Deacetylation of LAMP1 drives lipophagy-dependent generation of free fatty acids by Abrus agglutinin to promote senescence in prostate cancer.

Therapy-induced senescence in cancer cells is an irreversible antiproliferative state, which inhibits tumor growth and is therefore a potent anti-neoplastic mechanism. In this study, low doses of Abrus agglutinin (AGG)-induced senescence through autophagy in prostate carcinoma cells (PC3) and inhibited proliferation. The inhibition of autophagy with 3-methyl adenine reversed AGG-induced senescence, thus confirming that AGG-triggered senescence required autophagy. AGG treatment also led to lipophagy-mediated accumulation of free fatty acids (FFAs), with a concomitant decrease in the number of lipid droplets. Lalistat, a lysosomal acid lipase inhibitor, abrogated AGG-induced lipophagy and senescence in PC3 cells, indicating that lipophagy is essential for AGG-induced senescence. The accumulation of FFAs increased reactive oxygen species generation, a known facilitator of senescence, which was also reduced in the presence of lalistat. Furthermore, AGG upregulated silent mating type information regulator 2 homolog 1 (SIRT1), while the presence of sirtinol reduced autophagy flux and the senescent phenotype in the AGG-treated cells. Mechanistically, AGG-induced cytoplasmic SIRT1 deacetylated a Lys residue on the cytoplasmic domain of lysosome-associated membrane protein 1 (LAMP1), an autolysosomal protein, resulting in lipophagy and senescence. Taken together, our findings demonstrate a novel SIRT1/LAMP1/lipophagy axis mediating AGG-induced senescence in prostate cancer cells.

Terminalia bellirica extract induces anticancer activity through modulation of apoptosis and autophagy in oral squamous cell carcinoma.

Terminalia bellirica (TB) has been used in traditional Indian medical system, Ayurveda. However, the mechanism underlying the efficacy of the TB extract against oral squamous cell carcinoma (OSCC) is yet to be explored. The present study established a connecting link between the TB extract induced apoptosis and autophagy in relation to reactive oxygen species (ROS). Our study revealed, that gallic acid in the TB extract possess a strong free radical scavenging capacity contributing towards the selective anti-proliferative activity. Furthermore, TB extract markedly enhanced the accumulation of ROS that facilitated mitochondrial apoptosis through DNA damage, indicating ROS as the vital component in regulation of apoptosis. This effect was effectively reversed by the use of a ROS scavenger, N-acetyl cysteine (NAC). Moreover, it was observed to induce autophagy; however, it attenuated the autophagosome-lysosome fusion in Cal33 cells without altering the lysosomal activity. Pharmacological inhibitors of autophagy, namely, 3-methyladenine and chloroquine, were demonstarated to regulate the stage-specific progression of autophagy post treatment with the TB extract, favouring subsequent activation of apoptosis. These findings revealed, presence of gallic acid in TB extract below NOAEL value causes oxidative upset in oral cancer cells and promote programmed cell death which has a potential therapeutic value against oral squamous cell carcinoma.

Chemical Screening Approaches Enabling Drug Discovery of Autophagy Modulators for Biomedical Applications in Human Diseases.

Autophagy is an intracellular degradation pathway for malfunctioning aggregation-prone proteins, damaged organelles, unwanted macromolecules and invading pathogens. This process is essential for maintaining cellular and tissue homeostasis that contribute to organismal survival. Autophagy dysfunction has been implicated in the pathogenesis of diverse human diseases, and therefore, therapeutic exploitation of autophagy is of potential biomedical relevance. A number of chemical screening approaches have been established for the drug discovery of autophagy modulators based on the perturbations of autophagy reporters or the clearance of autophagy substrates. These readouts can be detected by fluorescence and high-content microscopy, flow cytometry, microplate reader and immunoblotting, and the assays have evolved to enable high-throughput screening and measurement of autophagic flux. Several pharmacological modulators of autophagy have been identified that act either via the classical mechanistic target of rapamycin (mTOR) pathway or independently of mTOR. Many of these autophagy modulators have been demonstrated to exert beneficial effects in transgenic models of neurodegenerative disorders, cancer, infectious diseases, liver diseases, myopathies as well as in lifespan extension. This review describes the commonly used chemical screening approaches in mammalian cells and the key autophagy modulators identified through these methods, and highlights the therapeutic benefits of these compounds in specific disease contexts.

