Low Levels Of Autophagy Research Articles

Identification of an Autophagy-Related Signature for Prognosis and Immunotherapy Response Prediction in Ovarian Cancer.

Ovarian cancer (OC) is one of the most malignant tumors in the female reproductive system, with a poor prognosis. Various responses to treatments including chemotherapy and immunotherapy are observed among patients due to their individual characteristics. Applicable prognostic markers could make it easier to refine risk stratification for OC patients. Autophagy is closely implicated in the occurrence and development of tumors, including OC. Whether autophagy -related genes can be used as prognostic markers for OC patients remains unclear. The gene transcriptome data of 374 OC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The correlation between the autophagy levels and outcomes of OC patients was identified through the single sample gene set enrichment analysis (ssGSEA). Recognized molecular markers of autophagy in different clinical specimens were detected by immunohistochemistry (IHC) assay. The gene set enrichment analysis (GSEA), ESTIMATE, and CIBERSORT analysis were applied to explore the correlation of autophagy with the tumor immune microenvironment (TIME). Single-cell RNA-sequencing (scRNA-seq) data from seven OC patients were included for characterizing cell-cell interaction patterns of autophagy-high or low tumor cells. Machine learning, Stepwise Cox regression and LASSO-Cox analysis were used to screen autophagy hub genes, which were used to establish an autophagy-related signature for prognosis evaluation. Four tumor immunotherapy cohorts were obtained from the GEO (Gene Expression Omnibus) database and the literature for autophagy risk score validation. The autophagy levels were closely related to the prognosis of the OC patients. Additionally, the autophagy levels were correlated with TIME status including immune score, and immune-cell infiltration. The scRNA-seq analysis found that tumor cells with high or low autophagy levels had different interactions with immune cells, especially macrophages. Eight autophagy-hub genes (ZFYVE1, AMBRA1, LAMP2, TRAF6, PDPK1, ATG2B, DAPK1 and TP53INP2) were screened for an autophagy-related signature. According to this signature, higher risk score was correlated with poor prognosis and better immunotherapy response in the OC patients. The autophagy-related signature is applicable to predict the prognosis and immune checkpoint inhibitors (ICIs) therapy efficiency in OC patients. It is possible to identify OC patients who will respond to ICIs therapy and have a favorable prognosis, although more verification is needed.

Chronic steatosis aggravates cold-storage induced acute ischemic injury in rat donor livers through the perturbation of lipophagy

In this study, we explored the effects of cold ischemia on chronic steatosis and lipid signaling in vivo. Sprague Dawley (SD) rat models of chronic steatosis were established. Pathological observations and liver indices were assessed through hematoxylin-eosin (HE)- and Oil Red O staining. Autophagy and metabolism in adipose tissue were analyzed under post-ischemia and hypoxic conditions via western blotting and immunofluorescent analysis.We found that cold ischemia treatment exacerbated hepatic steatosis and reduced lipid phagocytosis. This manifested as a loss of Microtubule-associated protein 1A/1B-light chain 3 (LC3) and Perilipin 2 (PLIN2), and lower levels of autophagy. Cold ischemia also inhibited lipophagy in transplanted rat livers, most notably in moderate-to-severe steatosis models. Ischemia and hypoxia inhibited lipid phagocytosis and increased lipid accumulation.Collectively, these data show that chronic steatosis aggravates cold storage induced acute ischemic injury in rat donor livers through the inhibition of lipophagy. Moderate-to-severe steatosis therefore influences the postoperative recovery of liver transplant recipients, which should be immediately transplanted to reduce the risk of cold ischemia.

The Role of Autophagy in Myocardial Ischemia and Reperfusion

Autophagy is a process in which lysosome-mediated intracellular damage or aging organelles and proteins are degraded to produce amino acids, fatty acids, ATP, etc., and then reused by cells. Under normal physiological conditions, cardiomyocytes maintain low levels of autophagy. However, autophagy is activated during myocardial ischemia-reperfusion (MI/R), and autophagosomes increase significantly, indicating that autophagy plays an important role in myocardial ischemia-reperfusion injury (MI/RI). Studies have shown that autophagy has protective and detrimental effects on MI/RI and is regulated by a variety of factors. This article reviews the relationship between autophagy and MI/RI. Keywords: Autophagy, cardiomyocytes, hypoxia-reoxygenation injury, myocardial ischemia-reperfusion injury

