Nme8 is essential for protection against chemotherapy drug cisplatin-induced male reproductive toxicity in mice.

Cisplatin (CP), a chemotherapy drug commonly used in cancers treatment, causes serious reproductive toxicity. With younger cancer patients and increasing survival rates, it is important to preserve their reproductive capacity. NME8 is highly expressed in testis and contains thioredoxin and NDPK domains, suggesting it may be a target against the CP-induced reproductive toxicity. We deleted exons 6-7 of the Nme8 in mice based on human mutation sites and observed impaired transcript splicing. In mice, Nme8 was not essential for spermatogenesis, possibly due to functional compensation by its paralog, Nme5. Nme8 expression was elevated and translocated to the nucleus in response to two weeks of CP treatment. Under CP treatment, Nme8 deficiency further impaired antioxidant capacity, induced lipid peroxidation and increased ROS level, and failed to activate autophagy, resulting in aggravated DNA damage in testes and sperm. Consequently, the proliferation and differentiation of spermatogonia and the meiosis of spermatocyte were almost completely halted, and sperm motility was impaired. Our research indicates that NME8 protects against CP-induced testis and sperm damage. This may provide new insights into the physiological functions of the Nme family and potential targets for preserving fertility in young male cancer patients.

Role of LGL1 in cerebellar primordium of embryonic mice.

Lethal giant larvae 1 (LGL1) is originally recognized as a tumor suppressor, implicated in maintaining cell polarity in Drosophila and mammalian cells. Cell polarity plays a crucial role in tumorigenesis. We previously established Pax2-LGL1 -/- conditional knockout mice but did not focus on the tumorigenesis in cerebellar primordium. HE staining was used to detect the morphological structure of the cerebellar primordium during early embryonic development in Pax2-LGL1 -/- mice. Immunofluorescence assays were used to detect the expression of polar molecules. TUNEL staining assessed tissue apoptosis. Our findings reveal that deletion of LGL1 leads to the emergence of neuroblastoma-like tissues within the cerebellum primordium during early embryogenesis. This outcome can be attributed to alterations in expression patterns of polar molecules Cdc42 and ß-catenin following early deletion of LGL1, resulting in loss of cell polarity among neuroepithelial cells and subsequent formation of tumor-like tissues. However, further histological examination demonstrated that these tumor-like tissues disappear from embryonic day 15.5 onwards within the cerebellar primordium of Pax2-LGL1 -/- mice due to apoptosis-mediated cellular compensation. Our data emphasize the importance of LGL1 in maintaining neuroepithelial cell polarity and reveal a novel role for LGL1 in regulating tumorigenesis and ablation in the cerebellar primordium.

Primary pure large cell neuroendocrine carcinoma of the urinary bladder: a case report and literature review.

Background: The large cell neuroendocrine carcinoma (LCNEC) of the urinary bladder is a rare malignancy. With its high aggressiveness and poor prognosis, the disease is often accompanied by metastasis or recurrence. The lack of specific clinical manifestations and imaging features causes considerable challenges for clinical diagnosis and treatment. Case presentation: We report a case of LCNEC of the urinary bladder. The patient was a 79-year-old male admitted to our hospital with recurrent episodes of asymptomatic gross hematuria. Based on the computed tomography (CT) scan findings, our patient presented with a bladder mass displaying invasion into the serosal layer, suggestive of muscle involvement and indicative of malignancy. The patient received a radical cystectomy, and the postoperative pathology confirmed primary, pure LCNEC of the urinary bladder. We gave him 16 cycles of toripalimab immunotherapy. As of follow-up, the patient was alive, and periodic CT reexamination showed no evidence of recurrence. Conclusions: We reviewed domestic and foreign literature and found no explicit treatment protocols exist for the disease. Surgical resection combined with chemotherapy were the most common treatments. Herein, we reported the first case of primary, pure LCNEC of the urinary bladder treated by radical cystectomy combined with pure immunotherapy, achieving sustained remission, which provides a new idea for the immunotherapy and integrative treatment of the disease.

