Inhibition And Mechanism Studies Of G31P: A Chemokine Receptor CXCR1/2Antagonist, In Human Prostate Cancer Cells

Objective: Prostate cancer occurrence development is a very complicated process,that the tumour cell energy supersession changes as well as angiogenesis creation aretwo big key factors of development happened in the tumour. The CXC chemokine andtheir receptors played various important effect in the metastasis, invasion as well asgrowth have aroused the home and abroad scholar gradually. CXCR1and CXCR2which over expressed in bladder, prostate cancer, breast cancer, intestinal cancer,stomach cancer, melanoma are the CXC chemokine receptors studied more. Thetumour’s growth and metastasis had been restrained by closing CXCR1and CXCR2antibody. Our lab has previously generated a broad-spectrum ELR-CXC chemokineantagonist, CXCL8(3-72) K11R/G31P (G31P) by Gene-directed mutagenesis. G31Pcan combine with CXCR1and CXCR2with High-affinity, but no biological activity,thus at the same time blocking all chemokine combination with CXCR1and CXCR2receptors. We investigate the effects of G31P on the proliferation, adhesion, apoptosis,metastasis and angiogenesis characteristics of the prostate cancer and its relatedmechanisms in vitro and in vivo by human androgen-independent prostate cancer celllines pc-3study.Methods: In our study, we investigated the effects of G31P on growth andapoptosis of PC-3cell by CCK-8and flow cytometry in vitro with theandrogen-independent prostate cancer cell line PC-3as the object of study. We studiedthe effects of G31P on adhesion and metastasis of PC-3cell by ECM matrix assay, Cellwound healing assay and Transwell chamber invasion assay. The effect of G31P on thevolume, weight, apoptosis, the vascular microvessel densities of human prostate tumorand body weight of nude mice were observed by building human androgen-independent prostate cancer PC-3(GFP-labeled) orthotopic transplantation tumor cells model innude mice. The effect of G31P on the prostate tumour apoptosis was analysized byTUNEL assay. The expression level of SURVIVIN, MMP-9, MMP-2, VEGF and NF-кBwere observed by immunohistochemistry (immunohistochemical) methods to study theeffect of G31P on the prostate tumor metastasis，angiogenesis and the process ofeffective mechanisms in vivo.Results:1. Cck-8results G31P inhibitory effect on the pc-3cells were increased withincreasing concentrations. Compared with the control group, g31p0.1ng/mL,1ng/mL,10ng/mL,100ng/mL for1day, the inhibition rates in the human prostate cancer tumoUrwere5.07%±4.4,20.29%±4.4,22.83%±4.1and38.04%±1.6; For three days, Theinhibition rates were2.27%±9.6,6.14%±9.4,3.87%±2.7and16.02%±6.8; For5days, the inhibition rates were6.08%±4.3,6.65%±1.9,10.13%±2.6and12.73%±3.2.There were some significant difference beteen100ng/mL G31P on PC-3cells for1day(p﹤0.01),3days (p﹤0.01)and5days(p﹤0.05) and the control group.2. AnnexinV-FITC staining results show that the normal cells of control group,G31P10ng/mL and G31P100ng/mL were greater than90%. There is a significantdifference (p﹤0.05) compared the number of cells of the control group with G31P100ng/ml treated group, while compared the number of cells of the control group withG31P10ng/mL treated group the result is opposite (p﹥0.05).3. PC-3cells pretreated with0ng/mL,0.1ng/mL,1ng/mL,10ng/mL and100ng/mL G31P for24h were inocubated on ECM-coated96well plate by ECM matrixadhesion assay. The relative adhesion rate calculated by CCK-8assay were100%±14.05,59.57±13.32(p﹤0.01),32.34%±23.35(p﹤0.01),40.85%±19.43(p﹤0.01)and51.49%±15.50(p﹤0.01)4. The migration rate of the PC-3cells of G31P0ng/mL,10ng/mL and100ng/mLtreatment for48h were62.83%±20.71,44.74%±21.2(p﹤0.05) and44.03%±14.14(p﹤0.05) by Cell wound healing assay; the migration rate of the PC-3cells of G31P0ng/mL,10ng/mL and100ng/mL treatment for72h were75.50%±23.64,52.53%±21.92(p﹤0.05)and50.17%±22.43(p﹤0.05) respectively.5. PC-3cells pretreated