CircRNF144B/miR-342-3p/FBXL11 axis reduced autophagy and promoted the progression of ovarian cancer by increasing the ubiquitination of Beclin-1

Circular RNAs (circRNAs) can regulate autophagy and ovarian cancer (OC) progression. However, autophagy-associated circRNAs involved in OC progression are largely unknown. Bioinformatics, RNA sequencing, and qRT-PCR were conducted to detect circRNF144B expression in OC as well as its relationship with patient prognosis. Functional experiments were used to determine the effects of circRNF144B on the proliferation, mobility and autophagy of OC. Double luciferase reporter assays, immunoprecipitation, and ubiquitination detection were performed to determine the molecular mechanisms of circRNF144B in autophagy and OC progression. CircRNF144B was elevated in OC tissues with low autophagy levels, and associated with poor prognosis. CircRNF144B promoted the malignant biological properties of OC cells, and inhibited the autophagy. Mechanistically, circRNF144B acts as a sponge for miR-342-3p and inhibits miR-342-3p-induced degradation of lysine demethylase 2 A (FBXL11) mRNA, leading to elevated FBXL11 protein levels. Elevated FBXL11 promoted the ubiquitination and degradation of Beclin-1, thus inhibiting autophagy. In conclusion, CircRNF144B increased FBXL11 level by sponging miR-342-3p, whereas elevated FBXL11 promoted the ubiquitination and protein degradation of Beclin-1, thus suppressing autophagy flux and promoting OC progression. Thus, circRNF144B may be an effective target for OC therapy.

Contribution of Adiponectin/Carnitine Palmityl Transferase 1A-Mediated Fatty Acid Metabolism during the Development of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that leads rapidly to death. The present study is aimed at discovering the in-depth pathogenesis of IPF, exploring the role of adiponectin/carnitine palmityl transferase 1A- (APN/CPT1A-) mediated fatty acid metabolism during the development of IPF, and excavating its potential mechanism. Here, THP-1 cells were differentiated into M0 macrophages, followed by polarization to M1 macrophages upon hypoxia. Subsequently, lung fibroblast HFL-1 cells were stimulated by M1 macrophages to simulate hypoxia-related IPF condition in vitro. It was discovered that the stimulation of M1 macrophages promoted fibroblast proliferation and fibrosis formation in vitro, accompanied with a disorder of the APN/CPT1A pathway, an overproduction of lipid peroxides, and a low level of autophagy in HFL-1 cells. Thereafter, APN treatment or CPT1A overexpression greatly suppressed above lipid peroxide accumulation, fibroblast proliferation, and fibrosis but activated autophagy in vitro. Furthermore, an in vivo IPF rat model was established by injection of bleomycin (BLM). Consistently, CPT1A overexpression exerted a protective role against pulmonary fibrosis in vivo; however, the antifibrosis property of CPT1A was partly abolished by 3-methyladenine (an autophagy inhibitor). In summary, APN/CPT1A-mediated fatty acid metabolism exerted its protective role in IPF partly through activating autophagy, shedding a new prospective for the treatment of IPF.

Comparative Study of Autophagy in Oxaliplatin-Sensitive and Resistant SNU-C5 Colon Cancer Cells.

Few studies have evaluated the role of autophagy in the development of oxaliplatin (OXT) resistance in colon cancer cells. In this study, we compared the role of autophagy between SNU-C5 colon cancer cells and OXT-resistant SNU-C5 (SNU-C5/OXTR) cells. At the same concentration of OXT, the cytotoxicity of OXT or apoptosis was significantly reduced in SNU-C5/OXTR cells compared with that in SNU-C5 cells. Compared with SNU-C5 cells, SNU-C5/OXTR cells exhibited low levels of autophagy. The expression level of important autophagy proteins, such as autophagy-related protein 5 (Atg5), beclin-1, Atg7, microtubule-associated proteins 1A/1B light chain 3B I (LC3-I), and LC3-II, was significantly lower in SNU-C5/OXTR cells than that in SNU-C5 cells. The expression level of the autophagy-essential protein p62 was also lower in SNU-C5/OXTR cells than in SNU-C5 cells. In SNU-C5/OXTR cells, the production of intracellular reactive oxygen species (ROS) was significantly higher than that in SNU-C5 cells, and treatment with the ROS scavenger N-acetylcysteine restored the reduced autophagy levels. Furthermore, the expression of antioxidant-related nuclear factor erythroid 2-related factor 2 transcription factor, heme oxygenase-1, and Cu/Zn superoxide dismutase were also significantly increased in SNU-C5/OXTR cells. These findings suggest that autophagy is significantly reduced in SNU-C5/OXTR cells compared with SNU-C5 cells, which may be related to the production of ROS in OXT-resistant cells.

