Plac8‐mediated autophagy regulates nasopharyngeal carcinoma cell function via AKT/mTOR pathway

To explore the relationship between autophagy and cell function, we investigated how PLAC8‐mediated autophagy influences proliferation, apoptosis and epithelial‐mesenchymal transition (EMT) in NPC. Colony formation analyses and CCK8 assays were used to assess the proliferative capacity of NPC cells. Transmission electron microscopy (TEM) was used to identify autophagosomes. Autophagic flux was monitored using the tandem monomeric RFP‐GFP‐tagged LC3 (tfLC3) assay. The rate of apoptosis in NPC cells was analysed by flow cytometry. Western blot analysis was used to evaluate the activation of autophagy and the signalling status of the AKT/mTOR pathway. Our study reveals that knocking out PLAC8 (koPLAC8) induces autophagy and apoptosis, while suppressing NPC cell proliferation and EMT. However, inhibition of autophagy with 3‐methyladenine or by knocking down Beclin‐1 reverses the cell proliferation, apoptosis and EMT influenced by koPLAC8. We find that koPLAC8 inhibits the phosphorylation of AKT and its downstream target, mTOR. Moreover, immunofluorescence and co‐immunoprecipitation reveal complete PLAC8/AKT colocalization and PLAC8/AKT interaction, respectively. Furthermore, knockout of PLAC8 induced autophagy and inactivated AKT/mTOR signalling pathway of NPC xenografts. Overall, our findings demonstrate that koPLAC8 induces autophagy via the AKT/mTOR pathway, thereby inhibiting cell proliferation and EMT, and promoting apoptosis in NPC cells.     

Phellodendrine promotes autophagy by regulating the AMPK/mTOR pathway and treats ulcerative colitis

To investigate the therapeutic effects of phellodendrine in ulcerative colitis (UC) through the AMPK/mTOR pathway. Volunteers were recruited to observe the therapeutic effects of Compound Cortex Phellodendri Liquid (Huangbai liniment). The main components of Compound Cortex Phellodendri Liquid were analysed via network pharmacology. The target of phellodendrine was further analysed. Caco-2 cells were cultured, and H₂O₂ was used to stimulate in vitro cell model. Expression levels of LC3, AMPK, p-AMPK, mTOR and p-mTOR were detected via Western blotting and through immunofluorescence experiments. The therapeutic effects of phellodendrine were analysed via expression spectrum chip sequencing. The sequencing of intestinal flora further elucidated the therapeutic effects of phellodendrine. Compared with the control group, Compound Cortex Phellodendri Liquid could substantially improve the healing of intestinal mucosa. Network pharmacology analysis revealed that phellodendrine is the main component of Compound Cortex Phellodendri Liquid. Moreover, this alkaloid targets the AMPK signalling pathway. Results of animal experiments showed that phellodendrine could reduce the intestinal damage of UC compared with the model group. Findings of cell experiments indicated that phellodendrine treatment could activate the p-AMPK /mTOR signalling pathway, as well as autophagy. Expression spectrum chip sequencing showed that treatment with phellodendrine could promote mucosal healing and reduce inflammatory responses. Results of intestinal flora detection demonstrated that treatment with phellodendrine could increase the abundance of flora and the content of beneficial bacteria. Phellodendrine may promote autophagy by regulating the AMPK-mTOR signalling pathway, thereby reducing intestinal injury due to UC.     

Hydrogen sulphide exacerbates acute pancreatitis by over‐activating autophagy via AMPK/mTOR pathway

