Influence Of Irradiated Hepatic NPCs On Metastatic Potential Of Sub-lethally Irradiated Hepatoma Cells And Liver Cell’s Radio-sensitivity

Part Ⅰ Separation, Culture and Detection Using Cytokine Array of Primary Rat Hepatic Non-parenchymal cells (NPCs)Purpose:Irradiation can induce hepatic NPCs release soluble mediators which can influence liver parenchyma cell’s radio-sensitivity and invasiveness and metastatic potential of sub-lethally irradiated hepatoma cells. Separation and culture hepatic NPC, will lay the foundation for the subsequent experiment.Methods and Materials:We obtained hepatic NPCs by means of irrigating and digesting with enzymes SD rats’ liver. After that, NPCs were cultured and subsequently irradiated or non-irradiated. Conditioned media from irradiated (SR) or non-irradiated (SnonR) cultures were collected, then they were detected using cytokine arrays.Results:SD rats’hepatic NPCs were separated and cultured successfully. The cultured NPCs in vitro did not express albumin that was a symbol of liver parenchyma cells. Cytokine Array analysis showed that expression of many cytokines related inflammation and metastasis such as IL-6, MMPs and TNF-α, were up-regulated in irradiated NPCs’ supernatants compared with un-irradiated NPCs’. Conclusions:Irradiation can induce hepatic NPCs cultured in vitro release cytokins related inflammation and tumor metastasis.Part Ⅱ Stimulation of Hepatoma Cell Invasiveness and Metastatic Potential by Proteins Secreted from Irradiated Nonparenchymal CellsPurpose:To determine whether factors secreted by irradiated liver NPCs may influence invasive and/or metastatic potential of hepatocellular carcinoma (HCC) cells and to elucidate a possible mechanism for such effect.Methods and Materials:Primary rat NPCs were cultured and divided into irradiated (lOGy X-ray) and non-irradiated groups. Forty-eight hours after irradiation, conditioned media from irradiated (SR) or non-irradiated (SnonR) cultures were collected and added to sub-lethally irradiated cultures of the hepatoma McA-RH7777cell line. Then hepatoma cells were continuously passaged for eight generations (RH10Gy-SR and RHIOGy-SnonR). The invasiveness and metastatic potential of McA-RH7777, RH10Gy-SnonR, and RH10Gy-SR were evaluated using an in vitro matrigel invasion and an in vivo metastasis assay. In addition, SR and SnonR were tested using by rat cytokine antibody arrays and an enzyme-linked immunosorbent assay (ELISA) test.Results:In vitro matrigel invasion assay indicated that the numbers of invading cells was significantly higher in RH10Gy-SR cells (40±4.74) than in RH10Gy-SnonR (30.6±3.85), and lowest in McA-RH7777cells (11.4±3.56). The same pattern was observed in vivo in a lung metastasis assay, as evaluated by number of metastatic lung nodules seen with RH10Gy-SR (28.83±5.38), RH10Gy-SnonR (22.17±4.26), and McA-RH7777cells (8.3±3.8). Rat cytokine antibody arrays and ELISA demonstrated that metastasis-promoting cytokines (tumor necrosis factor-a and interleukin-6), circulating growth factors (vascular endothelial growth factor and epidermal growth factor), and metalloproteinases (MMP-2and MMP-9) were upregulated in SR compared with SnonR.Conclusions:Radiation can increase invasiveness and metastatic potential of sublethally irradiated hepatoma cells, and soluble mediators released from irradiated NPCs further promote this potential. Increased secretion of metastasis-related cytokines and factors from NPCs after irradiation may be a possible mechanism for the radiation-induced invasiveness and metastatic potential of HCC. Part Ⅲ Sensitization of by liver parenchyma cell’s radio-sensitivity by Cytokines Secreted from Irradiated Nonparenchymal Cells and its MachanismsPurpose:Liver parenchyma cells are radio-resistance when they were cultured in vitro, while liver is an organ that is relatively radio-sensitive. This study is to determine whether irradiated hepatic NPCs could influence hepatic parenchymal cells’s radio-sensitivity and explore its possible mechanism.Methods and Materials:1. Conditioned media of SR or SnonR were collected and added to followed irradiated rats live cell line BRL3A. Colony formation were also used as estimation whether SR and SnonR influenced on BRL3A’s radio-sensitivity. BRL3A cells received one fraction of10Gy irradiation (BRL/SR group, BRL/SnonR group). Apoptosis of BRL3A cells then were evaluated with flow cytometry (FCM) using Annexin-V and propidium iodide (PI) double staining and JC-1fluorescent staining.2. BRL3A cell’s pro-apoptosis proteins Bid was silenced by SiRNA technology. Cells were divided into two groups, silence group (Si group) and negative control (NC group). The follow-up experiments were divided into four groups:(1) Si/SR group;(2) Si/SnonR group;(3) NC/SR group;(4) NC/SnonR group. Cells of each group received one fraction irradiation of10Gy. Accessing apoptosis of every group were employed nuclear fluorescent dyeing.3. Apoptosis related proteins (Bid, caspase-9, caspase-3) were detected with western blotting of these four groups cells after they received irradiation (10Gy)on the time point of6hours,12hours and24hours.Results:1. Colony formation assay showed that the number of colonies was significantly higher in BRL/SnonR group when they were irradiated6,8,1OGy (0.170±0.122vs0.085±0.112;0.103±0.006vs0.059±0.007;0.0516±0.005vs0.027±0.006, respectively. P<0.01) but that was not appeared in1,2,4Gy. BRL/SR group and BRL/SnonR group Percentage of normal cells in BRL/SnonR group was significantly more than that in BRL/SR group (0.561±0.091vs0.743±0.078, P=0.02) on the time point of24hours, by FCM detecting. Percentage of apoptotic cells in BRL/SR group was significantly more than that in BRL/SnonR group by JC-1staining assay (0.554±0.108vs0.388±0.082, P=0.025)2. SiRNA technoledge effectively knockdown BRL3A cell Bid protein. Percentage of apoptotic cells by using nuclear fluorescent dyeing in Si/SR group, Si/SnonR group, NC/SR group, NC/SnonR group was0.164±0.015,0.113±0.033,0.058±0.014,0.045±0.019, respectively. There was significant difference between NC/SR group and NC/SnonR group but there was no significant difference between Si/SR group. Si/SnonR group. Percentage of apoptotic cells in Si group was significantly lower than NC group.Expression of tBid, activated caspase-9, caspase3was higher in NC/SR group than SnonR group on some time points, but that was not appeared in Si/SR group and Si/SnonR group. All the above-mentioned proteins were lower expressed in Si groups. and there were no significant difference between Si/SR group and Si/SnonR group. Expression of tBid, activated caspase-9, caspase-3was higher in NC/SR group than SnonR group on some time points, but that was not appeared in Si/SR group and Si/SnonR group.Conclusions:This study suggests that supernatant of irradiated NPCs can increase hepatocytes’ radiation-induced apoptosis. The effect was related to activating TNF death receptor apoptotic signal pathway. BRL3A cells’radiation tolerability can be obviously improved through the silent Bid gene.