A molecular link between autophagy and circadian rhythm in plants.

Extremely high or low autophagy levels disrupt plant survival under nutrient starvation. Recently, autophagy has been reported to display rhythms in animals. However, the mechanism of circadian regulation of autophagy is still unclear. Here, we observed that autophagy has a robust rhythm and that various autophagy-related genes (ATGs) are rhythmically expressed in Arabidopsis. Chromatin immunoprecipitation (ChIP) and dual-luciferase (LUC) analyses showed that the core oscillator gene TIMING OF CAB EXPRESSION 1 (TOC1) directly binds to the promoters of ATG (ATG1a, ATG2, and ATG8d) and negatively regulates autophagy activities under nutritional stress. Furthermore, autophagy defects might affect endogenous rhythms by reducing the rhythm amplitude of TOC1 and shortening the rhythm period of CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1). Autophagy is essential for the circadian clock pattern in seedling development and plant sensitivity to nutritional deficiencies. Taken together, our studies reveal a plant strategy in which the TOC1-ATG axis involved in autophagy-rhythm crosstalk to fine-tune the intensity of autophagy.

Advances in the role of autophagy in the development of retinoblastoma.

Autophagy is a feedback regulatory mechanism of cells to external stress, which helps cells to adapt to changes in physiological conditions and environmental stress. Autophagy possesses a variety of target genes that control a wide range of signaling pathways. Maintenance of an appropriate level of autophagy is essential for the growth, metastasis and characteristics of tumors. Retinoblastoma (RB) is the most common primary intraocular malignant tumor found in the eyes of children following exposure to extreme environmental factors, such as mitochondrial defects, oxidative stress and excessive autophagy; this leads to the development of DNA damage and progressive loss of the function of the eye, which results in the occurrence of RB. Recent studies have documented the involvement of autophagy in the transformation, occurrence and metastasis of RB. High or low levels of autophagy exert notably promotive or repressive effects on the development, invasion, drug resistance and survival of RB, respectively. The present review reports the research progress on the association between autophagy and RB.

NF-\u03baB is involved in the regulation of autophagy in mutant p53 cells in response to ionizing radiation

Chemotherapy and ionizing radiation (IR) can induce autophagy in tumor cells. Here, we report that the level of autophagy in tumor cells was related to the background of p53 gene that NF-κB acts as a negative regulator of autophagy in mutant p53 (p53-R273H) cells, and that acetylation was involved in the IR-induced nuclear translocation of NF-κB. We found that autophagy-related proteins were highly expressed in wild-type p53 (wt-p53) cells and that IR increased their levels further. p53-R273H cells exhibited low levels of autophagy; there was no change following IR treatment. The nuclear translocation of p65 was upregulated in p53-R273H cells following IR; when p65 was competitively inhibited from entering the nucleus with SN50, the level of autophagy increased. The nuclear translocation of p65 was mediated by p300; this factor also regulates the nuclear behavior of NF-κB. The knockdown of p300 in p53-R273H cells led to an inhibition of p65 expression and an increase in autophagy. In addition, the inhibition of p300 or p65 not only activated autophagy, it also induced radiosensitivity in p53-R273H cells. The relationship between the p53 gene, NF-κB, and autophagy was further analyzed in a mouse model of xenograft tumors and in clinical tumor pathological specimens; the results were consistent with the in vitro experiments. Our findings indicate that autophagy may be regulated by NF-κB in p53-R273H cells. These findings may help to improve the therapeutic strategy adopted for tumors related to the mutant p53-R273H gene; such therapy would aim to target NF-κB to induce autophagy.

