Addition Of Chloroquine Research Articles

Synthesis and characterization of chloroquine-modified albumin-binding siRNA-lipid conjugates for improved intracellular delivery and gene silencing in cancer cells.

siRNA therapeutics have considerable potential as molecularly-targeted therapeutics in malignant disease, but identification of effective delivery strategies that mediate rapid intracellular delivery while minimizing toxicity has been challenging. Our group recently developed and optimized an siRNA conjugate platform termed "siRNA-L 2 ," which harnesses non-covalent association with endogenous circulating albumin to extend circulation half-life and achieve tumor-selective delivery without the use of traditional cationic lipids or polymers for transfection. To improve intracellular delivery and particularly the endosomal escape properties of siRNA-L 2 towards more efficient gene silencing, we report synthesis of siRNA-CQ-L 2 conjugates, in which chloroquine (CQ), an endosomolytic quinoline alkaloid, is covalently incorporated into the branching lipid tail structure. We accomplished this via synthesis of a novel CQ phosphoramidite, which can be incorporated into a modular siRNA-L 2 backbone using on-column solid-phase synthesis through use of asymmetric branchers with levulinyl-protected hydroxide groups that allow covalent addition of pendant CQ repeats. We demonstrate that siRNA-CQ-L 2 maintains the ability to non-covalently bind albumin, and with multiple copies of CQ, siRNA-CQ-L 2 mediates higher endosomal disruption, cellular uptake/retention, and reporter gene knockdown in cancer cells. Further, in mice, the addition of CQ did not significantly affect circulation kinetics nor organ biodistribution and did not produce hematologic or organ-level toxicity. Thus, controlled, multivalent conjugation of albumin-binding siRNA-L 2 to endosomolytic small molecule compounds holds promise in improving siRNA-L 2 knockdown potency while maintaining albumin-binding properties and overall safety.

Autophagy modulates the stability of Wee1 and cell cycle G2/M transition

The mammalian cell cycle is divided into four sequential phases, namely G1 (Gap 1), S (synthesis), G2 (Gap 2), and M (mitosis). Wee1, whose turnover is tightly and finely regulated, is a well-known kinase serving as a gatekeeper for the G2/M transition. However, the mechanism underlying the turnover of Wee1 is not fully understood. Autophagy, a highly conserved cellular process, maintains cellular homeostasis by eliminating intracellular aggregations, damaged organelles, and individual proteins. In the present study, we found autophagy deficiency in mouse liver caused G2/M arrest in two mouse models, namely Fip200 and Atg7 liver-specific knockout mice. To uncover the link between autophagy deficiency and G2/M transition, we combined transcriptomic and proteomic analysis for liver samples from control and Atg7 liver-specific knockout mice. The data suggest that the inhibition of autophagy increases the protein level of Wee1 without any alteration of its mRNA abundance. Serum starvation, an autophagy stimulus, downregulates the protein level of Wee1 in vitro. In addition, the half-life of Wee1 is extended by the addition of chloroquine, an autophagy inhibitor. LC3, a central autophagic protein functioning in autophagy substrate selection and autophagosome biogenesis, interacts with Wee1 as assessed by co-immunoprecipitation assay. Furthermore, overexpression of Wee1 leads to G2/M arrest both in vitro and in vivo. Collectively, our data indicate that autophagy could degrade Wee1—a gatekeeper of the G2/M transition, whereas the inhibition of autophagy leads to the accumulation of Wee1 and causes G2/M arrest in mouse liver.

Excessive ER-phagy contributes to ochratoxin A-induced apoptosis.

