (P<0.05), however, there was nodifference in the proliferation of EC1cells between untreated group and controlsiRNA group (P>0.05).(4) Gefitinib suppressed the proliferation of EC1cells in a dose-dependentmanner, and40M of gefitinib had the best inhibitory effect on ESCC EC1cells, andinhibitory ratio was92.14%, which was significantly higher than the other groups,and the differences were statistical significance (P<0.05). Additionally, the inhibitoryratios of1M and10M of gefitinib on ESCC EC1cells were42.5%and56.5%,respectively, compared with the other groups, the differences were statisticalsignificance (P<0.05). (5) The percentages of cell number in G0/G1phase in untreated group andcontrol siRNA group were45.390.64%and46.370.63%, respectively, and therewas no statistical difference between untreated group and control siRNA group(P>0.05). Compared with untreated group and control siRNA group, the percentageof cell number in G0/G1phase in HCCR-1siRNA group (58.330.83%), gefitinibgroup (57.630.76%) and combination group (71.580.87%) was significantlyincreased, and the differences were statistical difference (F=600.333, P=0.000). Mostimportantly, the percentage of cell number in G0/G1phase in combination groupreached the highest. Moreover, the percentages of cell number in S phase in untreatedgroup and control siRNA group were24.150.89%and22.021.45%, respectively,and there was no statistical difference between untreated group and control siRNAgroup (P>0.05), whereas the percentages of cell number in S phase in HCCR-1siRNA group and gefitinib group were29.301.99%and29.430.79%, and there wasno statistical difference (P>0.05), which was significantly higher than that inuntreated group and control siRNA group, and the differences were statisticalsignificance (P<0.05). Notably, the percentage of cell number in S phase was13.050.80%, which was evidently lower than that in the other groups (P<0.05).(6) The early apoptotic ratios of ESCC EC1cells in HCCR-1siRNA group,gefitinib group and combination group were26.731.73%,23.632.48%and31.792.04%, which was significantly higher than those in untreated group(11.391.62%) and control siRNA group (11.391.62%), and the differences werestatistical significances (F=71.196, P=0.000), whereas there was no difference inearly apoptotic ratio of ESCC EC1cells between untreated group and control siRNAgroup (P>0.05), in addition, compared with the other groups, the early apoptotic ratioof ESCC EC1cells in combination group was obviously elevated, and the differenceswere statistical significance (P<0.05).(7) The invading cell numbers of ESCC EC1cells in HCCR-1siRNA group,gefitinib group and combination group were139.227.37,160.626.75and48.822.77, which was significantly lower than those in untreated group(269.426.70) and control siRNA group (252.827.90), and the differences werestatistical significances (F=58.244, P=0.000), whereas there was no difference in invading cell numbers of ESCC EC1cells between untreated group and controlsiRNA group (P>0.05), in addition, compared with the other groups, the invading cellnumber of ESCC EC1cells in combination group was obviously reduced, and thedifferences were statistical significance (P<0.05).(8) There were no differences in the expressions of cell cycle related proteins(cyclin D1, cdk2and p21), cell apoptosis related proteins (bcl-2and bax), cellinvasion related proteins (MMP-2and MMP-9) and key proteins (p-Akt and total Akt)of Akt signaling pathway between untreated group and control siRNA group(P>0.05), however, compared with untreated group and control siRNA group, theexpressions of cyclin D1, cdk2, bcl-2, MMP-2, MMP-9and p-Akt proteins weresignificantly reduced in HCCR-1siRNA group and gefitinib group, whereas theexpressions of p21and bax were evidently elevated, and the differences werestatistical significance (P<0.05). Notably, compared with the other groups, theexpression changes of the proteins above were most notable, and the differences werestatistical significance (P<0.05).Part III The role of HCCR-1siRNA combinated with gefitinib inESCC implanted in nude miceMethods(1) Experimental grouping: untreated group: no any therapy; control siRNAgroup: control siRNA was injected into tumors, one time every three days; HCCR-1siRNA group: HCCR-1siRNA was injected into tumors, one time every three days;gefitinib group: oral administration gefitinib (150mg/kg); combination of HCCR-1siRNA and gefitinib group: HCCR-1siRNA was injected into tumors, one time everythree days, meanwhile, oral administration gefitinib (150mg/kg).