G31P, CXCR1/2 Antagonist,enhances The Effect Of PI3K Inhibitors In Breast Cancer Therapy

Objective:Chemokines are cytokine major secreted by the a variety of immune cells, contain tumor cells and other stromal cells, binding to the endothelial cell surface, mediating chemotaxis and activation of neutrophils, monocytes, lymphocytes and other cells.CXC chemokines play a important role in inflammation, angiogenesis and tumor microenvironment.CXCR1/2 active downstream in signaling pathway via binding with ligands. In addition to chemotaxis and activation of immune cells, chemokines also involve in pathological process as tumorigenesis,development,metastasis,pathogen infection and transplant rejection. Overexpression of IL-8 is found in various tumor, promoting angiogenesis, invasion and migration.G31 P,a chemokine receptor CXCR1/2 antagonist, can combine with CXCR1/CXCR2 with high affinity and block many chemokines which bind with CXCR1/2 receptors, including IL-8, and act as anti-inflammatory and antitumor agent.GDC-0941,an orally bioavailable class I PI3K/AKT/m TOR inhibitor, inhibits tumor proliferation and induce tumor apoptosis. At present,it’s in clinical test, well efficacy, low toxicity and well tolerated in patients. We searched the inhibition of human breast cancer through combination of GDC-0941 and G31 P,provid new evidences to clinic therapy. Methods: In vitro experiments:HCC1954、BT474 and 4T1 cultured in vitro, treated in divide. Testing methods:Cell wound healing assay,testing the impact of combination of GDC-0941 and G31 P to tumor cell migration. Western Blotting,testing the protein expression of cl-PARP、bcl-2 and p S6.Colony formation assay,testing the impact of combination to tumorigenesis and proliferation. Cell survival assay, detecting the effect of GDC-0941 and G31 P to cell survival. Transwell assay, detecting the effect to cell invasion ability of drug combination. Cell cycle testing: detecting the cell cycle distribution. In vivo assay:Establishing mouse model:5×104 4T1 inoculated in mammary fat pad into BALB/c female mice, random assignment into 4 group on the 10 th day, including GDC-0941+G31P group,GDC-0941,G31 P and Control.8 mice in each group. Intraperitoneal injection of G31 P,500μg/kg, to mice in GDC-0941+G31P and G31 P group in every other day, saline to other groups. Gavaging with GDC-0941,130mg/kg, to GDC-0941 and GDC-0941+G31P group each day, saline to other groups. Disposal of specimens, sacrifice all mice on the 24 th day after treatment. Stripping solid tumors and lungs. Pathological detecting after tissue fixing.Results:(1)In vivo experiments:Cell wound healing assay,in GDC-0941+G31P group, the wound “healing” less then other three groups, suggest combination of GDC-0941 and G31 P inhibit cell migration significantly. Western blots,results show cl-PARP overexpression in GDC-0941+G31P group then other groups,bcl-2 and p S6 protein are less expression, suggest that drug combination promotes tumor apoptosis, inhibits tumor proliferation. Colony formation assay,shows the colony number is reduced in GDC-0941 group,G31 P group and GDC-0941+G31P group in varying degrees, and colony number in GDC-0941+G31P group is the minimum(p<0.01),suggest combination drug therapy inhibits colony inhibition. Cell survival assay shows, compare with control, survival cells are less then control in GDC-0941, nonsenses between G31 P and Control, GDC-0941+G31P is the least(p<0.05), suggesting combination enhances the effect of GDC-0941 of cell-killing effect. Transwell assay shows crystal violet staining cells reduced in varying degrees compare with control, and the GDC-0941+G31P is the minimum( p<0.01), imply that combination inhibits the invasion ability significantly. Cell cycle testing,shows there’s no significant difference between GDC-0941,G31 P and Control, cells in GDC-0941+G31P group arrested in S phase(p<0.05),imply that combination inhibits cell cycle before G2/M phase,depress cell proliferation.(2)G31P combined with GDC-0941 could significantly inhibit tumor growth. The average tumor volume of GDC-0941+G31P group(188.4±23.32mm3) was significantly less than Control group(685.6±109.5)(p<0.01), and also less than the GDC-0941 group(461.2±50.70mm3)( p<0.05)or G31 P group(496±46.75mm3)(p<0.05),suggest combination could inhibit solid tumor volume. Metastasis tumor number and HE staining shows, combination could significantly inhibit tumor metastasis from mammary fad pad to lung. There’s no different among 4 groups, imply that combination is tolerated.Conclusion:(1)Combined with G31 P enhanced the efficacy of GDC-0941 on inhibit murine breast cancer cell proliferation.(5)GDC-0941 combined with G31 P is tolerated.(6)Combination significantly inhibits breast cancer metastasis, stronger than monotherapy.(7)The effect of GDC-0941 of inhibit tumorigenesis, progress, proliferation, migration and invasion, promoting tumor apoptosis and cell-killing are enhanced by combined with G31 P.