Mutations in the gene coding for connexin26 (Cx26) is the most common cause of human nonsyndromic hereditary deafness. To investigate deafness mechanisms underlying Cx26 null mutations, we generated three independent lines of conditional Cx26 null mice. Cell differentiation and gross cochlear morphology at birth seemed normal. However, postnatal development of the organ of Corti was stalled as the tunnel of Corti and the Nuel's space were never opened. Cell degeneration was first observed in the Claudius cells around P8. Outer hair cell loss was initially observed around P13 at middle turn when inner hair cells were still intact. Massive cell death occurred in the middle turn thereafter and gradually spread to the basal turn, resulting in secondary degeneration of spiral ganglion neurons in the corresponding cochlear locations. These results demonstrated that Cx26 plays essential roles in postnatal maturation and homoeostasis of the organ of Corti before the onset of hearing.

Mutations in the potassium channel subunit KCNQ1 cause the human severe congenital deafness Jervell and Lange-Nielsen (JLN) syndrome. We applied a gene therapy approach in a mouse model of JLN syndrome (Kcnq1−/− mice) to prevent the development of deafness in the adult stage. A modified adeno-associated virus construct carrying a Kcnq1 expression cassette was injected postnatally (P0–P2) into the endolymph, which resulted in Kcnq1 expression in most cochlear marginal cells where native Kcnq1 is exclusively expressed. We also found that extensive ectopic virally mediated Kcnq1 transgene expression did not affect normal cochlear functions. Examination of cochlear morphology showed that the collapse of the Reissner’s membrane and degeneration of hair cells (HCs) and cells in the spiral ganglia were corrected in Kcnq1−/− mice. Electrophysiological tests showed normal endocochlear potential in treated ears. In addition, auditory brainstem responses showed significant hearing preservation in the injected ears, ranging from 20 dB improvement to complete correction of the deafness phenotype. Our results demonstrate the first successful gene therapy treatment for gene defects specifically affecting the function of the stria vascularis, which is a major site affected by genetic mutations in inherited hearing loss.

We have shown previously that EM011, a synthetic compound, binds tubulin with a higher affinity than the founding compound, noscapine, without changing total microtubule polymer mass. Now we show that EM011 is potently effective against vinblastine-resistant human lymphoblastoid line CEM/VLB100 and its parental vinblastine-sensitive line CEM. The cytotoxicity is mediated by cell cycle arrest at

Connexin26 (Cx26) and connexin30 (Cx30) are two major protein subunits that co-assemble to form gap junctions (GJs) in the cochlea. Mutations in either one of them are the major cause of non-syndromic prelingual deafness in humans. Because the mechanisms of cochlear pathogenesis caused by Cx mutations are unclear, we investigated effects of Cx30 null mutation on GJ-mediated ionic and metabolic coupling in the cochlea of mice. A novel flattened cochlear preparation was used to directly assess intercellular coupling in the sensory epithelium of the cochlea. Double-electrode patch clamp recordings revealed that the absence of Cx30 did not significantly change GJ conductance among the cochlear supporting cells. The preserved electrical coupling is consistent with immunolabeling data showing extensive Cx26 GJs in the cochlea of the mutant mice. In contrast, dye diffusion assays showed that the rate and extent of intercellular transfer of multiple fluorescent dyes (including a non-metabolizable D-glucose analogue, 2-NBDG) among cochlear supporting cells were severely reduced in Cx30 null mice. Since the sensory epithelium in the cochlea is an avascular organ, GJ-facilitated intercellular transfer of nutrient and signaling molecules may play essential roles in cellular homeostasis. To test this possibility, NBDG was used as a tracer to study the contribution of GJs in transporting glucose into the cochlear sensory epithelium when delivered systemically. NBDG uptake in cochlear supporting cells was significantly reduced in Cx30 null mice. The decrease was also observed with GJ blockers or glucose competition, supporting the specificity of our tests. These data indicate that GJs facilitate efficient uptake of glucose in the supporting cells. This study provides the first direct experimental evidence showing that the transfer of metabolically-important molecules in cochlear supporting cells is dependent on the normal function of GJs, thereby suggesting a novel pathogenesis process in the cochlea for Cx-mutation-linked deafness.