Gsα Regulates Macrophage Foam Cell Formation During Atherosclerosis.

BACKGROUND: Many cardiovascular pathologies are induced by signaling through G-protein-coupled receptors via Gsα (G protein stimulatory α subunit) proteins. However, the specific cellular mechanisms that are driven by Gsα and contribute to the development of atherosclerosis remain unclear. METHODS: High-throughput screening involving data from single-cell and bulk sequencing were used to explore the expression of Gsα in atherosclerosis. The differentially expression and activity of Gsα were analyzed by immunofluorescence and cAMP measurements. Macrophage-specific Gsα knockout (Mac-GsαKO) mice were generated to study the effect on atherosclerosis. The role of Gsα was determined by transplanting bone marrow and performing assays for foam cell formation, Dil-ox-LDL (oxidized low-density lipoprotein) uptake, chromatin immunoprecipitation, and luciferase reporter assays. RESULTS: ScRNA-seq showed elevated Gnas in atherosclerotic mouse aorta's cholesterol metabolism macrophage cluster, while bulk sequencing confirmed increased GNAS expression in human plaque macrophage content. A significant upregulation of Gsα and active Gsα occurred in macrophages from human and mouse plaques. Ox-LDL could translocate Gsα from macrophage lipid rafts in short-term and promote Gnas transcription through ERK1/2 activation and C/EBPß phosphorylation via oxidative stress in long-term. Atherosclerotic lesions from Mac-GsαKO mice displayed decreased lipid deposition compared with those from control mice. Additionally, Gsα deficiency alleviated lipid uptake and foam cell formation. Mechanistically, Gsα increased the levels of cAMP and transcriptional activity of the cAMP response element binding protein, which resulted in increased expression of CD36 and SR-A1. In the translational experiments, inhibiting Gsα activation with suramin or cpGN13 reduced lipid uptake, foam cell formation, and the progression of atherosclerotic plaques in mice in vivo. CONCLUSIONS: Gsα activation is enhanced during atherosclerotic progression and increases lipid uptake and foam cell formation. The genetic or chemical inactivation of Gsα inhibit the development of atherosclerosis in mice, suggesting that drugs targeting Gsα may be useful in the treatment of atherosclerosis.

Identification of shared pathogenetic mechanisms between COVID-19 and IC through bioinformatics and system biology.

COVID-19 increased global mortality in 2019. Cystitis became a contributing factor in SARS-CoV-2 and COVID-19 complications. The complex molecular links between cystitis and COVID-19 are unclear. This study investigates COVID-19-associated cystitis (CAC) molecular mechanisms and drug candidates using bioinformatics and systems biology. Obtain the gene expression profiles of IC (GSE11783) and COVID-19 (GSE147507) from the Gene Expression Omnibus (GEO) database. Identified the common differentially expressed genes (DEGs) in both IC and COVID-19, and extracted a number of key genes from this group. Subsequently, conduct Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the DEGs. Additionally, design a protein-protein interaction (PPI) network, a transcription factor gene regulatory network, a TF miRNA regulatory network, and a gene disease association network using the DEGs. Identify and extract hub genes from the PPI network. Then construct Nomogram diagnostic prediction models based on the hub genes. The DSigDB database was used to forecast many potential molecular medicines that are associated with common DEGs. Assess the precision of hub genes and Nomogram models in diagnosing IC and COVID-19 by employing Receiver Operating Characteristic (ROC) curves. The IC dataset (GSE57560) and the COVID-19 dataset (GSE171110) were selected to validate the models' diagnostic accuracy. A grand total of 198 DEGs that overlapped were found and chosen for further research. FCER1G, ITGAM, LCP2, LILRB2, MNDA, SPI1, and TYROBP were screened as the hub genes. The Nomogram model, built using the seven hub genes, demonstrates significant utility as a diagnostic prediction model for both IC and COVID-19. Multiple potential molecular medicines associated with common DEGs have been discovered. These pathways, hub genes, and models may provide new perspectives for future research into mechanisms and guide personalised and effective therapeutics for IC patients infected with COVID-19.

