Induction Of Autophagic Cell Death Research Articles

Autophagic cell death induction in a clinical isolate of Acanthamoeba sp. exposed to methanolic extracts of Pereskia bleo Kuhn

Autophagic cell death induction in a clinical isolate of Acanthamoeba sp. exposed to methanolic extracts of Pereskia bleo Kuhn

Abstract 5937: KS20226, a first-in-class IRP2 inhibitor, preferentially induces reprogramming of iron metabolism and suppresses tumor growth in colorectal cancer

Abstract Background: Iron homeostasis plays a role in redox activity, mitochondrial function, and cell cycle regulation. Specifically, the dysregulation of iron metabolism is implicated in malignant transformation and cancer progression. Iron regulatory protein 2 (IRP2) is crucial for maintaining balance within iron levels and primarily controls genes associated with iron utilization. Our discovery of KS20226, a first-in-class IRP2 inhibitor, reveals remarkable potential as an effective anti-cancer drug candidate. We have elucidated the mechanisms underlying the therapeutic effects of KS20226, thereby highlighting the feasibility of targeting IRP2 in colorectal cancer (CRC). Methods: The proliferation inhibitory activity of KS20226 was assessed the CCK-8 and 3D Spheroid models. To investigate mitochondrial function, we conducted measurement of oxidative phosphorylation (OCR) and glycoPER. Furthermore, gene expression analysis was performed through RNA sequencing (RNA-seq) using the Illumina HiSeq 2500 system platform (Macrogen Inc.). Differential expressed gene (DEG) analysis was carried out using the R package DESeq2, and pathway enrichment analysis was analyzed using GSEA (v_4.2.3.). For the observation of autophagic morphology, we utilized transmission electron microscopy (TEM), immunofluorescence staining targeting the autophagy marker LC3B, and immunoblotting. Results: KS20226 exhibited a remarkable growth inhibitory effect in 10 different CRC cell lines, with GI50 values ranging from 1 to 10 μM. KS20226 suppressed IRP2 expression and the corresponding occupancy of the iron-responsive elements of ferritin H and transferrin receptor 1, resulting in iron deprivation, and efficiently controlled CRC growth. Pharmacologic inhibition of IRP2 induced reprogramming of mitochondrial respiration, reducing reliance on oxidative phosphorylation. The observed mitochondrial dysfunction induced by KS20226 was correlated with pathway analysis, revealing a decrease in the gene-set for HALLMARK_OXIDATIVE_PHOSPHORYLATION (NES = -1.38, p-value = 0.008) and an increase in the gene-set for POSITIVE_REGULATION_OF_AUTOPHAGY (NES=1.43, p-value=0.016). Immunoblotting demonstrated that KS20226 activated the phosphorylation of AMPK, ULK, and Beclin1 cascade, leading to the autophagic cell death of the CRC cells. Conclusions: The reliance of cancer cells on iron metabolism to drive proliferation has led to the development of therapeutic strategies targeting the inhibition of IRP2. KS20226, a first-in-class IPR2 inhibitor, exhibits distinct therapeutic properties, such as perturbation of mitochondrial iron metabolism and induction of autophagic cell death. Our findings represent important progress toward exploiting IRP2-mediated abnormal iron metabolism in cancer, an unexplored area of research with substantial unmet needs. Citation Format: Jieon Hwang, Areum Park, Chinwoo Kim, Chang Gon Kim, Jaesung Kwak, Byungil Kim, Hyunjin Shin, Minhee Ku, Jaemoon Yang, Ayoung Baek, Jiwon Choi, Hocheol Lim, Kyoung Tai No, Xianghua Zhao, Uyeong Choi, Tae Il Kim, Kyu-Sung Jeong, Hyuk Lee, Sang Joon Shin. KS20226, a first-in-class IRP2 inhibitor, preferentially induces reprogramming of iron metabolism and suppresses tumor growth in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5937.

