Nanoscopic Insights of Amphiphilic Peptide against the Oligomer Assembly Process to Treat Huntington's Disease.

Finding an effective therapeutic regimen is an urgent demand for various neurodegenerative disorders including Huntington's disease (HD). For the difficulties in observing the dynamic aggregation and oligomerization process of mutant Huntingtin (mHtt) in vivo, the evaluation of potential drugs at the molecular protein level is usually restricted. By combing lifetime-based fluorescence microscopies and biophysical tools, it is showcased that a designed amphiphilic peptide, which targets the mHtt at an early stage, can perturb the oligomer assembly process nanoscopically, suppress the amyloid property of mHtt, conformationally transform the oligomers and/or aggregates of mHtt, and ameliorate mHtt-induced neurological damage and aggregation in cell and HD mouse models. It is also found that this amphiphilic peptide is able to transport to the brain and rescue the memory deficit through intranasal administration, indicating its targeting specificity in vivo. In summary, a biophotonic platform is provided to investigate the oligomerization/aggregation process in detail that offers insight into the design and effect of a targeted therapeutic agent for Huntington's disease.

PSPC1 mediates TGF-ß1 autocrine signalling and Smad2/3 target switching to promote EMT, stemness and metastasis.

Activation of metastatic reprogramming is critical for tumour metastasis. However, more detailed knowledge of the underlying mechanism is needed to enable targeted intervention. Here, we show that paraspeckle component 1 (PSPC1), identified in an aberrant 13q12.11 locus, is upregulated and associated with poor survival in patients with cancer. PSPC1 promotes tumorigenesis, epithelial-to-mesenchymal transition (EMT), stemness and metastasis in multiple cell types and in spontaneous mouse cancer models. PSPC1 is the master activator for transcription factors of EMT and stemness and accompanies c-Myc activation to facilitate tumour growth. PSPC1 increases transforming growth factor-ß1 (TGF-ß1) secretion through an interaction with phosphorylated and nuclear Smad2/3 to potentiate TGF-ß1 autocrine signalling. Moreover, PSPC1 acts as a contextual determinant of the TGF-ß1 pro-metastatic switch to alter Smad2/3 binding preference from tumour-suppressor to pro-metastatic genes. Having validated the PSPC1-Smads-TGF-ß1 axis in various cancers, we conclude that PSPC1 is a master activator of pro-metastatic switches and a potential target for anti-metastasis drugs.

Erratum: USP11 regulates PML stability to control Notch-induced malignancy in brain tumours.

This corrects the article DOI: 10.1038/ncomms4214.

An intranasally delivered peptide drug ameliorates cognitive decline in Alzheimer transgenic mice.

Alzheimer's disease (AD) is the most common neurodegenerative disease. Imbalance between the production and clearance of amyloid ß (Aß) peptides is considered to be the primary mechanism of AD pathogenesis. This amyloid hypothesis is supported by the recent success of the human anti-amyloid antibody aducanumab, in clearing plaque and slowing clinical impairment in prodromal or mild patients in a phase Ib trial. Here, a peptide combining polyarginines (polyR) (for charge repulsion) and a segment derived from the core region of Aß amyloid (for sequence recognition) was designed. The efficacy of the designed peptide, R8-Aß(25-35), on amyloid reduction and the improvement of cognitive functions were evaluated using APP/PS1 double transgenic mice. Daily intranasal administration of PEI-conjugated R8-Aß(25-35) peptide significantly reduced Aß amyloid accumulation and ameliorated the memory deficits of the transgenic mice. Intranasal administration is a feasible route for peptide delivery. The modular design combining polyR and aggregate-forming segments produced a desirable therapeutic effect and could be easily adopted to design therapeutic peptides for other proteinaceous aggregate-associated diseases.

Intranasal Administration of a Polyethylenimine-Conjugated Scavenger Peptide Reduces Amyloid-ß Accumulation in a Mouse Model of Alzheimer's Disease.

Amyloid-ß (Aß) aggregation in the brain plays a central and initiatory role in pathogenesis and/or progression of Alzheimer's disease (AD). Inhibiting Aß aggregation is a potential strategy in the prevention of AD. A scavenger peptide, V24P(10-40), designed to decrease Aß accumulation in the brain, was conjugated to polyethylenimine (PEI) and tested as a preventive/therapeutic strategy for AD in this study. This PEI-conjugated V24P(10-40) peptide was delivered intranasally, as nasal drops, to four-month-old APP/PS1 double transgenic mice for four or eight months. Compared with control values, peptide treatment for four months significantly reduced the amount of GdnHCl-extracted Aß40 and Aß42 in the mice's hippocampus and cortex. After treatment for eight months, amyloid load, as quantified by Pittsburgh compound B microPET imaging, was significantly decreased in the mice's hippocampus, cortex, amygdala, and olfactory bulb. Our data suggest that this intranasally delivered scavenger peptide is effective in decreasing Aß accumulation in the brain of AD transgenic mice. Nasal application of peptide drops is easy to use and could be further developed to prevent and treat AD.

