Upregulation Of Atg5 Research Articles

Gubi decoction mitigates knee osteoarthritis via promoting chondrocyte autophagy through METTL3-mediated ATG7 m6A methylation.

Knee osteoarthritis (KOA) is a chronic joint disease that significantly affects the health of the elderly. As an herbal remedy, Gubi decoction (GBD) has been traditionally used for the treatment of osteoarthritis-related syndromes. However, the anti-KOA efficacy and mechanism of GBD remain unclear. This study aimed to experimentally investigate the anti-KOA efficacy and the underlying mechanism of GBD. The medial meniscus (DMM) mice model and IL-1β-stimulated chondrocytes were, respectively, constructed as invivo and invitro models of KOA to evaluate the osteoprotective effect and molecular mechanism of GBD. The UPLC-MS/MS analysis showed that GBD mainly contained pinoresinol diglucoside, rehmannioside D, hesperidin, liquiritin, baohuoside I, glycyrrhizic acid, kaempferol and tangeretin. Animal experiment showed that GBD could alleviate articular cartilage destruction and recover histopathological alterations in DMM mice. In addition, GBD inhibited chondrocyte apoptosis and restored DMM-induced dysregulated autophagy evidenced by the upregulation of ATG7 and LC3 II/LC3 I but decreased P62 level. Mechanistically, METTL3-mediated m6A modification decreased the expression of ATG7 in DMM mice, as it could be significantly attenuated by GBD. METTL3 overexpression significantly counteracted the protective effect of GBD on chondrocyte autophagy. Further research showed that GBD promoted proteasome-mediated ubiquitination degradation of METLL3. Our findings suggest that GBD could act as a protective agent against KOA. The protective effect of GBD may result from its promotion on chondrocyte autophagy by suppressing METTL3-dependent ATG7 m6A methylation.

Regulatory feedback loop between circ-EIF4A3 and EIF4A3 Enhances autophagy and growth in colorectal cancer cells

Recent studies indicate that circular RNAs (circRNAs) are crucial in the progression of colorectal cancer (CRC). Eukaryotic translation initiation factor 4A3 (EIF4A3) has been identified as a promoter of circRNA production. The biological roles and mechanisms of EIF4A3-derived circRNA (circEIF4A3) in CRC cell autophagy remain poorly understood. This study explores the effects of circEIF4A3 on CRC cell growth and autophagy, aiming to elucidate the underlying molecular mechanisms. We discovered that EIF4A3 and circEIF4A3 synergistically enhance CRC cell growth. CircEIF4A3 sequesters miR-3126–5p, consequently upregulating EIF4A3. Further, circEIF4A3 increases EIF4A3 expression, which promotes autophagy by stabilizing ATG5 mRNA and enhances ATG7 protein stability through the stabilization of USP14 mRNA, a deubiquitinating enzyme. Upregulation of ATG5 and ATG7 counteracts the growth-inhibitory effects of EIF4A3 knockdown on CRC cells. Moreover, our findings demonstrate that EIF4A3 induces the formation of circEIF4A3 in CRC cells. In conclusion, a positive feedback loop between circEIF4A3 and EIF4A3 supports CRC cell growth by facilitating autophagy.

CircCUL3 drives malignant progression of cervical cancer by activating autophagy through sponge miR-223-3p upregulation of ATG7

Circular RNA (circRNA) has emerged as a pivotal regulatory factor in cancer biology, yet its exact role in cervical cancer remains incompletely understood. In this study, we investigated the functional role of circCUL3 in cervical cancer and explored its potential as a therapeutic target. Functional gain and loss experiments were conducted in Hela and Siha cell lines to elucidate the biological functions of circCUL3 in cervical cancer. The results revealed that circCUL3 overexpression significantly enhanced cell viability, migration, and invasion while suppressing apoptosis, while circCUL3 knockout displayed the opposite effects. Mechanistically, we identified hsa-miR-223-3p as a target of circCUL3, with its expression being negatively regulated by circCUL3. Furthermore, we discovered that circCUL3 could sequester miR-223-3p, leading to the upregulation of ATG7 expression, and this was linked to the regulation of autophagy in cervical cancer cells. In vivo validation using a xenograft mouse model further supported our in vitro findings. Notably, we found that chloroquine (CQ), an autophagy inhibitor, restored miR-223-3p expression and counteracted the oncogenic effect of circCUL3 overexpression. In conclusion, circCUL3 potentially contributes to the malignant progression of cervical cancer by acting as a sponge for miR-223-3p, resulting in the upregulation of ATG7 and the activation of autophagy.

