| 【Background】Mucoepidermoid carcinoma (MEC) is the most common malignant tumor in salivary glands,accounting for approximately10%–15%of all salivary gland neoplasm and about30%of salivarymalignancies. Based on morphologic and cytologic features, MECs are divided into low-grade,intermediate-grade, and high-grade subtypes. Low-grade MECs have an indolent clinical course forwhich radical surgery is typically curative, whereas high-grade MECs have worse clinical outcome andlocal, locoregional and distant metastases of high-grade MECs are common. The average survival afterdiagnosis of metastatic disease is2to3years. To date, chemotherapy or systemic treatment has beenused largely for palliative treatment of metastatic disease but results are disappointing and responsescan be demonstrated only in a minority of high-grade MECs. The main obstacle is the low degree oftumor differentiation and multidrug resistance (MDR) potential of high-grade MECs. The developmentof new active substances or cytotoxic therapies for high-grade MECs is challenging. Although a largenumber of natural products have been tested demonstrated, animals and clinical studies have so farfailed to demonstrate similar efficacy. It is hoped that novel active substances or new cytotoxictherapeutics, including natural products, will significantly improve the outcome for these high-gradeMECs patients.【Objective】（1） To establish a multidrug resistant cell line MEC/5-FU cells of human salivarygland mucoepidermoid carcinoma and to study its characterization and multidrugresistance (MDR) mechanism.（2） To establish an xenograft model and a multidrug resistant model （MEC/5-FU/NU）of human mucoepidermoid carcinoma using nude mice forfuture therapeutic trials and provide evidence for studying MDR mechanism andfor screening of the reversal drugs in vivo.（3） To study the effects of baicalin on proliferation inhibition and apoptosis inductionon MC3cells and its nude mice xenograft，and to investigate the underlyingmechanism of its apoptosis action（4） To study the effects of baicalin on proliferation inhibition and apoptosis inductionon multidrug resistant MEC/5-FU cells and nude mice xenograft model. To studythe effect of baicalin on the expression of drug-resistance related genes onMEC/5-FU cells.【Methods】（1） Human mucoepidermoid carcinoma cell MC3cells were treated by pulsetreatments of high concentration combining with continuous gradually increasedconcentration exposed to5-fluorouracil (5-FU) to introduce its multidrugresistance cell line MEC/5-FU. MC3and MEC/5-FU cells were then exposed tovarious concentrations of5-FU, ADM, PADM, PYM, BLM, Ara-C, Cis-DDP,MMC, DNR and Tax for72h, the survival rates and resistance index of the cellswere determined by MTT assay.（2） Multidrug resistant MEC/5-FU cells were inoculated intradermally into nude mice.Solid tumors were locally measurable after14days and5-FU was repeatedintraperitoneal injected into tumor-bearing mice. MEC/5-FU/NU cells from tumorswere isolated, cultured and examined.（3） MC3cells and MEC/5-FU cells were in vitro cultured and treated with variouslyconcentration of baicalin. Its proliferation inhibition were detected by MTT assayThe cell apoptosis was tested by Annexin V FITC/PI double staining analysis, cellcycle analysis and DNA fragmentation. RT-PCR and immunofluorescence wereused to analyze the MDR related genes’mRNAand protein expression.（4） MC3cell and MEC/5-FU cell suspensions with a density of1×107cells/ml in PBS were subcutaneously injected (0.1ml) into the right flank of the nude mice. Aftertumor transplantation for14days, the tumors reached a volume of approximately300mm3.Then the xenograft tumor models of MC3cell and MEC/5-FU cell innude mice were established. Those mice were randomly assigned to designedgroups and treated with various concentration of baicalin combined with orwithout5-FU respectively. Nude mice growth state was observed. The length (a)and width (b) of the tumors were measured every two days by a vernier caliper andthe tumor volumes and inhibition rate of transplanted tumor were calculated. Theultramicrostructure change of xenografts cells were tested by transmission electronmicroscope.