Activation of AMP-activated protein kinase α1 mediates mislocalization of TDP-43 in amyotrophic lateral sclerosis.

TAR DNA-binding protein-43 (TDP-43) is a nuclear RNA-binding protein involved in many cellular pathways. TDP-43-positive inclusions are a hallmark of amyotrophic lateral sclerosis (ALS). The major clinical presentation of ALS is muscle weakness due to the degeneration of motor neurons. Mislocalization of TDP-43 from the nucleus to the cytoplasm is an early event of ALS. In this study, we demonstrate that cytoplasmic mislocalization of TDP-43 was accompanied by increased activation of AMP-activated protein kinase (AMPK) in motor neurons of ALS patients. The activation of AMPK in a motor neuron cell line (NSC34) or mouse spinal cords induced the mislocalization of TDP-43, recapitulating this characteristic of ALS. Down-regulation of AMPK-α1 or exogenous expression of a dominant-negative AMPK-α1 mutant reduced TDP-43 mislocalization. Suppression of AMPK activity using cAMP-simulating agents rescued the mislocalization of TDP-43 in NSC34 cells and delayed disease progression in TDP-43 transgenic mice. Our findings demonstrate that activation of AMPK-α1 plays a critical role in TDP-43 mislocalization and the development of ALS; thus, AMPK-α1 may be a potential drug target for this devastating disease.

Mutations in the ubiquitin-binding domain of OPTN/optineurin interfere with autophagy-mediated degradation of misfolded proteins by a dominant-negative mechanism.

OPTN (optineurin) is an autophagy receptor and mutations in the OPTN gene result in familial glaucoma (E50K) and amyotrophic lateral sclerosis (ALS) (E478G). However, the mechanisms through which mutant OPTN leads to human diseases remain to be characterized. Here, we demonstrated that OPTN colocalized with inclusion bodies (IBs) formed by mutant HTT/huntingtin protein (mHTT) in R6/2 transgenic mice and IBs formed by 81QNmHTT (nuclear form), 109QmHTT (cytoplasmic form) or the truncated form of TARDBP/TDP-43 (TARDBP(ND251)) in Neuro2A cells. This colocalization required the ubiquitin (Ub)-binding domain (UbBD, amino acids 424 to 511) of OPTN. Overexpression of wild-type (WT) OPTN decreased IBs through K63-linked polyubiquitin-mediated autophagy. E50K or 210 to 410Δ (with amino acids 210 to 410 deleted) whose mutation or deletion was outside the UbBD decreased the IBs formed by 109QmHTT or TARDBP(ND251), as was the case with WT OPTN. In contrast, UbBD mutants, including E478G, D474N, UbBDΔ, 411 to 520Δ and 210 to 520Δ, increased accumulation of IBs. UbBD mutants (E478G, UbBDΔ) retained a substantial ability to interact with WT OPTN, and were found to colocalize with polyubiquitinated IBs, which might occur indirectly through their WT partner in a WT-mutant complex. They decreased autophagic flux evidenced by alteration in LC3 level and turnover and in the number of LC3-positive puncta under stresses like starvation or formation of IBs. UbBD mutants exhibited a weakened interaction with MYO6 (myosin VI) and TOM1 (target of myb1 homolog [chicken]), important for autophagosome maturation, in cells or sorted 109QmHtt IBs. Taken together, our data indicated that UbBD mutants acted as dominant-negative traps through the formation of WT-mutant hybrid complexes to compromise the maturation of autophagosomes, which in turn interfered with OPTN-mediated autophagy and clearance of IBs.

Role of glycine N-methyltransferase in the regulation of T-cell responses in experimental autoimmune encephalomyelitis.

