Expression Of Aquaporin 1 And In Human Ovarian Carcinoma Tissue And Biological Effect

Water constitutes roughly 70% of the mass of most living organisms, 55%of the water is in the cell, 45% is outside the cell, The movement of wateracross cell membranes is fundamental to life. Aquaporin 1(AQP1) was firstdiscovered by Agre and expressed in many life forms. Aquaporin1 isexquisitely selective for the transport of water and related to the generation anddevelopment of many disorders. Researchers around the world have sought tolearn how AQP1 work. Recent research detect that various tumor tissuesexpress AQP1, it is highly expressed in the endothelial cells of capillary bloodvessel. AQP1 may closely related with the origin, growth and transfer etc ofthe tumor cells. Some researchers report that Acetazolamide inhibits the growthand migration of tumor by interacting with aquaporin-1.The pathologic mechanism of ovary malignant tumor is currently unclear.Ovarian carcinoma accompanied with ascites frequently, it is thought thatWater channel protein maybe related with ovarian carcinoma growth andtransfer. Nobody has reported whether AQP1 is expressed in ovarian carcinomatissue yet, say nothing of studies about influence on the ovary cancer cellgrowth by variety of AQP1 in the world. We aimed to determine the expressionof AQP1 in ovarian serous tumor and investigate how water channel proteininhibitor (acetazolamide) accelerate the apoptosis of ovary cancer cell byinhibit water channel protein in this study.1. Expression of Aquaporin 1 in human ovarian carcinomatissueMethods: A total of 66 frozen ovarian tissues of serous tumor and 10 ofnormal ovarian tissues were examined for the expression of AQP1 byimmunohistochemistry.Result: ⑴ positive rate of AQP1 expression was not statisticallydifferent between ovarian serous benign tumor (25%) and normal ovariantissue (20%), however, it was statistically difference between either ovarianserous borderline tumor (62.5%) or ovarian serous malignant tumor (82.6%)and normal ovarian tissue(20%)。⑵Expression of AQP1 in ovarian serousmalignant carcinoma has no relationship with its histological grade (p>0.05)and clinical stage (p>0.05). ⑶Expression of AQP1 was statistically differentbetween the cases of with and without ascites (p<0.05).2. Acetazolamide accelerated the apoptosis of ovary cancer cells(1) Apoptosis rate of ovarian tumor cells detected by flow cytometryMethod: After cultivating ovary cancer cell in vitro, adjusted theconcentration of cells to 1×105/ml, divided into 3 bottles of experiment groupand 1 bottle of comparison group, the culture fluid of experiment group wasreplaced by culture solution have different medicine concentration of 1×10-8mol/L,5×10-8 mol/L and 10×10-5 mol/L . The culture liquid of comparisongroup is invariant. Collected the cells on the 24h,48h,72h respectively fromeach bottles, adjusted the cell concentration to 1×106/ml, the PI dye, thenapoptosis rate of ovarian tumor cells rate in every group were detected 3 timesby flow cytometry.Result: ①Comparing with the comparison group, each experiment grouphas a statistically different apoptosis rate of ovarian tumor cells(P<0.05).②The apoptosis rate of ovarian tumor cells was statistically different betweenany two experiment groups (P<0.05).③The apoptosis rate of ovarian tumorcells increased along with the incubate time in every group. It was statisticallydifferent between 24h and 48h,24h and 72h,48h and 72h(P<0.05).(2)Examine cell survival rate by MTT methodMethod: After cultivating ovary cancer cell in vitro, adjusted theconcentration of cells to 1×105/ml, inoculated 12 bores with 50 μl of cellsuspension each and all in a 96 bore plastic plate. Join 50 μl of the reagentthing to nine of the 12 bores as the experiment group, ensuring that each threepores has a different patent medicine concentration of 1×10-8 mol/L L,5×10-8mol/L and 10×10-8 mol/L, the rest three bores didn't add the medicine as thecomparison group, three bores added culture solution only as zeroing bores.Then went along according to conventional MTT method.Result: ①Concentration of patent medicine affects the result. OD valueratio of experiment group and comparison group was inversely proportional topatent medicine concentration. It was statistically different between 1×10-5/Lgroup and each of the other two group(P<0.05), however, it was notstatistically different between 5×10-8 mol/L group and 10×10-8 mol/L group(P>0.05)。