LAMC2 regulates proliferation, migration, and invasion mediated by the Pl3K/AKT/mTOR pathway in oral

Background:Oral squamous cell carcinoma(OSCC)is a common malignant tumor.Recently,Laminin Gamma 2(LAMC2)has been shown to be abnormally expressed in OSCC;however,how LAMC2 signaling contributes to the occurrence and development of OSCC and the role of autophagy in OSCC has not been fully explored.This study aimed to analyze the role and mechanism of LAMC2 signaling in OSCC and the involvement of autophagy in OSCC.Methods:To explore the mechanism by which LAMC2 is highly expressed in OSCC,we used small interfering RNA(siRNA)to knock down LAMC2 to further observe the changes in the signaling pathway.Furthermore,we used cell proliferation assays,Transwell invasion assays,and wound-healing assays to observe the changes in OSCC proliferation,invasion,and metastasis.RFP-LC3 was used to detect the level of autophagy intensity.A cell line-derived xenograft(CDX)model was used to detect the effect of LAMC2 on tumor growth in vivo.Results:This study found that the level of autophagy was correlated with the biological behavior of OSCC.The downregulation of LAMC2 activated autophagy and inhibited OSCC proliferation,invasion,and metastasis via inhibiting the PI3K/AKT/mTOR pathway.Moreover,autophagy has a dual effect on OSCC,and the synergistic downregulation of LAMC2 and autophagy can inhibit OSCC metastasis,invasion,and proliferation via the PI3K/AKT/mTOR pathway.Conclusions:LAMC2 interacts with autophagy to regulate OSCC metastasis,invasion,and proliferation via the PI3K/AKT/mTOR pathway.LAMC2 down-regulation can synergistically modulate autophagy to inhibit OSCC migration,invasion,and proliferation.

Antitumor activity of miR-188-3p in gastric cancer is achieved by targeting CBL expression and inactivating the AKT/mTOR signaling

BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer and paired normal tissues were collected to analyze miR-188-3p and CBL expression.Normal and gastric cancer cells were used to manipulate miR-188-3p and CBL expression through different assays.The relationship between miR-188-3p and CBL was predicted bioinformatically and confirmed using a luciferase gene reporter assay.A Kaplan-Meier analysis was used to associate miR-188-3p or CBL expression with patient survival.A nude mouse tumor cell xenograft assay was used to confirm the in vitro data.RESULTS MiR-188-3p was found to be lower in the plasma of gastric cancer patients,tissues,and cell lines compared to their healthy counterparts.It was associated with overall survival of gastric cancer patients(P<0.001),tumor differentiation(P<0.001),lymph node metastasis(P=0.033),tumor node metastasis stage(I/II vs III/IV,P=0.024),and American Joint Committee on Cancer stage(I/II vs III/IV,P=0.03).Transfection with miR-188-3p mimics reduced tumor cell growth and invasion while inducing apoptosis and autophagy.CBL was identified as a direct target of miR-188-3p,with its expression antagonizing the effects of miR-188-3p on gastric cancer(GC)cell proliferation by inducing tumor cell apoptosis and autophagy through the inactivation of the Akt/mTOR signaling pathway.The in vivo data confirmed antitumor activity via CBL downregulation in gastric cancer.CONCLUSION The current data provides ex vivo,in vitro,and in vivo evidence that miR-188-3p acts as a tumor suppressor gene or possesses antitumor activity in GC.

Ligustilide protects PC12 cells from oxygen-glucose deprivation/reoxygenation-induced apoptosis via the LKB1-AMPK-mTOR signaling pathway