Synergic Induction of Autophagic Cell Death in Anaplastic Thyroid Carcinoma

Anaplastic thyroid carcinoma (ATC) has poor prognosis, high mortality rate and lack of effective therapy. A synergic combination of PD-L1 antibody together with cell death promoting substances like deacetylase inhibitors (DACi) and multi-kinase inhibitors (MKI) could sensitize ATC cells and promote decay by autophagic cell death. The PD-L1-inhibitor atezolizumab synergized with panobinostat (DACi) and sorafenib (MKI) leading to significant reduction of the viability, measured by real time luminescence, of three different patient-derived primary ATC cells, of C643 cells and follicular epithelial thyroid cells too. Solo administration of these compounds caused a significant over-expression of autophagy transcripts; meanwhile autophagy proteins were almost not detectable after the single administration of panobinostat, thus supporting a massive autophagy degradation process. Instead, the administration of atezolizumab caused an accumulation of autophagy proteins and the cleavage of the active caspases 8 and 3. Interestingly, only panobinostat and atezolizumab were able to exacerbate the autophagy process by increasing the synthesis, the maturation and final fusion with the lysosomes of the autophagosome vesicles. Despite ATC cells could be sensitized by atezolizumab via the cleavage of the caspases, no reduction of cell proliferation or promotion of cell death was observed. The apoptosis assay evidenced the ability of panobinostat alone and in combination with atezolizumab to induce the phosphatidil serine exposure (early apoptosis) and further the secondary necrosis. Instead, sorafenib was only able to cause necrosis. The increase of caspases activity induced by atezolizumab, the apoptosis and autophagy processes promoted by panobinostat synergize thus promoting cell death in well-established and primary anaplastic thyroid cancer cells. The combined therapy could represent a future clinical application for the treatment of such lethal and untreatable solid cancer.

Nitazoxanide and COVID-19: A review.

Coronavirus disease 2019 (COVID-19) is a current global illness triggered by severe acute respiratory coronavirus 2 (SARS-CoV-2) leading to acute viral pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and cytokine storm in severe cases. In the COVID-19 era, different unexpected old drugs are repurposed to find out effective and cheap therapies against SARS-CoV-2. One of these elected drugs is nitazoxanide (NTZ) which is an anti-parasitic drug with potent antiviral activity. It is effectively used in the treatment of protozoa and various types of helminths in addition to various viral infections. Thus, we aimed to elucidate the probable effect of NTZ on SARS-CoV-2 infections. Findings of the present study illustrated that NTZ can reduce SARS-CoV-2-induced inflammatory reactions through activation of interferon (IFN), restoration of innate immunity, inhibition of the release of pro-inflammatory cytokines, suppression of the mammalian target of rapamycin (mTOR), and induction of autophagic cell death. Moreover, it can inhibit the induction of oxidative stress which causes cytokine storm and is associated with ALI, ARDS, and multi-organ damage (MOD). This study concluded that NTZ has important anti-inflammatory and immunological properties that may mitigate SARS-CoV-2 infection-induced inflammatory disorders. Despite broad-spectrum antiviral properties of NTZ, the direct anti-SARS-CoV-2 effect was not evident and documented in recent studies. Then, in silico and in vitro studies in addition to clinical trials and prospective studies are needed to confirm the beneficial impact of NTZ on the pathogenesis of SARS-CoV-2 infection.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11033-022-07822-2.

F-box protein FBXO41 suppresses breast cancer growth by inducing autophagic cell death through facilitating proteasomal degradation of oncogene SKP2.