The nephrotoxic secondary fungal metabolite ochratoxin A (OTA) is ubiquitously existed in foodstuffs and feeds. Although our earlier research provided preliminary evidence that endoplasmic reticulum (ER) was crucial in OTA-induced nephrotoxicity, more research is necessary to understand the fine-tune mechanisms involving ER stress (ERS), ER-phagy, and apoptosis. In the present study, the cell viability and protein expressions of human proximal tubule epithelial (HK-2) cells in response to OTA and/or chloroquine/rapamycin/sodium phenylbutyrate/tunicamycin were determined via cell viability assay, apoptosis analysis, and Western blot analysis. The findings showed that a 24 h-treatment of 0.25-4μM OTA could significantly reduced the cell viability (P<0.05), which notably increased with the addition of chloroquine and sodium phenylbutyrate, while decreased with the addition of rapamycin and tunicamycin as compared to group OTA (P<0.05). A 24 h-treatment of 1-4μM OTA could markedly induce apoptosis via increasing the protein expressions of GRP78, p-eIF2α, Chop, LC3B-II, Bak, and Bax, and inhibiting the protein expressions of DDRGK1, UBA5, Lonp1, Tex264, FAM134B, p-mTOR, p62, and Bcl-2 in HK-2cells (P<0.05). In conclusion, OTA activated ERS, unfolded protein response, and subsequent excessive ER-phagy, thus inducing apoptosis, and the vicious cycle between excessive ER-phagy and ERS could further promote apoptosis in vitro.

Pharmacological inhibitors of autophagy have opposite effects in acute and chronic cisplatin-induced kidney injury.

The nephrotoxicity of cisplatin remains a major hurdle in the field of oncology. Thirty percent of patients treated with cisplatin develop acute kidney injury, and all patients are at risk for long-term impacts on kidney function. There are currently no Federal Drug Administration-approved agents to prevent or treat cisplatin-induced kidney injury. The dosing regimen used in preclinical models of nephrotoxicity may impact the success of therapeutic candidates in clinical trials. Here, we demonstrated that pharmacological inhibitors of autophagy have opposite effects when used as interventions in two different models of cisplatin-induced kidney injury. Eight-week-old male C57BL/6 mice were treated with either one dose of 20 mg/kg cisplatin or weekly doses of 9 mg/kg cisplatin for 4 wk or until body weight loss exceeded 30%. Concurrently, mice were administered multiple doses of 60 mg/kg chloroquine or 15 mg/kg 3-methyladenine attempting to globally inhibit autophagy. Mice that received a single high dose of cisplatin had worsened kidney function, inflammation, and cell death with the addition of chloroquine. 3-Methlyadenine did not impact the development of acute kidney injury in this model. In contrast, mice that received repeated low doses of cisplatin showed improved kidney function, reduced inflammation, and reduced fibrosis when treated with either chloroquine or 3-methyladenine. This study highlights how therapeutic candidates can have drastically different effects on the development of cisplatin-induced kidney injury depending on the dosing model used. This emphasizes the importance of choosing the appropriate model of injury for preclinical studies.NEW & NOTEWORTHY This study examined how inhibition of autophagy has opposite effects on the development of acute and chronic kidney injury. Autophagy inhibition exacerbated the development of acute kidney injury following a single high dose of cisplatin but prevented the development of injury and fibrosis following repeated low doses of cisplatin.

Selenium nanoparticles coupling with Astragalus Polysaccharides exert their cytotoxicities in MCF-7 cells by inhibiting autophagy and promoting apoptosis

BackgroundAstragalus Polysaccharides (APS) had been reported to exhibit antitumor activities. Given that nanoparticles possessed unique advantages in cancer treatment, APS was used as the modifier to prepare gold, silver and selenium nanoparticles (APS-Au, APS-Ag and APS-Se NPs) in the present study. MethodsThe three nanoparticles were synthesized via a green approach and characterized by DLS, TEM, XRD, FT-IR and UV-Vis. The inhibitory effects of these nanoparticles on various tumor cells proliferation were examined by MTT assay in vitro. Reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the expression of apoptosis and autophagy-related proteins were also detected. ResultsAmong these, APS-Se NPs displayed the most potent antitumor activities against MCF-7 cells in vitro. Flow cytometric analysis suggested that after cells were exposed to elevated concentrations of APS-Se NPs (10, 20 and 40 μmol/L), the rate of apoptosis was increasing (16.63 ± 0.89, 38.60 ± 3.46 and 44.38 ± 2.62%, respectively). Further analysis by immunofluorescence revealed an increase in intracellular ROS and a loss of MMP. This was accompanied by increased LC3-I to LC3-II conversion. Also, western blot analysis demonstrated that the ratios of Bax/Bcl-2 and cleaved caspase9/caspase 9 rose, and LC3-II and p62 protein levels increased. The addition of chloroquine, an inhibitor of autophagy, further enhanced protein expression of p62 and LC3-II. ConclusionAPS-Se NPs exerted their cytotoxic activity in MCF-7 cells by blocking autophagy and facilitating mitochondrial pathway-mediated apoptosis.

