The Biological Effects Of Gliotoxin On Fibroblasts Associated Tumor And Its Initially Search About Mechanism

Background and Objective:Hepatocellular carcinoma (HCC) is the most common form of adult liver cancer. In many countries, the incidence of HCC is steadily rising. At the same time, the mortality remains very high. Many studies indicated the transformation of liver microenvironment is one of the most important pathologic symbols, the majority of HCC patients suffer liver fibrosis and liver cirrhosis, accompanied by inflammation and ECM deposition, all of these complications altered liver microenvironment. In recent years, high-throughput genomic analysis has been gradually clarified the gene network and signal transduction pathways related to HCC. However, the recurrence and metastasis of HCC is still a obstacle, we should figure out new treatment targeting the tumor microenvironment.Tumor-associated fibroblasts (TAFs) are the most prominent cell type within the tumor stroma. TAFs participated in the maintenance of tumor characteristics. Also, they increase the resistance of cancer cells to chemotherapeutic drugs. Gliotoxin, a fungal metabolite, was reported to cause apoptosis of hepatic stellate cells (HSCs) to ameliorate liver fibrosis. Gliotoxin was found to lead apoptosis of HSCs at low concentration (100 nM) without affecting normal hepatocytes. The result demonstrated that Gliotoxin)(IC50=143.1 nM) could inhibit the proliferation of TAFs.Based on the background, we speculate that Gliotoxin could be a therapeutic drug for HCC by targeting TAFs. Our research will be expanded from two aspects. First, TAFs are isolated from human HCC tissue and identified by immunofluorescent, proliferation assay, migration assay and apoptosis assay are established to observe the effect of Gliotixin on TAFs. Then, the mechanism of Gliotoxin on TAFs is further investigated. Second, a DEN induced HCC model is established to investigate the role of Gliotoxin on HCC treatment. Liver tissue of HCC model will be evaluated by HE staining and immunohistochemical. These studies will provide new directions for clinical treatment of HCC.Methods:1. Effect of Gliotoxin on TAFs: ①Isolation and identification of TAFs. Human HCC tissues were cut into small pieces and enzymatically digested by collagenase Ⅳ. The suspension was centrifugated to isolate fibroblasts. TAFs were purified by using Anti-fibroblast beads. The expressions of a-SMA, Vimentin, Desmin in TAFs were’detected by immunofluorescent.②Effect of Gliotoxin on the biological characteristics of TAFs, To detect proliferation ability, TAFs were seeded into a 96-well plate with the number of 3×103 per well. Different concentration of Gliotoxin was added into TAF medium. Six days later, the viability of TAFs were detected by luciferase reporter asaay. 100nM Gliotoxin or DMSO was added into the lower 24-well plate. TAFs were digested by trypsin and suspended into serum-free DMEM, and then inoculated into the upper migration chamber (5×104 cells per well). The migrations of TAFs were detected after 24 hours.③Effect of Gliotoxin on promoting apoptosis of TAFs.1)100nM Gliotoxin or DMSO was added into TAF medium, Caspase fluorescent assay kit was used to measure Caspase3 activity.2) TAFs were treated with 100nM Gliotoxin or DMSO, cells were collected after four hours. Then TAFs were marked by Annexin V and PI, flow cytometry were used to detect apoptosis rate of TAFs.3) Different concentration of Gliotoxin was added into TAF medium, cellular ATP was measured using Cell viability assay kit-ATP.2. Mechanism of Gliotoxin on TAFs: ①100nM Gliotoxin or DMSO was added into TAF medium.48 hours later, RNA of TAFs was isolated then reversely transcripted into cDNA, the expressions of TAF related markers, such as a-SMA, Vimentin, Desmin, and the downstream genes of TGF-β pathway were detected by Real-time PCR.②Different concentration of Gliotoxin was added into TAF medium. After 4 hours, total protein of TAFs was isolated, then the expressions of Smad family protein was detected by western-blot. Other TAFs were tested for the expression of 3TP (TGF-β pathway reporter gene) luciferase.3. Effect of Gliotoxin on TAFs-HCC cell line coculture system: ①TAFs was treated with Gliotoxin, then normal medium, NF medium, TAF medium and TAF-Gliotoxin medium were collected as conditional medium. Hep3B cells were inoculated into 96-well plate(3×103 per well), the Hep3B cell medium was replaced by conditional medium, the proliferation abilities of HCC cells were detected by CCK-8.