Expression Of Caveolin-1 In Esophageal Squamous Cell Carcinoma And Effect Of Caveolin-1 On Multidrug Resistance Of ESCC Cell

Esophageal carcinoma is one of the malignant tumors in China. The incidence rates of esophageal cancer in Linzhou, located in the Taihang Mountain Area in Henan Province, were ranked first in the world. Esophageal squamous cell carcinoma(ESCC), the major histological form of esophageal cancer in China, has characters of strong invasiveness, dismal prognosis, rapid clinical progress, lymph node metastasis,and easy recurrence. The 5 year survival rate of ESCC is only about 10%.As a major marker of caveolae, Caveolin-1(Cav-1) is capable of interacting with some proteins depending on its several functional domains including caveolin scaffolding domain(CSD), transmembrane domain(TMD) and Tyrosine14. Cav-1can regulate activities of multiple signaling pathways to affect cellular proliferation,differentiation, migration and apoptosis. Cav-1 plays important roles in in oncogenic cellular transformation, hyperplasia and metastasis during tumor progression. Reports suggested that Cav-1 not only has abnormal expression patterns in many tumors, but also plays distinct roles in different tumor. For example, Cav-1 was overexpression in prostate, kidney and breast and acting as an oncogene; while in lung, colon, stomach and ovarian cancer, Cav-1 down-regulated and acts as tumor suppressors. Therefore,Cav-1 and its downstream signal transduction pathway have become one of the hot spots in the present research of cancer.Researchers discovered that Cav-1 was up-regulated in tumor-adjacent tissuesand ESCC, which suggested that Cav-1 can promote ESCC development. And there is almost no report on the correlation between Cav-1 expression and multidrug resistance of ESCC. In order to make up for the lack of research in this field, this study was first focus on checking the expression of Cav-1, P53, BDNF, P-gp, MRP1 and β-catenin in ESCC, matched adjacent tumor tissues and adjacent normal-looking tissues, then analyze the correlations between Cav-1 and P53, BDNF, P-gp, MRP1 and β-catenin; Second, the study investigate whether high expression of cav-1 can change the multidrug resistance in ESCC cell line Ec9706 using gene overexpression and drug efflux; at last, we further analyze whether knockdown cav-1 expression can change the multidrug resistance in ESCC cell line Ec9706 using RNAi and drug efflux. This study not only provides a new way for researching mechanism on the development of esophageal cancer, but also provides a new target for the clinical treatment and a scientific basis for reducing the drug resistance in ESCC.PartⅠ: The expression and correlation analysis of Caveolin-1 and proteins related multidrug resistance in esophageal squamous cell carcinomaMethods:1.Immunohistochemistry was use to check the expression of Cav-1, P-gp, MRP1,P53, β-catenin and BDNF in 86 samples of esophageal squamous cell carcinoma,matched adjacent tumor tissues and adjacent normal-looking tissues.2. Statistical analysis was performed using the Statistical Package for Social Sciences(SPSS, version 21.0). The univariate association was assessed the χ2 test and Spearman rank correlation coefficient analysis, P value of <0.05 was considered statistically significant.Results:1. In ESCC tissues, matched adjacent tumor tissues and adjacent normal-looking tissues, positive Cav-1 was mainly expressed in cytomembrane and cytoplasm and positive rates were 61.63%, 38.37%, 15.11%, respectively. The difference between the three groups were significant(P<0.05).2. In the adjacent normal-looking tissues, matched adjacent tumor tissues and ESCC tissues, the positive expression rate of MRP1, P-gp, P53, BDNF were increased, the positive rates were 17.44%, 39.53%, 63.95%; 22.09%, 44.18%,72.09%; 9.30%, 46.51%, 59.30%; 6.98%, 10.47%, 18.6%; respectively. And the differences between groups of MRP1, P-gp, P53 were significant(P<0.05), while no significant differences between groups of BDNF(P>0.05). No change of β-catenin expression was found in the adjacent normal-looking tissues, matched adjacent tumor tissues and ESCC tissues, while its positive expression rates in cytoplasm and nucleus were increased, the positive rates were8.14%、52.32%、80.23%, and the difference between groups were significant(P<0.05).3. Statistical analysis using SPSS21.0 showed that expression of Cav-1 during ESCC development was positively correlated with the expression of P-gp(γ=0.77,P=0.000), MRP1(γ= 0.453, P=0.000), and P53(γ= 0.581, P=0.000); Cav-1 has no correlation with BDNF( γ =0.000, P=0.391) or β-catenin, while has positively correlation with β-catenin, which expressed in cytoplasm and nucleus(γ=0.003;P=0.000).Part Ⅱ: The effect of the expression level of Caveolin-1 on proteins related to multidrug resistance in Ec9706 cellMethods:1. Gene overexpression and RNAi were used to study the role of Cav-1 in drug resistance of ESCC; And RT-PCR and Western blots were used to examine the m RNA and protein expression levels of Cav-1 when transfected with cav-1overexpression vector and cav-1 RNAi vector in Ec9706 cells.2. RT-PCR and Western blots were used to examine the m RNA and protein expression levels of MRP1、P-gp、P53、BDNF、β-catenin when cav-1overexpression and cav-1 knockdown in Ec9706 cells.3. Effects of Caveolin-1 