Chloroquine Diphosphate Research Articles

Abstract 297: Activity of carfilzomib (CFZ) alone and in combination with cytarabine and chloroquine diphosphate (CHQ) in acute myeloid leukemia (AML)

Abstract Background: Despite the recent advances in AML with the emergence of multiple targeted therapies, patient outcomes remain poor, particularly in the relapsed/refractory setting underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs) have antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis with multiple clinical trials showing promising activity of bortezomib particularly when combined with other cytotoxic agents (Csizmar 2016). CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib and is therefore an agent of interest in AML therapy (van der Helm 2015). Here we assessed the activity of CFZ alone and in combination with cytarabine and the autophagy inhibitor CHQ in AML cell lines. Methods: After screening 18 AML cell lines, 3 sensitive: KASUMI1, ML2, and MV411, and 3 resistant cell lines: AML193, THP1, and NOMO1, were selected for further analysis using an IC50 cutoff for CFZ of 10 nM. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM, cytarabine 200 nM, and CHQ 20 µM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA) and confirmed with western blot analysis. Results: Single-agent CFZ induced apoptosis in sensitive cell lines with apoptotic rates increasing by 85% to 95%; while apoptotic rates remained unchanged in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis and cell cycle arrest as the major mechanisms of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ 10 nM was combined with cytarabine (200 nM). RPPA revealed that AML cell lines with low expression of autophagy related proteins are more sensitive to proteasome inhibition with CFZ treatment compared to resistant cell lines. This was confirmed with western blot which showed upregulation of the autophagy related proteins in resistant cell lines upon exposure to CFZ. However, sensitive cell lines did not upregulate autophagy pathways which may lead to cell cycle arrest and apoptosis. Inhibiting autophagy with CHQ sensitized CFZ resistant lines to CFZ treatment. Conclusion: CFZ demonstrated single agent activity in human AML cell lines and upregulation of autophagy may contribute to resistance to CFZ which can be overcome using autophagy inhibitors such as CHQ. These results support the development of CFZ and autophagy inhibitor-based combinations for AML. Citation Format: Mao Yu Peng, Yasmin Abaza, Dylan Conklin, Erika Von Euw, Monica Mead, John Timmerman, Dennis Slamon, Sarah Larson. Activity of carfilzomib (CFZ) alone and in combination with cytarabine and chloroquine diphosphate (CHQ) in acute myeloid leukemia (AML) [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 297.

Cytotoxicity and in vitro evaluation of whey protein-based hydrogels for diabetes mellitus treatment

Obesity is the accumulation of excess body fat and the hallmark of type II diabetes mellitus, characterized by hyperglycemia. Glycemic control is very critical to reduce long-term vascular complications resulting from the progressive nature of hyperglycemia. In previous studies, thermally reduced graphene oxide (rGO)-based hydrogel biocomposites were prepared and in vitro drug release studies confirmed their potential as a biodegradable-targeted drug delivery system. Thus, the in vitro biological evaluation of these rGO-based hydrogels was investigated. The hydrogels were encapsulated with chloroquine diphosphate (CQ) and proguanil (P) drugs to investigate potential of combination therapy. The non-toxic nature of the hydrogels was investigated by the use of the MTT assay against 3T3-L1 and c2c12 cell lines. 3T3-L1 pre-adipocytes were grown, differentiated and treated with the drug-encapsulated hydrogels to detect the effect on the adipose tissue cells by quantitative real-time polymerase chain reaction (qPCR) identifying gene expression levels by utilizing gene markers specific for diabetes and obesity: cpt-1, glut-4, acc1, pgc-1, mef2a and nrf-1 with comparison to positive control metformin. The cytoxicity studies confirmed non-toxic nature of the hydrogels; identified dosage of drugs encapsulated were effective within investigated treatment time and qPCR revealed an upregulation of CPT-1, GLUT-4, PGC-1, MEF2A and NRF-1 marker genes, but a downregulation of ACC-1 marker gene. The results from the expression of investigated genes suggest the anti-obesity potential of drugs released from the hydrogels. There were identified positive effects employing combination therapy, but further studies are required to ascertain the actual effect of the drugs in combination, by further varying the ratios of drugs (instead of the presented 1:1 ratio) employed. Statistically, the results from the individual drug release treatments were not significantly different from positive control metformin treatments, but the combination therapy investigation showed more promise.