with0ng/mL,0.1ng/mL,1ng/mL,10ng/mL and100ng/mL G31P for24h were proceeded the Transwell chamber invasion assay.Thenumber of penetrating cells were34±6,35±4,33±5,33±4and31±6. With the gradual increase of the concentration G31P processing, the number of penetrating cells tendedto decrease, but there were not statistically significant (p﹥0.05) with the differences.6. The tumour volume in mice of the control group (100ul NS), G31P treatmentgroup (0.5mg/kg) and paclitaxel treatment group (15mg/kg) after administration0days,12days,18days and24days were measured in the fluorescence imaging systemby building human androgen-independent prostate cancer PC-3(GFP-labeled)orthotopic transplantation tumor cells model in nude mice in vivo.G31P treatment groupafter the12th day of administration inhibited significantly the prostate tumor volumecompared with control group(p﹤0.05), and this inhibition was more obvious (p﹤0.01)after the18days of administration.7.24days after dosing, the animals were sacrificed and the primary tumor were cut.The primary tumour weighted by electronic balance. The primary tumor weight ofcontrol group, G31P treatment group and the paclitaxel-treated group were2.31±1.08g,1.4±0.89g and1.38±0.87g. G31p treatment group (p﹤0.05) and paclitaxel treatmentgroup (p﹤0.05) were significantly inhibited prostate tumor weight compared with thecontrol group, but no significant difference between the two groups.8. The body weight of nude mice of the control group, G31P treatment group andthe paclitaxel treatment group administered at0days,7days,14days,21and24daysdid not find significant differences (P﹥0.05).9. Animals were sacrificed and the transfer of lymph nodes (lumbar lymph nodes,mesenteric lymph nodes, distant lymph node), pancreas, bone and other parts in theabdominal cavity was observed under a fluorescence imaging system. The transfer oflymph nodes (lumbar lymph nodes, mesenteric lymph nodes, distant lymph node) ofG31P and paclitaxel treatment group compared with the control group decreasedslightly,but significant differences (P﹥0.05) did not been found by χ2test. Thepancreas metastasis of G31P treatment group decreased obviously（P﹤0.05）.10. G31P treatment group compared with the control group significantly inhibitedprostate tumor angiogenesis (0.49±0.12, P﹤0.05). Paclitaxel treatment groupcompared with the control group could inhibit prostate tumor angiogenesis (0.41±0.18,P﹤0.05), but no significant difference between the two groups.11. The integrated optical density of each photo (IOD) of G31P treatment groupMMP-9(P﹤0.01), VEGF (P﹤0.01) and NF-кB (P<0.01) expression compared withthe control group using the Image-Pro6.0software were statistically significant byimmunohistochemical analysis. The IOD of G31P treatment group SURVIVIN （P﹥ 0.05）and MMP-2（P﹥0.05）expression compared with the control group were notstatistically significant.Conclusion:1. G31P could inhibit proliferation of the androgen-independent prostate cancercell line PC-3in a dose-dependent manner in vivo and in vitro. The inhibition effect ofG31P on PC-3cells was not caused by killing the PC-3cells directly or induce PC-3cells apoptosis.2. G31P could inhibit adhesion and metastasis of the androgen-independentprostate cancer cell line PC-3in a dose-dependent manner in vitro.3. G31P could significantly inhibit pancreas and the liver metastasis of theandrogen-independent prostate cancer cell line PC-3in vivo.4. There were not significant effect about symptoms and quality of life of G31P onthe nude mice.5. G31P could inhibit angiogenesis of the androgen-independent prostate cancercell line PC-3in vivo.6. The inhibition effect of G31P on metastasis of prostate cancer may be related toreduced expression of MMP-9, but not with MMP-2expression.7. The inhibition effect of G31P on angiogenesis of prostate cancer may be related to reducedexpression of VEGF and NF-кB, but not with SURVIVIN expression. This was the same withpaclitaxel.