A20 regulates inflammation through autophagy mediated by NF-κB pathway in human nucleus pulposus cells and ameliorates disc degeneration in vivo

Intervertebral disc degeneration (IDD) is closely related to loss of the extracellular matrix (ECM), apoptosis and inflammation in nucleus pulposus cells (NPCs). It has been reported that Zinc finger protein A20/TNFAIP3 (A20) can inhibit the activity of the NF-κB pathway and promote autophagy. Therefore, we speculated that A20 can regulate inflammation and ameliorate IDD through autophagy mediated by NF-κB in human NPCs. Our results indicated that the expression of A20 and inflammatory factors in IDD tissues was increased. A20 is an essential negative regulator in the NF-κB pathway. Constructed adenoviral shRNA and overexpression vectors for A20 could effectively regulate the inflammation, autophagy, and activity of NF-κB, which in turn affected the progression of IDD. Inhibition of NF-κB on the basis of knocking down A20 results in increased autophagy, suggesting that A20-regulated autophagy was mediated by NF-κB. In vivo, A20 overexpression could ameliorate the progression of IDD and promote autophagy at the same time, while deletion of A20 leads to low levels of autophagy and severe degeneration. In summary, A20 plays an important role in inhibiting inflammation through autophagy mediated by NF-κB in NPCs and ameliorating IDD.

Autophagy in T cells from aged donors is maintained by spermidine and correlates with function and vaccine responses.

Vaccines are powerful tools to develop immune memory to infectious diseases and prevent excess mortality. In older adults, however vaccines are generally less efficacious and the molecular mechanisms that underpin this remain largely unknown. Autophagy, a process known to prevent aging, is critical for the maintenance of immune memory in mice. Here, we show that autophagy is specifically induced in vaccine-induced antigen-specific CD8+ T cells in healthy human volunteers. In addition, reduced IFNγ secretion by RSV-induced T cells in older vaccinees correlates with low autophagy levels. We demonstrate that levels of the endogenous autophagy-inducing metabolite spermidine fall in human T cells with age. Spermidine supplementation in T cells from old donors recovers their autophagy level and function, similar to young donors' cells, in which spermidine biosynthesis has been inhibited. Finally, our data show that endogenous spermidine maintains autophagy via the translation factor eIF5A and transcription factor TFEB. In summary, we have provided evidence for the importance of autophagy in vaccine immunogenicity in older humans and uncovered two novel drug targets that may increase vaccination efficiency in the aging context.

Formation of organotypic testicular organoids in microwell culture†.

Three-dimensional (3D) organoids can serve as an in vitro platform to study cell-cell interactions, tissue development, and toxicology. Development of organoids with tissue architecture similar to testis in vivo has remained a challenge. Here, we present a microwell aggregation approach to establish multicellular 3D testicular organoids from pig, mouse, macaque, and human. The organoids consist of germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells forming a distinct seminiferous epithelium and interstitial compartment separated by a basement membrane. Sertoli cells in the organoids express tight junction proteins claudin 11 and occludin. Germ cells in organoids showed an attenuated response to retinoic acid compared to germ cells in 2D culture indicating that the tissue architecture of the organoid modulates response to retinoic acid similar to in vivo. Germ cells maintaining physiological cell-cell interactions in organoids also had lower levels of autophagy indicating lower levels of cellular stress. When organoids were treated with mono(2-ethylhexyl) phthalate (MEHP), levels of germ cell autophagy increased in a dose-dependent manner, indicating the utility of the organoids for toxicity screening. Ablation of primary cilia on testicular somatic cells inhibited the formation of organoids demonstrating an application to screen for factors affecting testicular morphogenesis. Organoids can be generated from cryopreserved testis cells and preserved by vitrification. Taken together, the testicular organoid system recapitulates the 3D organization of the mammalian testis and provides an in vitro platform for studying germ cell function, testicular development, and drug toxicity in a cellular context representative of the testis in vivo.

Autophagy induced by SAHA affects mutant P53 degradation and cancer cell survival.