Acbp is essential for decidualization during early pregnancy in mice.

Decidualization of uterine stromal cells plays an important role in the establishment of normal pregnancy. Previous studies have demonstrated that Acyl-CoA binding protein (Acbp) is critical to cellular proliferation, differentiation, mitochondrial functions, and autophagy. The characterization and physiological function of Acbp during decidualization remain largely unknown. In the present study, we conducted the expression profile of Acbp in the endometrium of early pregnant mice. With the occurrence of decidualization, the expression of Acbp gradually increased. Similarly, Acbp expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. We applied the mice pseudopregnancy model to reveal that the expression of Acbp in the endometrium of early pregnant mice was not induced by embryonic signaling. Moreover, P4 significantly upregulated the expression of Acbp, whereas E2 appeared to have no regulating effect on Acbp expression in uterine stromal cells. Concurrently, we found that interfering with Acbp attenuated decidualization, and that might due to mitochondrial dysfunctions and the inhibition of fatty acid oxidation. The level of autophagy was increased after knocking down Acbp. During induced decidualization, the expression of ACBP was decreased with the treatment of rapamycin (an autophagy inducer), while increased with the addition of Chloroquine (an autophagy inhibitor). Our work suggests that Acbp plays an essential role in the proliferation and differentiation of stromal cells during decidualization through regulating mitochondrial functions, fatty acid oxidation, and autophagy.

Canagliflozin protects against cisplatin-induced acute kidney injury by AMPK-mediated autophagy in renal proximal tubular cells

Sodium-glucose cotransporter 2 inhibitors, which are recently introduced as glucose-lowering agents, improve cardiovascular and renal outcomes in patients with diabetes mellitus. These drugs also have beneficial effects in various kidney disease models. However, the effect of SGLT2 inhibitors on cisplatin-induced acute kidney injury (AKI) and their mechanism of action need to be elucidated. In this study, we investigated whether canagliflozin protects against cisplatin-induced AKI, depending on adenosine monophosphate-activated protein kinase (AMPK) activation and following induction of autophagy. In the experiments using the HK-2 cell line, cell viability assay and molecular analysis revealed that canagliflozin protected renal proximal tubular cells from cisplatin, whereas addition of chloroquine or compound C abolished the protective effect of canagliflozin. In the mouse model of cisplatin-induced AKI, canagliflozin protected mice from cisplatin-induced AKI. However, treatment with chloroquine or compound C in addition to administration of cisplatin and canagliflozin eliminated the protective effect of canagliflozin. Collectively, these findings indicate that canagliflozin protects against cisplatin-induced AKI by activating AMPK and autophagy in renal proximal tubular cells.

Abstract PO-031: Lysosome inhibition overcomes resistance to CDK4/6 inhibition in PDA

Abstract The current FDA-approved chemotherapies for patients with Pancreatic Ductal Adenocarcinoma (PDA) demonstrate substantial toxicities with only a modest survival benefit highlighting the urgent need to better understand the molecular mechanisms driving PDA and how they interact in order to develop more precise and less toxic pathway-targeted therapies. PDA often harbors oncogenic mutations in KRAS and CDKN2A that drive a dysregulated cell cycle through activation of CDK4/6 (Cyclin-Dependent Kinases 4 and 6), which mono-phosphorylate RB1 to govern progression through G0◊G1◊S phases. Currently, there are three FDA-approved CDK4/6 inhibitors, palbo-, abema-, and ribociclib (X-ciclibs), that are predicted to have anti-cancer activity in PDA cells. However, the benefits of X-ciclib treatment in PDA patients are largely underwhelming. We found that inherent resistance to X-ciclib treatment in vitro is due to compensatory activation of protective autophagy, a critical lysosomal degradation process that generates a source of nutrients during periods of cellular stress. We observed that X-ciclib treatment induces autophagic flux in PDA cell lines. This pro-cancer process can be targeted with the lysosomal inhibitors, chloroquine and hydroxychloroquine. The addition of chloroquine sensitizes PDA cells to X-ciclib treatment, resulting in diminished proliferative ability. Additionally, a late-stage PDA patient was treated with the combination of an X-ciclib and hydroxychloroquine and displayed a dramatic decrease in the bloodborne PDA biomarker, Cancer Antigen 19-9. These data may suggest a novel combination treatment strategy for late-stage PDA patients, but the molecular mechanisms underlying are yet to be studied in PDA. Future work will explore the role RB1 plays in CDK4/6 regulated autophagy by investigating how phosphorylation at distinct RB1 sites modulates autophagy-related gene expression and the extent to which mTORC1 deactivation is necessary for CDK4/6 regulated autophagy. Citation Format: Dilru Silva, Conan Kinsey, Martin McMahon. Lysosome inhibition overcomes resistance to CDK4/6 inhibition in PDA [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-031.