Gene delivery to the rat retina by non-viral vectors based on chloroquine-containing cationic niosomes

The incorporation of chloroquine within nano formulations, rather than as a co-treatment of the cells, could open a new avenue for in vivo retinal gene delivery. In this manuscript, we evaluated the incorporation of chloroquine diphosphate into the cationic niosome formulation composed of poloxamer 188, polysorbate 80 non-ionic surfactants, and 2,3-di (tetradecyloxy) propan-1-amine (hydrochloride salt) cationic lipid, to transfect rat retina. Niosome formulations without and with chloroquine diphosphate (DPP80, and DPP80-CQ, respectively) were prepared by the reverse phase evaporation technique and characterized in terms of size, PDI, zeta potential, and morphology. After the incorporation of the pCMS-EGFP plasmid, the resultant nioplexes -at different cationic lipid/DNA mass ratios- were further evaluated to compact, liberate, and secure the DNA against enzymatic digestion. In vitro procedures were achieved in ARPE-19 cells to assess transfection efficacy and intracellular transportation. Both nioplexes formulations transfected efficiently ARPE-19 cells, although the cell viability was clearly better in the case of DPP80-CQ nioplexes. After subretinal and intravitreal injections, DPP80 nioplexes were not able to transfect the rat retina. However, chloroquine containing vector showed protein expression in many retinal cells, depending on the administration route. These data provide new insights for retinal gene delivery based on chloroquine-containing niosome non-viral vectors.

Gefitinib-mediated apoptosis is enhanced via inhibition of autophagy by chloroquine diphosphate in cutaneous squamous cell carcinoma cells.

The development of cutaneous squamous cell carcinoma (cSCC) is associated with activation of the epidermal growth factor receptor (EGFR). EGFR-targeting presents a promising strategy for improving therapeutic efficacy. However, recent studies have suggested that tumours overexpressing EGFR depend on autophagy for survival and exhibit resistance to EGFR-targeting drugs. Chloroquine diphosphate (CQ), an autophagy inhibitor that may enhance the cytocidal effect of gefitinib against cSCC, was used in the present study. Cytotoxicity assays were performed to determine the half-maximal inhibitory concentration values of gefitinib and CQ in A431 cells. Drug interaction was analysed using CompuSyn software, which also determined combination index and dose reduction index values. Apoptosis and autophagy of A431 cells were investigated via flow cytometry, western blotting analyses, acridine orange/ethidium bromide staining and monodansylcadaverine staining. Suppression of autophagy by CQ, which was demonstrated by an alteration in microtubule associated protein 1 light chain 3-B in CQ pre-treated A431 cells, significantly enhanced cell apoptosis, which suggested that gefitinib-induced autophagy is cytoprotective. Thus, CQ was demonstrated to exhibit a synergistic apoptotic effect when used in combination with gefitinib during cSCC therapy. Further in vivo investigations are required to confirm the results of the present study.

Phytochemical Analysis of <i>Guiera senegalensis</i> J.F. Gmel Extract and its Anti-Plasmodial Properties on Wister Albino Mice via Oral Route

Guiera senegalensis J.F. Gmel (Combretaceae) is acclaimed as a common herbal antipyretic and anti-malarial among some tribal groups in northern Nigeria. The aim of this study was to investigate the phytochemical constituents, antiplasmodial activity and the acute toxicity of G. senegalensis on mammalian cells. Aqueous ethanolic leaf extracts (AELE) of the plant were tested for the presence of phytochemicals including alkaloids, carbohydrates, flavonoids, cardiac glycoside, glycosides, phenolic acids, saponins and tannins using standard techniques. The AELE was then screened for acute toxicity on Wister albino mice (WAM) weighing between 30-35g and its antiplasmodial activity against Plasmodium berghei (the causative agent of malaria in some mammals). Phytochemical screening revealed the presence of carbohydrate, phenols, flavonoids, abundant tannins, alkaloid, saponins with the absence of cardiac glycosides in the AELE. Also, it was observed that G. senegalensis extracts had no toxic effect on the WAM across administered doses of 100, 200 and 400 mg/kg. From the prophylactic study, it was found that the AELE dosage of 200 mg/kg was most effective in the inhibition of P. berghei when compared with the control than other dosages tested (100 and 200 mg/kg). The extract also exhibited a better anti-plasmodial property (80% inhibition) in the curative study compared to the standard drug (Chloroquine diphosphate) (52%), inhibiting at the graded doses of AELE. It can, therefore, be concluded from this study that G. senegalensis extract possesses essential phytochemicals, resulting in antiplasmodial effect against Plasmodium berghei.

