Inhibition Of Protective Autophagy Research Articles

Hydrogel-immobilized nanotherapeutics: Inhibition of protective autophagy to amplify STING signals for postsurgical tumor immunotherapy

Activating the inherent stimulator of interferon gene (STING) pathway of cancer cells can inhibit tumor growth but can also induce protective autophagy of tumor cells to inhibit the STING-mediated immunotherapy. Therefore, amplification of STING activation and inhibition of autophagy could potentiate the cancer immunotherapy. Here, hydrogel-immobilized nanoparticles loaded with glucose oxidase (GOx), manganese ions (Mn2+) and hydroxychloroquine (HCQ) are prepared at the postsurgical site to enhance cancer therapy via activating the STING pathway and regulating the autophagy pathway. The delivered GOx and Mn2+ exert a chemo-dynamic therapy (CDT) to induce dsDNA damage, which can further initiate the STING pathway. HCQ can effectively inhibit protective autophagy associated with STING pathway activation, further amplify the antitumor immune responses. The synergistic delivery of CDT agents (i.e., GOx and Mn2+), STING agonists (i.e., Mn2+) and autophagy inhibitors (i.e., HCQ) results in a specific immune response, significantly inhibit tumor recurrence and metastatic tumor growth, which could extend the survival rate of mice suffered from the triple-negative breast cancer (TNBC).

ROS Responsive Nanoparticles Encapsulated with Natural Medicine Remodel Autophagy Homeostasis in Breast Cancer.

In photodynamic therapy (PDT), elevated reactive oxygen species (ROS) activate tumor cell protective autophagy, therefore attenuating the antitumor function of therapy. Hence, inhibition of protective autophagy in tumors can improve the antitumor effect of PDT. Herein, an innovative nanotraditional Chinese medicine system ((TP+A)@TkPEG NPs), which remodeled autophagy homeostasis, was fabricated. A photosensitizer aggregation inducing emission (AIE) and autophagy modulator triptolide (TP, an active ingredient of Tripterygium wilfordii Hook F) were encapsulated into ROS-responsive nanoparticles to improve antitumor effect of PDT in treatment of triple negative breast cancer. We proved that (TP+A)@TkPEG NPs effectively elevated intracellular ROS levels, activated ROS-responsive release of TP and inhibited the proliferation of 4T1 cells in vitro. More importantly, it sharply reduced autophagy related genes transcription and proteins expression in 4T1 cells, then promote cell apoptosis. In addition, this nanoherb therapeutic system effectively orientated to tumor sites, achieved efficient inhibition of tumor, and extended the survival time of 4T1-bearing mice in vivo. Further results confirmed that (TP+A)@TkPEG NPs remarkably inhibit the expression level of autophagy related initiation gene (becline-1) and elongation protein (light chain 3B) in tumor microenvironment and then block PDT induced protective autophagy. In brief, this system can remodel autophagy homeostasis and serve as an innovative approach for treatment of triple negative breast cancer.

Protective autophagy decreases lorlatinib cytotoxicity through Foxo3a-dependent inhibition of apoptosis in NSCLC

Lorlatinib is a promising third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) that has been approved for treating ALK-positive non-small-cell lung cancer (NSCLC) patients with previous ALK-TKI treatment failures. However, the inevitable emergence of acquired resistance limits its long-term efficacy. A more comprehensive understanding of the acquired resistance mechanisms to lorlatinib will enable the development of more efficacious therapeutic strategies. The efficacy of chloroquine (CQ) in combination with lorlatinib in ALK-positive NSCLC cells in vitro and in vivo was assessed using CCK-8, colony formation, immunofluorescence staining, flow cytometry analysis, western blot analysis, and xenograft implantation. Here, we show that lorlatinib induced apoptosis and protective autophagy in ALK-positive NSCLC cells. However, the protective autophagy can gradually lead to decreased cytotoxicity of loratinib in ALK-positive NSCLC cells. Meanwhile, we found that the combination of lorlatinib and CQ, an inhibitor of autophagy, inhibited autophagy and promoted apoptosis both in vitro and in vivo, which sensitized cells to lorlatinib through the dephosphorylation of Foxo3a and promoted nuclear translocation, then activation of Foxo3a/Bim axis. Taken together, our results suggest that inhibition of protective autophagy might be a therapeutic target for delaying the occurrence of acquired resistance to lorlatinib in ALK-positive NSCLC patients.