Mechanism by which Eucommia ulmoides leaves Regulate Nonalcoholic fatty liver disease based on system pharmacology

Ethnopharmacological relevanceEucommia ulmoides (E. ulmoides) leaves are included in the Chinese Pharmacopoeia, and are traditionally used to treat hypertension, obesity, diabetes, and other diseases. Numerous pharmacological studies have shown that E. ulmoides has a good effect on lowering blood lipids and can improve obesity and nonalcoholic fatty liver. AimTo study the mechanism of E. ulmoides leaves in regulating nonalcoholic fatty liver disease by combining prediction and validation. MethodsUsing network pharmacology, and molecular docking to predict E. ulmoides in regulating the action mechanism and potential active ingredients of nonalcoholic fatty liver, large hole adsorption resin enrichment active sites, in vitro experiments were performed to verify its fat-lowering effect and mechanism. ResultsThe major components of E. ulmoides leaves exhibited good combination with lipid metabolism-regulating core proteins, particularly flavonoids. EUL 50 significantly reduced lipid accumulation, and increased PPARγ. Compared with the control group, the autophagy level increased after the administration of EUL 50. PPARγ decreased significantly after the addition of chloroquine (CQ, autophagy inhibitor). ConclusionThe active ingredients in E. ulmoides leaves regulating nonalcoholic fatty liver disease are mainly flavonoids and phenolics. EUL 50 may play a role in lowering lipids by regulating PPARγ expression through inducing autophagy.

The addition of chloroquine and bevacizumab to standard radiochemotherapy for recurrent glioblastoma multiforme

Introduction Hypoxia-induced autophagy leads to an increase in vasculogenic-mimicry (VM) and the development of resistance of glioblastoma-cells to bevacizumab (BEV). Chloroquine (HCQ) inhibits autophagy, reduces VM and can thus produce a synergistic effect in anti-angiogenic-therapy by delaying the development of resistance to BEV. Purpose We retrospectively compared the combined addition of HCQ+BEV and adjuvant-radiochemotherapy (aRCT) to aRCT alone for recurrent-glioblastoma (rGBM) in regards of overall survival (OS). Methods Between 2006 and 2016, 134 patients underwent neurosurgery for rGBM at our institution. Forty-two patients (Karnofsky-Performance-Score>60%) with primary-glioblastoma underwent repeat-surgery and aRCT for recurrence. Four patients (9.5%) received aRCT+HCQ+BEV. Five patients received aRCT+BEV. Results In rGBM-patients who were treated with aRCT+HCQ+BEV, median OS was 36.57 months and median post-recurrence-survival (PRS) was 23.92 months while median PRS in the control-group was 9.63 months (p=0.022). In patients who received aRCT+BEV, OS and PRS were 26.83 and 12.97 months, respectively. Conclusions Although this study was performed on a small number of highly selected patients, it demonstrates a synergistic effect of HCQ+BEV in the treatment of rGBM which previously could be demonstrated based on experimental data. A significant increase of OS in patients who receive aRCT+HCQ+BEV cannot be ruled out and should be further investigated in randomised-controlled-trials.

