| BackgroundCancer is still the leading cause of human death.As one of the main methods of cancer treatment,chemotherapy has a very significant therapeutic effect.However,after repeated administration,the decrease of tumor sensitivity to chemotherapeutic drugs is still the biggest challenge hindering cancer treatment.Therefore,the continuous exploration and discovery of new antineoplastic drugs in the field of tumor therapy is still a vital task of drug research and development,and is of great significance for the treatment of cancer patients.Histone deacetylase(HDACs)includes 11 family members and is classified into class ⅠⅣ according to their homology with yeast homologous genes.HDACs is considered to be a histone modifier that modifies various proteins in cells that are not related to genetic processes.HDACs removes acetyl groups from lysine residues of histones and other proteins,resulting in down-regulation of gene transcription and subsequent changes in signal transduction.HDAC inhibitors(HDACi),as an epigenetic regulator,have become an epigenetic drug with potential therapeutic activity because of their effects on inflammation,cell differentiation and cytotoxicity.Many clinical trials have proved that HDACi is effective in the treatment of malignant hematological diseases,thymoma and melanoma,and HDACi has significant advantages in the treatment of advanced liver cancer in many preclinical studies.HDACi can regulate gene expression through a variety of mechanisms and play an antitumor effect.However,its mechanisms are different,mainly depending on factors such as the type of cancer,the structure and dose of HDACi,and so on.According to the existing research,the anti-tumor mechanism of most HDACi mainly includes the following aspects:regulating immune response,regulating the expression of non-coding RNA and some signal pathways,inducing autophagy,inhibiting angiogenesis,influencing cell cycle and apoptosis and so on.In addition,the results of in vivo and in vitro studies of different tumors also show that HDACi has synergistic or additive effects in combination with a variety of anticancer drugs or with radiotherapy.In a word,HDACi has great potential in anti-tumor therapy,which is worthy of further study.HXB-9L and HXB-9K,the novel HDACi used in this paper,have double target DNA and HDAC.They are designed by using the benzimidazole ring of Hoechst33258 as the cap group and hybridizing with the connecting group of SAHA by the major of pharmaceutical chemistry in our institute.Based on this,we used SAHA as a positive drug to evaluate the antitumor activity of dual-target HD AC inhibitors HXB-9L and HXB-9K of DNA/HDAC in vivo and in vitro.Through the study of the potential mechanism of the anti-tumor activity of the new HDACi,it provides new ideas and experimental basis for the clinical treatment of melanoma and HCC.Object1.To screen novel HDACi with strong anti-tumor activity by different tumor cells;2.To study the anti-tumor effects of new HDACi in vivo and in vitro;3.To analyze the effect of new HDACi on immune system in tumor-bearing mice;4.To provide experimental basis for HDACi drug research and development.Methods1.With SAHA as positive control,B16F10,MC38,Hepa 1-6,Huh-7 and Hep 3B cells were treated with novel HDACi for 48h,and the cell viability was detected by MTT(Methyl thiazolyl tetrazolium)method.2.With SAHA as positive control,B16F10,Hepa 1-6 and Huh-7 cells were treated with different concentrations of novel HDACi for 24 h.Apoptosis of different tumor cells were detected by Annexin V/7-AAD kit.3.With SAHA as positive control,B16F10,Hepa 1-6 and Huh-7 cells were treated with different concentrations of new HDACi for 24 h.The cell cycle changes were detected by PI flow cytometry.4.White gun tip was used to draw lines on the B16F10 cells covered with the bottom of the culture plate.With SAHA as positive control,B16F10 cells were treated with different concentrations of new HDACi for 24 hours.The degree of wound healing was observed and photographed by microscope,and the migration of B16F10 cells in the image was measured by ImageJ software.5.The correlation between HDAC1 or HDAC2 and the prognosis of HCC patients was analyzed by OncoLnc database.6.With SAHA as