Targeting the Cell Cycle, RRM2 and NF-κB for the Treatment of Breast Cancers.

A hallmark of cancer is the dysregulation of the cell cycle. The CDK4/6 inhibitor palbociclib is approved for treating advanced estrogen-receptor-positive breast cancer, but its success is limited by the development of acquired resistance owing to long-term therapy despite promising clinical outcomes. This situation necessitates the development of potential combination strategies. Here, we report that didox, an inhibitor of ribonucleotide reductase in combination with palbociclib, can overcome palbociclib resistance in ER-positive and ER-negative breast cancers. This study shows didox downregulates an element of the cell cycle checkpoint, cyclin D1, accompanied by a reduction in NF-κB activity in vitro and tumor growth inhibition of palbociclib-resistant ER positive breast cancer tumor growth in vivo. Furthermore, didox induces cell cycle arrest at G1 as well as reduces ROS generated by on-target effects of palbociclib on the cell cycle. Our current study also reports that the CCND1 and RRM2 upregulation associated with palbociclib-resistant breast cancers decreases upon ribonucleotide reductase inhibition. Our data present a novel and promising biomarker-driven combination therapeutic approach for the treatment of ER-positive and ER-negative breast cancers that involves the inhibition of the CDK4/6-cyclinD1/pRb cell cycle axis that merits further clinical investigation in human models.

Molecular Targeting of RRM2, NF-κB, and Mutant TP53 for the Treatment of Triple-Negative Breast Cancer.

Doxorubicin and other anthracycline derivatives are frequently used as part of the adjuvant chemotherapy regimen for triple-negative breast cancer (TNBC). Although effective, doxorubicin is known for its off-target and toxic side effect profile, particularly with respect to the myocardium, often resulting in left ventricular (LV) dysfunction and congestive heart failure when used at cumulative doses exceeding 400 mg/m2 Previously, we have observed that the ribonucleotide reductase subunit M2 (RRM2) is significantly overexpressed in estrogen receptor (ER)-negative cells as compared with ER-positive breast cancer cells. Here, we inhibited RRM2 in ER-negative breast cancer cells as a target for therapy in this difficult-to-treat population. We observed that through the use of didox, a ribonucleotide reductase inhibitor, the reduction in RRM2 was accompanied by reduced NF-κB activity in vitro When didox was used in combination with doxorubicin, we observed significant downregulation of NF-κB proteins accompanied by reduced TNBC cell proliferation. As well, we observed that protein levels of mutant p53 were significantly reduced by didox or combination therapy in vitro Xenograft studies showed that combination therapy was found to be synergistic in vivo, resulting in a significantly reduced tumor volume as compared with doxorubicin monotherapy. In addition, the use of didox was also found to ameliorate the toxic myocardial effects of doxorubicin in vivo as measured by heart mass, LV diameter, and serum troponin T levels. The data present a novel and promising approach for the treatment of TNBC that merits further clinical evaluation in humans.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IgE-mediated mast cell activation through attenuation of NFκB and AP-1 transcription.

Mast cell activation via the high-affinity IgE receptor (FcεRI) elicits production of inflammatory mediators central to allergic disease. As a synthetic antioxidant and a potent ribonucleotide reductase (RNR) inhibitor, Didox (3,4-dihyroxybenzohydroxamic acid) has been tested in clinical trials for cancer and is an attractive therapeutic for inflammatory disease. We found that Didox treatment of mouse bone marrow-derived mast cells (BMMC) reduced IgE-stimulated degranulation and cytokine production, including IL-6, IL-13, TNF and MIP-1a (CCL3). These effects were consistent using BMMC of different genetic backgrounds and peritoneal mast cells. While the RNR inhibitor hydroxyurea had little or no effect on IgE-mediated function, high concentrations of the antioxidant N-acetylcysteine mimicked Didox-mediated suppression. Furthermore, Didox increased expression of the antioxidant genes superoxide dismutase and catalase, and suppressed DCFH-DA fluorescence, indicating reduced reactive oxygen species production. Didox effects were not due to changes in FcεRI expression or cell viability, suggesting it inhibits signaling required for inflammatory cytokine production. In support of this, we found that Didox reduced FcεRI-mediated AP-1 and NFκB transcriptional activity. Finally, Didox suppressed mast cell-dependent, IgE-mediated passive systemic anaphylaxis in vivo. These data demonstrate the potential use for Didox asa means of antagonizing mast cell responses in allergic disease.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IL-33-induced cytokine production in primary mouse mast cells.

