Epigenetic Drugs Screening In Intrahepatic Cholangiocarcinoma Cells And The Effect Of HC Toxin In CCLP-1 Cell Line

Background and objectiveIntrahepatic cholangiocarcinoma (ICC) is an epithelial cell malignancy arising from cholangiocytes of the secondary bile duct within the liver and is of highly malignance, rapidly development, as well as a high mortality rate. Although ICC is rare, the morbidity and mortality rates have markedly increased over the last three decades, particularly in European and American countries, and the morbidity account for 5～10% of the primary liver cancer and 10% of the cholangiocarcinoma. Nowadays, Surgery is still the main treatment method. However, the ambiguous imaging features and the dormant clinical symptoms make it hard to be diagnosed at an early stage and therefore the effect of the curative resection is generally limited. For ICC patients can not received surgery, liver transplantation on the long-term effect have still been controversy. What’s more, due to its intricate tumor microenvironment, desmoplastic character and rich genetic heterogeneity, ICC is always resistant to the general chemotherapeutics. Combined chemotherapy protocols and various targeted therapeutics do not bring out desired results except for the cisplatin plus gemcitabine which achieves a median survival time of less than 12 months for patients can not be operated. So, it is of great significance to develop sensitive therapeutics and solve the problem of drug resistance for the treatment of ICC.Epigenetic mechanisms, including DNA methylation, non-coding RNA regulation, histone modifications, genes and protein phosphorylation process and bromine protein domain structure modification etc, play an important role in the occurrence and development of the environmentally linked diseases. Also, multiple epigenetic mechanisms have reflected in the pathophysiology of ICC. At present, the epigenetic drugs were thought to have a great potential in the treatment of cancer, chronic disease and mental illness, and there are a lot of epigenetic drugs entered into each phases of clinical researches. But it is still blank area for epigenetic drugs to be applied to the treatment of ICC or adjuvant therapy and even basic research. So, epigenetic drugs used in the treatment of intrahepatic bile duct carcinoma may have a very good prospect, and Histone acetylation enzyme (Histone deacetylase, HDAC) inhibitors HC toxin, a cyclic tetrapeptide first isolated from the secondary metabolite of Helminthosporium carbonum which is thought to be a type of HDAC inhibitor in plants, insects and mammals, could be one of them.Recent years, researches has found that the activation of Notch signaling pathways can make the mature liver cells into ICC precursor cells, which draws great attention on the Notch signaling pathway about the development of the ICC cells. At the same time, it said that the Notch signaling pathway is also regulated by the epigenetic mechanism, and it is still a controversy what is the affection about the HDAC modification on the Notch signaling pathway. And it suggest that the Notch pathway must be took in view when we use the HDAC related drugs in the treatment of ICC.Upon the background above, we designed three experiments and there are three objectives,Experiment one:To find out the sensitive and effective drugs for ICC by screening the epigenetic drugs library and provid primary experimental foundation for the chemotherapy of ICC.Experiment two:To explore the effects of the HDAC inhibitor HC toxin on the cell proliferation, cell cycle distribution and cell apoptosis in the ICC cells.Experiment three:To investigate the change the notch signaling pathway affected by the HDAC inhibitor HC toxin in the ICC cells.MethodsExperiment one:1. The epigenetic drug library (No.11076), provide by Cayman company, was primarily screened at only one time point for the sensitive and effective drugs in the ICC cells through the CCK-8 cell viability test on the RBE cell.2. The sensitive and effective drugs achieved by the trials above and gemcitabine were used to treat the HuCCTl、CCLP-1、TFK-1、RBE cells for 48 hours to compare the inhibitory effect with each other at multiple time points and the half maximal inhibitory concentration (IC 50) was calculated.Experiment two:1. Optical microscope was used to observe the Morphological changes of CCLP-1 cells with and without the giemsa staining after treated by HC toxin for 48 hours.2. Cell counting and plate clone formation assay were used to detect the cell proliferation and clone formation ability of the CCLP-1 cells.3. Flow cytometry with 7-AAD staining were used to check out the cell cycle distribution of the CCLP-1 cells after treated by HC toxin for 48 hours.4. Flow cytometry with 7-AAD and Annexin V FITC combining staining were used to evaluate the cell apoptosis after treated by HC toxin for 48 hours.5. Western Blot was used to detected the expression level of the apoptosis associated proteins caspase 3、bax、bcl-2 and cytochrome c after treated by HC toxin for 48 hours.Experiment three:1. CCK-8 cell viability test was used to detect the cell viability of CCLP-1 cells after treated by the notch signaling pathway inhibitor DAPT alone or combined with HC toxin.2. Real-time PCR was used to detect the mRNA level change of Notchl and some other important genes.3. Immunofluorescence and Western Blot was used