Smooth muscle liver kinase B1 inhibits foam cell formation and atherosclerosis via direct phosphorylation and activation of SIRT6.

Foam cell formation is a hallmark of the early phase of atherosclerosis. Growing evidence has demonstrated that vascular smooth muscle cells (VSMCs) comprise a considerable proportion of foam cells. Liver kinase B1 (LKB1) plays a crucial part in cardiovascular diseases. However, the role of LKB1 in VSMC-derived foam cell formation and atherosclerosis remains unclear. To explore the effects of LKB1 on VSMC-derived foam cell formation and atherosclerosis, we generated smooth muscle-specific LKB1 knockout (LKB1SMKO) mice by crossbreeding LKB1flox/flox mice with SM22α-CreERT2 mice. LKB1 expression decreased in plaque-loaded aortas and oxidized low-density lipoprotein (oxLDL)-treated VSMCs. Compared with controls, atherosclerosis development was exacerbated in LKB1SMKO mice via the promotion of VSMC-derived foam cell formation. Conversely, LKB1 overexpression inhibited lipid uptake and foam cell formation in VSMCs. Mechanistically, LKB1 binds to SIRT6 and directly phosphorylates and activates it, thereby reducing lectin-like oxLDL receptor-1 (LOX-1) via SIRT6-dependent histone deacetylation. Finally, adeno-associated virus (AAV)-mediated LOX-1 deficiency in smooth muscle ameliorated atherosclerosis in LKB1SMKO mice. Our findings suggest that LKB1 may modulate VSMC-derived foam cell formation and atherosclerosis via the phosphorylation and activation of SIRT6.

RhoGDIα regulates spermatogenesis through Rac1/cofilin/F-actin signaling.

Spermatogenesis is an extremely complex process, and any obstruction can cause male infertility. RhoGDIα has been identified as a risk of male sterility. In this study, we generate RhoGDIα knockout mice, and find that the males have severely low fertility. The testes from RhoGDIα-/- mice are smaller than that in WT mice. The numbers of spermatogonia and spermatocytes are decreased in RhoGDIα-/- testis. Spermatogenesis is compromised, and spermatocyte meiosis is arrested at zygotene stage in RhoGDIα-/- mice. Acrosome dysplasia is also observed in sperms of the mutant mice. At the molecular level, RhoGDIα deficiency activate the LIMK/cofilin signaling pathway, inhibiting F-actin depolymerization, impairing testis and inducing low fertility in mouse. In addition, the treatment of RhoGDIα-/- mice with Rac1 inhibitor NSC23766 alleviate testis injury and improve sperm quality by inhibiting the LIMK/cofilin/F-actin pathway during spermatogenesis. Together, these findings reveal a previously unrecognized RhoGDIα/Rac1/F-actin-dependent mechanism involved in spermatogenesis and male fertility.

Progressive degeneration of the retina in Loxl3 mutant mouse model of Stickler syndrome.

Stickler syndrome is a multisystem collagenopathy with affected individuals exhibiting a high rate of ocular complications. Lysyl oxidase-like 3 (LOXL3) is a human disease gene candidate with a critical role in catalyzing collagen crosslinking. A homozygous missense variant of LOXL3 was reported in Stickler syndrome with severe myopia. However, the underlying mechanisms of the LOXL3 missense mutation that causes Stickler syndrome are unknown. In this study, a mouse model of Stickler syndrome induced by LOXL3 mutation (c.2027G â€‹> â€‹A, p.Cys676Try) was obtained using CRISPR/Cas9 gene editing techniques. The Loxl3 mutant mice exhibited perinatal death, spinal deformity, and cleft palate, and Loxl3 mutation also induced skeletal dysplasia and progressive visual degeneration. Furthermore, we observed the damage of the bruch's membrane (BrM) and an increase in the levels of glial fibrillary acidic protein (GFAP) and Rpe65 in the Loxl3 mutant mice. Thus, we provided the critical in vivo evidence that Loxl3 possibly has a pivotal role in maintaining the eye function.

