I-kappa-kinase-2 (IKK-2) inhibition potentiates vincristine cytotoxicity in non-Hodgkin's lymphoma.

BACKGROUND: IKK-2 is an important regulator of the nuclear factor-κB (NF-κB) which has been implicated in survival, proliferation and apoptosis resistance of lymphoma cells. In this study, we investigated whether inhibition of IKK-2 impacts cell growth or cytotoxicity of selected conventional chemotherapeutic agents in non-Hodgkin's lymphoma.Two established model systems were used; Follicular (WSU-FSCCL) and Diffuse Large Cell (WSU-DLCL2) Lymphoma, both of which constitutively express p-IκB. A novel, selective small molecule inhibitor of IKK-2, ML120B (N-[6-chloro-7-methoxy-9H-ß-carbolin-8-yl]-2-methylnicotinamide) was used to perturb NF-κB in lymphoma cells. The growth inhibitory effect of ML120B (M) alone and in combination with cyclophosphamide monohydrate (C), doxorubicin (H) or vincristine (V) was evaluated in vitro using short-term culture assay. We also determined efficacy of the combination in vivo using the SCID mouse xenografts. RESULTS: ML120B down-regulated p-IκBα protein expression in a concentration dependent manner, caused growth inhibition, increased G0/G1 cells, but did not induce apoptosis. There was no significant enhancement of cell kill in the M/C or M/H combination. However, there was strong synergy in the M/V combination where the vincristine concentration can be lowered by a hundred fold in the combination for comparable G2/M arrest and apoptosis. ML120B prevented vincristine-induced nuclear translocation of p65 subunit of NF-κB. In vivo, ML120B was effective by itself and enhanced CHOP anti-tumor activity significantly (P = 0.001) in the WSU-DLCL2-SCID model but did not prevent CNS lymphoma in the WSU-FSCCL-SCID model. CONCLUSIONS: For the first time, this study demonstrates that perturbation of IKK-2 by ML120B leads to synergistic enhancement of vincristine cytotoxicity in lymphoma. These results suggest that disruption of the NF-κB pathway is a useful adjunct to cytotoxic chemotherapy in lymphoma.

Preclinical studies of Apogossypolone: a new nonpeptidic pan small-molecule inhibitor of Bcl-2, Bcl-XL and Mcl-1 proteins in Follicular Small Cleaved Cell Lymphoma model.

Elevated expression of anti-apoptotic Bcl-2 family proteins have been linked to a poor survival rate of patients with Follicular Lymphoma (FL). This prompted us to evaluate a very potent non-peptidic Small-Molecule Inhibitor (SMI) targeting Bcl-2 family proteins, Apogossypolone (ApoG2) using follicular small cleaved cell lymphoma cell line (WSU-FSCCL) and cell isolated from lymphoma patients. ApoG2 inhibited the growth of WSU-FSCCL significantly with a 50% growth inhibition of cells (IC50) of 109 nM and decreased cell number of fresh lymphoma cells. ApoG2 activated caspases-9, -3, and -8, and the cleavage of Poly (ADP-ribose) polymerase (PARP) and Apoptosis Inducing Factor (AIF). In the WSU-FSCCL-SCID xenograft model, ApoG2 showed a significant anti-lymphoma effect, with %ILS of 84% in the intravenous and 63% in intraperitoneal treated mice. These studies suggest that ApoG2 can be an effective therapeutic agent against FL.

Reduced hydroperoxidase (HPI and HPII) activity in the Deltafur mutant contributes to increased sensitivity to UVA radiation in Escherichia coli.

