| Epithelial ovarian cancer (EOC) is the fifth most common cause ofcancer deaths among women and is the leading cause of death fromgynecological neoplastic disease[1]. The standard treatment for advancedovarian cancer has been surgery and platinum-based combinationchemotherapy. Recurrence following primary platinum-based chemotherapyand high mortality remain give a challenge to the traditional maximumtolerated dose （MTD）chemotherapy schedule[2]. And the first thing we haveto do is to find the root cause of tumor recurrence and drug resistance.In recent years, the study found that the malignant tumor growth andmetastasis, the chemotherapy drug resistance and relapse are closely related tothe cancer stem cells (CSC). In terms of the experimental oncology, the CSCsis defined as a part of the malignant cells who get the ability of self-renewal orregeneration. Besides, they could succeed in xenotransplantation and presentthe same heterogeneity tumor cell surface markers or phenotype as the originalcells[3]. Paradoxically, although CSCs have the capacity for extensive orunlimited self-renewing proliferative cycles, in common with normal stemcells, they can adopt a quiescent, out-of-cycle state. CSCs could repopulate thecancer when the bulk of the tumour who at a state of proliferation is wiped outby anti-cancer drugs[4]. CSCs daughter cells could inherited the resistancecharacteristics of CSCs, which may lead to low chemotherapy reactivity ofrecurrent cancer. Chemotherapy treatment has been shown to be extremelyefficient in removing the bulk of the tumor mass, while leaving behind a coreof CSC-like cells which are not only very invasive but are able to causerelapse of the cancer[4]. The more numerous these cells are, the higherprobability there should be of treatment failure[5]. These results suggest thatchemotherapy leaves residual cells that are enriched in CSC-like traits, resulting in an increased metastatic potential[6]. Thus, we speculate thatreducing CSCs in the remnant tumor and making the cells who have nodifferentiation potential in a main position may effectively keep the sensitivityof chemotherapy. And it may become a breakthrough of reducing drugresistance, decreasing the recurrence rate and improving the prognosis.One such groundbreaking pre-clinical Chemotherapy schedule was firstlypublished by Browder[7]in2000, named low-dose metronomic(LDM)chemotherapy. Many studies indicate that metronomy chemotherapy affect thepancreatic CSC population, in contrast with standard doses of chemotherapy.Similar results have been obtained in a glioma subcutaneous modeland ahepatocar-cinoma orthotopic tumour model with cyclophosphamidemetronomic administration[8]. Altogether, metronomy-based schedules maybea more effective treatment focused on target CSCs, and provide new ideas forclinical treatment of ovarian cancer.Objective:1CSCs was considered a crucial cause of treatment failure, metastasis, andtumor recurrence. The present study aimed to examine the effects of differentchemotherapy schedule for ovarian epithelial carcinoma CSCs, throughestablishing mouse sub-cutaneous xenograft model and injecting cisplatin inMTD and LDM chemotherapy, so as to provide new ideas for clinicaltreatment.2In the fluorescence-activated cell sorting (FACS), CD133acts as a CSCsmarker. And following researches on stem cells characteristics of CD133positive cells explored the feasibility of CD133as a marker of ovarian cancerCSCs.Methods:1Establish a nude mice xenograft tumor models, Grouping, Adopt cisplatinchemotherapy and Regular monitoring of tumor growth.For subcutaneous (s.c.) tumor formation, SKOV3cells (1×107) weresuspended in0.2ml of serum-free medium and s.c. injected into the rightthigh of each nude mouse. Approximately2-3weeks later (average tumor volume150-200mm3), the mice were randomly separated into three groups, aMTD group, a LDM group and a control group.The first two groups weretreated of cisplatin intraperitoneal (i.p.) injection, the specific usage as follows:The mices in MTD group was treated of3mg/kg cisplatin once every3days,a total of6cycles. And the dosage in LDM group is1mg/kg, once a day for18days. The control group obtained the equal saline solution.The mices were closely monitored and measure body weights every day.The tumor volume and feeding were measured every3days. Extract the tailvenous blood to detect leucocyte every6days. During the experiment period,any side effects of the chemotherapy, such as weight loss, decreasingleucocyte, changes in behavior and feeding, reaction to stimulation and ruffingof fur were observed and recorded.2Isolation CD133positive cells of tumors by FACS.CD133fluorescent antibody, who is widely used for CSCs isolation andidentification, labeled the three groups of primary tumor cells who obtained in3-7days after chemotherapy finished. The cells could show differentfluorescence intensity, and were isolated by FACS and data were analyzedwith FACSDiva software. The stronger fluorescence cells were defined asCD133positive cells, while the relatively weak fluorescence cells were calledCD133negative cells.3Determination part of stem cell properties of CD133positive cells.3.1Clonogenicity assays in vitro were performed to determine the tumorinitiating capacity of the CD133positive cells and CD133negative cells.The CD133positive cells obtained by FACS are less and part of them aredamaged in the experiment, hence the CD133positive cells from the threegroups were mixed culture. Replace medium at the third day after FACS andselect active well adherent cells for in vitro clonogenicity assays. CD133negative cells were given the same treatment.The two kinds of cells were inoculated in the six orifice plate at the sametime, and each one holded three holes. Terminate the experiment at themoment that clonogenicity could be seen by naked eyes, and compaire the clone formation rate between CD133positive cells