While IgE is considered the primary mediator of mast cell activation, IL-33 contributes substantially in asthma, allergic rhinitis, and atopic dermatitis. To develop effective treatments for allergic disease, it is important to understand the role of therapeutic agents on IL-33 activation. We examined the effect of Didox (3,4-dihydroxybenzohydroxamic acid), an antioxidant and ribonucleotide reductase (RNR) inhibitor, on IL-33-mediated mast cell activation. Didox suppressed IL-6, IL-13, TNF, and MIP-1α (CCL3) production in bone marrow derived mast cells following IL-33 activation. This suppression was observed in different genetic backgrounds and extended to peritoneal mast cells. The antioxidant N-acetylcysteine mimicked the suppression of Didox, albeit at a much higher dose, while the RNR inhibitor hydroxyurea had no effect. Didox substantially suppressed IL-33-mediated NFκB and AP-1 transcriptional activities. These results suggest that Didox attenuates IL-33-induced mast cell activation and should be further studied as a potential therapeutic agent for inflammatory diseases involving IL-33.

Targeting Ribonucleotide Reductase M2 and NF-κB Activation with Didox to Circumvent Tamoxifen Resistance in Breast Cancer.

Tamoxifen is widely used as an adjuvant therapy for patients with estrogen receptor (ERα)-positive tumors. However, the clinical benefit is often limited because of the emergence of drug resistance. In this study, overexpression of ribonucleotide reductase M2 (RRM2) in MCF-7 breast cancer cells resulted in a reduction in the effectiveness of tamoxifen, through downregulation of ERα66 and upregulation of the 36-kDa variant of ER (ERα36). We identified that NF-κB, HIF1α, and MAPK/JNK are the major pathways that are affected by RRM2 overexpression and result in increased NF-κB activity and increased protein levels of EGFR, HER2, IKKs, Bcl-2, RelB, and p50. RRM2-overexpressing cells also exhibited higher migratory and invasive properties. Through time-lapse microscopy and protein profiling studies of tamoxifen-treated MCF-7 and T-47D cells, we have identified that RRM2, along with other key proteins, is altered during the emergence of acquired tamoxifen resistance. Inhibition of RRM2 using siRRM2 or the ribonucleotide reductase (RR) inhibitor didox not only eradicated and effectively prevented the emergence of tamoxifen-resistant populations but also led to the reversal of many of the proteins altered during the process of acquired tamoxifen resistance. Because didox also appears to be a potent inhibitor of NF-κB activation, combining didox with tamoxifen treatment cooperatively reverses ER-α alterations and inhibits NF-κB activation. Finally, inhibition of RRM2 by didox reversed tamoxifen-resistant in vivo tumor growth and decreased in vitro migratory and invasive properties, revealing a beneficial effect of combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance.

The efficacy of the ribonucleotide reductase inhibitor Didox in preclinical models of AML.

Acute Myeloid Leukemia (AML) is an aggressive malignancy which leads to marrow failure, and ultimately death. There is a desperate need for new therapeutics for these patients. Ribonucleotide reductase (RR) is the rate limiting enzyme in DNA synthesis. Didox (3,4-Dihydroxybenzohydroxamic acid) is a novel RR inhibitor noted to be more potent than hydroxyurea. In this report we detail the activity and toxicity of Didox in preclinical models of AML. RR was present in all AML cell lines and primary patient samples tested. Didox was active against all human and murine AML lines tested with IC50 values in the low micromolar range (mean IC50 37 µM [range 25.89-52.70 µM]). It was active against primary patient samples at concentrations that did not affect normal hematopoietic stem cells (HSCs). Didox exposure resulted in DNA damage and p53 induction culminating in apoptosis. In syngeneic, therapy-resistant AML models, single agent Didox treatment resulted in a significant reduction in leukemia burden and a survival benefit. Didox was well tolerated, as marrow from treated animals was morphologically indistinguishable from controls. Didox exposure at levels that impaired leukemia growth did not inhibit normal HSC engraftment. In summary, Didox was well tolerated and effective against preclinical models of AML.