In Escherichia coli, Deltafur (ferric uptake regulator) mutants are hypersensitive to various oxidative agents, including UVA radiation (400-315 nm). Studies suggest that UVA radiation mediates its biological effects on bacteria via oxidative mechanisms that lead to reactive oxygen species, including the superoxide anion radical (O2.-), hydroxyl radical (HO.), hydrogen peroxide (H2O2) and singlet oxygen (1O2). There is accumulating evidence that Fur may play an important role in the defense against UVA radiation. In addition to regulating almost all genes directly involved in iron acquisition, Fur also regulates the expression of manganese and iron superoxide dismutase (MnSOD, FeSOD), key enzymes in the defense against oxygen toxicity in E. coli. In Deltafur mutants, there is a complete absence of FeSOD. Previous results suggest that the native iron chelating agent, enterobactin, which exists in increased levels in Deltafur mutants, is an endogenous chromophore for UVA, releasing Fe2+ into the cytoplasm to catalyze the production of highly reactive hydroxyl radicals. We now report that the hypersensitivity of Deltafur mutants to UVA irradiation is associated with reduced hydroperoxidase I (HPI) and hydroperoxidase II (HPII) activity, and is associated with a decrease in the transcription of katE and katG genes. The observed decrease in HPII activity in Deltafur mutants is also associated with reduced rpoS gene transcription. This study provides additional evidence that the Fur gene product, in addition to its known regulatory effect on the expression of SOD and iron uptake mechanisms, also regulates HPI and HPII activity levels in E. coli. An H2O2-inducible antioxidant defense system leading to an increase in HPI activity, is unaltered in Deltafur mutants.

Effects of sublethal UVA irradiation on activity levels of oxidative defense enzymes and protein oxidation in Escherichia coli.

When bacterial cells are stressed by a change in the environment, they respond by changing the activity of enzymes at both the transcriptional and post-transcriptional levels. The UVA component (400-315 nm) of solar radiation reaching the Earth's surface is one of the most common stresses encountered by bacteria in their environment. Bacteria have evolved various antioxidant defense systems to increase survival when subjected to the deleterious effects of UVA irradiation. Recently, UVA-induced cytotoxicity and oxidative damage have been shown to be dependent on radiation intensity and dose distribution, not just total energy dose. We now report that when Escherichia coli is subjected to continuous sublethal, low-fluence UVA irradiation (7.4 W/m(2)) while growing to stationary phase, it responds by changing the activity levels of hydroperoxidases (HPI, HPII), glutathione reductase and manganese superoxide dismutase. This leads to an attenuation of the growth-delay response and an increase resistance to lethal UVA irradiation. When E. coli is given a UVA dose of 135 kJ/m(2) delivered at a fluence rate of 50 W/m(2), extensive protein oxidation occurs which may contribute to the inhibition of key cellular enzymes, leading to cellular dysfunction, DNA damage and eventually death. Changes in antioxidant enzymes induced by low-fluence UVA irradiation do not confer greater protection from protein oxidation after a challenge dose of UVA irradiation delivered at a fluence rate of 50 W/m(2).

Apogossypolone, a nonpeptidic small molecule inhibitor targeting Bcl-2 family proteins, effectively inhibits growth of diffuse large cell lymphoma cells in vitro and in vivo.

Apogossypolone (ApoG2) is a semi-synthesized derivative of gossypol. The principal objective of this study was to compare stability and toxicity between ApoG2 and gossypol, and to evaluate anti-lymphoma activity of ApoG2 in vitro and in vivo. ApoG2 shows better stability when compared with a racemic gossypol and can be better tolerated by mice compared to gossypol. ApoG2 showed significant inhibition of cell proliferation of WSU-DLCL(2) and primary cells obtained from lymphoma patients, whereas it displayed no toxicity on normal peripheral blood lymphocytes. For a treatment of 72 h, the IC(50) of ApoG2 was determined to be 350 nM against WSU-DLCL2 cells. Treatment with ApoG2 at 600 mg/kg resulted in significant growth inhibition of WSU-DLCL(2) xenografts. When combined with CHOP, ApoG2 displayed even more complete inhibition of tumor growth. ApoG2 binds to purified recombinant Bcl-2, Mcl-1 and Bcl-X(L) proteins with high affinity and is shown to block the formation of heterodimers between Bcl-X(L) and Bim. For a treatment of 72 h, ApoG2 induced a maximum of 32% of apoptotic cell death. Western blot experiments showed that treatment with ApoG2 led to cleavage of caspase-3, caspase-9 and PARP. Moreover, pretreatment of DLCL(2) cells with caspase-3, -9 and broad spectrum caspase inhibitors significantly blocked growth inhibition induced by ApoG2. In conclusion, ApoG2 effectively inhibits growth of DLCL(2) cells at least partly by inducing apoptosis. It is an attractive small molecule inhibitor of the Bcl-2 family proteins to be developed further for the treatment of diffuse large cell lymphoma.