Anti-proliferative activity of the quassinoid NBT-272 in childhood medulloblastoma cells

BACKGROUND: With current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to correlate with anaplasia and unfavorable prognosis. In neuroblastoma – an embryonal tumor with biological similarities to MB – the quassinoid NBT-272 has been demonstrated to inhibit cellular proliferation and to down-regulate c-MYC protein expression. METHODS: To study MB cell responses to NBT-272 and their dependence on the level of c-MYC expression, DAOY (wild-type, empty vector transfected or c-MYC transfected), D341 (c-MYC amplification) and D425 (c-MYC amplification) human MB cells were used. The cells were treated with different concentrations of NBT-272 and the impact on cell proliferation, apoptosis and c-MYC expression was analyzed. RESULTS: NBT-272 treatment resulted in a dose-dependent inhibition of cellular proliferation (IC50 in the range of 1.7 – 9.6 ng/ml) and in a dose-dependent increase in apoptotic cell death in all human MB cell lines tested. Treatment with NBT-272 resulted in up to 90% down-regulation of c-MYC protein, as demonstrated by Western blot analysis, and in a significant inhibition of c-MYC binding activity. Anti-proliferative effects were slightly more prominent in D341 and D425 human MB cells with c-MYC amplification and slightly more pronounced in c-MYC over-expressing DAOY cells compared to DAOY wild-type cells. Moreover, treatment of synchronized cells by NBT-272 induced a marked cell arrest at the G1/S boundary. CONCLUSION: In human MB cells, NBT-272 treatment inhibits cellular proliferation at nanomolar concentrations, blocks cell cycle progression, induces apoptosis, and down-regulates the expression of the oncogene c-MYC. Thus, NBT-272 may represent a novel drug candidate to inhibit proliferation of human MB cells in vivo

Both Stereoselective (R)- and (S)-1-Methyl-1,2,3,4-tetrahydroisoquinoline Enantiomers Protect Striatal Terminals Against Rotenone-Induced Suppression of Dopamine Release

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) is present in the human and rodent brain as a mixture of stereospecific (R)- and (S)-1MeTIQ enantiomers. The racemate, (R,S)-1MeTIQ, exhibits neuroprotective activity as shown in the earlier study by the authors, and In addition, it was suggested to play a crucial physiological role in the mammalian brain as an endogenous regulator of dopaminergic activity. In this article, we investigated the influence of stereospecific enantiomers of 1MeTIQ, (R)- and (S)-1MeTIQ (50 mg/kg i.p.) on rotenone-induced (3 mg/kg s.c.) behavioral and neurochemical changes in the rat. In behavioral study, in order to record dynamic motor function of rats, we measured locomotor activity using automated locomotor activity boxes. In biochemical studies, we analyzed in rat striatum the concentration of dopamine (DA) and its metabolites: intraneuronal DOPAC, extraneuronal 3-MT, and final HVA using HPLC with electrochemical detection. Otherwise, DA release was estimated by in vivo microdialysis study. The behavioral study has demonstrated that both acute and repeated (3 times) rotenone administration unimportantly depressed a basic locomotor activity in rat. (R)- and (S)-1MeTIQ stereoisomers (50 mg/kg i.p.) produced a modest behavioral activation both in naïve and rotenone-treated rats. The data from ex vivo neurochemical experiments have shown stereospecificity of 1MeTIQ enantiomers in respect of their effects on DA catabolism. (R)-1MeTIQ significantly increased both the level of the final DA metabolite, HVA (by about 70%), and the rate of DA metabolism (by 50%). In contrast to that, (S)-1MeTIQ significantly depressed DOPAC, HVA levels (by 60 and 40%, respectively), and attenuated the rate of DA metabolism (by about 60%). On the other hand, both the enantiomers increased the concentrations of DA and its extraneuronal metabolite, 3-MT in rat striatum. In vivo microdialysis study has shown that repeated but not acute administration of rotenone produced a deep and significant functional impairment of striatal DA release. Both (R)- and (S)- stereospecific enantiomers of 1MeTIQ antagonized rotenone-induced suppression of DA release; however, the effect of (R)-1MeTIQ was more strongly expressed in microdialysis study. In conclusion, we suggest that both chiral isomers of 1MeTIQ offer neuroprotection against rotenone-induced disturbances in the function of dopaminergic neurons and (R,S)-1MeTIQ will be useful as a drug with marked neuroprotective activity in the brain

RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells

BACKGROUND: With current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis. METHODS: To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA) and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR) and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425). RESULTS: siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT) expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector. CONCLUSION: In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly

Role of Matrix Metalloproteinases and Therapeutic Benefits of Their Inhibition in Spinal Cord Injury

This review will focus on matrix metalloproteinases (MMPs) and their inhibitors in the context of spinal cord injury (SCI). MMPs have a specific cellular and temporal pattern of expression in the injured spinal cord. Here we consider their diverse functions in the acutely injured cord and during wound healing. Excessive activity of MMPs, and in particular gelatinase B (MMP-9), in the acutely injured cord contributes to disruption of the blood-spinal cord barrier, and the influx of leukocytes into the injured cord, as well as apoptosis. MMP-9 and MMP-2 regulate inflammation and neuropathic pain after peripheral nerve injury and may contribute to SCI-induced pain. Early pharmacologic inhibition of MMPs or the gelatinases (MMP-2 and MMP-9) results in an improvement in long-term neurological recovery and is associated with reduced glial scarring and neuropathic pain. During wound healing, gelatinase A (MMP-2) plays a critical role in limiting the formation of an inhibitory glial scar, and mice that are genetically deficient in this protease showed impaired recovery. Together, these findings illustrate complex, temporally distinct roles of MMPs in SCIs. As early gelatinase activity is detrimental, there is an emerging interest in developing gelatinase-targeted therapeutics that would be specifically tailored to the acute injured spinal cord. Thus, we focus this review on the development of selective gelatinase inhibitors

Metalloproteinases and their inhibitors—diagnostic and therapeutic opportunities in orthopedics

Matrix metalloproteinases (MMPs) and related enzymes (ADAMs, ADAMTS) and their inhibitors control matrix turnover and function. Recent advances in our understanding of musculoskeletal conditions such as tendinopathy, arthritis, Dupuytren's disease, degenerative disc disease, and bone and soft tissue healing suggest that MMPs have prominant roles. Importantly, MMPs are amenable to inhibition by cheap, safe, and widely available drugs such as the tetracycline antibiotics and the bisphosphonates. This indicates that these MMP inhibitors, if proven effective for any novel indication, may be quickly brought into clinical practice