The Study On The Effects And Mechanisms Of Triptolide And Uroacitides To Dex-resistant And Sensitive Human Multiple Myeloma Cell Lines

Section 1 Triptolide(TPL) induces MM.1R and MM.1S cells into apoptosis via cell survival and glucocorticoid receptor signaling pathwayObjective:The aim of this section was to investigate the antitumor activity of TPL against MM.1R and MM.1S cells and to determine if TPL-induced apoptosis of drug-resistant MM cells was associated with the PI3K/Akt and NF-κB survival signaling pathways, and if the activity of TPL-increasing the sensitivity of dexamethasone in MM.1R and MM.1S cells was associated with the glucocortioid receptor.Methods:We used MTT assay to investigate the effects of TPL on the MM.1R and MM.1S cells.We examine the effects of TPL on the MM.1R and MM.1S cells of induction of growth arrest and apoptosis.Apoptosis was measured by DNA Ladder and flow cytometry using the Annexin V-FITC apoptosis detection kit,according to the manufacturer's instructions.Alterations in the mitochondrial membrane potential were analyzed by flow cytometry using the specific dye JC-1.The cell-cycle analysis was done according to the PI staining by flow cytometry.Apoptotic proteins including Caspase-9,-8,-3,members of Bcl-2 family and IAPs family were studied by western-blot.Phosphoinositide 3-kinase(PI3K)/Akt and NF-κB survival signaling pathways were also examined using western-blot analysis.The caspase-3 inhibitor Z-DEVD-FMK was used to determine the involvement of caspase-3 and PARP.PI3K inhibitor LY294002 and NF-κB inhibitor PDTC were used to examine the involvement of PI3K/Akt and NF-κB signaling pathways.Immunofluorescent staining was used for observing the distribution of NF-κB in cells.The IGF-1R and PIK3CA genes,which expressed the IGF-1R and PI3Kp110αprotein,were examined by using RT-PCR to identify the target of TPL-induced apoptosis.We evaluate whether TPL-inhibited cell growth can be prevented by exogenous IL-6.We also evaluate the expression of miR181a,miR142-5p and the regulation of glucocorticoid receptor induced by TPL on MM.1R and MM.1S cells.Results:①TPL could inhibit the growth of MM.1R and MM.1S cell lines and the tumor cells isolated from three relapsed multiple myeloma patients.The cell growth was inhibited in a time- and dose-dependent manner,corresponding to the reduced cell viability.②TPL induced apoptosis of MM.1R and MM.1S cell lines in a dose-dependent manner.It showed that MM.1R and MM.1S cells after treated with 5ng/ml,10ng/ml TPL for 24h,a typical DNA Ladder was observed.③Treatment with TPL induced marked activation of caspase-9,-3 in a dose-dependent manner,however, caspase-8 was activated only at TPL concentrations greater than 10ng/mL.Pretreatment with Z-DEVD-FMK significantly decreased the apoptotic cells following TPL treatment. TPL also induced a loss in mitochondria transmembrane potential as evidence of mitochondria-mediated apoptosis.④Bcl-x1,Mcl-1 and p-Bad proteins were decreased, however,Bax and Smac proteins were increased in a dose-dependent manner after different concentrations of TPL treatment for 24h.⑤The xIAP,cIAP1 and Survivin proteins were down- regulated in a dose-dependent manner after TPL treatment.⑥TPL inhibited IκBαphosphorylation and prevented NF-κB(p65) nuclear translocation.⑦TPL significantly reduced expression of PI3K/Akt signaling pathway in a dose-dependent manner;the results were further confirmed by PIK3CA gene at mRNA level in a time- and dose-dependent manner,and TPL combined with LY294002 significantly inhibited the expression of p-IκBα;TPL reduced the expression of IGF-1R expression in a time-dependent and dose-dependent manner.