Entrapped chemically synthesized gold nanoparticles combined with polyethylene glycol and chloroquine diphosphate as an improved antimalarial drug

Objective(s): Drug delivery is an engineering technology to control the release and delivery of therapeutic agents to target organs, tissues, and cells. Metallic nanoparticles, such as gold nanoparticles (AuNPs) have exceptional properties which enable efficient drug transport into different cell types with reduced side effects and cytotoxicity to other tissues.Materials and Methods: AuNPs were synthesized by adopting the Turkevich method to reduce tetra chloroauric (III) acid (HAuCl4) solution with sodium citrate. A factorial design of 24 was used to investigate the influence of temperature, stirring speed, and the volume of citrate and gold salt on the size of AuNPs synthesis. The produced chemical-AuNPs (CN-AuNPs) were characterized using ultraviolet-visible spectroscopy and dynamic light scattering (DLS) which was conjugated with polyethylene glycol (PEG) loaded with chloroquine diphosphate. The latter were characterized with transmission electron microscopy (TEM), Energy dispersive x-ray spectroscopy (EDS), selected area electron diffraction (SAED) patterns and Fourier transmission infrared spectroscopy. The antimalarial activities of the three formulations were tested on Plasmodium-infected mice. Moreover, the evaluation of curative potentials of the formulations was carried out via parasite counts. The anemic and pathological conditions of nano-encapsulation were investigated for their cytotoxicity level. Results: The CN-AuNPs show surface plasmon resonance absorption ranging from 526 to 529 nm with smaller particle size at the lower citrate volume. The TEM image of CN-AuNPs with polyethylene glycol (PEG) and CN-AuNPs-PEG encapsulated with chloroquine diphosphate revealed spherical shape with EDS showing the appearance of gold (Au) at 2.0, 2.1, and 9.9 KeV. The SAED also revealed that the AuNPs were crystalline in nature. The in vitro time-dependent encapsulation release showed an extension of time release, compared to CN-AuNPs-PEG with parasitemia clearance at the same level of cytotoxicity. Conclusion: Therefore, although improved activity of the CN-AuNPs-PEG encapsulating was achieved but its cytotoxicity still is a limitation.

Inhibition of Autophagy Promoted Apoptosis and Suppressed Growth of Hepatocellular Carcinoma Upon Photothermal Exposure.

Photothermal therapy (PTT) is a recently developed and promising strategy for the treatment of hepatocellular carcinoma (HCC). However, apoptosis has been extensively investigated as the mechanism of the underlying effect of PTT on cancer to date. Here, we explored alternative mechanisms of these therapeutic effects, including the activation of cell-cycle arrest and autophagy during PTT in addition to apoptosis under mild temperature. We treated the HCC cell line HepG2 with polydopamine (PDA)-coated branched Au-Ag nanoparticles at various concentrations along with PTT using an 808-nm laser. Apoptosis was evaluated based on flow cytometry, western blot analysis of apoptosis related proteins (BAX, BCL2, caspase 3), Hoechst staining, and TUNEL staining. To explore the role of autophagy, we treated cells with the autophagy inhibitor chloroquine diphosphate. Enhancement of apoptosis by PTT with nanoparticle treatment was observed after autophagy was inhibited. Moreover, inhibition of autophagy markedly enhance the suppression of tumor growth in vivo in a HepG2 mouse xenograft model. These results suggest that further exploration of the mechanism of PTT can help guide its clinical application, and that autophagy inhibition combined with PTT could be a promising strategy for HCC treatment.