F-box proteins form SCF (Cullin1, SKP1 and F-box-protein) ubiquitin ligase complexes to ubiquitinate cellular proteins. They play key role in several biological processes, including cell cycle progression, cellular signaling, stress response and cell death pathways. Therefore, deregulation of F-box proteins is closely associated with cancer progression. However, the role of most of the F-box proteins, including FBXO41, in cancer progression remains elusive. Here, we unravel the role of FBXO41 in cancer progression. We show that FBXO41 suppresses cancer cell proliferation and tumor growth by inducing autophagic cell death through an alternative pathway. Results revealed that FBXO41-mediated autophagic cell death induction is dependent on accumulation of cell cycle checkpoint protein p21. We found that FBXO41 increases the expression levels of p21 at the post-translational level by promoting the proteasomal degradation of SKP2, an oncogenic F-box protein. Mechanistically, FBXO41 along with p21 disrupts the inhibitory BCL2 (anti-apoptotic protein)-Beclin1 (autophagy initiating factor) complex of autophagy induction to release Beclin1, thereby inducing autophagy. Overall, the present study establishes a new FBXO41-SKP2-p21 axis for induction of autophagic cell death to prevent cancer growth, which could be explored to develop promising cancer therapeutics.

F-box protein FBXO41 plays vital role in arsenic trioxide-mediated autophagic death of cancer cells

Arsenic trioxide (ATO), a potent anti-neoplastic drug, is known to prevent cancer cell growth through induction of autophagic cell death. However, importance of cellular factors in ATO-mediated autophagic cell death is poorly understood. In this study, using biochemical and immunofluorescence techniques, we show that F-box protein FBXO41 plays a critical role in anti-proliferative activity of ATO. Our study reveals the importance of FBXO41 in induction of autophagic death of cancer cells by ATO. Further, we show that the autophagic cell death induced by FBXO41 is distinct and independent of apoptosis and necrosis, showing that FBXO41 may play vital role in inducing autophagic death of apoptosis resistant cancer cells. Overall, our study elucidates the importance of FBXO41 in ATO induced autophagic cell death to prevent cancer progression, which could be explored to develop promising cancer therapeutic strategy.

Synergistic inhibitory effect of berberine and icotinib on non-small cell lung cancer cells via inducing autophagic cell death and apoptosis.

Resistance to epidermal growth factor receptor-tyrosin kinase inhibitors (TKIs, e.g. icotinib) remains a major clinical challenge. Non-small cell lung cancer patients with wild-type EGFR and/or K-RAS mutation are primary resistance to EGFR-TKIs. Berberine has been found to have potent anticancer activities via distinct molecular mechanism. In this study, we sought to investigate the therapeutic utility of BBR in combination with icotinib to overcome icotinib resistance in NSCLC cells, and explore the molecular mechanism of synergism of icotinib and BBR to EGFR-resistant NSCLC cells. We used the two EGFR-resistant NSCLC cell lines H460 and H1299 for testing the inhibitory effect of icotinib and/or BBR on them. Moreover, xenograft mouse model was applied for assessing the anti-tumor activities of BBR and icotinib in combination. Results showed that BBR and icotinib have a synergistic inhibitory effect on H460 and H1299 cells through induction of autophagic cell death and apoptosis. Accordingly, the anti-cancer effect of BBR plus icotinib was further confirmed in the NSCLC xenograft mouse models. Combination of BBR and icotinib significantly inhibited the protein expression and the activity of EGFR by inducing autophagic EGFR degradation. BBR plus icotinib resulted in intracellular ROS accumulation, which could mediated autophagy and apoptosis and involved in the suppression of cell migration and invasion. In conclusions, combination application of BBR and icotinib could be an effective strategy to overcome icotinib resistance in the treatment of NSCLC.

Itraconazole improves survival outcomes in patients with colon cancer by inducing autophagic cell death and inhibiting transketolase expression.