⑧The effect of TPL-inhibited cell growth could not be affected by exogenous IL-6,and TPL could enhance the growth inhibitory effect of Dex and PS-341.⑨TPL reduced the expression of miR-181a,miR142-5p.And low expression of miR-181a and miR-142-5p resulted in an increased GR protein expression.TPL also enhanced the expression of glucocorticoid-induced leucine zipper(GILZ),a key component of GC-signaling.Section 2:The mechanisms and the effects of Uroacitides(CDA-Ⅱ) on the multiple myelomaObjective:The aim of this section was to investigate the antitumor activity of CDA-Ⅱagainst MM cells and to determine if CDA-Ⅱ-induced apoptosis of MM cells was associated with NF-κB nuclear translocation.Methods:We used MTT assay to investigate the effects of CDA-Ⅱon the MM cells.We examine the effects of CDA-Ⅱon the MM cells of induction of apoptosis.Apoptosis was measured by DNA Ladder and flow cytometry using the Annexin V-FITC apoptosis detection kit,according to the manufacturer's instructions.Alterations in the mitochondrial membrane potential were analyzed by flow cytometry using the specific dye JC-1.Apoptotic proteins including Caspase-9,-3,Bcl-2 family members and IAPs family members were studied by western-blot.The caspase-3 inhibitor Z-DEVD-FMK was used to determine the involvement of caspase-3 and PARP.We exanmine the inhibition effect of CDA-Ⅱon the translocation of NF-κB using immunofluorescence analysis.Results:①CDA-Ⅱcould inhibit the growth of MM cells in a time- and dose-dependent manner,corresponding to the reduced cell viability.In contrasts,CDA-Ⅱdid not induce cytotoxicity in BMMCs from normal volunteers.②CDA-Ⅱinduced apoptosis of MM cells in a dose-dependent manner.It showed that RPMI8226 cells after treated with CDA-Ⅱfor 24h,a typical DNA Ladder was observed.③Treatment with CDA-Ⅱinduced marked activation of caspase-9,-3 in a dose-dependent manner. Pretreatment with Z-DEVD-FMK significantly decreased the apoptotic cells following CDA-Ⅱtreatment.CDA-Ⅱalso induced a loss in mitochondria transmembrane potentia as evidence of mitochondria-mediated apoptosis.④Bcl-2,Mcl-1 proteins were decreased,however,Bax protein was increased in a dose-dependent manner after CDA-Ⅱtreatment for 24h.The xIAP and Survivin proteins were down-regulated in a dose-dependent manner after CDA-Ⅱtreatment.⑤CDA-Ⅱprevented NF-κB nuclear translocation.Conclusions:CDA-Ⅱcould induce growth arrest and apoptosis of MM cells.The mechanism was related to the prevention NF-κB nuclear translocation,downregulation of IAPs family protein as well as involvement of Bcl-2 family members and triggered the mitochondrial pathway mediated caspase-3-dependent apoptosis. Summary:TPL can induce growth arrest and apoptosis in MM.1R and MM.1S cell lines.TPL is an effective chinese medical herb with multiple targets,including involvement of PI3K/Akt/NF-κB survival signaling pathway directly,downregulation of the miRNA181a and miRNA142-5p,upregulation of the phosphorylated glucocorticoid receptor protein.TPL can enhance the expression of glucocorticoid-induced leucine zipper(GILZ),a key component of GC-signaling.These probably are the mechanisms of dexamethasone-induced apoptosis in dexamethasone-resistant multiple myeloma.CDA-Ⅱcan induce growth arrest and apoptosis in MM cells,but not in normal BMMCs.CDA-Ⅱcan prevent NF-κB nuclear translocation and downregulate the expression of Bcl-2,Mcl-1 and IAPs family proteins,upregulation the expression of Bax. Different mechanisms of action acted concurrently to contribute to the TPL and CDA-Ⅱ-induced MM cells,especially Dex-resistant MM cells apoptosis.This research work may provide new insights for the treatment of drug-resistant MM.