Synthesis and Heme Polymerization Inhibitory Activity (HPIA) Assay of Chalcone, Flavone and Flavanone Derivatives

Chalcone, flavone, and flavanone had been synthesized as candidate of the antimalarial compounds. The chalcone 1was synthesised from 2,4-dihydroxyacetophenone and 4-metoxybenzaldehyde as starting materials using base catalyst KOH via Claisen-Schmidt Condensation. The synthesis flavone 2was carried out by cyclization of chalcone 1using I2 in DMSO, while the flavanone 3was produced by cyclization of chalcone 1using NaOAc as a base catalyst. An in vitro antimalarial activity of the compound 1, 2and 3have been evaluated with the chloroquine diphosphate as a positive control in various concentration, and this assay was carried out according to the Basilico method. The IC50value of compound 1, 2, 3and a positive control were 0.25; 0.62; 1.33 and 0.35 mM, respectively. The result showed that chalcone1with the lowestIC50value is a potential candidate as an antimalarial agent.

AKTIVITAS PENGHAMBATAN POLIMERISASI HEM DARI FRAKSI ETIL ASETAT DAUN MANURAN, Coptosapelta tomentosa Valeton ex K.Heyne (Rubiaceae)

Malaria is a serious disease caused by Plasmodium parasites and is transmitted by the salivary glands of female Anopheles mosquito. The manuran (Coptosapelta tomentosa Valeton ex K.Heyne) is empirically used as a malarial treatment. The study aimed to determine the IC 50 value of ethyl acetate fraction and the coumpounds contained on the C. tomentosa Valeton ex K. Heyne leaf ethyl acetat fraction. The identification of chemical composition used tube test method. The inhibitory activity of hem e polymerization in vitro did by Basillico modified method . The identification of chemical contents on the ethyl acetate fraction of C. tomentosa leaf Valeton ex K. Heyne showed positive results containing flavonoid, tannin, saponin, terpenoid, and anthraquinone. The average percentage heme polymerization inhibition of C. tomentosa valeton ex K. Heyne leaf ethyl acetate fraction respectively from large to small concentrations of 97.94; 96.94; 95.01; 91.63; 86.19; 76.12; and 44.83 % . Result of probit analysis, that it has HPIA IC 50 value of 0.252±0.009 mg/mL and chloroquine diphosphate was 0.214±0.012 mg/mL. The independent sample T-test showed that there was significant difference between IC 50 value of them. The ethyl acetate fraction of C. tomentosa leaf Valeton ex K. Heyne has heme polymerization inhibition activity.

Chondroprotection of PPARα activation by WY14643 via autophagy involving Akt and ERK in LPS-treated mouse chondrocytes and osteoarthritis model.

Autophagy maintains cellular homoeostasis. The enhancement of autophagy in chondrocytes could prevent osteoarthritis (OA) progression in articular cartilage. Peroxisome proliferator‐activated receptor α (PPARα) activation may also protect articular chondrocytes against cartilage degradation in OA. However, whether the protective effect of activated PPARα is associated with autophagy induction in chondrocytes is not determined. In this study, we investigated the effect of PPARα activation by its agonist, WY14643, on the protein expression level of Aggrecan and ADAMTS5, and the protein expression level of autophagy biomarkers, including LC3B and P62, using Western blotting analysis in isolated mouse chondrocytes pre‐treated with lipopolysaccharides (LPS, mimicking OA chondrocytes) with or without the autophagy inhibitor chloroquine diphosphate salt. Furthermore, Akt and ERK phosphorylation was detected in LPS‐treated chondrocytes in response to WY14643. In addition, the effect of intra‐articularly injected WY14643 on articular cartilage in a mouse OA model established by the destabilization of the medial meniscus was assessed using the Osteoarthritis Research Society International (OARSI) histopathology assessment system, along with the detection of Aggrecan, ADAMTS5, LC3B and P62 protein levels using immunohistochemistry assay. The results indicated that PPARα activation by WY14643 promoted proteoglycan synthesis by autophagy enhancement in OA chondrocytes in vivo and in vitro concomitant with the elevation of Akt and ERK phosphorylation. Therefore, autophagy could contribute to the chondroprotection of PPARα activation by WY14643, with the implication that PPARα activation by WY14643 may be a potential approach for OA therapy.