and CD133negative cells.3.2The expression of resistance protein in CD133positive cells and CD133negative cells were analyzed by Western blot.The cells number at least reach105for the Western blot experiment, butthe CD133positive cells obtained by FACS are not enough for the proteindetermination in three groups. Thus, CD133positive cells from three groupswere mixed to extract protein, and then determine the resistance andproliferation protein. CD133negative also cells should be blended toexperiment. Repeat the experiment for3times.3.3The expression of Ki-67protein in CD133positive cells and CD133negative cells were analyzed by Western blot.4Statistical methods: Data were evaluated using SPSS13.0statisticalsoftware, 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:1The LDM chemotherapy showed a more significant inhibition than MTDin tumor growth and chemotherapy side effects can be tolerated.For tumor formation,1×107SKOV3cells were s.c injected into thesubcutaneous of mice (n=30). The tumor formation rate is100%. Engraftedmice (average tumor volume150-200mm3) were randomly assigned intothree groups after injection for2-3weeks, that are a MTD group, a LDMgroup and a control group. There is no significant difference in xenografttumor volumes between the three groups （P>0.05）. At the end ofchemotherapy, the average tumor volume of the above three group were564.72±43.89mm3,382.25±28.09mm3,782.01±34.24mm3，and there havestatistical significance between any two of the three groups.According to the formulat, Tumor inhibiting rates=(The average tumorvolum of control group–The average tumor volum of MTD group or LDMgroup)/The average tumor volum of control group×100%. The result showsthat the tumor inhibiting rates in MTD group was27.78%, and51.12%in LDM group. The LDM chemotherapy showed more effective inhibition oftumor growth.The process of chemotherapy, compaired with control group, the nudemices in MTD group and LDM group all appeared weight loss (P<0.05),feeding decrease (P<0.05), leucocyte decrease (P<0.05),depression and otheradverse reactions. However，chemotherapy side effects in the two treatmentgroup nude mices are classified to Ⅰgrade, which suggests that at least totolerance of chemotherapy. However, the above indicators of side effectsbetween the LDM group and MTD group mices had found no obviousdifference.2MTD chemotherapy could enrich the CD133positive cells in the tumor,while LDM chemotherapy could reduce the expression rate of CD133positivecells.The CD133positive cells obtained by FACS are less and part of them aredamaged in the experiment, hence the CD133positive cells from the threegroups were mixed culture. Replacing medium at the third day after FACS andselecting active good adherent cell for in vitro clonogenicity assays. CD133negative cells were given the same treatment.CD133positive cells proportion in LDM group tumors by FACS is（0.247%±0.021%）,significantly lower than the control group（0.413%±0.021%，P=0.001）.However, the ratio of CD133positive cells inMTD group tumor （1.463%±0.208%）is higher than the control groupobviously(P=0.000). It turned out that the LDM chemotherapy schedule couldreduced the CD133positive cells more significantly than traditional MTDmode, and is a more effective treatment focused on target CSCs.3CD133positive cells showed obvious stem cell properties.3.1CD133positive cells showed stronger colony formation capability thanCD133negative cells.The two kinds of cells were prepared to single cell suspension andinoculated in the six orifice plate at the same time, and each one hold threeholes, getting500cells every hole. Terminate the experiment at the moment that clonogenicity could be seen by naked eyes. Count the cloned balls whosecell number>30by the microscopy. Repeat test three times.Clone formation rate (%)=Colony formation numbers/The originalcells number(500)×100%. Colony formation in vitro experimental resultsdisplay that both CD133positive and CD133negative cells demonstratedclonogenic capability and the clone formation rate of CD133positive cells(48.26%±1.33%) was significantly higher than that of CD133negative cells（3.37%±0.76%，P<0.05）. CD133positive cells have stronger cloneformation ability, that is one of the important conditions for the stem cellidentification.3.2CD133positive cells have stronger drug resistance than CD133negativecells.The Breast Cancer Resistance Protein (BCRP) could independently orcombined classic resistant protein P-gp, MRP mediated multi-drug resistance.The detection of BCRP could evaluate the drug tolerance of different cells.The expression of BCRP in CD133positive cells and CD133negativecells is（0.677±0.032）vs (0.228±0.018)，P<0.05.3.3CD133positive cells demonstrated poorer proliferation activity thanCD133negative cellsKi-67is a kind of proliferating related cell nuclear antigen, and isindispensable in cell proliferation. It expressed only in the proliferous cellsand had no express in still cells. The detection of Ki-67protein could evaluatethe malignant tumor cell proliferation activity.The result of expression of Ki-67protein by Western blot in the twokinds of cells as follows: CD133positive cells（0.215±0.001）vs CD133negative cells（0.739±0.009）,P<0.05. It prompted that the proliferationactivity of CD133positive cells is relatively poor, which accord with thedormant existence state of CSCs.Conclusions:1The cisplatin LDM chemotherapy showed a more significant inhibitionthan MTD in tumor growth and chemotherapy side effects could be tolerated. 2CD133positive cells showed obvious stem cell properties.3The LDM chemotherapy could reduce the cancer stem-like cells insubcutaneous tumors derived from SKOV3cells，which maybe expected to bea way to targeted therapy of ovarian cancer stem cells，and in the help tolessen drug resistance, reduce the recurrence rate, thus improve the prognosisof patients. |
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