②Time of incubating also affects the result. OD value ratio ofexperiment group and comparison group was inversely proportional toincubation time,it was statistically different between 24h and 72h(P<0.05)but not between 48h and either of the other two time points(P>0.05).(3)Detect apoptosis of ovarian tumor cells by TUNEL methodMethod: After cultivating ovary cancer cell in vitro, adjusted theconcentration of cells to 1×105/ml, divided into 3 bottles of experiment groupand 1 bottle of comparison group, joined to 6 bore plates covered with apretreated cover glasses, change the liquid every three days. When cells growto logarithmic phase on the cover glasses, replace the liquid in experimentgroup bottles by culture fluid with a patent medicine concentration of 5×10-8mol/L. The culture fluid of comparison group is constantly, take out the coverglasses on 24h, 48h and 72h respectively, fix by 4% formaldehyde confectedwith DEPC water. Operated according to the specification of a in situhybridization kit. Negative comparison groups were set in all groups.Apoptosis ratio of ovarian tumor cells(AI)was detected by ordinary opticalmicroscope.Result: ①Apoptosis ratios of ovarian tumor cells(AI)are differentbetween the experiment groups and the comparison group, the difference isstatistically(P<0.05)。was statistically different②Apoptosis ratios of ovariantumor cells was direct proportional to incubation time, it is statisticallydifferent between any two points of time(P<0.05). That is to say it wasstatistically different between 24h and 48h,24h and 72h,48h and 72h.(4)Detected the expression of AQP1 by RT-PCR methodMethod: After cultivating ovary cancer cell in vitro, adjusted theconcentration of cells to 1×105/ml, joined to 6 bore plates, divided into 9bores of experiment group and 9 bores of comparison group. Changed theliquid every three days, When cells grow to logarithmic phase, replace theliquid in experiment group by culture fluid with a patent medicineconcentration of 5×10-8 mol/L, The culture fluid of comparison group isconstantly. Collected cells of experiment group and comparison group on 1day,2 day, 3day, 4day and 5day. Operated according to conventional RT-PCRapproach subsequently.Result: ① The content of AQP1 mRNA was statistically differentbetween the experiment group and the comparison group(P<0.05).②Thecontent of AQP1 mRNA in experiment group and comparison group weredifferent at different time points. The content of AQP1mRNA declined in allgroups along with time. The difference was not statistically in the comparisongroup(P>0.05). The content of AQP1mRNA in the experiment group declinedremarkably from either 1day or 2day to 5day, and the difference wasstatistically. However, the content of AQP1mRNA in the experiment groupdeclined from 3day to 5day, the difference was not statistically(P>0.05).Conclusion:1)Positive incidence of AQP1 in either ovarian serousborderline tumor (62.5%) or ovarian serous malignant (82.6%) is statisticallyhigher than normal ovarian tissue(20%);Expression of AQP1 in ovarian serousmalignant carcinoma has no relationship with its histological grade (p>0.05)nor clinical stage(p>0.05);Expression of AQP1 in the cases with ascites isstatistically higher than the cases without ascites (p<0.05).2)After cultivatingovary cancer cell in vitro, it was proved by flow cytometry, MTTmethod,TUNEL method and RT-PCR method that acetazolamide canaccelerated the apoptosis of ovary cancer cells by inhibiting water channelprotein .This study first proved that AQP1 is expressed in ovarian serousmalignant carcinoma and expression of AQP1 is higher in the ovary cancerwith ascites, which may provide theoretical evidence for the pathologicmechanism of ovary malignant tumor and emergence , increase of ascites. Thisstudy also first proved that acetazolamide can effectively accelerate theapoptosis of ovary cancer cells by inhibit water channel protein. It is presumedthat AQP inhibitor can at least partly inhibit the growth and transfer of ovarycancer, which may become a new target of clinical treatment to ovary cancer.