Postsynaptic receptors regulate presynaptic transmitter stability through transsynaptic bridges.

Stable matching of neurotransmitters with their receptors is fundamental to synapse function and reliable communication in neural circuits. Presynaptic neurotransmitters regulate the stabilization of postsynaptic transmitter receptors. Whether postsynaptic receptors regulate stabilization of presynaptic transmitters has received less attention. Here, we show that blockade of endogenous postsynaptic acetylcholine receptors (AChR) at the neuromuscular junction destabilizes the cholinergic phenotype in motor neurons and stabilizes an earlier, developmentally transient glutamatergic phenotype. Further, expression of exogenous postsynaptic gamma-aminobutyric acid type A receptors (GABAA receptors) in muscle cells stabilizes an earlier, developmentally transient GABAergic motor neuron phenotype. Both AChR and GABAA receptors are linked to presynaptic neurons through transsynaptic bridges. Knockdown of specific components of these transsynaptic bridges prevents stabilization of the cholinergic or GABAergic phenotypes. Bidirectional communication can enforce a match between transmitter and receptor and ensure the fidelity of synaptic transmission. Our findings suggest a potential role of dysfunctional transmitter receptors in neurological disorders that involve the loss of the presynaptic transmitter.

Clarithromycin overcomes stromal cell-mediated drug resistance against proteasome inhibitors in myeloma cells via autophagy flux blockage leading to high NOXA expression.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

Glucocorticoid receptor-NECAB1 axis can negatively regulate insulin secretion in pancreatic ß-cells.

The mechanisms of impaired glucose-induced insulin secretion from the pancreatic ß-cells in obesity have not yet been completely elucidated. Here, we aimed to assess the effects of adipocyte-derived factors on the functioning of pancreatic ß-cells. We prepared a conditioned medium using 3T3-L1 cell culture supernatant collected at day eight (D8CM) and then exposed the rat pancreatic ß-cell line, INS-1D. We found that D8CM suppressed insulin secretion in INS-1D cells due to reduced intracellular calcium levels. This was mediated by the induction of a negative regulator of insulin secretion-NECAB1. LC-MS/MS analysis results revealed that D8CM possessed steroid hormones (cortisol, corticosterone, and cortisone). INS-1D cell exposure to cortisol or corticosterone increased Necab1 mRNA expression and significantly reduced insulin secretion. The increased expression of Necab1 and reduced insulin secretion effects from exposure to these hormones were completely abolished by inhibition of the glucocorticoid receptor (GR). NECAB1 expression was also increased in the pancreatic islets of db/db mice. We demonstrated that the upregulation of NECAB1 was dependent on GR activation, and that binding of the GR to the upstream regions of Necab1 was essential for this effect. NECAB1 may play a novel role in the adipoinsular axis and could be potentially involved in the pathophysiology of obesity-related diabetes mellitus.

Azithromycin, a potent autophagy inhibitor for cancer therapy, perturbs cytoskeletal protein dynamics.

BACKGROUND: Autophagy plays an important role in tumour cell growth and survival and also promotes resistance to chemotherapy. Hence, autophagy has been targeted for cancer therapy. We previously reported that macrolide antibiotics including azithromycin (AZM) inhibit autophagy in various types of cancer cells in vitro. However, the underlying molecular mechanism for autophagy inhibition remains unclear. Here, we aimed to identify the molecular target of AZM for inhibiting autophagy. METHODS: We identified the AZM-binding proteins using AZM-conjugated magnetic nanobeads for high-throughput affinity purification. Autophagy inhibitory mechanism of AZM was analysed by confocal microscopic and transmission electron microscopic observation. The anti-tumour effect with autophagy inhibition by oral AZM administration was assessed in the xenografted mice model. RESULTS: We elucidated that keratin-18 (KRT18) and α/ß-tubulin specifically bind to AZM. Treatment of the cells with AZM disrupts intracellular KRT18 dynamics, and KRT18 knockdown resulted in autophagy inhibition. Additionally, AZM treatment suppresses intracellular lysosomal trafficking along the microtubules for blocking autophagic flux. Oral AZM administration suppressed tumour growth while inhibiting autophagy in tumour tissue. CONCLUSIONS: As drug-repurposing, our results indicate that AZM is a potent autophagy inhibitor for cancer treatment, which acts by directly interacting with cytoskeletal proteins and perturbing their dynamics.