Paradoxical response to prophylactic Didox (N-3, 4 trihydroxybenzamide) treatment in murine cytomegalovirus-infected mice.

BACKGROUND: In this study, we investigated the effect of Didox (DX) on the pathogenicity of and host responses to murine cytomegalovirus (MCMV) infection. METHODS: In vitro efficacy of DX against MCMV was determined using plaque reduction assays. For in vivo studies, mice infected with a sublethal dose (10(4) PFU) of MCMV were treated daily with DX (200 mg/kg) using either a prophylactic or delayed protocol. At predetermined intervals, target organs were removed for histopathology. Cytokine transcription and viral load were performed using real-time PCR. Serum cytokine levels were determined by ELISA, and T-cell markers by real-time PCR. RESULTS: DX (0.5-50 µM) inhibited MCMV plaque formation in vitro. However, in vivo, prophylactic DX treatment did not decrease viral load and prolonged hepatic proinflammatory cytokine transcription at days 3 and 5 post-infection, which corresponded with more severe histopathological changes observed in the liver. Significant CD8(+) T-cell marker suppression was seen, in accordance with DX-induced inhibition of lymphocyte proliferation observed in vitro. DX prolonged the recovery of MCMV-infected mice when given after infection was established. CONCLUSIONS: Despite promising MCMV inhibition in vitro, DX had no beneficial effect on MCMV disease in our model and paradoxically had adverse effects when administered prophylactically. The lack of correlation between in vitro activity and in vivo efficacy emphasizes the importance of selecting appropriate antiviral targets and of using animal models when testing new drugs.

Inhibition of allogeneic inflammatory responses by the Ribonucleotide Reductase Inhibitors, Didox and Trimidox.

BACKGROUND: Graft-versus-host disease is the single most important obstacle facing successful allogeneic stem cell transplantation (SCT). Even with current immunosuppressive therapies, morbidity and mortality rates are high. Current therapies including cyclosporine A (CyA) and related compounds target IL-2 signaling. However, although these compounds offer great benefit, they are also associated with multiple toxicities. Therefore, new compounds with a greater efficacy and reduced toxicity are needed to enable us to overcome this hurdle. METHODS: The allogeneic mixed lymphocyte reaction (MLR) is a unique ex vivo method to study a drug's action on the initial events resulting in T-cell activation and proliferation, synonymous to the initial stages of tissue and organ destruction by T-cell responses in organ rejection and Graft-versus-host disease. Using this approach, we examined the effectiveness of two ribonucleotide reductase inhibitors (RRI), Didox and Trimidox, to inhibit T-cell activation and proliferation. RESULTS: The compounds caused a marked reduction in the proliferative responses of T-cells, which is also accompanied by decreased secretion of cytokines IL-6, IFN-gamma, TNF-alpha, IL-2, IL-13, IL-10 and IL-4. CONCLUSIONS: In conclusion, these data provide critical information to justify further investigation into the potential use of these compounds post allogeneic bone marrow transplantation to alleviate graft-versus-host disease thereby achieving better outcomes.

Ribonucleotide reductase inhibitors reduce atherosclerosis in a double-injury rabbit model.

Atheroproliferative disorders such as atherosclerosis are an important health problem and one of the leading causes of morbidity and mortality in the United States. Minimally invasive therapeutic procedures, including angioplasty with stent deployment, are used frequently for obstructive coronary artery disease. However, restenosis, a proliferative vascular response, is a common sequela to this procedure. The current study investigated the effect of inhibiting ribonucleotide reductase (RR), an enzyme necessary for cellular proliferation, in an attempt to ameliorate the proliferative response. Two RR inhibitors, didox and hydroxyurea, were chosen for their potent antiproliferative properties. Studies were carried out by using a double-injury rabbit model, in which endothelial denudation was followed by the administration of a high-fat diet. At 4 wk after initial endothelial denudation, the developing atherosclerotic lesion was subjected to transluminal balloon dilation to simulate clinical intervention with percutaneous transluminal angioplasty. The degree of restenosis and atheroproliferation was assessed at 8 wk. Histologic evaluation of the lesion demonstrated that treatment with didox and hydroxyurea significantly decreased lesion area and lumen loss. These results suggest that RR inhibition may be an effective new tool for the treatment of atheroproliferative disorders.