Previously, we have shown that hydrogen sulphide (H₂S) might be pro‐inflammatory during acute pancreatitis (AP) through inhibiting apoptosis and subsequently favouring a predominance of necrosis over apoptosis. In this study, we sought to investigate the detrimental effects of H₂S during AP specifically with regard to its regulation on the impaired autophagy. The incubated levels of H₂S were artificially intervened by an administration of sodium hydrosulphide (NaHS) or DL‐propargylglycine (PAG) after AP induction. Accumulation of autophagic vacuoles and pre‐mature activation of trypsinogen within acini, which indicate the impairment of autophagy during AP, were both exacerbated by treatment with NaHS but attenuated by treatment with PAG. The regulation that H₂S exerted on the impaired autophagy during AP was further attributed to over‐activation of autophagy rather than hampered autophagosome–lysosome fusion. To elucidate the molecular mechanism that underlies H₂S‐mediated over‐activation of autophagy during AP, we evaluated phosphorylations of AMP‐activated protein kinase (AMPK), AKT and mammalian target of rapamycin (mTOR). Furthermore, Compound C (CC) was introduced to determine the involvement of mTOR signalling by evaluating phosphorylations of downstream effecters including p70 S6 kinase (P70S6k) and UNC‐51‐Like kinase 1 (ULK1). Our findings suggested that H₂S exacerbated taurocholate‐induced AP by over‐activating autophagy via activation of AMPK and subsequently, inhibition of mTOR. Thus, an active suppression of H₂S to restore over‐activated autophagy might be a promising therapeutic approach against AP‐related injuries.     

PSPH induces cell autophagy and promotes cell proliferation and invasion in the hepatocellular carcinoma cell line Huh7 via the AMPK/mTOR/ULK1 signaling pathway

Phosphoserine phosphatase (PSPH), a key enzyme of the l -serine synthesis pathway, has been involved in cancer progression and survival. However, limited evidence revealed the PSPH influence on hepatocellular carcinoma (HCC). Herein, we observed that PSPH expression was upregulated in both HCC tissues and cell lines, which was determined by western blotting. TCGA database showed that the PSPH protein levels were significantly upregulated and affected patient survival rates in HCC. Then gain- and loss-of-function manipulations were performed by transfection with a pcDNA-PSPH expression vector or a specific short interfering RNA against PSPH in Huh7 cells. Huh7 cell proliferation, stemness, invasion, and apoptosis were assessed by using CCK-8 test, colony formation assay, Transwell assay, and Flow cytometry analysis, respectively, and levels of autophagy-related proteins were detected by using western blotting. The results showed that PSPH could induce Huh7 cell autophagy, promote cell proliferation and invasion, and inhibit apoptosis. The knockdown of PSPH could inhibit Huh7 cell proliferation, invasion, and autophagy. Furthermore, PSPH activated Liver kinase B1 (LKB1) and TGF beta-activated kinase 1 (TAK1), affected the adenosine 5′-monophosphate-activated protein kinase (AMPK)/mTOR/ULK1 signaling pathway, but could not activate calcium/calmodulin-dependent protein kinase kinase (CaMKK) in Huh7 cells. Inhibition of either LKB1, TAK1, or AMPK could eliminate the effect of PSPH overexpression on Huh7 cell behaviors. However, inhibition of CaMKK could not influence the effect of PSPH overexpression on Huh7 cell behaviors. In conclusion, PSPH could induce autophagy, promote proliferation and invasion, and inhibit apoptosis in HCC cells via the AMPK/mTOR/ULK1 signaling pathway.     

MCM7 silencing promotes cutaneous melanoma cell autophagy and apoptosis by inactivating the AKT1/mTOR signaling pathway