Base-modified aptamers obtained by cell-internalization SELEX facilitate cellular uptake of an antisense oligonucleotide

Intracellular delivery of oligonucleotides is important for their use as therapeutic drugs. The conjugation of molecules interacting with cell membrane proteins to enhance their internalization into cells is an effective strategy for delivering oligonucleotides. In the present study, we focused on creating aptamers, which are single-stranded oligonucleotides that bind target molecules with high affinity and specificity, as membrane protein-binding molecules. With an evolutionary selection approach using a random DNA library containing a uracil derivative with a hydrophobic functional group at the 5 position, we successfully obtained aptamers that are efficiently internalized into A549 cells. The efficacies of the aptamers were tested by further conjugation with MALAT1-targeting antisense oligonucleotides (ASOs), and the expression levels of MALAT1 RNA were examined. The aptamer-ASO conjugates were taken up by A549 cells, although there was no observable reduction in MALAT1 RNA levels. In contrast, the activity of the aptamer-ASO conjugate was potentiated when endosomal/lysosomal escape was enhanced by the addition of chloroquine. Thus, we showed that the hydrophobic modification of the nucleobase moiety is useful for developing highly internalizing aptamers and that endosomal/lysosomal escape is important for the intracellular delivery of ASOs by aptamers.

Formation of Higher Structural Levels in λ-Carrageenan Induced by the Antimalarial Drug Chloroquine.

The linear polysaccharide λ-carrageenan is the only one among the carrageenans not forming secondary, tertiary, and quaternary structures in the presence of inorganic ions. Chloroquine (CQ) is a well-established antimalaria drug also recently discussed in therapeutics against the COVID-19 pandemic. The interaction of this polysaccharide-ionic drug pair was investigated by combining UV-vis spectrophotometry and atomic force microscopy (AFM) imaging. A decrease of the UV peak assigned to free CQ and the occurrence of isosbestic points indicate the formation of complexes. High-resolution AFM height images revealed an increasing height of the single polysaccharide chains in the random coil state upon addition of CQ, indicating the formation of a secondary structure, followed by higher hierarchical aggregates. The disappearance of higher-ordered structures and the recovery of polysaccharide chains with primary structure were observed by introducing inorganic cations (Na+, K+, Ca2+), replacing the condensed CQ and paving the way to reversible ion-induced drug release.

RETRACTED ARTICLE: Inhibition of autophagic flux differently modulates cannabidiol-induced death in 2D and 3D glioblastoma cell cultures

Radiotherapy combined with chemotherapy is the major treatment modality for human glioblastoma multiforme (GBM). GBMs eventually relapse after treatment and the average survival of GBM patients is less than two years. There is some evidence that cannabidiol (CBD) can induce cell death and increases the radiosensitivity of GBM by enhancing apoptosis. Beside initiation of death, CBD has been demonstrated as an inducer of autophagy. In the present study, we address the question whether CBD simultaneously induces a protective effect in GBM by upregulating autophagy. Addition of chloroquine that suppressed autophagic flux to 2D GBM cultures increased CBD-induced cell death, presenting proof for the protective autophagy. Blockage of autophagy upregulated radiation-induced cytotoxicity but only modestly affected the levels of cell death in CBD- or CBD/γ-irradiated 3D GBM cultures. Furthermore, CBD enhanced the pro-apoptotic activities of JNK1/2 and MAPK p38 signaling cascades while partially downregulated the pro-survival PI3K-AKT cascade, thereby changing a balance between cell death and survival. Suppression of JNK activation partially reduced CBD-induced cell death in 3D GBM cultures. In contrast, co-treatment of CBD-targeted cells with inhibitors of PI3K-AKT-NF-κB, IKK-NF-κB or JAK2-STAT3 pathways killed surviving GBM cells in both 2D and 3D cultures, potentially improving the therapeutic ratio of GBM.

Eriocalyxin B Induces Apoptosis and Autophagy Involving Akt/Mammalian Target of Rapamycin (mTOR) Pathway in Prostate Cancer Cells.

BackgroundEriocalyxin B (EriB), a diterpenoid isolated from the plant Isodon eriocalyx, has been shown to possess anti-tumor properties. However, few systematic studies of the mechanism underlying the anti-tumor activity of Eriocalyxin B in prostate cancer cells have been published. The aim of this study was to investigate the effect of Eriocalyxin B on prostate cancer cells.Material/MethodsIn the present study, the PC-3 (androgen-independent) and 22RV1 (androgen-dependent) human prostate cancer cell lines were cultured with and without increasing doses of Eriocalyxin B. MTT assay was used to measure cell viability. Western blotting was performed to measure levels of proteins associated with apoptosis and autophagy. Flow cytometry was used to assess changes in cell apoptosis and cycle. Fluorescence microscopy was used to capture images of autophagy-related proteins.ResultsTreatment of human prostate cancer cells with Eriocalyxin B resulted in apoptosis in a dose- and time-dependent manner. Eriocalyxin B also induced autophagy, with elevated LC3B-II protein expression and punctuate patterns. Additionally, autophagy protected prostate cancer cells from apoptosis induced by Eriocalyxin B, which was demonstrated by addition of chloroquine (CQ). Moreover, the results indicated that Eriocalyxin B could inhibit the phosphorylation of Akt and mTOR. Eriocalyxin B induced apoptosis and autophagy by inhibition of the Akt/mTOR pathway.ConclusionsEriocalyxin B induces apoptosis and autophagy involving the Akt/mTOR pathway in prostate cancer cells in vitro. These findings provide evidence for Eriocalyxin B as a potent therapeutic for the treatment of prostate cancer.