Inhibiting autophagy flux and DNA repair of tumor cells to boost radiotherapy of orthotopic glioblastoma

Radio-resistance of glioblastoma (GBM) remains a leading cause of radiotherapy failure because of the protective autophagy induced by X-Ray irradiation and tumor cells’ strong capability of repairing damaged DNA. It is of great importance to overcome the radio-resistance for improving the efficacy of radiotherapy. Herein, we report the novel mechanism of core-shell copper selenide coated gold nanoparticles (Au@Cu2-xSe NPs) inhibiting the protective autophagy and DNA repair of tumor cells to drastically boost the radiotherapy efficacy of glioblastoma. We reveal that the core-shell Au@Cu2-xSe NPs can inhibit the autophagy flux by effectively alkalizing lysosomes. They can increase the SQSTM1/p62 protein levels of tumor cells without influencing their mRNA. We also reveal that Au@Cu2-xSe NPs can increase the ubiquitination of DNA repair protein Rad51, and promote the degradation of Rad51 by proteasomes to prevent the DNA repair. The simultaneous inhibition of protective autophagy and DNA repair significantly suppress the growth of orthotopic GBM by using radiotherapy and our novel Au@Cu2-xSe NPs. Our work provides a new insight and paradigm to significantly improve the efficacy of radiotherapy by rationally designing theranostic nano-agents to simultaneously inhibit protective autophagy and DNA repair of tumor cells.

New insights of RA-V cyclopeptide as an autophagy inhibitor in human COLO 320DM cancer cell lines

Colon cancer is the leading cause for the malignancy in the gastrointestinal tract. Autophagy is a self-degradation process of the unnecessary, injured and aged organelles and proteins in the cell, which is followed by recovering of degraded products. Apoptosis is a programmed cell death which is characterized by membrane blebbing, chromosome condensation and nuclear fragmentation. Apoptosis and autophagy can occur frequently in a cell, predominantly in a series preceding apoptosis through autophagy by the formation of autophagosomes. In current research, the impact of autophagy inhibition and apoptosis activation were found to be the targeted strategies to treat colon cancer. This study is focused on the apoptotic potential of RA-V, a natural cyclopeptide through the inhibition of protective autophagy in colon cancer cells. Growth inhibitory properties were observed in the RA-V treated (125 μM) colo 320DM cells using cell viability assay. RA-V induced apoptosis of colo 320DM cells at the maximum concentration of 125 μM, which was observed using DAPI and Annexin - PI staining methods. In this study we also examined the mechanistic role of RA-V (125 μM) incolo 320DM cells in the presence of Rapamycin (mTOR inhibitor) and chloroquine (autophagy inhibitor) using MDC and AO staining methods.

Cardiac glycoside sensitized hepatocellular carcinoma cells to TRAIL via ROS generation, p38MAPK, mitochondrial transition, and autophagy mediation.

A major concern in the clinical application of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) in tumors is the development of resistance. Therefore, agents that can potentially restore TRAIL sensitivity are important therapeutic targets for cancer treatment. Herein, we evaluated lanatoside c and digoxin, both of which are widely used cardiac glycosides (CGs), for their ability to sensitize human hepatocellular carcinoma cells (Huh-7 and HepG2) through TRAIL-induced apoptosis. CGs functionalize TRAIL as shown by its effect on intracellular reactive oxygen species (ROS) generation, which damages mitochondrial integrity and thereby confers intrinsic apoptotic caspase cascade during combined treatment. Caspase activation is dependent on ROS as shown by the ability of CGs to generate ROS and the ROS-N-acetylcysteine (NAC) relationship, which inhibits apoptosis during cotreatment by preventing the formation of caspase-8 and -3. Furthermore, CGs triggered p38MAPK phosphorylation and NAC pre-exposure blocked p38MAPK phosphorylation, which demonstrated that p38MAPK was dependent upon ROS generation. Additionally, CGs were found to be potent inducers of AMPK-mediated protective autophagy as pharmacological and genetic autophagy inhibition reached the higher threshold of TRAIL-mediated apoptosis. Finally, CGs downregulated the expression of the antiapoptotic protein Bcl-2 and increased the translocation of proapoptotic protein cytochrome c, thereby inducing apoptosis. Collectively, these results indicate that CGs potentiate the enhanced cytotoxic capacity to TRAIL through ROS generation, p38MAPK phosphorylation, cell survival protein downregulation, and protective autophagy inhibition.