Activity-dependent Organization of Topographic Neural Circuits.

Sensory information in the brain is organized into spatial representations, including retinotopic, somatotopic, and tonotopic maps, as well as ocular dominance columns. The spatial representation of sensory inputs is thought to be a fundamental organizational principle that is important for information processing. Topographic maps are plastic throughout an animal's life, reflecting changes in development and aging of brain circuitry, changes in the periphery and sensory input, and changes in circuitry, for instance in response to experience and learning. Here, we review mechanisms underlying the role of activity in the development, stability and plasticity of topographic maps, focusing on recent work suggesting that the spatial information in the visual field, and the resulting spatiotemporal patterns of activity, provide instructive cues that organize visual projections.

Ricolinostat enhances adavosertib­induced mitotic catastrophe in TP53­mutated head and neck squamous cell carcinoma cells.

TP53 mutation is one of the most frequent gene mutations in head and neck squamous cell carcinoma (HNSCC) and could be a potential therapeutic target. Recently, the WEE1 G2 checkpoint kinase (WEE1) inhibitor adavosertib (Adv) has attracted attention because of its selective cytotoxicity against TP53­mutated cells and has shown promising activity in early phase clinical trials. In the present study, it was demonstrated that combined treatment with Adv and a selective histone deacetylase 6 (HDAC6) inhibitor, ricolinostat (RCS), synergistically enhanced cell death induction in four out of five HNSCC cell lines with TP53 mutation (CAL27, SAS, HSC­3, and OSC­19), one HNSCC cell line with impaired TP53 function by HPV­infection (UPCI­SCC154), and TP53­knockout human lung cancer cell line (A549 TP53­KO), but not in TP53 wild­type A549 cells. Time­lapse imaging showed that RCS enhanced the Adv­induced mitotic catastrophe. Consistent with this, RCS treatment suppressed checkpoint kinase 1 (Chk1) (Ser345) phosphorylation and co­administration of RCS with Adv suppressed cyclin­dependent kinase 1 (Tyr15) phosphorylation along with increased expression of γ­H2A.X, a marker of DNA double­strand breaks in CAL27 cells. These data showed that RCS enhanced Adv­induced premature mitotic entry and cell death induction in the mitotic phase. However, although HDAC6 knockdown enhanced Adv­induced cell death with γ­H2A.X elevation, HDAC6 knockdown did not repress Chk1 phosphorylation in CAL27 cells. Our data demonstrated that the co­administration of RCS with Adv in HNSCC cells resulted in the suppression of Chk1 activity, leading to synergistically enhanced apoptosis via mitotic catastrophe in a p53­dependent manner. This enhanced cell death appeared to be partially mediated by the inhibition of HDAC6 activity by RCS.

Lysosome­targeted drug combination induces multiple organelle dysfunctions and non­canonical death in pancreatic cancer cells.

Pancreatic cancer is one of the leading causes of cancer­related mortality and has the lowest 5­year survival rate. Therefore, novel strategies are urgently required to treat pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) cells rely on enhanced lysosomal function for survival and proliferation to facilitate the degradation of contents accumulated via autophagy and macropinocytosis. Previously, we have reported that the combination of epidermal growth factor receptor/HER2 inhibitor lapatinib and sphingosine analog fingolimod (FTY720) confers a significant cytostatic effect in lung cancer cells. In the present study, the combined effects of these drugs on PDAC cell lines, BxPC­3, KP­4, PANC­1 and MIA PaCa­2, were examined. It was observed that FTY720 enhanced the lapatinib­induced cytotoxic effect and caused non­canonical and lysosome­dependent death in PDAC cells. Lapatinib and FTY720 induced lysosomal swelling and inhibited lysosomal acidification. Combination treatment with lapatinib and FTY720 increased lysosomal membrane permeability, induced mitochondrial depolarization, induced endoplasmic reticulum stress and disturbed intracellular calcium homeostasis. Additionally, the cytotoxic effect of lapatinib was enhanced by hydroxychloroquine or the CDK4/6 inhibitor abemaciclib, both of which induce lysosomal dysfunction. Collectively, these results indicated that the lysosome­targeted drug combination induces multiple organelle dysfunction and exerts a marked cytotoxic effect in PDAC cells.