positive control,Hepa1-6 and Huh-7 cells were treated with novel HDACi for 7 days,fixed with paraformaldehyde and stained with crystal violet.Then the colony formation of Hepa1-6 and Huh-7 cells was observed under microscope.7.With SAHA as positive control,Hepa 1-6 cells and Huh-7 cells were treated with new HDACi for 24 h,and the expression of stemness related molecules was detected by qPCR.8.Hepa1-6 cells and Huh-7 cells were cultured with ultra-low adhesion culture plate.Then with SAHA as positive control,HCC cells were treated with novel HDACi for 7 days.The sphere formation of tumor was photographed using microscope.9.Hepa1-6 cells and Huh-7 cells were cultured in ultra-low adhesion culture plate for 7 days to form HCC cell spheroids.Then with SAHA as positive control,HCC cells were treated with novel HDACi for 24 hours,the morphological changes of tumor spheroids were observed using microscope.10.With SAHA as positive control,HCC cell spheriods were treated with novel HDACi for 48 hours.The viability of HCC cell spheroids viability were detected by MTT assay.11.With SAHA as the positive control,HCC cell spheriods were treated with novel HDACi for 24 hours.qPCR was used to detect the expression of tumor stem relative molecules.12.Tumor cells with different cell numbers were subcutaneously injected into the armpit of 6week-old C57BL/6J mice.After the tumor grew to a certain size,Vehicle and different HDACi were given.The body weight curve and tumor growth curve of mice during treatment were plotted.Representative images of subcutaneous tumors of mice in each group were taken after euthanasia,and the tumor weight of mice was weighed.13.After euthanasia,lymphocytes were isolated from different tissues and tumor tissues of tumor-bearing mice,and the proportion of immune cells and the expression of related molecules were detected by flow cytometry.14.The tumor tissues,kidney,heart,liver,lung and spleen of tumor-bearing mice were fixed,embedded in paraffin,cut into 5 μm sections and stained with hematoxylin-eosin(H&E)for histopathological evaluation.Results1.The novel HDACi inhibits tumor cell proliferationThe IC50 values of HDACi HXB-9L and HXB-9K in different tumor cells were determined by MTT assay.The results showed that the IC50 values of compounds HXB-9L and HXB-9K were significantly decreased in B16F10 and Hepa 1-6,Huh-7 and Hep 3B cells compared with SAHA,but there was no significant difference in the IC50 values of the three compounds in MC38 cells.2.The novel HDACi promotes apoptosis of tumor cellsAnnexinV-APC/7-AAD-PerCP double staining assay was used to detect the effect of the novel HDACi on apoptosis of different tumor cells.The results showed that HXB-9L and HXB9K showed a stronger pro-apoptotic effect on B16F10 cells than SAHA,while Hepa1-6 and Huh-7 cells promoted the apoptosis of HCC cells to a similar degree as SAHA.3.The novel HDACi induces cell cycle arrestPI staining flow cytometry was used to determine the effect of HDACi HXB-9L and HXB9K on cell cycles of B16F10,Hepa 1-6 and Huh-7 cell lines.The results showed that compounds HXB-9L and HXB-9K could induce cell cycle arrest in B16F10 cells at low or high concentrations,and their effect was similar to that of positive drug SAHA.The proportion of Hepa1-6 cells in G0/G1 phase was significantly upregulated,while the proportion of S phase cells was significantly decreased.The proportion of Huh7 cells in G2 phase upregulated,and the proportion of S phase cells decreased.HCC cell lines Hepa1-6 and Huh-7 cells treated with HXB-9L and HXB-9K for 24 hours could also induce HCC cell cycle arrest,which was similar to the positive drug SAHA,showing that the proportion of cells in G0/G1 phase was significantly up-regulated and the proportion of cells in S phase decreased,while that of Huh-7 cells in G2 phase was significantly upregulated and the proportion of cells in S phase was significantly decreased.4.The novel HDACi inhibits B16F10 cell migrationScratch test was used to detect the effect of novel HDACi on the migration ability of B16F10 cells.Compared with SAHA,compounds HXB-9L and HXB-9K had stronger inhibitory effect on B16F10 cell migration.5.HCC patients with low HDAC1 and HDAC2 have a better prognosisThe correlation