Antitumor Activity of Human Urine Extract Cda-2 against Multiple Myeloma Cell Lines Via PI3K/Akt Signaling Pathway in a Caspase-3-Dependent Manner

Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment although a number of new agents have recently been shown to be effective. There were still some patients who did not respond to GCs or develop resistance. The aim of this study was to investigate the antitumor activity of human urine extract against MM and Dexamethasone-resistant and sensitive MM cell lines. These cell lines were used to examine the effects of a human urine preparation CDA-2 on the induction of growth arrest and apoptosis. Apoptotic proteins including caspase family, Bcl-2 family were studied. Phosphoinositide 3-kinase (PI3K)/Akt survival signaling pathway was also examined. The caspase-3 inhibitor Z-DEVD-FMK was used to examine the involvement of caspase-3 and PARP. In current study we found that CDA-2 could induce growth arrest and apoptosis of MM cells in vitro. Using MTT assay, we determined the effects of CDA-2 on RPMI8226, U266, MM.1R and MM.1S cells as well as peripheral blood monouclear cells(PBMCs) from three normal volunteers. Fifty percents of growth inhibition (IC50) was measured in these cells treated with CDA-2 at concentrations ranging from 2 to 16 mg/ml for 24 to 72h. CDA-2 exerted substantial growth inhibition in RPMI8226, U266, MM.1R and MM.1S cell lines, while it did not induce cytotoxicity in normal PBMCs. The 24h and 48h IC50 mean values of CDA-2 showed 4.61 and 3.47 mg/ml in RPMI8226 cells(P=0.043<0.05),6.09 and 5.71 mg/ml in U266 cells(P>0.05),5.07and 4.11 mg/ml in MM.1R cells(P=0.002<0.01), 3.26 and 2.13mg/ml in MM.1S cells(P=0.007<0.01). The results indicated that cell viability in the presence of CDA-2 decreased almostly in a dosedependent manner. The inhibitory rates of cell growth were positively correlated with CDA-2 concentrations except U266 cell lines. While in contrasts, CDA-2 did not induce cytotoxicity in PBMCs from three normal volunteers (IC50=103.92 mg/ml, p=0.006<0.01). The mechanism of CDA-2 in MM and Dexmethasone-resistant and sensitive MM cell lines was related to the inhibition of PI3Kp110α expression in protein level, which inactivated the phosphorylation of Akt involving dephosphorylation of Bad protein, downregulation of Bcl-xl protein, and triggered the activation of caspase cascades. We contrasted the grey scale of the PI3Kp110αprotein level after treated with the different concentrations of CDA-2. The grey scale values of 8226 cells and MM.1R cells after treated with 4mg/ml CDA-2 were 3.71 A2.11 respectively, while the grey scale values of the untreated control cells of 8226 and MM.1R were 6.92 A7.46 respectively (P=0.001<0.01). This phenomenon could be inhibited by the caspase-3 inhibitor Z-DEVD-FMK. In addition, CDA-2 could also downregulate the level of DNA maintenance methylation enzyme, DNMT-1 in MM and Dexamethasone-sensitive and resistant MM cell lines. Therefore, our results demonstrate the presence of active components in the human urine extract that can induce growth arrest and apoptosis of Multiple Myeloma and Dexamethasone-resistant and sensitive Multiple Myeloma cell lines and may involve the PI3K/Akt signaling pathway in a caspase-3 dependent manner, associated with downregulation the level of the DNA maintenance methylation enzyme. This may provide new insights for the treatment of MM, especially the drug-resistant Multiple Myeloma.