Missense mutations in the TP53 gene produce mutant p53 (mutp53) proteins which may acquire oncogenic properties favoring chemoresistance, cell migration, and metastasis. The exploitation of cellular pathways that promote mutp53 degradation may reduce cell proliferation and invasion as well as increase the sensitivity to anticancer drugs, with a strong impact on current cancer therapies. In the last years, several molecules have been characterized for their ability to induce the degradation of mutp53 through the activation of autophagy. Here, we investigated the correlation between autophagy and mutp53 degradation induced by suberoylanilide hydroxamic acid (SAHA), an FDA-approved histone deacetylase inhibitor. In the human cancer lines MDA-MB-231 (mutp53-R280K) and DLD1 (mutp53-S241F), SAHA induced a significant mutp53 degradation. However, such degradation correlated with autophagy induction only in MDA-MB-231 cells, being counteracted by autophagy inhibition, which also increased SAHA-induced cell death. Conversely, in DLD1 cells SAHA triggered a low level of autophagy despite promoting a strong decrease in mutp53 level, and autophagy inhibition did not change either mutp53 levels or sensitivity to this drug. We conclude that autophagy can be a relevant pathway for mutp53 degradation induced by SAHA, but its contribution to mutp53 destabilization and the consequences on cell death are likely context-dependent.

Human tissue kallikrein 1 ameliorates erectile function via modulation of macroautophagy in aged transgenic rats.

Previously, we have demonstrated that human tissue kallikrein 1 (hKLK1) improves age-related erectile dysfunction (ED). Autophagy has been implicated in age-related diseases, including ED. However, the molecular mechanisms underlying hKLK1-mediated amelioration of age-related ED via regulation of autophagy remains unknown. To explore the potential mechanism, male wild-type Sprague-Dawley rats (WTR) and transgenic rats harboring human KLK1 (TGR) were bred till 4 or 18months of age and divided into three groups: young WTR (yWTR) as the control group, aged WTR (aWTR) group, and aged TGR (aTGR) group. The erectile function of each rat was evaluated using cavernous nerve electrostimulation. The ratio of intracavernous pressure/mean arterial pressure (ICP/MAP) and total ICP were also measured. Western blotting, immunohistochemistry, and transmission electron microscopy were performed to detect the levels of autophagy. The expression levels of related signaling pathways were determined by western blotting and immunohistochemistry. We found that hKLK1 improved the impaired erectile function of aged rats. Compared to the yWTR and aTGR groups, the aWTR group showed reduced smooth muscle/collagen ratio, fewer autophagosomes, and lower expression of Beclin 1 and LC3-II, which indicate impaired smooth muscle function and low level of autophagy in the smooth muscle cells. Moreover, the PI3K/Akt/mTOR signaling pathway, which is considered to be a negative regulator of autophagy, was upregulated in the aWTR group. hKLK1 may partially restore erectile function in aged transgenic rats by upregulating protective autophagy via the PI3K/Akt/mTOR pathway. These observations indicate that hKLK1 is a potential gene therapy candidate for age-related ED.

Protein translation, proteolysis and autophagy in human skeletal muscle atrophy after spinal cord injury.

Spinal cord injury-induced loss of skeletal muscle mass does not progress linearly. In humans, peak muscle loss occurs during the first 6 weeks postinjury, and gradually continues thereafter. The aim of this study was to delineate the regulatory events underlying skeletal muscle atrophy during the first year following spinal cord injury. Key translational, autophagic and proteolytic proteins were analysed by immunoblotting of human vastus lateralis muscle obtained 1, 3 and 12 months following spinal cord injury. Age-matched able-bodied control subjects were also studied. Several downstream targets of Akt signalling decreased after spinal cord injury in skeletal muscle, without changes in resting Akt Ser473 and Akt Thr308 phosphorylation or total Akt protein. Abundance of mTOR protein and mTOR Ser2448 phosphorylation, as well as FOXO1 Ser256 phosphorylation and FOXO3 protein, decreased in response to spinal cord injury, coincident with attenuated protein abundance of E3 ubiquitin ligases, MuRF1 and MAFbx. S6 protein and Ser235/236 phosphorylation, as well as 4E-BP1 Thr37/46 phosphorylation, increased transiently after spinal cord injury, indicating higher levels of protein translation early after injury. Protein abundance of LC3-I and LC3-II decreased 3 months postinjury as compared with 1 month postinjury, but not compared to able-bodied control subjects, indicating lower levels of autophagy. Proteins regulating proteasomal degradation were stably increased in response to spinal cord injury. Together, these data provide indirect evidence suggesting that protein translation and autophagy transiently increase, while whole proteolysis remains stably higher in skeletal muscle within the first year after spinal cord injury.

