Ability Of MDR Reversing Effects Treated With Arsenic Trioxide In Gastric Cancer

Multidrug resistance ( MDR) is a major factor leading to the failure of many forms of chemotherapy. Of several different molecular mechanisms in MDR cells, the most investigated one is: P - glycoprotein ( P - gp) , encoded by mdrl, which can pump some simple - lipid anti - tumor drugs out of cells, degrade the concentration of these drugs in cells and induce MDR - the classic MDR path. Some ADM resistant cell lines mainly appearance over - expression of P - gp. So, it becomes important how to reverse the MDR of tumors now. Although some MDR - reversing agents had been discovered, such as Ca2 + channel antagonists, calmodulin inhibitors, cyclosporines and so on, side effects appeared seriously when they achieve the effective concentration in vivo, which limited their clinical application. Contrastly, some traditional Chinese medicines may become the good MDR - reversing agents because of their universal pharma-cologic actions and a few side effect.Arsenic trioxide, a kind of Chinese traditional medicine, had been confirmed the excellent effects on treatment of APL( acute promyelocytic leukemia) in clinic and anti - solid - tumor effects in vitro, by the way of apoptosis inducing in tumor cells. Recently some investigators reported that arsenic trioxide showed MDR reversing effects on the tumor cell lines resisted to ADM (adriamy-cin) or VCR (vincristin), but its mechanism was not so clear yet, especially the relationship between MDR reversing effects and signal transduction systems in tumor cells.Some studies showed that Ras/MAPK signal pathway and Caspases protein family play an important role in tumors apoptosis and MDR reversing.Ras/MAPK process is; stimulating factor-mitogen—+Ras-+Raf -1-MEK -1/-2-+ERK1/2-+EIR CPLA2-c -fos. Finally, this path regulates genes' expression and cells'proliferation or differentiation. Once it was activated abnormally, persistent proliferation would happened and apoptosis inducing ability would be down - regulated in tumor cells.Caspase family ( Cysteinyl aspartate - specific proteinase) plays an important role in apoptosis courses of mammalian cells. Caspase3, member of caspase family, is an important effective enzyme that triggers apoptosis protease cascade.Materials and MethodsAgentsRPMI1640( Hyclone company, American) ; standard fetal calf serum( TBD biotechnology limited company, Tianjin) ; As2O3( Sigma company; American) , prepared to 50|xmol/L depoting liquid with phosphate buffer (PBS) ; Adriamycin ( ADM, Haizheng limited company, Zhejiang) , mouse anti - human monoclonal antibody of P - gp, Ras, phosphorylated ERK1/2 and immuno-hischemistry kit (Zhongshan biotechnology limited company, Beijing).Cell cultureHuman gastric cell line SGC7901/S (parental generation) and adriamycin - resistant cell line SGC7901/ADM, benefited by Digestion Disease Institute of the Forth Medic University. The cell line of SGC7901/S was cultured in the RP-MI 1640 culture fluid containing 10% FCS. SGC7901/ADM cell line was cultured in the liquid above with 1 jxg/ml ADM, and two weeks before experiment, cultured in the liquid without ADM. The two cell lines in exponential phase of growth were used at experiments.Determin MDR Multiple and sensitivity to As2O3 of SGC7901/ADM by MTT MethodDetermin MDR Multiple to ADM: Inoculated SGC7901/ADM and SGC7901/S (final concentration:2 x 105/ml) in 96 - pore plates ,200jjd per pore. Added ADM ,the final concentration range from 0.1 to 50mg/L. The control groups included blank control group (without cells) and negative control group (cell growing normally). After incubating for 48h, added MTT per poreand incubated for4h. Finally added DMSO lOOjxl per pore, rocked so as to well - distributing. Determined the absorbance values and obtained ADM% 50% inhibiting concentration ( IC50 ) by the automatic enzyme - scale instrument (AR2010 Type). MDR Multiple = (IC50 to SGC7901/ADM)/ (IC50 to SGC7901/S).According to these procedures, the two cell lines'sensitivity to As2O3 and the MDR Multiple to ADM were determined after intervented by IC50, Leubene andZ-DEVD-fmk.Analysis cell cycle and detect apoptosis rate by flow cytometrySGC7901/ADM and SGC7901/S (final concentration of 5x105/ml) were inoculated in 6 -pore platqs, 5ml per pore. As2O3 in final concentrations of 0. 