| Background : Multiple myeloma(MM)is still an incurable hematological malignant disorder of bone marrow.The proliferation of clonal plasma cells with abnormal origin in the bone marrow microenvironment(BM)leads to a progression of various complications,including the inevitable feature of relapse with no cure and,eventually death.Various efficient therapeutic regimens have made dramatic progress in treatment options for MM as compared to other cancer types.The current therapeutic regimens such as the first approved proteasome inhibitor bortezomib(BTZ)and immunomodulatory drugs(thalidomide and lenalidomide)for cancer treatment have been considered the crucial regimen for treating MM patients.However,MM patients are still facing major clinical issues of relapse and BTZ resistance due to the complex system of the BM,extracellular matrix,cell adhesion molecules,and heterogeneous genetic/epigenetic basis of MM.Therefore,constant research of identifying novel effective therapeutic agents or strategies is necessary for the developmental progress of MM treatment.Cell adhesion-mediated drug resistance(CAM-DR)is a well-known mechanism of resistance to chemotherapy in MM which originates in BM of MM patients.The BM is a diverse system of different cell types,soluble factors,adhesion molecules,and signaling pathways.The interaction of MM cells with various adhesion molecules and extracellular matrix in BM supports MM pathogenesis,drug resistance,and cell migration.In MM pathogenesis,CAMs induce cell-adhesion-mediated signaling which is a major drug resistant characteristic of MM.The participation of CAMs in cancer stemness and drug resistance of MM has been reported in the last decades.The phenomenon of CAMs in drug resistance of MM was initially reported by the resistance of MM cells against melphalan and doxorubicin treatments due to the adhesion of fibronectin(FN)to MM cells via cell adhesion molecules CD49 d and CD49 e.In previous studies,various CAMs were investigated including CD44,CD138,ICAM-1,VCAM-1,CD11 A,CD49D,α4β1,and CXC motif chemokine receptors(CXCR)integrin,which has been reported for adhesion of MM cells to bone marrow stromal cells(BMSC),which leads to MM stemness and participate in BTZ resistance of MM.It has also been found that inhibition of CAMs enhances the sensitivity of several anticancer agents and induces cell death in MM.Currently,the inhibition of CAMs expression is a promising approach to overcoming CAM-DR.recently,targeting CAMs signaling pathways has been shown to sensitize cancer cells to chemotherapy-induced apoptosis.However,proper inhibiting or degrading agents against CAMs have still not been discovered to overcome the BTZ resistance capability of MM.Thus,a chemotherapeutic agent which can inhibit the growth of MM to overcome BTZ resistance through molecular targets as CAMs can be an important regimen to develop in the future.Chemotherapies that use natural compounds in anticancer regimens often originate the basis for effective development in anticancer treatment options.Over decades,numerous natural compounds have been investigated for the effective development of anticancer therapies and many of them have been approved for integration into modern anticancer medicine.Exploring natural compounds against cancer is the most common way to novel anticancer drug discovery.Various natural compounds alone or in synergy with anticancer drugs have been reported for efficient target approaches in different cancers.Currently,more than25% of the approved anticancer agents originated from natural compounds,38% were imitated from organic synthesis,and even the existing commercial anticancer drugs are more than 60% of natural origin.Therefore,the identification of potential natural anticancer compounds with a different mechanism of action might play a role in novel drug discovery against MM.Periplocin(PP)is a cardiac glycoside monomer compound,derived from cortex periplocae which is generally used for the treatment of rheumatoid arthritis and the reinforcement of bones and tendons in traditional Chinese medicine.Cortex periplocae is the dried root bark of Periplocasepium Bge,referred to as Xiangjiapi in Chinese.Cortex periplocae possesses various biological activities such as antiradiation,antitumor,anti-inflammatory,and immune function enhancement effects,while most studies have reported that cortex periplocae could be used in the treatment of various heart conditions.Anyhow,PP from the Cortex periplocae expressed an inhibitory effect on diverse cancer cell lines including colon carcinoma cells,hepatocellular carcinoma cells,lung carcinoma cells,and breast carcinoma cells basically through blocking the cell cycle and inducing apoptosis.Researchers reported the PP-induced apoptosis and inhibition