The incidence of colon cancer continues to increase annually, and it is the leading cause of cancer-associated mortality worldwide. Altering cell metabolism and inducing autophagic cell death have recently emerged as novel strategies in preventing tumor growth. Autophagy plays an essential role in energy production by degrading damaged cellular components and is also associated with tumor proliferation suppression. Itraconazole is an FDA-approved drug used as an antifungal medication and has been reported to induce autophagic cell death in breast cancer. However, the effects of itraconazole on cell metabolism and induction of apoptosis in colon cancer remain unclear. The present study analyzed extensive data from patients diagnosed with colon cancer using itraconazole between January 2011 and December 2015, from the Taiwanese National Health Insurance Research Database. The underlying molecular mechanisms of itraconazole in autophagy-induced cell death were also investigated. The results demonstrated that the 5-year survival rate was significantly higher in patients with colon cancer who received itraconazole treatment. In addition, itraconazole decreased the viability and cell colony formation, and induced cleaved caspase-3 expression and G1 cell cycle arrest of COLO 205 and HCT 116 cells. Notably, itraconazole induced autophagy by enhancing LC3B and p62 expression. Following LC3 knockdown, the viability of itraconazole-treated COLO 205 and HCT 116 cells notably improved. Taken together, the results of the present study suggest that itraconazole may have a beneficial effect on patients with colon cancer, and its underlying molecular mechanisms may be associated with the induction of autophagic cell death.

Biochemical studies of the effect of arsenic trioxide on Ehrlich ascites tumor: Thesis Abstract

Thesis Abstract Hexokinase 2 enzyme catalyzes the rate-limiting step in glycolysis. It is overexpressed in several carcinomas including breast cancer to sustain energy for rapidly dividing cells and associates with chemoresistance. However, impact of chemo drugs alone or in combination on hexokinase activity and autophagic cell death is unclear. Arsenic trioxide (As2O3) (ATO) is a metalloid with potent antineoplastic effects in several types of cancer especially acute promyelocytic leukemia and breast cancer. Due to the cytotoxicity and low efficacy of ATO when it is used alone, a combination of ATO with other chemotherapeutic drugs may provide a rational basis for novel therapeutic combinations. Despite these efforts, the specific cellular events that account for differential effects of ATO on cancer cells’viability and the possible therapeutic combinations remain to be well‐defined. In this report, we used an in vivo murine adenocarcinoma model to validate the effects of As2O3 and cisplatin on hexokinase activity and autophagic cancer cell death. We found that the two drugs inhibit hexokinase activity and induceautophagic marker, beclin 1 expression. Interestingly, combining As2O3 with cisplatin synergistically enhanced these effects and alleviated oxidative stress often encountered in As2O3 treatment. Altogether, our data provide direct evidence that inhibition of hexokinase activity and induction of autophagic cell death are mediating the antineoplastic effects of As2O3 and cisplatin. Our findings raise the potential of combiningAs2O3 with cisplatin as an approach to augment cisplatin-induced cell death and combat cisplatin chemo-resistance in cancer.

Targeting AKT/mTOR and Bcl-2 for Autophagic and Apoptosis Cell Death in Lung Cancer: Novel Activity of a Polyphenol Compound.

Autophagic cell death (ACD) is an alternative death mechanism in resistant malignant cancer cells. In this study, we demonstrated how polyphenol stilbene compound PE5 exhibits potent ACD-promoting activity in lung cancer cells that may offer an opportunity for novel cancer treatment. Cell death caused by PE5 was found to be concomitant with dramatic autophagy induction, as indicated by acidic vesicle staining, autophagosome, and the LC3 conversion. We further confirmed that the main death induction caused by PE5 was via ACD, since the co-treatment with an autophagy inhibitor could reverse PE5-mediated cell death. Furthermore, the defined mechanism of action and upstream regulatory signals were identified using proteomic analysis. Time-dependent proteomic analysis showed that PE5 affected 2142 and 1996 proteins after 12 and 24 h of treatment, respectively. The crosstalk network comprising 128 proteins that control apoptosis and 25 proteins involved in autophagy was identified. Protein–protein interaction analysis further indicated that the induction of ACD was via AKT/mTOR and Bcl-2 suppression. Western blot analysis confirmed that the active forms of AKT, mTOR, and Bcl-2 were decreased in PE5-treated cells. Taken together, we demonstrated the novel mechanism of PE5 in shifting autophagy toward cell death induction by targeting AKT/mTOR and Bcl-2 suppression.

