Induction Of Apoptosis In Bortezomib-resistant Multiple Myeloma Cells By The Novel Targeting Molecule BY4015 And Its Related Mechanisms

Objective:Multiple myeloma(MM)is a plasma cell malignancy that is currently difficult to cure.Bortezomib is the first-line agent in the treatment of multiple myeloma,and most patients with myeloma have a poor prognosis for relapsed disease progression due to resistance to bortezomib.The aim of this study is to design and synthesize a novel pyrimidine compound to induce apoptosis in bortezomib-resistant myeloma cells,to overcome bortezomib resistance,and to explore its pharmacological activity and mechanism of action on bortezomib-resistant multiple myeloma cells.Methods:In the first part of this experiment,a new pyrimidine compound BY4015 was designed and synthesized,and its molecular structure was verified by high-resolution mass spectrometry and NMR.The second part detects the survival of compound BY4015 against bortezomib-sensitive human multiple myeloma cell line(KM3)and bortezomibresistant human multiple myeloma cell line(KM3/BTZ)by CCK-8 assay and the toxicity to human normal hepatocytes L-02.Effect of BY4015 on KM3 and KM3/BTZ cell morphology by natural light photography.The effect of compound BY4015 on apoptosis and cell cycle of KM3/BTZ cells was also detected by AO/EB staining,DAPI staining and flow cytometry.Transwell assay was performed to assess the effect of BY4015 on the migratory ability of KM3/BTZ cells.Finally,the effect of the compound on STAT3 signaling pathway proteins was detected by immunohistochemical staining and Western blot to verify the targeting effect of compound BY4015 on STAT3 signaling pathway.Results:1.The molecular structure of compound BY4015 was verified by highresolution mass spectrometry and NMR.2.The results of CCK-8 method proved that compound BY4015 could effectively inhibit KM3/BTZ cell growth and showed time-concentration dependence,and the inhibitory effect of BY4015 on KM3/BTZ was significantly better than that of bortezomib;BY4015 was less toxic to human normal hepatocytes L-02.3.Natural light photography experiment,AO/EB staining and DAPI experiment all showed that the cell morphology changed,the number of tumor cells decreased and the number of apoptotic cells increased,and the pro-apoptotic effect of BY4015 on KM3/BTZ was significantly better than that of bortezomib.4.Transwell assay results showed that KM3/BTZ cells had reduced migratory capacity after treatment with BY4015.5.Flow cytometry showed that BY4015 blocked the growth of KM3/BTZ cells in G2/M phase and promoted KM3/BTZ cell apoptosis,and the pro-apoptotic effect was superior to that of bortezomib.6.The results of immunohistochemical staining and Western blot showed that BY4015 could inhibit the phosphorylation of STAT3 and the expression of its downstream target gene Bcl-2 protein,and promote the expression of apoptotic protein Bax.Conclusion: BY4015,a novel pyrimidine compound synthesized in this experiment,has significant anti-proliferative activity against multiple myeloma cells with low cytotoxicity.It is especially effective against bortezomib-resistant KM3/BTZ cells and is superior to the reference drug bortezomib.BY4015 can reduce the migration ability of KM3/BTZ cells.It inhibits cell proliferation by blocking the cell growth cycle in the G2/M phase.BY4015 exerted its anti-KM3/BTZ cellular activity by inhibiting STAT3 phosphorylation.