| Objective:To investigate the impact of low-dose metronomic chemotherapy (LDM)versus maximum tolerated dose chemotherapy (MTD) administration ofcisplatin over an18-day period to Balb/c nude mice bearing SKOV3ovariancancer xenografts. And to explore the influence of the two chemotherapymodels on the expression level of P-glycoprotein（P-gp）, breast cancerresistant protein (BCRP), lung resistance protein (LRP), proliferating cellnuclear antigen KI-67and cancer stem cell marker CD133.Methods:1Animal housing, generation of tumors, and cisplatin treatmentFor subcutaneous (s.c.) tumor formation, SKOV3cells (1×107) weresuspended in0.2ml phosphate-buffered saline (PBS) and s.c. injected into theright thigh of each nude mouse. Approximately two weeks later (averagetumor volume150mm3-200mm3), the mice were randomly divided into threegroups as follows: MTD-DDP group (intraperitoneal injection of3mg/kgcisplatin once every three days for six cycles), LDM-DDP group(intraperitoneal injection of1mg/kg cisplatin daily for eighteen days) andcontrol group, nude mice were treated with the equal saline solution.2Tumor growth and side effectsThe mice were closely monitored every day and the body weight, dietand xenograft tumor volumes were measured every3days. White blood cellsfrom tail vein were counted every6days. During the experiment period, anyside effects of the chemotherapy, such as weight loss, changes in behavior andfeeding, skin color and subcutaneous fat were observed and recorded.3Western Blotting was used to measure the expression levels of P-gp, BCRP,LRP, Ki-67and CD133in three groups. 4Hematoxylin and eosin staining was performed to observe small metasticlesion and histomorphology changes of internal organs in three groups.5Statistical analysisA Statistical Package (SPSS17.0) was used for statistical analysis. Toassess statistical significance between two groups, a two-sample t-test wasused. For comparisons among three or more groups, one-way analysis ofvariance (ANOVA) was used. Multiple comparisons with LSD and DunnettT3were performed for comparisons showing statistical significance based onthe ANOVA F-test. Significance was assigned as P<0.05. Reported results areexpressed as mean±standard error of the mean.Results:1Tumor growth and mouse condition assessmentDuring the1st day to18th day of the chemotherapy, subcutaneousxenograft tumors of control group, MTD-DDP group and LDM-DDP groupwere increasing constantly. Among the three groups, the xenograft tumors onday18were significantly larger than those on day9(P<0.05). On day6(micewere treated with cisplatin for6days), tumors in LDM-DDP group weresmaller than those in control group (P=0.022), whereas tumors in MTD-DDPgroup were not statistically different from control group (P=0.215). On day9(mice were treated with cisplatin for9days), compared with control group,tumor growth in MTD-DDP and LDM-DDP group was significantly slowedand tumors were smaller (P=0.047, P=0.002). Tumor volumes showed nodifference between LDM-DDP group control group (P=0.158). The tumorgrowth suppression rate in MTD-DDP and LDM-DDP was31.32%and47.39%, respectively. On day18(mice were treated with cisplatin for18days),tumor volumes in MTD-DDP group and LDM-DDP group were smaller thancontrol group (P=0.020, P=0.000) and those in LDM-DDP group weresignificantly smaller than MTD-DDP group (P=0.004). The tumor growthsuppression rate for MTD-DDP group and LDM-DDP group was27.00%and54.29%, respectively.2General situation and side effects assessment 2.1General situation assessment of nude miceDuring the first day to18th day of the chemotherapy, nude mice were ingood condition, without physical activity decrease and constipation/diarrhea.Compared with control group, nude mice in MTD-DDP group and LDM-DDPgroup exhibited diet decline, mild skin discoloration and subcutaneous fatdecrease. On day9, food-intake of mice in MTD-DDP group and LDM-DDPgroup were less than control group (P=0.001, P=0.000), and the food weightsof mice in MTD-DDP group and LDM-DDP group showed no statisticaldifference, yet (P=0.657). On day18, food-intake of mice in MTD-DDP groupand LDM-DDP group were both less than control group (P=0.000, P=0.000),whereas food-intake exhibited no difference between MTD-DDP group andLDM-DDP group (P=0.834).2.2Side effects assessmentDuring the first day to18th day of the chemotherapy, mice in MTD-DDPgroup and LDM-DDP group exhibited weight loss. On day9, average