The Mechanism Of Human Umbilical Cord Mesenchymal Stem Cells Suppress Cholangiocarcinoma Cell Growth

Background and ObjectiveIntrahepatic cholangiocarcinoma(ICC), also known as peripheral cholangiocarcinoma, is a malignancy whose pathogenesis involves abnormal biliary epithelial differentiation and is originated from the secondary bile duct epithelium branch. It is one kind of primary liver cancer. World widely, ICC accounted for about10%-15%of the primary liver malignant tumor, and it is the second common form of primary liver cancer next to that of hepatocellular carcinoma. Nowadays, it has been rising for primary tumor form among the liver cancer which leads to death. Presently, it is reported that the incidence of ICC is closely related with bile duct calculi, HBV/HCV infection and some other bile duct inflammation. ICC possess the following characteristics:hidden onset, atypical clinical symptoms, high malignant degree, rapid development, and easy to transfer. Despite advances in diagnosis and treatment, most patients present with advanced metastatic lesions that are not amenable to surgical excision or liver transplantation and the poor prognosis can not be changed. So, to find a new and effective method to apply to diagnose and treat ICC is our priority. With the development of molecular biology and the deep understanding to tumor molecular mechanism, researchers have been aware of that malignant tumor is one kind of systemic diseases involved by multiple factors and multiple genes. Therefore, in the recent10years, biological therapy and gene therapy have been become the hot spot in tumor treatment.Mesenchymal stem cells (MSCs) is one kind of stem cells, possessing a multiple-differentiation potential which permits these cells to differentiate into a variety of mesodermal cell lineages, including bone, cartilage, fat, muscle and tendon. Therefore, MSCs have been widely used in studies of cell therapy and tissue regeneration. In contrast to hMSCs from other resources, human umbilical cord mesenchymal stem cells(hUC-MSCs), as a new source of seed cells, get more and more attention due to their availability, low immunogenicity, as well as strong tropism for tumors. It is reported that hUC-MSCs not only have strong proliferation ability, but also have the ability to differentiate to some other cells (such as neurons, astrocytes, fat cells and oligodendrocytes, etc). Because of its strong tropism for tumors, many researchers have been focus on its relationship with the tumor cells. Nowadays, stem cell transplantation technology has been applied in several kinds of hematologic and non-hematologic malignancies. Previous studies have shown that the development and growth of some human solid malignancies can be inhibited by mesenchymal stem cells. And other studies have demonstrated that human mesenchymal stem cells may inhibit tumor cell phenotypes by secreting certain soluble factors.Because the mechanism of human mesenchymal stem cells effects on human intrahepatic cholangiocarcinoma has not been reported, in the current study, we sought to shed light on this phenomenon. Co-culture systems, MTT assays and DNA fragmentation assays were used to determine the intrahepatic cholangiocarcinoma HCCC-9810cells proliferation and apoptosis. Western Blot and immunofluorescence staining methods were used to determine the expression level of proteins which were closely related to several pathways in intrahepatic cholangiocarcinoma to identify the possible molecular mechanism.Part IEffect of human umbilical cord mesenchymal stem cells on intrahepatic cholangiocarcinoma cellsObjective1. To detect the effect of hUC-MSCs on human intrahepatic cholangiocarcinoma HCCC-9810cells.2. To detect the effect of various concentrations of conditioned media from hUC-MSCs (10%,25%,50%and75%) on human intrahepatic cholangiocarcinoma HCCC-9810cells.3. To evaluate whether the effect of conditioned media from hUC-MSCs on HCCC-9810cells was mediated by apoptosis.4. To detect the effect of hUC-MSCs on tumor formation in BALB/c Nude Mice.Methods1. Co-culture