Loss of FoxO1 activates an alternate mechanism of mitochondrial quality control for healthy adipose browning.

Browning of white adipose tissue is hallmarked by increased mitochondrial density and metabolic improvements. However, it remains largely unknown how mitochondrial turnover and quality control are regulated during adipose browning. In the present study, we found that mice lacking adipocyte FoxO1, a transcription factor that regulates autophagy, adopted an alternate mechanism of mitophagy to maintain mitochondrial turnover and quality control during adipose browning. Post-developmental deletion of adipocyte FoxO1 (adO1KO) suppressed Bnip3 but activated Fundc1/Drp1/OPA1 cascade, concurrent with up-regulation of Atg7 and CTSL. In addition, mitochondrial biogenesis was stimulated via the Pgc1α/Tfam pathway in adO1KO mice. These changes were associated with enhanced mitochondrial homeostasis and metabolic health (e.g., improved glucose tolerance and insulin sensitivity). By contrast, silencing Fundc1 or Pgc1α reversed the changes induced by silencing FoxO1, which impaired mitochondrial quality control and function. Ablation of Atg7 suppressed mitochondrial turnover and function, causing metabolic disorder (e.g., impaired glucose tolerance and insulin sensitivity), regardless of elevated markers of adipose browning. Consistently, suppression of autophagy via CTSL by high-fat diet was associated with a reversal of adO1KO-induced benefits. Our data reveal a unique role of FoxO1 in coordinating mitophagy receptors (Bnip3 and Fundc1) for a fine-tuned mitochondrial turnover and quality control, underscoring autophagic clearance of mitochondria as a prerequisite for healthy browning of adipose tissue.

Circadian Clock REV-ERBs Agonist SR9009 Induces Synergistic Antitumor Activity in Multiple Myeloma by Suppressing Glucose-Regulated Protein 78-Dependent Autophagy and Lipogenesis.

Proteasome inhibitors, such as bortezomib, have demonstrated efficacy in the therapeutic management of multiple myeloma (MM). However, it is important to note that these inhibitors also elicit endoplasmic reticulum stress, which subsequently triggers the unfolded protein response (UPR) and autophagy, which have been shown to facilitate the survival of tumor cells. The disruption of the circadian clock is considered a characteristic feature of cancer. However, how disrupted circadian clock intertwines with tumor metabolism and drug resistance is not clearly clarified. This work explores the antitumor effectiveness of bortezomib and the circadian clock agonist SR9009, elucidating their impact on glucose-regulated protein 78 (GRP78), the autophagy process, and lipogenesis. The antitumor effects of bortezomib and SR9009 were evaluated using human MM cell lines (RPMI8226 and U266) in vitro and in vivo nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft MM model. The assessment of cell viability was conducted using the cell counting kit-8 (CCK8) method, whereas the measurement of cell proliferation was performed with the inclusion of EdU (5-ethynyl-2'-deoxyuridine). Apoptosis was assessed by flow cytometry. The cells were transduced using adenovirus-tf-LC3, which was labeled with dual fluorescence. Subsequently, confocal imaging was employed to observe and examine the autophagosomes. REV-ERBα knockdown leads to upregulation of ATG5 and BENC1 at the protein level with immunoblot. Changes in the expression levels of GRP78, LC3, stearoyl-CoA desaturase 1 (SCD1), and fatty acid synthase (FASN) were assessed through the utilization of quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Our results showed that both bortezomib and circadian clock REV-ERBs agonist SR9009 decreased MM viability, proliferation rate and induced an apoptotic response in a dose-dependent manner in vitro. However, the two differ greatly in their mechanisms of action. Bortezomib upregulated GRP78 and autophagy LC3, while circadian clock agonist SR9009 inhibited GRP78 and autophagy LC3. Combined SR9009 with bortezomib induced synergistic cytotoxicity against MM cells. REV-ERBα knockdown lead to upregulation of ATG5, BENC1 and significant upregulation of FASN, and SCD1. Mechanically, SR9009 inhibited the core autophagy gene ATG5 and BECN1, and two essential enzymes for de novo lipogenesis FASN and SCD1. SR9009 had synergistic effect with bortezomib and slowed down murine xenograft models of human MM tumor growth in vivo. Taken together, these results demonstrated that the circadian clock component REV-ERBs agonist SR9009 could inhibit GRP78-induced autophagy and de novo lipogenesis processes and had a synergistic effect with proteasome inhibitors in both in vitro and in vivo models of MM. Our findings shed light on how a disrupted circadian clock interacts with metabolic mechanisms to shape proteasome inhibitor drug resistance and suggest that SR9009 may be able to overcome the inherent drug resistance of proteasome inhibitors.

