Monitoring autophagy in glioblastoma with antibody against isoform B of human microtubule-associated protein 1 light chain 3

Autophagy, an evolutionarily conserved response to stress, has recently been implicated in cancer initiation and progression, but the detailed mechanisms and functions have not yet been fully elucidated. One major obstacle to our understanding is lack of an efficient and robust method to specifically monitor autophagic cells in cancer specimens. To identify molecular events associated with autophagy, we performed cDNA microarray analysis of autophagic glioblastoma cell lines. Based on the analysis, we raised a polyclonal antibody against isoform B of human microtubule-associated protein 1 light chain 3 (LC3B). Application of the anti-LC3B antibody revealed the presence of autophagic cells in both in vitro and in vivo settings. Of the 65 glioblastoma tissues, 31 had highly positive cytoplasmic staining of LC3B. The statistical interaction between cytoplasmic staining of LC3B and Karnofsky Performance Scale score was significant. High expression of LC3B was associated with an improved outcome for patients with poorer performance, whereas, for patients with normal performance, survival was better for patients with low staining than with high staining of LC3B. Anti-LC3B antibody provides a useful tool for monitoring the induction of autophagy in cancer cells and tissues.     

Prodigiosin stimulates endoplasmic reticulum stress and induces autophagic cell death in glioblastoma cells

Prodigiosin, a secondary metabolite isolated from marine Vibrio sp., has antimicrobial and anticancer properties. This study investigated the cell death mechanism of prodigiosin in glioblastoma. Glioblastoma multiforme (GBM) is an aggressive primary cancer of the central nervous system. Despite treatment, or standard therapy, the median survival of glioblastoma patients is about 14.6 month. The results of the present study clearly showed that prodigiosin significantly reduced the cell viability and neurosphere formation ability of U87MG and GBM8401 human glioblastoma cell lines. Moreover, prodigiosin with fluorescence signals was detected in the endoplasmic reticulum and found to induce excessive levels of autophagy. These findings were confirmed by observation of LC3 puncta formation and acridine orange staining. Furthermore, prodigiosin caused cell death by activating the JNK pathway and decreasing the AKT/mTOR pathway in glioblastoma cells. Moreover, we found that the autophagy inhibitor 3-methyladenine reversed prodigiosin induced autophagic cell death. These findings of this study suggest that prodigiosin induces autophagic cell death and apoptosis in glioblastoma cells.     

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy.     

Autophagic cell death induced by TrkA receptor activation in human glioblastoma cells

The neurotrophin receptor tropomyosin-related kinase A (TrkA) and its ligand nerve growth factor (NGF) are expressed in astrocytomas, and an inverse association of TrkA expression with malignancy grade was described. We hypothesized that TrkA expression might confer a growth disadvantage to glioblastoma cells. To analyze TrkA function and signaling, we transfected human TrkA cDNA into the human glioblastoma cell line G55. We obtained three stable clones, all of which responded with striking cytoplasmic vacuolation and subsequent cell death to NGF. Analyzing the mechanism of cell death, we could exclude apoptosis and cellular senescence. Instead, we identified several indications of autophagy: electron microscopy showed typical autophagic vacuoles; acridine orange staining revealed acidic vesicular organelles; acidification of acidic vesicular organelles was prevented using bafilomycin A1; cells displayed arrest in G2/M; increased processing of LC3 occurred; vacuolation was prevented by the autophagy inhibitor 3-methyladenine; no caspase activation was detected. We further found that both activation of ERK and c-Jun N-terminal kinase but not p38 were involved in autophagic vacuolation. To conclude, we identified autophagy as a novel mechanism of NGF-induced cell death. Our findings suggest that TrkA activation in human glioblastomas might be beneficial therapeutically, especially as several of the currently used chemotherapeutics also induce autophagic cell death.     