Electroretinogram as an early detection of chloroquine retinal toxicity in pigmented rabbits

Objective Chloroquine (CQ) is a useful drug. It has less systemic hazards than many of the other medications used for immune diseases, but may cause retinopathy. The ophthalmologists do not discover the retinopathy till the appearance of bull’s eye. The aim of the work is to try to use electroretinogram (ERG) and its b/a ratio in early detection of CQ retinopathy for pigmented rabbits chronically treated with CQ. Materials and methods Twenty adult pigmented rabbits (1.5–2 kg) were classified into four groups (five each): the first group served as control and the other three groups of rabbits were intramuscularly injected with CQ diphosphate (14 mg/kg body weight) three times/week for 50, 70, and 90 days, respectively. Each group is subjected to ERG recording, and then decapitation of animal and measurement of oxidant, antioxidant parameters, and histopathological examination in the retinal tissue were done. Results All results show no change by the 50th day after CQ administration. All the changes appear by the 70th day. The data indicated there is a deformation in ERG waves and significant decrease (P Conclusion Our data showed the possibility of using b/a ratio as an early indicator to CQ retinal toxicity in pigmented rabbits. It is also suggested that CQ increases the oxidative stress levels, so supplementation by antioxidants should be a part of the treatment regime.

Anti-Human Rhinovirus 1B Activity of Dexamethasone viaGCR-Dependent Autophagy Activation.

ObjectivesHuman rhinoviruses (HRVs) are the major cause of the common cold. Currently there is no registered, clinically effective, antiviral chemotherapeutic agent to treat diseases caused by HRVs. In this study, the antiviral activity of dexamethasone (DEX) against HRV1B was examined.MethodsThe anti–HRV1B activity of DEX was assessed by sulforhodamine B assay in HeLa cells, and by RT-PCR in the lungs of HRV1B-infected mice. Histological evaluation of HRV1B-infected lungs was performed and a histological score was given. Anti-HRV1B activity of DEX via the glucocorticoid receptor (GCR)-dependent autophagy activation was assessed by blocking with chloroquine diphosphate salt or bafilomycin A1 treatment.ResultsIn HRV1B-infected HeLa cells, treatment with DEX in a dose-dependent manner, resulted in a cell viability of > 70% indicating that HRV1B viral replication was reduced by DEX treatment. HRV1B infected mice treated with DEX, had evidence of reduced inflammation and a moderate histological score. DEX treatment showed antiviral activity against HRV1B via GCR-dependent autophagy activation.ConclusionThis study demonstrated that DEX treatment showed anti-HRV1B activity via GCR-dependent autophagy activation in HeLa cells and HRV1B infected mice. Further investigation assessing the development of topical formulations may enable the development of improved DEX effectiveness.

Potential ceRNA networks involved in autophagy suppression of pancreatic cancer caused by chloroquine diphosphate: A study based on differentially‑expressed circRNAs, lncRNAs, miRNAs and mRNAs.

Autophagy has been reported to be involved in the occurrence and development of pancreatic cancer. However, the mechanism of autophagy-associated non-coding RNAs (ncRNAs) in pancreatic cancer remains largely unknown. In the present study, microarrays were used to detect differential expression of mRNAs, microRNAs (miRNAs), long ncRNAs (lncRNAs) and circular RNAs (circRNAs) post autophagy suppression by chloroquine diphosphate in PANC-1 cells. Collectively, 3,966 mRNAs, 3,184 lncRNAs and 9,420 circRNAs were differentially expressed. Additionally, only two miRNAs (hsa-miR-663a-5p and hsa-miR-154-3p) were underexpressed in the PANC-1 cells in the autophagy-suppression group. Furthermore, miR-663a-5p with 9 circRNAs, 8 lncRNAs and 46 genes could form a prospective ceRNA network associated with autophagy in pancreatic cancer cells. In addition, another ceRNA network containing miR-154-3p, 5 circRNAs, 2 lncRNAs and 11 genes was also constructed. The potential multiple ceRNA, miRNA and mRNA associations may serve pivotal roles in the autophagy of pancreatic cancer cells, which lays the theoretical foundation for subsequent investigations on pancreatic cancer.