in the proliferation of human hepatocarcinoma cell line SMMC-7721 via restraining Stat3 signal transduction.By the downregulation of the Wnt/β-catenin signaling pathway,PP markedly inhibited the proliferation of the human colon carcinoma cell line SW840.The probable mechanism by which PP induced apoptosis in the human lung cancer cell line A549 was related to the downregulation of the survivin m RNA and protein.Furthermore,PP had an effect on breast carcinoma MCF-7 cells,which was associated with the increased expression of the m RNA and protein of gene p21WAF1/CIP1.Additionally,PP displayed the ability to suppress the growth of human(A549)and mouse lung cancer cells in vitro and in vivo by blocking the protein kinase B/extracellular signal-regulated kinase signal pathway.Aims : The current anti-MM chemotherapeutic treatment options including bortezomib are still facing challenges of drug resistance.Therefore,it is necessary to continue the treatment options for MM to find a strong novel effective therapeutic option against the BTZ resistance of MM.To elevate the treatment against MM BTZ resistance,we processed a library including 2370 natural bioactive compounds,after screening,we explored periplocin(PP)as the most effective compound among them against MM which was not reported in MM till this study.Thus,the study will demonstrate whether a newly discovered anti-MM natural compound(PP)can overcome bortezomib resistance of myeloma or not in vitro and in vivo.Furthermore,the anticancer effect cannot only be induced by pathways,genes,or proteins reported previously in the literature of PP against cancers.Therefore,it is also the purpose of this study to find the molecular mechanism of PP in MM and to discover new anticancer targets of PP.Experimental Design : In order to investigate the most effective anti-MM agents in library of 2370 natural compounds,the compounds were screened for BTZ sensitive ARP1 cell lines and BTZ resistant ARP1-BR MM cell lines using the CCK-8 method.We determined that PP was the most effective against ARP1 and ARP1-BR cell lines.Subsequently,the growth inhibition effect of PP on MM cells was confirmed in different cell lines and CD138+ cells isolated from MM patient samples using CCK-8 assay.Furthermore,this study used colony forming assay,flow cytometry,q RT-PCR,and transwell assay to identify the effects of PP on MM cell proliferation,apoptosis,migration,and dryness.Then the molecular effects of PP were analyzed by transcriptome sequencing of ARP1 and ARP1-BR cells treated with PP.Furthermore,the molecular effects of sequencing were confirmed through q RT-PCR and Western blot analysis.Meanwhile,the growth inhibitory effect of PP was also confirmed in MM mouse models of ARP1 and ARP1-BR cell lines.The predicted molecular effect of PP was also confirmed in tissue samples of MM mouse models treated with PP after tumorigenesis in ARP1 and ARP1-BR cell lines using q RT-PCR and Western blot analysis.Results:This study identified PP as an effective anti-MM compound with an IC50<1 μM on ARP1 and ARP1-BR cell lines.PP also showed a significant selective growth inhibitory effect in different MM cell lines,and in CD138+ by in related assays,while at the same concentrations,PP neither affected the cell viability of B cells nor affected the cell viability of Peripheral blood mononuclear cells(PBMCs)isolated from healthy donors.Furthermore,PP inhibited cell proliferation,induced apoptosis,decreased the expressions of stemness-related genes,and suppressed the migration ability of ARP1 and ARP1-BR cell lines.These results demonstrated that PP is a strong anti-MM agent which can electively inhibit MM cell growth,induce apoptosis,decrease MM stemness and suppress the migration ability of MM cells irrespective of BTZ sensitivity or BTZ-resistance.Transcriptome sequencing was performed for PP-treated ARP1 and ARP1-BR cell lines to investigate molecular targets of PP in MM.The results of the transcriptome sequencing analysis showed that 386 genes were commonly downregulated in PP-treated ARP1 and ARP1-BR cells by using a differential gene volcano-map of two-fold changes.Furthermore,KEGG analysis results revealed that Cell Adhesion Molecules(CAMs)were the most co-enriched differential genes in both ARP1 and ARP1-BR cell lines.As CAMs contribute in cell to cell adhesion and participate in cell migration and metastasis of cancer,therefore,the results of KEGG analysis are consistent with the experimental results of inhibiting cell migration under PP treatment.In addition,the Dcook Thor server was used to dock PP drugs with CAM such as CDH5,NRCAM,and CD2,indicating that PP compounds have a high binding affinity with CDH5,NRCAM,and CD2.These results indicate that PP downregulates CAM in MM.According to the