Cutaneous melanoma (CM) has become a major public health concern. Studies illustrate that minichromosome maintenance protein 7 (MCM7) participate in various diseases including skin disease. Our study aimed to study the effects of MCM7 silencing on CM cell autophagy and apoptosis by modulating the AKT threonine kinase 1 (AKT1)/mechanistic target of rapamycin kinase (mTOR) signaling pathway. Initially, microarray analysis was used to screen the CM-related gene expression data as well as differentially expressed genes. Subsequently, MCM7 expression vector and lentivirus RNA used for MCM7 silencing (LV-shRNA-MCM7) were constructed, and these vectors, dimethyl sulfoxide (DMSO) and AKT activator SC79 were then introduced into CM cell line SK-MEL-2 to validate the role of MCM7 in cell autophagy, viability, apoptosis, cell cycle, migration, and invasion. To further investigate the regulatory mechanisms of MCM7 in CM progress, the expression of MCM7, AKT1, mTOR, cyclin D1, as well as autophagy and apoptosis relative factors, such as LC3B, SOD2, DJ-1, p62, Bcl-2, Bax, and caspase-3 in melanoma cells was determined. MCM7 might mediate the AKT1/mTOR signaling pathway to influence the progress of melanoma. MCM7 silencing contributed to the increased expression of Bax, capase-3, and autophagy-related genes (LC3B, SOD2, and DJ-1), but decreased the expression of Bcl-2, which suggested that MCM7 silencing promoted autophagy and cell apoptosis. At the same time, MCM7 silencing also attenuated cell viability, invasion, and migration, and reduced the cyclin D1 expression and protein levels of p-AKT1 and p-mTOR. Taken together, MCM7 silencing inhibited CM via inactivation of the AKT1/mTOR signaling pathway.     

Larotrectinib induces autophagic cell death through AMPK/mTOR signalling in colon cancer

Larotrectinib (Lar) is a highly selective and potent small‐molecule inhibitor used in patients with tropomyosin receptor kinase (TRK) fusion‐positive cancers, including colon cancer. However, the underlying molecular mechanisms specifically in patients with colon cancer have not yet been explored. Our data showed that Lar significantly suppressed proliferation and migration of colon cancer cells. In addition, Lar suppressed the epithelial–mesenchymal transition (EMT) process, as evidenced by elevation in E‐cadherin (E‐cad), and downregulation of vimentin and matrix metalloproteinase (MMP) 2/9 expression. Furthermore, Lar was found to activate autophagic flux, in which Lar increased the ratio between LC3II/LC3I and decreased the expression of p62 in colon cancer cells. More importantly, Lar also increased AMPK phosphorylation and suppressed mTOR phosphorylation in colon cancer cells. However, when we silenced AMPK in colon cancer cells, Lar‐induced accumulation of autolysomes as well as Lar‐induced suppression of the EMT process were significantly diminished. An in vivo assay also confirmed that tumour volume and weight decreased in Lar‐treated mice than in control mice. Taken together, this study suggests that Lar significantly suppresses colon cancer proliferation and migration by activating AMPK/mTOR‐mediated autophagic cell death.     

Fibroblast growth factor 21 alleviates acute pancreatitis via activation of the Sirt1‐autophagy signalling pathway

Fibroblast growth factor 21 (FGF21), a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity, alleviates the process of acute pancreatitis (AP). However, its mechanism remains elusive. The pathological and physiological characteristics of FGF21 are observed in both patients with AP and cerulein‐induced AP models, and the mechanisms of FGF21 in response to AP are investigated by evaluating the impact of autophagy in FGF21‐treated mice and cultured pancreatic cells. Circulating levels of FGF21 significantly increase in both AP patients and cerulein‐induced AP mice, which is accompanied by the change of pathology in pancreatic injury. Replenishment of FGF21 distinctly reverses cerulein‐induced pancreatic injury and improves cerulein‐induced autophagy damage in vivo and in vitro. Mechanically, FGF21 acts on pancreatic acinar cells to up‐regulate Sirtuin‐1 (Sirt1) expression, which in turn repairs impaired autophagy and removes damaged organs. In addition, blockage of Sirt1 accelerates cerulein‐induced pancreatic injury and weakens the regulative effect in FGF21‐activated autophagy in mice. These results showed that FGF21 protects against cerulein‐induced AP by activation of Sirtuin‐1‐autophagy axis.     