1,0.25,0.5,0.75|xmol/L was added. The negative control group was normal -growing cells, and the positive control group, 4μg/mlCsA. After cultured for 24h,48h and 72h, cells were collected respectively, fixed over night by alchole at -20℃ , washed and centrifuged, 10ul/ml PI and 0.1% Rnase were added, dyed for 30min, protecting from light, , the cell cycles and the apoptosis rates were detected by flow cytometry ( B. D. Vantage type) and analysised with Modf-it 2.0 Systems Software. Every sample was determined three times.Repeated these procedures after intervented by As2O3, Leubene and Z -DEVD-fink.Detect the express of P - gp, Ras, phosphorylated ERK1/2 and Caspase3 in the two lines by Immunocytochemistry (P - V Method).As2O3, Leubene and Z - DEVD - fmk. intervented the cell lines samely as above. SGC7901/ADM and SGC7901/S (final concentration:5x105/ml)were inoculated in 6 - hole - culture - plates, 5ml per hole. As2O3 (final concentration: 0.10, 0.25, 0.50, 0.75μmol/L) was added, 4μg/ml cyclosporine A as the positive control group,cells in normal - growing,negative control group. Incubated for 24h,48h and 72h, fixed by dimethyl ketone at 4℃, soaked in 3% H2O2 for 10 min, the first antibody of P - gp, Ras, phosphorylated ERK1/2 and Caspase3 (50fμl/piece) were dropwised respectively, incubated for 2 hour at 37℃ ,and polymer assistant(polymer helper) were added, incubated for 30min at 37℃ , then the second antibody of horseradish enzyme labeled was added.Colored with DAB liquor, afterstained with hematoxylin, mounted with neutro -gum. The-results were analizied with VIDAS image analysator quantitative analysis according to absolute gray scales. Chose 3 different fields in every pieces.Western blot analysis of P - gp and caspase3The drugs intervented the cell lines same as above. Cells were collected and lysed them in 200μl RIPA buffer. Then samples were sonicated on ice, lysed 40min at 4℃ , transferred the supernatant to a new trip on ice ,then calculated the amount of the protein by extra - violet quantitative method. Protein samples were separated by gel electrophoresis, transferred to NC membranes. The membranes were probed with mouse anti - human P - gp and Caspase3 respectively, incubated for 2h at 37℃ ,and the membrane were stained by P — V Method. The results were analyzed with VIDAS image analysator quantitative analysis according to absolute gray scales.Statistics analysisThe variance analysis and Student - Newman - Keult methods in PEMS 3.1 edition statistics software were adopted.ResultsSensitivity research to ADM and As2O3 in human gastric cancer cell line SGC7901/ADMThe IC50 of ADM or As2O3 was detected by MTT Method. (1)ADM IC50 to SGS7901/ADM and SGC7901/S were 31.67 ±4. 86mg/L and 3.93 ±0.52mg/ L, the difference was 8.1 times( tTest,t'=9.8301 ,P <0.01) ; (2)The sensitivity to As2O3 of SGC7901/ADM and SGC7901/S were similar;the dose of As2O3 that didnt appear cytotoxicity was 0.1 μmol/L and that of low - cytotoxicity was 0.5jxmol/L; (DAfter interfered by As2O3, ADM's IC50 to SGC7901/ADM became smaller,and the decreasing level was related to the dose and actiing time of As2O3; The maximum reversing multiple was 6.29 at As2O3 0. 5μmol/L,48h; (4)After interfered by Leubene or Z - DEVD - fink, the maximum reversing multiple of As2O3 were 4.72 and 1.37 respectively, smaller than that before interfered.Cell cycle and apoptosis analysis in human gastric cancer cell line SGC7901/ADM(1)After interfered by As2O3, the cells in GO - G1 period and apoptosis rates increased, the degree of changes was related to the dose and acting time of As2O3; (DAfter interfered by Leubene or Z - DEVD - fmk, the cells in GO - Gl period decreased, and apoptosis rates became mailer than that of As2O3 interfering.Express of P - gp, Ras, phosphorylated ERK1/2 and Caspase3 in SGC7901/ADM and SGC7901/S1. P - gp: P - gp expressed at cell membranes. The expression of P - gp in SGC7901/ADM cells was more obviously than that in SGC7901/S cells (t = 11. 