between HDAC1 or HDAC2 and prognosis of patients with liver cancer was analyzed by OncoLnc database(http://www.oncolnc.org/).Compared with HCC patients with higher HDAC1 and HDAC2 expression,patients with lower HDAC1 and HDAC2 expression had longer survival.6.The novel HDACi inhibits the colony-forming ability of hepatocellular carcinoma cellsThe effect of novel HDACi on the colony-forming ability of HCC cells was evaluated by colony formation experiment.The results showed that compared with the positive drug SAHA,the number of clones formed in compound HXB-9L and HXB-9K groups decreased significantly.7.The novel HDACi inhibits the expression of stemness related molecules in HCC cellsFluorescence quantitative PCR was used to detect the effect of novel HDACi on the expression of stemness related molecules c-Myc,Nanog,Klf4 and Epcam in hepatocellular carcinoma cells.The results showed that compared with the positive drug SAHA,HXB-9L and HXB-9K significantly down-regulated the expression of c-Myc,Nanog,Klf4 in Hepa 1-6 cells and the expression of c-Myc,Nanog and Epcam in Huh-7 cells.8.The novel HDACi inhibits the sphere formation ability of HCC cellsWe explore the effect of novel HDACi on the sphere formation of HCC cells by observing the morphology and number of Hepa1-6 and Huh-7 cell spheres formed.The results showed that compared with the drug SAHA,the number of HCC cell spheres formed in compound HXB-9L and HXB-9K groups decreased significantly.9.The novel HDACi inhibits cell viability of HCC cell spheriodsThe real-time monitoring by JuLITM Stage instrument showed that the treatment of compounds HXB-9L and HXB-9K could significantly destroy the morphology of HCC spheriods induced by Hepa1-6 cells and Huh-7 cells,resulting in obvious cleavage of the spheriods.Furthermore,the cell viability of HCC cell spheriods treated with compounds was assayed.The results showed that compared with positive drug SAHA,compounds HXB-9L and HXB-9K could significantly inhibit the viability of HCC cell spheriods in a dose-dependent manner.10.The novel HDACi inhibits the expression of stemness related molecules in HCC cell spheriodsFluorescence quantitative PCR was used to detect the expression of stemness related molecules in HCC cell spheriods treated with novel HDACi compounds at mRNA level.The results showed that compared with positive drug SAHA,compound HXB-9L and HXB-9K could significantly down-regulate the expression of stemness related molecules such as Epcam,Nanog and c-Myc in Hepa1-6 cells and Huh-7 cells.11.The novel HDACi inhibits tumor growth in subcutaneous grafted tumor models in mice11.1 The novel HDACi inhibits tumor growth in B16F10 subcutaneous grafted tumor modelIn the subcutaneous tumor model of B16F10 cells,compared with positive drug SAHA,the tumor volume decreased significantly after treatment with HXB-9L and HXB-9K,and in the process of continuous administration,the range of weight fluctuation of mice in HXB-9L and SAHA group was the smallest,followed by HXB-9K group.In the tumor tissue,the heterogeneity of the HXB-9L and HXB-9K treatment groups decreased,which was similar to the positive drug SAHA treatment group.11.2 The novel HDACi inhibits tumor growth in Hepa 1-6 subcutaneous grafted tumor modelsCompared with vehicle group,compounds HXB-9L,HXB-9K and SAHA had no obvious effect on the body weight of mice,and all had significant anti-tumor activity.The anti-tumor effect of compound HXB-9K was similar to that of SAHA,and the tumor volume decreased significantly,while the therapeutic effect of HXB-9L was weak.12.Effect of novel HDACi on anti-tumor immunity in tumor-bearing mice12.1 Effect of novel HDACi on anti-tumor immunity in melanoma tumor-bearing miceCompared with the vehicle group,the proportion of T cells,CD8+T cells and CD4+T cells in the spleen of HXB-9L and HXB-9K treatment groups were up-regulated,while the expression of activation molecules CD107a,ICOS,CD69 and IFNy were significantly upregulated in CD8+T cells and CD4+T cells.Among them,HXB-9L up-regulated and activated T cells more significantly.The proportion of macrophages and the expression of costimulatory molecule CD86 were also significantly up-regulated in spleen.The proportion of DCs did not change