Exploring The Mechanism Of Cell Apoptosis Induced By Prodigiosin Based On Omics Technology In Multiple Myeloma

Objective:This paper intends to investigate the apoptosis inducing effect of prodigiosin（PG）in multiple myeloma（MM）cells and its related mechanisms,provide theoretical and experimental basis for the treatment and prevention of MM.Methods:The cytotoxicity assay was used to detect the killing effect of PG on MM cell line and normal B cell;soft agar clone formation assay was used to explore the inhibitory effect of PG on MM cell proliferation;flow cytometry was used to investigate the effect of PG induce MM cells apoptosis;WB was used to detect the expression level of MM cell apoptosis related molecules after PG treatment;In vivo experiment to explore the effect of PG on the proliferation of MM cells in bone marrow of mice;transcriptomics techniques were used to explore the effects of low,medium and high concentrations of PG on the m RNA transcriptional expression profile of MM cells,and the signaling pathways involved in PG induced apoptosis in MM cells were explored by bioinformatics analysis methods such as GO enrichment and the KEGG pathway;the role of amino acid metabolism in PG-induced apoptosis of MM cells was explored using targeted amino acid metabolomics techniques combined with amino acid treatment;The mechanism of PG-induced apoptosis of MM cells further investigated by WB and RT-q PCR.Results:（1）The IC₅₀ of PG on normal B cells GM12878 was 3,010.0nmol/L and the IC₅₀ values of PG on MM1.s,H929,ARP1,OCI-MY5,XG1,U266 and 8226 were 485.0,426.9,409.7,408.7,779.4,595.6,209.7nmol/L,respectively.（2）The clone numbers of H929 cell in the control and 100 nmol/L and 200 nmol/L PG-treated groups were 120.0±10.0,31.0±3.6,16.3±2.5,respectively.（3）PG induced apoptosis of H929,ARP1,XG1,OCI-MY5 and the expression of apoptotic-associated activated Caspase-3 and PARP proteins.（4）The fluorescence intensity of mice in the PG-dosed group was significantly lower than the fluorescence intensity value of control group in both the treatment and prevention groups.（5）RNA-seq assay of 100 nmol/L and 200 nmol/L PG-treated ARP1 cells obtained 591 and 1165 differential genes,respectively.Correlation analysis and RT-q PCR assay confirmed that the sequencing results were plausible.（6）Transcriptomic results showed that differential genes were enriched to the amino acid metabolic pathway after PG treatment;targeted amino acid metabolomic results showed that the content of various free amino acid in intracellular and culture supernatant of ARP1 cells was increased after PG treatment;meanwhile,the lack of glycine,serine and valine in the culture medium could lead to the apoptosis of ARP1 and OCI-MY5 cells.（7）After PG treatment,the TP53 pathway was altered but the m RNA and protein expression levels of TP53 were unchanged in H929 cells.（8）The IC₅₀ of PG on H929-TP53^KO-C1 and H929-TP53^KO-C2 cells were 166.4 nmol/L and 163.3 nmol/L,respectively.The clonal numbers of H929-TP53^KO-C1and H929-TP53^KO-C2 cells in the control group were 144.33±9.61 and155.00±14.11,respectively;the clonal numbers of cells after 100 nmol/L PG treatment were 25.33±5.69 and 14.00±4.58,respectively;the clonal numbers of cells after 200 nmol/L PG treatment were 10.33±2.52 and 5.67±1.53,respectively.（9）PG induced apoptosis in H929-TP53^KO-C1 and H929-TP53^KO-C2 cells and the expression of apoptosis-associated activated Caspase-3 and PARP proteins.（10）Expression of TP53 down-stream molecule p21 was elevated in TP53 knockdown MM cell lines and TP53 wild-type MM cell lines.Conclusion:（1）Within the IC₅₀ range of PG on MM cells,PG has almost no killing effect on B cells.（2）PG inhibits MM cell proliferation in vitro and vivo and induces MM cell apotosis.（3）Pre-tumorigenic supplementation with PG inhibited MM cell homing in the bone marrow of mice.（4）Transcriptomics and targeted amino acid metabolomics confirmed that PG could induce MM cell apoptosis by regulating amino acid metabolism.（5）PG upregulated p21 expression in a TP53 non-dependent manner and regulated the TP53 signaling pathway to exert tumor suppressive effects.