Hadh deficiency induced oligoasthenoteratozoospermia through the TNF-α/Bcl-2 pathway in male mice.

The process of spermatogenesis is a complex and delicate process that is still not fully understood. In this study, we examined the role of fatty acid oxidase 3-hydroxy acyl CoA dehydrogenase (HADH) in maintaining normal spermatogenesis in mice. In male mice, ablation of the Hadh gene using CRISPR/Cas9 technology arrested spermatocyte meiosis, increased multinucleated giant germ cells and vacuoles in seminiferous tubules, and accompanied with acrosomal dysplasia. Hadh-/- male mice showed the typical features of oligoasthenoteratozoospermia (OAT), including decreased sperm concentration and motility and increased sperm abnormalities. Next, we explored the molecular events in the testes of the mutant mice. We found fatty acids accumulated in the testis of Hadh-/- mice. And also, inflammatory factors TNF-α, IL-1ß, and IL-6 were significantly increased, apoptosis-related protein Bcl-2 was decreased, and Bax and cleaved-Caspase3 were increased in Hadh-/- male mice testis. After using etanercept, a specific inhibitor of TNF-α, testis injury caused by Hadh knockout was significantly alleviated, the sperm quality and motility were improved, and germ cell apoptosis was reduced. So our study demonstrated that Hadh deletion caused an increase in fatty acids. The accumulated fatty acids further induced testicular inflammation and germ cell apoptosis through the TNF-α/Bcl-2 signaling pathway, finally resulting in OAT in the Hadh-/- mice. Inhibiting TNF-α may be used as a new treatment approach for testicular inflammation and OAT.

Endothelial G protein stimulatory α-subunit is a critical regulator of post-ischemic angiogenesis.

Post-ischemic angiogenesis is a vital pathophysiological process in diseases such as peripheral arterial disease (PAD), heart ischemia, and diabetic retinopathy. The molecular mechanisms of post-ischemic angiogenesis are complicated and not fully elucidated. The G protein stimulatory alpha subunit (Gsα) is essential for hormone-stimulated cyclic adenosine monophosphate (cAMP) production and is an important regulator for many physiological processes. In the present study, we investigated the role of endothelial Gsα in post-ischemic angiogenesis by generating adult mice with endothelial-specific Gsα deficiency (GsαECKO). GsαECKO mice had impaired blood flow recovery after hind limb ischemic injury, and reduced neovascularization in allograft transplanted tumors. Mechanically, Gsα could regulate the expression of angiogenic factor with G patch and FHA domains 1 (AGGF1) through cAMP/CREB pathway. AGGF1 plays a key role in angiogenesis and regulates endothelial cell proliferation as well as migration. Knockdown of CREB or mutation of the CRE site on the AGGF1 promoter led to reduced AGGF1 promoter activity. In addition, knockdown of AGGF1 reduced the proangiogenic effect of Gsα in endothelial cells, and overexpression of AGGF1 reversed the impaired angiogenesis in GsαECKO mice in vivo. The finding may prove useful in designing new therapeutic targets for treatments of post-ischemic angiogenesis-related diseases.

Stimulatory G-Protein α Subunit Modulates Endothelial Cell Permeability Through Regulation of Plasmalemma Vesicle-Associated Protein.

Endothelial cell leakage occurs in several diseases. Intracellular junctions and transcellular fashion are involved. The definite regulatory mechanism is complicated and not fully elucidated. The alpha subunit of the heterotrimeric G-stimulatory protein (Gsα) mediates receptor-stimulated production of cyclic adenosine monophosphate (cAMP). However, the role of Gsα in the endothelial barrier remains unclear. In this study, mice with knockout of endothelial-specific Gsα (GsαECKO) were generated by crossbreeding Gsαflox/flox mice with Cdh5-CreERT2 transgenic mice, induced in adult mice by tamoxifen treatment. GsαECKO mice displayed phenotypes of edema, anemia, hypoproteinemia and hyperlipoproteinemia, which indicates impaired microvascular permeability. Mechanistically, Gsα deficiency reduces the level of endothelial plasmalemma vesicle-associated protein (PLVAP). In addition, overexpression of Gsα increased phosphorylation of cAMP response element-binding protein (CREB) as well as the mRNA and protein levels of PLVAP. CREB could bind to the CRE site of PLVAP promoter and regulate its expression. Thus, Gsα might regulate endothelial permeability via cAMP/CREB-mediated PLVAP expression.