139 P<0.001). After interfered by drugs,the changes were following:(1)After interfered by As2O3, the expression of P - gp in SGC7901/ADM cells became fewer, the degree of degression was related to the dose and acting time of As2O3; (DAfter interfered by Leubene or Z - DEVD - fmk, although the expression of P -gp in SGC7901/ADM cells became fewer, the degree of degression was smaller obviously.2. Ras: Ras expressed at cell membranes or in cytoplasm. The expression of Ras in SGC7901/ADM cells was more obviously than that in SGC7901/S cells. After interfered by drugs, the changes were following: (1)After interfered by As2O3, the expression of Ras in SGC7901/ADM cells became fewer, the degree of degression was related to the dose and action time of As2O3; (2)After interfered by Leubene, although the expression of Ras in SGC7901/ADM cells became fewer, the degree of degression became smaller(t =5.3324,p =0.0060).3. phosphorylated ERK1/2; It expressed in cell nucleus. The expression of phosphorylated ERK1/2 in SGC7901/ADM cells and in SGC7901/S cells is similar. After interfered by drugs, the changes were following ;'(1)After interfered by As2O3 0. 1μmol/L, The expression of phosphorylated ERK1/2 in SGC7901/ ADM cells had no changes; but after interfered by As2O3 0. 5μmol/L and 0. 75μmol/L, the expression of phosphorylated ERK1/2 in SGC7901/ADM cells became fewer, the degree of degression was related to the dose and acting time of As2O3; (DAfter interfered by Leubene, the degree of degression became smal-ler.4. Caspase3: It expressed in cytoplasm. The expression of Caspase3 in SGC7901/ADM cells and that in SGC7901/S cells is similar. After interfered by drugs, the changes were following :(1)After interfered by As2O3, the expression of Caspase3 in SGC7901/ADM cells became more, the degree of increasing was related to the dose and acting time of As2O3; (2)After interfered by Z - DEVD -fmk, and the expression of Caspase3 in SGC7901/ADM cells became fewer ( q = 14.3333,p<0.01).DiscussionResearch indicated that MDR phenomenon of gastric cancer and other tumor cells is one of the main cause of chemotherapy failure in clinic. MDR mechanisms are: (1). The sensitivity to anti - tumor drugs of gastric cancer cells is lower than that of leukaemia and lymphoma, which is called intrinsic resistance ; (2). The drug resistance of gastric cancer cells induced by chemotherapy is called acquired drug - resistance, which becomes the major obstacle to chemotherapy success. P - glycoprotein (P - gp) , encoded by mdrl, can pump some simple - lipid anti - tumor drugs out of cells and degrades drugs'concentration in cells, inducing MDR. The substrates that P - gp effects are maily natural hydro-phobicitied anti - tumor drugs , such as drugs including anthracene nucleus, kinds of vincristine or actinomycin. SGC7901/ADM appeared MDR to ADM and over - expression of P - gp at this experiment.As the main component of arsenicum sublimatum - one of the traditional Chinese Medicine, As2O3 had been purified to be used for injection in vein. It had be used in clinic to treat APL, and have a significant curative effect. This experiment discovered that SGC7901/ADM and SGC7901/S had the same sensitivity to As2O3. As2O3 can reverse MDR of SGC7901/ADM and down -regulate the expression of P - gp, regardless of the dose of no - cytotoxicity or low - cyto-toxicity. The MDR - reversing ability of As2O3 appeared dose and time dependence. These results showed that As2O3 is not only an effective anti - tumor drug, but also a MDR reversing agentAbout As2O3's MDR reversing mechanisms, it was found that As2O3 can...