| Objective: Chemokines,secreted by many kinds of immunocyte,are a family of small heparin-binding proteins that can mediate leukocyte recruitment to sites of inflammation.There are four subgroups involve in chemokine family:CC,CXC,CX3 C and C chemokine ligands(C-cysteine,X-any amino acid).CXCL8,belongs to CXC chemokine family,can behave in the regulation of tumor invasion,angiogenesis,and metastasis by combining with CXCR1/2 selectively.A CXCR1/2 antagonist,iP10,can binding to CXCR1/2 and simultaneously block CXCL8 integrate with CXCR1/2 receptor.During the whole procedure,iP10 action as an anti-inflammatory and antineoplastic factor.In this research,we work with the aim of finding whether iP10 can intensify the antineoplastic affect of Cisplatin and alleviate Cisplatin toxicity in mice breast cancer.Methods:1.In vivo: Animal model: Inoculated 3×105 4T1 cells into Bal B/C mice mammary fat pad.On the 7th day after inoculation,divided 32 mice into 4 groups randomly,including Control group,iP10 group,DDP group and iP10+DDP group.There were 8 mice in each group.DDP group and iP10+DDP group treated with Cisplatin,12.5mg/kg,only once time,and the other groups are treated with isovolumic saline simultaneously.iP10 group and iP10+ DDP group are treated with iP10,500?g/kg,in every other day,and other groups are treated with isovolumic saline simultaneously.Disposal of samples: Mice were put to death 21 days after treatment,stripping solid tumors.Measuring tumor diameters and calculating volumes.Detective methods:(1)Compare tumor volumes among 4 groups.(2)Compare the variation of weight among 4 groups.(3)Compare the survival percent among 4 groups.(4)The expression of EGFR in tumor tissue was detected by immunohistochemistry.(5)The mRNA expression of CXCL6,CXCL8,TNF-? in tumor tissue were detected by RT-qPCR.(6)The protein expression of VEGF and NF-?B were detected by western blotting.(7)Tumor tissue MPO activity detection.2.In vitro: 4T1 was cultured in vitro,divided into 4 groups randomly,treated respectively.Detective methods:(1)Wound healing assay: Testing whether iP10 can enhance the inhibition of Cisplatin in tumor cell migration.(2)Soft agar colony formation assay: Testing whether iP10 can enhance the inhibition of Cisplatin in tumor cell proliferation.Results:1.In vivo:(1)Compare tumor volumes,the average volume of iP10+ DDP group(295.90±52.57mm3)is significantly less than iP10 group(526.14±72.54mm3)(P<0.05),DDP group(457.65±89.96mm3)(P<0.05),and Control group(747.86±106.38mm3)(P<0.05).(2)Compare the variation of weight: There is no difference among 4 groups,imply the side effect is tolerated.(3)Survival percent: Control group 10%,iP10 group 10%,DDP group30% and iP10+ DDP group with no death.(4)Immunohistochemistry: Compared with Control group,the level of EGFR in iP10 group,DDP group and iP10+ DDP group are less expressed(P<0.05),and among them the iP10+ DDP group is the lowest one.(5)RT-qPCR: The mRNA level of CXCL6,CXCL8 and TNF-? of Control group is highest(P<0.05),and that of iP10 group,DDP group and iP10+ DDP group are lower than Control group(P<0.05),the lowest is iP10+ DDP group(P<0.05).(6)Western blotting: The protein expression of VEGF and NF-?B in Control group is highest(P<0.05),followed are iP10 group,DDP group and iP10+ DDP group(P<0.05).(7)MPO activity: Control group(0.53±0.09),iP10 group(0.28±0.08),DDP group(0.72±0.13),iP10+ DDP group(0.43±0.06)?2.In vitro:(1)Wound healing assay: The wound healing velocity of iP10+ DDP group is slowest among 4 groups(P<0.05),followed are DDP group,iP10 group and Control group.(2)Soft agar colony formation assay: The colony amount of iP10 group,DDP group and iP10+ DDP group are decreased to various extent(P<0.05),and that of iP10+ DDP group is the minimum.Conclusion:1.iP10 enhance the antineoplastic effect of Cisplatin,and the coalition efficacy is much better than individually.2.iP10 can alleviate the toxicity of Cisplatin.3.iP10 heighten the ability of Cisplatin in inhibiting tumor cells proliferation and migration. |
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