Gallic acid inhibits ribonucleotide reductase and cyclooxygenases in human HL-60 promyelocytic leukemia cells.

Gallic acid (GA) is a naturally occurring polyhydroxyphenolic compound and an excellent free radical scavenger. In this study, we examined its cytotoxic and biochemical effects on the human HL-60 promyelocytic leukemia cell line. GA caused a significant imbalance of deoxynucleosidetriphosphate (dNTP) pool sizes, indicating ribonucleotide reductase inhibition. Moreover, GA induced dose-dependent apoptosis in HL-60 cells (80microM GA led to the induction of apoptosis in 39% of cells) and attenuated progression from G0/G1 to the S phase of the cell cycle (60microM GA doubled the number of cells in G0/G1 phase from 22 to 44% when compared to untreated controls). We further determined IC(50) values of 3.5 and 4.4nM for the inhibition of cyclooxygenases I and II, respectively. When cells were simultaneously treated with GA and trimidox, another inhibitor of RR, highly synergistic growth inhibitory effects could be observed. Taken together, we identified novel biochemical effects of GA which could be the basis for further preclinical and in vivo studies.

Potentiation of the activity of cisplatin and cyclophosphamide by trimidox, a novel ribonucleotide reductase inhibitor, in leukemia-bearing mice.

We describe the use of the new ribonucleotide reductase inhibitor, trimidox (TDX), in combination chemotherapy under in vitro and in vivo conditions with cisplatin and cyclophosphamide. In vitro, the combination of TDX and cisplatin was tested in L1210 cells. The combination caused concentration dependent antagonistic or additive effects. However, the combination of TDX-cisplatin-cyclophosphamide in vivo is highly synergistic in both, the L1210 and P388D1 leukemia mouse models. Both combinations, TDX with cisplatin or TDX with cyclophosphamide were also synergistic in the L1210 and P388D1 leukemia animal models.

Oxygen carriers: a selected review.

The most common and widely transplanted tissue world wide is blood, which in 2000 resulted in the transfusion of 12.5 million units of blood in the US alone [Goodnough LT, Shander A, Brecher ME. Transfusion medicine: looking to the future. Lancet 2003;361:161-9]. The current use of donated blood products is relatively safe; however, there are inherent problems with allogeneic blood transfusions. The wide spread use of blood in procedures results in problems involving inadequate supply exacerbated in times of war and disasters and by the limited storage life of blood donations (30-42 days). Blood contamination due to patient pre-disposition, poor collection, sterilization, or storage is the second most common cause of death from transfusion in the US [Hillyer CD, Josephson CD, Blajchman MA, Vostal JG, Epstein JS, Goodman JL. Bacterial contamination of blood components: risks, strategies, and regulation: joint ASH and AABB educational session in transfusion medicine. Hematology (Am Soc Hematol Educ Program) 2003:575-89]. Blood is a complex tissue involved in a plethora of homeostatic roles, including immunity, wound healing and the transport of nourishment, electrolytes, hormones, vitamins, heat, oxygen and the removal of metabolic waste products. However, by far the principle role of blood transfusions is the replacement of red cell volume and the maintenance of oxygen levels within the circulation. Creation of investigational new drugs (INDs) which would function as oxygen carriers and prolong shelf life is now a very active arena of scientific research. Several such IND products are now in clinical trials. This article gives an easy to follow concise evaluation of major areas of focus and current testing for each type of blood substitution molecule.

Inhibition of ribonucleotide reductase reduces neointimal formation following balloon injury.

