The Tumor Micro-vascular Normalization Window Related To Endostar Treatment And Combination Of Endostar And Cisplatin For The Treatment Of Lung Carcinoma

Objective: The aim of this study was to observe the changes of tumormicro-vascular density, pericyte maturation and hypoxia markers of LewisLung Carcinoma(LLC) Xenografts in mice after treatment with Endostar(recombinant human endostatin, rh-ES) in different periods, and to find outthe tumor micro-vascular normalization window. Then the anti-tumor effectof Endostar and cisplatin(DDP) was to be evaluated according to thecombination at different time windows, to explore the optimal schedule ofcombination of both drugs.Methods:1. The observation of the tumor micro-vascular normalizationwindow after treatment with Endostar in different periods:(1) LLC xenograftswere established in C57BL/6mice. Then the mice were randomly assigned totwo groups, with20in each. The Endostar group received intra-abdominalinjection of Endostar at20mg/kg/d on d1-12, while the control groupreceived normal saline at0.2ml/d on the same period. Five mice were put todeath on d2, d5, d8and d12in each group, respectively.(2) The tumormicro-vascular density, pericyte maturation and hypoxia markers wereobserved by immunohistochemical staining, real-time quantitative PCR andELISA, respectively.2. The effect of Endostar combination with cisplatinduring the different time windows:(1)30mice with LLC xenografts wererandomly assigned to six groups, with5in each. The control group receivedintra-abdominal injection of normal saline at0.2ml/d on d1-12, the Endostar group Endostar20mg/kg/d on d1-12, the DDP group at DDP3mg/kg/d ond1-4, the Endostar+DDP(d1-4) at Endostar20mg/kg/d on d1-12+DDP3mg/kg/d on d1-4, the Endostar+DDP(d5-8) at Endostar20mg/kg/d on d1-12+DDP3mg/kg/d on d5-8, the Endostar+DDP(d9-12) at Endostar20mg/kg/don d1-12+DDP3mg/kg/d on d9-12.(2) The tumor growth curves weredrawn by measuring the changes of tumor volumes in vivo every other day,and the tumor-inhibition rates were calculated by stripped tumor weight aftertreatment. The necrosis of tumor, lung metastasis, tumor micro-vasculardensity and apoptosis marker were observed by HE staining,Immunofluorescence staining and ELISA, respectively.Results:1. The observation of the tumor vascular normalization windowby Endostar:(1) The brownish yellow districts in the Endostar groups,which indicated the tumor micro-vascular density(MVD), were discontinuedand interrupted, and the CD34expression was decreased on d5, d8and d12,with significant difference compared with those in the control group.(2) Theexpression of RGS5in the Endostar group, which indicated the pericytematuration, was significantly lower than that in control group on d5and d8(P<0.05). RGS5expression was increased after d8and showed no significantdifference compared with control on the same period (P>0.05).(3) Theexpression of hypoxia markers, CA9and HIF-1α, were consistently decreasedon d5and d8in tumor tissue in the Endostar group, with significant differencecompared with control (P<0.05). The expression of CA9and HIF-1α wereincreased on d12, with no significant difference compared with control on thesame period (P>0.05);(4) A significant positive correlation was seen amongMVD, RGS5, CA9and HIF-1ɑ (correlation coefficient0<r <1, P <0.05).2.The effect of Endostar combination with cisplatin during the different time windows:(1) Tumor grew most slowly and the tumor-inhibition rate was thehighest in the Endostar+DDP(d5-8) group, which was statistically significantcompared with each other group (P <0.05).(2) Necrosis of tumor was themost apparent in the Endostar+DDP(d5-8) group, and the lest in the normalsaline group.(3) Lung metastasis was seen in every group except the Endostar+DDP(d5-8) group. The most invasive growth of metastatic tumors in lungswas seen in the control group, with little normal lung tissue remaining.(4)MVD was the lowest in Endostar+DDP(d5-8) group after therapy, withsignificant difference compared with each other group (P<0.05).(5) Theexpression of survivin was the lowest in Endostar+DDP(d5-8) group aftertherapy, with significant difference compared with each other group (P<0.05).Conclusion:1. After treatment with Endostar in mice with LLCXenografts, the tumor microvascular and expression of RGS5was inhibited,which made the tumor micro-vascular had a short time window ofnormalization during d5-8. The hypoxic state of tumor was partially improvedwithin the time window.2. Different time pattern of combination of Endostarand DDP might influence the therapeutic effect. During the micro-vascularnormalization window induced by Endostar, an optimal effect could beexpected.