Streptococcus uberis induced expressions of pro-inflammatory IL-6, TNF-α, and IFN-γ in bovine mammary epithelial cells associated with inhibited autophagy and autophagy flux formation

Autophagy is a highly conserved cellular defensive mechanism that can eliminate bacterial pathogens such as Streptococcus uberis, that causes mastitis in cows. However, S. uberis induced autophagy is still unclear. In this study, we tested if certain inflammatory cytokines such as IL-6, TNF-α, and IFN-γ, critical in mastitis due to S. uberis infection, regulate autophagy activation in bovine mammary epithelial cells (bMECs). Using Western blot and laser scanning confocal microscope in bMECs challenged by S. uberis, showed that the expression of IL-6, TNF-α, IFN-γ oscillated with the expressions of autophagic Atg5, ULK1, PTEN, P62, and LC3ӀӀ/LC3Ӏ. S. uberis infection induced autophagosomes and LC3 puncta in bMECs with upregulation of Atg5, ULK1, PTEN, LC3ӀӀ/LC3Ӏ, and downregulation of P62. The levels of IL-6, TNF-α, and IFN-γ increased during autophagy flux formation to decrease during autophagy induction. Autophagy inhibition increased the expression of IL-6, TNF-α, and IFN-γ and increased S. uberis burden. This study indicates autophagy is induced during S. uberis infection and IL-6, TNF-α, and IFN-γ contribute to autophagy and autophagy flux formation.

Altered transcriptional levels of autophagy-related genes, induced by oxidative stress in fish Channa punctatus exposed to chromium.

Chromium has been detected in various water bodies as a harmful metallic stressor to aquatic organisms. This study aimed to investigate the mechanism associated with autophagy, oxidative stress, and genotoxicity after chromium (Cr6+) exposure (1/20th of 96h-LC50, 1/10th of 96h-LC50, and 1/5th of 96h-LC50 of Cr6+) of common food fish Channa punctatus. The mRNA levels of autophagy-related genes ATG5, LC3, GABARAP, and mTOR were assessed in the liver and kidney tissue of fish. An upregulation of ATG5, LC3, and GABARAP was observed in both liver and kidney tissue samples, while mTOR showed transcriptional downregulation in both the tissue samples. This depicts autophagic vesicle formation due to stress signals. All the studied oxidative stress markers SOD, CAT, GSH, GR, and GPx showed an increase in the activity level of treated groups in a dose-dependent manner. Particularly, SOD and CAT have shown a significant elevation in activity level. ROS levels in blood cells increased significantly (p < 0.05) in all the treated groups (groups II, III, and IV) in a time-dependent manner as compared to the control (group I). There was a significant induction in MN frequency in all the treated groups. The highest frequency of micronuclei induced by Cr6+ was recorded in group IV after 28days of exposure period. Collectively, it can be concluded that the information about Cr6+-induced oxidative stress-mediated autophagy in vital organs of fish Channa punctatus remains largely obscure hitherto; to fill the aforesaid gap, this study was undertaken, which gives a snapshot for the mechanisms of autophagy induced by Cr6+ in fish. HIGHLIGHTS: • Chronic exposure to Cr6+ induces eco-toxicological manifestations in a fish Channa punctatus. • Altered transcriptional profile of autophagy-related genes suggests autophagic vesicle formation due to stress signals. • Increased activity levels of oxidative stress biomarkers reveal that Cr6+ annihilates antioxidative defense system in fish. • Genotoxicity due to chromium exposure is evident by increased frequency of MN in red blood cells of fish. • The information presented in this study is helpful to get an insight into the mechanism of Cr6+-induced oxidative stress-mediated induction of autophagy in the liver and kidney of Channa punctatus.

Long non-coding RNA WAC antisense RNA 1 mediates hepatitis B virus replication in vitro by reinforcing miR-192-5p/ATG7-induced autophagy.