Sufentanil impairs autophagic degradation and inhibits cell migration in NCI-H460 in vitro.

Metastasis, which involves the spread of cancer cells to distant tissues and organs, is a major cause of cancer-associated mortality. Although the use of anesthetics and analgesics may affect cancer cell metastasis, the underlying molecular mechanism remains unclear. Autophagy is a lysosome-based dynamic intracellular catabolic process that serves a crucial role in cancer cell metastasis. In order to investigate the role of autophagy in the migration of cancer cells treated with analgesics, immunofluorescence, western blotting, wound healing assay and cell invasion assay were performed in the present study. The results from immunofluorescence and western blotting demonstrated that the opioid analgesic sufentanil stimulated LC3 induction in NCI-H460 cells. Furthermore, sufentanil increased LC3 and Beclin1 protein levels, but inhibited the fusion of autophagosomes and lysosomes. In addition, sufentanil decreased cathepsin D protein level and increased p62 protein level. The addition of chloroquine (CQ) to sufentanil did not induce a further increase in LC3-II protein levels in NCI-H460 cells, suggesting the impairment of autophagic degradation. Furthermore, treatment with trehalose stimulated the migration of sufentanil-treated cells, whereas additional treatment with CQ did not further decrease the migration of sufentanil-treated cells. In addition, sufentanil co-treatment with trehalose significantly increased the invasion of lung cancer cells, whereas, additional treatment with CQ did not further reduce the invasion of sufentanil-treated cells. These results indicated that autophagy may be involved in the inhibition of NCI-H460 cell migration by sufentanil, and that sufentanil may be considered as a favorable analgesic for patients with lung cancer.

425P - CHLOROBRAIN phase IB trial: The addition of chloroquine, an autophagy inhibitor, to concurrent radiation and temozolomide for newly diagnosed glioblastoma

BackgroundTreatment of glioblastoma (GBM) xenografts with chloroquine (CQ) has been shown to inhibit autophagy, thereby reducing the hypoxic fraction and sensitizing tumours to radiation. Preclinical evidence shows that EGFRvIII+ GBM may benefit most from CQ because of autophagy dependency. This study explores the safety, pharmacokinetics and maximum tolerated dose (MTD) of CQ in combination with radiotherapy (RT) and concurrent daily temozolomide (TMZ) in patients with a newly diagnosed GBM. MethodsThis study is a single-centre, open label, dose-finding phase I trial (3+3 design). Patients received oral CQ once daily one week before the course of concurrent chemoradiation (TMZ 75mg/m2/day) until the end of RT. Toxicity was scored according to the CTCAE 4.0. Molecular markers were identified on paraffin embedded tissue. ResultsThirteen patients were included in the study (n=6:200mg, n=3:300mg, n=4:400mg CQ). Tumour characteristics: 7/13 MGMT promotor hypermethylation, 12/13 IDH wildtype, 0/13 1p/19q co-deletion and 7/13 EGFRvIII expression. A total of 44 adverse events possibly/likely related to CQ were registered. Serious adverse events are presented in Table. In the 400mg cohort 2 patients developed ECG QTc prolongation and 1 patient developed irreversible blurred vision. Three patients developed grade III nausea/vomiting (n=2 300mg, n=1 200mg) resulting in cessation of TMZ or delay of adjuvant TMZ cycles. Median overall survival is 8,1 months for EGFRvIII – and 13,4 months for EGFRvIII + patients; with 7 patients currently still alive at a median follow-up of 9 months (range 2 – 21).Table: 425PSerious adverse events grade I - V - Adverse events possibly/likely related to chloroquine are presented with an *.Table: 425PToxicityGrade 1Grade 2Grade 3Grade 4Grade 5Blurred Vision*1Confusion1Electrocardiogram (ECG) QT corrected interval prolonged*11Fall (trauma capitis)*1Hypercalcemia1Nausea/vomiting*33Seizure1Thromboembolic event11 ConclusionsA daily dose of 200mg CQ was established as the MTD when combined with RT and concurrent TMZ for newly diagnosed GBM. Favorable tolerability and extensive pre-clinical evidence on anti-tumour activity support further clinical phase II and III studies. Clinical trial identificationThe study was approved by the Medical Review Ethics Committee Maastricht UMC+: #NL52723.068.15/METC153051. (NCT02378532). Legal entity responsible for the studyThe authors. FundingStichting StopHersentumoren. DisclosureAll authors have declared no conflicts of interest.

