Inhibitory Effect Of Hedgehog Signaling Pathway Blocking On Metastatic Ability Of EC109Cells And Its Mechanism

Research background:At present the incidence of esophageal cancer worldwide are relativelyhigh, especially in our country, is the highest incidence of a disease.Esophageal cancer is a malignant tumor of high degree, and since most of theclinically diagnosed late, the prognosis is very poor, although for patientswith early surgery is currently the best way to treat, but with a greaterincidence of postoperative recurrence and distant metastasis. Early cancereasily occurs metastasis is the main reason, Lack of postoperativeesophageal cancer therapy drugs, and there is no clear effect of targeteddrugs also is the important reasons of decreased esophageal cancer survivalrates. Hedgehog (Hh) signaling pathway by Hh-Ptch-Smo-Gli cascade,When Hh protein ligand and receptor Ptch, can eliminate the inhibitoryeffect of the membrane receptor Ptch on Smo, the activation of the Smo andfurther activate the nuclear transcription factors Gli, induced expression ofdownstream target genes. In recent years, studies have shown that the Hhsignaling pathway is closely related to the occurrence and metastasis of human breast cancer, prostate cancer, liver cancer, stomach cancerandpancreatic cancer etc. Esophageal cancer activation of the Hh pathwayabnormalities have been confirmed, But the Hh pathway and the study on therelationship between the esophageal cancer cell metastasis ability is littlereported. The vascular endothelial growth factor (VEGF) and matrixmetalloproteinase-9(MMP-9) is closely related to the occurrence,development, and metastasis of esophageal cancer Had confirmed, alsostudies have shown that both is closely related with the Hh pathway, but thespecific mechanism of action research is still in its infancy.Objective:This experiment by esophageal cancer cell line EC109as the researchobject, After specific blocking the Hh signaling pathway by Cyclopamine,toobserve the effect of cyclopamine on metastatic ability and transfer relatedfactor to MMP-9, VEGF protein expression level of human esophagealcancer EC109cells and explore the possible mechanism, Provide theoreticaland experimental basis for clinical targeted treatment of esophageal cancer.Methods：1. Cultivate EC109cells as experiment object, After specific blockingthe Hh signaling pathway by Cyclopamine, determined by MTT method todetect Cyclopamine inhibitory effect on the esophageal cancer cells, andcalculate the corresponding half inhibition rate (IC50). 2. Transwell Chamber assay were used to examine the change ofmigration and invasion ability of EC109cells treated with cyclopamine.3. Angiogenesis in vitro assay were used to examine the angiogenesisability of EC109cells treated with cyclopamine.4. The expression changes of Gli-1mRNA on EC109cells treated withcyclopamine was detected by RT-PCR.5. Western blotting was used to examine the protein expressionschanges of Gli-1, matrixmetalloproteainse-9(MMP-9) and vascularendothelial growth factor (VEGF) of EC109cells treated with cyclopamine.Results：1. EC109cells can be inhibited the proliferation and growth afterCyclopamine treated, cell transformed the full fusiform adherent growthstate in outline of obtuse state of suspension, Under certain concentration ofinhibition rate and concentration were positively correlated, and over time,the inhibition rate also increased, subsequently presents a certain amount oftime and dose dependent. Role in48hours after half of the inhibition rate of12mu mol/L.2. In Transwell chamber assay and angiogenesis assay we found thatinhibition of the hedgehog signaling pathway by cyclopamine suppressedthe migration, invasion, and angiogenesis of EC109cells, compared with control group, the migration out of the small room of the microporousmembrane cells was significantly reduced (32.80±6.77) vs (98.40±10.08),the attack in membrane cells was also significantly reduced (26.20±4.58) vs(83.40±8.65); Reduced angiogenesis (8.60±2.70) vs (19.40±4.86),(p <0.05).3. In RT-PCR assay we found that cyclopmine treatment significantlylowered the expression of Gli-1mRNA(2.89±0.34)vs(5.58±0.89),(P<0.05).4. By Western blot method to detect protein levels found that theexperimental group compared with control group, Gli-1, MMP-9, andrelative expression of VEGF protein were decreased (P <0.05).ConclusionsHigh expression of EC109cells exist in the Hh signaling pathway, andcan be blocked by cyclopmine, blocking the Hh signaling pathway couldsignificantly inhibit the growth of EC109cells and metastatic ability,possibly by down-regulating MMP-9and VEGF expression as a result ofGli-1inhibition.