Beclin1‑armed oncolytic Vaccinia virus enhances the therapeutic efficacy of R‑CHOP against lymphoma invitro and invivo.

Non-Hodgkin lymphoma (NHL) is a form of lymphoid malignancy, with diffuse large B cell lymphoma (DLBCL) being the most common NHL isoform. Approximately half of patients with DLBCL are successfully cured via first-line Rituximab, Cyclophosphamide, Epirubicin, Vindesine, Prednisolone (R-CHOP) treatment. However, 30–40% of patients with DLBCL ultimately suffer from treatment-refractory or relapsed disease. These patients often suffer from high mortality rates owing to a lack of suitable therapeutic options, and all patients are at a high risk of serious treatment-associated dose-dependent toxicity. As such, it is essential to develop novel treatments for NHL that are less toxic and more efficacious. Oncolytic Vaccinia virus (OVV) has shown promise as a means of treating numerous types of cancer. Gene therapy strategies further enhance OVV-based therapy by improving tumor cell recognition and immune evasion. Beclin1 is an autophagy-associated gene that, when upregulated, induces excess autophagy and cell death. The present study aimed to develop an OVV-Beclin1 therapy capable of inducing autophagic tumor cell death. OVV-Beclin1 was able to efficiently kill NHL cells and to increase the sensitivity of these cells to R-CHOP, thereby decreasing the dose-dependent toxic side effects associated with this chemotherapeutic regimen. The combination of OVV-Beclin1 and R-CHOP also significantly improved tumor growth inhibition and survival in a BALB/c murine model system owing to the synergistic induction of autophagic cell death. Together, these findings suggest that OVV-Beclin1 infection can induce significant autophagic cell death in NHL, highlighting this as a novel means of inducing tumor cell death via a mechanism that is distinct from apoptosis and necrosis.

Two species of Ulva inhibits the progression of cervical cancer cells SiHaby means of autophagic cell death induction.

Edible green algal seaweeds, namely Ulva intestinalis and Ulva lactuca constitute a significant repository of popular herbal medicines in the Traditional Chinese Medicinal system. The present study aimed to assess the anticancer potential of these algal members and its mode of action in cervical cancer cells SiHa. The algal samples primarily extracted in methanol was fractionated into hexane, chloroform, and aqueous algal fractions, which inhibited the proliferation of SiHa cells in a dose-dependent manner, with the algal chloroform fractions harbouring the lowest IC50 dose of 141.38µg/ml in U. intestinalis and 445.278µg/ml in U. lactuca. These algal chloroform fractions when studied for their in-depth mode of action, were found to damage and pulverise the nuclei, resulting in a concomitant increase in subG0-phase of SiHa cells, studied by flow cytometry. The algal treatment also caused an increase in the number of acidic vesicles and enhanced the expression of LC3BII, p62 and atg12 proteins, which together pointed out autophagy as the induced mode of cell death. Upregulated Bax and p53 expression along with decreased Bcl2 expression also correlated to autophagic cell death. Decreased expression of E6 viral oncogene was noted as a significant response to algal fractions. Lastly, these potent algal fractions when characterised pharmacologically through GC-MS analysis were found to be rich in unsaturated fatty acids, majorly palmitic acid. Hence, this study concludes that the two species of Ulva successfully decreased the proliferation of SiHa cervical cancer cells through autophagy, hinting at palmitic acid being the major responsible bioactive compound in both.

Metabolome-Driven Regulation of Adenovirus-Induced Cell Death.

