| BackgroudEsophageal carcinoma is one of the gastrointestinal tumors with high incidence and poor prognosis in our country. Studies have reported that tumor angiogenesis play a key role in recurrence and metastasis. When the tumor grows to 2mm3, angiogenesis and vasculogenic mimicry(VM) have been occurred to maintain blood supply for tumor. Studies have shown that the VM could be found in a variety of solid tumors, such as invasive ovarian cancer, inflammatory breast cancer, malignant glioma, laryngeal squamous cell carcinoma, and suggests that VM has an important significance in tumor tissue. While, the PI3 K signaling pathway plays an important role in the VM formation. PI3 K and its downstream MMPs family can regulate the growth of vascular mimicry, which is related to the proliferation and migration of malignant tumor.All trans retinoic acid(ATRA) as an important differentiation-inducing agent has been successfully applied to clinical, and research suggests that ATRA can inhibit the migration and proliferation of esophageal cancer cells, which may be involved in the inhibition of VEGF. The blood supply of the tumor depends on angiogenesis, in the meanwhile vasculogenic mimicry also plays an important role, but the signaling pathway is not clear, there may be exist multiple signal pathways and targets. In this study we investigated the effect of all-trans retinoic acid on proliferation, migration and expression of PI3 K and MMP-2 protein and m RNA in EC1 cells. This study could provide the theory basis for anti-angiogenesis therapy.Objective To investigate effects of all-trans retinoic acid(ATRA) and LY294002 alone or combined on proliferation, migration and expression of phosphoinositide3-kinase and matrix metalloproteinase-2 in EC1 cells.Methods The cultured EC1 cells were randomly devided into four groups: ATRA group, LY294002 group, ATRA plus LY294002 group and control group. The proliferation rate of treated cells was detected by CCK-8 assay. Wound healing assay was used to observe the migration rate. The m RNA and protein expressions of PI3 K and MMP-2 in the cells were detected by reverse transcriptase polymerase chain reaction(RT-PCR) and western blot assay.Results 1.CCK-8 results showed that after 24 h, in the experimental groups, namely ATRA(10 ?mol/L)?LY294002(30 ?mol/L)?ATRA plus LY294002(ATRA 10 ?mol/L?LY294002 30 ?mol/L), the proliferation inhibition rate was(45.81±1.25)%,(46.79±0.93)% and(53.10±0.87)% respectively; After 48 h, the proliferation rate of experimental groups was(52.76±1.17)%,(55.29±2.24)%, and(65.55±2.50)% respectively; After 72 h, the proliferation inhibition rate of each experimental group was(66.77±1.10)%,(70.26±2.35)% and(84.24±2.38)% respectively. While the proliferation inhibition rate of control group was 0.00% at three time points. According to statistic analysis, as compared with control group, the differences of the experimental groups had statistical significance(P<0.05), and compared with the single drug group, the difference of the two combination group was statistically significant(P<0.05).2.Wound healing assay showed that when the EC1 cells were incubated for 24 h with each drug after scratching, the migration rate of experimental groups was(50.43±1.36) %,(50.36±0.79) % and(24.63±1.74) %, respectively; while the control group was(74.50±1.06) %. After statistics analysis, there was a statistically significant difference between experimental group and control group(P<0.05), and compared with the single, the difference of the combination group was statistically significant(P<0.05).3.Western blot showed that after 48 h, the relative expression of PI3 K protein was(72.53±1.42) ×10-2 in control group, and(54.08±0.97) ×10-2,(54.08±0.97) ×10-2,(25.08±0.58) ×10-2 in the experimental groups, respectively; Relative expression of MMP-2 protein was(58.43±0.49) ×10-2 in control group,(43.07±1.08) ×10-2,(39.78±0.36) ×10-2,(20.48±0.56) ×10-2 in the experimental groups, respectively. The results show that the expression of PI3 K and MMP-2 protein decreased in EC1 cell under the influence of drugs. Satistically significant was observed between experimental groups and control group(P<0.05). Further more, there was statistically significant between the combination groups and the single one(P < 0.05).4.RT-PCR assay showed that when the EC1 cells were incubated for 48 h, PI3 K m RNA expression of experimental groups were(25.45±3.45) ×10-2,(24.08±1.48) ×10-2 and(12.45±1.77) ×10-2 respectively. while, control group was(46.48±0.78) ×10-2; MMP-2 m RNA expression were(20.39±1.97)×10-2,(19.78±4.17) ×10-2 and(9.74±2.25) ×10-2 in the experimental groups, respectively; while the control group was(38.09±1.81) ×10-2. From the results, we can see that the m RNA expression of PI3 K and MMP-2 gene was weakened by drugs. There was statistical significance between experimental groups and control group(P < 0.05). Also, the difference of the combination groups was statistically significant which compared with the single one(P < 0.05).Conclusion1. All trans retinoic acid(ATRA)and LY294002 have inhibitory effect on EC1 cells? proliferation, the inhibitory effect is more significant with the extension of time, and the combined group was more better than that of the single one.2. ATRA and LY294002 can inhibit the migration of EC1 cells, also depress the protein and m RNA expressions of PI3 K and MMP-2, and the combined effect of the two drugs is more significant than the single action.3. ATRA and LY294002 are able to inhibit the proliferation and migration of EC1 cells, which may be related to down-regulating the expression levels of PI3 K and MMP-2 gene. The down-regulation effect of combined drugs is more obvious than the single, showing that the two drugs can synergistically inhibit the PI3 K signaling pathway and suppress tumor vascular mimicry. |
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