Improving Encapsulation of Hydrophilic Chloroquine Diphosphate into Biodegradable Nanoparticles: A Promising Approach against Herpes Virus Simplex-1 Infection

Chloroquine diphosphate (CQ) is a hydrophilic drug with low entrapment efficiency in hydrophobic nanoparticles (NP). Herpes simplex virus type 1 (HSV-1) is an enveloped double-stranded DNA virus worldwide known as a common human pathogen. This study aims to develop chloroquine-loaded poly(lactic acid) (PLA) nanoparticles (CQ-NP) to improve the chloroquine anti- HSV-1 efficacy. CQ-NP were successfully prepared using a modified emulsification-solvent evaporation method. Physicochemical properties of the NP were monitored using dynamic light scattering, atomic force microscopy, drug loading efficiency, and drug release studies. Spherical nanoparticles were produced with modal diameter of <300 nm, zeta potential of −20 mv and encapsulation efficiency of 64.1%. In vitro assays of CQ-NP performed in Vero E6 cells, using the MTT-assay, revealed different cytotoxicity levels. Blank nanoparticles (B-NP) were biocompatible. Finally, the antiviral activity tested by the plaque reduction assay revealed greater efficacy for CQ-NP compared to CQ at concentrations equal to or lower than 20 µg mL−1 (p < 0.001). On the other hand, the B-NP had no antiviral activity. The CQ-NP has shown feasible properties and great potential to improve the antiviral activity of drugs.

Chloroquine Is Effective for Maintenance of Remission in Autoimmune Hepatitis: Controlled, Double-Blind, Randomized Trial.

Between 50% and 86% of patients with autoimmune hepatitis (AIH) relapse after immunosuppression withdrawal; long‐term immunosuppression is associated with increased risk of neoplasias and infections. Chloroquine diphosphate (CQ) is an immunomodulatory drug that reduces the risk of flares in rheumatologic diseases. Our aims were to investigate the efficacy and safety of CQ for maintenance of biochemical remission of AIH in a double‐blind randomized trial and to define a subgroup that obtained a greater benefit from its use. A total of 61 patients with AIH in histologic remission (90.1% AIH type 1 [AIH‐1]) were randomized to receive CQ 250 mg/day or placebo for 36 months. Of the 61 patients, 31 received CQ and 30 placebo. At baseline, clinical, laboratory, histologic findings, and human leukocyte antigen (HLA) profile were similar between the two groups. Relapse‐free survival was significantly higher in the CQ group compared to the placebo group (59.3% and 19.9%, respectively P = 0.039). For those patients completing 3‐year treatment, relapse rates were 41.6% and 0% after CQ and placebo withdrawal, respectively. Factors associated with a higher risk of relapse in multiple Cox regression were placebo use (hazard ratio, 2.4; 95% confidence interval [CI], 1.055.5; P = 0.039) and anti‐soluble liver antigen/liver‐pancreas (anti‐SLA/LP) seropositivity (hazard ratio, 5.4; 95% CI, 1.91‐15.3; P = 0.002). Although it was not possible to define a subgroup that obtained a greater benefit from CQ according to anti‐SLA/LP reactivity or HLA profile, 100% of patients who were anti‐SLA/LP‐positive (+) relapsed with placebo compared to 50% with CQ (P = 0.055). In the CQ group, 54.8% had side effects and 19.3% interrupted the drug regimen. Conclusion: CQ safely reduced the risk of relapse of AIH, but it was not possible to define a subgroup that obtained a greater benefit with CQ use, probably because of sample size.

Evaluation of toxic retinopathy caused by antimalarial medications with spectral domain optical coherence tomography.