Long non-coding RNA WAC antisense RNA 1 (lncRNA WAC-AS1) is involved in the replication of the hepatitis B virus (HBV). The purpose of this study was to determine its functions and specific mechanism. The levels of lncRNA WAC-AS1, RNA (miR)-192-5p and were examined in serum of HBV-infected patients and in HepG2.2.15 cells using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. Using the database starBase, the target binding sites of lncRNA WAC-AS1 and miR-192-5p were predicted and confirmed by dual-luciferase reporter assay and RNA pull-down assay. The expression of pgRNA and HBV DNA was determined by qRT-PCR, while the levels of HBeAg and HBsAg were measured by enzymelinked immunosorbent assay (ELISA). Using laser scanning confocal microscopy, the light chain 3 (LC3) expression was analyzed. qRT-PCR and Western blotting were used to assess the expression of beclin-1, p62, and LC3I/II. Overexpression of lncRNA WAC-AS1, upregulation of ATG7. and downregulation of miR-192-5p were observed in the serum of HBV-infected patients and the in vitro model. miR-192-5p directly targets lncRNA WAC-AS1. LncRNA WAC-AS1 was downregulated in lncRNA WAC-AS1-shRNA‒transfected cells. miR-192-5p was upregulated in lncRNA WAC-AS1-shRNA-transfected cells and downregulated in cells transfected with a miR-192-5p inhibitor. In HepG2 2.15 cells, the downregulation of lncRNA WAC-AS1 inhibited HBV replication and autophagy. In contrast, the miR-192-5p inhibitor-transfected group exhibited the opposite results, and ATG7 overexpression reversed the effects of miR-192-5p mimic or lncRNA WAC-AS1-shRNA on HBV replication and cell autophagy. Our findings indicate that lncRNA WAC-AS1 regulates HBV replication by reinforcing the autophagy induced by miR-192-5p/ATG7. Consequently, lncRNA WAC-AS1 may serve as a therapeutically- promising target in HBV patients.

Sec62 promotes gastric cancer metastasis through mediating UPR-induced autophagy activation.

Sec62 is a membrane protein of the endoplasmic reticulum that facilitates protein transport. Its role in cancer is increasingly recognised, but remains largely unknown. We investigated the functional role of Sec62 in gastric cancer (GC) and its underlying mechanism. Bioinformatics, tissue microarray, immunohistochemistry (IHC), western blotting (WB), quantitative polymerase chain reaction (qPCR), and immunofluorescence were used to examine the expression of target genes. Transwell, scratch healing assays, and xenograft models were used to evaluate cell migration and invasion. Transmission electron microscopy and mRFP-GFP-LC3 double-labeled adenoviruses were used to monitor autophagy. Co-immunoprecipitation (CO-IP) was performed to evaluate the binding activity between the proteins. Sec62 expression was upregulated in GC, and Sec62 upregulation was an independent predictor of poor prognosis. Sec62 overexpression promoted GC cell migration and invasion both in vitro and in vivo. Sec62 promoted migration and invasion by affecting TIMP-1 and MMP2/9 balance. Moreover, Sec62 could activate autophagy by upregulating PERK/ATF4 expression and binding to LC3II with concomitant FIP200/Beclin-1/Atg5 activation. Furthermore, autophagy blockage impaired the promotive effects of Sec62 on GC cell migration and invasion, whereas autophagy activation rescued the inhibitory effect of Sec62 knockdown on GC metastasis. Notably, Sec62 inhibition combined with autophagy blockage exerted a synergetic anti-metastatic effect in vitro and in vivo. Sec62 promotes GC metastasis by activating autophagy and subsequently regulating TIMP-1 and MMP2/9 balance. The activation of autophagy by Sec62 may involve the unfolded protein response (UPR)-related PERK/ATF4 pathway and binding of LC3II during UPR recovery involving FIP200/Beclin-1/Atg5 upregulation. Specifically, the dual inhibition of Sec62 and autophagy may provide a promising therapeutic strategy for GC metastasis.

Dysregulated Autophagy and Mitophagy in a Mouse Model of Duchenne Muscular Dystrophy Remain Unchanged Following Heme Oxygenase-1 Knockout.

Dysregulation of autophagy may contribute to the progression of various muscle diseases, including Duchenne muscular dystrophy (DMD). Heme oxygenase-1 (HO-1, encoded by Hmox1), a heme-degrading enzyme, may alleviate symptoms of DMD, inter alia, through anti-inflammatory properties. In the present study, we determined the role of HO-1 in the regulation of autophagy and mitophagy in mdx animals, a commonly used mouse model of the disease. In the gastrocnemius of 6-week-old DMD mice, the mRNA level of mitophagy markers: Bnip3 and Pink1, as well as autophagy regulators, e.g., Becn1, Map1lc3b, Sqstm1, and Atg7, was decreased. In the dystrophic diaphragm, changes in the latter were less prominent. In older, 12-week-old dystrophic mice, diminished expressions of Pink1 and Sqstm1 with upregulation of Atg5, Atg7, and Lamp1 was depicted. Interestingly, we demonstrated higher protein levels of autophagy regulator, LC3, in dystrophic muscles. Although the lack of Hmox1 in mdx mice influenced blood cell count and the abundance of profibrotic proteins, no striking differences in mRNA and protein levels of autophagy and mitophagy markers were found. In conclusion, we demonstrated complex, tissue, and age-dependent dysregulation of mitophagic and autophagic markers in DMD mice, which are not affected by the additional lack of Hmox1.

