Inhibition of the mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase by doxorubicin and brequinar sensitizes cancer cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent in selectively killing tumor cells. However, TRAIL monotherapy has not been successful as many cancer cells are resistant to TRAIL. Chemotherapeutic agents, such as doxorubicin have been shown to act synergistically with TRAIL, but the exact mechanisms of actions are poorly understood. In this study, we performed high-throughput small interfering RNA screening and genome-wide gene expression profiling on doxorubicin-treated U1690 cells to explore novel mechanisms underlying doxorubicin-TRAIL synergy. The screening and expression profiling results were integrated and dihydroorotate dehydrogenase (DHODH) was identified as a potential candidate. DHODH is the rate-limiting enzyme in the pyrimidine synthesis pathway, and its expression was downregulated by doxorubicin. We demonstrated that silencing of DHODH or inhibition of DHODH activity by brequinar dramatically increased the sensitivity of U1690 cells to TRAIL-induced apoptosis both in 2D and 3D cultures, and was accompanied by downregulation of c-FLIPL as well as by mitochondrial depolarization. In addition, uridine, an end product of the pyrimidine synthesis pathway was able to rescue the sensitization effects initiated by both brequinar and doxorubicin. Furthermore, several other cancer cell lines, LNCaP, MCF-7 and HT-29 were also shown to be sensitized to TRAIL by brequinar. Taken together, our findings have identified a novel protein target and its inhibitor, brequinar, as a potential agent in TRAIL-based combinatorial cancer therapy and highlighted for the first time the importance of mitochondrial DHODH enzyme and pyrimidine pathway in mediating TRAIL sensitization in cancer cells.

Bcl-2 overexpression is associated with resistance to paclitaxel, but not gemcitabine, in multiple myeloma cells.

Multiple myeloma (MM) is an incurable disease despite an initial response-rate of >60% with conventional or high-dose chemotherapy using glucocorticosteroids (i.e. dexamethasone), or alkylating agents (i.e. melphalan). Although these agents are capable of inducing complete remission (CR) in >50% of MM patients, resistance develops rapidly, in >90% of patients, within 2 years of treatment. Therefore, there is a need for new drugs for the treatment of relapsing and refractory MM patients. Gemcitabine (GEM) is a pyrimidine analog that blocks DNA synthesis, whereas, paclitaxel (TAX) is a mitotic spindle poison that promotes microtubular aggregation. Since it appears that these two drugs have different cellular targets, we examined the effect of each drug individually for several parameters and for possible synergy. We studied the cytotoxic effect of TAX and GEM on MM cells expressing varying levels of the antiapoptotic protein bcl-2, which is overexpressed in the majority of myeloma cell from MM patients. We found that both drugs are cytotoxic by inducing apoptosis, however, the extent of apoptosis with TAX, but not with GEM was dependent on the levels of bcl-2 expression. We further investigated the effect of TAX and GEM on the cell cycle distribution and on the levels of bcl-2. The results indicate that the two drugs have different modes of action with respect to each parameter tested. TAX induced arrest of the cells in the G2/M phase of the cell cycle, regardless of bcl-2 levels, however, apoptosis was induced in mitotic cells expressing relatively low levels of bcl-2. In contrast, GEM caused apoptosis of cells in the S-phase, regardless of level of bcl-2 expression. A major difference between TAX and GEM was in their effects on the levels of bcl-2. Whereas, TAX induced an early downregulation of bcl-2 (only in the cells with relatively low levels of bcl-2), treatment with GEM did not affect bcl-2 levels. The effects of TAX on both the cell cycle and bcl-2 were detected very early (4-8 h) and preceded the onset of apoptosis. GEM and TAX act synergistically, at low doses (IC50 of 0.5 microM for GEM and 0.025 microM for TAX), to effectively kill bcl-2 overexpressing cells that are resistant to higher doses (0.25 microM) of TAX alone. Therefore, we have initiated a phase II clinical trial of TAX and GEM for MM patients refractory to current therapy.

Bcl-2 overexpression is associated with resistance to dexamethasone, but not melphalan, in multiple myeloma cells.

Multiple myeloma (MM) is an incurable disease despite an initial response-rate of >60% with conventional or high-dose chemotherapy. Glucocorticosteroids such as dexamethasone (DEX) and alkylating agents such as melphalan (MEL) are capable of inducing complete responses (CR) in >50% of MM patients, however, resistance to these drugs develop rapidly, in >90% of patients, within 2 years of treatment. The exact mechanism of resistance to these drugs is not known. We investigated the mechanism of resistance to DEX and MEL. In particular, we investigated the role of bcl-2 in development of resistance to these two drugs. We tested the role of bcl-2 by transfecting 2 low bcl-2-expressing myeloma cell lines, ARP-1 and 8226, with a bcl-2 expression vector and compared the effects of DEX and MEL on apoptosis, cell cycle distribution and the levels of proapoptotic (bax) and antiapoptotic (bcl-2, bclx) proteins. The results indicate that the two drugs act by a different mechanism with respect to all the parameters tested. While DEX-induced apoptosis was dependent on the level of bcl-2 expression, MEL-induced apoptosis was independent of bcl-2 levels. Treatment with DEX of the low bcl-2-expressing cells (DEX-sensitive) resulted in a rapid apoptosis from all phases of the cell cycle. In contrast, treatment with MEL blocked the cells in late-S/G2 phase of the cell cycle and caused substantial apoptosis, regardless of bcl-2 expression. Major differences between DEX and MEL were also observed with respect to their effects on the levels of bcl-2 and p53. Whereas DEX induced an early (day 1) downregulation of bcl-2 (only in the cells with low bcl-2), treatment with MEL did not affect bcl-2 levels. The levels of bclx and bax remained unchanged following treatment with either MEL or DEX. These results, taken together, suggest that the two drugs target different cellular components and induce apoptosis by different pathways, and that resistance to DEX is associated with low levels of bcl-2, whereas resistance to MEL is independent of bcl-2, and therefore, in vivo resistance to MEL, observed in MM patients, might involve other mechanisms rather than bcl-2.

Defective LN cell proliferative response to Sm antigen in mice bearing the lpr gene.

Several normal and lpr/lpr congenic mouse strains were immunized with an autoantigen (Sm) and a conventional antigen (PPD) and tested for their respective in vitro proliferative responses. All normal strains of mice demonstrated a brisk proliferative response to Sm and PPD on days 3 and 5 of culture. Two month old mice bearing the lpr gene demonstrated a proliferative response to the two antigens only on day 3, while by day 5 of culture, the proliferative response was markedly diminished. Mixing experiments between +/+ and lpr/lpr Sm primed lymph node cells ruled out the influence of a suppressor cell within the lpr/lpr population at day 5. The inability to sustain an in vitro proliferative response to antigen in young lpr/lpr mice was not due to an overall T cell defect nor to in vitro cell death since Con A stimulation and viability were the same in cultures containing lpr/lpr and +/+ LN, cells, respectively. The primary defect may be partly attributed to a defect in interleukin-2 (IL-2) production and response. By day 5 of culture, lpr/lpr supernatants contained much lower levels of IL-2 activity compared to supernatants from +/+ cultures. The addition of exogenous IL-2 was only moderately successful in improving the response falling far below that seen when IL-2 was added to +/+ cultures. This study demonstrates a decrease in autoantigen-induced T cell proliferation in mice that spontaneously produce autoantibodies to that antigen.