Glycine N-methyltransferase (GNMT) is known for its function as a tumor suppressor gene. Since 100% of female Gnmt(-/-) mice developed hepatocellular carcinoma, we hypothesized that Gnmt(-/-) mice may have defective immune surveillance. In this study, we examined the immune modulation of GNMT in T-cell responses using experimental autoimmune encephalomyelitis (EAE). The results showed that EAE severity was reduced significantly in Gnmt(-/-) mice. Pathological examination of the spinal cords revealed that Gnmt(-/-) mice had significantly lower levels of mononuclear cell infiltration and demyelination than the wild-type mice. In addition, quantitative real-time PCR showed that expression levels of proinflammatory cytokines, including interferon (IFN)-γ and interleukin (IL)-17A, were much lower in the spinal cord of Gnmt(-/-) than in that of wild-type mice. Accordingly, myelin oligodendrocyte glycoprotein (MOG)-specific T-cell proliferation and induction of T-helper (Th)1 and Th17 cells were markedly suppressed in MOG(35-55)-induced Gnmt(-/-) mice. Moreover, the number of regulatory T (Treg) cells was increased significantly in these mice. When the T-cell receptor was stimulated, the proliferative capacity and the activation status of mTOR-associated downstream signaling were decreased significantly in Gnmt(-/-) CD4(+) T cells via an IL-2- and CD25-independent manner. Moreover, GNMT deficiency enhanced the differentiation of Treg cells without affecting the differentiation of Th1 and Th17 cells. Furthermore, the severity of EAE in mice adoptive transferred with GNMT-deficient CD4(+) T cells was much milder than in those with wild-type CD4(+) T cells. In summary, our findings suggest that GNMT is involved in the pathogenesis of EAE and plays a crucial role in the regulation of CD4(+) T-cell functions.

Full-length TDP-43 forms toxic amyloid oligomers that are present in frontotemporal lobar dementia-TDP patients.

Proteinaceous inclusions are common hallmarks of many neurodegenerative diseases. TDP-43 proteinopathies, consisting of several neurodegenerative diseases, including frontotemporal lobar dementia (FTLD) and amyotrophic lateral sclerosis (ALS), are characterized by inclusion bodies formed by polyubiquitinated and hyperphosphorylated full-length and truncated TDP-43. The structural properties of TDP-43 aggregates and their relationship to pathogenesis are still ambiguous. Here we demonstrate that the recombinant full-length human TDP-43 forms structurally stable, spherical oligomers that share common epitopes with an anti-amyloid oligomer-specific antibody. The TDP-43 oligomers are stable, have exposed hydrophobic surfaces, exhibit reduced DNA binding capability and are neurotoxic in vitro and in vivo. Moreover, TDP-43 oligomers are capable of cross-seeding Alzheimer's amyloid-ß to form amyloid oligomers, demonstrating interconvertibility between the amyloid species. Such oligomers are present in the forebrain of transgenic TDP-43 mice and FTLD-TDP patients. Our results suggest that aside from filamentous aggregates, TDP-43 oligomers may play a role in TDP-43 pathogenesis.

Immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of enterovirus 71.

UNLABELLED: Like poliovirus infection, severe infection with enterovirus 71 (EV71) can cause neuropathology. Unlike poliovirus, EV71 is often associated with hand-foot-and-mouth disease (HFMD). Here we established three mouse models for experimental infection with the same clinical isolate of EV71. The NOD/SCID mouse model is unique for the development of skin rash, an HFMD-like symptom. While the NOD/SCID mice developed limb paralysis and death at near-100% efficiency, the gamma interferon receptor knockout (ifngr KO) and stat-1 knockout mice exhibited paralysis and death rates near 78% and 30%, respectively. Productive infection with EV71 depends on the viral dose, host age, and inoculation route. Levels of infectious EV71, and levels of VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared side by side between the NOD/SCID and stat-1 knockout models before, during, and after disease onset. Spleen fibrosis and muscle degeneration are common in the NOD/SCID and stat-1 knockout models. The main differences between these two models include their disease manifestations and cytokine/chemokine profiles. The pathology of the NOD/SCID model includes (i) inflammation and expression of viral VP1 antigen in muscle, (ii) increased neutrophil levels and decreased eosinophil and lymphocyte levels, and (iii) hair loss and skin rash. The characteristic pathology of the stat-1 knockout model includes (i) a strong tropism of EV71 for the central nervous system, (ii) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brain stem, and spinal cord, (iii) amplification of microglial cells, and (iv) dystrophy of intestinal villi. Our comparative studies on these new models with oral or intraperitoneal (i.p.) infection underscored the contribution of host immunity, including the gamma interferon receptor, to EV71 pathogenesis. IMPORTANCE: In the past decade, enterovirus 71 (EV71) has emerged as a major threat to public health in the Asia-Pacific region. Disease manifestations include subclinical infection, common-cold-like syndromes, hand-foot-and-mouth disease (HFMD), uncomplicated brain stem encephalitis, severe dysregulation of the autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse. To date, no effective vaccine or treatment is available. A user-friendly and widely accessible animal model for researching EV71 infection and pathogenesis is urgently needed by the global community, both in academia and in industry.