Targeted disruption of GAK stagnates autophagic flux by disturbing lysosomal dynamics.

The autophagy­lysosome system allows cells to adapt to environmental changes by regulating the degradation and recycling of cellular components, and to maintain homeostasis by removing aggregated proteins and defective organelles. Cyclin G­associated kinase (GAK) is involved in the regulation of clathrin­dependent endocytosis and cell cycle progression. In addition, a single nucleotide polymorphism at the GAK locus has been reported as a risk factor for Parkinson's disease. However, the roles of GAK in the autophagy­lysosome system are not completely understood, thus the present study aimed to clarify this. In the present study, under genetic disruption or chemical inhibition of GAK, analyzing autophagic flux and observing morphological changes of autophagosomes and autolysosomes revealed that GAK controlled lysosomal dynamics via actomyosin regulation, resulting in a steady progression of autophagy. GAK knockout (KO) in A549 cells impaired autophagosome­lysosome fusion and autophagic lysosome reformation, which resulted in the accumulation of enlarged autophagosomes and autolysosomes during prolonged starvation. The stagnation of autophagic flux accompanied by these phenomena was also observed with the addition of a GAK inhibitor. Furthermore, the addition of Rho­associated protein kinase (ROCK) inhibitor or ROCK1 knockdown mitigated GAK KO­mediated effects. The results suggested a vital role of GAK in controlling lysosomal dynamics via maintaining lysosomal homeostasis during autophagy.

Induction of synergistic non-apoptotic cell death by simultaneously targeting proteasomes with bortezomib and histone deacetylase 6 with ricolinostat in head and neck tumor cells.

Following surgery and chemoradiation, ~50% of patients with locally advanced head and neck tumors experience relapse within the first two years, with a poor prognosis. Therefore, a novel therapeutic approach is required. The aim of the present study was to investigate the effect of combination treatment with the proteasome inhibitor bortezomib (BTZ), and ricolinostat (RCS), a specific inhibitor of histone deacetylase 6 (HDAC6), on CAL27 and Detroit562 head and neck cancer cells. BTZ and RCS exhibited cytotoxicity in a dose- and time-dependent manner. Simultaneous treatment with BTZ and RCS resulted in the synergistic enhancement of non-apoptotic cell death and autophagy. The receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitor, necrostatin, but not the autophagy inhibitor, 3-methyladenine, attenuated the cytotoxicity of combined BTZ and RCS treatment. Thus, necroptosis [type-III programmed cell death (PCD)], but not autophagic cell death (type-II PCD), appeared to contribute to the pronounced cytotoxicity. However, no phosphorylation of RIPK1 or mixed lineage kinase domain-like protein was detectable in response to BTZ or RCS. Furthermore, RCS induced α-tubulin acetylation and inhibited BTZ-induced aggresome formation along with endoplasmic reticulum stress loading. Combined treatment with BTZ and RCS enhanced the production of reactive oxygen species (ROS). The ROS scavenger, N-acetyl cysteine, abrogated the increase in cytotoxicity. These results suggest the potential therapeutic value of the dual targeting of the proteasome and HDCA6 for head and neck cancers through the induction of necroptosis-like cell death along with ROS generation.

Azithromycin enhances the cytotoxicity of DNA-damaging drugs via lysosomal membrane permeabilization in lung cancer cells.

Cancer cells use autophagy for growth, survival, and cytoprotection from chemotherapy. Therefore, autophagy inhibitors appear to be good candidates for cancer treatment. Our group previously reported that macrolide antibiotics, especially azithromycin (AZM), have potent autophagy inhibitory effects, and combination treatment with tyrosine kinase inhibitors or proteasome inhibitors enhances their anti-cancer activity. In this study, we evaluated the effect of combination therapy with DNA-damaging drugs and AZM in non-small-cell lung cancer (NSCLC) cells. We found that the cytotoxic activities of DNA-damaging drugs, such as doxorubicin (DOX), etoposide, and carboplatin, were enhanced in the presence of AZM in NSCLC cell lines, whereas AZM alone exhibited almost no cytotoxicity. This enhanced cell death was dependent on wild-type-p53 status and autophagosome-forming ability because TP53 knockout (KO) and ATG5-KO cells attenuated AZM-enhanced cytotoxicity. DOX treatment upregulated lysosomal biogenesis by activating TFEB and led to lysosomal membrane damage as assessed by galectin 3 puncta assay and cytoplasmic leakage of lysosomal enzymes. In contrast, AZM treatment blocked autophagy, which resulted in the accumulation of lysosomes/autolysosomes. Thus, the effects of DOX and AZM were integrated into the marked increase in damaged lysosomes/autolysosomes, leading to prominent lysosomal membrane permeabilization (LMP) for apoptosis induction. Our data suggest that concomitant treatment with DNA-damaging drugs and AZM is a promising strategy for NSCLC treatment via pronounced LMP induction.