systems were established by using transwell6-well plates to detect cell proliferation.2. HCCC-9810cells were treated with various concentrations of conditioned media from hUC-MSCs or HUVECs(10%、25%、50%and75%) to detect cell proliferation using MTT and colony-forming assay.3. Hoechst33258assay were used to determine cell apoptosis.4. BALA/c nude mice transplantation was established to detect tumor formation.5. SPSS16.0software was used for all statistical analysis. Statistical significance was assessed by comparing mean values (±SD) using the Student’s t-test for independent groups.Results1. Effect of human umbilical cord mesenchymal stem cells on cholangiocarcinoma cellsThe numbers of HCCC-9810cells co-cultured with hMSCs and HUVECs for48h were7.64X105个/ml and11.98X105个/ml, respectively. The hMSC-induced inhibitory effects on tumor cell proliferation were significantly greater than in controls. Compared to that of the control, the cell proliferation rate decreased34.4%(P<0.05). In order to determine whether the cultured hUC-MSCs microenvironment could inhibit tumor cell growth, we tested the effect of conditioned media from hUC-MSCs on HCCC-9810cells. HCCC-9810cells treated with various concentrations hUC-MSC-conditioned media resulted in dose-dependent and time-dependent inhibition of cell proliferation. The proliferation inhibition rate increased from6.21%to49.86%when HCCC-9810cells were cultured with50%hUC-MSC-conditioned media for24h. Treatment of HCCC-9810cells with50%hUC-MSCs conditioned media led to a significant reduction in the number of colony-forming units relative to control cells. The mean numbers of colony-forming units in the hUC-MSC media treatment group, and in the HUVEC media control group were14.7and35.7, respectively (P<0.01).2. The hUC-MSCs conditioned media treatment resulted in an induction of apoptosis in HCCC-9810cellsTo further evaluate whether the inhibitory effect was mediated by inducing cell apoptosis, DNA staining assay was used. Treatment of HCCC-9810cells with50%conditioned media for48h resulted in apoptosis. We counted the apoptotic cells depending on the presence of cell rounding, detachment, and nuclear fragmentation. The means±SEM for the percentage of apoptotic cells in the treatment group and the control groups were (48.1±2.98)%,(9.3±3.05)%, and (9.6±3.51)%, respectively. Differences between the treatment group and control groups were statistically significant (P<0.05).3. Tumor formation was inhibited by hUC-MSCs in BALB/c nude miceResults showed that on the50th day after injection, the mice injected with HCCC-9810cells and hUC-MSCs had a lower tumor incidence than the control groups, and the mean volume of tumors of the mice injected with tumor cells and MSCs was dramatically lower than that of control groups:7mice developed detectable tumors on day35-40(average tumor volume=1.3cm3on day50), and3mice had not developed any tumors when they were killed on day50. By contrast, mice injected with HCCC-9810only, or a mixture of HCCC-9810and HUVEV, formed detectable tumors (average tumor volumes were2.6cm3and2.5cm3, respectively on day50). From day50to day70, mice in the three subgroups derived from Group4were injected with conditioned media from hUC-MSCs or HUVECs in the tumor sites, or received no treatment. The mean tumor volume in the hUC-MSCs group was significantly smaller compared with that of the control. The average tumor volume decreased to1.4cm3in the MSCs group, whereas it continued to increase to3.5cm3in the HUVEC control group by day70. Taken together, these observations suggested that hUC-MSCs may inhibit tumor growth in animals.Conclusion1、Human umbilical cord mesenchymal stem cells could inhibit the growth of HCCC-9810cells. 