To investigate the frequency of toxic retinopathy in patients with lupus erythematosus and rheumatoid arthritis with long-term use of chloroquine diphosphate or hydroxychloroquine through spectral domain optical coherence tomography and the outcomes of ophthalmological exams (visual acuity - Snellen's table, color vision test - Ishihara's table, fundoscopy, and retinography - red-free). A cross-sectional study was carried out involving the ophthalmologic evaluation of patients using regular chloroquine diphosphate or hydroxychloroquine for a period of 1 year or longer. The patients completed a questionnaire on their opinions and treatment regularity. The same patients underwent ophthalmologic examination and spectral domain optical coherence tomography. The prevalence of toxic retinopathy caused by antimalarials was 4.15% (9 of 217 patients), 7.4% (4 of 54 patients) following chloroquine diphosphate usage, and 0.82% (1 of 121 patients) following hydroxychloroquine usage. Only patients with advanced stage maculopathy presented abnormalities during the ophthalmologic exam: the color vision test was altered in 11.1%, and visual acuity and fundoscopy were altered in 33.3%. Identification of early toxic retinopathy, detected in six patients, was possible using spectral domain optical coherence tomography. The mean duration of antimalarial drug usage among patients with toxic retinopathy was 10.4 years. Only 31% of the patients reported some symptoms during treatment, and although 24% were afraid to use the medication, they did so as prescribed. Use of spectral domain optical coherence tomography was essential for the diagnosis of early-stage antimalarial toxic retinopathy in patients with the following characteristics: asymptomatic, antimalarial use 7 days a week for a period of more than 5 years, and normal clinical ophthalmologic examination.

Hypoxia-induced autophagy of stellate cells inhibits expression and secretion of lumican into microenvironment of pancreatic ductal adenocarcinoma.

Lumican is secreted by pancreatic stellate cells and inhibits cancer progression. Extracellular lumican inhibits cancer cell replication and restrains growth of early-stage pancreatic adenocarcinoma (PDAC) such that patients with tumors containing stromal lumican experience a three-fold longer survival after treatment. In the present study, patient tumor tissues, ex-vivo cultures of patient-derived xenografts (PDX), PDAC stellate and tumor cells were used to investigate whether hypoxia (1% O2) within the tumor microenvironment influences stromal lumican expression and secretion. We observed that hypoxia significantly reduced lumican expression and secretion from pancreatic stellate cells, but not cancer cells. Although hypoxia enhanced lactate dehydrogenase A (LDHA) expression and lactate secretion from all cells, neither hypoxia-induced nor exogenous lactate influenced lumican expression. Autophagy was induced by hypoxia in ex vivo cultures of PDX and pancreatic stellate cells, but not cancer cells cultured in 2D. Autophagic flux inhibitors, bafilomycin A1, chloroquine diphosphate salt, and ammonium chloride prevented hypoxia-mediated reduction in lumican expression in stellate cells. Furthermore, inhibition of AMP-regulated protein kinase (AMPK) phosphorylation or hypoxia-inducible factor (HIF)-1α expression within hypoxic stellate cells restored lumican expression levels. Hypoxia did not affect lumican mRNA expression, indicating that hypoxia-induced reduction of lumican occurs post-transcriptionally; in addition, AMPK inhibition prevented hypoxia-reduced phosphorylation of the mTOR/p70S6K/4EBP signaling pathway, a key contributor to protein synthesis. Taken together, these findings demonstrate that hypoxia reduces stromal lumican in PDAC through autophagy-mediated degradation and reduction in protein synthesis within pancreatic cancer stellate cells.

The role of autophagy in the radiosensitivity of the radioresistant human nasopharyngeal carcinoma cell line CNE-2R

PurposeThe present study aimed to study the role of autophagy in the radiosensitivity of the radioresistant human nasopharyngeal carcinoma cell line CNE-2R.MethodsBefore being irradiated, CNE-2R cells were treated with the autophagy inhibitor chloroquine diphosphate (CDP) or the autophagy inducer rapamycin (RAPA). Microtubule-associated protein light chain 3 (LC3-II) and p62 were assessed using Western blotting analysis 48 hours after CNE-2R cells were irradiated. The percentage of apoptotic cells was assessed via flow cytometry. CNE-2R cell viability was evaluated using the Cell Counting Kit-8 (CCK8). The radiosensitivity of cells was assessed via clone formation analysis.ResultsThe level of autophagy in CNE-2R cells improved as the radiation dose increased, reaching the maximum at a dose of 10 Gy. Autophagy was most significantly inhibited by 60 µmol/L CDP in CNE-2R cells, but was obviously enhanced by 100 nmol/L RAPA. Compared with the irradiation (IR) alone group, in the IR + CDP group, autophagy was significantly inhibited, viability was low, the rate of radiation-induced apoptosis was increased, and radiosensitivity was upregulated. In contrast, cells of the IR + RAPA group exhibited greater autophagy, higher viability, a lower rate of radiation-induced apoptosis, and downregulated radiosensitivity.ConclusionThe autophagy level is negatively correlated with radiosensitivity for the radio-resistant human nasopharyngeal carcinoma cell line CNE-2R.