Reliable LC3 and p62 Autophagy Marker Detection in Formalin Fixed Paraffin Embedded Human Tissue by Immunohistochemistry

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and nonneoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60-0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.Key words: Immunohistochemistry, immunofluorescence, formalin fixed paraffin embedded tissue, LC3B, p62, cancer     

Autophagosome maturation is impaired in Fabry disease

Fabry disease is a lysosomal storage disorder (LSD) caused by a deficiency in α-galactosidase A. The disease is characterized by severe major organ involvement, but the pathologic mechanisms responsible have not been elucidated. Disruptions of autophagic processes have been reported for other LSDs, but have not yet been investigated in Fabry disease. Renal biopsies were obtained from 5 adult male Fabry disease patients before and after 3 years of enzyme replacement therapy (ERT) with agalsidase alfa. Vacuole accumulation was seen in renal biopsies from all patients compared with control biopsies. Decreases in the number of vacuoles were seen after 3 years of ERT primarily in renal endothelial cells and mesangial cells. Measurement of the levels of LC3, a specific autophagy marker, in cultured cells from Fabry patients revealed increased basal levels compared to cells from non-Fabry subjects and a larger increase in response to starvation than seen in non-Fabry cells. Starvation in the presence of protease inhibitors did not result in a significant increase in LC3 in Fabry cells, whereas a further increase in LC3 was observed in non-Fabry cells, an observation that is consistent with impaired autophagic flux in Fabry disease. Overexpression of LC3 mRNA in Fabry fibroblasts compared to control cells is consistent with an upregulation of autophagy. Furthermore, LC3 and p62/SQSTM1 (that binds to LC3) staining in renal tissues and in cultured fibroblasts from Fabry patients supports impairment of autophagic flux. These findings suggest that Fabry disease is linked to a deregulation of autophagy.     

A quantitative TR-FRET plate reader immunoassay for measuring autophagy

Autophagy involves the isolation and targeting of unwanted cellular components to lysosomes for their digestion and reuse. Autophagic dysregulation has recently been implicated in a wide range of disease processes, yet facile methods for quantifying autophagy have been lacking in the field. Here we describe the generation of a quantitative plate reader assay for measuring the autophagic activity of cells. One of the best characterized autophagy markers is the protein LC3B, which normally resides in the cytosol (LC3B-I) but upon induction of autophagy becomes lipidated and embedded in autophagosomal membranes (LC3B-II). To quantify autophagy, we reasoned that GFP-tagged LC3B could serve as a time-resolved fluorescence resonance energy transfer (TR-FRET) acceptor upon cell lysis in the presence of terbium-labeled LC3B antibodies. Using this TR-FRET immunoassay approach, we screened a panel of LC3B antibodies and identified an antibody that exhibits strong preferential affinity toward autophagosome-associated LC3B-II and thereby facilitates specific detection of autophagic activity. The plate reader format provides both a quantitative and an objective result, thus overcoming some of the key limitations of the traditional immunoblotting and imaging approaches used to monitor autophagy. Moreover, since the assay step requires only a single addition of cell lysis buffer containing the detection antibody its simple workflow is both automation-friendly and scalable, which renders it suitable for high-throughput screening. We demonstrate how this TR-FRET immunoassay for GFP-tagged LC3B-II can be applied to quantitatively detect changes in the autophagy activity of cells, including estimating effects on autophagic flux.     

The natural compound oblongifolin C inhibits autophagic flux and enhances antitumor efficacy of nutrient deprivation

Metabolic stress induces autophagy as an alternative source of energy and metabolites. Insufficient autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy. Here, we performed a functional screen in search of novel autophagy regulators from natural products. We showed that oblongifolin C (OC), a natural small molecule compound extracted from Garcinia yunnanensis Hu, is a potent autophagic flux inhibitor. Exposure to OC results in an increased number of autophagosomes and impaired degradation of SQSTM1/p62. Costaining of GFP-LC3B with LysoTracker Red or LAMP1 antibody demonstrates that autophagosome-lysosome fusion is blocked by OC treatment. Furthermore, OC inhibits lysosomal proteolytic activity by altering lysosomal acidification and downregulating the expression of lysosomal cathepsins. Importantly, OC can eliminate the tolerance of cancer cells to nutrient starvation. Starvation dramatically increases the susceptibility of cancer cells to OC-induced CASP3-dependent apoptosis in vitro. Subsequent studies in xenograft mouse model showed that OC has anticancer potency as revealed by increased staining of cleaved CASP3, LC3 puncta, and SQSTM1, as well as reduced expression of lysosomal cathepsins. Combined treatment with OC and caloric restriction potentiates anticancer efficacy of OC in vivo. Collectively, these data demonstrated that OC is a novel autophagic flux inhibitor and might be useful in anticancer therapy.     