2、When HCCC-9810cells were cultured with50%hUC-MSCs-conditioned media for24h, the half inhibitory rate could be up to.3、hUC-MSCs could inhibit the tumor formation in BALB/c Nude mice, and the formed tumors shrank in size after injection of conditioned media from hUC-MSCs.4、The inhibitory effect of hUC-MSCs on HCCC-9810cells was mediated by inducing HCCC-9810cells apoptosis. Part IIThe molecular mechanism of human umbilical cord mesenchymal stem cells suppress cholangiocarcinoma cellsObjective1. To examine the effects of conditioned media from hUC-MSCs on the PI3K/Akt signaling pathway.2. To investigate the effects of conditioned media from hUC-MSCs on the Wnt/β-catenin signaling pathway.3. To detect the changes of HCCC-9810cell proliferation ability after activation or inhibitation Akt or Wnt signaling pathway.Methods1. Western Blot was used to observe the change of p-PI3K、p-PDK1、p-Akt、Akt、 p-GSK-3β、GSK-3β、β-Catenin、c-Myc. Cyclin D1in HCCC-9810cells after treatment with hUC-MSCs conditioned media.2. Immunofluorescence was used to detect the change of p-Akt、p-GSK-3β、β-Catenin in HCCC-9810cells after treatment with hUC-MSCs conditioned media.3. MTT assay was used to detect the changes of cell proliferation ability after activation or inhibition GSK-3β in HCCC-9810cells.4. MTT assay was used to detect the changes of cell proliferation ability with or without IGF-1preincubation for15min. Results 1. Inhibition of Akt Signaling Using Conditioned Media from hUC-MSC Cultures in tumor cells(1) Immunoblot analysis showed that, compared with control groups, treatment of HCCC-9810cells with hUC-MSC conditioned media resulted in reduced phosphorylation of PI3K、PDK1、Akt and GSK-3P, while the total Akt and GSK-3β level did not change. However, HUVEC conditioned media failed to down-regulate the expression of these proteins. In the HUVEC control group and MSCs group, the ratios of phospho-to total Akt were0.65±0.04and0.19±0.02, respectively, while the ratios of phospho-to total Gsk-3β was0.48±0.03and0.22±0.05, respectively. There were substantial differences in the phospho-to total Akt and Gsk-3β ratios between the treated group and control group (P<0.001;P<0.01, versus controls, respectively).(2) The expression of phospho-Akt(Ser473) and phospho-GSK-3β (Ser9) were examined through indirect immunofluorescence staining. Compared to the controls, the relative luciferase activities for p-Akt and p-GSK-3β were37.3±0.8%and35.7±1.2%, respectively. In contrast to the control, the immunofluorescence staining of phospho-Akt (Ser473) and phospho-GSK-3β (ser9) in the treatment group was significantly weaker than that of control(P<0.01, p-Akt;P<0.01, p-GSK-3p)2. Down-regulation of Wnt signaling in tumor cells by conditioned media from hUC-MSC cultures(1) Immunoblot results showed that treatment of HCCC-9810cells with hUC-MSC conditioned media resulted in the down-regulation of β-catenin、c-Myc and cyclin-D1. However, conditioned media from HUVEC failed to down-regulate the expression of these proteins.(2) Immunofluorescence cytology showed that treatment of HCCC-9810cells with conditioned media from hUC-MSC resulted in a decrease in β-catenin nuclear assembly.3. The hUC-MSC conditioned media decreased β-catenin expression and inhibited Wnt signaling by regulating GSK-3β activity by the Akt signaling pathwayThe GSK-3β inhibitors CHIR99021significantly rescued HCCC-9810cells from the inhibitory effects of hUC-MSCs conditioned media (P<0.001), whereas the GSK-3β activator SNP significantly increased the inhibitory effects of hUC-MSCs conditioned media (P<0.001). Treatment with CHIR99021increased β-catenin protein levels in the conditioned media-treated group, while SNP treatment resulted in decreased β-catenin protein levels. We further treated with hUC-MSC conditioned media for24h, with or without IGF-1(200ng/mL) preincubation for15min. The results showed that exposure to IGF-1significantly blocked the inhibitory effects of hUC-MSC conditioned media on HCCC-9810cells (P<0.05).Conclusion1、Human umbilical cord mesenchymal stem cells could inhibit the growth of cholangiocarcinoma cells via PI3K/Akt signaling pathway2、Human umbilical cord mesenchymal stem cells could inhibit the growth of cholangiocarcinoma cells via Wnt/β-catenin signaling pathway.3、Suppression of cholangiocarcinoma cell growth by human umbilical cord mesenchymal stem cells:A possible role of Wnt and Akt signaling...