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Following surgery, the hormone dependence of breast tumors is exploited for therapy using antagonists such as tamoxifen, although occasional hormone-resistant clones do appear. Another chemotherapeutic strategy uses microtubule inhibitors such as taxanes. Unfortunately, these agents elicit toxicities such as leukocytopenia, diarrhea, alopecia, and peripheral neuropathies and are also associated with the emergence of drug resistance. We have previously described a tubulin-binding, natural compound, noscapine, that was nontoxic and triggered apoptosis in many cancer types albeit at 10 Mmol/L or higher concentrations depending on the cell type. We now show that a synthetic analogue of noscapine, 9-bromonoscapine, is f10-fold to 15-fold more potent than noscapine in inhibiting cell proliferation and induces apoptosis following G 2 -M arrest in hormone-insensitive human breast cancers (MDA-MB-231). Furthermore, a clear loss of mitochondrial membrane potential, release of cytochrome c, activation of the terminal caspase-3, and the cleavage of its substrates such as poly(ADP-ribose) polymerase, suggest an intrinsic apoptotic mechanism. Taken together, these data point to a mitochondrially mediated apoptosis of hormone-insensitive breast cancer cells. Human tumor xenografts in nude mice showed significant tumor volume reduction and a surprising increase in longevity without signs of obvious toxicity. Thus, our data provide compelling evidence that 9-bromonoscapine can be useful for the therapy of hormone-refractory breast cancer. [Mol Cancer Ther 2006;5(9):2366 -77]

We studied in silico docking of noscapine onto tubulin, combined with calculations of surface charge, P-P, van der Waals, and hydrogen bonding interactions, to rationally design a new compound, EM015. This tubulin-binding semisynthetic compound is a selective and potent anti-breast cancer agent and displays a 20-fold lower IC 50 against many tumor cells compared with our founding compound, (S)-6,7-dimethoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro[1,3]-dioxolo-[4,5-g]isoquinolin-5-yl)isobenzo-furan-1(3H

Several DNA-and microtubule-binding agents are used to manage hematologic malignancies in the clinic. However, drug resistance has been a challenge, perhaps due to a few surviving cancer stem cells. Toxicity is another major impediment to successful chemotherapy, leading to an impoverished quality of life. Here, we show that a semisynthetic nontoxic tubulin-binding agent, 9-bromonoscapine (EM011), effectively inhibits growth and regresses multidrug resistance-associated protein (MRP)-overexpressing teniposide-resistant T-cell lymphoma xenografts and prolongs longevity. As expected, teniposide treatment failed to regress teniposide-resistant xenografts, rather, treated mice suffered tremendous body weight loss. Mechanistically, EM011 displays significant antiproliferative activity, perturbs cell cycle progression by arresting mitosis, and induces apoptosis in teniposide-resistant lymphoblastoid T cells both in vitro and in vivo. EM011-induced apoptosis has a mitochondriallymediated component, which was attenuated by pretreatment with cyclosporin A. We also observed alterations of apoptosisregulatory molecules such as inactivation of Bcl2, translocation of BAX to the mitochondrial membrane, cytochrome c release, and activation of downstream apoptotic signaling. EM011 caused DNA degradation as evident by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling staining of the increased concentration of 3 ¶-DNA ends. Furthermore, the apoptotic induction was caspase dependent as shown by cleavage of the caspase substrate, poly(ADP)ribose polymerase. In addition, EM011 treatment caused a suppression of natural survival pathways such as the phosphatidylinositol-3 ¶-kinase/Akt signaling. These preclinical findings suggest that EM011 is an excellent candidate for clinical evaluation.