to detect the expression level of the notch signaling pathway associated proteins notchl and HES1 after treated by HC toxin for 48 hours.ResultsExperiment one:1. In the primary screening, all of the compounds at a concentration of 27 μM were co-cultured with the REB cells for 48 h and cell viability was assessed by the CCK-8 assay. The results revealed that 22 of the 95 types of epigenetic drugs showed inhibitory activities with cell viability<60%. Including 12 HDAC inhibitors (HC Toxin, TSA, SAHA,4-iodo-SAHA, SB939, CAY10398, M344, ITF 2257, Oxamflatin, Scriptai, CBHA, CAY 10603), two Histone demethylation transferase (HDMT) inhibitors (PFI-1, GSK-J4), three Histone demethylation transferase (HMT) inhibitors (Chaetocin, GSK 343, BIX01294), two phosphorylation inhibitors (Bromosporine, JQ1), one bromodomain accociated (phthalazinone pyrazole) and DNA demethylation inhibitor (Neplanocin A). Among these, the inhibitory effects of HC toxin, TSA, SAHA,4-iodo-SAHA and Chaetocin were the most intensive with cell viability <60% at the drug dose 3 uM and the first four were HDAC inhibitors.2. In the comparing trials, HC toxin was the most effective followed by TSA, and all three HDAC inhibitors exhibited stronger HC toxin was the most effective followed by TSA, and all three HDAC inhibitors exhibited stronger anti-ICC activities than gemcitabine in the four ICC cell lines. And the IC 50 of HC toxin in CCLP-1. HuCCT1, TFK-1, RBE were 297.6+80.4 nM,720.0+43.0 nM,854.6+86.9 nM,713.7+27.3 nM respectively.Experiment two:1. Cell counting test showed that HC toxin inhibit the proliferation of CCLP-1 at the manner of dose- and time-dependence. And In the colony formation assay, HC toxin can inhibit the cell colony formation ability dependent on the dose of HC toxin. The rate of colony formation of the 50 nM group was modestly increased when compared with the 0 nM group, but the difference was not statistically significant.2. With the increasing doses of HC toxin, the cells gradually decreased in density with single cells dwindling in size, dendrite-like structures appearing and gradually became longer and multinucleated and cellular atypia were reduced, mitotic figures decreased, apoptotic bodies appeared and progressively increased with the increasing concentration of the HC toxin. The number of cells with apoptotic bodies in the 200 and 400 nM groups was much higher than that in the 0 and 100 nM groups, and the difference between 0 and 100 nM groups was not statistical significance.3. In the cell cycle detection test, the results showed that HC toxin arrests the cell cycle in the G0/G1 stage. Compared with the 0 nM group, the 200 and 400 nM groups exhibited remarkable significantly lower percentages of cells in the G2/M stage and the percentages of cells in the G0/G1 stage were significantly inceased in a concentration-dependent manner. No difference in the percentage of cells in the S stage was observed among the groups.4. Apoptosis detection illustrated that the percentages of the total apoptosis, early apoptosis and late apoptosis were increased with the increasing HC toxin concentration by flow cytometry with 7-AAD staining.5. In the western blot assay tested the apoptosis associated protein, the expression of caspase 3 did not show a significant increase until the concentration of HC toxin increased to 400 nM with only an increase of ～1.5-fold. The levels of bax/bcl-2 and cytochrome c did not change in our experimental environment.Experiment three:1. In the real time PCR assay used to detect the mRNA level change, Notch and six other genes showed a considerable mRNA level change while STAT3 and ten other genes showed an mRNA change without statistic significance.2. Notch signaling pathway inhibitor DAPT did not showed a inhibitory effect on the CCLP-1 cell when the DAPT dose less than 50 uM, however it could enhance the inhibitory effect of HC toxin in 200 nM remarkable. When the DAPT dose passed 50 uM, it would significantly inhibit the proliferation of CCLP-1 and showed a synergistic effect with HC toxin.3. The expression level of Notch 1 and Hesl increased significantly dependent on the dose of HC toxin compared with the control group in the immunofluorescence and Western blot assay. It illustrate that HC toxin will activate the Notch signaling pathwayConclusions1. Multiple epigenetic drugs can inhibit the proliferation of ICC in vitro, especially the HDAC inhibitors and this illustrated that HDAC may be the sensitive treat target of ICC cells though these trials did not provide enough evidence to support it and they also could not deny the other epigenetic targets’sensibility.2. HC toxin can intensively inhibit the proliferation of ICC cells at a manner of dose-dependent and is much stronger than the other HDAC inhibitors and gemcitbine. HC toxin was the most effective among various HDAC inhibitors and gemcitabine on the inhibitory of ICC cells in vitro. This inhibitory effect is associated with cell proliferation inhibition, cell cycle arrest and cell apoptosis boost.3. The Notch signaling pathway inhibitor DAPT can inhibit the cell viability of CCLP-1 while HC toxin can activate the Notch signaling pathway, it illustrate that the effect HC toxin shows on CCLP-1 could be a two-side sword and the inhibit effect is stronger than the promote effect and Notch signaling pathway inhibitors can be used to block the promoting effect of HC toxin.