USP11 regulates PML stability to control Notch-induced malignancy in brain tumours.

The promyelocytic leukaemia (PML) protein controls multiple tumour suppressive functions and is downregulated in diverse types of human cancers through incompletely characterized post-translational mechanisms. Here we identify USP11 as a PML regulator by RNAi screening. USP11 deubiquitinates and stabilizes PML, thereby counteracting the functions of PML ubiquitin ligases RNF4 and the KLHL20-Cul3 (Cullin 3)-Roc1 complex. We find that USP11 is transcriptionally repressed through a Notch/Hey1-dependent mechanism, leading to PML destabilization. In human glioma, Hey1 upregulation correlates with USP11 and PML downregulation and with high-grade malignancy. The Notch/Hey1-induced downregulation of USP11 and PML not only confers multiple malignant characteristics of aggressive glioma, including proliferation, invasiveness and tumour growth in an orthotopic mouse model, but also potentiates self-renewal, tumour-forming capacity and therapeutic resistance of patient-derived glioma-initiating cells. Our study uncovers a PML degradation mechanism through Notch/Hey1-induced repression of the PML deubiquitinase USP11 and suggests an important role for this pathway in brain tumour pathogenesis.

Inhibition of TDP-43 aggregation by nucleic acid binding.

The aggregation of TAR DNA-binding protein (TDP-43) has been shown as a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) since 2006. While evidence has suggested that mutation or truncation in TDP-43 influences its aggregation process, nevertheless, the correlation between the TDP-43 aggregation propensity and its binding substrates has not been fully established in TDP-43 proteinopathy. To address this question, we have established a platform based on the in vitro protein expression system to evaluate the solubility change of TDP-43 in response to factors such as nucleotide binding and temperature. Our results suggest that the solubility of TDP-43 is largely influenced by its cognate single-strand DNA (ssDNA) or RNA (ssRNA) rather than hnRNP, which is known to associate with TDP-43 C-terminus. The direct interaction between the refolded TDP-43, purified from E.coli, and ssDNA were further characterized by Circular Dichroism (CD) as well as turbidity and filter binding assay. In addition, ssDNA or ssRNA failed to prevent the aggregation of the F147L/F149L double mutant or truncated TDP-43 (TDP208-414). Consistently, these two mutants form aggregates, in contrast with the wild-type TDP-43, when expressed in Neuro2a cells. Our results demonstrate an intimate relationship between the solubility of TDP-43 and its DNA or RNA binding affinity, which may shed light on the role of TDP-43 in ALS and FTLD.

A homozygous NOTCH3 mutation p.R544C and a heterozygous TREX1 variant p.C99MfsX3 in a family with hereditary small vessel disease of the brain.

BACKGROUND: Mutations in the TREX1 and NOTCH3 genes cause retinal vasculopathy with cerebral leukodystrophy (RVCL) and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), respectively. Both are hereditary small vessel diseases of the brain (HSVDB). METHODS: We performed mutational analyses of TREX1 in genomic DNA from 39 unrelated patients who were NOTCH3-negative in genetic testing, selected out of 72 unrelated consecutive patients with HSVDB. RESULTS: Only one patient had a TREX1 sequence variation, a heterozygous TREX1 c.294dupA, putatively resulting in a truncated protein, p.C99MfsX3. The medical history of the patient's family was scrutinized, which revealed that heterozygous TREX1 p.C99MfsX3 was not segregating with the HSVDB. Re-examination of the NOTCH3 sequence data of the proband led to the identification of a homozygous NOTCH3 c.1630C>T (p.R544C) mutation, which segregated with the HSVDB in the family. The proband had a slightly more severe phenotype in comparison with her heterozygous p.R544C sister. CONCLUSION: TREX1 mutation is not a common cause of HSVDB. TREX1 p.C99MfsX3 is not a dominant mutation. Homozygosity of the NOTCH3 p.R544C has a modestly deleterious effect on the CADASIL phenotype. The NOTCH3 mutation may cause CADASIL through a gain-of-toxic function effect, which can be modified by other genetic or environmental factors and results in the phenotypic variation of CADASIL.