Maduramicin inactivation of Akt impairs autophagic flux leading to accumulated autophagosomes-dependent apoptosis in skeletal myoblast cells.

It has been clinically documented that maduramicin (Mad), a polyether ionophore antibiotic widely used in the control of coccidiosis in poultry worldwide, can elicit skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. Here, we show that Mad induced apoptosis dose-dependently, which was associated with impaired autophagic flux in skeletal myoblast (C2C12 and L6) cells. This is supported by the findings that Mad treatment resulted in increase of autophagosomes with a concomitant elevation of LC3-II and p62 in the cells. Also, Mad increased co-localization of mCherry and GFP tandem-tagged LC3 puncta in the cells, suggesting a blockage of autophagic flux. Furthermore, addition of chloroquine (CQ) strengthened the basic and Mad-enhanced LC3-II and p62 levels, autophagosome formation and cell apoptosis, whereas pretreatment with rapamycin alleviated the effects in the cells exposed to Mad. Moreover, we noticed that Mad treatment inactivated Akt dose-dependently. Inhibition of Akt with inhibitor X potentiated Mad-induced decrease in phosphorylated Akt, and increases in LC3-II and p62 levels, autophagosome formation and cell apoptosis, whereas ectopic expression of constitutively active Akt rendered resistance to these events. Collectively, these results indicate that Mad inactivation of Akt impairs autophagic flux leading to accumulated autophagosomes-dependent apoptosis in skeletal myoblast cells. Our findings suggest that manipulation of Akt activity to improve autophagic flux is a promising strategy against Mad-induced myotoxicity.

Abstract 5278: Separate and combined effects of metabolic reprogramming interventions, autophagy inhibition, and carboplatin on murine triple-negative breast cancer cells

Abstract Triple-negative breast cancers (TNBC) are an aggressive BC subset currently lacking targeted therapies. TNBCs exhibit aberrant fuel metabolism, including increased flux through glycolysis and the pentose phosphate pathway, increased lactate production (Warburg metabolism), and elevated dependence on glutamine (glutaminolysis). While dietary interventions such as caloric restriction, intermittent fasting, or ketogenic diets have demonstrated benefits (e.g, reduced tumor incidence, progression, and sensitization to some chemotherapeutic agents) in preclinical BC models, translation to humans has proven challenging, and concerns remain regarding tolerance and long-term safety of extreme diets in cancer patients. We hypothesized that targeted disruption of select metabolic pathways could recapitulate the beneficial effects of the aforementioned dietary interventions and improve the efficacy of carboplatin, part of first-line chemotherapy for primary and metastatic TNBCs. Moreover, we posited that blocking autophagy would further enhance the anticancer effects of metabolic reprogramming interventions (MRIs) alone and in combination with carboplatin. To test these hypotheses, we are using a high-throughput screening approach employing several MRIs with or without the addition of chloroquine and/or carboplatin in vitro. Serum starvation (an in vitro model of caloric restriction), rapamycin, BMS-754807 (a dual IGF-1R/insulin receptor), and nicotinamide significantly reduced cell viability (assessed by MTT assay) and clonogenicity (assessed by colony formation assay) of M-Wnt and M6 cells, both well-characterized TNBC cell lines derived from mammary tumors of MMTV-Wnt-1 and C3-TAg transgenic mice, respectively. The growth suppressive effects of these MRIs were further enhanced when combined with autophagy inhibition via chloroquine (CQ). Treatment of M-Wnt and M6 cells with CQ (20-80 µM) also increased in vitro cytotoxicity of carboplatin. Ongoing analyses and planned in vivo studies will confirm whether co-treatment with MRIs and/or CQ are an effective strategy for increasing the efficacy of carboplatin in TNBC cells. Citation Format: Alyssa J. Cozzo, Michael F. Coleman, Ciara H. O'Flanagan, Jane B. Pearce, Magdalena A. Rainey, Stephen D. Hursting. Separate and combined effects of metabolic reprogramming interventions, autophagy inhibition, and carboplatin on murine triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5278.