Mixture designs to investigate adverse effects upon co-exposure to environmental cyanotoxins

The goal of this study was to implement powerful mixture design techniques, commonly used in process optimization, to investigate enhanced adverse effects upon co-exposure to environmental cyanotoxins. Exposure to cyanobacteria, which are found ubiquitously in environmental water reservoirs, have been linked to several neurodegenerative diseases. Despite the known co-occurrence of various cyanotoxins, the majority of studies investigating this link have focused on the investigation of a single cyanotoxin, a noncanonical amino acid called β-methylamino-L-alanine (BMAA), which poorly recapitulates an actual environmental exposure. Interactions amongst cyanotoxic compounds is an area of great concern and remains poorly understood. To this end, we describe the use of a simplex axial mixture design to screen for interactive adverse effects of cyanotoxic mixtures. Using a combination of basic toxicity assays coupled with contemporary proteomic techniques, our results show the existence of a significant (p ≤ 0.01) interaction between BMAA and its isomers aminoethyl glycine (AEG) and 2,4-diaminobutyric acid (2,4DAB). Cyanotoxic mixtures significantly decreased cell viability by an average of 19% and increased caspases 3/7 activities by an average of 110% when compared to individual cyanotoxins (p ≤ 0.05). Cyanotoxic mixtures perturbed various biological pathways associated with neurodegeneration, including inhibition of protective autophagy and activation of mitochondrial dysfunction (z-score >|2|). Additionally, exposure to mixtures perturbed important upstream regulators involved in cellular dysfunction, morbidity, and development. Taken together, our results highlight: (1) the need to study combinations of cyanotoxins when investigating the link between cyanobacteria and neurodegenerative pathologies and (2) the application of design of experiment (DoE) as an efficient methodology to study mixtures of relevant environmental toxins.

Targeting V-ATPase Isoform Restores Cisplatin Activity in Resistant Ovarian Cancer: Inhibition of Autophagy, Endosome Function, and ERK/MEK Pathway.

Ovarian cancer (OVCA) patients often develop tolerance to standard platinum therapy that accounts for extensive treatment failures. Cisplatin resistant OVCA cells (cis-R) display enhanced survival mechanisms to cope with therapeutic stress. In these cells, increased autophagy process assists in chemoresistance by boosting the nutrient pool under stress. To improve the treatment response, both protective autophagy inhibition and its overactivation are showing efficacy in chemosensitization. Autophagy requires a tightly regulated intracellular pH. Vacuolar ATPases (V-ATPases) are proton extruding nanomotors present on cellular/vesicular membranes where they act as primary pH regulators. V-ATPase ‘a2' isoform (V0a2), the major pH sensing unit, is markedly overexpressed on the plasma membrane and the early endosomes of OVCA cells. Previously, V0a2 inhibition sensitized cis-R cells to platinum drugs by acidifying cytosolic pH that elevated DNA damage. Here, we examined how V0a2 inhibition affected endosomal function and the autophagy process as a possible factor for cisplatin sensitization. Clinically, V0a2 expression was significantly higher in tissues from drug nonresponder OVCA patients compared to treatment responders. In vitro V0a2 knockdown in cis-R cells (sh-V0a2-cisR) significantly reduced the tumor sphere-forming ability and caused complete disintegration of the spheres upon cisplatin treatment. The apoptotic capacity of sh-V0a2-cisR improved substantially with potentiation of both intrinsic and extrinsic apoptotic pathway when treated with cisplatin. Unlike the chemical V-ATPase inhibitors that acutely induce autophagy, here, the stable V0a2 inhibition dampened the protective autophagy process in sh-V0a2-cisR cells with downregulated expression of proteins beclin-1, ATG-7, and LC3B and low autophagosome numbers compared to control cis-R cells. These cells showed downregulated ERK/MEK pathway that is known to repress autophagy. Interestingly, upon cisplatin treatment of sh-V0a2-cisR, the autophagy initiation proteins (LC3B, ATG7, and Beclin 1) were found upregulated as a stress response compared to the untreated cells. However, there was a concomitant downstream autophagosome accumulation and an enhanced P62 protein levels indicating the overall block in autophagy flux. Mechanistically, V0a2 knockdown caused defects in early endosome function as the transferrin internalization was impaired. Taken together, this study provides a novel insight into the mechanism by which V-ATPase-isoform regulates autophagy that assists in chemoresistance in ovarian cancer. We conclude that V-ATPase-V0a2 is a potent target for developing an effective treatment to enhance patient survival rates in ovarian cancer.