Concanavalin-A-induced autophagy biomarkers requires membrane type-1 matrix metalloproteinase intracellular signaling in glioblastoma cells

Pre-clinical trials for cancer therapeutics support the anti-neoplastic properties of the lectin from Canavalia ensiformis (Concanavalin-A, ConA) in targeting apoptosis and autophagy in a variety of cancer cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP), a plasma membrane-anchored matrix metalloproteinase, is a glycoprotein strongly expressed in radioresistant and chemoresistant glioblastoma that mediates pro-apoptotic signalling in brain cancer cells, we investigated whether MT1-MMP could also signal autophagy. Among the four lectins tested, we found that the mannopyranoside/glucopyranoside-binding ConA, which is also well documented to trigger MT1-MMP expression, increases autophagic acidic vacuoles formation as demonstrated by Acridine Orange cell staining. Although siRNA-mediated MT1-MMP gene silencing effectively reversed ConA-induced autophagy, inhibition of the MT1-MMP extracellular catalytic function with Actinonin or Ilomastat did not. Conversely, direct overexpression of the recombinant Wt-MT1-MMP protein triggered proMMP-2 activation and green fluorescent protein-microtubule-associated protein light chain 3 puncta indicative of autophagosomes formation, while deletion of MT1-MMP's cytoplasmic domain disabled such autophagy induction. ConA-treated U87 cells also showed an upregulation of BNIP3 and of autophagy-related gene members autophagy-related protein 3, autophagy-related protein 12 and autophagy-related protein 16-like 1, where respective inductions were reversed when MT1-MMP gene expression was silenced. Altogether, we provide molecular evidence supporting the pro-autophagic mechanism of action of ConA in glioblastoma cells. We also highlight new signal transduction functions of MT1-MMP within apoptotic and autophagic pathways that often characterize cancer cell responses to chemotherapeutic drugs.     

Elongation factor-2 kinase: its role in protein synthesis and autophagy

Elongation factor-2 kinase (eEF-2 kinase; Ca(2+)/calmodulin-dependent kinase III) controls the rate of peptide chain elongation. The activity of eEF-2 kinase is increased in many malignancies, yet its precise function in carcinogenesis remains unknown. Autophagy, a well-defined survival pathway in yeast, may also play an important role in oncogenesis. Furthermore, the autophagic response to nutrient deprivation is regulated by the mammalian target of rapamycin (mTOR). eEF-2 kinase lies downstream of mTOR and is regulated by several kinases in this pathway. Therefore, we studied the role of eEF-2 kinase in autophagy. Knockdown of eEF-2 kinase by RNA interference inhibited autophagy in several cell types as measured by light chain 3 (LC3)-II formation, acidic vesicular organelle staining, and electron microscopy. In contrast, overexpression of eEF-2 kinase increased autophagy. Furthermore, inhibition of autophagy markedly decreased the viability of glioblastoma cells grown under conditions of nutrient depletion. These results suggest that eEF-2 kinase plays a regulatory role in the autophagic process in tumor cells and may promote cancer cell survival under conditions of nutrient deprivation. Therefore, eEF-2 kinase activation may be a part of a survival mechanism in glioblastoma, and targeting this kinase may represent a novel approach to cancer treatment.     

Autophagy in endometrial carcinomas and prognostic relevance of 'stone-like' structures (SLS): what is destined for the atypical endometrial hyperplasia?

Autophagy, as an intracellular adaptation mechanism for oxygen and nutrient deprivation, is associated with tumor cell survival and aggressiveness. This was reaffirmed in a series of 360 endometrial carcinomas, using a standard immunohistochemical technique and the LC3A antibody, capable of recognizing both the soluble (LC3A-I) and the membrane-bound form (LC3A-II) of the protein. LC3A reactivity was recognized in three basic patterns-diffuse cytoplasmic, cytoplasmic/juxta-nuclear, and the so-called "stone-like" structures (SLS). The latter has emerged as the: hallmark of autophagic activity, being detected exclusively in endometrial carcinomas and their immediate precursor lesions, namely the atypical hyperplasias, albeit in small numbers. Other forms of hyperplasia without cytological atypia and normal endometrial tissues expressed only cytoplasmic staining patterns. High SLS counts, presumed to reflect excessive levels of autophagic activity, were associated with tumors of extremely poor prognosis. In contrast, a basal level of autophagic activity, as exemplified by the diffuse cytoplasmic and the cytoplasmic/juxta-nuclear patterns, had no impact on prognosis. Survival, according to tumor cell types, showed that serous papillary, clear cell and the high-grade endometrioid carcinomas had the worst prognosis compared to low-grade endometrioid carcinomas, but interestingly, within this tumor group, those having high-SLS counts had a much worse survival rate than those that did not. It is concluded that an assessment of autophagic activity, particularly in the form of SLS, is useful for evaluating tumor aggressiveness and, in the absence or an excess of SLS, it may also prove valid for differentiating grade 1 endometrioid adenocarcinomas from their precursor lesions.     

