The Inhibition Effects Of Anti-heparanase Monoclonal Antibody In Combination With Paclitaxel On Lung Cancer

OBJECTIVE:To investigate the effects of anti-heparanase monoclonal antibody(anti-HPA m Ab) on HPA m RNA transcription, HPA protein expression level and HPA activity of A549 cell, to investigate the effects of anti-HPA m Ab in combination with paclitaxel(PTX) on cell proliferation,migration and invasion of A549 cell. In vitro experiments show that anti-HPA m Ab in combination with PTX inhibited cell proliferation, migration and invasion of A549 cell. On this basis, subcutaneous Lewis,lung cancer xenograft models in C57BL/6 mice were established, and then investigate the inhibition effect of anti-HPA m Ab in combination with PTX on tumor growth and its possible mechanism in vivo. METHODS:A549 cell was treated with anti-HPA m Ab, HPA m RNA transcription level and HPA protein expression level were detected by RT-PCR and western blot. HPA activity was examined by colorimetry and polyacrylamide gel electrophoresis(PAGE). A549 cell was treated with anti-HPA m Ab and PTX alone or in combination, cell proliferation was tested by MTT assay, the effect between anti-HPA m Ab and PTX was evaluated by Jin’s description; cell cycle was detected by flow cytometry(FCM), cell migration and invasion abilities was examined by wound scratch assay and Transwell assay, respectively. Establish subcutaneous Lewis lung cancer xenograft models in C57BL/6 mice, and the 25 C57BL/6 mice were randomized into 5 groups, normal saline group, mouse Ig G group, anti-HPA m Ab group, PTX group, and anti-HPA m Ab in combination with PTX group. Tumor volume was measured and tumor growth curve was draw, tumor weight was weighed and tumor inhibitory rate was calculated, morphological changes of tumor were examined with HE staining. and micro vessel density(MVD) was detected by immunohistochemistry. RESULTS:Compared with solvent control group and mouse Ig G group, HPA m RNA transcription level in A549 cell was down-regulated after treatment with anti-HPA m Ab(100μg/ml, 200μg/ml). HPA activity was decreased in solvent control group after treatment with anti-HPA m Ab(100μg/ml, 200μg/ml). Anti-HPA m Ab and PTX alone inhibited the proliferation A549 cell in a concentration dependent manner. When anti-HPA m Ab(100μg/ml) and PTX(10nmol/L) were applied simultaneously, the growth inhibition rate was significantly higher than anti-HPA m Ab and PTX alone(both P<0.05), and the q value was greater than 1.15. Compared with solvent control group, the ratio of G2-M phase increased significantly(P<0.01), the apoptosis ration increased. The migration capacity of A549 cell was decreased obviously compared with solvent control group and mouse Ig G group(P<0.05), and was lower than anti-HPA m Ab and PTX alone. After treatment with anti-HPA m Ab(50μg/ml) and PTX(5nmol/L), the invasion ability of A549 cell was declined compared with solvent control group(P<0.01), and was lower than that of anti-HPA m Ab and PTX alone(P<0.05).Compared with normal saline group and mouse Ig G group, tumor growth was slower, tumor volume was reduced and tumor weight was lighter in anti-HPA m Ab group and PTX group. When anti-HPA m Ab and PTX were applied simultaneously, the inhibition rate of tumor growth was more significant than that of normal saline group and mouse Ig G group, the tumor volume and the tumor weight were reduced than that of anti-HPA m Ab group and PTX group. HE staining showed that tumor cells growth was inhibited, the necrosis areas in treatment groups were more than those in normal saline group and mouse Ig G group. Immunohistochemical staining indicated that MVD in anti-HPA m Ab group was less than normal saline group, when anti-HPA m Ab and PTX were applied simultaneously, MVD was less than normal saline group. CONCLUSION:HPA m RNA, HPA protein expression level and HPA activity were decreased after treatment with anti-HPA m Ab on A549 cell. Anti-HPA m Ab in combination with PTX had synergistic inhibitory effect on A549 cell, and inhibited the invasion and migration abilities of A549 cell. The mechanism was probably associated with the arrestment in G2-M phase. The tumor inhibition rate of anti-HPA m Ab in combination with PTX was higher than anti-HPA m Ab group or PTX group. Anti-HPA m Ab may be a protein chemosensitizer for lung cancer.