p73 induction by Abrus agglutinin facilitates Snail ubiquitination to inhibit epithelial to mesenchymal transition in oral cancer.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT), a key step in oral cancer progression, is associated with invasion, metastasis, and therapy resistance, thus targeting the EMT represents a critical therapeutic strategy for the treatment of oral cancer metastasis. Our previous study showed that Abrus agglutinin (AGG), a plant lectin, induces both intrinsic and extrinsic apoptosis to activate the tumor inhibitory mechanism. OBJECTIVE: This study aimed to investigate the role of AGG in modulating invasiveness and stemness through EMT inhibition for the development of antineoplastic agents against oral cancer. METHODS: The EMT- and stemness-related proteins were studied in oral cancer cells using Western blot analysis and fluorescence microscopy. The potential mechanisms of Snail downregulation through p73 activation in FaDu cells were evaluated using Western blot analysis, immunoprecipitation, confocal microscopy, and molecular docking analysis. Immunohistochemical staining of the tumor samples of AGG-treated FaDu-xenografted nude mice was performed. RESULTS: At the molecular level, AGG-induced p73 suppressed Snail expression, leading to EMT inhibition in FaDu cells. Notably, AGG promoted the translocation of Snail from the nucleus to the cytoplasm in FaDu cells and triggered its degradation through ubiquitination. In this setting, AGG inhibited the interaction between Snail and p73 in FaDu cells, resulting in p73 activation and EMT inhibition. Moreover, in epidermal growth factor (EGF)-stimulated FaDu cells, AGG abolished the upregulation of extracellular signal-regulated kinase (ERK)1/2 that plays a pivotal role in the upregulation of Snail to regulate the EMT phenotypes. In immunohistochemistry analysis, FaDu xenografts from AGG-treated mice showed decreased expression of Snail, SOX2, and vimentin and increased expression of p73 and E-cadherin compared with the control group, confirming EMT inhibition as part of its anticancer efficacy against oral cancer. CONCLUSION: In summary, AGG stimulates p73 in restricting EGF-induced EMT, invasiveness, and stemness by inhibiting the ERK/Snail pathway to facilitate the development of alternative therapeutics for oral cancer.

PUMA dependent mitophagy by Abrus agglutinin contributes to apoptosis through ceramide generation.

PUMA, a BH3-only pro-apoptotic Bcl2 family protein, is known to translocate from the cytosol into the mitochondria in order to induce apoptosis. Interestingly, the induction of PUMA by p53 plays a critical role in DNA damage-induced apoptosis. In this study, we reported mitophagy inducing potential of PUMA triggered by phytolectin Abrus agglutinin (AGG) in U87MG glioblastoma cells and established AGG-induced ceramide acts as the chief mediator of mitophagy dependent cell death through activation of both mitochondrial ROS as well as ER stress. Importantly, AGG upregulates PUMA expression in U87MG cells with the generation of dysfunctional mitochondria, with gain and loss of function of PUMA is shown to alter mitophagy induction. At the molecular level, our study identified that the LC3 interacting region (LIR) located at the C-terminal end of PUMA interacts with LC3 in order to stimulate mitophagy. In addition, AGG is also found to trigger ubiquitination of PUMA which in turn interacted with p62 for prompting mitophagy suggesting that AGG turns on PUMA-mediated mitophagy in U87MG cells in both p62-dependent as well as in p62-independent manner. Interestingly, AGG-triggered ceramide production through activation of ceramide synthase-1 leads to induction of ER stress and ROS accumulation to promote mitochondrial damage as well as mitophagy. Further, upon pre-treatment with Mdivi-1, DRP1 inhibitor, AGG exposure results in suppression of apoptosis in U87MG cells indicating AGG-induced mitophagy switches to apoptosis that can be exploited for better cancer therapeutics.

