The Study Of The Anti-angiogenic Effect Of ZR30 In Glioma

[Background] Glioblastoma(GBM)is the most common type of central nervous system malignant tumor,which originates from glial cells.Although the study of its occurrence and development and treatment strategies have been carried out worldwide,the current treatment effect is remaining frustrating.and the average overall survival time of patients is less than 2 years because of its characteristic likes diffuse growth,unclear boundaries with normal brain tissue,resistance to chemoradiotherapy and recurrence.Conventional treatment methods of GBM include surgical resection,combining radiotherapy and chemotherapy.Finding new therapeutic targets such as anti-tumor angiogenesis is critical at present.Our group has confirmed that ZR30,epidermal growth factor-containing fibulin-like extracellular matrix protein 1(EFEMP1)-derived tumor suppressor protein,has a reasonable inhibitory effect on glioma cell lines such as U251,U87 in vitro and in vivo.In the current study,we focus on the anti-angiogenic effect of ZR30 on human primary cells.[Objective] To elucidate the antiangiogenic effect of ZR30 on angiogenesis of human glioma primary culture cells in vitro and in vivo,and to investigate its possible mechanism.[Methods] Human glioma primary cell lines 51 B,97B,and 98 B were used in our study.Cell growth and microtubule formation were monitored to test the role of ZR30 on the proliferation and antiangiogenic ability in vitro.Westernblotting was used to detect proteins level related to the vascular endothelial growth factor(VEGF)/ vascular endothelial growth factor receptor 2(VEGFR2)signaling pathway.The in vivo mice model was applied to evaluate the efficacy of ZR30 on glioma proliferation.Microvascular density formed by tumor specimens were analyzed to determine the antiangiogenic ability of ZR30 on glioma.[Results] ZR30 inhibited the proliferation ability of primary glioma cells 51 B,97B and 98 B.In the microtubule formation experiment,the number of microtubules formed by 51 B cells were significantly different in the ZR30 treated groups(both low concentration group and high concentration group)and the control group(n=3,P<0.01).The average number of microtubules was 54% in ZR30 low concentration group which less than that of the control group,the ZR30 high-concentration group had an average of 78% fewer microtubules than that of control group.The number of microtubules in the high concentration group and the low concentration group was also statistically significant(n=3,P <0.05).Compared with the ZR30 low concentration group,the ZR30 high concentration group had an average of 57% fewer microtubules.The ZR30 lowconcentration group of 97 B had an average of 32% less than the control group (n=3,P<0.05),and the high-concentration group had an average of 36% less.In the 98 B cell,ZR30 low-concentration group had an average of 34% less microtubules than the control group %,high-concentration group was 48% less on average(n=3,P<0.05).The expression of VEGFR2 and VEGFA was detected by Western blot.After 12 hours of treatment with ZR30,the expression of VEGFR2 in 97 B cells was significantly reduced(down to the 30% of the untreated control group),and then returned to 60% of the control group after 24 hours.However,the reduction of VEGFR2 upon the treatment of ZR30 was not repeated in the 51 B cell line.Animal experiments showed that ZR30 prolonged the median survival time of 51B-51A(1: 9)glioma in situ tumor formation model by 12.5 days,with short-term difference in efficacy ZR30 prolongs 97B-97A(1: 9)glioma in situ tumor formation model with a median survival time of 14.5 days,with long-term difference in efficacy;the number of micro-vessels in the ZR30 treatment group were significantly reduced compared to the PBS treatment group,and 51B-51 A transplantation.The vascular density of the tumor formed by 97B-97 A with the treatment of ZR30 was 37% of the control group(n=20,P<0.01).The vascular density of the tumor formed by 97B-97 A with the treatment of ZR30 was 30% of the untreated control group(n=20,P<0.01).[Conclusions] EFEMP1-derived tumor protein ZR30 can inhibit angiogenesis of gliomas.The molecular mechanism of how ZR30's inhibits the glioma angiogenesis might be related to the down-regulated VEGFR2.Our results therefore revealed that the synthetic variant ZR30 of EFEMP1 inhibited glioma angiogenesis efficiently through the downregulation of VEGFA-VEGFR2 signal pathway.