| Epithelial ovarian cancer (EOC) is one of the most frequent malignanttumors in the female population and the mortality rate is the highest in theovarian tumors. Current standards of clinical treatment is optimalcytoreductive surgery followed by systemic paclitaxel-and platinum-basedchemotherapy, usually adopt cisplatin (DPP) maximum tolerated dose (MTD).However, tumor recurrence is still inevitable, the5-years survival rate hoversat30%.Cancer stem cells (CSC) make up a small proportion of malignant cellswithin a tumor. CSC, which have an unlimited proliferation, self-renewal andmultipotent differentiation, were thought to play a crucial role in tumordevelopment, recurrence, chemoresistance and metastasis. However, aconventional schedule based on MTD shows several side effects andfrequently allows the development of drug resistance. In recent years, a newmodel of chemotherapy-metronomic chemotherapy that low-dose metronomicchemotherapy(LDM), based on the frequent administration of low orintermediate doses of chemotherapeutics, with lower grade of side effects, isgradually applied in tumor treatment, may be better than or as efficient asMTD.Aldehyde dehydrogenase1(ALDH1) is a kind of enzymes that exists inthe cytoplasm, widely expressed in different organizations, with highoxidation activity, and is responsible for tissue specific irreversible oxidationof retinal to the signaling molecule, retinoic acid. High ALDH activity isdetected in stem and progenitor cells of various lineages including lung, liver,colon, prostate, mammary, head and neck, melanoma, ovary, ect.Accumulating evidence suggests that ALDH was the only potential stem cellmaker expressed in all primary tumor specimens and detected in limitedcellular sub-population of human primary tumor cells. In the present study, we established mouse xenograft models with epithelial ovarian cancer SKOV3cells, adopted MTD-DDP chemotherapy and LDM-DDP chemotherapy, andobserved the influence of different chemotherapy model of xenograft tumor innude mice. We got ALDH1positive cells by fluorescence-activated cellsorting and identified whether ALDH1positive cells have the characteristicsof CSC. While, in the present study, we also analyzed the expression ofALDH1positive cells in MTD-DDP chemotherapy and LDM-DDPchemotherapy. Then we could seek a more effective treatment of ovariancancer, providing a new method for the clinic.Objective: To isolate and identify the CSC characteristics of ALDH1positive cells and the influence of MTD-DDP chemotherapy and LDM-DDPchemotherapy in xenograft tumor and in ALDH1positive cells.Methods:1The establishment of xenograft tumor models: For subcutaneous (s.c.)tumor formation, SKOV3cells (1×107) were suspended in0.2ml ofphosphate-buffered saline (PBS) and s.c. injected into the right thigh of eachnude mouse. The mice were randomly separated into three groups as follows:MTD-DDP group of mice was treated with cisplatin (intraperitoneal injectionof3mg/kg cisplatin every3days,6times); LDM-DDP group of mice wastreated with cisplatin (intraperitoneal injection of1mg/kg cisplatin every day,18times); Control group of mice was not treated.2ALDH1positive cells and ALDH1negative cells were detected and sortedby fluorescence-activated cell sorting. The primary cells of xenograft tumor inMTD-DDP group, LDM-DDP group and control group were labeled byALDH1antibody. These cells expressed strong fluorescence were ALDH1positive cells; these cells expressed weak fluorescence were ALDH1negativecells.3Clonogenicity assays were performed to determine the initiating tumorcapacity of ALDH1positive cells and ALDH1negative cells. ALDH1positivecells and ALDH1negative cells were got from the primary cells of xenografttumors in all groups sorted by fluorescence-activated cell sorting. 