Peritumoral monocytes induce cancer cell autophagy to facilitate the progression of human hepatocellular carcinoma

Macroautophagy/autophagy is an important catabolic process mediating cellular homeostasis and plays critical roles in cancer development. Whereas autophagy has been widely studied in various pathological models, little is known about the distribution, clinical significance and regulatory mechanism of this process in human hepatocellular carcinoma (HCC). In the present study, we found that tumor tissues exhibited significantly increased levels of autophagy compared with non-tumor tissues, and cancer cells with higher levels of autophagy were predominantly enriched in the invading edge regions of human HCC. Increased MAP1LC3B/LC3B expression in the invading edge regions was significantly correlated with a higher density of closely located monocytes, and TNF and IL1B derived from tumor-activated monocytes synergistically induced cancer cell autophagy in the invading edge regions of HCC. Monocyte-elicited autophagy induced the epithelial-mesenchymal transition (EMT) of cancer cells and promoted tumor metastasis by activating the NFKB-SNAI1 signaling pathway. Moreover, the increase of LC3B⁺ cancer cells in the invading edge areas was associated with high mortality and reduced survival of patients with HCC. These findings indicated that cancer cell autophagy is regulated by a collaborative interaction between tumor and immune cell components in distinct HCC microenvironments, thus allowing the inflammatory monocytes to be rerouted in a tumor-promoting direction.     

Novel monofunctional platinum (II) complex Mono-Pt induces apoptosis-independent autophagic cell death in human ovarian carcinoma cells,distinct from cisplatin

Failure to engage apoptosis appears to be a leading mechanism of resistance to traditional platinum drugs in patients with ovarian cancer. Therefore, an alternative strategy to induce cell death is needed for the chemotherapy of this apoptosis-resistant cancer. Here we report that autophagic cell death, distinct from cisplatin-induced apoptosis, is triggered by a novel monofunctional platinum (II) complex named Mono-Pt in human ovarian carcinoma cells. Mono-Pt-induced cell death has the following features: cytoplasmic vacuolation, caspase-independent, no nuclear fragmentation or chromatin condensation, and no apoptotic bodies. These characteristics integrally indicated that Mono-Pt, rather than cisplatin, initiated a nonapoptotic cell death in Caov-3 ovarian carcinoma cells. Furthermore, incubation of the cells with Mono-Pt but not with cisplatin produced an increasing punctate distribution of microtubule-associated protein 1 light chain 3 (LC3), and an increasing ratio of LC3-II to LC3-I. Mono-Pt also caused the formation of autophagic vacuoles as revealed by monodansylcadaverine staining and transmission electron microscopy. In addition, Mono-Pt-induced cell death was significantly inhibited by the knockdown of either BECN1 or ATG7 gene expression, or by autophagy inhibitors 3-methyladenine, chloroquine and bafilomycin A₁. Moreover, the effect of Mono-Pt involved the AKT1-MTOR-RPS6KB1 pathway and MAPK1 (ERK2)/MAPK3 (ERK1) signaling, since the MTOR inhibitor rapamycin increased, while the MAPK1/3 inhibitor U0126 decreased Mono-Pt-induced autophagic cell death. Taken together, our results suggest that Mono-Pt exerts anticancer effect via autophagic cell death in apoptosis-resistant ovarian cancer. These findings lead to increased options for anticancer platinum drugs to induce cell death in cancer.     

Autophagy-independent function of MAP-LC3 during intracellular propagation of Chlamydia trachomatis