BRCA1 degradation in response to mitochondrial damage in breast cancer cells.

BRCA1 is a well-studied tumor suppressor involved in the homologous repair of DNA damage, whereas PINK1, a mitochondrial serine/threonine kinase, is known to be involved in mitochondrial quality control. Genetic mutations of PINK1 and Parkin cause autosomal recessive early-onset Parkinson's disease. We found that in breast cancer cells, the mitochondrial targeting reagents, which all induce mitochondrial depolarization along with PINK1 upregulation, induced proteasomal BRCA1 degradation. This BRCA1 degradation was dependent on PINK1, and BRCA1 downregulation upon mitochondrial damage caused DNA double-strand breaks. BRCA1 degradation was mediated through the direct interaction with the E3 ligase Parkin. Strikingly, BRCA1 and PINK1/Parkin expression were inversely correlated in cancerous mammary glands from breast cancer patients. BRCA1 knockdown repressed cancer cell growth, and high BRCA1 expression predicted poor relapse-free survival in breast cancer patients. These observations indicate a novel mechanism by which mitochondrial damage is transmitted to the nucleus, leading to BRCA1 degradation.

DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation.

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.

Tetrode Recording in the Xenopus laevis Visual System Using Multichannel Glass Electrodes.

The Xenopus tadpole visual system shows an extraordinary extent of developmental and visual experience-dependent plasticity, establishing sophisticated neuronal response properties that guide essential survival behaviors. The external development and access to the developing visual circuit of Xenopus tadpoles make them an excellent experimental system in which to elucidate plastic changes in neuronal properties and their capacity to encode information about the visual scene. The temporal structure of neural activity encodes a significant amount of information, access to which requires recording methods with high temporal resolution. Conversely, elucidating changes in the temporal structure of neural activity requires recording over extended periods. It is challenging to maintain patch-clamp recordings over extended periods and Ca2+ imaging has limited temporal resolution. Extracellular recordings have been used in other systems for extended recording; however, spike amplitudes in the developing Xenopus visual circuit are not large enough to be captured by distant electrodes. Here we describe a juxtacellular tetrode recording method for continuous long-term recordings from neurons in intact tadpoles, which can also be exposed to diverse visual stimulation protocols. Electrode position in the tectum is stabilized by the large contact area in the tissue. Contamination of the signal from neighboring neurons is minimized by the tight contact between the glass capillaries and the dense arrangement of neurons in the tectum. This recording method enables analysis of developmental and visual experience-dependent plastic changes in neuronal response properties at higher temporal resolution and over longer periods than current methods.

Precisely controlled visual stimulation to study experience-dependent neural plasticity in Xenopus tadpoles.

Studies on visual experience-dependent plasticity can benefit tremendously from experimental protocols in which sensory stimulation is precisely controlled for extended periods over which neuronal, circuit, and behavioral plasticity occurs. Small vertebrates, such as Xenopus tadpoles and zebrafish, are excellent systems for studying brain plasticity. Here, we present a detailed protocol to perform controlled visual stimulation for extended time periods. These methods have been used to study structural plasticity induced by temporally controlled visual stimulation in Xenopus tadpoles. For further details on the use and execution of this protocol, please refer to Hiramoto and Cline (2014, 2020).

Macrolide antibiotics enhance the antitumor effect of lansoprazole resulting in lysosomal membrane permeabilization­associated cell death.

