The BRD4 Inhibitor dBET57 Exerts Anticancer Effects by Targeting Superenhancer-Related Genes in Neuroblastoma.

Neuroblastoma (NB) is the most common solid tumor of the neural crest cell origin in children and has a poor prognosis in high-risk patients. The oncogene MYCN was found to be amplified at extremely high levels in approximately 20% of neuroblastoma cases. In recent years, research on the targeted hydrolysis of BRD4 to indirectly inhibit the transcription of the MYCN created by proteolysis targeting chimaera (PROTAC) technology has become very popular. dBET57 (S0137, Selleck, TX, USA) is a novel and potent heterobifunctional small molecule degrader based on PROTAC technology. The purpose of this study was to investigate the therapeutic effect of dBET57 in NB and its potential mechanism. In this study, we found that dBET57 can target BRD4 ubiquitination and disrupt the proliferation ability of NB cells. At the same time, dBET57 can also induce apoptosis, cell cycle arrest, and decrease migration. Furthermore, dBET57 also has a strong antiproliferation function in xenograft tumor models in vivo. In terms of mechanism, dBET57 targets the BET protein family and the MYCN protein family by associating with CRBN and destroys the SE landscape of NB cells. Combined with RNA-seq and ChIP-seq public database analysis, we identified the superenhancer-related genes TBX3 and ZMYND8 in NB as potential downstream targets of dBET57 and experimentally verified that they play an important role in the occurrence and development of NB. In conclusion, these results suggest that dBET57 may be an effective new therapeutic drug for the treatment of NB.

The effects of simultaneous operation on dissociated vertical deviation with horizontal and torsional strabismus.

AIM: To investigate the therapeutic effects of simultaneous horizontal and vertical operations on dissociated vertical deviation (DVD) associated with other deviations. METHODS: Forty-five cases of DVD with horizontal and torsional strabismus underwent combined operation were collected retrospectively. All clinical records were analyzed. All patients were followed up for 6 to 24mo. Wilcoxon signed-ranks test was performed to evaluate the changes of vertical and horizontal deviation. χ 2 test was used to evaluate the changes of binocular visual function. RESULTS: Forty-five cases included 36 patients with intermittent exotropia and binocular inferior oblique overaction (IOOA), 5 patients with concomitant esotropia and binocular IOOA, 4 patients with intermittent exotropia and monocular superior oblique palsy. The superior rectus recession (SRR) combined with horizontal rectus recession and the myectomy of inferior oblique or anterior transposition were operated simultaneously to correct all types of strabismus. There were 43 cases who achieved normal eye position in vertical direction, while 2 cases were with undercorrection of 5Δ to 6Δ. In patients with horizontal strabismus, 2 cases of exotropia were with overcorrection of 6Δ to 8Δ, 1 case of esotropia was with undercorrection of 6Δ, and 1 case of monocular superior oblique palsy with compensatory head posture was not significantly improved. The binocular visual function of most patients recovered after operation. The difference of the binocular visual function and eye position were significant compared with that before operation (P<0.05). CONCLUSION: The simultaneous operation on DVD with horizontal and torsional strabismus is successful.

Hoechst 33342-induced autophagy protected HeLa cells from caspase-independent cell death with the participation of ROS.

Autophagy, an evolutionarily-conserved intracellular organelle and protein degradation process, may exhibit drastically different effects on cell survival depending on the particular environmental and culturing conditions. Hoechst 33342 (HO), a fluorescent dye widely used for staining DNA, has been reported to induce apoptosis in mammalian cells. Here we showed that, in addition to caspase-independent cell death, HO also induced autophagy in HeLa cells, as evidenced by the accumulation of autophagosomes, LC3 form conversion and LC3 puncta formation in a cell line stably expressing GFP-LC3. HO treatment led to generation of reactive oxygen species (ROS), and inhibition of ROS with N-acetyl-l-cysteine (NAC) abrogated both autophagy and caspase-independent cell death. Finally, autophagy played a protective role against caspase-independent cell death, as cell death induced by HO was enhanced under pharmacological and siRNA-mediated genetic inhibition of autophagy.

Autophagy-mediated chemosensitization by cysteamine in cancer cells.