Targeting autophagy for combating chemoresistance and radioresistance in glioblastoma.

Autophagy is an evolutionarily conserved catabolic process that plays an essential role in maintaining cellular homeostasis by degrading unneeded cell components. When exposed to hostile environments, such as hypoxia or nutrient starvation, cells hyperactivate autophagy in an effort to maintain their longevity. In densely packed solid tumors, such as glioblastoma, autophagy has been found to run rampant due to a lack of oxygen and nutrients. In recent years, targeting autophagy as a way to strengthen current glioblastoma treatment has shown promising results. However, that protective autophagy inhibition or autophagy overactivation is more beneficial, is still being debated. Protective autophagy inhibition would lower a cell's previously activated defense mechanism, thereby increasing its sensitivity to treatment. Autophagy overactivation would cause cell death through lysosomal overactivation, thus introducing another cell death pathway in addition to apoptosis. Both methods have been proven effective in the treatment of solid tumors. This systematic review article highlights scenarios where both autophagy inhibition and activation have proven effective in combating chemoresistance and radioresistance in glioblastoma, and how autophagy may be best utilized for glioblastoma therapy in clinical settings.

Action of Gold Nanospikes-Based Nanoradiosensitizers: Cellular Internalization, Radiotherapy, and Autophagy.

A major challenge to achieve effective X-ray radiation therapy is to use a relatively low and safe radiation dose. Various radiosensitizers, which can significantly enhance the radiotherapeutic performance, have been developed. Gold-based nanomaterials, as a new type of nanoparticle-based radiosensitizers, have been extensively used in researches involving cancer radiotherapy. However, the cancer therapeutic effect using the gold nanoparticle-based radiotherapy is usually not significant because of the low cellular uptake efficiency and the autophagy-inducing ability of these gold nanomaterials. Herein, using gold nanospikes (GNSs) as an example, we prepared a series of thiol-poly(ethylene glycol)-modified GNSs terminated with methoxyl (GNSs), amine (NH2-GNSs), folic acid (FA) (FA-GNSs), and the cell-penetrating peptide TAT (TAT-GNSs), and evaluated their effects on X-ray radiotherapy. For the in vitro study, it was found that the ionizing radiation effects of these GNSs were well correlated with their cellular uptake amounts, with the same order of GNSs < NH2-GNSs < FA-GNSs < TAT-GNSs. The sensitization enhancement ratio (SER), which is commonly used to evaluate how effectively radiosensitizers decrease cell proliferation, reaches 2.30 for TAT-GNSs. The extremely high SER value for TAT-GNSs indicates the superior radiosensitization effect of this nanomaterial. The radiation enhancement mechanisms of these GNSs involved the increased reactive oxygen species (ROS), mitochondrial depolarization, and cell cycle redistribution. Western blotting assays confirmed that the surface-modified GNSs could induce the up-regulation of autophagy-related protein (LC3-II) and apoptosis-related protein (active caspase-3) in cancer cells. By monitoring the degradation of the autophagy substrate p62 protein, GNSs caused impairment of autolysosome degradation capacity and autophagosome accumulation. Our data demonstrated that autophagy played a protective role against caner radiotherapy, and the inhibition of protective autophagy with inhibitors would result in the increase of cell apoptosis. Besides the above in vitro experiments, the in vivo tumor growth study also indicated that X-ray + TAT-GNSs treatment had the best tumor growth inhibitory effect, which confirmed the highest radiation sensitizing effect of TAT-GNSs. This work furthered our understanding on the interaction mechanism between gold nanomaterials and cancer cells and should be able to promote the development of nanoradiosensitizers for clinical applications.

CISD2 enhances the chemosensitivity of gastric cancer through the enhancement of 5-FU-induced apoptosis and the inhibition of autophagy by AKT/mTOR pathway.