Percutaneous transluminal coronary angioplasty (PTCA) has greatly benefited patients with occluded coronary arteries, but its benefits have been undermined by a high incidence of restenosis. The introduction of coronary stents has significantly improved the short and long term outcome but restenosis still occurs in approximately 15 to 30% of patients within 6 months. Research efforts are now being directed toward combination stenting and drug delivery. Among the therapeutic targets being pursued are agents that can impede smooth muscle cell migration and proliferation, as these processes are critical components of restenosis injury. We propose that inhibiting the conversion of ribonucleotides to deoxyribonucleotides will impede cell proliferation and, as such, limit the degree of restenosis. Therefore, we tested whether the potent ribonucleotide reductase inhibitors Didox (3,4-dihydroxybenzohydraxamic acid) and Imidate (ethyl-3,4,5-hydroxybenzimidate) can limit the neointimal proliferation associated with restenosis using a rat carotid model of balloon dilatation injury. Results demonstrated that both Didox and Imidate significantly reduced intimal thickening, resulting in a 71 and 62% decrease in the intima/media ratio, respectively. Similar efficacy was seen with the commercially available ribonucleotide reductase inhibitor hydroxyurea, demonstrating the importance of this enzyme in vascular remodeling. Results from cell proliferation studies suggest that the mechanism of protection is inhibition of smooth muscle cell (SMC) proliferation. In addition, Didox and Imidate (100 microM) are potent inhibitors of SMC migration, which may also contribute to their vascular protective effects. These results suggest that inhibition of ribonucleotide reductase may provide a potent strategy to prevent post-PTCA restenosis.

Combination of inhibitors of lymphocyte activation (hydroxyurea, trimidox, and didox) and reverse transcriptase (didanosine) suppresses development of murine retrovirus-induced lymphoproliferative disease.

The ribonucleotide reductase inhibitor hydroxyurea (HU) has demonstrated some benefit as a component of drug cocktails for the treatment of HIV-1 infection. However, HU is notoriously myelosuppressive and often administered only as salvage therapy to patients with late-stage disease, potentially exacerbating the bone marrow toxicity of HU. In this report we have compared the antiviral effects of HU and two novel RR inhibitors trimidox (3,4,5-trihydroxybenzamidoxime) and didox (3,4-dihydroxybenzohydroxamic acid) in combination with didanosine (2,3-didoxyinosine; ddI) in the LPBM5 MuLV retrovirus model (murine AIDS). We also evaluated the effects of these drug combinations on the hematopoietic tissues of LPBM5 MuLV-infected animals. The combination of RR inhibitors and ddI was extremely effective (DX>TX>HU) in inhibiting development of retrovirus-induced disease (splenomegaly, hypergammaglobulinemia, activated B-splenocytes and loss of splenic architecture). In addition, relative levels of proviral DNA were significantly lower in combination drug-treated animals compared to infected controls. Evaluation of femur cellularity, numbers of marrow-derived myeloid progenitor cells (CFU-GM and BFU-E) and peripheral blood indices revealed that TX and DX in combination with ddI were well-tolerated. However, treatment with HU and ddI induced moderate myelosuppression. These data demonstrate that RR inhibitors in combination with ddI provide significant protection against retroviral disease in murine AIDS. Moreover, the novel RR inhibitors TX and DX appear to be more effective and less myelosuppressive than HU when administered with ddI in this model.

Synergistic cytotoxicity of the ribonucleotide reductase inhibitor didox (3,4-dihydroxy-benzohydroxamic acid) and the alkylating agent carmustine (BCNU) in 9L rat gliosarcoma cells and DAOY human medulloblastoma cells.