TUSC3 inhibits cell proliferation and invasion in cervical squamous cell carcinoma via suppression of the AKT signalling pathway

The decreased expression of tumour suppressor candidate 3 (TUSC3) is associated with proliferation in several types of cancer, leading to an unfavourable prognosis. The present study aimed to assess the cellular and molecular function of TUSC3 in patients with cervical squamous cell carcinoma (CSCC). Levels of mRNA expressions of TUSC3 were analysed in CSCC tissues and six cell lines using qRT‐PCR. Immunohistochemistry(IHC) was used to evaluate the protein expression level of TUSC3 in four paired specimens, 220 paraffin‐embedded CSCC specimens and 60 cases of normal cervical tissues(NCTs), respectively. Short hairpin RNA interference was employed for TUSC3 knockdown. Cell proliferation, migration and invasion were evaluated using growth curve, MTT assay, wound healing, transwell assay and xenograft tumour model, respectively. The results demonstrated that TUSC3 mRNA and protein expression levels were downregulated in CSCC samples. Multivariate and univariate analyses indicated that TUSC3 was an independent prognostic factor for patients with CSCC. Decreased TUSC3 expression levels were significantly associated with proliferation and an aggressive phenotype of cervical cancer cells both in vitro and in vivo. Moreover, the knockdown of TUSC3 promoted migration and invasion of cancer cells, while the increased expression of TUSC3 exhibited the opposite effects. The downregulation of TUSC3 facilitated proliferation and invasion of CSCC cells through the activation of the AKT signalling pathway. Our data demonstrated that the downregulation of TUSC3 promoted CSCC cell metastasis via the AKT signalling pathway. Therefore, TUSC3 may serve as a novel prognostic marker and potential target for CSCC.     

Polycystin‐1 affects cancer cell behaviour and interacts with mTOR and Jak signalling pathways in cancer cell lines

Polycystic Kidney Disease (PKD), which is attributable to mutations in the PKD1 and PKD2 genes encoding polycystin‐1 (PC1) and polycystin‐2 (PC2) respectively, shares common cellular defects with cancer, such as uncontrolled cell proliferation, abnormal differentiation and increased apoptosis. Interestingly, PC1 regulates many signalling pathways including Jak/STAT, mTOR, Wnt, AP‐1 and calcineurin‐NFAT which are also used by cancer cells for sending signals that will allow them to acquire and maintain malignant phenotypes. Nevertheless, the molecular relationship between polycystins and cancer is unknown. In this study, we investigated the role of PC1 in cancer biology using glioblastoma (GOS3), prostate (PC3), breast (MCF7), lung (A549) and colorectal (HT29) cancer cell lines. Our in vitro results propose that PC1 promotes cell migration in GOS3 cells and suppresses cell migration in A549 cells. In addition, PC1 enhances cell proliferation in GOS3 cells but inhibits it in MCF7, A549 and HT29 cells. We also found that PC1 up‐regulates mTOR signalling and down‐regulates Jak signalling in GOS3 cells, while it up‐regulates mTOR signalling in PC3 and HT29 cells. Together, our study suggests that PC1 modulates cell proliferation and migration and interacts with mTOR and Jak signalling pathways in different cancer cell lines. Understanding the molecular details of how polycystins are associated with cancer may lead to the identification of new players in this devastating disease.     

VDAC1 promotes cardiomyocyte autophagy in anoxia/reoxygenation injury via the PINK1/Parkin pathway

Ischemia/reperfusion (I/R) is a well-known injury to the myocardium, but the mechanism involved remains elusive. In addition to the well-accepted apoptosis theory, autophagy was recently found to be involved in the process, exerting a dual role as protection in ischemia and detriment in reperfusion. Activation of autophagy is mediated by mitochondrial permeability transition pore (MPTP) opening during reperfusion. In our previous study, we showed that MPTP opening is regulated by VDAC1, a channel protein located in the outer membrane of mitochondria. Thus, upregulation of VDAC1 expression is a possible trigger to cardiomyocyte autophagy via an unclear pathway. Here, we established an anoxia/reoxygenation (A/R) model in vitro to simulate the I/R process in vivo. At the end of A/R treatment, VDAC1, Beclin 1, and LC3-II/I were upregulated, and autophagic vacuoles were increased in cardiomyocytes, which showed a connection of VDAC1 and autophagy development. These variations also led to ROS burst, mitochondrial dysfunction, and aggravated apoptosis. Knockdown of VDAC1 by RNAi could alleviate the above-mentioned cellular damages. Additionally, the expression of PINK1 and Parkin was enhanced after A/R injury. Furthermore, Parkin was recruited to mitochondria from the cytosol, which suggested that the PINK1/Parkin autophagic pathway was activated during A/R. Nevertheless, the PINK1/Parkin pathway was effectively inhibited when VDAC1 was knocked-down. Taken together, the A/R-induced cardiomyocyte injury was mediated by VDAC1 upregulation, which led to cell autophagy via the PINK1/Parkin pathway, and finally aggravated apoptosis.     