②Conditional medium was added into the lower 24-well plate. SMMC-7721 cells (5×104 cells per well)were seeded into transwell chamber, the migrations of TAFs were detected after 24 hours.4. Effect of Gliotoxin on DEN induced HCC model: ①Evaluation of Gliotoxin effection on HCC treatment, The rats used in HCC model were sacrificed at the end of 22 week. The livers were perfused with normal saline through the portal vein. The treatment effect was evaluated by HE staining.②Masson’s trichrome and Sirius Red staining was utilized to detect the expression of collagens. Immunohistochemical staining was taken to detect the expression of a-SMA.5. Statistical analysis:SPSS 16.0 software package was used for statistical analysis. The results were expressed as with X±SD. One-Way ANOVA was used for inter-group analysis. P<0.05 was considered significantly different.Results:1. Gliotoxin present inhibitory effect on TAFs:①The immunofluorescence results showed that α-SMA, Vimentin and Desmin were expressed in TAFs. All the TAFs showed the presence of α-SMA and Vimentin, however,70% of TAFs expressed Desmin.②Gliotoxin caused no effect on TAF viability when the concentration was lower than 50 nM. If the concentration of Gliotoxin was between 50 nM and 400 nM, the TAF viability was decreased with the up-regulation of the dosage. The IC50 of Gliotoxin to TAFs was about 147.1nM according to the drug sensitive curve. Gliotoxin presented inhibitory effect on TAF migration. The inhibitory effect was shown on the concentration of 30 nM, when the concentration of Gliotoxin was 100 nM. The migration rate of TAFs was decrease a half. The migration inhibitory rate was 90% by Gliotoxin (300 nM) treatment.③Gliotoxin causes apoptosis of TAFs.1) In Gliotoxin-treated TAFs, Caspase activity was increased time-dependently from 15 min. The Caspase3 activity was 25 fold higher than control group at 3 hours.2) Flow cytometry detection showed the early apoptosis rate was increased 6% by 100nM Gliotoxin treatment.3) ATP content did not decrease significantly with low-concentration Gliotoxin. However,100 nM Gliotoxin caused rapid and progressive decrease in ATP. Robust ATP depletion occurred in TAFs treated with 300 nM Gliotoxin even at 30 min.2. Gliotoxin inhibit the activation of TGF-β pathway in TAFs: ①The western-blot showed that the total content of Smad 2 and Smad 3 did not change by Gliotoxin treatment. However, the phosphorylation of Smad 2/3 was decreased significantly. In addition, TGF-P pathway reporter assay indicated the luciferase activity was reduced by 100 nM Gliotoxin.②The expression of down-stream genes, such as C-myc, Bcl-2, AP-1, were decreased by Gliotoxin administration. At the same time, the expression of TAF related genes (α-SMA, Vimentin, FAP) were reduced.3. Effect of Gliotoxin on TAFs-HCC cell line coculture system:It was found that both NFs and TAFs could promote the proliferation of HCC cells, but the promoting effect of TAFs was significantly higher than that in NFs. In addition, Gliotoxin-treated TAFs conditional medium was co-cultured with Hep3B cells, leading to decrease in Hep3B proliferation.The migration assay showed that the TAFs conditional medium could promote SMMC-7721 migration, and the promoting ability was higher than that in the NF group. Gliotoxin could partially decrease the promoting ability of TAFs.4. Gliotoxin presents inhibitory effect on TAFs:①Effect of Gliotoxin on DEN induced HCC model, At the end of 22 week, the liver in model group and DMSO group showed multiple tumor nodules or neoplasms, accompanied with severe liver cirrhosis. No tumor neoplasm was found in Gliotoxin group. HE staining indicated that:6 rats of model group were diagnosed of HCC, the remaining were diagnosed of atypical hyperplasia.In DMSO group,5 rats were diagnosed of HCC. However, there are only 2 rat along with HCC in Gliotoxin group(each group has one), the others were diagnosed of atypical hyperplasia or cirrhosis.②Effect of Gliotoxin on liver fibrosis, There was no difference between model group and DMSO group on the collagen expression, but the Gliotoxin group showed significant decrease on collagen expression. The result of IHC showed the expression of a-SMA was reduced in Gliotoxin group, which was localized the portal area, whereas the expression of a-SMA in model group was extended to hepatocyte area.Conclusions:1. Gliotoxin suppress proliferation and migration of TAFs. Meanwhile, Gliotoxin promote apoptosis of TAFs.2. The inhibitory effect of Gliotoxin on TAFs was related to TGF-β pathway.3. Gliotoxin inhibit the development of experimental liver cancer.