expression on efflux of Calcein-AM and Rh123 in Eca109 cells were measured by drug efflux and photo by fluorescence microscope.4. Statistical analysis was performed using the Statistical Package for Social Sciences(SPSS, version 21.0). All data were shown as mean±SD. Differences among the groups were tested by Student’s t test, the comparison of multiple sample mean using One-way ANOVA. Densitometric analyses of Western blots and Fluorescence intensity were performed using software of Image-Pro Plus 6.0. P value of <0.05 was considered statistically significant.Results:1. Cav-1 overexpression vectors and cav-1 RNAi vectors are successfully constructed:Compared to control vector groups, the relative expression amount of Cav-1m RNA and protein in Ec9706 cell transfected with Cav-1 overexpression vector after48 h and 72 h are 221.46 ± 25.14, 290.13 ± 43.36; 3.19, 5.97; respectively. The expression level are significant increased(P<0.01) and suggested the cav-1overexpression vector was successfully constructed.The relative expression amount of Cav-1 m RNA and protein in Ec9706 cell transfected with Cav-1 si RNA vector of P3-1 and P3-3 after 72 h, has no difference with that transfected with control vector groups(P > 0.05), while the relative expression amount of Cav-1 m RNA and protein were obviously decreased in Ec9706 transfected with Cav-1 si RNA vector of P3-2 after 72h(P<0.01). The relative expression amount of cav-1 m RNA and protein were 0.454±0.17, 0.52,respectively. The result suggested P3-2 vector is successfully constructed for cav-1knockdown.2. The effect of cav-1 expression level on the expression of multidrug resistance associated protein P-gp, MRP1, P53, BDNF, and β-catenin in Eca9706 cells(1) The effect of cav-1 expression level on the expression of P-gpThere was no difference of expression of P-gp m RNA and protein in cav-1overexpression groups(0.84 ±0.31, 0.72±0.20; 1.01, 0.91, respectively) and control vector groups(P>0.05), while expression of P-gp m RNA and protein(0.56±0.07, 0.71) were obviously decreased in Cav-1 RNAi groups compared to control vector groups(P<0.05).(2) The effect of Cav-1 expression level on the expression of MRP1Compared to control vector groups, the expression of MRP1 m RNA and protein in Cav-1 overexpression groups(1.58 ±0.18, 3.55±0.21; 1.76, 3.51; respectively)were significant increased(P<0.05). And the expression of MRP1 m RNA and protein(0.33 ±0.09, 0.62) were obviously decreased in Cav-1 RNAi groups compared to control vector groups(P<0.05).(3) The effect of Cav-1 expression level on the expression of P53The expression of P53 m RNA and protein in Cav-1 overexpression groups were0.90 ±0.13, 2.30±0.11; 1.14, 0.95; respectively. And compared to control vector groups, the P53 m RNA was significantly increased transfection after 72h(P<0.05),while the protein has no change; the expression of P53 m RNA and protein(0.63±0.14, 0.42) were obviously decreased in Cav-1 RNAi groups compared to control vector groups(P<0.05).(4) The effect of Cav-1 expression level on the expression of β-cateninCompared to control vector groups, the expression of β-catenin m RNA and protein in Cav-1 overexpression groups(0.98±0.10, 1.27±0.07; 1.20, 0.98) and in Cav-1 RNAi groups(0.92 ±0.08、0.89), and there was no difference existed(P>0.05).(5) The effect of Cav-1 expression level on the expression of BDNFNo m RNA and protein of BDNF was detected in Ec9706, control vector groups,Cav-1 RNAi groups and Cav-1 overexpression groups.3. Effect of Caveolin-1 on the efflux of Calcein-AM and Rhodamine123In Cav-1 overexpression cells at 48 h and 72 h, the fluorescence intensity of Calcein-AM(790540.2±1145.1, 714629.4±1788.4) showed no difference with that in control groups(844086.6±762.7, 687789.1±2906.2)(P>0.05); And the fluorescence intensity of Rhodamine123(720408.4±5210.1, 737540.7±3353) also has no difference with that in compared cells(768775±1106.5, 846575±5651.4)(P>0.05).In Cav-1 knockdown cells, fluorescence intensity of Calcein-AM(1291559±4326.5)were obviously increased than that in control vector groups(875417±731.5)(P <0.01), and the fluorescence intensity of Rhodamine123(1119115±794.6) was also increased than that in compared cells(809929.7±256.4)(P<0.01).Conclusions:1. The overexpression of Cav-1, P53, P-gp and MRP1 maybe promote ESCC development. There is no correlation between expression level of β-catenin and ESCC development, while β-catenin translocation from cytomembrane to cytoplasm and nucleus maybe has close relation with ESCC occurrence.2. During tumorgenesis of ESCC, the expression of Cav-1 has close correlation with P-gp, MRP1, P53 and β-catenin expressed in cytoplasm and nucleus; while has no correlation with BDNF.3. Cav-1 overexpression displays no role in the m RNA and protein expression of P-gp and P53; while cav-1 knockdown inhibits the m RNA and protein expression of P-gp and P53. This result indicated that the regulatory effect of Cav-1 on the expression of P-gp and P53 in ESCC maybe has dose effect.4. Cav-1 has close relation with tumor drug resistance in ESCC: The change of expression level of Cav-1 can affect the role of drug efflux in ESCC cells by regulating expression of P-gp and MRP1. Therefore, Cav-1 can be used as an effective target for reversing multidrug resistance in esophageal squamous cell carcinoma.