Involvement Of PLGF In Small Cell Lung Cancer Migration Through Human Brain Microvascular Endothelial Cells

From the epidemiology invest, cancer patiens account for approximately 10%～20% of brain metastases cases, and in Chinese, the latest brain metastases cases is about 17000 per year. In all kinds of brain metastases cases, patients with lung cancer account for approximately 30% of brain metastases cases. More specially, small cell lung cancer (SCLC) has the propensity to progress aggressively and early metastasize to brain. Brain metastases are a source of significant morbidity and mortality in patients with SCLC. So, to investe the mechanism of how SCLC cell metastases to brain is very significance to SCLC's prevention and cure.However, cancer metastases are complected patho-stepes. Cancer cells flowed in blood get to distant organ, which firstly extravasate from blood vessels to parenchyma. Thus, the vascellum' permeability effectes cancer cells metastses. A key event of brain metastases is that SCLC cells migrate across the blood-brain barrier (BBB), which separates blood from the brain parenchyma. We had observed that the activation of Rho/ROCK signaling in human brain micrivascular endothelial cells (HBMECs) was required for SCLC cells transendothelial migration, and the increase of brain endothelial cell permeability(FEBS Letters,2006,580:4252-4260). However, the precise molecular mechanism of SCLC cells penetrating the BBB that mainly consists of endothelial cells with tight junction (TJ) is poorly defined.Recently, the relationship between vascular endothelial growth factor(VEGF) and cancer was focused by people. VEGF has been shown to increase vascular permeability, and exerted a pro-angiogennic influence in a variety of human tumours. PLGF is one of the VEGF family, and is known to specifically bind with Flt-1. The logical function of VEGF and PLGF are similar, including stimulation of the growth of vascular endothelial cells. But now, people haven't known that the PLGF is whether expressed in SCLC, and which can be induced to migrate through human brain microvascular by PLGF.So, we first detected PLGF expressed in lung cancer specimen and six kinds of lung cancer cells. Besides, we silenced PLGF in SCLC cells, and observed it transmigrated through HBMEC. Finally, we identificated the signaling pathway induced by PLGF in HBMEC. It is expectable that these works might provide the target to interfere SCLC transmigration through HBMECs.Methods1. Identification the expression in lung cancer by immunohistochemical staining and real-time PCRa. S-P immunohistochemical staining for PLGF in turnoutsb. Determination of PLGF expression in six kinds of lung cancer cells by real-time PCR2. Silencing PLGF contributes to SCLCs transmigration through human brain microvascular endothelial cellsa. Construction of recombinant adenovirus(a) Construction of si-PLGF shuttle(b) Analysis of recombinant shuttle plasmid: shuttle(si PLGF).(c) Cotransformed linearized shuttle and adenoviral backbone(d) Construction of recombinant adenovirus(e) Identification the recombinant adenovirusb. Packing virus(a) HEK 293 cell culture (b) Primary virus harvested(c) Second time virus harvested(d) Third time virus harvested(e) Purified adeno virusc. Infected NCI-H 250 and analysised the effect RNA interference by real-time PCRd. Analysised the infected cells: NCI-H 250 with and without PLGF silenced transmigration through HBMEC monlayer, the effection to the integrity of BBB and ZO-1 protein of TJ in vitro.3. Identification the signaling pathway induced by PLGF in HBMECa. Detection Transendothelial electrical resistance(TEER), and measurement of permeability-HRP fluxb. Observe the distribution of TJ protein (ZO-1) and changes of another TJ protein (occludin)c. Effects of inhibitors of ROCK, Rho, PI3K, PKC, ERK on HBMECs by PLGF induced.d. Dectection activatied RhoA-GTPe. Immumofluorescence for f-actinResults1. PLGF was expressed in lung cancer patients.a. PLGF was expressed in three kinds of lung cancer patients, including adenocarcinoma, squamous cell carcinoma and SCLC. PLGF was high-expressed in adenocarcinoma, and moderate-expressed in squamous cell carcinoma and SCLC.b. PLGF was expressed in small cell lung cancer patients. About 27.8% of SCLC patients, turnouts displayed higher PLGF. And, the lymph nodal status between high-level expression of PLGF and the low-level's was highly significant, p＜0.05.c. PLGF was expressed in six kinds of lung cancer cells. SCLC cells came from brain metastases (HCI-H 250) stained positively for PLGF highly; SCLC cell came from bone marrow metastases (NCI-H 209) stained positively for PLGF highly; SCLC cell came from plenral fluid of lung tumours (NCI-H 446) expressed low-level PLGF; Lung adenocarcinoma (A 549) cell and large cell carcinoma (BE1 and LH 7) expressed low-level PLGF.2. Silencing PLGF contributes to SCLCs transmigration through human brain microvascular endothelial cells.a. Successed construction of si-PLGEb. Successed construction of recombinant asdenovirus.c. Gained purified high titer virus particle.d. After virus infected, about 80% NCI-H 250 cells could be transfected.e. PLGF gene was efficiently silenced in HCI-H 250 by adenovirus infected.f. PLGF silenced NCI-H 250 cell transmigrated through HBMEC monlayer less than the normal NCI-H 250. And the damage of the integrity of BBB was restored in PLGF silencing NCI-H 250 cells transendothelial migration.3. PLGF induced TJ's opening between HBMECs in virto by activation Rho/ROCK signaling.a. PLGF induced a decrease of TEER in a time or dose-dependent manner, and TEER reached lowest point at 30 min after PLGF tratement. As followed, PLGF induced an increased of HRP flux in a time or dose-dependent.b. The recombinant protein PLGF caused TJ disassembly in HBMECs and an increased peak of soluble occluding after 30 min.c. Only the inhibitor of ROCK (Y27632) blocked the HRP flux induced by PLGF.d. RhoA-GTP was decteced in HBMEC after PLGF tratement, and the peak point is 30 min. Besides, PLGF induced changes of TEER and HRP flux in ROCK-WT,ROCK-KDIA and pCAG transfected HBMECs.e. The recombinant protein PLGF induced cofilin changing to p-cofilin. f. The recombinant protein PLGF induced the regulation of action cytoskeleton in HBMECs, and the peak point is 30min.Conclusions1. PLGF was high-expressed in SCLC tumour tissues and SCLC cells came from brain metastases.2. Silencing PLGF contributes to SCLCs transmigration through human brain microvascular endothelial cells.3. PLGF induced TJ's opening between HBMECs in virto by activation Rho/ROCK signaling pathway.