Autophagy regulates cisplatin-induced stemness and chemoresistance via the upregulation of CD44, ABCB1 and ADAM17 in oral squamous cell carcinoma.

OBJECTIVE: We inspected the relevance of CD44, ABCB1 and ADAM17 in OSCC stemness and deciphered the role of autophagy/mitophagy in regulating stemness and chemoresistance. MATERIAL AND METHODS: A retrospective analysis of CD44, ABCB1 and ADAM17 with respect to the various clinico-pathological factors and their correlation was analysed in sixty OSCC samples. Furthermore, the stemness and chemoresistance were studied in resistant oral cancer cells using sphere formation assay, flow cytometry and florescence microscopy. The role of autophagy/mitophagy was investigated by transient transfection of siATG14, GFP-LC3, tF-LC3, mKeima-Red-Mito7 and Western blot analysis of autophagic and mitochondrial proteins. RESULTS: In OSCC, high CD44, ABCB1 and ADAM17 expressions were correlated with higher tumour grades and poor differentiation and show significant correlation in their co-expression. In vitro and OSCC tissue double labelling confirmed that CD44+ cells co-expresses ABCB1 and ADAM17. Further, cisplatin (CDDP)-resistant FaDu cells displayed stem-like features and higher CD44, ABCB1 and ADAM17 expression. Higher autophagic flux and mitophagy were observed in resistant FaDu cells as compared to parental cells, and inhibition of autophagy led to the decrease in stemness, restoration of mitochondrial proteins and reduced expression of CD44, ABCB1 and ADAM17. CONCLUSION: The CD44+ /ABCB1+ /ADAM17+ expression in OSCC is associated with stemness and chemoresistance. Further, this study highlights the involvement of mitophagy in chemoresistance and autophagic regulation of stemness in OSCC.

Oral Cancer Stem Cells Microenvironment.

Cancer stem cells (CSCs) play important role in tumor growth and metastasis coupled with increased recurrences and acquired therapeutic resistance in oral cancer. The tumor microenvironment imposes intense pressure in cancer evolution in response to adverse growth conditions, resource limitation and immune predation. Here, we discussed the dynamic interplay between cancer stem cells and tumor microenvironment in the formation of intratumoral heterogeneity to modulate tumor progression. The CSCs niche provide a special microhabitat for survival, maintenance of stemness and tumor re-propagation. Moreover, adaptive cellular behavior might be driven by tough tumor microenvironmental selective forces which highly regulate alterations in the gene expression leading to the reprogramming of signaling pathways generating stem-like characteristics, adaptive metabolic plasticity and energy fueling with autophagy to permit the CSCs to sustain in the ever changing microenvironments during tumor progression. On the other hand, CSCs also direct the tumor microenvironment modulation and remodeling in its favour. The cytokines, chemokines and growth factors released from CSCs regulates neoangiogensis, differentiation, degradation of matrix protein and immune suppression favoring tumor-promoting conditions and initiates multiple signaling cascades augmenting the tumor progression.

DNA damage by 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced p53-mediated apoptosis through activation of cytochrome P450/aryl hydrocarbon receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; polycyclic aromatic hydrocarbon) is a persistent and ubiquitous environmental contaminant that causes a wide variety of deleterious effects. In this study, the DNA damage and apoptotic activity induced by TCDD was examined using in silico and in vitro approaches. In silico study showed that conformational changes and energies involved in the binding of TCDD to cytochrome P450 1B1 (CYP1B1) were crucial for its target proteins. Moreover, activated TCDD had high affinity to bind with aryl hydrocarbon receptor (AhR), with a binding energy of -564.7 Kcal/mol. Further, TCDD-CYP1B1 complex showed strong binding affinity for caspase 3, showing a binding energy of -518.5 Kcal/mol, and the docking of caspase inhibitors in the complex showed weak interaction with low binding energy as compared to TCDD-CYP1B1 caspase complexes. Interestingly, TCDD-induced apoptosis was significantly suppressed in Ac-DEVD-CMK-pretreated cells. The DNA damage activity of TCDD was quantified by comet tail formation and γ-H2AX foci formation in HaCaT cells. The role of CYP1B1 and AhR in DNA damage and apoptosis was demonstrated, and clotrimazole as well as knockdown of CYP1B1 and AhR could inhibit TCDD activation and suppress DNA damage followed by apoptosis in HaCaT cells. Moreover, TCDD increased expression of p53 and PUMA and our data showed that TCDD induced DNA damage followed by p53-mediated apoptosis. This study highlights the critical role of CYP1B1 and AhR in TCDD activity and proposes that inhibition of these key molecules might serve as a potential therapeutic approach for treatment of allergy and cancer.

