Palbociclib Reverses Doxorubicin Resistance In Liver Cancer Cells By Inhibiting The Rb/E2F-1 Pathway

Objective:Doxorubicin is a usual chemotherapy drug used in the clinical treatment of liver cancer that can upgrade patients’quality of life and prolong their survival.Although there have been many studies on the drug resistance mechanism of Doxorubicin,there is still a lack of effective methods to overcome its drug resistance.Through clinical specimens and cell lines,we found that Doxorubicin can up-regulate the expression of retinoblastoma protein(Rb),and the sensitivity of liver cancer to Doxorubicin is negatively correlated with Rb.It is speculated that Rb is the mediator of Doxorubicin resistance in liver cancer key factor.The study also found that Doxorubicin can release the transcription factor E2F-1 by phosphorylating Rb and promote the transcription of Siglec-15.However,the detailed molecular mechanism has not yet been elucidated.In this study,clinical specimens,tumor-bearing animal models and cell models were used to clarify the specific molecular mechanism of Doxorubicin resistance in liver cancer,and to evaluate the curative effect of Palbociclib and Doxorubicin combined on liver cancer.These studies provide new ideas and strategies for solving Doxorubicin resistance and liver cancer.Methods:1.Evaluation of the synergistic effect of Doxorubicin and Palbociclib:Calculation of synergy index of combined use of Doxorubicin and Palbociclib by MTT method;Evaluating the effect of combined ingestion of Doxorubicin and Palbociclib on cell proliferation by means of colony formation assay;The function of Palbociclib and Doxorubicin on the cell cycle were measured through flow cytometry;To establish animal models of hepatocellular carcinoma and evaluate the synergistic antitumor effect of Doxorubicin and Palbociclib in vivo.Pathological detection of the effect of Palbociclib in reducing the cardiotoxicity of adriamycin;biochemical indicators analysis of the synergistic and attenuating effect of the combination of the two drugs.2.To elucidate the function of Rb in promoting Doxorubicin resistance in hepatocellular carcinoma cells:According to the human gene expression data set,the expression difference of Rb in normal samples and liver cancer was contrasted;MTT and clonogenic assays to detect changes in the sensitivity of hepatoma cells to Doxorubicin after knockdown/overexpression of Rb;Construct liver cancer cell lines with stable over/low expression of Rb,establish animal models,use Palbociclib as a tool drug,and verify the role of Rb in promoting Doxorubicin resistance in vivo.3.To elucidate the molecular mechanism of Doxorubicin regulating Siglec-15-mediated tumor immune escape through Rb:Western blotting and Real time PCR to detect the effect of Doxorubicin on Rb and Siglec-15 protein and mRNA;Immunofluorescence and Co-IP experiments demonstrate the effect of Doxorubicin on the interaction between Rb and E2F;Dual-luciferase reporter assay and Ch-IP assay to clarify the regulation of E2F-1 on Siglec-15;Effect of Doxorubicin on tumor CD8~+T cells in mice detected by flow cytometry;Effect of Doxorubicin on immune-related result in mouse serum evaluated through enzyme-linked immunoso Rbent assay;Evaluate clinical specimens from patients with liver cancer,verify the relationship between Rb and Siglec-15 and liver cancer,and clarify the immunosuppressive effect of Siglec-15.Results:1.Combination of Doxorubicin and Palbociclib has a good synergistic anti-tumor effect in vivo and vitro,and Palbociclib can significantly reduce the cardiotoxicity caused by Doxorubicin.2.HepG2 Doxorubicin-resistant(HepG2/DOX)cell line has been successfully constructed.Compared with the sensitive strain,Rb was abnormally activated in the HepG2/DOX cells,and the expression of Rb was negatively correlated with the sensitivity of hepatocellular carcinoma cells to Doxorubicin.The use of Palbociclib can increase the sensitivity of hepatocellular carcinoma cells to Doxorubicin.These results indicated that Rb is a key factor leading to Doxorubicin resistance in HCC cells.3.Clinical specimens and data sets showed that contrast to normal parts,the content of Rb and Siglec-15 was up-regulated in liver cancer.Relevance analysis showed that the two were noticeable positive correlation,and inverse correlated with the lifetime of liver cancer patients;After incubating liver cancer cells with Doxorubicin for 24 hours,the interaction between Rb and E2F-1 was significantly reduced,promoting the release of E2F1;The tests of dual luciferase reporter genes manifest that E2F-1 can enhance the function of Siglec-15 promoter;The number of CD8~+T cells was significantly suppressed.These results show that Doxorubicin can activate the Rb/E2F1 pathway to regulate the expression of Siglec-15 and inhibit the activity of CD8~+T cells.Conclusions:The combination of Palbociclib and Doxorubicin can immensely reverses the drug resistance of liver cancer cells to Doxorubicin.