4Tumorigenicity assays were performed to determine the tumorigenicity ofALDH1positive cells and ALDH1negative cells. ALDH1positive cells andALDH1negative cells were got from the primary cells of xenograft tumors inall groups sorted by fluorescence-activated cell sorting.5Expression of Ki-67, breast cancer resistance protein (BCRP), CD133protein of ALDH1positive cells and ALDH1negative cells were analyzed byWestern blot. ALDH1positive cells and ALDH1negative cells were got fromthe primary cells of xenograft tumors in all groups sorted byfluorescence-activated cell sorting.6Statistical methods: Data were evaluated using SPSS17.0statisticalsoftware, and data were expressed as mean±standard deviation. Thesignificance difference was determined using analysis of variance, LSDmethod and the t test. P <0.05was considered significant.Results:1Influence of different chemotherapy model of nude mice s.c. xenografttumorMTD-DDP group and LDM-DDP group had a significant inhibition inxenograft tumor. The average volume of xenograft tumor in MTD-DDP groupand LDM-DDP group were significantly less than that in control group(P<0.05, P<0.05). The tumor inhibition rate of MTD-DDP group was22.05%;the tumor inhibition rate of LDM-DDP group was44.02%. There was asignificant difference of the average volume of xenograft tumor betweenMTD-DDP group and LDM-DDP group (P<0.05).2Expression of ALDH1positive cells in the primary cells of xenografttumor in MTD-DDP group, LDM-DDP group and control group.The percentage of ALDH1positive cells (0.72%) in the primary cells ofxenograft tumor in MTD-DDP group was significantly higher than that incontrol group (0.43%, P<0.05). However, the percentage of ALDH1positivecells (0.25%) in the primary cells of xenograft tumor in LDM-DDP group wassignificantly lower than that in control group (0.43%, P<0.05). It showed thatthe ALDH1positive cells were enriched in MTD-DDP group; however, LDM-DDP chemotherapy reduced the percentage of ALDH1positive cells.3Clonogenic capability of the ALDH1positive cells and the ALDH1negative cellsALDH1positive cells and ALDH1negative cells were got from theprimary cells of xenograft tumors in all groups sorted byfluorescence-activated cell sorting.The colony formation rate of ALDH1positive cells is48.53%; and thecolony formation rate of ALDH1negative cells is6.53%. There was asignificant difference between the colony formation rates of ALDH1positivecells and ALDH1negative cells (P<0.05). So, in vitro, ALDH1positive cellsdemonstrated clonogenic capability.4Tumorigenicity of ALDH1positive cells and ALDH1negative cellsALDH1positive cells and ALDH1negative cells were got from theprimary cells of xenograft tumors in all groups sorted byfluorescence-activated cell sorting.The tumorigenic rate of5000ALDH1positive cells was40%(2/5); thetumorigenic rate of10000ALDH1positive cells was80%(4/5). Whereastumorigenicity was totally absent in ALDH1negative cells at both celldensities injected.5Expression of Ki-67, BCRP and CD133in ALDH1positive cells andALDH1negative cells were analyzed by Western blot.ALDH1positive cells and ALDH1negative cells were got from theprimary cells of xenograft tumor in all groups sorted by fluorescence-activatedcell sorting.The protein expression of Ki-67in ALDH1positive cells wassignificantly lower than that in ALDH1negative cells. The proteins expressionof BCRP and CD133were high expressed in ALDH1positive cells, while,these proteins were low expressed in ALDH1negative cells. There werestatistically significant differences in the proteins expression of Ki-67, BCRPand CD133in ALDH1positive cells and ALDH1negative cells (P <0.05). Conclusions:1ALDH1positive cells demonstrated clonogenic capability andtumorigenicity, expressed highly BCRP and CD133. Ki-67protein was lowexpression in ALDH1positive cells. It suggested that ALDH1positive cellshad the CSC characteristics. ALDH1positive cells may be used as reliablemaker of ovarian cancer stem cells.2MTD chemotherapy may increase ALDH1positive cells; it showed thatCSC-like cells become enriched when tumors are subjected to chemotherapy.It may be associated with tumor recurrence and drug resistance. LDMchemotherapy may decrease ALDH1positive cells; it showed that LDMchemotherapy may reduce the percentage of CSC-like cells. Therefore, LDMchemotherapy may reduce the resistance and recurrence rate and improve theprognosis of ovarian cancer patients. |
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