Evaluation Of The Effect Of Berberine Combined With Venetoclax On Reversing Acquired Venetoclax Resistance In Acute Leukemia Cells

Objective:Leukemia is a common hematological malignant tumor.Patients with leukemia are often accompanied by a series of complications,which seriously affect their quality of life and endanger their life and health.China is rich in Chinese herbal medicine resources,so researchers pay special attention to finding effective traditional Chinese medicine ingredients from medicinal plants for remission and treatment of leukemia.Berberine is an effective component of the traditional Chinese medicine Coptis chinensis,which is often used in digestive system-related diseases.High-dose single drug can inhibit the proliferation of a variety of leukemia cell lines,and can also improve tumor drug resistance when combined with chemotherapeutic drugs.The main purpose of this study is to study the effect and mechanism of Berberine(BBR)combined with Venetoclax(VEN)in inducing apoptosis of VEN-resistant human acute lymphoblastic leukemia cell line RS4;11(RS4;11-VENR).At the same time,it also provides ideas for the combination of traditional Chinese medicine and chemical drugs to exert anti-tumor activity.Methods:1.Study on the effect of Berberine on reversing VEN resistance in RS4;11 cellsCell proliferation experiments and anti-apoptotic protein expression tests were carried out on acute leukemia cell lines often reported in the literature.Finally,RS4;11 cells were used as acute leukemia cell model,and the molecular mechanism of drug resistance after chronic administration of VEN was studied by subcutaneous transplant tumor model of RS4;11 in mice.Finally,RS4;11-VENR was obtained by continuously exposing human acute lymphoblastic leukemia cell line RS4;11 to VEN,and the follow-up study based on drug resistance model was carried out.Venetoclax of the mechanism of RS4;11-VENR resistance:(1)the effect of VEN on the proliferation of RS4;11-VENR was tested by MTS method;(2)the apoptosis-inducing effect of VEN on RS4;11-VENR was detected by Annexin V-FITC/PI double staining;(3)the level of RS4;11-VENR anti-apoptotic protein was detected by Western blot method;(4)the transcriptional level of RS4;11-VENR anti-apoptotic protein gene was detected by qRT-PCR method;(5)the combined effect of MCL-1 inhibitor S63845 and VEN in RS4;11-VENR was evaluated based on ZIP model.Screening of Chinese herbal monomers with reversing drug resistance:(1)the effect of each monomer on the proliferation of RS4;11-VENR was tested by MTS method;(2)the toxic effect of each monomer on human normal peripheral blood mononuclear cells(PBMC)was tested by CTG method;(3)the combined effect of each monomer and VEN in RS4;11-VENR was evaluated based on ZIP model.2.Study on the mechanism of Berberine reversing VEN resistance in RS4;11 cells(1)the apoptosis induction of 25 μM BBR and 1 μM VEN was observed by Hoechst33342/PI staining;(2)the apoptosis induction of RS4;11-VENR was detected by Annexin V-FITC/PI double staining;(3)the effect of the combination on RS4;11-VENR cell cycle was detected by PI staining;(4)the effects of 25 μM BBR and 1 μM VEN on apoptosis related signals and anti-apoptotic proteins were detected by Western blot method;(5)the transcriptional level of RS4;11-VENR antiapoptotic protein gene was detected by qRT-PCR method.Results:1.RS4;11-VENR has the characteristic that compensatory upregulation of MCL-1 protein leads to VEN resistance in patients with leukemia.Apoptosis induction and proliferation inhibition experiments showed that VEN was not sensitive to RS4;11-VENR(IC50>10 μM),Western blot results showed that MCL-1 was up-regulated in RS4;11-VENR compared with wild-type RS4;11 cells,qRT-PCR results showed that MCL-1 gene transcription was also up-regulated,and MCL-1 inhibitor combined with VEN could effectively reverse RS4;11-VENR drug resistance.2.BBR combined with VEN synergistically inhibits the proliferation of RS4;11-VENR cells.CTG assay showed that BBR had no significant effect on human PBMC,and the combination evaluation based on ZIP model showed that 12-50 μM BBR and 0.1-10 μM VEN synergistically inhibited the proliferation of RS4;11-VENR cells.3.The combination of BBR and VEN can induce apoptosis of RS4;11-VENR cells through mitochondrial pathway,and BBR can reduce the protein levels of MCL-1 and BCL-XL in drug-resistant cells.The results of Hoechst 33342/PI staining and flow cytometry showed that 25 μM BBR combined with 1 μM VEN induced apoptosis,Western blot results showed that BBR alone decreased the protein levels of MCL-1 and BCL-XL,and qRT-PCR assay showed that 25 μM BBR alone decreased the expression of MCL-1 and BCL-XL genes.Conclusion:BBR combined with VEN synergistically kills RS4;11-VENR cells,and BBR may induce apoptosis by reducing the levels of MCL-1 and BCL-XL proteins and increasing the sensitivity of RS4;11-VENR cells to VEN.