Elimination of dysfunctional mitochondria through mitophagy suppresses benzo[a]pyrene-induced apoptosis.

Mitophagy, a special type of autophagy, plays an important role in the mitochondria quality control and cellular homeostasis. In this study, we examined the molecular mechanism of mitophagy induction with benzo[a]pyrene (B[a]P), a ubiquitous polycyclic aromatic hydrocarbon, which acts as a prosurvival response against apoptotic cell death. Our study showed that B[a]P displayed higher cytotoxicity in autophagy-deficient HaCaT cells as compared to control. Further, we showed that B[a]P triggered the Beclin-1-dependent autophagy through the mammalian target of rapamycin (mTOR)/AMP-activated protein kinase (AMPK) pathway. Moreover, our study indicated that the B[a]P-induced autophagy was initiated through the activation of cytochrome P450 1B1 (CYP1B1) and the aryl hydrocarbon receptor (AhR) in HaCaT cells. Intriguingly, the B[a]P-induced Beclin-1-mediated mitophagy was suppressed in CYP1B1 and AhR knockdown HaCaT cells, indicating a crucial role of B[a]P activation in the mitophagy induction to regulate cell death. B[a]P was shown to increase the mitochondrial dysfunction and decrease the mitochondrial membrane potential, resulting in depletion of ATP level along with the inhibition of the oxygen consumption rate in HaCaT cells. Importantly, the supplementation of methyl pyruvate compensated for the B[a]P-induced drop in the ATP level and mitigated the reactive oxygen species burden and autophagy. Mechanistically, B[a]P inhibited the manganese superoxide dismutase (MnSOD) activity and we found that the activated mitochondrial CYP1B1 interacted with MnSOD, inflicting mitophagy to protect from B[a]P-induced apoptosis. In summary, our study reveals mitophagy induction as a cellular protection mechanism against B[a]P-triggered toxicity and carcinogenesis.

Abrus agglutinin targets cancer stem-like cells by eliminating self-renewal capacity accompanied with apoptosis in oral squamous cell carcinoma.

The accumulating evidences show that Abrus agglutinin, a plant lectin, displays a broad range of anticancer activity including cancer-specific induction of apoptosis; however, the underlying molecular mechanism of Abrus agglutinin-induced oral cancer stem cell elimination remains elusive. Our data documented that Abrus agglutinin effectively downregulated the CD44+ expression with the increased CD44- population in different oral cancer cells. After 24-h Abrus agglutinin treatment, FaDu cells were quantified for orosphere formation in ultra-low attachment plates and data showed that Abrus agglutinin inhibited the number and size of orosphere in a dose-dependent manner in FaDu cells. Furthermore, Abrus agglutinin hindered the plasticity of FaDu orospheres as supported by reduced sphere formation and downregulated the self-renewal property via inhibition of Wnt-ß-catenin signaling pathway. Introduction of LiCl, a glycogen synthase kinase 3ß inhibitor, rescued the Abrus agglutinin-stimulated inhibition of ß-catenin and phosphorylated glycogen synthase kinase 3ß in FaDu cell-derived orospheres confirming importance of Wnt signaling in Abrus agglutinin-mediated inhibition of stemness. In this connection, our data showed that Abrus agglutinin restrained proliferation and induced apoptosis in FaDu-derived cancer stem cells in dose-dependent manner. Moreover, western blot data demonstrated that Abrus agglutinin increased the Bax/Bcl-2 ratio with activation of poly(adenosine diphosphate-ribose) polymerase and caspase-3 favoring apoptosis induction in orospheres. Abrus agglutinin induced reactive oxygen species accumulation in orospheres and pretreatment of N-acetyl cysteine, and a reactive oxygen species scavenger inhibited Abrus agglutinin-mediated caspase-3 activity and ß-catenin expression indicating reactive oxygen species as a principal regulator of Wnt signaling and apoptosis. In conclusion, Abrus agglutinin has a potential role as an integrative therapeutic approach for combating oral cancer through targeting self-renewability of orospheres via reactive oxygen species-mediated apoptosis.

