Amlodipine inhibits Synaptotagmin-4's oncogenic activity on gastric cancer proliferation by targeting calcium signaling.

BACKGROUND: Gastric cancer (GC) remains a leading cause of cancer mortality globally. Synaptotagmin-4 (SYT4), a calcium-sensing synaptic vesicle protein, has been implicated in the oncogenesis of diverse malignancies. PURPOSE: This study delineates the role of SYT4 in modulating clinical outcomes and biological behaviors in GC. METHODS: We evaluated SYT4 expression in GC specimens using bioinformatics analyses and immunohistochemistry. Functional assays included CCK8 proliferation tests, apoptosis assays via flow cytometry, confocal calcium imaging, and xenograft models. Western blotting elucidated MAPK pathway involvement. Additionally, we investigated the impact of the calcium channel blocker amlodipine on cellular dynamics and MAPK pathway activity. RESULTS: SYT4 was higher in GC tissues, and the elevated SYT4 was significantly correlated with adverse prognosis. Both univariate and multivariate analyses confirmed SYT4 as an independent prognostic indicator for GC. Functionally, SYT4 promoted tumorigenesis by fostering cellular proliferation, inhibiting apoptosis, and enhancing intracellular Ca2+ influx, predominantly via MAPK pathway activation. Amlodipine pre-treatment attenuated SYT4-driven cell growth and potentiated apoptosis, corroborated by in vivo xenograft assessments. These effects were attributed to MAPK pathway suppression by amlodipine. CONCLUSION: SYT4 emerges as a potential prognostic biomarker and a pro-oncogenic mediator in GC through a Ca2+-dependent MAPK mechanism. Amlodipine demonstrates significant antitumor effects against SYT4-driven GC, positing its therapeutic promise. This study underscores the imperative of targeting calcium signaling in GC treatment strategies.

Synaptotagmin XIII expression and peritoneal metastasis in gastric cancer.

BACKGROUND: Peritoneal metastasis is a frequent cause of death in patients with gastric cancer. The aim of this study was to identify molecules responsible for mediating peritoneal metastasis of gastric cancer. METHODS: Transcriptome and bioinformatics analyses were conducted to identify molecules associated with peritoneal metastasis. The therapeutic effects of intraperitoneally administered small interfering (si) RNA were evaluated using mouse xenograft models. Expression of mRNA and protein was determined in gastric tissues from patients with gastric cancer. RESULTS: Synaptotagmin XIII (SYT13) was expressed at significantly higher levels in patients with peritoneal recurrence, but not in those with hepatic or distant lymph node recurrence. Inhibition of SYT13 expression in a gastric cancer cell line transfected with SYT13-specific siRNA (siSYT13) was associated with decreased invasion and migration ability of the cells, but not with proliferation and apoptosis. Intraperitoneal administration of siSYT13 significantly inhibited the growth of peritoneal nodules and prolonged survival in mice. In an analysis of 200 patients with gastric cancer, SYT13 expression in primary gastric cancer tissues was significantly greater in patients with peritoneal recurrence or metastasis. A high level of SYT13 expression in primary gastric cancer tissues was an independent risk factor for peritoneal recurrence. CONCLUSION: SYT13 expression in gastric cancer is associated with perioneal metatases and is a potential target for treatment.

Pharmacological Bypass of Cockayne Syndrome B Function in Neuronal Differentiation.

Cockayne syndrome (CS) is a severe neurodevelopmental disorder characterized by growth abnormalities, premature aging, and photosensitivity. Mutation of Cockayne syndrome B (CSB) affects neuronal gene expression and differentiation, so we attempted to bypass its function by expressing downstream target genes. Intriguingly, ectopic expression of Synaptotagmin 9 (SYT9), a key component of the machinery controlling neurotrophin release, bypasses the need for CSB in neuritogenesis. Importantly, brain-derived neurotrophic factor (BDNF), a neurotrophin implicated in neuronal differentiation and synaptic modulation, and pharmacological mimics such as 7,8-dihydroxyflavone and amitriptyline can compensate for CSB deficiency in cell models of neuronal differentiation as well. SYT9 and BDNF are downregulated in CS patient brain tissue, further indicating that sub-optimal neurotrophin signaling underlies neurological defects in CS. In addition to shedding light on cellular mechanisms underlying CS and pointing to future avenues for pharmacological intervention, these data suggest an important role for SYT9 in neuronal differentiation.

Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles.

In forebrain neurons, Ca(2+) triggers exocytosis of readily releasable vesicles by binding to synaptotagmin-1 and -7, thereby inducing fast and slow vesicle exocytosis, respectively. Loss-of-function of synaptotagmin-1 or -7 selectively impairs the fast and slow phase of release, respectively, but does not change the size of the readily-releasable pool (RRP) of vesicles as measured by stimulation of release with hypertonic sucrose, or alter the rate of vesicle priming into the RRP. Here we show, however, that simultaneous loss-of-function of both synaptotagmin-1 and -7 dramatically decreased the capacity of the RRP, again without altering the rate of vesicle priming into the RRP. Either synaptotagmin-1 or -7 was sufficient to rescue the RRP size in neurons lacking both synaptotagmin-1 and -7. Although maintenance of RRP size was Ca(2+)-independent, mutations in Ca(2+)-binding sequences of synaptotagmin-1 or synaptotagmin-7--which are contained in flexible top-loop sequences of their C2 domains--blocked the ability of these synaptotagmins to maintain the RRP size. Both synaptotagmins bound to SNARE complexes; SNARE complex binding was reduced by the top-loop mutations that impaired RRP maintenance. Thus, synaptotagmin-1 and -7 perform redundant functions in maintaining the capacity of the RRP in addition to nonredundant functions in the Ca(2+) triggering of different phases of release.

Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes.

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MßCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MßCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MßCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.

Synaptotagmin XI regulates phagocytosis and cytokine secretion in macrophages.

Synaptotagmins (Syts) are a group of type I membrane proteins that regulate vesicle docking and fusion in processes such as exocytosis and phagocytosis. All Syts possess a single transmembrane domain, and two conserved tandem Ca(2+)-binding C2 domains. However, Syts IV and XI possess a conserved serine in their C2A domain that precludes these Syts from binding Ca(2+) and phospholipids, and from mediating vesicle fusion. Given the importance of vesicular trafficking in macrophages, we investigated the role of Syt XI in cytokine secretion and phagocytosis. We demonstrated that Syt XI is expressed in murine macrophages, localized in recycling endosomes, lysosomes, and recruited to phagosomes. Syt XI had a direct effect on phagocytosis and on the secretion of TNF and IL-6. Whereas small interfering RNA-mediated knockdown of Syt XI potentiated secretion of these cytokines and particle uptake, overexpression of an Syt XI construct suppressed these processes. In addition, Syt XI knockdown led to decreased recruitment of gp91(phox) and lysosomal-associated membrane protein-1 to phagosomes, suggesting attenuated microbicidal activity. Remarkably, knockdown of Syt XI ensued in enhanced bacterial survival. Our data reveal a novel role for Syt XI as a regulator of cytokine secretion, particle uptake, and macrophage microbicidal activity.

Synaptotagmin I and IX function redundantly in controlling fusion pore of large dense core vesicles.

Synaptotagmins (Syts) constitute a large family of at least 16 members and individual Syt isoforms exhibit distinct Ca(2+)-binding properties and subcellular localization. It remains to be demonstrated whether multiple Syt isoforms can function independently or cooperatively on certain type of vesicle. In the current study, we have developed NPY-pHluorin to specifically assess exocytosis of large dense core vesicles (LDCVs) and studied the requirement of Syt I and Syt IX for LDCV exocytosis in PC12 cells. We found that down-regulation of both Syt I and Syt IX resulted in a significant loss of Ca(2+)-dependent LDCV exocytosis. Moreover, our results suggest Syt I and Syt IX play redundant role in controlling the choice of fusion modes. Down-regulation of both Syt I and Syt IX renders more fusion in the kiss-and-run mode. We conclude that Syt I and Syt IX function redundantly in Ca(2+)-sensing and fusion pore dilation on LDCVs in PC12 cells.