| BackgroudEsophageal squamous cell carcinoma(ESCC) is one of the most frequently diagnosed and highly incident cancers in developing countries. When the diameter of primary tumor is 1-2mm, angiogenesis has been occurred to provide nutrition for tumor and promote tumor growth, invasion and metastasis. A variety of growth factors which are secreted by tumor cells regulate tumor angiogenesis. Among those, VEGF has been studied extensively and is the main regulator of tumor angiogenesis. It plays an important role in the process of malignant tumor growth and metastasis[1,2,3]. FGF family is other one of the largest known growth factor family, b FGF is first identified as a promote angiogenesis molecule[4] and plays an important role in angiogenesis.The concept of differentiation therapy provides a new and effective treatment for cancer treatment. More and more cancer experts begin to pay attention to the research progress of differentiation therapy. ATRA is the most important differentiation agent which has been applied in the leukemia and some solid tumors. In this study we investigated that effect of all-trans retinoic acid on expressions of VEGF、b FGF protein and m RNA in Eca109 cells. This study could provide the theory basis for anti-angiogenesis therapy.ObjectiveTo investigate the effect of all-trans retinoic acid(ATRA) with different concentrations on expressions of vascular endothelial growth factor(VEGF) and basic fibroblast factor(b FGF) in esophageal cancer Eca109 cells in vitro. MethodsEca109 cells were processed by different concentrations(0、1、5、10μmol/L)of ATRA. The proliferation of the treated cells in different time was determined by MTT assay. Wound healing assay was used to observe the mobility rate after 48 h. The protein and m RNA expressions of VEGF、b FGF in the cells were detected by immunohistochemical assay and reverse transcriptase polymerase chain reaction(RT-PCR). Results1. MTT results showed that: After 24 h, experimental groups, namely 1μmol/L,5μmol/L,10μmol/L, the proliferation inhibition rate was respectively:(44.57±1.36)%,(50.26±1.64)%,(54.24±1.39)%; After 48 h, the proliferation inhibition rate of experimental groups was respectively:(54.29±0.15)%,(59.52±0.74)%,(65.45±4.33)%; After 48 h, the proliferation inhibition rate of experimental groups was respectively:(66.39±0.87)%,(74.33±2.69)%,(85.24±1.49)%. The proliferation inhibition rate of control group, 0 mol/L group, was 0.00%. After statistics analysis, compared with control group, the differences of the experimental groups were statistically significant(P<0.05), and the two comparison between experimental groups, the difference is statistically significant(P<0.05).2.Wound healing assay results showed that: 1, 5, 10μmol/L ATRA on Eca109 after 48 h, cell mobility was(50.82±1.61) %,(46.80±5.12) %,(32.31±3.25) %, respectively; the control group was(68.81±2.64) %.0 h cell migration rate was 0.00%. After statistics analysis, statistically significant difference was observed in experimental groups compared with control group(P<0.05), and the two comparison between experimental groups, the difference was statistically significant(P<0.05).3.Immunocytochemistry results showed that: Intracellular yellow or brown granular can be considered to be related protein positive expression. VEGF was expressed in the cytoplasm and nuclear membrane; While b FGF was expressed in the nuclei, cytoplasm and nuclear membrane. 0, 1, 5, 10μmol/L ATRA on Eca109 after 48 h, the relative expression of VEGF protein in the cells was:(85.43±0.53) ×10-2,(77.45±0.51) ×10-2,(51.43±0.34) ×10-2,(21.74±0.11) ×10-2; Relative expression of b FGF protein was:(71.41 ± 0.52) ×10-2,(51.63±0.46) ×10-2,(38.44±0.32) ×10-2,(11.24±0.21) ×10-2. Different concentrations of ATRA on Eca109 after 48 h, The intensity of intracellular VEGF and b FGF protein expression gradually was weakened with the increase of concentration. Satistically significant difference was observed in experimental groups compared with control group(P<0.05). Also, the difference among the experimental groups was statistically significant(P<0.05).4.RT-PCR results showed that: 0, 1, 5, 10μmol/L ATRA on Eca109 cells after 48 h, VEGF m RNA expression was:(12.40±1.11) ×10-2,(7.27±0.71) ×10-2,(4.63±0.45) ×10-2,(1.62±0.25) ×10-2; b FGF m RNA expression was:(17.96±0.46) ×10-2,(15.32±0.33) ×10-2,(12.04±0.50) ×10-2,(9.13±0.30) ×10-2. The bands of VEGF and b FGF gene which not treated by ATRA were brightness. The m RNA of VEGF and b FGF gene expression wasgradually weakened with the increase of concentration. Satistically significant difference was observed in experimental groups compared with control group(P < 0.05). Also, the difference among the experimental groups was statistically significant(P < 0.05). Conclusion1. In a certain concentration range, ATRA can inhibit the proliferation of Eca109 cells, and its inhibitory effect is more obvious with the increase of drug concentration and the extension of time. Thus, its inhibition of ATRA on Eca109 cells has a dose and time dependent manner.2. In a certain concentration range, ATRA can inhibit cell migration, also inhibit the protein and m RNA expressions of VEGF and b FGF, and its inhibitory effect is more obvious with the increase of drug concentration.3. ATRA may influence esophageal Eca109 tumor angiogenesis by down-regulation expression levels of VEGF and b FGF gene, thus inhibiting the proliferation of Eca109 cells. |
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