A viral infection that involves virus invasion, protein synthesis, and virion assembly is typically accompanied by sharp fluctuations in the intracellular levels of metabolites. Under certain conditions, dramatic metabolic shifts can result in various types of cell death. Here, we review different types of adenovirus-induced cell death associated with changes in metabolic profiles of the infected cells. As evidenced by experimental data, in most cases changes in the metabolome precede cell death rather than represent its consequence. In our previous study, the induction of autophagic cell death was observed following adenovirus-mediated lactate production, acetyl-CoA accumulation, and ATP release, while apoptosis was demonstrated to be modulated by alterations in acetate and asparagine metabolism. On the other hand, adenovirus-induced ROS production and ATP depletion were demonstrated to play a significant role in the process of necrotic cell death. Interestingly, the accumulation of ceramide compounds was found to contribute to the induction of all the three types of cell death mentioned above. Eventually, the characterization of metabolite analysis could help in uncovering the molecular mechanism of adenovirus-mediated cell death induction and contribute to the development of efficacious oncolytic adenoviral vectors.

New Insight into Triple-Negative Breast Cancer Therapy: The Potential Roles of Endoplasmic Reticulum Stress and Autophagy Mechanisms.

Breast cancer is accounted as the fifth leading cause of mortality among the other cancers. Notwithstanding, Triple Negative Breast Cancer (TNBC) is responsible for 15-20% of breast cancer mortality. Despite many investigations, it remains incurable in part due to insufficient understanding of its exact mechanisms. A literature search was performed in PubMed, SCOPUS and Web of Science databases using the keywords autophagy, Endoplasmic Reticulum (ER) stress, apoptosis, TNBC and the combinations of these keywords. It was found that autophagy plays a dual role in cancer, so that it may decrease the viability of tumor cells or act as a cytoprotective mechanism. It then appears that using compounds having modulatory effects on autophagy is of importance in terms of induction of autophagic cell death and diminishing the proliferation and metastasis of tumor cells. Also, ER stress can be modulated in order to stimulate apoptotic and autophagic cell death in tumor cells. Perturbation in the signaling pathways related to cell survival leads to the initiation and progression of cancer. Regarding the advancement in the cancer pathology, it seems that modulation of autophagy and ER stress are promising.

Acquired tamoxifen resistance is surmounted by GW8510 through ribonucleotide reductase M2 downregulation-mediated autophagy induction

Tamoxifen resistance is a major roadblock in the treatment of patients with breast cancer. Ribonucleotide reductase M2 (RRM2) was found to be involved in acquired resistance of breast cancer cells (BCCs) to tamoxifen. Here, we used GW8510, which has been identified as a potential RRM2 inhibitor, to evaluate the effect of RRM2 inhibition on reversing resistance of BCCs to tamoxifen and investigate its mechanisms. We showed that RRM2 overexpression played a key role in the development of acquired tamoxifen resistance in BCCs through downregulation of autophagy level. Combination treatment with tamoxifen and GW8510 significantly inhibited survival of the tamoxifen-resistant BCCs through induction of autophagic cell death compared to either of the two drugs. Furthermore, combination of tamoxifen and GW8510 resulted in marked growth inhibition of tamoxifen-resistant BBC xenograft tumor in vivo compared to tamoxifen or GW8510 alone. In conclusion, tamoxifen in combination with GW8510 can overcome acquired tamoxifen resistance in BCCs and may be a rational therapeutic approach against breast cancer with high RRM2 expression.

Possible Selective Cytotoxicity of Vanadium Complex on Breast Cancer Cells Involving Pathophysiological Pathways.