The proton pump inhibitor lansoprazole (LPZ) inhibits the growth of several cancer cell lines, including A549 and CAL 27. We previously reported that macrolide antibiotics such as azithromycin (AZM) and clarithromycin (CAM) potently inhibit autophagic flux and that combining AZM or CAM with the epidermal growth factor receptor inhibitors enhanced their antitumor effect against various cancer cells. In the present study, we conducted the combination treatment with LPZ and macrolide antibiotics against A549 and CAL 27 cells and evaluated cytotoxicity and morphological changes using cell proliferation and viability assays, flow cytometric analysis, immunoblotting, and morphological assessment. Combination therapy with LPZ and AZM greatly enhanced LPZ­induced cell death, whereas treatment with AZM alone exhibited negligible cytotoxicity. The observed cytotoxic effect was not mediated through apoptosis or necroptosis. Transmission electron microscopy of A549 cells treated with the LPZ + AZM combination revealed morphological changes associated with necrosis and accumulated autolysosomes with undigested contents. Furthermore, the A549 cell line with ATG5 knockout exhibited complete inhibition of autophagosome formation, which did not affect LPZ + AZM treatment­induced cytotoxicity, thus excluding the involvement of autophagy­dependent cell death in LPZ + AZM treatment­induced cell death. A549 cells treated with LPZ + AZM combination therapy retained the endosomal Alexa­dextran for extended duration as compared to untreated control cells, thus indicating impairment of lysosomal digestion. Notably, lysosomal galectin­3 puncta expression induced due to lysosomal membrane permeabilization was increased in cells treated with LPZ + AZM combination as compared to the treatment by either agent alone. Collectively, the present results revealed AZM­induced autolysosome accumulation, potentiated LPZ­mediated necrosis, and lysosomal membrane permeabilization, thus suggesting the potential clinical application of LPZ + AZM combination therapy for cancer treatment.

Sequestosome 1 (p62) accumulation in breast cancer cells suppresses progesterone receptor expression via argonaute 2.

Sequestosome 1 (p62) is a multifunctional adapter protein involved in various physiological functions, such as selective autophagy and oxidative stress response. Hence, aberrant expression and defective regulation of p62 are thought to lead to the onset of various diseases, including cancer. The expression of p62 has been shown to be increased in breast cancer tissues, and is correlated with a poor prognosis. However, the role of p62 in the breast cancer pathophysiology is still unclear. Here, we aimed to analyze the effect of changes in p62 expression on breast cancer cell lines. DNA microarray analysis revealed that the expression of progesterone receptor (PR), which is one of the indices for the classification of breast cancer subtypes, was markedly suppressed by forced expression of p62. The protein expression of PR was also decreased by forced expression of p62, but increased by knockdown of p62. Moreover, we found that p62 knockdown induced the protein expression of argonaute 2 (AGO2). Luciferase reporter assay results showed that the gene expression of PR was promoted by AGO2. Furthermore, results revealed that overexpression of AGO2 partially rescued the decrease in PR expression induced by forced expression of p62. Collectively, our findings indicated that p62 accumulation suppressed the expression of AGO2, which in turn decreased the expression of PR, suggesting that p62 may serve as a marker of aggressive breast cancer and poor prognosis. Moreover, the p62-AGO2-PR axis was identified as a crucial signaling cascade in breast cancer progression.

NMDARs Translate Sequential Temporal Information into Spatial Maps.

Spatial representations of the sensory world are important for brain function. Timing is an essential component of sensory information. Many brain circuits transform the temporal sequence of input activity into spatial maps; however, the mechanisms underlying this transformation are unclear. Different N-methyl-D-aspartate receptor (NMDAR) response magnitudes result in synaptic potentiation or depression. We asked whether NMDAR response magnitude also affects the transformation of temporal information into directional spatial maps. We quantified retinotectal axon branch dynamics in Xenopus optic tectum in response to temporal sequences of visual stimulation. Reducing NMDAR responses by 50% inverts the spatial distribution of branch dynamics along the rostrocaudal axis in response to temporal patterns of input, suggesting that the magnitude of NMDAR signaling encodes the temporal sequence of inputs and translates the temporal code into a directional spatial map using structural plasticity-based branch dynamics. We discuss how this NMDAR-dependent decoding mechanism retrieves spatial information from sequential afferent activity.

UGGT1 retains proinsulin in the endoplasmic reticulum in an arginine dependent manner.