PURPOSE: Ribonucleotide reductase (RR) is the rate-limiting enzyme of de novo DNA synthesis and has been shown to be upregulated linked with proliferation and malignant transformation. It was therefore identified as an excellent target for antitumor therapy. In the present study we investigated the biochemical and cytotoxic effects of didox, an inhibitor of RR, as a single agent and in combination with BCNU, an alkylating anticancer drug, in 9L rat gliosarcoma cells and DAOY human medulloblastoma cells. METHODS: The effect of didox on the intracellular concentrations of deoxynucleosidetriphosphates (dNTPs) was studied in 9L cells. Pool sizes were determined by HPLC. In addition, the cytotoxic effects of didox and BCNU as single drugs and in equimolar combination were tested in 9L and in DAOY cells. Combination effects were determined according to the equation of Chou and Talalay. The expression of DNA repair-related genes was determined after exposure of 9L cells to BCNU, didox and a combination of the two compounds, using a cDNA array. RESULTS: Incubation of 9L cells with 30 microM didox for 24 h significantly decreased the intracellular concentrations of the DNA precursors dCTP (61% of control) and dGTP (17% of control), and significantly increased the concentration of dATP (155% of control). This dNTP imbalance compromised DNA synthesis and repair and might therefore have been, at least in part, responsible for the highly synergistic cytotoxic effects seen when BCNU was used simultaneously with didox in 9L and in DAOY cells. With almost all combinations tested, highly synergistic effects were seen, as indicated by combination indices of <1 according to the equation of Chou and Talalay. In 9L cells, BCNU upregulated the expression of DNA repair-associated genes, whereas coincubation of the cells with didox reduced overexpression of some of these repair-related genes. CONCLUSION: A combination of BCNU and didox was proven to act in a synergistic manner in two cell lines, 9L rat gliosarcoma and DAOY human medulloblastoma cells. Further in vivo tests using these two compounds systemically and/or locally at the tumor site might be warranted.

In vivo examination of hydroxyurea and the novel ribonucleotide reductase inhibitors trimidox and didox in combination with the reverse transcriptase inhibitor abacavir: suppression of retrovirus-induced immunodeficiency disease.

Inhibition of ribonucleotide reductase (RR) has gained attention as a potential strategy for HIV-1 therapy through the success of hydroxyurea (HU) to potentiate the activity of the nucleoside reverse transcriptase inhibitor (NRTI) didanosine (ddI) in clinical trials. However, the use of HU has been limited by its development of hematopoietic toxicity. In this study, the novel RR inhibitors didox (DX; 3,4-dihydroxybenzohydroxamic acid), and trimidox (TX; 3,4,5-trihydroxybenzamidoxime) were evaluated along with HU for anti-retroviral efficacy in LPBM5-induced retro-viral disease (MAIDS) both as monotherapeutic regimens and in combination with the guanine containing NRTI abacavir (ABC). Anti-retroviral drug efficacy was determined by measuring inhibition of splenomegaly, hypergammaglobulinemia, and splenic levels of proviral DNA. In this study, all RRIs tested showed the ability to improve the efficacy of ABC in the MAIDS model by reducing splenomegaly, hypergammaglobulinemia, and splenic proviral DNA levels.

Biochemical modulation of Ara-C effects by amidox, an inhibitor of ribonucleotide reductase in HL-60 promyelocytic human leukemia cells.

Amidox, a new polyhydroxy-substituted benzoic acid derivative, is a potent inhibitor of the enzyme ribonucleotide reductase (RR), which catalyses the de novo synthesis of DNA. RR is considered to be an excellent target for anti cancer chemotherapy. We investigated the biochemical and antineoplastic effects of amidox as a single agent and in combination with Ara-C in human HL-60 promyelocytic leukemia cells. Amidox inhibited the growth of HL-60 cells in a growth inhibition assay with an IC50 of 25 microM. In a soft agar colony forming assay, amidox yielded a 50% inhibition of colony formation at 13 microM. We also investigated the effects of amidox treatment on the formation of deoxynucleosidetriphosphates. Amidox (50 and 75 microM for 24 hours) could significantly decrease intracellular concentrations of dCTP, dATP and dGTP pools, whereas dTTP levels increased. We then tested the combination effects of amidox with Ara-C; this combination yielded additive cytotoxic effects both in growth inhibition and in soft agar colony formation assays. This effect was due to the increased formation of Ara-CTP, the active metabolite of Ara-C, after preincubation with amidox. Preincubation of HL-60 cells with 75 and 100 microM amidox for 24 hours caused an increase in the intracellular Ara-CTP concentrations by 576% and 1143%, respectively. Therefore amidox might offer an additional option for the treatment of leukemia and thus be further investigated in in vivo studies as a single agent and in combination with Ara-C.