ATG14 facilitated lipophagy in cancer cells induce ER stress mediated mitoptosis through a ROS dependent pathway.

Understanding the dynamics of autophagy and apoptosis crosstalk in cancer progression remains a challenging task. Here, we reported how the autophagy protein ATG14 induces lipophagy-mediated mitochondrial apoptosis. The overexpression of ATG14 in HeLa cells inhibited cell viability and increased mitochondrial apoptosis and endoplasmic reticulum (ER) stress. Furthermore, inhibition of this ATG14-induced autophagy promoted apoptosis. ATG14 overexpression resulted in the accumulation of free fatty acids (FFA), with a concomitant decrease in the number of lipid droplets. Our data showed that ER stress induced by ATG14 was due to the lipophagy-mediated FFA accumulation, which resulted in ROS-dependent mitochondrial stress leading to apoptosis. Inhibition of lipophagy in HeLa-ATG14 cells enhanced the cellular viability and rescued them from lipotoxicity. Mechanistically, we found that ATG14 interacted with Ulk1 and LC3, and knock down of Ulk1 prevented the lipidation of LC3 and autophagy in HeLa-ATG14 cells. We also identified a phosphatidylethanolamine (PE) binding region in ATG14, and the addition of Ulk1 to Hela-ATG14 cells decreased the ATG14-PE interaction. Lastly, confocal microscopy studies showed that the decrease in ATG14-PE binding was concomitant with the increase in LC3 lipidation over time, confirming the importance of Ulk1 to sort PE to LC3 during ATG14 mediated lipophagy induction. In conclusion, ATG14 and Ulk1 interact to induce lipophagy resulting in FFA accumulation leading to ER stress-mediated apoptosis.

Phytotherapeutic approach: a new hope for polycyclic aromatic hydrocarbons induced cellular disorders, autophagic and apoptotic cell death.

Polycyclic aromatic hydrocarbons (PAHs) comprise the major class of cancer-causing chemicals and are ranked ninth among the chemical compounds threatening to humans. Moreover, interest in PAHs has been mainly due to their genotoxic, teratogenic, mutagenic and carcinogenic property. Polymorphism in cytochrome P450 (CYP450) and aryl hydrocarbon receptor (AhR) has the capacity to convert procarcinogens into carcinogens, which is an imperative factor contributing to individual susceptibility to cancer development. The carcinogenicity potential of PAHs is related to their ability to bind to DNA, thereby enhances DNA cross-linking, causing a series of disruptive effects which can result in tumor initiation. They induce cellular toxicity by regulating the generation of reactive oxygen species (ROS), which arbitrate apoptosis. Additionally, cellular toxicity-mediated apoptotic and autophagic cell death and immune suppression by industrial pollutants PAH, provide fertile ground for the proliferation of mutated cells, which results in cancer growth and progression. PAHs play a foremost role in angiogenesis necessary for tumor metastasization by promoting the upregulation of metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and hypoxia inducible factor (HIF) in human cancer cells. This review sheds light on the molecular mechanisms of PAHs induced cancer development as well as autophagic and apoptotic cell death. Besides that authors have unraveled how phytotherapeutics is an alternate potential therapeutics acting as a savior from the toxic effects of PAHs for safer and cost effective perspectives.

Abrus Agglutinin, a type II ribosome inactivating protein inhibits Akt/PH domain to induce endoplasmic reticulum stress mediated autophagy-dependent cell death.