Duvelisib attenuates bleomycin‐induced pulmonary fibrosis via inhibiting the PI3K/Akt/mTOR signalling pathway

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that seriously threatens the health of patients. The pathogenesis of IPF is still unclear, and there is a lack of effective therapeutic drugs. Myofibroblasts are the main effector cells of IPF, leading to excessive deposition of extracellular matrix (ECM) and promoting the progression of fibrosis. Inhibiting the excessive activation and relieving autophagy blockage of myofibroblasts is the key to treat IPF. PI3K/Akt/mTOR pathway plays a key regulatory role in promoting fibroblast activation and autophagy inhibition in lung fibrosis. Duvelisib is a PI3K inhibitor that can simultaneously inhibit the activities of PI3K‐δ and PI3K‐γ, and is mainly used for the treatment of relapsed/refractory chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma tumour (SLL). In this study, we aimed to examine the effects of Duvelisib on pulmonary fibrosis. We used a mouse model of bleomycin‐induced pulmonary fibrosis to evaluate the effects of Duvelisib on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of Duvelisib in lung fibroblasts in vitro. The in vivo experiments showed that Duvelisib significantly alleviated bleomycin‐induced collagen deposition and improved pulmonary function. In vitro and in vivo pharmacological experiments showed that Duvelisib dose‐dependently suppressed lung fibroblast activation and improved autophagy inhibition by inhibiting the phosphorylation of PI3K, Akt and mTOR. Our results indicate that Duvelisib can alleviate the severity of pulmonary fibrosis and provide potential drugs for the treatment of pulmonary fibrosis.     

Long non‐coding RNA TNRC6C‐AS1 promotes methylation of STK4 to inhibit thyroid carcinoma cell apoptosis and autophagy via Hippo signalling pathway

The role of long non‐coding RNAs (lncRNAs) in thyroid carcinoma (TC), the most frequent endocrine malignancy, has been extensively examined. This study investigated effect of interaction among lncRNA TNRC6C‐AS1, serine/threonine‐protein kinase 4 (STK4) and Hippo signalling pathway on TC. Initially, lncRNA TNRC6C‐AS1 expression in TC tissues was detected. To explore roles of lncRNA TNRC6C‐AS1, STK4 and Hippo signalling pathway in TC progression, their expressions were altered. Interaction between lncRNA TNRC6C‐AS1 and STK4, STK4 promoter methylation, or Hippo signalling pathway was verified. After that, a series of experiments were employed to evaluate in vitro ability of apoptosis, proliferation and autophagy of TC cells and in vivo tumorigenicity, and tumour growth of TC cells. lncRNA TNRC6C‐AS1 was highly expressed while STK4 was poorly expressed in TC tissues. LncRNA TNRC6C‐AS1 promoted the STK4 methylation and down‐regulated STK4 expression, which further activated the Hippo signalling pathway. STK4 silencing was observed to promote the proliferation ability of TC cells, inhibit the apoptosis and autophagy abilities, as well as enhance the tumorigenicity and tumour growth. Moreover, the in vitro proliferation ability as well as the in vivo tumorigenicity and tumour growth of TC cells were inhibited after the blockade of Hippo signalling pathway, while the apoptosis and autophagy abilities were promoted. The results demonstrate that the lncRNA TNRC6C‐AS1 increases STK4 promoter methylation to down‐regulate STK4 expression, thereby promoting the development of TC through activation of Hippo signalling pathway. It highlights that lncRNA TNRC6C‐AS1 may be a novel therapeutic target for the treatment of TC.