Inhibition of Hsp90 in the spinal cord enhances the antinociceptive effects of morphine by activating an ERK-RSK pathway.

Morphine and other opioids are commonly used to treat pain despite their numerous adverse side effects. Modulating µ-opioid receptor (MOR) signaling is one way to potentially improve opioid therapy. In mice, the chaperone protein Hsp90 mediates MOR signaling within the brain. Here, we found that inhibiting Hsp90 specifically in the spinal cord enhanced the antinociceptive effects of morphine in mice. Intrathecal, but not systemic, administration of the Hsp90 inhibitors 17-AAG or KU-32 amplified the effects of morphine in suppressing sensitivity to both thermal and mechanical stimuli in mice. Hsp90 inhibition enabled opioid-induced phosphorylation of the kinase ERK and increased abundance of the kinase RSK in the dorsal horns of the spinal cord, which are heavily populated with primary afferent sensory neurons. The additive effects of Hsp90 inhibition were abolished upon intrathecal inhibition of ERK, RSK, or protein synthesis. This mechanism downstream of MOR, localized to the spinal cord and repressed by Hsp90, may potentially be used to enhance the efficacy and presumably decrease the side effects of opioid therapy.

Evaluation of mitochondrial redox status and energy metabolism of X-irradiated HeLa cells by LC/UV, LC/MS/MS and ESR.

To evaluate the metabolic responses in tumour cells exposed to ionizing radiation, oxygen consumption rate (OCR), cellular lipid peroxidation, cellular energy status (intracellular nucleotide pool and ATP production), and mitochondrial reactive oxygen species (ROS), semiquinone (SQ), and iron-sulphur (Fe-S) cluster levels were evaluated in human cervical carcinoma HeLa cells at 12 and 24 h after X-irradiation. LC/MS/MS analysis showed that levels of 8-iso PGF2α and 5-iPF2α-VI, lipid peroxidation products of membrane arachidonic acids, were not altered significantly in X-irradiated cells, although mitochondrial ROS levels and OCR significantly increased in the cells at 24 h after irradiation. LC/UV analysis revealed that intracellular AMP, ADP, and ATP levels increased significantly after X-irradiation, but adenylate energy charge (adenylate energy charge (AEC) = [ATP + 0.5 × ADP]/[ATP + ADP + AMP]) remained unchanged after X-irradiation. In low-temperature electron spin resonance (ESR) spectra of HeLa cells, the presence of mitochondrial SQ at g = 2.004 and Fe-S cluster at g = 1.941 was observed and X-irradiation enhanced the signal intensity of SQ but not of the Fe-S cluster. Furthermore, this radiation-induced increase in SQ signal intensity disappeared on treatment with rotenone, which inhibits electron transfer from Fe-S cluster to SQ in complex I. From these results, it was suggested that an increase in OCR and imbalance in SQ and Fe-S cluster levels, which play a critical role in the mitochondrial electron transport chain (ETC), occur after X-irradiation, resulting in an increase in ATP production and ROS leakage from the activated mitochondrial ETC.

Synergistic effects of rmhTRAIL and 17-AAG on the proliferation and apoptosis of multiple myeloma cells.

OBJECTIVE: This study aimed to investigate synergistic effects of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmhTRAIL) and heat-shock protein 90 (HSP90) inhibitor (geldanamycin derivative 17 -allylamino- 17-demethoxy -geldanamycin, 17-AAG) on the proliferation and apoptosis of multiple myeloma (MM) cells. METHODS: MTT assays evaluated inhibitory effects of rmhTRAIL and 17-AAG in different concentrations and treatment durations on the proliferation of RPMI8226 and U266 cells. The half maximal inhibitory concentration was calculated using OriginPro7.5. Synergistic effects of rmhTRAIL and 17-AAG on apoptosis of MM cells were detected using flow cytometry at 24 and 48 h post-treatment. To evaluate synergistic effects of rmhTRAIL and 17-AAG, the Q-value was calculated using King's formula. RESULTS: rmhTRAIL exhibited significant inhibitory effects on the proliferation of RPMI8226 cells in a dose- and time-dependent manner (>50%), whereas U266 cells were not sensitive to rmhTRAIL (<50%). 17-AAG inhibited the proliferation of RPMI8226 and U266 cells in a dose-dependent manner (>80%). Significant synergistic effects of rmhTRAIL and 17-AAG on the proliferation of RPMI8226 cells were revealed (Q-value > 1.15), whereas synergistic effects were not evident on the proliferation of U266 cells (Q-value < 1.15). rmhTRAIL and 17-AAG exhibited significant synergistic effects on apoptosis of RPMI8226 and U266 cells (Q-value > 1.15). CONCLUSION: The combined application of rmhTRAIL and 17-AAG revealed favorable synergistic effects in the treatment of MM.

Dasatinib inhibits B cell receptor signalling in chronic lymphocytic leukaemia but novel combination approaches are required to overcome additional pro-survival microenvironmental signals.

As antigenic stimulation of the B cell antigen receptor (BCR) is key to chronic lymphocytic leukaemia (CLL) pathogenesis, targeting dysregulated kinases involved in BCR signalling is an attractive therapeutic approach. We studied the effects of the Src/c-Abl tyrosine kinase inhibitor dasatinib on BCR signal transduction in CLL cells. Treatment of CLL cells with 100 nmol/l dasatinib induced apoptosis by an average reduction in viability of 33·7% at 48 h, with dasatinib sensitivity correlating with inhibition of Syk(Y348) phosphorylation. Dasatinib inhibited calcium flux, phosphatidylinositol-3-kinase and mitogen-activated protein kinase activation following BCR crosslinking, and blocked the Mcl-1-dependent increase in CLL cell survival on prolonged BCR stimulation. However, the pro-apoptotic effect of dasatinib was abrogated by stromal cell contact alone or in the presence of CD154 and interleukin (IL)-4 (CD154L/IL-4 system). Whilst dasatinib retained the ability to sensitize CLL cells in stromal co-culture to both fludarabine and chlorambucil, the addition of CD154 and IL-4 rendered cells resistant to these drug combinations. We demonstrate that the HSP90 inhibitor 17-DMAG exhibited synergy with dasatinib in vitro, and moreover, induced apoptosis of CLL cells in the CD154L/IL-4 system. Our data provide evidence that dasatinib would be most clinically effective in combination with agents able to target antigen-independent microenvironmental signals.

Alpha1-adrenergic receptors and their significance to chemical-induced nephrotoxicity--a brief review.

Stimulation of alpha-adrenergic receptors by cold stress or adrenergic agents has been shown to potentiate the toxicity of numerous toxicants. Several lines of evidence indicate that this interaction is dependent on glutathione depression and increased cytosolic Ca2+ concentrations produced by alpha1-adrenergic compounds. In this report, evidence is provided in support of the mechanism of adrenoreceptor-mediated potentiation of nephrotoxicity. Alpha1-adrenergic agonists are shown to potentiate the toxicity of nephrotoxicants that exert their toxic effects via glutathione conjugation or Ca2+ deregulation. This review summarizes the effects of the alpha1-adrenergic agonist, phenylephrine, at enhancing the toxicity of 2-bromohydroquinone, 1,2-dibromoethane, and cis-diammineplatinum(II) dichloride.