Evaluation Of Triptonide On Proliferation Of Human Multiple Myeloma Cells

Multiple myeloma（MM）is a malignant hematological tumor characterized by abnormal accumulation of clonal plasma cells in the bone marrow and overproduction of monoclonal immunoglobulin.Lenalidomide（LENA）,the second-generation immunomodulatory drugs（IMi Ds）,is the first-line drug for MM,which can degrade substrate proteins IKZF1 and IKZF3 through ubiquitin-proteasome pathway,thereby inhibiting MM cell proliferation.There is not only primary drug resistance,but also acquired drug resistance after long-term use in the clinical use of IMi Ds.The reason for drug resistance is often accompanied by the degradation ability of substrates such as IKZF1 and IKZF3,which play a key role in tumor cell proliferation of IMi Ds,thus reducing the therapeutic effect of drugs.Therefore,for MM patients who are insensitive to and resistant to IMi Ds,it is necessary to focus on the substrate proteins of IMi Ds to research and develop more effective drugs.Triptonide（TPN）is one of the main effective components of Tripterygium wilfordii,which has pharmacological activities such as anti-tumor,contraception and immunosuppression.It was found that TPN has a strong inhibitory effect on MM cell proliferation.Further studies have shown that it can reduce expression of related proteins by inhibiting transcription of IKZF1 and IKZF3 genes in MM cells,which may overcome the resistance of MM cells to immunomodulators.From the perspective of clinically existing MM resistance to IMi Ds,this thesis evaluates the effects of TPN on proliferation,cycle,apoptosis and protein expression of primary and acquired drug-resistant multiple myeloma cells,in order to provide experimental data support for solving the current clinical problem of MM patient resistance to immunomodulators.ObjectiveTo investigate the effects and mechanism of TPN on proliferation and apoptosis of primary resistant RPMI-8226 cells and acquired resistant NCI-H929R cellsMethodsMTS colorimetric assay to detect the effect of components on the proliferative capacity of MM.1S,RPMI-8226,NCI-H929 and NCI-H929R cells by MTS colorimetry;the effect of LENA,POMA and TPN on the cell cycle and apoptosis of MM.1S,RPMI-8226,NCI-H929 and NCI-H929R cells by Western blot to detect the effects of different concentrations of LENA,POMA and TPN on the expression of IKZF1,IKZF3,Caspase3 and Cleaved-caspase3 proteins in MM.1S,RPMI-8226,NCI-H929 and NCI-H929R cells.;RT-q PCR assay to detect the effect of TPN on the transcription of IKZF1and IKZF3 genes in MM.1S cells;the effect of LENA,POMA and TPN on the cell cycle and apoptosis of MM.1S,RPMI-8226,NCI-H929 and NCI-H929R cells by flow cytometry.Results1.TPN has a strong ability to inhibit the proliferation of IMi Ds sensitive MM.1S cells,and can reduce protein expression by inhibiting gene transcription of IKZF1 and IKZF3 in cells.Based on phenotype screening strategy,the anti-proliferation activity of some components of traditional Chinese medicine was tested.From the point of view of clinical safety,TPN was finally selected as the main research object.The results showed that TPN had a strong inhibitory effect on the proliferation of MM.1S cells.This is consistent with the characteristics of IMi Ds.Further mechanism studies found that TPN inhibited the expression of related proteins by downregulating m RNA transcription levels of IKZF1 and IKZF3,which confirmed that TPN had a different mechanism from IMi Ds in inhibiting the expression of IKZF1 and IKZF3 proteins.These results suggest that TPN may also inhibit the expression of related proteins in drug-resistant cell lines.2.TPN inhibited proliferation of primary resistant RPMI-8226 cells and promoted apoptosis of RPMI-8226 cells.LENA and POMA can inhibit the proliferation of MM.1S cells by degrading IKZF1 and IKZF3 in sensitive MM.1S cells,blocking the G₀/G₁ phase,and promoting MM apoptosis.However,the degradation effect of IKZF1 and IKZF3 in RPMI-8226 cells were weakened and proliferation inhibition effect was not evident,cell cycle and apoptosis were not significantly affected.Compared to IMi Ds,TPN had a stronger inhibition of proliferation on MM.1S and RPMI-8226 cells,and inhibited protein expression of IKZF1 and IKZF3,promoting apoptosis.At low concentrations,TPN blocked G₀/G₁ phase of MM.1S and promoted Cleaved-caspase3 expression.It is suggested that TPN can inhibit cell proliferation and promote cell apoptosis by inhibiting the expression of proteins IKZF1and IKZF3,indicating that TPN can not only strongly inhibit the proliferation of multiple myeloma cells sensitive to IMi Ds,but also overcome the problem of primary drug resistance to IMi Ds.3.TPN inhibited the proliferation of acquired resistant NCI-H929R cells and promoted apoptosis of NCI-H929R cells.NCI-H929 POMA resistant cell line NCI-H929R was constructed by long-term administration of 200n M POMA.Western blot results showed that CRBN expression in cells was significantly reduced after resistance to POMA.In NCI-H929 cells,POMA promoted apoptosis of NCI-H929 cells and inhibited proliferation of NCI-H929 cells by degrading IKZF1 and IKZF3.In NCI-H929R cells,POMA had no degradation effect on IKZF1 and IKZF3 and had no obvious effect on cell proliferation.Meanwhile,Poma had no significant effect on cell cycle and apoptosis of NCI-H929R cells.TPN had strong proliferation inhibition on both NCI-H929 and NCI-H929R cells,and inhibited the expression of proteins IKZF1 and IKZF3,promoted cell apoptosis,and promoted intracellular Cleaved-caspase3expression.It is suggested that TPN can inhibit cell proliferation and promote cell apoptosis by inhibiting the expression of proteins IKZF1 and IKZF3,and overcome acquired resistance to IMi Ds in multiple myeloma..ConclusionTPN can inhibit MM cell proliferation and promote MM cell apoptosis by downregulating the transcription of IKZF1 and IKZF3 genes in MM cells and reducing the expression of related proteins,which can overcome the problem of primary and acquired drug resistance to MM IMiDs.