(2) ESCC EC1cells were harvested, and5×106cells were subcutaneouslyinjected into the right blank of back in nude mice. When tumor volume reached about100mm3, the treatment began according to experimental grouping, long diameter a(mm) and short diameter b (mm) were measured using caliper every three days, and formula V=1/2ab2(mm3) was used to obtain the tumor volume of each nude mice,meanwhile, tumor growth curve was made. When the treatment was finished, thenude mice was euthanasiaed, tumor was peeled, the weight of tumor was measured,and inhibitory ratio of tumor was calculated according the formula: tumor inhibitoryratio=[(tumor weight in control group-in experimental group)/tumor weight in controlgroup]×100%.(3) Expression of Ki-67protein was detected by immunohistochemistry methodin different treatment nude mice tumor tissues.(4) HCCR-1mRNA expression was investigated by real-time quantitative PCRmethod in different treatment nude mice tumor tissues.(5) Expressions of HCCR-1, p-Akt and total Akt proteins were examined byWestern blotting in different treatment nude mice tumor tissues.(6) Statistical analysis: statistical analysis was performed by SPSS17.0software,all data were expressed as x s. The comparison of mutiple sample tests werecarried out by One-way ANOVA and a P value less than0.05was considered asstatistical significance.Results(1) There was no difference in nude mice tumor volume between untreated goupand control siRNA (P>0.05), however, compared with untreated group and controlsiRNA group, tumor volume was significantly reduced in HCCR-1siRNA group andgefitinib group, and the differences were statistical significance (P>0.05). Notably,tumor volume in combination group was signicantly lower than those in the othergroups, and the differences were statistical significances (P<0.05).(2) There was no difference in nude mice tumor weight between untreated goupand control siRNA (P>0.05), however, compared with untreated group and controlsiRNA group, tumor inhibitory ratios in HCCR-1siRNA group and gefitinib groupwere38.83%and43.92%, and their tumor weight was markedly reduced, and thedifferences were statistical significance (P<0.05). Furthermore, tumor inhibitoryratios in combination group was77.25%, and the differences were statistical significance (P<0.05).(3) There was no difference in the expressions of HCCR-1mRNA and protein innude mice tumor tissues between untreated goup and control siRNA (P>0.05),however, compared with untreated group and control siRNA group, expressions ofHCCR-1mRNA and protein were significantly down-regulated in HCCR-1siRNAgroup, gefitinib group and combination group, in which expressions of HCCR-1mRNA and protein were lowest.(4) Ki-67proliferation indexs in untreated group and control siRNA group was85.77±2.40%and84.87±2.42%, respectively, and there was no difference betweenuntreated group and control siRNA group (P>0.05), in addition, Ki-67proliferationindexs in HCCR-1siRNA group, gefitinib group and combination group were47.85±3.23%,56.52±3.73%and31.26±2.69%, respectively, and there were statisticaldifferences (P<0.05), in which Ki-67proliferation indexs in combination groupreached the lowest.(5) There was no difference in the expressions of p-Akt and total Akt proteins innude mice tumor tissues between untreated goup and control siRNA (P>0.05),however, compared with untreated group and control siRNA group, expressions ofp-Akt and total Akt proteins were significantly down-regulated in HCCR-1siRNAgroup, gefitinib group and combination group, in which expressions of p-Akt andtotal Akt proteins were lowest, and the differences were statistical significances(P<0.05).Conclusion(1) HCCR-1mRNA and protein at high level may be tightly associated with theoccurrence and development of ESCC.(2) HCCR-1mRNA and protein levels may be the molecular marker for themetastasis and prognosis of ESCC.(3) Combination of HCCR-1siRNA and gefitinib significantly suppresses ESCCcell proliferation, accumulates cell number in G0/G1phase, induces cell apoptosisand reduces cell invasion, which may be closely correlated with the expression changes of cell cycle related proteins (cyclin D1, cdk2and p21), cell apoptosis relatedproteins (bcl-2and bax), cell invasion related proteins (MMP-2and MMP-9) as wellas the activated status of Akt signaling pathway.(4) Combination of HCCR-1siRNA and gefitinib significantly inhibits thegrowth of ESCC implanted in nude mice, which may be tightly associated with thedecrease of Ki-67proliferation index and the inactivated status of Akt signalingpathway.(5) HCCR-1siRNA and gefitinib have synergistic effect in anti-ESCCproliferation in vitro and invo. |
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