Abrus agglutinin (AGG), a type II ribosome-inactivating protein has been found to induce mitochondrial apoptosis. In the present study, we documented that AGG-mediated Akt dephosphorylation led to ER stress resulting the induction of autophagy-dependent cell death through the canonical pathway in cervical cancer cells. Inhibition of autophagic death with 3-methyladenine (3-MA) and siRNA of Beclin-1 and ATG5 increased AGG-induced apoptosis. Further, inhibiting apoptosis by Z-DEVD-FMK and N-acetyl cysteine (NAC) increased autophagic cell death after AGG treatment, suggesting that AGG simultaneously induced autophagic and apoptotic death in HeLa cells. Additionally, it observed that AGG-induced autophagic cell death in Bax knock down (Bax-KD) and 5-FU resistant HeLa cells, confirming as an alternate cell killing pathway to apoptosis. At the molecular level, AGG-induced ER stress in PERK dependent pathway and inhibition of ER stress by salubrinal, eIF2α phosphatase inhibitor as well as siPERK reduced autophagic death in the presence of AGG. Further, our in silico and colocalization study showed that AGG interacted with pleckstrin homology (PH) domain of Akt to suppress its phosphorylation and consequent downstream mTOR dephosphorylation in HeLa cells. We showed that Akt overexpression could not augment GRP78 expression and reduced autophagic cell death by AGG as compared to pcDNA control, indicating Akt modulation was the upstream signal during AGG's ER stress mediated autophagic cell death. In conclusion, we established that AGG stimulated cell death by autophagy might be used as an alternative tumor suppressor mechanism in human cervical cancer. © 2016 Wiley Periodicals, Inc.

Implications of cancer stem cells in developing therapeutic resistance in oral cancer.

Conventional therapeutics are often frequented with recurrences, refraction and regimen resistance in oral cavity cancers which are predominantly manifested by cancer stem cells (CSCs). During oncoevolution, cancer cells may undergo structural and functional reprogramming wherein they evolve as highly tolerant CSC phenotypes with greater survival advantages. The CSCs possess inherent and exclusive properties including self-renewal, hierarchical differentiation, and tumorigenicity that serve as the basis of chemo-radio-resistance in oral cancer. However, the key mechanisms underlying the CSC-mediated therapy resistance need to be further elucidated. A spectrum of dysfunctional cellular pathways including the developmental signaling, apoptosis, autophagy, cell cycle regulation, DNA damage responses and epigenetic regulations protect the CSCs from conventional therapies. Moreover, tumor niche shelters CSCs and creates an immunosuppressive environment favoring the survival of CSCs. Maintenance of lower redox status, epithelial-to-mesenchymal transition (EMT), metabolic reprogramming and altered drug responses are the accessory features that aid in the process of chemo-radio-resistance in oral CSCs. This review deals with the functional and molecular basis of cancer cell pluripotency-associated resistance highlighting the abrupt fundamental cellular processes; targeting these events may hold a great promise in the successful treatment of oral cancer.

Serum starvation induces anti-apoptotic cIAP1 to promote mitophagy through ubiquitination.

Mitophagy is a highly specialised type of autophagy that plays an important role in regulating mitochondrial dynamics and controls cellular quality during stress. In this study, we established that serum starvation led to induction of cellular inhibitor of apoptosis protein-1 (cIAP1), which regulates mitophagy through ubiquitination. Importantly, gain and loss of function of cIAP1 resulted in concomitant alteration in mitophagy confirming the direct implication of cIAP1 in induction of mitophagy. Interestingly, it was observed that cIAP1 translocated to mitochondria to associate with TOM20, Ulk1, and LC3 to initiate mitophagy. Further, cIAP1-induced mitophagy led to dysfunctional mitochondria that resulted in abrogation of mitochondrial oxygen consumption rate along with the decrease in ATP levels. The ubiquitination of cIAP1 was found to be the critical regulator of mitophagy. The disruption of cIAP1-ubiquitin interaction by PYR41 ensured the abrogation of cIAP1-LC3 interaction and mitophagy inhibition. Our study revealed an important function of cIAP1 as a crucial molecular link between autophagy and apoptosis for regulation of mitochondrial dynamics to mitigate cellular stress.