results of transcriptome sequencing analysis,we further detected the changes of CAM molecules at m RNA and protein levels in the ARP1 cell line and ARP1-BR cell line after PP treatment.The q RT-PCR results showed that PP significantly inhibited the expression levels of CDH5,NRCAM,and CD2 genes.Western blot also confirmed a significant decrease in the expression of CDH5,NRCAM,and CD2 genes at the protein level.These results indicate that PP downregulates CAM in MM.In previous studies,it has been reported that CAM is involved in cell adhesion mediated MM drug resistance.These reports are based on the adhesion of MM cells to FN through CD49 d and CD49 e,mediating MM cell survival and anti-apoptosis.Recently,research has also reported close relationships between CAMs and cancer drug resistance.Therefore,considering the preliminary results of KEGG analysis of ARP1 and ARP1-BR cell lines in the current study,we subsequently examined the effect of PP on CAM expression levels,including CD2,CDH5,and NRCAM.We found that after PP treatment,q RT-PCR detection showed a significant decrease in the expression levels of CD2,CDH5,and NRCAM in ARP1 and ARP1-BR cell lines.In addition,protein immunoblotting also confirmed a significant decrease in the expression of CD2,CDH5,and NRCAM at the protein level in ARP1 and ARP1-BR cells treated with PP.In addition,it was confirmed that PP treatment significantly reduced the expression of CD2,CDH5,and NRCAM at the protein level in the tissues of allogeneic tumor mice inoculated with ARP1 and ARP1-BR cells.Although PP has been shown to be an effective natural compound against MM in this study,which can overcome the BTZ resistance of MM and downregulate CAMs in ARP1 and BTZ resistance ARP1-BR,it must be noted that under PP treatment,the resistance of CAMs to MM-BTZ still requires further investigation of its detailed mechanism.Further research is needed in the future to clarify how PP can overcome BTZ resistance through CAMs.Discussion:In this study,we explored for the first time the significant inhibition of MM cell proliferation by PP in vitro and in vivo,as well as overcoming BTZ resistance.The anti-MM effect of PP is related to the downregulation of CAMs at the molecular level,which is crucial for the previously reported cell-cell adhesion,stemness,and cell adhesion mediated resistance(CAMs)of CAMs to MM.In the past decade,with the development of chemotherapy regimens in the field of myeloma treatment,the survival rate of MM patients has improved.However,the current clinical problems related to MM are drug resistance and relapse,which are mainly due to the interaction of the complex system of bone marrow microenvironment(BM),extracellular matrix,and adhesion molecules,as well as the heterogeneous genetic/epigenetic basis of MM.The mechanism of MM recurrence is still unclear.According to reports,the BTZ resistance of MM can lead to recurrence,which poses a challenge to the effective treatment of MM.Multiple factors are involved in the resistance of anti-MM drugs,and BTZ resistance is the most common type of resistance in MM,which has been identified as the culprit for MM recurrence in most cutting-edge studies.Therefore,there is an urgent need for new treatment strategies to treat MM’s resistance to BTZ.In this study,we identified PP as the most crucial anti-MM drug in the drug library by the screening of library including 2370 natural anti-cancer compounds,and explored their potential mechanisms in MM.We found that PP can inhibit the proliferation of BTZ sensitive and BTZ resistant MM cells,induce apoptosis,inhibit MM stemness,and reduce the ability of cell migration.In addition,we demonstrated the regulation of CAMs after PP treatment in two MM cell lines.The growth inhibition effect of PP in MM cell xenograft tumor mice and the downregulation of CAMs expression in vivo have also been confirmed.The development of anti-MM treatment strategies has made impressive achievements in the field of MM,and the discovery of new anti-MM drugs,especially those from natural sources,is considered a key method for treating MM.Research has shown that PP is a natural anticancer drug that exhibits inhibitory effects on tumor cell proliferation and induction of apoptosis in certain cancers through different mechanisms at the cellular level,both in vivo and in vitro.In clinical practice,PP is used due to its cardiotonic effect.A study reported the clinical observation of 147 patients with chronic congestive heart failure treated with Xiangfu Pi Mixture(including the main component PP),which showed high efficacy and safety.According to reports,high-dose or prolonged use of polypropylene can lead to complications of arrhythmia,thereby limiting its clinical application.Anyway,the combination therapy of PP and