Gastric cancer (GC) is a prevalent upper gastrointestinal tumor characterized by high morbidity and mortality due to imperfect screening systems and the rapid development of resistance to 5‐fluorouracil (5‐FU). CDGSH iron sulfur domain 2 (CISD2) has been recently regarded as a candidate oncogene in several types of tumors. It is, therefore, necessary to investigate its biological function and clinical significance in gastric cancer. In this study, the down‐regulated expression level of CISD2 in GC compared with adjacent normal tissues was evaluated by quantitative RT‐PCR and Western blotting. An immunohistochemical analysis indicated that CISD2 expression in GC was significantly correlated with age (P = 0.002), Lauren's classification (P = 0.001), and differentiation (P = 0.049). Two cell lines, MKN1 and BGC823, were used to analyze the role of CISD2 in gastric carcinogenesis and response to 5‐FU through CCK‐8 assays, the RT‐CES system, Transwell assays, flow cytometry, and confocal fluorescence microscopy. The overexpression of CISD2 resulted in reduced cellular growth and proliferation, inhibition of metastatic ability, and increased apoptosis. 5‐FU treatment increased endogenous as well as exogenous overexpression of CISD2 in GC cells. Further investigation revealed that CISD2 enhanced sensitivity to 5‐FU via an increase in apoptosis and inhibition of protective autophagy through the activation of the AKT/mTOR pathway. In conclusion, CISD2 is down‐regulated in gastric cancer, and its effects on the inhibition of cellular proliferation, metastatic ability, and increased chemotherapy sensitivity are mediated by antagonism to 5‐FU‐induced autophagy through the AKT/mTOR pathway.

Reactive oxygen species acts as executor in radiation enhancement and autophagy inducing by AgNPs

Malignant glioma is one of the most common intracranial tumor with a dismal prognosis. The radiosensitizing effect of silver nanoparticles (AgNPs) on glioma both in vitro and in vivo were demonstrated in the previous studies of our group. However, the underlying mechanism is still unclear. In this present study, the use of antioxidants is employed for the regulating of reactive oxygen species (ROS) in U251 cells treated with various agents, and the results shows that ROS played an essential role in the autophagy inducing and radiosensitization effect of AgNPs. Moreover, the inhibition of protective autophagy with 3-MA is another way to increase ROS, resulting in the increasing of cell death and apoptosis. Taken together, understanding the relationship between the elevated ROS and autophagy and the effect of ROS should be useful to the clinical applications of AgNPs. These findings could potentially be exploited for new therapeutic strategies in glioma radiotherapy.

Prospective impact of 5-FU in the induction of endoplasmic reticulum stress, modulation of GRP78 expression and autophagy in Sk-Hep1 cells

Hepatocellular carcinoma (HCC) is one of the most aggressive malignant diseases and is highly resistant to conventional chemotherapy. Therefore, HCC requires more effective prevention and treatment strategies. 5-fluorouracil (5-FU) remains the most widely used chemotherapeutic drug for the treatment of gastrointestinal, breast, head and neck, and ovarian cancers. In pursuit of a novel effective strategy, we have evaluated the potential of 5-FU to promote endoplasmic reticulum (ER) stress and autophagy in Sk-Hep1 HCC cells. We found that 5-FU profoundly induces ER stress in Sk-Hep1 cells and upregulates p53 and activates CHOP/GADD153 and caspase-12. Activation of CHOP/GADD153 and caspase-12 promotes mitochondrial cell death in Sk-Hep1 cells followed by ER stress. Changes in calcium homeostasis and the protein folding machinery cause stress in the ER, leading to apoptotic cell death. Stress in the ER activates autophagy to remove the misfolded protein aggregates and recover from the stress environment. Our study demonstrates that 5-FU-induced ER stress suppresses autophagy and also downregulates GRP78 expression. Activation of autophagy followed by ER stress facilitates the cell survival response. Therefore, the inhibition of protective autophagy may provide a useful pharmacological target. Taken together, these results indicate that 5-FU-induced ER stress activates the mitochondrial apoptotic cell death pathway by downregulating GRP78 and protective autophagy proteins in Sk-Hep1 cells, raising the possibility of using 5-FU as a therapeutic agent to target human HCC.