Triple-Negative Breast Cancers (TNBC) are among the most aggressive and therapyresistant breast tumors. Development of new treatment strategies that target pathways involved in cancer cells resistance is an attractive candidate to overcome therapeutic resistance. To clarify the antitumor activity of [VO (bpy)2 Cl] Cl complex as a new therapeutic agent through studying the interplay between apoptosis, autophagy and notch signaling pathways. Proliferation of MDA-MB-231 cells and IC50 value of the vanadium complex were assessed by MTT assay. Flow cytometry was utilized to detect cell cycle distribution, apoptosis assay, LC3 levels and Acid Vascular Organelles (AVOs). Caspase 3 levels were detected by ELISA. Changes in Notch1 gene expression were assessed by real-time PCR. AVOs qualitative detection was assessed by a fluorescence microscope. The growth of MDA-MB-231 cells was suppressed after treatment with [VO (bpy)2 Cl] Cl complex, in a dose-dependent manner. The affinity for apoptotic cell death induction was shown through the increase in the sub G0 peak, the percentage of early and late apoptotic phases, and the elevation in caspase 3 levels. The affinity for autophagic cell death induction was observed through the increase in the G0/G1 phase, G2/M arrest, the increase of AVOs red fluorescence and elevated LC3 levels. The affinity for notch pathway inhibition was shown through the suppression of Notch 1 gene expression. [VO (bpy)2 Cl] Cl complex could be a promising candidate as therapeutic agent targeting different therapeutic targets including apoptosis, autophagy and notch signaling pathways.

STF-62247 and pimozide induce autophagy and autophagic cell death in mouse embryonic fibroblasts

Induction of autophagy can have beneficial effects in several human diseases, e.g. cancer and neurodegenerative diseases (ND). Here, we therefore evaluated the potential of two novel autophagy-inducing compounds, i.e. STF-62247 and pimozide, to stimulate autophagy as well as autophagic cell death (ACD) using mouse embryonic fibroblasts (MEFs) as a cellular model. Importantly, both STF-62247 and pimozide triggered several hallmarks of autophagy in MEFs, i.e. enhanced levels of LC3B-II protein, its accumulation at distinct cytosolic sites and increase of the autophagic flux. Intriguingly, autophagy induction by STF-62247 and pimozide resulted in cell death that was significantly reduced in ATG5- or ATG7-deficient MEFs. Consistent with ACD induction, pharmacological inhibitors of apoptosis, necroptosis or ferroptosis failed to protect MEFs from STF-62247- or pimozide-triggered cell death. Interestingly, at subtoxic concentrations, pimozide stimulated fragmentation of the mitochondrial network, degradation of mitochondrial proteins (i.e. mitofusin-2 and cytochrome c oxidase IV (COXIV)) as well as a decrease of the mitochondrial mass, indicative of autophagic degradation of mitochondria by pimozide. In conclusion, this study provides novel insights into the induction of selective autophagy as well as ACD by STF-62247 and pimozide in MEFs.

Naringin Induces Lysosomal Permeabilization and Autophagy Cell Death in AGS Gastric Cancer Cells.

Autophagy is a process of active programmed cell death, where a dying cell induces autophagosomes and subsequently regulated by degradative machinery. The aim of this study was to investigate the mechanism behind induction of autophagic cell death by Naringin flavonoid in AGS cancer cells. Growth inhibition of AGS cells showed downregulation of PI3K/Akt/mTOR signaling by Naringin treatment. Transmission electron microscopy observation showed swollen mitochondria and lysosome near peri-nuclear zone fused with autophagic vacuoles. Rapamycin pre-treatment with Naringin showed significant decrease in mTOR phosphorylation and increase in LC3B activation in AGS cells. Decrease in mTOR phosphorylation is associated with lysosomal function activation was observed by time-dependent treatment of Naringin. Induction of lysosomal membrane permeabilization (LMP) was observed by LAMP1 activation leading lysosomal cell death by releasing Cathepsin D from lysosomal lumen to cytosol. Naringin treated AGS cells showed up-regulating BH3 domain Bad, down-regulating Bcl-xL, and Bad phosphorylation and significant mitochondrial fluorescence intensity expression. Significant localization of mitochondria and LC3B activation was examined by person coefficient correlation. Activation of ERK1/2-p38 MAPKs and production of intracellular ROS has been observed over Naringin treatment. It has also been elucidated that pre-treatment with NAC inhibited mitochondria-LC3B colocalization, where ROS acted as upstream of ERK1/2-p38 MAPKs activation. Lysosomal cell death involvement has been evaluated by BAF A1 pre-treatment, inhibiting LAMP1, Cathepsin D, ROS, and blocking autophagolysosome in AGS cell death. Taken together, these findings show that, Naringin induced autophagy cell death involves LMP mediated lysosomal damage and BH3 protein Bad activation in AGS cancer cells.