Loss of BAF (mSWI/SNF) chromatin-remodeling ATPase Brg1 causes multiple malformations of cortical development in mice.

Mutations in genes encoding subunits of the BAF (BRG1/BRM-associated factor) complex cause various neurodevelopmental diseases. However, the underlying pathophysiology remains largely unknown. Here, we analyzed the function of Brahma-related gene 1 (Brg1), a core ATPase of BAF complexes, in the developing cerebral cortex. Loss of Brg1 causes several morphological defects resembling human malformations of cortical developments (MCDs), including microcephaly, cortical dysplasia, cobblestone lissencephaly and periventricular heterotopia. We demonstrated that neural progenitor cell renewal, neuronal differentiation, neuronal migration, apoptotic cell death, pial basement membrane and apical junctional complexes, which are associated with MCD formation, were impaired after Brg1 deletion. Furthermore, transcriptome profiling indicated that a large number of genes were deregulated. The deregulated genes were closely related to MCD formation, and most of these genes were bound by Brg1. Cumulatively, our study indicates an essential role of Brg1 in cortical development and provides a new possible pathogenesis underlying Brg1-based BAF complex-related neurodevelopmental disorders.

Gstm1/Gstt1 is essential for reducing cisplatin ototoxicity in CBA/CaJ mice.

Cisplatin is a widely used chemotherapeutic agent. However, its clinical utility is limited because of cisplatin-induced ototoxicity. Glutathione S-transferase (GST) was found to play a vital role in reducing cisplatin ototoxicity in mice. Deletion polymorphisms of GSTM1 and GSTT1, members of the GST family, are common in humans and are presumed to be associated with cisplatin-induced hearing impairment. However, the specific roles of GSTM1 and GSTT1 in cisplatin ototoxicity are not completely clear. Here, under cisplatin treatment, simultaneous deletion of Gstm1 and Gstt1 lead to a more profound hearing loss in CBA/CaJ mice (Gstm1/Gstt1-DKO) than in wild-type mice. The Gstm1/Gstt1-DKO mice, in which phase II detoxification genes were upregulated, exhibited more severe oxidative stress and higher outer hair cell apoptosis in the cochleae than the control mice. Thus, our study revealed that Gstm1 and Gstt1 protect auditory hair cells from cisplatin-induced ototoxicity in the CBA/CaJ mice, and genetic screening for GSTM1 and GSTT1 polymorphisms could help determine a standard cisplatin dose for cancer patients undergoing chemotherapy.

Activation of Rictor/mTORC2 signaling acts as a pivotal strategy to protect against sensorineural hearing loss.

The Food and Drug Administration­approved drug sirolimus, which inhibits mechanistic target of rapamycin (mTOR), is the leading candidate for targeting aging in rodents and humans. We previously demonstrated that sirolimus could treat ARHL in mice. In this study, we further demonstrate that sirolimus protects mice against cocaine-induced hearing loss. However, using efficacy and safety tests, we discovered that mice developed substantial hearing loss when administered high doses of sirolimus. Using pharmacological and genetic interventions in murine models, we demonstrate that the inactivation of mTORC2 is the major driver underlying hearing loss. Mechanistically, mTORC2 exerts its effects primarily through phosphorylating in the AKT/PKB signaling pathway, and ablation of P53 activity greatly attenuated the severity of the hearing phenotype in mTORC2-deficient mice. We also found that the selective activation of mTORC2 could protect mice from acoustic trauma and cisplatin-induced ototoxicity. Thus, in this study, we discover a function of mTORC2 and suggest that its therapeutic activation could represent a potentially effective and promising strategy to prevent sensorineural hearing loss. More importantly, we elucidate the side effects of sirolimus and provide an evaluation criterion for the rational use of this drug in a clinical setting.