Cysteamine (CS) has many biomedical and clinical applications because of its excellent water solubility, low cytotoxicity and good biocompatibility. A previous study by Brawer et al. reported the occurrence of many Gomori inclusion bodies in CS-treated astrocytes, which would suggest the induction of autophagy. Here we provided a comprehensive line of evidence demonstrating that CS caused autophagosome accumulation in cancer cells. CS exerted a biphasic effect on the autophagy process, increasing the formation of autophagosomes in the early phase and blocking the autophagic degradation in a later phase. Furthermore, we showed that CS sensitized doxorubicin-elicited chemotherapeutic killing in HeLa, B16 melanoma and doxorubicin-resistant MCF-7 cells and also enhanced chemotherapeutic efficacy of doxorubicin in a mouse melanoma model. Finally, we demonstrated that the chemosensitizing effect of CS was at least partly dependent on its ability to modulate autophagy. Our results revealed a novel biological function for CS in enhancing the chemotherapeutic effect of doxorubicin through autophagy modulation and pointed to the potential use of CS in adjunct cancer chemotherapy.

Minimally invasive botulinum toxin type A injection from the ocular surface to extraocular muscles.

AIM: To investigate a new, safe and effective injection method for strabismus patients. Botulinum toxin type A (BTXA) was injected by pulling the extraocular muscles with a minimally-invasive technique into the ocular surface, and it was ensured that the extraocular muscles was maintained in the suspended state. METHODS: A total of 32 patients with different types of strabismus were treated at our institution from February to October 2010. A small conjunctival incision (≤2mm) was made under a microscope. The extraocular muscles were pulled out with a hook to ensure an elevated position compared with the wall of eyeball. The muscle fiber was clearly seen through the conjunctiva and BTXA was injected at a small angle under the microscope. The deviation angles before and after the injection were recorded. All patients were followed up at 5 and 30 days after the operation. Recovery was defined as abolition of diplopia in straight-ahead gaze and anteroinferior gaze and the symptoms of giddiness disappeared thoroughly. Eyeball position was essentially normal. Improvement was defined as basic disappearance of diplopia in straight-ahead gaze and anteroinferior gaze; restriction of action of paralytic muscle improved. If most of the symptoms and signs still existed and disturbed normal work and life, the treatment was determined to be invalid. The injection dose for patients of 5 to 10 prism diopter (PD), 11 to 20PD, and ≥21PD was 1u, 3u and 4u to 5u, respectively. RESULTS: Of the 32 treated patients, 11(34.4%) were cured, and 18(56.3%) were improved at 5 days after the operation; 12(40%) were cured, and 15(46.9%) were improved at 30 days. Five patients (15.6%) who had unsatisfactory response after BTXA injection at 30 days received repeated injections or underwent strabismus surgery. Ptosis was present in 2.5% of the injected eyes. No retrobulbar hemorrhage or ocular perforation was found in any eye. CONCLUSION: It is safe and efficient to inject BTXA by pulling extraocular muscles with a minimally-invasive technique under the microscope to make the muscles separated from the wall of eyeball.

Identification of nose-to-brain homing peptide through phage display.

Brain delivery of drug molecules through the nasal passage represents a viable approach for bypassing the blood-brain barrier (BBB) but remains a major challenge due to lack of efficient homing carriers. To screen for potential peptides with the ability to transport into the brain via the nasal passage, we applied a C7C phage peptide display library (Ph.D.-C7C) intra-nasally to anesthetized rats and recovered phage from the brain tissue 45 min after phage administration. After three rounds of panning, 10 positive phage clones were selected and sequenced. Clone7, which exhibited highest translocation efficiency, was chosen for further studies. After nasal administration, Clone7 entered the brain within 30 min and exhibited translocation efficiency about 50-fold higher than a random phage. A 11-amino acid synthetic peptide derived from the displayed sequence of Clone7 (ACTTPHAWLCG) efficiently inhibited the nasal-brain translocation of Clone7. Both phage recovery results and fluorescent microscopy images revealed the presence of many more Clone7 phage in the brain than in the liver, kidney and other internal organs after the nasal administration, suggesting that Clone7 bypassed the BBB and entered brain directly. Furthermore, both Clone7 and the ACTTPHAWLCG peptide were found to be heavily distributed along the olfactory nerve after the nasal administration, further suggesting a direct passage route into the brain via the olfactory region. These results demonstrated the feasibility of using the in vivo phage display approach for selecting peptides with the nose-to-brain homing capability and may have implications for the development of novel targeting carriers useful for brain delivery.