Microtubule-associated protein 1 (MAP1) light chain 3 (LC3) has proven useful as autophagosomal marker in studies on the interaction between pathogens and the host autophagic machinery. However, the function of LC3 is known to extend above and beyond its role in autophagosome formation. We previously reported that intrinsic LC3 is associated with the intracellular Chlamydia trachomatis inclusion in human epithelial cells. Here we show that LC3, most likely the cytoplasmic nonlipidated form, interacts with the C. trachomatis inclusion as a microtubule-associated protein rather than an autophagosome-associated component. In contrast, N-terminally GFP-tagged LC3 exclusively targets autophagosomes rather than chlamydial inclusions. Immunofluorescence analysis revealed an association of LC3 and MAP1 subunits A and B with the inclusion as early as 18 h post infection. Inclusion-bound LC3 was connected with the microtubular network. Depolymerization of the microtubular architecture disrupted the association of LC3/MAP1s with the inclusion. Furthermore, siRNA-mediated silencing of the MAP1 and LC3 proteins revealed their essential function in the intracellular growth of C. trachomatis. Interestingly, defective autophagy remarkably enhanced chlamydial growth, suggesting a suppressive effect of the autophagic machinery on bacterial development. However, depletion of LC3 in autophagy-deficient cells noticeably reduced chlamydial propagation. Thus, our findings demonstrate a new function for LC3, distinct from autophagy, in intracellular bacterial pathogenesis.     

Interconnections between autophagy and the coagulation cascade in hepatocellular carcinoma

Autophagy has an important role in tumor biology of hepatocellular carcinoma (HCC). Recent studies demonstrated that tissue factor (TF) combined with coagulation factor VII (FVII) has a pathological role by activating a G-protein-coupled receptor called protease-activated receptor 2 (PAR2) for tumor growth. The present study aimed to investigate the interactions of autophagy and the coagulation cascade in HCC. Seventy HCC patients who underwent curative liver resection were recruited. Immunohistochemical staining and western blotting were performed to determine TF, FVII, PAR2 and light chain 3 (LC3A/B) expressions in tumors and their contiguous normal regions. We found that the levels of autophagic marker LC3A/B-II and coagulation proteins (TF, FVII and PAR2) were inversely correlated in human HCC tissues. Treatments with TF, FVII or PAR2 agonist downregulated LC3A/B-II with an increased level of mTOR in Hep3B cells; in contrast, knockdown of TF, FVII or PAR2 increased LC3A/B. Furthermore, mTOR silencing restored the impaired expression of LC3A/B-II in TF-, FVII- or PAR2-treated Hep3B cells and activated autophagy. Last, as an in vivo correlate, we administered TF, FVII or PAR2 agonist in a NOD/severe combined immunodeficiency xenograft model and showed decreased LC3A/B protein levels in HepG2 tumors with treatments. Overall, our present study demonstrated that TF, FVII and PAR2 regulated autophagy mainly via mTOR signaling. The interaction of coagulation and autophagic pathways may provide potential targets for further therapeutic application in HCC.     

Interleukin-6

Autophagy reallocates nutrients and clears normal cells of damaged proteins and organelles. In the context of metastatic disease, invading cancer cells hijack autophagic processes to survive and adapt in the host microenvironment. We sought to understand how autophagy is regulated in the metastatic niche for prostate cancer (PCa) cells where bone marrow stromal cell (BMSC) paracrine signaling induces PCa neuroendocrine differentiation (NED). In PCa, this transdifferentiation of metastatic PCa cells to neuronal-like cells correlates with advanced disease. Because autophagy provides a survival advantage for cancer cells and promotes cell differentiation, we hypothesized that autophagy mediates PCa NED in the bone. Thus, we determined the ability of paracrine factors in conditioned media (CM) from two separate BMSC subtypes, HS5 and HS27a, to induce autophagy in C4-2 and C4-2B bone metastatic PCa cells by characterizing the autophagy marker, LC3. Unlike HS27a CM, HS5 CM induced LC3 accumulation in PCa cells, suggesting autophagy was induced and indicating that HS5 and HS27a secrete a different milieu of paracrine factors that influence PCa autophagy. We identified interleukin-6 (IL-6), a cytokine more highly expressed in HS5 cells than in HS27a cells, as a paracrine factor that regulates PCa autophagy. Pharmacological inhibition of STAT3 activity did not attenuate LC3 accumulation, implying that IL-6 regulates NED and autophagy through different pathways. Finally, chloroquine inhibition of autophagic flux blocked PCa NED; hence autophagic flux maintains NED. Our studies imply that autophagy is cytoprotective for PCa cells in the bone, thus targeting autophagy is a potential therapeutic strategy.     