Botulinum toxin type A induces protective autophagy in human dermal microvascular endothelial cells exposed to an in vitro model of ischemia/reperfusion injury.

Botulinum toxin type A (BTXA) has been reported to increase the survival of ischemic skin flaps; however, the exact mechanism underlying this effect remains unclear and needs to be further established. The present study aimed to elucidate whether autophagy caused by BTXA functions as a protection mechanism and to identify the mechanisms of its regulation by BTXA in human dermal microvascular endothelial cells (HDMECs) subjected to hypoxia/reoxygenation (H/R)-induced injury. HDMECs were harvested from the upper eyelid tissues of female blepharoplasty patients. HDMECs were exposed to BTXA treatment for 12 h and then subjected to hypoxia for 8 h, followed by reoxygenation for 24 h. Chloroquine diphosphate salt (CQ) was used as an autophagy inhibitor. H/R led to extreme injury to the HDMECs as indicated by the rise in the apoptosis rate, which was significantly attenuated by BTXA pretreatment. The outcomes demonstrated that H/R caused autophagy, as evidenced by a higher type II/type I ratio of light chain 3 (LC3), increased expression of Beclin-1 and increased autophagosome formation. BTXA enhanced autophagy and attenuated apoptosis in a dose-dependent manner, whereas CQ attenuated the BTXA antiapoptotic effects and inhibited the formation of autophagolysosomes, which caused clustering of the LC3-II in cells. In conclusion, autophagy promoted by BTXA serves as a potential protective effect on ischemia/reperfusion injury.

Irisin alleviates pressure overload-induced cardiac hypertrophy by inducing protective autophagy via mTOR-independent activation of the AMPK-ULK1 pathway

In hypertrophic hearts, autophagic flux insufficiency is recognized as a key pathology leading to maladaptive cardiac remodeling and heart failure. This study aimed to illuminate the cardioprotective role and mechanisms of a new myokine and adipokine, irisin, in cardiac hypertrophy and remodeling. Adult male wild-type, mouse-FNDC5 (irisin-precursor)-knockout and FNDC5 transgenic mice received 4 weeks of transverse aortic constriction (TAC) alone or combined with intraperitoneal injection of chloroquine diphosphate (CQ). Endogenous FNDC5 ablation aggravated and exogenous FNDC5 overexpression attenuated the TAC-induced hypertrophic damage in the heart, which was comparable to the protection of irisin against cardiomyocyte hypertrophy induced by angiotensin II (Ang II) or phenylephrine (PE). Accumulated autophagosome and impaired autophagy flux occurred in the TAC-treated myocardium and Ang II- or PE-insulted cardiomyocytes. Irisin deficiency caused reduced autophagy and aggravated autophagy flux failure, whereas irisin overexpression or supplementation induced protective autophagy and improved autophagy flux, which were reversed by autophagy inhibitors Atg5 siRNA, 3-MA and CQ. Irisin boosted the activity of only AMPK but not Akt and MAPK family members in hypertrophic hearts and cultured cardiomyocytes and further activated ULK1 at Ser555 but not Ser757 and did not affect the mTOR-S6K axis. Blockage of AMPK and ULK1 with compund C and SBI-0206965, respectively, both abrogated irisin's protection against cardiomyocyte hypertrophic injury and reversed its induction of both autophagy and autophagy flux. Our results suggest that irisin protects against pressure overload-induced cardiac hypertrophy by inducing protective autophagy and autophagy flux via activating AMPK-ULK1 signaling.