TRAIL or PP and Panax notoginseng saponins can reduce the cardiotoxicity of PP.The role of PP in inducing cell apoptosis has been reported,but the effect of PP on MM has not been reported yet.Therefore,in the current study,we found that PP is the most significant anti-MM drug in a library of 2370 natural anticancer compounds by inhibiting the growth of MM BTZ sensitive ARP1 cell lines and BTZ resistant ARP1 cell lines.In addition,we studied the effect of PP on the cell viability of more MM cell lines,B cells,CD138+ cells isolated from MM patients,and PBMCs from healthy donors through CCK-8 assay.As a result,we found that PP significantly inhibited the cell viability of MM,but had no significant effect on B cells or PBMC.These results indicate that PP can selectively inhibit the cell viability of MM.In addition,we further observed the ability of PP to inhibit colony formation in colony formation experiments and found PP-induced apoptosis in relevant MM cell lines through flow cytometry.Next,we investigated the impact of PP on the MM stemness characteristics related to BTZ resistance and conducted Aldfluor assays on ARP1 and ARP1-BR cells to examine whether PP affects ALDH.The results showed that PP reduced the proportion of ALDH positive cells in both types of cell lines.In addition,we also found through q RT-PCR that after PP treatment,the expression of ALDH1A1 and i PSC genes decreased at the m RNA level in ARP1 and ARP1-BR cells,such as NANOG,BMI 1,LIN28,and OCT2.Overall,these results indicate that PP treatment can inhibit the growth and dryness of MM cells to overcome BTZ resistance in MM cell lines.Next,to investigate the molecular mechanism of the anti-MM effect of PP,we performed RNA-seq on ARP1 and ARP1-BR cells after PP treatment at concentrations of 0 μM and 0.5 μM for 24 h to determine the molecular effect of PP in MM.The differential gene volcano map and Venn diagram analysis by transcriptome sequencing showed that 386 genes downregulated in the RNA-seq analysis of ARP1 and ARP1-BR cells after PP treatment.In particular,the co-downregulation of CAMs was predicted through KEGG enrichment analysis in ARP1 and ARP1-BR cells.It has been reported that CAMs are mainly involved in cell adhesion,inducing cell migration and cancer metastasis.It can be seen that KEGG enrichment analysis shows that the co-downregulation of CAMs by PP in the two cell lines is consistent with the results of the transwell experiment,indicating that the migration ability of MM cell lines ARP1 and ARP1-BR is inhibited after PP treatment.In previous studies,various researchers have reported that CAMs are involved in cell-adhesion-mediated drug resistance of MM.Anyhow,it was reported initially based on the adhesion of MM cells to FN through CD49 d and CD49 e which mediates MM cell survival and anti-apoptosis.Recently,studies also reported the emerging role and close relation of CAMs with cancer drug resistance.Therefore,keeping in mind the initial results and the prediction of KEGG analysis of ARP1 and ARP1-BR cell lines in the current study,we then examined the effect of PP on the expression level of CAMs including CD2,CDH5,and NRCAM.We found significantly decreased expression levels of CD2,CDH5,and NRCAM in ARP1 and ARP1-BR cell lines through q RT-PCR after PP treatments.Furthermore,the significantly reduced expression at protein levels of CD2,CDH5,and NRCAM were also verified through Western blot in ARP1 and ARP1-BR cells after PP treatments.In addition,the significantly reduced protein levels of CD2,CDH5,and NRCAM by PP treatment were also verified in the tissue of ARP1 and ARP1-BR cells inoculated xenograft mice.Although PP has been investigated as a strong anti-MM natural compound to overcome BTZ-resistance of MM and downregulate CAMs in both wild-type ARP1 and BTZ-resistant type ARP1-BR in this study,it must be noted that MM BTZ resistance executed by CAMs under PP treatment still needs further investigation for its detailed mechanism.Conclusions:1.PP can significantly inhibit the growth of MM BTZ sensitive ARP1 and BTZ resistant ARP1-BR cell lines,induce cell apoptosis,reduce MM stemness,and inhibit the migration ability of MM cells.2.In the RNA-seq analysis of ARP1 and ARP1-BR cells treated with PP,386 genes were downregulated,and KEGG enrichment analysis revealed a common downregulation of the CAMs pathway in both cell lines.3.After PP treatment,the expression levels of CD2,CDH5,and NRCAM in ARP1 and ARP1-BR cell lines were significantly reduced at m RNA and protein levels.4.PP can significantly reduce the growth of BTZ sensitive and BTZ resistant MM cell line xenograft tumors in vivo,and reduce the expression of CAMs.There are 36 figures,13 tables,195 references... |
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