Flubendazole elicits anti-cancer effects via targeting EVA1A-modulated autophagy and apoptosis in Triple-negative Breast Cancer.

Background: Triple-negative breast cancer (TNBC) is one of the most prevalent neoplastic diseases worldwide, but efficacious treatments for this pathological condition are still challenging. The lack of an effective targeted therapy also leads to a poor prognosis for patients affected by TNBC. In the present study, we repurposed the distinctive inhibitory effects of flubendazole, a traditional anthelmintic drug, towards the putative modulation of proliferation and migration of TNBC in vitro and in vivo.Methods: According to a series of experimental approaches, including immunofluorescence (IF), immunoblotting (IB), siRNA and GFP-mRFP-LC3 plasmid transfection, respectively, we have found that flubendazole is capable of inducing autophagic cell death and apoptosis, thus exerting some anti-proliferative and anti-migration activity in TNBC cells. The therapeutic effects of flubendazole were evaluated by xenograft mouse models, followed by immunohistochemistry (IHC), IF and IB. Changes in the gene expression profiles of flubendazole-treated TNBC cells were analyzed by RNA sequencing (RNA-seq) and validated by IB. The potential binding mode of flubendazole and EVA1A was predicted by molecular docking and demonstrated by site-directed mutagenesis.Results: We have presently found that flubendazole exhibits a considerable anti-proliferative activity in vitro and in vivo. Mechanistically, the induction of autophagic cell death appears to be pivotal for flubendazole-mediated growth inhibition of TNBC cells, whereas blocking autophagy was able to improve the survival rate and migration ability of flubendazole-treated TNBC cells. Specifically, RNA-seq analysis showed that flubendazole treatment could promote the up-regulation of EVA1A. Flubendazole may regulate autophagy and apoptosis by targeting EVA1A, thus affecting the mechanisms of TNBC proliferation and migration. Furthermore, Thr113 may be the key amino acid residues for the binding of flubendazole to EVA1A.Conclusion: Our results provide novel insights towards the putative anti-cancer efficacy of flubendazole. Furthermore, here we show that flubendazole could serve as a potential therapeutic drug in TNBC. Altogether, this study highlights the possibility of this repurposed autophagic inducer for future cancer treatments.

Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation

Recently, novel therapeutic strategies have been designed with the aim of killing cancer stem-like cells (CSCs), and considerable interest has been generated in the development of specific therapies that target stemness-related marker of CSCs. In this study, nonsteroidal anti-inflammatory drugs (NSAIDs) significantly potentiated Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-mediated cytotoxicity through apoptotic and autophagic cell death induction, but COX-2-inhibitory function was not required for NSAID-induced autophagy in CD44-overexpressing human chronic myeloid leukemia K562 (CD44highK562) cells. Importantly, we found that treatment with NSAIDs resulted in a dose-dependent increase in LC3-II level and decrease in p62 level and simultaneous reduction in multiple stemness-related markers including CD44, Oct4, c-Myc, and mutant p53 (mutp53) in CD44highK562 cells, suggesting that NSAIDs could induce autophagy, which might mediate degradation of stemness-related marker proteins. Activation of AMPK and inhibition of Akt/mTOR/p70S6K/4EBP1 participated in NSAID-induced autophagy in CD44highK562 cells. In addition, treatment of CD44highK562 cells with NSAIDs inhibited expression of HSF1/Hsps, which resulted in suppression of 17-AAG-induced activation of Hsp70, leading to reversal of 17-AAG resistance and sensitization of CD44highK562 cells to 17-AAG by NSAIDs. In conclusion, combining NSAIDs with Hsp90 inhibitor may offer one of the most promising strategies for eradication of CD44-overexpressing CSCs.