significantly,but the expression of CD86 was significantly up-regulated.In addition,the proportion of Treg cells infiltrating in tumor was reduced.The expression of CD200R in MDSC cells was down-regulated,and the immunosuppressive activity of tumor infiltrating immunosuppressive cells was inhibited.Moreover,HXB-9L and HXB-9K can participate in the phenotypic transformation of macrophages,significantly reducing the proportion of macrophages in tumor,especially M2-type macrophages,resulting in inhibition of their tumorpromoting function.12.2 Effect of novel HDACi on antitumor immunity of HCC tumor-bearing miceCompared with vehicle group,the number of tumor infiltrating lymphocytes increased significantly in HXB-9K and SAHA treatment group.Among them,compound HXB-9K and SAHA treatment could promote the infiltration of DCs and macrophages in tumor,and the number of antigen presenting cells expressing MHCII,IL-12 and CD 86 increased significantly.At the same time,in tumor microenvironment,the proportion of CD4+T cells and CD8+T cells treated with HXB-9K and SAHA were up-regulated,and the expression of functional molecules was also significantly increased,including proliferation marker molecule Ki67,activationrelated molecules CD107a,ICOS,CD69 and effector cytokine TNFα.The above-mentioned effect of compound HXB-9K was stronger than that of positive drug SAHA,while compound HXB-9L had no similar effect.In addition,compared with vehicle group,compound HXB-9K and SAHA treatment group could also significantly increase the number of infiltration of NK cells and NKT cells,and up-regulate the expression of activation-related molecules ICOS and TNFα.In lymph nodes,compared with vehicle group,the proportion of CD4+T cells and CD8+T cells in HXB-9L and HXB-9K treatment group also increased significantly,and the expression of T cell effector factor TNF-α was up-regulated,and this phenomenon was similar to that in positive drug SAHA group.13.Effect of novel HDACi on main organs in miceThe results showed that compared with the vehicle group,compounds HXB-9L,HXB-9K and SAHA had no significant effect on the heart,liver and spleen of mice after treatment.In lung tissue,the lung structure of mice showed obvious damage after tumor bearing,and the degree of lung injury was alleviated after HXB-9L and SAHA treatment,but this damage was not alleviated in HXB-9K treatment group.In renal tissue,the glomerular structure was significantly damaged after HXB-9L and SAHA treatment,while the degree of damage was slight in the HXB-9K treatment group.Conclusion and significance1.The novel HDACi HXB-9L and HXB-9K can inhibit tumor cell proliferation by inducing apoptosis and cell cycle arrest in B16F10,Hepa 1-6 and Huh-7 cells.2.The novel HDACi HXB-9L and HXB-9K in Hepa 1-6 and Huh-7 cells can inhibit clonogenesis,tumor stemness,and the expression of stemness related molecules.It can also destroy the morphology of HCC cell spheriods and inhibit the viability and stemness related molecules expression of HCC cell spheriods.3.B16F10 subcutaneous tumor-bearing model was used to demonstrate the antitumor effects of HDACi HXB-9L and HXB-9K in two aspects:1)directly killing tumor cells;2)it is involved in the regulation of anti-tumor immunity,including promoting the activation of antigen presenting cells and T cells,inhibiting the infiltration of immunosuppressive cells,and participating in the phenotypic transformation of macrophages.4.The anti-tumor effects of the novel HDACi HXB-9L and HXB-9K on HCC cells were proved by Hepa 1-6 subcutaneous tumor-bearing model.Firstly,it can kill tumor cells directly;Secondly,it can activate anti-tumor immunity including promoting the infiltration and activation of antigen presenting cells,NK cells,T cells and NKT cells.5.Through the toxicity test of organs in tumor-bearing mice,HXB-9L has certain renal toxicity and HXB-9K has certain pulmonary toxicity,which provides experimental basis for the further optimization of the novel HDACi structure.6.This study preliminarily explored the potential mechanism of antitumor activity of the novel HDACi,and provided a new idea and experimental basis for the clinical treatment of melanoma and HCC. |
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