（5） Gene express profile of MC3/5-FU cells and MC3cells were examined with Genechip Agilent which containing45,015human genes. The data were analysed withDAVID Bioinformatics Resources. The differently expressed genes with theEnrichment Score (ES)1were analysed by “GO”. And the differentely expressedgenes related to MDR were tested by RT-PCR and immunofluorescence.Rhodamine123assays were used for functional activity study of ATP-bindingcassette (ABC) transporters proteins.（6） The total miRNA expression levels between MEC/5-FU and MC3cells, as well asthat between MEC/5-FU cells and baicalin treated MEC/5-FU cells, were tested byMicroRNAs chip. And the differentely expressed miRNAs related to MDR andapoptosis were classified by realtime-PCR.【Results】（1） After14pulse treatments of high concentration of5-FU combining withcontinuous gradually increased concentration, a multidrug resistance cell lineMEC/5-FU was established. The resistance index of the MEC/5-FU cells to5-FUwas36.11. And they also showed remarkable resistance to multiple antitumordrugs. And the cell morphology and growth characteristics were similar with itsparental cell line MC3cells. （2） After tumor transplantation for14days, the tumors reached a volume ofapproximately300mm3. Then,15mg/kg of5-FU was intraperitoneal injected intotumor-bearing mice for two weeks. MEC/5-FU/NU cells from the xenografts wereisolated, cultured and examined. The xenografts were similar histologically to theoriginal mucoepidermoid carcinoma from which the cell line was derived. Theresistance index (RI) of the MEC/5-FU/NU cells to5-FU was27.82. Compared tothe MEC, the expressions of ABCB1, ABCB11and GSTA1genes and MDR-1protein were significant increased in the MEC/5-FU/NU cells by RT-PCR andimmunocytochemistry.（3） Baicalin exerted dose-and time-dependent antiproliferative potential against MC3cells as assessed by MTT assay, with an IC50value of40μg/ml. Treatment ofMC3cells with100and320μg/mL baicalin for72h caused75.42%and91.58%reduction in cell viability respectively. Baicalin treatment of MC3cells resulted ina significant accumulation of cells at the G0/G1and G2/M phase with aconcomitant decrease in cells processing to S phase as assessed by flow cytometry.Furthermore, baicalin induced apoptosis of MC3cells as determined by annexin Vbinding and PI dual staining, DNA fragmentation, nuclear condensation. Byannexin V binding and PI dual staining, we found that baicalin treatment (40μg/mlfor72h) greatly enhanced the apoptosis of MC3cells with an apoptotic rate of14.47%in baicalin-treated group and2.80%in the control group, respectively.Rhodamine123assay indicated that baicalin caused cytotoxicity and inducedapoptosis through decreasing the mitochondrial membrane potential in Mc3cells.Characteristic morphological changes of apoptosis, including nuclear condensation,boundary aggregation and DNA fragmentation in baicalin treated MEC/5-FU cellswere observed by Hoechst33342-stained.（4） For the MC3cell xenograft groups, the average tumor volume was1233.02mm3inthe200mg/kg baicalin treated group, whereas it was2682.67mm3in the controlgroup (P=0.0348), demonstrating the significant tumor growth inhibitory effects ofbaicalin in nude mice. In addition, no evidence of drug-related toxicity was observed in mice treated with the highest dosage of baicalin (200mg/kg) bycomparing the body weight, organ histology, complete blood count, liver chemistry,or kidney function of the experimented group with those of controls, suggestingthat baicalin can be well tolerated by the animals.（5） Baicalin also exerted dose-and time-dependent antiproliferative potential againstMEC/5-FU cells as assessed by MTT assay, with an IC50value of68.75μg/ml.For the MEC/5-FU cell xenograft groups, the average tumor volume was1682.0mm3in the200mg/kg baicalin treated group, whereas it was3259.0mm3in thecontrol group, demonstrating the significant tumor growth inhibitory effects ofbaicalin in nude mice. RT-PCR and Rhodamine123assay results revealed thatthere was no significant influence on expression of the MDR related genes bybaicalin treatment on MEC/5-FU cells.（6） According to the gene chip,7516genes were up-regulated (Ratio up to2.0),7275genes were down-regulated (Ratio down to0.5) in MEC/5-FU cells compare to itsparental MC3cells. Up-regulated genes in the cells with MDR include thoserelated to growth factor, extracellular matrix, regulation of cell adhesion,angiogenesis and so on. Down-regulated in the cells with MDR includes thoserelated to response to endogenous stimulus or drug, apoptosis, and so on.Compared to the MC3cells, the expressions of ABCB1、ABCB11、ABCC4andGSTA1were increased in the MEC/5-FU cells according to the RT-PCR results.Rh123accumulation in MEC/5-FU cells was far less than that in MC3cells.