Platelet factor 4 induces cell apoptosis by inhibition of STAT3 via up-regulation of SOCS3 expression in multiple myeloma

Abstract

© F e r r a t a S t o r t i F o u n d a t i o nH929 cells using increasing doses over a period of 96 h. Results of WST-1 assays ( Figure 1A) and trypan blue exclusion ( Figure 1B) showed that PF4 markedly inhibited the growth of these cell lines in time-and dose-dependent manners. A significant decrease in cell number was observed for OPM2, NCI-H929 and U266 after 24, 72 and 96 h of incubation with PF4. The inhibitory concentration at 50% (IC 50 ) for these three cell lines were approximately 2, 4 and 4 μM, respectively. Next, we investigated whether the observed inhibitory effects of PF4 on cell growth were due to cell cycle arrest, apoptosis, or both. The effect of PF4 on the cellular DNA content was determined using flow cytometric analysis in U266 and NCI-H929 cell lines. While changes in G0/G1, S, and G2/M phases were not distinctively different, we observed a population of cells in the sub-G1 phase indicative of increased apoptosis after PF4 treatment (data not shown). To further confirm that apoptosis was induced by PF4, we treated U266, OPM2 and NCI-H929 cells with increasing doses of PF4 and determined the percentage of apoptotic cells by flow cytometric analysis of annexin V and 7-amino-actinomycin D (7-AAD). Results showed that PF4 led to an increase in apoptotic cells (annexin V+ and/or 7AAD+) in all three of these MM cell lines ( Figure 1C). Pretreatment of cells with cycloheximide, a protein synthesis inhibitor, inhibited PF4-induced apoptosis of MM cells (P=0.001) (Online Supplementary Figure S1), indicating that the induction of apoptosis by PF4 is likely dependent on upregulation of pro-apoptotic proteins. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays in U266 and OPM2 cells further confirmed that PF4 induced apoptosis in MM cells, as evidenced by the observed increase in staining of nuclear DNA fragments (Online Supplementary Figure S2). In addition, treatment of OPM2 and U266 cells with PF4 triggered a marked increase in proteolytic cleavage of PARP, a signature event during apoptosis ( Figure 1D). Similarly, PF4 increased caspase-3 activity, an upstream activator of PARP, by 2.6-fold in U266 cells and by 3.2-fold in OPM2 cells ( Figure 1E).We also examined the effect of PF4 on purified cells from patients with MM. CD138 + plasma cells were isolated from 26 patients diagnosed with MM as described in Online Supplementary Table S2. Cells were treated with PF4 for 48 h and the levels of apoptosis were measured by annexin V-7AAD staining. To compare the cytotoxicity of PF4 in MM and normal cells, normal plasma cells from the bone marrow of healthy donors and normal mononuclear cells from the peripheral blood of healthy donors were obtained. We found minimal changes and a significant increase of mean percentages of apoptotic cells in PF4-treated normal (bone marrow plasma cells and peripheral blood mononuclear cells) and patients' MM cells, respectively (0.01±2.78%, 0.06±1.36%, and 15.16±2.52%) ( Figure 1F). Taken together, our findings suggest that PF4 inhib...

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