We sought to clarify a pathway by which L- and dD-arginine simulate insulin secretion in mice and cell lines and obtained the following novel two findings. (1) Using affinity magnetic nanobeads technology, we identified that proinsulin is retained in the endoplasmic reticulum (ER) through UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) when arginine availability is limited. (2) L- and d-arginine release proinsulin from UGGT1 through competition with proinsulin and promote exit of proinsulin from the ER to Golgi apparatus. The ability of arginine to release proinsulin from UGGT1 closely correlates with arginine-induced insulin secretion in several models of ß cells indicating that UGGT1-proinsulin interaction regulates arginine-induced insulin secretion.

Abemaciclib induces atypical cell death in cancer cells characterized by formation of cytoplasmic vacuoles derived from lysosomes.

In the cell cycle, the G1 /S transition is controlled by the cyclin-dependent kinase (CDK) 4/6-cyclin D complex. Constitutive activation of CDK4/6 dysregulates G1 /S transition, leading to oncogenic transformation. We found that 3 CDK4/6 inhibitors, abemaciclib, ribociclib, and palbociclib, exerted a cytocidal effect as well as a cytostatic effect at the G1 phase in cancer cell lines, including A549 human non-small cell lung cancer cells. Among these inhibitors, abemaciclib exhibited the most potent cytotoxic effect. The cell-death phenotype induced by abemaciclib, which entailed formation of multiple cytoplasmic vacuoles, was not consistent with apoptosis or necroptosis. Abemaciclib blocked autophagic flux, resulting in accumulation of autophagosomes, however vacuole formation and cell death induced by abemaciclib were independent of autophagy. In addition, methuosis, a cell-death phenotype characterized by vacuole formation induced by excessive macropinocytosis, was excluded because the vacuoles did not incorporate fluorescent dextran. Of note, both formation of vacuoles and induction of cell death in response to abemaciclib were inhibited by vacuolar-type ATPase (V-ATPase) inhibitors such as bafilomycin A1 and concanamycin A. Live-cell imaging revealed that the abemaciclib-induced vacuoles were derived from lysosomes that expanded following acidification. Transmission electron microscopy revealed that these vacuoles contained undigested debris and remnants of organelles. Cycloheximide chase assay revealed that lysosomal turnover was blocked by abemaciclib. Furthermore, mTORC1 inhibition along with partial lysosomal membrane permeabilization occurred after abemaciclib treatment. Together, these results indicate that, in cancer cells, abemaciclib induces a unique form of cell death accompanied by swollen and dysfunctional lysosomes.

Comparison of autophagy inducibility in various tyrosine kinase inhibitors and their enhanced cytotoxicity via inhibition of autophagy in cancer cells in combined treatment with azithromycin.

Tyrosine kinase inhibitors (TKIs) induce autophagy in many types of cancer cells. We previously reported that gefitinib (GEF) and imatinib (IMA) induce autophagy in epidermal growth factor receptor (EGFR) knock-out A549 and non-BCR-ABL-expressing leukemia cell lines, respectively. This evidence suggests that TKI-induced autophagy is independent of the original target molecules. The present study compared the autophagy-inducing abilities of various TKIs, regardless of their targets, by quantitative autophagy flux assay. We established stable clones expressing the GFP-LC3-mCherry-LC3ΔG plasmid in A549, PC-9, and CAL 27 cell lines and assessed autophagy inducibility by monitoring the fluorescent ratios of GFP-LC3 to mCherry-LC3ΔG using an IncuCyte live cell imaging system during exposure to TKIs viz; GEF, osimertinib (OSI), lapatinib (LAP), lenvatinib (LEN), sorafenib (SOR), IMA, dasatinib (DAS), and tivantinib (TIV). Among these TKIs, DAS, GEF, and SOR exhibited prominent autophagy induction in A549 and PC-9 cells. In CAL 27 cells, IMA, SOR, and LEN, but not GEF, TIV, or OSI, exhibited autophagy induction. In the presence of azithromycin (AZM), which showed an inhibitory effect on autophagy flux, TKIs with prominent autophagy inducibility exhibited enhanced cytotoxicity via non-apoptotic cell death relative to effects of TKI alone. Therefore, autophagy inducibility of TKIs differed in the context of cancer cells. However, once induced, they appeared to have cytoprotective functions. Thus, blocking TKI-induced autophagy with AZM may improve the therapeutic effect of TKIs in cancer cells.