（7） There was no significant quantitative difference of MRP1mRNA and proteinexpression was found between MEC/5-FU cells and its parental cell line MC3cellsas determined by gene chip and RT-PCR. However, immunofluorescence stainingrevealed that MRP1was translocated from the cytoplasmic membrane of the MC3cells to the nuclei of MEC/5-FU cells. And down-regulation of MRP1inMEC/5-FU by RNAinterference increased the drug sensitivity of the cells to5-FU.Furthermore, MRP1down-regulation mainly decreased the nuclear expression ofMRP1rather than the cytoplasmic membrane expression. （8） Compared to the MC3cells,19miRNAs were up-regulated and11miRNAs weredown-regulated according to the miRNAs chip assay. And, baicalin treatment ledto162miRNAs up-regulation and13miRNAs down-regulation on MEC/5-FUcells. The expression of miR-34, miR-107, miR-202and miR-125a, which werereported to relate to MDR and apoptosis, were tested by realtime-PCR. However,results showed there were no significant difference between MC3cells andMEC/5-FU cells.【Conclusions】（1） A multidrug resistance cell line (MEC/5-FU) and xenograft tumor models ofhuman mucoepidermoid carcinoma has been established. The resistance index ofthe MEC/5-FU cells to5-FU was36.11. And they also showed remarkableresistance to multiple antitumor drugs.（2） Baicalin could efficiently inhibit proliferation and induce apoptosis in MC3cells,which could be correlated with the down-regulated expressions of bcl-2, andup-regulated expressions of caspase3and bad. Meanwhile, baicalin could inhibitgrowth and induce apoptosis of MC3cell xenografts in nude mice.（3） Baicalin could efficiently inhibit proliferation and induce apoptosis in MEC/5-FUcells, inhibit growth and induce apoptosis of MEC/5-FU cell xenografts in nudemice.（4） Compared to the MC3cells, the expressions of ABC transporter genes and P-gpprotein were increased in the MEC/5-FU cells, which play an important role forthe multidrug resistance of MEC/5-FU cells.（5） The expressions of ABCB1、ABCC1in the MEC/5-FU cells were not changed bybaicalin treatment. And there wasn’t any difference of the accumulation ofrhodamine123between baicalin treated MEC/5-FU cells and MEC/5-FU cellswhich indicated that baicalin have no effection on the P-gp protein in MEC/5-FUcells.（6） Although there was no signifcant quantitative difference of MRP1mRNA expression between MEC/5-FU cells and its parental cell line MC3cells, MRP1was translocated from the cytoplasmic membrane of the MC3cells to the nuclei ofMEC/5-FU cells. And down-regulation of MRP1in MEC/5-FU cells by RNAinterference increased the drug sensitivity of the cells to5-FU. Our resultssuggested that MRP1was involved in the chemoresistance of MEC and MRP1may confer drug-resistance by a mechanism associated with its nucleartranslocation.（7） The MicroRNAs chip result showed us that series of miRNAs expression levelswere changed between MEC/5-FU cells and its parental cell line MC3cells, whichsuggested that dysregulation of miRNAs may be involved in the chemoresistanceof MEC. The expressions of miR-202（ratio3.179）and miR-125a-5p（ratio0.0013）between MEC/5-FU cells and its parental cell line were classified byrealtime-PCRThere. However, there were no significant differences of theexpressions of them between MEC/5-FU cells and MC3cells according to ourrealtime-PCR results, which was not consort with the results of miRNAs chipassay. The expressions and founctions of miR-202and miR-125a-5p, which wererelated to MDR according to previous reports, in MEC/5-FU cells warrant furtherinvestigation.（8） The MicroRNAs chip result showed us that series of miRNAs expression levelswere changed between baicalin treated MEC/5-FU cells and MEC/5-FU cells. Theexpressions of miR-34a（3.8891048）and miR-107（ratio0.0009556）were changedsignificantly However, there were no significant differences of the expressions ofthem according to our realtime-PCR results. The expressions and founctions ofmiR-34a and miR-107in the process of baicalin induce MEC/5-FU cells apoptosiswarrant further investigation. |
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