Objective To prepare a kind of P-selectin antibody loaded-targeted ultrasound contrast agent,and to investigate its ability of targeting in vitro.Methods P-selectin antibody loaded-targeted ultrasound contrast agent was prepared via an avidin-biotin bridge.Its basic property was determined.Human umbilical vein endothelial cells (HUVECs) were stimulated to express P-selectin by applying different doses of recombinant human interleukin-4 (rhIL-4) and histamine.The expression level of P-selectin was detected by immunofluorescence technique,and explored rhIL-4 optimal stimulation dose.The experiment group was divided into 3 groups,including targeted ultrasound contrast agent,isotype control ultrasound contrast agent and blank ultrasound contrast agent.Targeting abilities of 3 groups were observed by adhering to treated HUVECs and untreated HUVECs,respectively.Results P-selectin antibody loaded targeted ultrasound contrast agent was prepared successfully.The average diameter was (2.24 ± 0.71)μm.The average Zeta potential was (-2.75 ± 0.84)mV.The concentration was (3.0 ± 0.3) × 109/ml.The rate of antibody binding was as high as 99.80％.RhIL-4 and histamine could stimulate HUVECs to express P-selectin,and optimal stimulation dose was obtained.Targeting experiment showed that targeted ultrasound contrast agent could preferably adhere to HUVECs stimulated by optimal dose of rhIL-4 and histamine.Compared with other groups,there were significant differences.Conclusions P-selectin antibody loaded targeted ultrasound contrast agent was prepared successfully via an avidin-biotin bridge.The targeted ultrasound contrast agent could effectively adhere to HUVECs stimulated by rhIL-4 and histamine.

OBJECTIVETo observe the impact of concurrent administration of recombinant human p53 adenovirus (Ad-p53) with EGFR inhibitor gefitinib on breast cancer MDA-MB-468 cells.

METHODSMDA-MB-468 cells were treated with Ad-p53 and/or gefitinib. The effect of Ad-p53 and gefitinib on the growth of MDA-MB-468 cells was evaluated by MTT assay. Cell apoptosis was detected by flow cytometry. Western blot analysis was used to detect the alteration of p53,EGFR, phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and apoptosis-related proteins. Ad-p53 combined with gefitinib was used in vivo to explore their effect on tumor xenograft in nude mice. Immunohistochemistry was used to detect the p53 expression in vivo.

RESULTSThe MTT assay showed a stronger inhibitory effect of gefitinib on MDA-MB-468 cells infected with Ad-p53 than on the control cells. Cell apoptosis assay revealed that the apoptosis rates of MDA-MB-468 cells in vehicle-treated group, Ad-p53 group, gefitinib group, and combination group were 8.5%, 17.4%, 20.5% and 32.6%, respectively. The apoptosis rate of MDA-MB-468 cells in the combination group was higher than that in other groups (P < 0.05, for all) . Western blot analysis revealed that the expression of p53 was significantly up-regulated in the presence of Ad-p53. The combination of Ad-p53 and gefitinib significantly down-regulated p-Akt (S473)(P < 0.01) and up-regulated caspase-9 and cleaved caspase-3 (P < 0.01 for both). Tumor inhibition rates (TIR) in the Ad-p53, gefitinib, and combination groups were 35.7%, 28.7% and 74.4%, respectively. Ad-p53 and gefitinib combination therapy significantly reduced the tumor burden in nude mice (P < 0.05 for all).Immunohistochemistry showed that after intratumoral administration of Ad-p53, wild-type p53 was increased (P < 0.01). p53 expressions in the vehicle-treated, Ad-p53, gefitinib and combination groups were 45.2%, 80.1%, 50.7% and 90.6%, respectively.

CONCLUSIONSWild-type p53 may reverse the sensitivity of MDA-MB-468 cells to gefitinib through down-regulation of the PI3K/Akt pathway. The apoptotic activity induced by this combination treatment might be regulated through caspase cascade.

Adenoviridae ; Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Breast Neoplasms ; Caspase 3 ; Caspase 9 ; Cell Line, Tumor ; Down-Regulation ; Genes, p53 ; Humans ; Mice ; Mice, Nude ; Phosphatidylinositol 3-Kinases ; Quinazolines ; pharmacology