Melatonin Potentiates Sensitivity to 5-Fluorouracil in Gastric Cancer Cells by Upregulating Autophagy and Downregulating Myosin Light-Chain Kinase.

5-Fluorouracil is an effective chemotherapeutic drug for gastric cancer. However, the acquisition of chemotherapeutic resistance remains a challenge in treatment. Melatonin can enhance the therapeutic effect of 5-fluorouracil; however, the underlying mechanisms are not well understood. We investigated the effects of combinations of melatonin and 5-fluorouracil on the proliferation, migration and invasion of gastric cancer cells. Melatonin significantly potentiated the 5-fluorouracil-mediated inhibition of proliferation, migration and invasion in gastric cancer cells, which potentiates sensitivity to 5-FU by promoting the activation of Beclin-1-dependent autophagy and targeting the myosin light-chain kinase (MLCK) signaling pathway. Previous studies have shown that autophagy might be associated with the MLCK signaling pathway. The autophagy inhibitor, 3-methyladenine, effectively rescued the migratory and invasive capabilities of gastric cancer cells, while also reducing expression level of MLCK and the phosphorylation level of MLC. This indicates that autophagy is involved in tumor metastasis, which may be related to inhibition of the MLCK signaling pathway. Our findings indicate that melatonin can improve the effectiveness of 5-fluorouracil in gastric cancer and could be used as a supplemental agent in the treatment of gastric cancer with 5-fluorouracil.

Thin and open vessel windows for intra-vital fluorescence imaging of murine cochlear blood flow.

Normal microvessel structure and function in the cochlea is essential for maintaining the ionic and metabolic homeostasis required for hearing function. Abnormal cochlear microcirculation has long been considered an etiologic factor in hearing disorders. A better understanding of cochlear blood flow (CoBF) will enable more effective amelioration of hearing disorders that result from aberrant blood flow. However, establishing the direct relationship between CoBF and other cellular events in the lateral wall and response to physio-pathological stress remains a challenge due to the lack of feasible interrogation methods and difficulty in accessing the inner ear. Here we report on new methods for studying the CoBF in a mouse model using a thin or open vessel-window in combination with fluorescence intra-vital microscopy (IVM). An open vessel-window enables investigation of vascular cell biology and blood flow permeability, including pericyte (PC) contractility, bone marrow cell migration, and endothelial barrier leakage, in wild type and fluorescent protein-labeled transgenic mouse models with high spatial and temporal resolution. Alternatively, the thin vessel-window method minimizes disruption of the homeostatic balance in the lateral wall and enables study CoBF under relatively intact physiological conditions. A thin vessel-window method can also be used for time-based studies of physiological and pathological processes. Although the small size of the mouse cochlea makes surgery difficult, the methods are sufficiently developed for studying the structural and functional changes in CoBF under normal and pathological conditions.

A differentially amplified motion in the ear for near-threshold sound detection.

The ear is a remarkably sensitive pressure fluctuation detector. In guinea pigs, behavioral measurements indicate a minimum detectable sound pressure of ∼20 µPa at 16 kHz. Such faint sounds produce 0.1-nm basilar membrane displacements, a distance smaller than conformational transitions in ion channels. It seems that noise within the auditory system would swamp such tiny motions, making weak sounds imperceptible. Here we propose a new mechanism contributing to a resolution of this problem and validate it through direct measurement. We hypothesized that vibration at the apical side of hair cells is enhanced compared with that at the commonly measured basilar membrane side. Using in vivo optical coherence tomography, we demonstrated that apical-side vibrations peaked at a higher frequency, had different timing and were enhanced compared with those at the basilar membrane. These effects depend nonlinearly on the stimulus sound pressure level. The timing difference and enhancement of vibrations are important for explaining how the noise problem is circumvented.

The influence of NF-kappaB signal-transduction pathways on the murine inner ear by acoustic overstimulation.

Nuclear factor-kappa B (NF-kappaB) comprises a family of inducible transcription factors that serve as important regulators of the host immune and inflammatory responses. The NF-kappaB signals are activated via the canonical and/or noncanonical pathways in response to diverse stimuli. The excessive action of NF-kappaB signal-transduction pathways frequently causes self-injurious phenomena such as allergic diseases, vascular disorders, and ischemia-reperfusion neuronal damage. In the inner ear, the role of NF-kappaB has not been clarified because the activated NF-kappaB signals potentially induce both cytoprotective and cytotoxic target genes after ototoxic stimulation. In the present study, we investigated the response of NF-kappaB in both the canonical and noncanonical pathways to acoustic overstimulation (117 dB/SPL/2 hr) and followed the change of inflammatory factors (inducible nitric oxide synthase [iNOS], intracellular adhesion molecule-1 [ICAM-1], and vascular cell adhesion molecule-1 [VCAM-1]) in the cochlear lateral wall (CLW) and the rest of cochlea (RoC). By means of immunohistochemistry combined with confocal microscopy and reverse transcriptase-polymerase chain reaction techniques, we found the response of NF-kappaB family members (NF-kappa B1, 2, RelA, and RelB) at the transcription level. After the NF-kappaB signaling, the inflammatory factors were significantly increased in the CLW and the RoC. Additionally, at the protein level, the prominent expression of adhesion molecules (ICAM-1 and VCAM-1) was observed in the tissue around the capillaries in the stria vascularis. These results show that acoustic overstimulation causes the NF-kappaB signaling to overexpress the inflammatory factors in the inner ear, and the up-regulation of the adhesion molecules (ICAM-1 and VCAM-1) and iNOS potentially influence the hemodynamics and the cellular integrity in the stria vascularis.

Altered expression of inducible nitric oxide synthase (iNOS) in the cochlea.

Using immunohistochemistry and Western blot, the expression of inducible nitric oxide synthase (iNOS) in the lateral wall and organ of Corti was examined in normal (unstimulated) and stimulated mice and guinea pigs. The stimuli were: (1). injection of bacterial lipopolysaccharide (LPS, 5 mg/ml) into the middle ear through the tympanic membrane and (2). exposure to a 110 dB SPL (A-weighted) broadband noise, 3 h/day, for three consecutive days. For the unstimulated condition, weak iNOS expression was found in the vascular endothelium, marginal cells, nerve fibers, stereocilia of hair cells and Hensen's cells of the organ of Corti. More intense iNOS fluorescence signals were observed in cochlear tissues (particularly in hair cells and stria vascularis marginal cells) in animals exposed to loud sound or treated with LPS. Although the precise roles of iNOS expression in normal cochlear function have yet to be determined, enhanced iNOS expression following noise exposure and LPS suggests its participation in cochlear pathophysiology, including noise- and inflammatory factor-induced hearing loss.