Mohammed S. Inayat

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 (SF2=0.84; D0=437cGy), the PC-3/vector cells (SF2=0.4; D0=235cGy) were significantly sensitive to ionizing radiation (p

Farnesyltransferase inhibitor (L-744,832) restores TGF-β type II receptor expression and enhances radiation sensitivity in K-ras mutant pancreatic cancer cell line MIA PaCa-2

Activated ras is known to dysregulate TGF-β signaling by altering the expression of TGF-β type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-β signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 μM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 μM concentrations (P>0.004). The TGF-β effector gene p21waf1/cip1 was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-β responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-β signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT–PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-β signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.

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

BackgroundGraft-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.MethodsThe allogeneic mixed lymphocyte reaction (MLR) is a unique ex vivo method to study a drugs 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.ResultsThe compounds caused a marked reduction in the proliferative responses of T-cells, which is also accompanied by decreased secretion of cytokines IL-6, IFN-γ, TNF-α, IL-2, IL-13, IL-10 and IL-4.ConclusionsIn 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.

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.