Solamargine Inhibits The Growth Of Multiple Myeloma By Activating Autophagy And Exerts A Synergistic Effect With Bortezomib

Multiple myeloma(MM)is the second most common hematologic tumor.More than 32,000 new cases are diagnosed in the United States each year,and nearly 13,000 patients die of the disease.The incidence of MM increased significantly in China from 2006 to 2016,which has attracted the attention of hematologists in China.The median age of onset of MM is about 65 years,although many younger patients are also diagnosed.In the last decade or so,new drugs and treatment technologies,such as proteasome inhibitors,immune modulators,histone deacetylase inhibitors,monoclonal antibodies,autologous stem cell transplantation,chimeric antigen receptor T cells(CAR-T)have made remarkable progress in the treatment of MM.The prognosis of MM patients was significantly improved,but MM remained incurable.Therefore,many domestic and foreign scholars and clinical experts have been exploring new strategies for the treatment of MM to improve the efficacy.The treatment of malignant tumor with Chinese herbal medicine is one of the hot spots in anti-tumor research.Clinical practice has shown that the compound with solanum nigrum as the main component is often used in the treatment of malignant tumors,and the effect is obvious.Through pharmacy study and analysis,the whole plant of Solanum nigrum L.contains many anti-tumor components.Solamargine(SM)is one of the main components of Solanum solanum,which has anti-tumor effects on various solid tumor cells and plays synergistic anti-tumor effect with other chemotherapy drugs to enhance the therapeutic effect.However,the effect and mechanism of SM on MM have not been reported.Newly diagnosed multiple myeloma(NDMM)patients from Jingjiang People’s Hospital were systematically analyzed to evaluate the survival of these patients under the current treatment regimen.SM was selected as a candidate therapeutic agent to explore the activity of SM against MM cells and its possible mechanism through cytological studies in vitro and animal experiments in vivo,and to further observe the efficacy of SM combined with Bortezomib(BTZ)in the treatment of MM.In this study,a total of 85 NDMM cases admitted to the Hematology Department of Jingjiang People’s Hospital from 2012 to 2021 were collected,and the treatment and survival of the patients were statistically analyzed according to clinical characteristics and different treatment schemes.A predicted network of SM was built with SymMap database.ARP1 and H929 cells were treated with different concentrations of SM.Cell viability and proliferation inhibition of ARP-1 and H929 cells by SM were detected by CCK-8 assay.The cell apoptosis was determined using flow cytometry(FCM).Western blotting was used to detect the expression levels of autophagy and apoptosis-related proteins in ARP-1 and H929 cells after SM intervention.The regulation of SM on gene expression in H929 cells was analyzed by RNA sequencing(RNA-Seq).In addition,the effects of SM combined with BTZ on MM cell growth and proliferation were investigated.CompuSyn software was used to calculate the combined index(CI).Trypan blue staining was used to evaluate cell survival rate.FCM was used to evaluate the inhibitory effect of SM combined with BTZ on CD 13 8+ cells in primary bone marrow mononuclear cells(BM-MNCs)obtained from NDMM patients.Finally,the MM model of non-obese diabetes/server combined immune deficiency(NOD/SCID)mice was constructed.The in vivo efficacy of SM monotherapy and its combination with BTZ in MM was preclinical validated.Clinical studies showed that the number of new NDMM cases from Jingjiang People’s Hospital increased significantly in recent years.The estimated 3-year and 5-year survival rate and estimated median survival time of the new regimen group were significantly better than that of the traditional regimen group,and the survival rate of the untreated group was the shortest.Due to economic reasons,many patients could not adopt or adhere to the treatment of new regimen with adequate course and ASCT,and the overall estimated survival rate and estimated median survival time were low.The basic research part showed that SM had a good inhibitory effect on MM,and the main results were as follows:1.The inhibitory effect of SM on ARP-1 and H929 cells was concentration-dependent.The IC50 values of SM on ARP-1 and H929 cells were 5.36 ?μM and 5.23 μM,respectively,so 5 μM was selected as the concentration for subsequent experiments.In addition,SM inhibited the proliferation of ARP-1 and H929 cells in a time-dependent manner.Compared with the Control group,the apoptosis rate of ARP-1 and H929 cells treated with SM was significantly increased,and the expression of anti-apoptotic protein Bcl-2 was downregulated,while the expression of pro-apoptotic protein Bax and apoptotic indicator Cleaved caspase-3 was upregulated(P<0.01).2.By RNA-Seq analysis of SM-treated H929 cells,667 differentially expressed genes were identified,including 547 upregulated genes and 120 downregulated ones.GO and KEGG analysis showed that,in addition to cell death related genes,autophagy related genes were also enriched among the upregulated genes.At the protein level,SM significantly increased the expression of autophagy related proteins LC3-Ⅱ and Beclin 1 in ARP-1 and H929 cells,while the autophagy inhibitor 3-MA effectively antagonized the above effects of SM,and weakened the inhibitory effect of SM on MM cell viability and cell apoptosis induced by SM.3.The inhibitory effect of SM combined with BTZ on ARP-1 and H929 cells was significantly enhanced.When different concentrations of SM combined with different concentrations of BTZ were applied to MM cell line,the combination index(CI)was<1,and the higher the concentration was,the lower the CI value was.Compared with Control group,SM and BTZ alone reduced the survival rate of ARP-1 and H929 cells(P<0.01),while SM+BTZ had a more significant inhibitory effect on the survival rate(P<0.01).Compared with the Control group,both SM and BTZ effectively reduced the percentages of CD 13 8+cells in primary BM-MNCs obtained from NDMM patients(P<0.01),while the percentages of CD138+cells in SM+BTZ group decreased more significantly(P<0.01).4.Both SM and BTZ alone could inhibit the tumor volume,tumor weight,tumor cell density and Ki-67 expression of tumors in NOD/SCID mice MM model,and SM+BTZ had more significant inhibition on the above indexes.In this study,the therapeutic regimen and survival of NDMM were retrospectively analyzed,and the efficacy of existing drugs and regimen needed to be improved.Based on this,this study further explored new drugs and regimens for the treatment of MM.It is suggested that SM can be used as an autophagy activator for the development of MM drugs,and the combination of SM and BTZ is a new treatment strategy for MM.This study provides a research basis and scientific basis for SM as a candidate therapeutic drug for MM and its use strategy,which is helpful to promote the clinical application of Chinese herbal active ingredients in the treatment of MM and is expected to effectively improve the survival rate of patients.