Deletion of Smooth Muscle Lethal Giant Larvae 1 Promotes Neointimal Hyperplasia in Mice.

Vascular smooth muscle cell (VSMC) proliferation and migration contribute to neointimal hyperplasia after injury, which causes vascular remodeling related to arteriosclerosis, hypertension, and restenosis. Lethal giant larvae 1 (LGL1) is a highly conserved protein and plays an important role in cell polarity and tumor suppression. However, whether LGL1 affects neointimal hyperplasia is still unknown. In this study, we used smooth muscle-specific LGL1 knockout (LGL1SMKO) mice generated by cross-breeding LGL1flox/flox mice with α-SMA-Cre mice. LGL1 expression was significantly decreased during both carotid artery ligation in vivo and PDGF-BB stimulation in vitro. LGL1 overexpression inhibited the proliferation and migration of VSMCs. Mechanistically, LGL1 could bind with signal transducer and activator of transcription 3 (STAT3) and promote its degradation via the proteasomal pathway. In the carotid artery ligation animal model, smooth muscle-specific deletion of LGL1 accelerated neointimal hyperplasia, which was attenuated by the STAT3 inhibitor SH-4-54. In conclusion, LGL1 may inhibit neointimal hyperplasia by repressing VSMC proliferation and migration via promoting STAT3 proteasomal degradation.

Deficiency of Klc2 Induces Low-Frequency Sensorineural Hearing Loss in C57BL/6 J Mice and Human.

The transport system in cochlear hair cells (HCs) is important for their function, and the kinesin family of proteins transports numerous cellular cargos via the microtubule network in the cytoplasm. Here, we found that Klc2 (kinesin light chain 2), the light chain of kinesin-1 that mediates cargo binding and regulates kinesin-1 motility, is essential for cochlear function. We generated mice lacking Klc2, and they suffered from low-frequency hearing loss as early as 1 month of age. We demonstrated that deficiency of Klc2 resulted in abnormal transport of mitochondria and the down-regulation of the GABAA receptor family. In addition, whole-genome sequencing (WGS) of patient showed that KLC2 was related to low-frequency hearing in human. Hence, to explore therapeutic approaches, we developed adeno-associated virus containing the Klc2 wide-type cDNA sequence, and Klc2-null mice delivered virus showed apparent recovery, including decreased ABR threshold and reduced out hair cell (OHC) loss. In summary, we show that the kinesin transport system plays an indispensable and special role in cochlear HC function in mice and human and that mitochondrial localization is essential for HC survival.

Liver kinase B1 inhibits smooth muscle calcification via high mobility group box 1.

Vascular calcification is a common pathological feature of atherosclerosis, chronic kidney disease, vascular injury, and aging. Liver kinase B1 (LKB1) plays pivotal roles in cellular processes such as apoptosis, metabolism, and cell cycle regulation. In addition, growing evidence has indicated that LKB1 functions as a tumor suppressor gene. However, its role in vascular calcification has not been reported. LKB1flox/flox mice were hybridized with SM22-CreERT2 transgenic mice and adult mice received tamoxifen to obtain smooth muscle-specific LKB1-knockout (LKB1SMKO) mice. LKB1 expression was decreased under calcifying conditions, and LKB1 overexpression had a protective effect on vascular calcification. However, high mobility group box 1 (HMGB1) overexpression partially counteracted the promotion of vascular calcification induced by LKB1 overexpression. Mechanically, LKB1 could bind to HMGB1 to promote HMGB1 degradation. Furthermore, LKB1SMKO mice showed intensified vascular calcification, which was alleviated by treatment with the HMGB1 inhibitor glycyrrhizic acid. Based on our results, LKB1 may inhibit vascular calcification via inhibiting HMGB1 expression.