Silver nanoparticles induce protective autophagy via Ca2+/CaMKKβ/AMPK/mTOR pathway in SH-SY5Y cells and rat brains

Silver nanoparticles (AgNPs) are widely used for manufacturing products containing antibacterial agents, as well as food technologies such as edible films and food packaging. Routes of AgNPs exposure are principally derived by contacting with certain medical sprays, food, toothpaste, and purification products. Previously, we showed that AgNPs induce endoplasmic reticulum (ER) stress and promote apoptosis progression in SH-SY5Y cells; however, whether AgNP-induced ER stress is able to trigger autophagy in vivo and in vitro, and the role of autophagy in AgNP-induced cytotoxicity remain unclear. In the present study, we found that increased intracellular calcium (Ca2+) levels arising from AgNP-induced-ER stress resulted in activation of calmodulin-dependent protein kinase kinase β (CaMKKβ) and adenosine 5′-monophosphate-activated protein kinase (AMPK), which downregulated the level of mammalian target of rapamycin (mTOR) and upregulated Beclin-1 to activate autophagy in SH-SY5Y cells. Specifically, inhibition of autophagy by the addition of chloroquine (CQ) or silencing of Beclin-1 significantly enhanced the cytotoxicity of AgNPs, suggesting that autophagy plays a protective role in AgNP-induced cell apoptosis. Furthermore, we showed that oral administration of AgNPs for 28 continuous days induced ER stress-mediated apoptosis and autophagy in rats via activation of CaMKKβ and AMPK. In summary, this study is the first to report that AgNPs induce protective autophagy via a Ca2+/CaMKKβ/AMPK/mTOR pathway in vivo and in vitro. Therefore, public exposure to AgNPs should arouse concerns regarding environmental safety and human health.HighlightSilver nanoparticle-induced ER stress elicits protective autophagy via a Ca2+-dependent mechanism in SH-SY5Y cells.The Ca2+/CaMKKβ/AMPK/mTOR pathway is involved in autophagy.Orally administered silver nanoparticles induce ER stress-mediated autophagy and apoptosis in rats.

Chloroquine Protects Human Corneal Epithelial Cells from Desiccation Stress Induced Inflammation without Altering the Autophagy Flux.

Dry eye disease (DED) is a multifactorial ocular surface disorder affecting millions of individuals worldwide. Inflammation has been associated with dry eye and anti-inflammatory drugs are now being targeted as the alternate therapeutic approach for dry eye condition. In this study, we have explored the anti-inflammatory and autophagy modulating effect of chloroquine (CQ) in human corneal epithelial and human corneal fibroblasts cells exposed to desiccation stress, (an in-vitro model for DED). Gene and protein expression profiling of inflammatory and autophagy related molecular factors were analyzed in HCE-T and primary HCF cells exposed to desiccation stress with and without CQ treatment. HCE-T and HCF cells exposed to desiccation stress exhibited increased levels of activated p65, TNF-α, MCP-1, MMP-9, and IL-6. Further, treatment with CQ decreased the levels of active p65, TNF-α, MCP-1, and MMP-9 in cells underdesiccation stress. Increased levels of LC3B and LAMP1 markers in HCE-T cells exposed to desiccation stress suggest activation of autophagy and the addition of CQ did not alter these levels. Changes in the phosphorylation levels of MAPKinase and mTOR pathway proteins were found in HCE-T cells under desiccation stress with or without CQ treatment. Taken together, the data suggests that HCE-T cells under desiccation stress showed NFκB mediated inflammation, which was rescued through the anti-inflammatory effect of CQ without altering the autophagy flux. Therefore, CQ may be used as an alternate therapeutic management for dry eye condition.