Unraveling the human protein atlas of metastatic melanoma in the course of ultraviolet radiation-derived photo-therapy

To explore the photo-therapeutic capacity of UV radiation in solid tumors, we herein employed an nLC-MS/MS technology to profile the proteomic landscape of irradiated WM-266-4 human metastatic-melanoma cells. Obtained data resulted in proteomic catalogues of 5982 and 7280 proteins for UVB- and UVC-radiation conditions, respectively, and indicated the ability of UVB/C-radiation forms to eliminate metastatic-melanoma cells through induction of synergistically operating programs of apoptosis and necroptosis. However, it seems that one or more WM-266-4 cell sub-populations may escape from UV-radiation's photo-damaging activity, acquiring, besides apoptosis tolerance, an EMT phenotype that likely offers them the advantage of developing resistance to certain chemotherapeutic drugs. Low levels of autophagy may also critically contribute to the selective survival and growth of UV-irradiated melanoma-cell escapers. These are the cells that must be systemically targeted with novel therapeutic schemes, like the one of UV radiation and Irinotecan herein suggested to be holding strong promise for the effective treatment of metastatic-melanoma patients. Given the dual nature of UV radiation to serve as both anti-tumorigenic and tumorigenic agent, all individuals being subjected to risk factors for melanoma development have to be appropriately informed and educated, in order to integrate the innovative PPPM concept in their healthcare-sector management. SignificanceThis study reports the application of nLC-MS/MS technology to deeply map the proteomic landscape of UV-irradiated human metastatic-melanoma cells. Data bioinformatics processing led to molecular-network reconstructions that unearthed the dual nature of UV radiation to serve as both anti-tumorigenic and tumorigenic factor in metastatic-melanoma cellular environments. Our UV radiation-derived “photo-proteomic” atlas may prove valuable for the identification of new biomarkers and development of novel therapies for the disease. Given that UV radiation represents a major risk factor causing melanoma, a PPPM-based life style and clinical practice must be embraced by all individuals being prone to disease's appearance and expansion.

Reduced Autophagy in 5-Fluorouracil Resistant Colon Cancer Cells.

We investigated the role of autophagy in SNUC5/5-FUR, 5-fluorouracil (5-FU) resistant SNUC5 colon cancer cells. SNUC5/5-FUR cells exhibited low level of autophagy, as determined by light microscopy, confocal microscopy, and flow cytometry following acridine orange staining, and the decreased level of GFP-LC3 puncta. In addition, expression of critical autophagic proteins such as Atg5, Beclin-1 and LC3-II and autophagic flux was diminished in SNUC5/5-FUR cells. Whereas production of reactive oxygen species (ROS) was significantly elevated in SNUC5/5-FUR cells, treatment with the ROS inhibitor N-acetyl cysteine further reduced the level of autophagy. Taken together, these results indicate that decreased autophagy is linked to 5-FU resistance in SNUC5 colon cancer cells.

Abstract 1151: IL-8, VEGF and IGF-1 play the important role in ARHI mediated-tumor dormancy in ovarian cancer