Autophagy has been shown to have a protective effect against brain damage.Ligustilide(LIG)is a bioactive substance isolated from Ligusticum chuanxiong,a traditional Chinese medicine.LIG has a neuroprotective effect;however,it is unclear whether this neuroprotective effect involves autophagy.In this study,PC12 cells were treated with 1×10^-5–1×10^-9 M LIG for 0,3,12 or 24 hours,and cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay.Treatment with 1×10^-6 M LIG for 3 hours had the greatest effect on cell proliferation,and was therefore used for subsequent experiments.PC12 cells were pre-treated with 1×10^-6 M LIG for 3 hours,cultured in 95%N2/5%CO2 in Dulbecco’s modified Eagle’s medium without glucose or serum for 4 hours,and then cultured normally for 16 hours,to simulate oxygen-glucose deprivation/reoxygenation(OGD/R).Cell proliferation was assessed with the MTS assay.Apoptosis was detected by flow cytometry.The expression levels of apoptosis-related proteins,Bcl-2 and Bax,autophagy-related proteins,Beclin 1 and microtubule-associated protein l light chain 3B(LC3-II),and liver kinase B1(LKB1)-5′-adenosine monophosphate-activated protein kinase(AMPK)-mammalian target of rapamycin(mTOR)signaling pathway-related proteins were assessed by western blot assay.Immunofluorescence staining was used to detect LC3-II expression.Autophagosome formation was observed by electron microscopy.LIG significantly decreased apoptosis,increased Bcl-2,Beclin 1 and LC3-II expression,decreased Bax expression,increased LC3-II immunoreactivity and the number of autophagosomes,and activated the LKB1-AMPK-mTOR signaling pathway in PC12 cells exposed to OGD/R.The addition of the autophagy inhibitor 3-methyladenine or dorsomorphin before OGD/R attenuated the activation of the LKB1-AMPK-mTOR signaling pathway in cells treated with LIG.Taken together,our findings show that LIG promotes autophagy and protects PC12 cells from apoptosis induced by OGD/R via the LKB1-AMPK-mTOR signaling pathway.

Quercetin modulates ovarian autophagy-related molecules and stereological parameters in a rat model of PCOS

Objective:To examine the effect of quercetin on stereological parameters and autophagy-related genes in ovaries of polycystic ovary syndrome(PCOS)rats.Methods:Fifty female Sprague-Dawley rats were randomly divided into five groups:the control group,the ethanol group,the quercetin group(15 mg/kg/day),the PCOS group,as well as the PCOS+quercetin group.After the induction of PCOS,quercetin was administered orally for 30 days.Histological,stereological and real-time PCR analyses were carried out to evaluate the effect of quercetin on PCOS rats.Results:Stereological analysis revealed that quercetin significantly increased the number of ovarian follicles and the volume of corpus luteum and induced a significant decrease in atretic follicles in comparison to the PCOS group.In addition,quercetin markedly increased mTOR gene expression while decreasing Beclin-1 and LC3 gene expression.Conclusions:Quercetin strongly modulates the expression of ovarian autophagy-related genes and stereological parameters in PCOS rats.Therefore,it can be considered as an ameliorative component for ovarian follicular impairments.

Autophagy: novel insights into therapeutic target of electroacupuncture against cerebral ischemia/reperfusion injury

Electroacupuncture is known as an effective adjuvant therapy in ischemic cerebrovascular disease. However, its underlying mechanisms remain unclear. Studies suggest that autophagy, which is essential for cell survival and cell death, is involved in cerebral ischemia reperfusion injury and might be modulate by electroacupuncture therapy in key ways. This paper aims to provide novel insights into a therapeutic target of electroacupuncture against cerebral ischemia/reperfusion injury from the perspective of autophagy. Here we review recent studies on electroacupuncture regulation of autophagy-related markers such as UNC-51-like kinase-1 complex, Beclin1, microtubule-associated protein-1 light chain 3, p62, and autophagosomes for treating cerebral ischemia/reperfusion injury. The results of these studies show that electroacupuncture may affect the initiation of autophagy, vesicle nucleation, expansion and maturation of autophagosomes, as well as fusion and degradation of autophagolysosomes. Moreover, studies indicate that electroacupuncture probably modulates autophagy by activating the mammalian target of the rapamycin signaling pathway.This review thus indicates that autophagy is a therapeutic target of electroacupuncture treatment against ischemic cerebrovascular diseases.