bodyweights in treated mice were significantly lower than control mice (P=0.023,P=0.002), whereas body weights in LDM-DDP group were not different fromMTD-DDP group (P=0.322). On day18, body weights in MTD-DDP andLDM-DDP were lower than control group (P=0.015, P=0.002), whereas therewas no difference between LDM-DDP group and MTD-DDP group(P=0.399).During the first day to18th day of the chemotherapy, white blood cells ofmice in MTD-DDP group showed a trend of constant decline., white bloodcells showed no statistical differences among the three groups (P=0.930,F=0.073). On day12, white blood cells in MTD-DDP group was lower thancontrol group (P=0.006), while there was no significantly difference of WBCbetween LDM-DDP group and control group (P=0.192). On day18, ascompared to LDM-DDP group and control group, WBC in MTD-DDP groupwas lower than that in LDM-DDP group and control group (P=0.010,P=0.029).During the chemotherapy, any small metastase lesion was not observed in organs of the three groups. Until mice were treated with cisplatin for9days,one mouse in MTD-DDP group was not observed hepatocellular fattydegeneration. On day18, the number increased to two. But no signs ofdamage in organs were observed in LDM-DDP group and control group.3Expression of proliferating cell nuclear antigen Ki-67Ki-67was used for assessing tumor proliferation. On day9, comparedwith control group, the expression of Ki-67was less in MTD-DDP andLDM-DDP groups (P=0.004, P=0.000), whereas Ki-67expression inLDM-DDP group was not statistically different from MTD-DDP group(P=0.080).On day18, the expression level of Ki-67in LDM-DDP group wassignificantly lower than control group (P=0.003), while Ki-67expression inMTD-DDP group was not different from LDM-DDP group and control group(P=0.126, P=0.079).4Expression of cancer stem cell marker CD133On day9, there was no statistical difference of CD133expression amongcontrol group, MTD-DDP group and LDM-DDP group (P=0.190, F=1.858).On day18, as compared to control group, the expression level of CD133was significantly higher in MTD-DDP group and LDM-DDP group (P=0.000,P=0.037). But CD133expression in LDM-DDP group was lower thanMTD-DDP group (P=0.011).5Expression of drug resistantce related protein5.1Expression of BCRPOn day9, the expression level of BCRP in MTD-DDP group andLDM-DDP group was higher than control group (P=0.000, P=0.001), whereasno statistical difference was observed between LDM-DDP group andMTD-DDP group (P=0.098).On day18, comparing with control group, MTD-DDP group andLDM-DDP group demonstrated more BCRP expression (P=0.000, P=0.004),while LDM-DDP resulted in lower BCRP expression than MTD-DDP(P=0.001). Among the three groups, the expression of BCRP on day18were all increased compared with which on day9(P<0.05).5.2Expression of LRPOn day9, as compared to control group, LRP expression in MTD-DDPgroup and LDM-DDP group was higher (P=0.000, P=0.000), whereas theexpression of LRP in LDM-DDP group was not significantly different fromMTD-DDP group (P=0.060).On day18, the expression level of LRP in MTD-DDP group andLDM-DDP group was higher than control group (P=0.000, P=0.003), whereasLRP expression showed no difference between LDM-DDP group andMTD-DDP group (P=0.274).5.3Expression of P-gpOn day9, compared with control group, the expression of P-gp inMTD-DDP group and LDM-DDP group was higher (P=0.000, P=0.006),while P-gp expression was not significantly different from MTD-DDP groupin LDM-DDP group (P=0.075).On day18, the expression level of P-gp in MTD-DDP group andLDM-DDP group was higher than control group (P=0.002, P=0.038), whereasthere’s no difference of P-gp expression between LDM-DDP group andMTD-DDP group (P=0.171).Conclusion:1LDM-DDP resulted in greater tumor growth inhibition and less bonemarrow suppression compared with MTD-DDP, at the same total cumulativedose.2The expression of tumor stem cell marker and multi-drug resistantrelated protein in LDM-DDP were significantly lower than MTD-DDP,suggesting that tumor cells with partial tumor stem cell properties and highexpression of drug resistance related protein were easier to be enriched inMTD model. That is to say, in MTD model, maybe the chemotherapyresistance of residual tumors is stronger and the tumors are more difficult tocure, whereas LDM is a new potential model that could reduce the recurrenceand increase the sensitivity to chemotherapy. |
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