Effect of Calomelanone, a Dihydrochalcone Analogue, on Human Cancer Apoptosis/Regulated Cell Death in an In Vitro Model.

Calomelanone, 2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone, possesses anticancer activities. This study was conducted to investigate the cytotoxic effect of calomelanone, a dihydrochalcone analogue, on human cancer cells and its associated mechanisms. The cytotoxic effect of calomelanone was measured by MTT assay. Annexin V-FITC/propidium iodide and DiOC6 staining that employed flow cytometry were used to determine the mode of cell death and reduction of mitochondrial transmembrane potential (MTP), respectively. Caspase activities were measured using specific substrates and colorimetric analysis. The expression levels of Bcl-2 family proteins were determined by immunoblotting. Reactive oxygen species were also measured using 2′,7′-dihydrodichlorofluorescein diacetate and dihydroethidium (fluorescence dyes). Calomelanone was found to be toxic towards various human cancer cells, including acute promyelocytic HL-60 and monocytic leukemic U937 cells, in a dose-dependent manner at 24 h and human hepatocellular HepG2 cells at 48 h. However, the proliferation of HepG2 cells increased at 24 h. Calomelanone was found to induce apoptosis in HL-60 and U937 at 24 h and HepG2 apoptosis at 48 h via the intrinsic pathway by inducing MTP disruption. This compound also induced caspase-3, caspase-8, and caspase-9 activities. Calomelanone upregulated proapoptotic Bax and Bak and downregulated antiapoptotic Bcl-xL proteins in HepG2 cells. Moreover, signaling was also associated with oxidative stress in HepG2 cells. Calomelanone induced autophagy at 24 h of treatment, which was evidenced by staining with monodansylcadaverine (MDC) to represent autophagic flux. This was associated with a decrease of Akt (survival pathway) and an upregulation of Atg5 (the marker of autophagy). Thus, calomelanone induced apoptosis/regulated cell death in HL-60, U937, and HepG2 cells. However, it also induced autophagy in HepG2 depending on duration, dose, and type of cells. Thus, calomelanone could be used as a potential anticancer agent for cancer treatment. Nevertheless, acute and chronic toxicity should be further investigated in animals before conducting investigations in human patients.

Pimozide Suppresses the Growth of Brain Tumors by Targeting STAT3-Mediated Autophagy.

Brain tumors are considered as one of the most aggressive and incurable forms of cancer. The majority of the patients with brain tumors have a median survival rate of 12%. Brain tumors are lethal despite the availability of advanced treatment options such as surgical removal, chemotherapy, and radiotherapy. In this study, we have evaluated the anti-cancer effects of pimozide, which is a neuroleptic drug used for the treatment of schizophrenia and chronic psychosis. Pimozide significantly reduced the proliferation of U-87MG, Daoy, GBM 28, and U-251MG brain cancer cell lines by inducing apoptosis with IC50 (Inhibitory concentration 50) ranging from 12 to 16 μM after 48 h of treatment. Our Western blotting analysis indicated that pimozide suppressed the phosphorylation of STAT3 at Tyr705 and Src at Tyr416, and it inhibited the expression of anti-apoptotic markers c-Myc, Mcl-1, and Bcl-2. Significant autophagy induction was observed with pimozide treatment. LC3B, Beclin-1, and ATG5 up-regulation along with autolysosome formation confirmed the induction of autophagy with pimozide treatment. Inhibiting autophagy using 3-methyladenine or LC3B siRNA significantly blocked the apoptosis-inducing effects of pimozide, suggesting that pimozide mediated its apoptotic effects by inducing autophagy. Oral administration of 25 mg/kg pimozide suppressed the intracranially implanted U-87MG tumor growth by 45% in athymic nude mice. The chronic administration of pimozide showed no general signs of toxicity, and the behavioral activity of the mice remained unchanged. Taken together, these results indicate that pimozide inhibits the growth of brain cancer by autophagy-mediated apoptosis.

Docosahexaenoic acid protection against palmitic acid-induced lipotoxicity in NGF-differentiated PC12 cells involves enhancement of autophagy and inhibition of apoptosis and necroptosis.