Schwann cells differentiated from skin-derived precursors provide neuroprotection via autophagy inhibition in a cellular model of Parkinson’s disease

Autophagy has been shown to play an important role in Parkinson’s disease.We hypothesized that skin-derived precursor cells exhibit neuroprotective effects in Parkinson’s disease through affecting autophagy.In this study,6-hydroxydopamine-damaged SH-SY5Y cells were pretreated with a culture medium containing skin-derived precursors differentiated into Schwann cells(SKP-SCs).The results showed that the SKP-SC culture medium remarkably enhanced the activity of SH-SY5Y cells damaged by 6-hydroxydopamine,reduced excessive autophagy,increased tyrosine hydroxylase expression,reducedα-synuclein expression,reduced the autophagosome number,and activated the PI3K/AKT/mTOR pathway.Autophagy activator rapamycin inhibited the effects of SKP-SCs,and autophagy inhibitor 3-methyladenine had the opposite effect.These findings confirm that SKP-SCs modulate the PI3K/AKT/mTOR pathway to inhibit autophagy,thereby exhibiting a neuroprotective effect in a cellular model of Parkinson’s disease.This study was approved by the Animal Ethics Committee of Laboratory Animal Center of Nantong University(approval No.S20181009-205)on October 9,2018.

Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.

Leptin Modulates the Differentiation of Keratinocytes via Autophagy in Psoriasis Patients With Metabolic Syndrome

Objective:Psoriasis is associated with a high prevalence of metabolic syndrome(MS),and patients with concomitant psoriasis and MS are more severely affected and less responsive to treatment.However,the molecular mechanisms behind these effects are unknown.Recent studies have shown that leptin may serve as a molecular link between psoriasis and MS,suggesting that high leptin concentrations may exacerbate psoriasis.However,the molecular mechanism of this effect is still unclear.We aimed to investigate the effect of leptin on autophagy in patients with psoriasis.Methods:From January 2021 to June 2022 in PLA General Hospital,we enrolled 51 patients with psoriasis,including 21 patients with MS and 30 without MS,and 30 healthy controls who had undergone nevus surgery.We measured the epidermal leptin,P62,and LC3B concentrations of patients by immunohistochemistry,and measured the serum leptin concentration by enzyme-linked immunosorbent assay.We then performed correlation analyses to compare these proteins’concentrations between patients with concomitant psoriasis and MS,patients with psoriasis alone,and healthy control groups.Additionally,we performed western blotting after in vitro culture of HaCaT cells with different concentrations of leptin and measured the expression levels of the autophagy markers Beclin1,LC3B,and P62;the differentiation markers K10,K16,and K17;and PI3K/AKT/mTOR signaling pathway-related proteins of HaCat cells.Next,we transfected ATG5 into HaCaT cells to revert autophagy and used the specific PI3K inhibitor LY294002 to block PI3K/AKT/mTOR signaling.The expression levels of K10,K16,and K17 of HaCat cells were again measured.One-way analysis of variance was used for the comparison of means of multiple samples,and LSD-t post hoc test was used for comparison between the 2 groups.The counting data were analyzed by the chisquare test.Correlations were evaluated by Pearson correlation analysis.Results:The serum leptin concentration was significantly higher in patients with concomitant psoriasis and MS than in patients with psoriasis alone,and healthy controls(1,330.0±244.2 pg/mL,1,041.0±282.7 pg/mL,and 760.4±361.1 pg/mL,P<0.001).Optical density of epidermal leptin concentration was significantly higher in patients with psoriasis and MS than in patients with psoriasis alone and healthy controls(0.59±0.15,0.39±0.12,and 0.27±0.19,P<0.001).The level of the autophagy marker LC3B was strongly reduced and that of P62 was strongly increased in the epidermis of patients with concomitant psoriasis and MS compared with patients with psoriasis alone and healthy controls(optical density value:LC3B:0.27±0.11,0.29±0.13,and 0.46±0.17,P<0.001;P62:0.18±0.08,0.13±0.03,and 0.10±0.03,P<0.001).We also observed a positive correlation between leptin and P62 concentrations in the blood(r=0.40,P<0.001)and epidermis(r=0.27,P=0.017),and a negative correlation between serum leptin concentrations and epidermal LC3B concentrations(r=−0.39,P<0.001).In vitro,leptin significantly decreased Beclin1 and LC3B and increased P62.Western blotting showed that leptin treatment resulted in decreased expression of K10,and increased expressions of K16 and K17;when the decrease in autophagy was restored by ATG5,this phenomenon was reversed.In addition,leptin treatment significantly upregulated the expressions of phosphorylated PI3K,AKT,and mTOR in HaCaT cells compared with the control treatment;when the expression of phosphorylated PI3K was significantly inhibited by LY294002,leptin did not reverse the decreased expression of these proteins.Conclusion:Leptin is negatively associated with autophagy in psoriasis,and leptin markedly decreased autophagy and affected keratinocyte differentiation by downregulating autophagy via the PI3K/AKT/mTOR pathway.Our study enhances the understanding of leptin as the link between MS and psoriasis and provides potential therapeutic targets for patients with concomitant psoriasis and MS.

