| Background and objective of studyLung cancer is one of the most common malignant tumor worldwide, radiation therapy is an important means to treatment of lung cancer. Undergoing radiotherapy, more than60%of patients still have residual tumor, residual tumor of lung cancer is the source of recurrence and metastasis. The distant metastasis is the leading cause of death in patients with lung cancer, therefore, it is very important to study the change of the ability to tumor cell invasion and metastasis. Most studies shown that residual tumor cells'invasive ability enhanced after radiotherapy, but there were a few studies found that radiotherapy reduced the invasiveness of cancer cells, therefore, the impact of radiation on the tumor ce11invasion and metastasis is still controversial. In order to know whether the residual lung tumor cells'ability of invasion and metastasis increase after radiation, we selected the lung adenocarcinoma A549cell line with high metastatic potential as a research object. We observed the change of potential of metastasis by the adhesion, invasion, migration experiments in vitro, and lung metastasis model in vivo. And then its mechanism was studied. This study will provide a theoretical basis to us to find ways reduce radiation-induced metastasis ability increasing.Through years of research and clinical application, we found Angelica sinensis is very effective in the prevention and treatment of radiation pneumonitis. The use of Angelica sinensis is very frequent during radiotherapy and after radiotherapy. Many studies have found that the extract of Angelica sinensis have anti-tumor effect through inducing tumor cells apoptosis, many other studies found the extract of Angelica sinensis could inhibit tumor invasion and metastasis. Our previous study found one of the mechanisms of Angelica sinensis to treat radiation pneumonitis was it could inhibit radiation-induced TGF-β1increasing. So we speculate that Angelica sinensis might decrease residual tumor cells invasion and metastasis after radiation by affecting TGF-β1expression. But Angelica sinensis belongs to Chinese medicine of blood circulation. Many studies found that these kinds of Chinese medicine can promote tumor cell metastasis, Chinese medicine of blood circulation in general should not be alone to use during the tumor treatment. So, whether Angelica sinensis is able to inhibit invasion and metastasis of tumor cells after radiation is unknown. In this study, we first study the effects of Angelica sinensis on invasion and metastasis of lung adenocarcinoma cell line A549after radiation in vitro and in vivo, and then we further explore its mechanism. This study will provide a theoretical basis for the clinical application of Angelica sinensis and have great significance.Methods and results1. We examined the change of A549tumor cells after radiation in capacity of adhesion, invasion and migration by MTT cell adhesion assay, Tranwell invasion assay and Transwell migration assay in vitro. We examined the change of the A549tumor cells after radiation in the capacity of lung metastasis using an animal model in vitro system. Compared with A549tumor cells before radiation, residual cells after radiation in the capacity of adhesion, invasion and migration were significantly increased approximately1.46times,1.4times and1.45times in vitro. Lung metastasis ability increased about1.2times in vivo.2. We found that residual A549cells after radiation undergo phenotypic changes characteristic of EMT:spindle-cell shape, loss of polarity, intercellular separation and pseudopodia formation through the microscope. Quantitative real-time PCR with SYBR Green was employed to analyze the mRNA transcriptional expressions of E-Cadherin and Vimentin. The protein levels from whole-cell lysates of E-Cadherin and Vimentin were assessed using West blot. The expression of E-cadherin mRNA of residual cells after radiation decrease approximately59.4%compare with the control group; E-cadherin protein expression was also reduced about74.6%compare with the control group. The expression of Vimentin mRNA of residual cells after radiation increase approximately1.37times compare with the control group, Vimentin protein expression was also increased about1.6times compare with the control group. Residual A549cells after radiation metastasis ability increased may be related to the radiation-induced EMT.3. We found TGF-B1mRNA expression levels of residual cells after radiation were increased approximately2.37times,TGF-β1secreted into the extracellular increased approximately2.34times, intracellular proteins corresponding increased approximately1.8times compare with the control group by Quantitative real-time PCR with SYBR Green, West blot and ELISA method. Residual A549cells after radiation metastasis ability increased may be related to the radiation-induced TGF-β1expression that resulting in the occurrence of EMT.4. We examined MMP-2and MMP-9mRNA expression, intracellular MMP-2and MMP-9protein expression by Quantitative real-time PCR with SYBR Green and West blot. The results showed that MMP-2and MMP-9mRNA expression of residual cells after radiation were significantly increased about1.8times and1.5times compare with the control group. The MMP-2and MMP-9protein expression of residual cells after radiation were increased accordingly, about1.1times and1.2times compare with the control group. Residual A549cells after radiation metastasis ability increased may be related to the radiation-induced MMP-2and MMP-9expression.5. In this study, we examined the effects of Angelica sinensis on the proliferation of residual A549cells after radiation using the CCK-8assay. The cells were treated with Angelica sinensis at various concentrations (0,2.5,10,25mg/ml) for12,24,48and72h. Comparing to that of control (untreated with Angelica sinensis), the viability of A549cells was not significantly affected at the dosage2.5mg/ml and10mg/ml. At the period of48h, we observed weak growth inhibition even at the maximum concentration of25mg/ml, the values was not much greater than those observed for the untreated control. These results imply that Angelica sinensis in low concentration didn't affect proliferation of residual A549cells after radiation, Angelica sinensis in high concentration has a weak inhibitory effect on the proliferation of A549cells.6. We examined the effect of Angelica sinensis on capacity of adhesion, invasion and migration of residual A549cells after radiation by MTT cell adhesion assay, Tranwell invasion assay and Transwell migration assay in vitro. The results showed that Angelica sinensis induced a dose-dependent decrease in invasion and migration with the concentration increasing. We examined effect of Angelica sinensis on capacity of lung metastasis using an animal model in vitro system. These results showed that Angelica sinensis in low concentration didn't affect lung metastasis of residual A549cells after radiation, Angelica sinensis reduced the metastatic ability only at high concentration.7. We detected the effect of Angelica sinensis on expression of EMT markers E-cadherin and Vimentin in A549cells. The results showed that Angelica sinensis have no significant effect at various concentrations on the mRNA expression and intracellular protein levels of E-cadherin. Angelica sinensis can inhibit Vimentin mRNA expression and intracellular protein levels only at the high concentration (25mg/ml), but it had no significant effect on Vimentin at low concentrations. It means Angelica sinensis at high concentration can inhibit tumor metastasis may be related to inhibit the expression of Vimentin. It indicated that composition of Angelica sinensis is complex, the regulatory mechanism of Angelica sinensis on Vimentin maybe involve multiple levels, multiple mechanisms. It Also suggest that Angelica sinensis inhibit tumor cell metastasis maybe not by EMT, the cell morphology also confirmed this point, the cells morphological changes was not obvious after treated by Angelica sinensis.8. In order to know the effect of Angelica sinensis on the expression of TGF-β1,TGF-β1mRNA and protein levels were measured. The protein levels from whole-cell lysates of TGF-β1were assessed using West blot, the protein levels in the culture medium was measured by an ELISA. The mRNA expression was measured by Quantitative real-time PCR with SYBR Green. The results showed mRNA expression,the intracellular and extracellular protein levels of TGF-β1all decreased in a dose-dependent manner with the concentration increasing of Angelica sinensis. It means Angelica sinensis can inhibit the expression and secretion of TGF-β1, its inhibition may above the transcriptional level. 9. To examine the possible anti-metastatic mechanisms of Angelica sinensis, we determined the activity of MMP-2and MMP-9in culture media of A549cells by zymographic analysis. The result showed MMP-2and MMP-9activity were suppressed in a dose-dependent manner by treatment of Angelica sinensis. To investigate whether the MMP-2and MMP-9activity inhibitory effect of Angelica sinensis resulted from the suppression of MMP-2and MMP-9expression, MMP-2and MMP-9protein and mRNA levels were measured. The protein levels from whole-cell lysates of MMP-2and MMP-9were assessed using West blot, and the protein levels in the culture medium was measured by an ELISA respectively. The result showed the intracellular protein levels of MMP-2and MMP-9by West blot decreased in a dose-dependent manner, and extracellular protein levels of MMP-2and MMP-9by an ELISA decreased in a dose-dependent manner too indicating that Angelica sinensis might regulate MMP-2and MMP-9expressions at the transcriptional level.10. Activity of MMP-2and MMP-9are significantly related to their inhibition TIMPs, especially TIMP-1and TIMP-2. In order to know the effects of Angelica sinensis on the inhibitor of MMP-2and MMP-9, the expression of TIMP-1and TIMP-2mRNA and protein levels were measured. The result showed Angelica sinensis obviously increased mRNA and protein levels of TIMP-2, the mRNA expression of TIMP-2increased to2.4fold even at the lowest dose2.5mg/ml, and protein levels of TIMP-2increased to1.7fold by treatment of2.5mg/ml Angelica sinensis. In contrast to TIMP-2, TIMP-1decreased significantly. TIMP-1mRNA reduced to76%and protein levels of TIMP-1reduced to50%by treatment of25mg/ml Angelica sinensis. These results suggest that Angelica sinensis inhibit the enzyme activity of MMPs (especially MMP-2) is related to the increasing of TIMP-2but not TIMP-1.Conclusion1. The metastasis ability of residual A549cells after radiation Significantly enhanced compared to before radiation, its mechanism may be related to radiation-induced TGF-B1increasing, EMT and radiation-induced MMP-2and MMP-9expression upregulating.2. Angelica sinensis in low concentration didn't affect proliferation of residual A549cells after radiation, Angelica sinensis in high concentration has a weak inhibitory effect on the proliferation of A549cells.3. Angelica sinensis exert anti-growth and anti-metastasis activity against lung cancer cells through decreasing of TGF-β1,MMP-2and MMP-9, and increasing TIMP-2. There was no relationship of Angelica sinensis exerting anti-metastasis activity with EMT and TIMP-1.4. It is safe to lung cancer patient to use Angelica sinensis during the radiation, it don't promote tumor grow, it maybe reduce the risk of tumor metastasis. |
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