Didox (a novel ribonucleotide reductase inhibitor) overcomes Bcl-2 mediated radiation resistance in prostate cancer cell line PC-3.

In this study, we investigated the influence of Bcl-2 overexpression on the radiosensitizing potential of Didox (DX; 3,4-Dihydroxybenzohydroxamic acid), a novel ribonucleotide reductase inhibitor, in p53-null prostate cancer cell line PC-3. The PC-3 cells were transfected with vector alone or ectopically overexpressed with CMV-Bcl-2 construct. The effect of radiation (IR) or DX alone and in combination (pre and post IR exposure of DX) on cell survival was determined by colony-forming assay. The impact of these two treatments on the cell cycle was determined by flow cytometry. To further understand the molecular mechanism of DX-mediated radiosensitization, induction of pro-survival and pro-apoptotic factors were determined by Western blot and gel-shift assays respectively. When compared to PC-3/Bcl-2 cells (SF(2)=0.84; D(0)=437cGy), the PC-3/vector cells (SF(2)=0.4; D(0)=235cGy) were significantly sensitive to ionizing radiation (p<0.001). Exposure of DX at 5 microM concentration prior or post to radiation in both PC-3/vector and PC-3/Bcl-2 transfectants caused an increase in radiation enhancement ratios. A significant reduction in G(2)M phase was observed in cells exposed to DX post IR when compared to cells exposed to IR alone. Exposure to DX after radiation in PC-3/vector significantly abrogated radiation-induced Bcl-2 upregulation, with a concomitant induction of bax protein. In PC-3/Bcl-2 transfectants, DX exposure after IR caused an induction of bax protein. Gel shift assays indicated that in PC-3/vector cells when exposed to IR caused an induction of NFkappa-B activity however, DX down regulated the NFkappa-B activity. Radiation-induced NFkappa-B activity was abrogated in pre and post DX exposure in combination with IR. These findings indicate that DX mediates a potent radiosensitizing effect in p53 null prostate cancer cells by overcoming radiation induced NFkappa-B activity and Bcl-2 expression.

Suppression of retrovirus-induced immunodeficiency disease (murine AIDS) by trimidox and didox: novel ribonucleotide reductase inhibitors with less bone marrow toxicity than hydroxyurea.

Recently, the use of the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU) in combination with nucleoside analogs has gained attention as a potential strategy for anti-HIV-1 therapy. However, appeal for the long-term use of HU in HIV-1 infection may be limited by its propensity to induce hematopoietic toxicity. We report a comparison of the efficacy and bone marrow toxicity of HU (400 and 200 mg/kg/day) with the novel RR inhibitors and free radical-scavenging compounds didox (DX; 3,4-dihydroxybenzohydroxamic acid; 350 mg/kg/day) and trimidox (TX; 3,4,5-trihydroxybenzamidoxime; 175 mg/kg/day) in the murine AIDS (LPBM5 MuLV) model of retrovirus infection. Infected mice received daily drug treatment for 8 weeks. Efficacy was determined by measuring drug effects on retroviral-induced disease progression (i.e. development of splenomegaly and hypergammaglobulinemia) and by evaluating splenic levels of proviral DNA. Bone marrow toxicity was evaluated by measuring peripheral blood indices (WBC, hematocrit and reticulocyte counts), femoral cellularity and by determining the numbers of hematopoietic progenitor cells (CFU-GM, BFU-E) per femur and spleen. Compared to infected controls receiving no drug treatment, disease progression was significantly suppressed by TX, DX and HU. However, HU was associated with mortality and induced significant hematopoietic toxicity in a time- and dose-dependent manner. Conversely, TX and DX effectively inhibited retrovirus-induced disease but did not induce hematopoietic toxicity. These results suggest that due to their reduced hematopoietic toxicity and ability to inhibit disease progression in murine AIDS, TX and DX may offer effective alternatives to HU therapy in HIV-1 infection.