Insulin enhances apoptosis induced by cisplatin in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy

Background Chemoresistance is common among patients with esophageal squamous cell carcinoma （ESCC）.We investigated the effect and mechanism of insulin on enhancing anticancer functions of cisplatin in human esophageal cancer cell line EC9706.Methods The viability of EC9706 cells exposed to cisplatin was assessed using MTT assay.The times T1,when the number of living cells reached a plateau and T2,when the number of living cells reached a new plateau after the addition of insulin were found.T1 and T2 plateau cells were stained by Annexin V-FITC/PI and monodansylcadaverin （MDC）.Fluorescent microscopy was used to observe the expression of apoptosis and autophagy intuitively.Apoptotic ratio and fluorescent intensity were analysed by flow cytometry （FCM） quantitatively.Western blotting analysis was used to estimate the protein expression levels of AKT,mTOR,PI3K,PTEN,autophage related indicator LC3-Ⅱ and autophage related protein Beclin1 changes that occurred in the course of treatment.Results A larger number of typical autophagosomes were detected in EC9706 cells exposed to cisplatin.Insulin can increase the apoptosis induced by cisplatin.Apoptotic ratio of T1 plateau cells （（32.6±4.3）％） is significantly less than T2 plateau （（47.5±5.6）％）.MDC fluorescent intensity at T1 plateau （104.9±13.2） was significantly higher than intensity at T2 plateau （82.6±10.3）.After cotreatment with insulin,the expression level of LC3-Ⅱ,Beclin1 and PTEN in T2 plateau cells were significantly downregulated,but AKT,mTOR and PI3K expressions significantly upregulated compared with T1 plateau.Conclusions Insulin could enhance cisplatin-induced apoptosis in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy.The activation of PI3K/Akt/mTOR signaling pathway induced by insulin resulted in the suppression of autophagy in EC9706 cells,which may be attributed to the anticancer effects of cisplatin.

Prostate cancer cells at a therapeutic gunpoint of the autophagy process

In a normal prostate,the process of controling cell death is essential to maintain tissue homeostasis and its inhibition may lead to the development of cancer.Androgen receptor signaling plays pivotal roles in the prostate development and homeostasis as well as in the progression of prostate cancer.The main treatment for prostate cancer is a combination of androgen deprivation therapy(ADT)using anti-androgens and docetaxil administration.However,ADT eventually fails due to a pathological unbalance of cell death processes,in particular apoptosis and autophagy.As a result prostate tumors may re-grow and progress into the castration resistant stage.The role of autophagy in tumorigenesis is complex and it could be a double-edged sword process,as autophagy defects promote cancer progression in association with various dangerous cellular processes,while functional autophagy enables cancer cell survival under stress and likely contributes to the resistance of treatment.Autophagy is often impaired in prostate cancer,due to either activation of the Akt/mTOR pathway,which normally inhibits autophagy,or through allelic loss of Beclin-1(BECN1),an essential autophagy gene.In particular,elucidating the interplay between autophagy and tumor cell metabolism will provide unique opportunities to identify new therapeutic targets and to develop synthetically lethal treatment strategies that preferentially target cancer cells,while sparing normal tissues.