Lipotoxicity (LTx) leads to cellular dysfunction and cell death and has been proposed to be an underlying process during traumatic and hypoxic injuries and neurodegenerative conditions in the nervous system. This study examines cellular mechanisms responsible for docosahexaenoic acid (DHA 22:6 n‐3) protection in nerve growth factor‐differentiated pheochromocytoma (NGFDPC12) cells from palmitic acid (PAM)‐mediated lipotoxicity (PAM‐LTx). NGFDPC12 cells exposed to PAM show a significant lipotoxicity demonstrated by a robust loss of cell viability, apoptosis, and increased HIF‐1α and BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 gene expression. Treatment of NGFDPC12 cells undergoing PAM‐LTx with the pan‐caspase inhibitor ZVAD did not protect, but shifted the process from apoptosis to necroptosis. This shift in cell death mechanism was evident by the appearance of the signature necroptotic Topo I protein cleavage fragments, phosphorylation of mixed lineage kinase domain‐like, and inhibition with necrostatin‐1. Cultures exposed to PAM and co‐treated with necrostatin‐1 (necroptosis inhibitor) and rapamycin (autophagy promoter), showed a significant protection against PAM‐LTx compared to necrostatin‐1 alone. In addition, co‐treatment with DHA, as well as 20:5 n‐3, 20:4 n‐6, and 22:5 n‐3, in the presence of PAM protected NGFDPC12 cells against LTx. DHA‐induced neuroprotection includes restoring normal levels of HIF‐1α and BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 transcripts and caspase 8 and caspase 3 activity, phosphorylation of beclin‐1, de‐phosphorylation of mixed lineage kinase domain‐like, increase in LC3‐II, and up‐regulation of Atg7 and Atg12 genes, suggesting activation of autophagy and inhibition of necroptosis. Furthermore, DHA‐induced protection was suppressed by the lysosomotropic agent chloroquine, an inhibitor of autophagy. We conclude that DHA elicits neuroprotection by regulating multiple cell death pathways including enhancement of autophagy and inhibiting apoptosis and necroptosis.

The Acetone Indigo Red Dehydrating Agent IF203 Induces HepG2 Cell Death Through Cell Cycle Arrest, Autophagy and Apoptosis.

BackgroundIsatin derivatives have extensive biological activities, such as antitumor. IF203, a novel isatin derivative, has not previously been reported to have antitumor activity.MethodsAcid phosphatase assays (APAs) and Ki-67 immunohistochemistry were used to detect the proliferation of HepG2 cells. Transmission electron microscope (TEM) was applied to detect ultrastructural changes. Flow cytometry (FCM) was used to detect cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) of HepG2 cells in vitro. TUNEL, MMP and ROS immunofluorescence assays were applied to assess apoptosis, MMP, and ROS of HepG2 cells in vivo. Western Blotting was applied to assess the levels of apoptosis- and autophagy-related proteins.ResultsIn this study, in vivo and in vitro experiments showed that IF203 possesses antitumor activity. The results of APAs and Ki-67 immunohistochemistry demonstrated that IF203 could inhibit the proliferation of HepG2 cells. Cell cycle assays, downregulation of Cyclin B1 and Cdc2, and upregulation of P53 suggested that IF203 could lead to G2/M cell cycle arrest. In addition, ultrastructural changes, apoptosis assays, TUNEL immunofluorescence results, upregulated expression of Bax, and downregulated expression of Bcl-2 suggest that IF203 can induce apoptosis in HepG2 cells. After IF203 treatment, intracellular ROS levels increased, MMP decreased, JC-1 green fluorescence was enhanced, and the levels of Caspase-9, Caspase-3 and Cytochrome C expression were upregulated, suggesting that IF203 could induce apoptosis of HepG2 cells through the mitochondrial apoptosis pathway. Moreover, characteristic apoptotic ultrastructural changes were accompanied by the appearance of many autophagy bubbles and upregulation of Atg5, Atg12, ULK1, Beclin-1 and LC3-II proteins, suggesting that IF203 could induce autophagy in HepG2 cells.ConclusionThis study showed that IF203 leads to the death of HepG2 cells through cell cycle arrest, apoptotic induction, and autophagy promotion.

Cordycepin Enhances Radiosensitivity in Oral Squamous Carcinoma Cells by Inducing Autophagy and Apoptosis Through Cell Cycle Arrest.

Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide and accounts for over 90% of malignant neoplasms of the oral cavity, with a 5-year survival rate of less than 50%. The long-term survival rate of OSCC patients has not markedly improved in recent decades due to its heterogeneous etiology and treatment outcomes. We investigated the anticancer effect of the combination of irradiation (IR) and cordycepin in the treatment of human OSCC cells in vitro. The type of cell death, especially autophagy and apoptosis, and the underlying mechanisms were examined. We found synergistic effects of cordycepin and IR on the viability of human oral cancer cells. The combination of cordycepin and IR treatment induced apoptosis, cell cycle arrest, and autophagic cell death. Furthermore, cordycepin induced S-phase arrest and prolonged G2/M arrest in the cells that received the combination treatment compared with those that received irradiation alone. Combined treatment induced the upregulation of ATG5 and p21 in an autophagy cascade-dependent manner, arrested the cell cycle in the G2/M phase, and repressed cell proliferation. Thus, we conclude that the combination of cordycepin and IR treatment could be a potential therapeutic strategy for OSCC.

Atiprimod induce apoptosis in pituitary adenoma: Endoplasmic reticulum stress and autophagy pathways.

Pituitary adenoma is the most common tumor with a high recurrence rate due to a hormone-dependent JAK/signal transducer and activator of transcriptions (STAT) signaling. Atiprimod, a novel compound belonging to the azaspirane class of cationic amphiphilic drugs, has antiproliferative, anticarcinogenic effects in multiple myeloma, breast, and hepatocellular carcinoma by blocking STAT3 activation. Therapeutic agents' efficiency depends on endoplasmic reticulum (ER) stress-autophagy regulation during drug-mediated apoptotic cell death decision. However, the molecular machinery of dose-dependent atiprimod treatment regarding ER stress-autophagy has not been investigated yet. Thus, our aim is to investigate the ER stress-autophagy axis in atiprimod-mediated apoptotic cell death in GH-secreting rat cell line (GH3) pituitary adenoma cells. Dose-dependent atiprimod treatment decreased GH3 cell viability, inhibited cell growth, and colony formation. Upregulation of Atg5, Atg12, Beclin-1 expressions, cleavage of LC-3II and formation of autophagy vacuoles were determined only after 1 µM atiprimod exposure.In addition, atiprimod-triggered ER stress was evaluated by BiP, C/EBP-homologous protein (CHOP), p-PERK upregulation, and Ca+2 release after 1 µM atiprimod exposure. Concomitantly, increasing concentration of atiprimod induced caspase-dependent apoptotic cell death via modulating Bcl2 family members. Moreover, by N-acetyl cycteinc pretreatment, atiprimod triggered reactive oxygen species generation and prevented apoptotic induction. Concomitantly, dose-dependent atiprimod treatment decreased both GH and STAT3 expression in GH3 cells. In addition, overexpression of STAT3 increased atiprimod-mediated cell viability loss and apoptotic cell death through suppressing autophagy and ER stress key molecules expression profile. In conclusion, a low dose of atiprimod exposure triggers autophagy and mild-ER stress as a survival mechanism, but increased atiprimod dose induced caspase-dependent apoptotic cell death by targeting STAT3 in GH3 pituitary adenoma cells.

Cytotoxic and Antitumor Activity of Lactaptin in Combination with Autophagy Inducers and Inhibitors.

Autophagy is a degradative process in which cellular organelles and proteins are recycled to restore homeostasis and cellular metabolism. Autophagy can be either a prosurvival or a prodeath process and remains one of the most fundamental processes for cell vitality. Thus autophagy modulation is an important approach for reinforcement anticancer therapeutics. Earlier we have demonstrated that recombinant analog of human milk protein lactaptin (RL2) induced apoptosis of various cultured cancer cells and activated lipidation of microtubule-associated protein 1 light chain 3 (LC3). In this study we investigated whether autophagy inhibitors—chloroquine (CQ), Ku55933 (Ku), and 3-methyladenine (3MA)—or inducer—rapamycin (Rap)—can enhance cytotoxic activity of lactaptin analog in cancer cells and its anticancer activity in the mice model. Western Blot analysis revealed that RL2 induced short-term autophagy in MDA-MB-231 and MCF-7 cells at early stages of incubation and that these data were confirmed by the transmission electron microscopy of autophagosome/autophagolysosome formation. RL2 stimulates reactive oxygen species (ROS) production, autophagosomes accumulation, upregulation of ATG5 with processing of LC3I to LC3II, and downregulation of p62/sequestosome 1 (p62). We have shown that autophagy modulators, CQ, Ku, and Rap, synergistically increased cytotoxicity of RL2, and RL2 with CQ induced autophagic cell death. In addition, CQ, Ku, and Rap in combination with RL2 decreased activity of lysosomal protease Cathepsin D. More importantly, combining RL2 with CQ, we improved antitumor effect in mice. Detected synergistic cytotoxic effects of both types of autophagy regulators, inhibitors, and inducers with RL2 against cancer cells allow us to believe that these combinations can be a basis for the new anticancer approach. Finally, we suppose that CQ and Rap promoting of short-term RL2-induced autophagy interlinks with final autophagic cell death.

