Research Of Qi Lan Capsule Inhibiting VM Formation In Prostate Cancer Based On VEGF

Background and Aim:Prostate cancer is a common malignancy in male reproductive system, which ranks first in male cancer incidence in Europe and America. In China, with the aging population and the popularity of medical screening of middle-aged men, the incidence of prostate cancer and the detection rate increased significantly in recent years. For a long time, tumor angiogenesis has been known as the only way to get their blood supply, but angiogenesis probably not the only mechanism of microcirculation of blood supply to the tumor. Some highly malignant tumor cells can be simulated endothelium-dependent blood (Edothelium dependent vessel, EDV) morphological structure to form a new blood supply mode, called "vascular mimicry" (Vasculogenic mimicry, VM). It has been reported that VM can be found in many cancers, such as liver cancer, prostate cancer, ovarian cancer and many more. VM plays an important role in prostate cancer progression. VEGF can promote the formation of prostate tumor growth and metastasis by increasing vascular permeability and stimulate angiogenesis. Increased VEGF expression in prostate cancer tissue by stimulating angiogenesis, cancer cells can supply more nutrients, promote sustainable growth and progressive carcinoma. Recent studies have found that VEGF can induce the formation of VM in prostate tumor cells via HIF-1?VEGF?EphA2 ?MMPs?Laminin 5 y 2?VM pathways,especially in the reproductive system cancer, only high expression of VEGF cells have the potential formation of VM. Therefore, we participate in the selection of VEGF-mediated involved in the formation of VM pathways; discuss the mechanism of Qilan capsule inhibiting VM formation in prostate cancer based on VEGF. This study intends to analyze the impact of Qilan capsule in VM formation, and to investigate the role of Qilan capsul in intervention VM formation and "dose-response" relationship.In 3D culture system, we use RNA interference technology to silence the expression of each site on the VEGF pathway, with RT-PCR and Western-blotting technology to detect the changes of gene and protein expression in each point on the VEGF path, aim to explore the action target of Qilan capsule in inhibiting VM formation in prostate cancer.Methods:1. Remove the PC-3 cells frozen Vials from the liquid nitrogen tank, rapidly melting, transfer it to a centrifuge tube, add 2ml DMEM/F12 medium, mix by pipetting cells. The supernatant was discarded after centrifugation; add 1ml DMEM/F12 medium, mix by pipetting cells, made into single cell suspension. Transfer the cell suspension to a 25ml cell culture flask; add in containing 10% FBS in DMEM/F-12 medium 5ml to conduct cells 'passage culture. Cell counts until a sufficient number of cells for experiment. Developped cells culture again by BeaverNanoTM 3D cell culture methods, the 3D cultured cells were randomly divided into:control group, low dose group, middle dose group and high dose group, and each group with 3 holes. Qilan containing serum at 10%,5%, and 2.5%(culture system volume ratio) is set high, medium and low dose group administered, less than 10% of the group with fetal calf serum complement. The blank group was administered with 10% blank serum, reaction time 48h. Finally, the microscope was used to observe the cell VM in the culture medium.2. When PC-3 cells in culture flasks in logarithmic growth phase, containing 10% FBS in DMEM/F-12 medium 2ml was added into it after treatment. Pipetting the cells form a single cell suspension,6 holes plate was inoculated by 2.5* 105 cells/hole concentration, after mixing at 37?,5% CO2 incubator 24 hours. Transfection of siRNA after culture, the transfection mixture was added dropwise to 6 well plates, after mixing, incubation for 6 hours in incubator. Then determine the transfection, estimate transfection efficiency, if the transfection efficiency was more than 80%, continue follow-up experiment. The fluorescent labeling group was no longer in the follow-up experiment. Grouping cells, Including blank control group, HIF-1? siRNA group, VEGF-? siRNA group, EphA2 siRNA group, Qilan capsule group, Qilan capsule+HIF-1? siRNA group, Qilan capsule+VEGF-? siRNA group, Qilan capsules+EphA2 siRNA group. Use RNA interference technology to silence the expression of each site on the VEGF pathway.Finally use RT-PCR and Western-blotting technology to detect the changes of gene and protein expression on the path.Results:1.Various types of VM formation performance were seen in the blank control group, Qilan capsule low dose group showed straight, arranged in parallel, crossed, rainbow-like (not fully closed), rainbow-like branched channels, but the quantity is less than the blank group. In the middle dose group, only a small amount of straight, parallel aligned, cross shaped channels; the high dose group of only a very small amount of straight channel.2. The gene expression of HIF-1?, VEGF-? and MMP-1 can be reduced by the Qilan capsule, but there was no significant effect on EphA2. After transfection with HIF-1? siRNA, HIF-1?, MMP-1 gene expressions were significantly inhibited, VEGF-?, EphA2 gene expressions were not significantly inhibited. After transfection with VEGF-? siRNA, HIF-1?, VEGF-? gene expression was not significantly inhibited, but EphA2 and MMP-1 was obviously inhibited. After transfection with EphA2 siRNA, no significant inhibition of gene expression of HIF-1? and VEGF-?, but MMP-1 and EphA2 was obviously inhibited. The expression of HIF-1? protein was significantly inhibited in all 4 groups adding Qilan capsule. VEGF-? protein expression was inhibited in 4 Qilan capsule groups, HIF-1? siRNA group, VEGF-? siRNA group, EphA2 siRNA group. No effect of Qilan capsule showed in EphA2 protein expression.Conclusions:1. Serum containing Qilan capsule can inhibit VM formation in 3D culture model by prostate cancer cell PC-3 in vitro. The effect is more obvious in the middle and high dosage group.2. The relationship between VEGF-a, EphA2 and MMP-1 was significantly related to the upper and lower reaches, to be important upstream factors in the formation of VM. The relationship confirmed the existence of VEGF-??-EphA2?MMP-1 pathway in the formation of VM. No significant relationship found between HIF-1? and VEGF-? or EphA2, probably the upstream of VEGFa gene is regulated by other more factors. The expression of MMP-1 gene was inhibited after HIF-1? silence, suggested that HIF-1? might inhibit the expression of MMP-1 through other pathways, thus affecting the formation of VM, then further research is needed.3. Qilan capsule can inhibit the formation of VM in prostate cancer by inhibiting the gene and protein expression of HIF-1?, VEGF-? and MMP-1. By this way, the growth and development process of the blood supply system in the tumor tissues were affected, and the therapeutic effect was appeard.