Overexpressed-eIF3I interacted and activated oncogenic Akt1 is a theranostic target in human hepatocellular carcinoma.

UNLABELLED: Eukaryotic translation initiation factor 3 subunit I (eIF3I) with transforming capability is often overexpressed in human hepatocellular carcinoma (HCC) but its oncogenic mechanisms remain unknown. We demonstrate that eIF3I is overexpressed in various cancers along with activated Akt1 phosphorylation and kinase activity in an eIF3I dose-dependent manner. A novel eIF3I and Akt1 protein interaction was identified in HCC cell lines and tissues and was required for eIF3I-mediated activation of Akt1 signaling. Expression of either antisense eIF3I or dominant negative Akt1 mutant suppressed eIF3I-mediated Akt1 oncogenic signaling and various other tumorigenic effects. Oncogenic domain mapping of the eIF3I and Akt1 interaction suggested that the C-terminal eIF3I interacted with the Akt1 kinase domain and conferred the majority of oncogenic functions. In addition, eIF3I interaction with Akt1 prevented PP2A dephosphorylation of Akt1 and resulted in constitutively active Akt1 oncogenic signaling. Importantly, concordant expression of endogenous eIF3I and phospho-Akt1 was detected in HCC cell lines and tissues. Treatment of eIF3I overexpressing HCC cells with the Akt1 specific inhibitor API-2 suppressed eIF3I-mediated tumorigenesis in vitro and in vivo. CONCLUSION: We describe a constitutive Akt1 oncogenic mechanism resulting from interaction of overexpressed eIF3I with Akt1 that prevents PP2A-mediated dephosphorylation. Overexpression of eIF3I in HCC is oncogenic and is a surrogate marker and therapeutic target for treatment with Akt1 inhibitors.

A critical role of astrocyte-mediated nuclear factor-κB-dependent inflammation in Huntington's disease.

Huntington's disease (HD) is an autosomal disease caused by a CAG repeat expansion in the huntingtin (HTT) gene. The resultant mutant HTT protein (mHTT) forms aggregates in various types of cells, including neurons and glial cells and preferentially affects brain function. We found that two HD mouse models (Hdh(150Q) and R6/2) were more susceptible than wild-type (WT) mice to lipopolysaccharide-evoked systemic inflammation and produced more proinflammatory cytokines in the brain. Such an enhanced inflammatory response in the brain was not observed in N171- 82Q mice that express mHTT only in neurons, but not in glial cells. Thus, HD glia might play an important role in chronic inflammation that accelerates disease progression in HD mice. Intriguingly, enhanced activation of nuclear factor (NF)-κB-p65 (p65), a transcriptional mediator of inflammatory responses, was observed in astrocytes of patients and mice with HD. Results obtained using primary R6/2 astrocytes suggest that these cells exhibited higher IκB kinase (IKK) activity that caused prolongation of NF-κB activation, thus upregulating proinflammatory factors during inflammation. R6/2 astrocytes also produced a more-damaging effect on primary R6/2 neurons than did WT astrocytes during inflammation. Blockage of IKK reduced the neuronal toxicity caused by R6/2 astrocytes and ameliorated several HD symptoms of R6/2 mice (e.g. decreased neuronal density, impaired motor coordination and poor cognitive function). Collectively, our results indicate that enhancement of the p65-mediated inflammatory response in astrocytes contributes to HD pathogenesis. Therapeutic interventions aimed at preventing neuronal inflammation may be an important strategy for treating HD.

Exome sequencing identifies GNB4 mutations as a cause of dominant intermediate Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of inherited neuropathies. Mutations in approximately 45 genes have been identified as being associated with CMT. Nevertheless, the genetic etiologies of at least 30% of CMTs have yet to be elucidated. Using a genome-wide linkage study, we previously mapped a dominant intermediate CMT to chromosomal region 3q28-q29. Subsequent exome sequencing of two affected first cousins revealed heterozygous mutation c.158G>A (p.Gly53Asp) in GNB4, encoding guanine-nucleotide-binding protein subunit beta-4 (Gß4), to cosegregate with the CMT phenotype in the family. Further analysis of GNB4 in an additional 88 unrelated CMT individuals uncovered another de novo mutation, c.265A>G (p.Lys89Glu), in this gene in one individual. Immunohistochemistry studies revealed that Gß4 was abundant in the axons and Schwann cells of peripheral nerves and that expression of Gß4 was significantly reduced in the sural nerve of the two individuals carrying the c.158G>A (p.Gly53Asp) mutation. In vitro studies demonstrated that both the p.Gly53Asp and p.Lys89Glu altered proteins impaired bradykinin-induced G-protein-coupled-receptor (GPCR) signaling, which was facilitated by the wild-type Gß4. This study identifies GNB4 mutations as a cause of CMT and highlights the importance of Gß4-related GPCR signaling in peripheral-nerve function in humans.

A hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic ALS in Taiwan.

A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene was recently identified as an important cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in Caucasian populations. The role of the C9ORF72 repeat expansion in ALS in Chinese populations has received little attention. We therefore performed mutation analyses in a Taiwanese cohort of 22 unrelated familial ALS (FALS) patients and 102 sporadic ALS patients of Han Chinese descent. The C9ORF72 mutation was found in 4 FALS (18.2%; 4/22) and 2 sporadic ALS patients (2.0%; 2/102). The C9ORF72 repeat numbers in the 300 healthy controls and the 118 ALS patients without the C9ORF72 mutation ranged from 3 to 15. Needle biopsy in the left frontal cortex of 1 patient with FALS-frontotemporal dementia revealed numerous cytoplasmic TAR DNA-binding protein 43 (TDP-43) inclusions and minimal neuritis, consistent with type B frontotemporal lobar degeneration with TDP-43 (FTLD-TDP) pathology. This study clearly demonstrates the existence and importance of the C9ORF72 hexanucleotide repeat expansion in a Taiwanese ALS cohort of Chinese origin, and supports the global presence of the C9ORF72 repeat expansion in ALS.

A combined DNA-affinic molecule and N-mustard alkylating agent has an anti-cancer effect and induces autophagy in oral cancer cells.

Although surgery or the combination of chemotherapy and radiation are reported to improve the quality of life and reduce symptoms in patients with oral cancer, the prognosis of oral cancer remains generally poor. DNA alkylating agents, such as N-mustard, play an important role in cancer drug development. BO-1051 is a new 9-anilinoacridine N-mustard-derivative anti-cancer drug that can effectively target a variety of cancer cell lines and inhibit tumorigenesis in vivo. However, the underlying mechanism of BO-1051-mediated tumor suppression remains undetermined. In the present study, BO-1051 suppressed cell viability with a low IC(50) in oral cancer cells, but not in normal gingival fibroblasts. Cell cycle analysis revealed that the tumor suppression by BO-1051 was accompanied by cell cycle arrest and downregulation of stemness genes. The enhanced conversion of LC3-I to LC3-II and the formation of acidic vesicular organelles indicated that BO-1501 induced autophagy. The expression of checkpoint kinases was upregulated as demonstrated with Western blot analysis, showing that BO-1051 could induce DNA damage and participate in DNA repair mechanisms. Furthermore, BO-1051 treatment alone exhibited a moderate tumor suppressive effect against xenograft tumor growth in immunocompromised mice. Importantly, the combination of BO-1051 and radiation led to a potent inhibition on xenograft tumorigenesis. Collectively, our findings demonstrated that BO-1051 exhibited a cytotoxic effect via cell cycle arrest and the induction of autophagy. Thus, the combination of BO-1051 and radiotherapy may be a feasible therapeutic strategy against oral cancer in the future.

Targeting protective autophagy exacerbates UV-triggered apoptotic cell death.

Autophagy is activated by various stresses, including DNA damage, and previous studies of DNA damage-induced autophagy have focused on the response to chemotherapeutic drugs, ionizing radiation, and reactive oxygen species. In this study, we investigated the biological significance of autophagic response to ultraviolet (UV) irradiation in A549 and H1299 cells. Our results indicated that UV induces on-rate autophagic flux in these cells. Autophagy inhibition resulting from the knockdown of beclin-1 and Atg5 reduced cell viability and enhanced apoptosis. Moreover, we found that ATR phosphorylation was accompanied by microtubule-associated protein 1 light chain 3B II (LC3B-II) expression during the early phases following UV irradiation, which is a well-established inducer of ATR. Knocking down ATR further attenuated the reduction in LC3B-II at early stages in response to UV treatment. Despite the potential role of ATR in autophagic response, reduced ATR expression does not affect autophagy induction during late phases (24 and 48 h after UV treatment). The result is consistent with the reduced ATR phosphorylation at the same time points and suggests that autophagic response at this stage is activated via a distinct pathway. In conclusion, this study demonstrated that autophagy acts as a cytoprotective mechanism against UV-induced apoptosis and that autophagy induction accompanied with apoptosis at late stages is independent of ATR activation.