Posttranscriptional regulation of colonic epithelial repair by RNA binding protein IMP1/IGF2BP1.

RNA binding proteins, including IMP1/IGF2BP1, are essential regulators of intestinal development and cancer. Imp1 hypomorphic mice exhibit gastrointestinal growth defects, yet the specific role for IMP1 in colon epithelial repair is unclear. Our prior work revealed that intestinal epithelial cell-specific Imp1 deletion (Imp1ΔIEC ) was associated with better regeneration in mice after irradiation. Here, we report increased IMP1 expression in patients with Crohn's disease and ulcerative colitis. We demonstrate that Imp1ΔIEC mice exhibit enhanced recovery following dextran sodium sulfate (DSS)-mediated colonic injury. Imp1ΔIEC mice exhibit Paneth cell granule changes, increased autophagy flux, and upregulation of Atg5. In silico and biochemical analyses revealed direct binding of IMP1 to MAP1LC3B, ATG3, and ATG5 transcripts. Genetic deletion of essential autophagy gene Atg7 in Imp1ΔIEC mice revealed increased sensitivity of double-mutant mice to colonic injury compared to control or Atg7 single mutant mice, suggesting a compensatory relationship between Imp1 and the autophagy pathway. The present study defines a novel interplay between IMP1 and autophagy, where IMP1 may be transiently induced during damage to modulate colonic epithelial cell responses to damage.

Lycium barbarum polysaccharide prevents cisplatin-induced MLTC-1 cell apoptosis and autophagy via regulating endoplasmic reticulum stress pathway.

BackgroundLycium barbarum polysaccharide (LBP) has been reported to contribute to the recovery of male hypogonadism and infertility.AimThe aim of current study was to investigate the underlying mechanisms of LBP on male infertility recovery.MethodsRecently, it is reported that cell apoptosis mediated by endoplasmic reticulum stress (ERS) was distinguished from that mediated by death reporters and mitochondria pathway, which could induce cell apoptosis independently. The possible signaling mechanisms were investigated using diversified molecular biology techniques, such as flow cytometry, western blotting, and immunofluorescence.ResultsIn this study, we found that LBP protected Leydig MLTC-1 cells against cisplatin (DDP) by regulating ERS-mediated signal pathway, which was evidenced by downregulation of phosphorylation PERK, phosphorylation of eukaryotic translation-initiation factor 2α and activating transcription factor 4. Meanwhile, LBP decreased DDP-induced MLTC-1 cell apoptosis via reducing ERS apoptosis-relative proteins caspase 3, caspase 7, and caspase 12. In addition, the result of monodansylcadaverine staining indicated that LBP significantly inhibited DDP-induced autophagosome formation in MLTC-1 cells. Moreover, immunofluorescences and Western blot assays demonstrated that LBP reversed DDP-induced LC3II and Atg5 upregulation in MLTC-1 cells. Finally, the data of enzyme-linked immunosorbent assay showed that LBP markedly recovered MLTC-1 cells testosterone level even in the presence of DDP.ConclusionThus, we suggest that LBP protected MLTC-1 cells against DDP via regulation of ERS-mediated apoptosis and autophagy.

Inhibition of autophagy significantly increases the antitumor effect of Abiraterone in prostate cancer.

Abiraterone acetate (AA) plus prednisone is an approved treatment of advanced prostate cancer (PCa). Autophagy is linked to drug resistance in numerous types of cancers. We hypothesized, that upregulation of autophagy is one of the mechanisms by which PCa cells survive AA anti-tumor treatment and therefore evaluated the potential effect of a combination with autophagy inhibition. Human PCa LNCaP cell lines were cultured in steroid-free medium and treated with AA. Autophagy was inhibited by 3-methyladenine, chloroquine and ATG5 siRNA knock-down. Cell viability and apoptosis was assessed by flow cytometry and fluorescence microscopy, and autophagy was monitored by immunohistochemistry, AUTOdot and Western blotting. Western blot revealed upregulation of ATG5 and LC3 II with a reduction of p62 protein expression in AA-treated cells, indicating upregulation of autophagy. These data were supported by results obtained with immunocytochemistry and AUTOdot assays. Using flow cytometry, we showed that combining AA with autophagy inhibition significantly impaired cell viability (1.3-1.6-fold, p < 0.001) and increased apoptosis (1.4-1.5-fold, p < 0.001) compared to AA treatment alone. AA activates autophagy as a cytoprotective mechanism in LNCaP prostate cancer cells and targeting of autophagy enhances the antitumor effect of the compound.