Interleukin-6: a bone marrow stromal cell paracrine signal that induces neuroendocrine differentiation and modulates autophagy in bone metastatic PCa cells

Autophagy reallocates nutrients and clears normal cells of damaged proteins and organelles. In the context of metastatic disease, invading cancer cells hijack autophagic processes to survive and adapt in the host microenvironment. We sought to understand how autophagy is regulated in the metastatic niche for prostate cancer (PCa) cells where bone marrow stromal cell (BMSC) paracrine signaling induces PCa neuroendocrine differentiation (NED). In PCa, this transdifferentiation of metastatic PCa cells to neuronal-like cells correlates with advanced disease. Because autophagy provides a survival advantage for cancer cells and promotes cell differentiation, we hypothesized that autophagy mediates PCa NED in the bone. Thus, we determined the ability of paracrine factors in conditioned media (CM) from two separate BMSC subtypes, HS5 and HS27a, to induce autophagy in C4-2 and C4-2B bone metastatic PCa cells by characterizing the autophagy marker, LC3. Unlike HS27a CM, HS5 CM induced LC3 accumulation in PCa cells, suggesting autophagy was induced and indicating that HS5 and HS27a secrete a different milieu of paracrine factors that influence PCa autophagy. We identified interleukin-6 (IL-6), a cytokine more highly expressed in HS5 cells than in HS27a cells, as a paracrine factor that regulates PCa autophagy. Pharmacological inhibition of STAT3 activity did not attenuate LC3 accumulation, implying that IL-6 regulates NED and autophagy through different pathways. Finally, chloroquine inhibition of autophagic flux blocked PCa NED; hence autophagic flux maintains NED. Our studies imply that autophagy is cytoprotective for PCa cells in the bone, thus targeting autophagy is a potential therapeutic strategy.     

Autophagy in normal tissues of camel (Camelus dromedarius) with focus on immunoexpression of LC3 and LC3B

Autophagy is a highly regulated intracellular pathway for degradation and recycling of cytoplasmic protein aggregates and entire organelles. The autophagic pathway is stimulated by nutrient starvation, which prompted us to study the desert camel. Various organs of the camel undergo ecological and physiological stress due to food and water deprivation, dehydration and long exposure to solar radiation. We investigated the immunohistochemical expression of specific biomarkers of autophagy under normal conditions as a baseline for later work on stressed individuals. The autophagy-specific biomarkers, microtubule-associated protein1 light chain 3 (LC3), and its cleaved variant, LC3B, were strongly expressed in the cytosol of all tissues examined. The cytosolic immunoreactivity of LC3 was relatively weak, diffuse and vacuolar, while that of LC3B was stronger, punctate and at lower levels. LC3 appears to be associated with the autophagosomal membranes, either free or lysosome-bounded. LC3B is specific for the autophagosome-lysosome complexes and their degraded, granular contents. Autophagy was strongly expressed in CNS neurons and intestinal neural elements, which suggests a protective function for the nervous system. Autophagic markers also were seen in deformed immune-competent cells with fragmented nuclei in lymph nodes, spleen and gut-associated lymphoid tissue (GALT), which suggests a “suicidal” activity of eliminating unneeded cells. Autophagy, as measured by LC3 and LC3B expression, may participate in a general regulatory mechanism in tissues of the desert camel.     

Autophagy-independent function of MAP-LC3 during intracellular propagation of Chlamydia trachomatis

Microtubule-associated protein 1 (MAP1) light chain 3 (LC3) has proven useful as autophagosomal marker in studies on the interaction between pathogens and the host autophagic machinery. However, the function of LC3 is known to extend above and beyond its role in autophagosome formation. We previously reported that intrinsic LC3 is associated with the intracellular Chlamydia trachomatis inclusion in human epithelial cells. Here we show that LC3, most likely the cytoplasmic nonlipidated form, interacts with the C. trachomatis inclusion as a microtubule-associated protein rather than an autophagosome-associated component. In contrast, N-terminally GFP-tagged LC3 exclusively targets autophagosomes rather than chlamydial inclusions. Immunofluorescence analysis revealed an association of LC3 and MAP1 subunits A and B with the inclusion as early as 18 h post infection. Inclusion-bound LC3 was connected with the microtubular network. Depolymerization of the microtubular architecture disrupted the association of LC3/MAP1s with the inclusion. Furthermore, siRNA-mediated silencing of the MAP1 and LC3 proteins revealed their essential function in the intracellular growth of C. trachomatis. Interestingly, defective autophagy remarkably enhanced chlamydial growth, suggesting a suppressive effect of the autophagic machinery on bacterial development. However, depletion of LC3 in autophagy-deficient cells noticeably reduced chlamydial propagation. Thus, our findings demonstrate a new function for LC3, distinct from autophagy, in intracellular bacterial pathogenesis.