The Experimental Study Of The Effect Of Human Mesenchymal Stem Cells On The Proliferation, Apoptosis And Invasion Of Tumor Cells A549 And Eca-109

Background and objective:The statistical data of WHO showed that at present, there could be a case of death from cancer in every eight dead patients. No matter in the developed countries or the developing countries. Cancer has all been one of the most "killer". Though the level of medical treatment has being elevated, the prognosis of patients with cancer has not been obviously improved. To this day, the therapy of malignant tumors mostly depends on the operation, radiotherapy and chemotherapy. Therefore, we all actively search the new and more effective therapeutic methods to treat tumor. With the development of molecular biology and the further knowledge on the molecular mechanism of tumor, we have thought that malignant tumor is a kind of systemic disease that resulted from multiple factors, procedures and genes. In the last ten years, the biological therapy and gene therapy of tumor have been thought as the focus and new hope of tumor therapy strategies.Mesenchymal stem cells (MSCs), the stromal progenitor stem cells found in the bone marrow, which can be defined according to its ability to self-renew and differentiate into tissue of mesodermal and non-mesodermal cell lineages. So, MSCs have been widely used in the researches of cell therapy and tissue regeneration fields. Human mesenchymal stem cells (hMSCs) used in the experiment are mostly acquired from the adult bone marrow. Recently, more and more attention has been paid to the relationship between hMSCs and tumor cells. The studies found that along with their property of migrating to tumor sites, hMSCs have been regarded as the most promising delivery vehicle for a cell-based targeted cancer gene therapy in the future. However, at present, the the relationship between hMSCs and tumor cells has not arrived at the concordant conclusion. In order to make clear the effect of hMSCs on tumor growth and explore the applied safe of hMSCs in the malignant conditions, we investigated the effect of hMSCs on the proliferation, apoptosis and invasion of lung cancer cell line A549 and esophageal cancer cell line Eca-109 in vitro and the influence of hMSCs on tumor cells A549 and Eca-109 growth in vivo.Part 1 The Effect of Human Mesenchymal Stem Cells on the Proliferation, Apoptosis and Invasion of Tumor cells A549 and Eca-109 in vitroMethods:hMSCs and hMSCs growth medium were bought from Cyagen, USA. Human esophageal cancer cell line Eca-109 and human lung cancer cell line A549 were bought from Chinese SIBCB. At first, the medium that had cultured hMSCs was collected, filtered and stored at-80?until used as the hMSCs-conditioned medium. Then, the influence of hMSCs on the proliferation of A549 and Eca-109 was observed by using transwell co-culture system and treatment with hMSCs-conditioned medium. Flow cytometric analysis and assay of caspase-3 activity were used to test the cell cycle and apoptosis of tumor cells A549 and Eca-109 treated with the hMSCs-conditioned medium. The effect of hMSCs on the invasive ability of tumor cells was tested by use of transwll chambers. Moreover, in order to explore the molecular mechanism of the effect of hMSCs on tumor cells in vitro, we investigated the expressions of related proteins, such as PCNA, Cyclin D1, Cyclin E, pRb, phospho-pRb, P27, Bcl-2, Bcl-xL, Bax and MMP2 by western blotting and the activity of Cyclin E-CDK2 complexes by immunoprecipitation in tumor cells A549 and Eca-109 treated with the hMSCs-conditioned medium.Results:Our results revealed that hMSCs inhibited the proliferation and invasion of A549 and Eca-109 cells, arrested tumor cells in the G1 phase of the cell cycle and induced the apoptosis of tumor cells in vitro by using a co-culture system and the hMSCs-conditioned medium. Western blotting and immunoprecipitation data showed that the expressions of PCNA, Cyclin E, pRb, Bcl-2, Bcl-xL and MMP-2 were downregulated and the formation of CyclinE-CDK2 complexes was inhibited in the tumor cells treated with the hMSCs-conditioned medium.Conclusion:In vitro, hMSCs inhibited the proliferation and invasion of A549 and Eca-109 cells, arrested tumor cells in the G1 phase of the cell cycle and induced the apoptosis of tumor cells. The inhibitory effect of hMSCs on tumor cells in vitro was related with the downregulation of the expressions of PCNA, Cyclin E, pRb, Bcl-2, Bcl-xL and MMP-2 and the inhibition of the formation of Cyclin E-CDK2 complexes in the tumor cells treated with the hMSCs-conditioned medium via some soluble factores secreted by hMSCs.Part 2 The Effect of Human Mesenchymal Stem Cells on Tumor cells A549 and Eca-109 growth in vivoMethods:The animal experiments used the six-week-old BALB/c nude mice. Tumor cells and hMSCs were prepared either as single-cell type suspensions (5×106 A549 or Eca-109 cells in 0.1ml PBS) or a mix of cells (5×106 tumor cells and 1x106 hMSCs in 0.1ml PBS). Cells were injected subcutaneously at one site of the back of nude mice. Mice were examined one time at 5 days and tumor growth was evaluated by measuring the length and width of tumor mass. At the end of the experiment, animals were killed, and tumor masses were removed and fixed in 10% neutral buffered formalin solution for histologic preparations. MVD was assessed by immunohistochemical analysis with antibodies to the endothelial marker CD34.Results:Animal study showed that the mice co-injected with hMSCs and tumor cells had the higher tumor incidence and the bigger mean volume of tumors than that of control groups injected only with tumor cells. These results suggested that hMSCs enhanced tumor formation and growth in vivo. According to the observation of tumor mass and the result of MVD, we found that the promoting role of hMSCs on tumor growth was related with the increase of tumor vessel formation.Conclusion:In vivo, hMSCs enhanced tumor formation and growth by promoting tumor vessel formation.SUMMARYIn order to make clear the effect of hMSCs on tumor cell growth and explore the applied safe of hMSCs in the malignant conditions, we investigated the influence of hMSCs on lung cancer cell line A549 and esophageal cancer cell line Eca-109 growth in vitro and in vivo. Our results shows, in vitro, hMSCs inhibited the proliferation and invasion of A549 and Eca-109 cells, arrested tumor cells in the G1 phase of the cell cycle and induced the apoptosis of tumor cells. The inhibitory effect of hMSCs on tumor cells in vitro was related with the downregulation of the expressions of PCNA, Cyclin E, pRb, Bcl-2, Bcl-xL and MMP-2 and the inhibition of the formation of Cyclin E-CDK2 complexes in the tumor cells treated with the hMSCs-conditioned medium via some soluble factores secreted by hMSCs. However, in vivo, hMSCs enhanced tumor formation and growth by promoting tumor vessel formation.According to our studies, we come to a conclusion that hMSCs have dual characters:on the one hand, hMSCs have the ability to inhibit the proliferation and invasion of tumor cells in vitro; on the other hand, in vivo, hMSCs can favor tumor formation and growth in the tumor microenvironment. This difference of results between in vitro with in vivo was possible related with the differentiation of mesenchymal stem cells themselves in the tumor microenvironment. Therefore, we suggest that the exploitation of hMSCs in new therapeutic strategies should be cautious under the malignant conditions.