| Primary liver cancer is an aggressive malignant tumor characterized by a high incidence,high recurrence rate,and high mortality rate.Its incidence is second only to lung cancer,prostate cancer,colorectal cancer,and gastric cancer,and it seriously threatens human health.Systemic doses of chemotherapeutic drugs as a conventional method for clinical treatment of advanced liver cancer may cause non-specific cytotoxic reactions in patients,leading to poor patient prognosis.How to increase the sensitivity of liver cancer to chemotherapeutic drugs,thereby reducing the dose of drugs and reducing side effects has become the focus of current research on the treatment of liver cancerDocetaxel(DTX)is a taxane anticancer drug.It is widely used in clinical treatment of non-small cell cancer,breast cancer,liver cancer and other malignant tumors.Docetaxel and paclitaxel have a similar mechanism of action,can be combined with free tubulin,promote microtubule polymerization and inhibit its disaggregation,and regulate cell mitosis.In vitro and in vivo experiments prove that docetaxel has a good killing effect on liver cancer cells.Although docetaxel alone has a certain effect on liver cancer,its adverse reactions have limited the use of high doses.Therefore,it is necessary to explore the mechanism of tumor cells,response to docetaxel in order to obtain tumor cells that can enhance docetaxel.The new molecular mechanism of sensitivity provides innovative therapeutic strategies for its clinical antitumor application.Histone Deacetylase 6(HDAC6)is a class Ilb histone acetylase located in the cytoplasm.It has two tandem and independent catalytic domains DDI and DD2.HDAC6 not only deacetylates histones,but also interacts with non-histone substrates in the cytoplasm,which is different from other members of the histone deacetylase family(HDAC).HDAC6 is strongly expressed in a variety of tumor tissues.Some studies have reported that HDAC6 can combine with ubiquitin-labeled misfolded proteins and dynein to form trimers,and trimers transport misfolded proteins to aggregates along microtubules,thereby degrading misfolded proteins.Interestingly,?-tubulin,a microtubule subunit,is one of the non-histone substrates of HDAC6.After deacetylation by HDAC6,it can regulate the dynamics of microtubules in autophagosomes and lysosomes.It plays a major role in the integration process.Therefore,when HDAC6 is inhibited,it can lead to a large accumulation of misfolded proteins in tumor cells,which increase cell toxicity and induce cell death.This is one of the principal mechanisms for tumor cells to fight chemotherapy drugs,so HDAC6 may be a potential target for increasing the sensitivity of chemotherapy point.Tubacin is the primary HDAC6 specific inhibitor to be developed.It has been observed in laboratory studies that it has a synergistic effect in combination with multiple chemotherapeutics.For example,the combination of tubacin and bortezomib for the treatment of EB virus-related lymphomas and the combination of vincristine can inhibit the proliferation of malignant gliomas.The possible mechanism is to regulate the acetylation level of lysine residues in a-tubulin.Microtubule dynamics to achieve anti-tumor effects,but the specific mechanism is not completely clear.In summary,the anticancer mechanisms of HDAC6 specific inhibitor tubacin and docetaxel are related to the regulation of microtubule dynamics.Can the combined effect of tubacin and docetaxel synergistically regulate microtubules to maximize the therapeutic effect?Therefore,this study is based on a human hepatocellular carcinoma cell model,first verifying the anticancer effects of docetaxel and tubacin,and then experimentally characterizing the biological role of docetaxel and tubacin in human hepatocellular carcinoma cells,and further exploring docetaxel and Antitumor effect and mechanism of tubacin combined application.Experimental method:(1)Western blot and HDAC6 enzyme activity kits were used to detect the effects of different concentrations of tubacin on the expression of Ac-?-tubulin and enzyme activity in human hepatocellular carcinoma SNU449 cells and SNU387 cells(2)Detection of cell survival rate:MTT method was used to detect the effect of DTX,tubacin,DTX+tubacin on the survival rate of human hepatocellular carcinoma SNU387 cells and SNU449 cells(3)Detection of cell proliferation capacity:Real-time cell-free cell analysis technology(RealTime Cell ?L ar Analysis,RTCA)monitors cell survival status in real time and detects the effect of tubacin combined with DTX on the proliferation capacity of human hepatocellular carcinoma SNU387 cells and SNU449 cells(4)Detection of cell invasion and migration ability:Transwell invasion test and cell scratch test respectively detect the effect of tubacin combined with DTX on the invasion and migration of human hepatocellular carcinoma SNU387 cells and SNU449 cells(5)Cell cycle:Flow cytometry was used to detect the effect of DTX combined with tubacin on the cell cycle of human hepatocellular carcinoma cells SNU387 and SNU449(6)Western blot method and HDAC6 enzyme activity kit to detect the effect of DTX combined with tubacin on the expression level and enzyme activity of Ac-a-tubulin in human hepatocellular carcinoma SNU387 cells and SNU449 cells;indirect immunofluorescence test to detect SNU387 cells and SNU449 cells Changes in the expression of Ac-?-tubulin(7)Nuclear morphology:Hoechst staining was used to detect the effect of DTX combined with tubacin on the nuclear morphology of human hepatocellular carcinoma cells SNU387 and SNU449Results:(1)The gradient concentration of tubacin acts on human hepatocellular carcinoma SNU449 cells and SNU387 cells for 24h.The results show that 10 ?mol/L tubacin can significantly increase the expression of Ac-?-tubulin and significantly inhibit the enzyme activity of HDAC6(2)In SNU449 cells,compared with the control group,the cell survival rate of the DTX group was significantly reduced;compared with the DTX group,the cell survival rate of the DTX+tubacin group was further reduced.In SNU387 cells,compared with the control group,the cell survival rate of the DTX group did not change significantly;compared with the DTX group,the cell survival rate of the DTX+Tubacin group decreased significantly(3)In SNU449 cells and SNU387 cells,compared with the control group,the cell doubling time of the DTX group increased,and compared with the DTX group,the cell doubling time of the DTX+tubacin group further increased(4)In SNU449 cells and SNU387 cells,compared with the control group,the ability of DTX group cells to invade and migrate was significantly reduced.Compared with the DTX group,the ability of DTX+tubacin group cells to invade and migrate was further reduced(5)In SNU449 cells and SNU387 cells,compared with the control group,there was no significant change in the amount of cells in the S phase and G2/M phase of the DTX group.Compared with the DTX group,the S phase and G2/M phase of the DTX+tubacin group The cell volume decreased significantly(6)In SNU449 cells and SNU387 cells,compared with the control group,the expression of Ac-a-tubulin in the DTX group increased,and the activity of HDAC6 decreased.Compared with the DTX group,the expression of Ac-a-tubulin in the DTX+tubacin group increased significantly,HDAC6 activity is further reduced(7)In SNU449 cells and SNU387 cells,compared with the control group,the degree of abnormal nuclear division in the DTX group increased,compared with the DTX group,the degree of abnormal nuclear division in the DTX+tubacin group further increasedConclusion:In human liver cancer SNU449 cells and SNU387 cells,docetaxel and tubacin have a positive synergistic effect.The combined use can significantly inhibit the proliferation and invasion and metastasis of liver cancer cells.The combination of docetaxel and tubacin can affect the nucleus of the tubulin by affecting the acetylation of tubulin.Abnormal division,which in turning blocks the cell cycle and increases anticancer sensitivity.This study may provide new ideas for increasing the sensitivity of docetaxel... |
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