Abstract Ovarian cancer is the most lethal gynecologic malignancy. One of the most important factors contributing to poor outcomes is the ability of drug resistant ovarian cancer cells to remain dormant for years after treatment, only to grow progressively and kill their host. Judging from sites of recurrence, most dormant ovarian cancer cells are found on the surface of the peritoneal cavity, often in small, poorly vascularized collagenous scars observed at “second look” operations. Previously, we reported that more than 80% of persistent, dormant drug resistant ovarian cancers express ARHI and are undergoing autophagy. Autophagy can protect or kill tumor cells depending upon the context. Our group has found that autophagy and tumor dormancy can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), which is downregulated in 60% of ovarian cancers. Re-expression of ARHI in cell culture produces cell death within 72 hours, whereas re-expression of ARHI in xenografts produces cell growth arrest and tumor dormancy. When ARHI levels are reduced after 6 weeks of induction, xenografts grow promptly. Our current experiments were designed to determine whether survival of autophagic cells in vivo requires permissive levels of VEGF, IL-8 and IGF-1 in the tumor microenvironment. In cell culture, co-incubation of VEGF, IGF-1 and IL-8 with SKOv3-ARHI cells, increased AKT activity, reduced ARHI-mediated autophagy formation and decreased autophagic cell death. SKOv3-ARHI cells grown subcutaneously in nude mice exhibited higher levels of AKT activity and lower levels of autophagy than cells grown in cell culture. When cytoplasmic and nuclear fractions were obtained from xenografts, the nuclear fractions showed increased phospho-AKT, and decreased levels of a transcription factor EB (TFEB) and low mRNA expression of LC3, suggesting survival factors in tumor environment may stimulate AKT, downregulate expression of TFEB and inhibit formation of autophagy. Treatment with anti-VEGF, anti-IL-8 and anti-IGF-1 antibodies, individually and in combination, inhibited survival factors-mediated AKT activation and slowed the outgrowth of dormant autophagic ovarian cancer cells. Treatment of dormant xenografts with the same monoclonal antibodies against VEGF, IL-8 and IGFR not only delayed outgrowth when DOX is withdrawn, but cured a fraction of mice. Re-expression of ARHI reduced expression of IGF-1 receptor, but not VEGF and IL8 receptors. Treatment ovarian cancer cells with VEGF, IL-8 and IGF-1 reduced ARHI's ability to induce autophagy and to inhibit AKT. These preclinical studies support that the possibility that treatment with a combination of anti-VEGF, anti-IL8 and anti-IGF-1 antibodies could prevent outgrowth of dormant cells in patients with ovarian cancer. Furthermore, these results suggest that survival factors are required to limit autophagic death. Citation Format: Zhen Lu, Aaron Orozco, Weiqun Mao, Yan Wang, Margie Nicole Sutton, Hailing Yang, Douglas A. Levine, Robert C. Bast Jr.. IL-8, VEGF and IGF-1 play the important role in ARHI mediated-tumor dormancy in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1151.

Effects of Bone Marrow Mesenchymal Stem Cells on the Biological Characteristics of Migrating and Homing in Multiple Myeloma Cells

To construct a co-culture system for bone marrow mesenchymal stem cells (BMMSC) and multiple myeloma (MM) cells, and to investigate the effects of co-cultured BMMSC on the migrating and homing of multiple myeloma cells. The BMMSC from the transgenic mice with green fluorescent protein (GFP) fetal bone were cultured by adherent screening. A co-culture system of BMMSC and MM cell line XG-7 cells was constracted, the proliferation and apoptosis of cells were determined by trypan blue exclusion and Annexin V/PI, respectively, MDC staining was employed to detect the autophagy. The moving direction distribution of molecule in BMMSC and XG-7 cells labeled with PE-CD138 in co-culture process were observed dinamically by confocal microscopy. After co-culture with GFP-BMMSC, the resistance of XG-7 cells to apoptosis and autophagy were enhanced; at the same time, their proliferation increased. Apoptosis rates of XG-7 cells directly and indirectly co-cultured with BMMSC were (6.23 ± 0.12)% and (6.97 ± 0.03)% respectively, which were lower than that of XG-7 cells cultured alone (17.90 ± 1.46)% (P < 0. 01). There was low level of autophagy in XG-7 cells co-cultured with BMMSC. XG-7 cells are highly polarized and contained a specialized membrane domain with specific protein and lipid components to contact with BMMSC under confocal microscope. After methyl-β-cyclodextrin treatment, the molecules were normally enriched in the specialized domain. BMMSC can protect XG-7 cells from apoptosis and autophagy, and obviously promote the proliferation of XG-7 cells, and can influence the migrating and homing of multiple myeloma cells.

IL-1β induces low levels of autophagy which clears dysfunctional mitochondria and inhibits ROS, MMP-13 and ADAMTS5 expression in OA chondrocytes

IL-1β induces low levels of autophagy which clears dysfunctional mitochondria and inhibits ROS, MMP-13 and ADAMTS5 expression in OA chondrocytes