Responsive hyaluronic acid-gold cluster hybrid nanogel theranostic systems.

Tumor microenvironment responsive and self-monitored multimodal synergistic theranostic strategies can significantly improve therapeutic efficacy by overcoming biological barriers. Herein, we report a type of smart fluorescent hyaluronic acid nanogel that can respond to the reducing microenvironment and activate tumor targeting with light-traceable monitoring in cancer therapy. First, the derivative of hyaluronic acid (HA) with a vinyl group and cystamine bisacrylamide were used to synthesize bioreducible HA based nanogels via copolymerization in aqueous medium. Then, multifunctional mHA-gold cluster (mHA-GC) hybrid nanogels were successfully prepared by the in situ reduction of gold salt in the HA nanogels. The HA matrix turns the nanogels into a capsule for effective drug loading with excellent colloidal stability. Interestingly, the reducing tumor microenvironment dramatically enhanced the fluorescence signal of gold clusters in the hybrid nanogels. The highly selective cancer cell uptake and efficient intratumoral accumulation of the hybrid nanogels were demonstrated by fluorescence tracking of these nanogels. Responsive disassembly of the hybrid nanogels and drug release were triggered by excess glutathione presence in cancer cells. Moreover, in vivo and in vitro tumor suppression assays revealed that the doxorubicin-loaded hybrid nanogels exhibited significantly superior tumor cell inhibition abilities compared to free DOX. Overall, the mHA-GC hybrid nanogels emerge as a promising theranostic nanoplatform for the targeted delivery and controlled release of antitumor drugs with light-traceable monitoring in cancer treatment.

WWOX promotes apoptosis and inhibits autophagy in paclitaxel­treated ovarian carcinoma cells.

Although paclitaxel (PTX) is a first­line chemotherapeutic agent for the treatment of epithelial ovarian cancer (EOC), its clinical use is restricted by chemoresistance. Autophagy is believed to promote drug resistance, and WW domain­containing oxidoreductase (WWOX) has been predicted to serve an essential role in apoptosis induction and to suppress autophagy in various tumor cell types. In the present study, the role of WWOX was demonstrated using PTX­treated EOC cells. Cell viability and apoptosis were detected using Cell Counting Kit­8, morphological and flow cytometric analyses. WWOX and phosphorylated (p)­WWOX were highly expressed in PTX­treated sensitive EOC cells (A2780), which was accompanied by activation of the apoptosis­related proteins caspase­3 and poly (ADP­ribose) polymerase (PARP). Conversely, PTX­resistant EOC cells (A2780/T) were characterized by reduced WWOX expression and constant phosphorylation levels, as well as undetectable levels of activated caspase­3 and PARP when cells were treated with PTX. The altered expression of WWOX between the two cell types was further validated by reverse transcription­-quantitative PCR. The apoptosis­inducing role of WWOX was also confirmed by flow cytometry after WWOX overexpression was induced in PTX­treated A2780 cells. These findings indicated that WWOX activation may inhibit chemoresistance and trigger cancer cell death. The upregulated expression levels of the autophagy­related protein 12­5 complex, Beclin­1 and LC3, as well as the downregulation of P62, were also detected following PTX treatment, suggesting that PTX induced autophagic flux in both types of EOC cells. This conclusion was further supported by visualizing the accumulation of autophagosome and autolysosome vesicles, using confocal microscopy and transmission electron microscopy. PTX was also shown to inhibit mTOR signaling, indicated by a decreased level of p­mTOR and increased expression of eukaryotic translation initiation factor 4E­binding protein 1. Finally, the interaction between WWOX, mTOR and autophagy was investigated via WWOX transfection experimentation, and indicated that WWOX activated mTOR whilst inhibiting autophagy. These data indicated that WWOX may serve a critical role in PTX­induced apoptosis and could suppress autophagy by downregulating essential autophagic effectors in EOC cells via mTOR signaling.