Effects Of New Anti-Cancer Compounds Salinomycin In Tumor Growth Of Human PC-3Transplanted Into NOD/SCID Mice And The Gene Expression Of Prostate Cancer Stem Cells

Objective To observe effects of Salinomycin in tumor growth of Human PC-3transplantedinto NOD/SCID mice. Analysis changes in the all gene expression of prostate cancer stem cellswith microarray technology. To discuss its possible mechanism of salinomycin anti-prostate cancerandanti-prostate cancer stem cells in vivo.Methods Animal model: subcutaneous injection of PC-3cells that are on the time of logarithmicphasesuspension(1×106/0.2ml) into303-4week-old NOD/SCID mice, and observe the growth of thexenograft tumor, then screening the neoplasia mice4weeks later. Second, twenty-four NOD/SCID malemice with basically the same tumor size were randomly divided into4groups: A:the negative controlgroup;B:the vehicle control group(DMSO);C:Salinomycin5mg/kg.dgroup;D:Salinomycin10mg/kg.dgroup.Every group were pour the medicine by intraperitoneal injection twice a day.Third, after usedmedicine for3weeks, sacrificed the mice.①Tumor volumes were measured and inhibition tumor rateswere calculated.②Take out the transplant tumors:Some parts of the specimen for pathologicalexamination and immunohistochemistry;Some parts of the tumor were prepared into single cells, toidentify the proportion of prostate cancer stem cells and sorting out the proportion of CD133+CD44+prostate cancer stem cells with flow cytometric.③Then obviously different genes were analyzed bycomparing the host cell genome bwteen B.C and D groups respectively at transcriptional level ofNimbleGen whole human genome, with fold change≥2as obvious up-regulated genes and fold change≤0.5as obvious down-regulated genes.Then we make Go analysis and gene functional pathway analysis ofdifferentially expressed genes.Results The salinomycin can effectively inhibit the growth of PC-3transplanted tumors in NOD/SCID mice:Inoculated30mice,29into the tumor，and the success rate is96.7%.①The average tumorvolume in A,B,C, D, is (164.44±76.60) mm3、(162.72±61.94)mm3、(149.85±59.72) mm3、(149.21±78.14) mm3.Compared with the negative control group, the tumor volume and tumor weight of each salinomycingroup was significantly inhibited. The two treatment groups tumor volume inhibition rates were60.6%and72.4%tumor weight inhibition rate of （50.4±0.10）%and（55.8±0.11）%, respectively. Tumor volumeand tumor weight of the statistical indicators are statistically significant.②HE staining andimmunohistochemistry confirmed the animal model was successfully established.③Flow cytometryanalysis revealed that the proportion of cancer stem cellsof each group is A（3.54±0.19）%，B（3.72±0.52）%，C（1.24±0.13）,D、（0.52±0.11）%.Thereweregreatstatisticalsignificationbetweenvaries salinomycin group and negative control group (P<0.05). There were great statistical significationbetween varies salinomycin group and vehicle control group (P<0.05).There were also great statisticalsignification between salinomycin group each other(P<0.05). But, There were no great statisticalsignification between the vehicle control group and negative control group(P>0.05). With increasing dosesof salinomycin, the proportion of cancer stem cells decreased.④Microarray technology test results:Compared salinomycin treated group with the vehicle control group of tumor stem cell gene expressionprofile, there are6genes expression were upregulatedin2salinomycin treated group.:AGTR1，AKR1B10， ATP2A3， CXCR4， CNTFR， CCL14;and16genes expression weredownregulated:CACNG6，ALOX15，CYP3A4，PLA2G1B，PTGS1，PTGS2，CCL20，FOS，IL8，MAP3K1，MAP3K5，MAP3K8，LAMA4，THBS1，CXCL3，FGF3.⑤C ompared salinomycin treatedgroup with the vehicle control group of tumor stem cell gene expression profile, the upregulated gene in2salinomycin treated group, which are related to the tumor signaling pathway include:“MAPK signalingpathway”，“Pathways in cancer”，“VEGF signaling pathway”，“TGF-beta signaling pathway”；thedownregulated gene in2salinomycin treated group,which are related to the tumor signaling pathwayinclude:“Calcium signaling pathway”，“Cytokine-cytokine receptor interaction”，“Bladder cancer”.Indicated that salinomycin can affect the signal transduction pathway-related genes to inhibit and killprostate cancer CSC effectively.Conclusion①P C-3cell(1×106/0.2ml) can plant and growth in the back of NOD/SCID malemice.②Salinomycin can inhibit the growth of tumor of human PC-3transplanted into NOD/SCIDmice,and the effects associated with the concentration of Salinomycin.③Salinomycin can selectively acton NOD/SCID male mice transplanted tumor of prostate cancer CSC in vivo.④Salinomycin role inprostate cancer stem cells refers to multiple genes and pathways participated. Salinomycin can multi-target effect in prostate cancer stem cells.