PLoS 1
PLoS 1. addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells as well as tumor growth of DLBCL cells xenografted in mice at concentrations that may be attainable in human being sera having a restorative dose of the drug, identifying doxycycline like a potential low-cost and safe restorative agent for DLBCL and possibly additional NHLs. Additionally, our work uncovers CSN5 like a novel target of doxycycline and as a potential target in CB 300919 DLBCL therapy. RESULTS Connectivity map analysis uncovers doxycycline as an inhibitor of NF-B signaling To identify potential inhibitors of NF-B signaling that may be exploited as restorative providers for DLBCL treatment, we queried the Connectivity Map with a set of known NF-B focuses on. Notably, among the top hit compounds that potentially inhibit NF-B signaling from this analysis are members of the tetracycline family of antibiotics, including doxycycline (Table ?(Table11). Table 1 Connectivity map database analysis identifies tetracycline family antibiotics as potential NF-B signaling inhibitors [11, 13C15], suggesting that doxycycline affects other pathways in addition to NF-B signaling. Open in VCL a separate windowpane Number 2 Doxycycline inhibits the proliferation and survival of DLBCL cellsA. The DLBCL cell lines were treated with the indicated concentrations of doxycycline for 96 hrs. The viable cells were counted from the trypan blue exclusion assay. Demonstrated are the mean and SD from at least three self-employed experiments. The mean from your samples without exposure to doxycycline was arranged at 100. B. Main tumor cells from DLBCL individuals were plated at 5 105 cells/ml for patient samples ACC or at 3 105 cell/ml for patient samples DCG and treated with the indicated concentrations of doxycycline for 96 hrs. The live cells were measured as explained in (A). The cells from patients ACC were subjected to doxycycline treatment without prior passage for 3C5 doublings before being treated with doxycycline. Samples DCF and G were classified as GCB and non-GCB subtypes, respectively, by Hans staining. The subtypes for samples A-C were CB 300919 unknown. Mean and SD from triplicate samples are depicted. C. The estimated IC50 values of doxycycline against DLBCL cell lines and main cells. The IC50 values were calculated from your dose response at 96 hours in experiments explained in 2A and 2B. D. The Burkitt lymphoma cell lines and E. the mantle cell lymphoma cell lines were treated as explained in (A). Results from triplicate samples are depicted. F. Doxycycline inhibits cell cycle progression. OCI-Ly7 (top panels) and OCI-Ly10 (bottom panels) cells were treated with the indicated concentrations of doxycycline for 48 hrs. Ethynyl-deoxyuridine (EdU) was added into the culture medium for 2 hr before the cells were harvested for cell-cycle distribution analysis. G. Doxycycline induces apoptosis of DLBCL cells. OCI-Ly7 (top panels) and OCI-Ly10 cells (bottom panels) were treated with the indicated concentrations of doxycycline for 66 hrs. The apoptotic (annexin V-positive) cells were measured by circulation cytometry. H. DLBCL cells were treated with doxycycline for the indicated time. The cleavage of PARP1 was analyzed by western blotting. As main DLBCL cells may have different requirements for growth than established cell lines, we examined the effect of doxycycline around the survival of main DLBCL samples. The viability of main DLBCL cells was also inhibited by doxycycline, indicating that the cytotoxic effect of CB 300919 doxycycline is not limited to the established cell lines (Determine ?(Physique2B2B and ?and2C2C). We also examined the effects of doxycycline around the growth of other types of B-lymphoma cells. We found that the growth of Burkitt CB 300919 lymphoma (Daudi and Ramos) and mantle cell lymphoma (Granta, JEKO-1, Mino and Rec-1) cells were also inhibited by doxycycline at comparable concentrations observed for DLBCL cells (Physique ?(Physique2D2D and ?and2E),2E), suggesting that doxycycline inhibits the growth of a broad range of aggressive B-lymphoma cells in culture. The average peak concentration of doxycycline in human serum is usually 3C6.